Session I: Muscle: Mechanics

    The role of stretch-induced structural changes of the thick filament in length-dependent activation in cardiac muscle

    S.-J. Park-Holohan, E. Brunello, T. Kampourakis, M. Rees, M. Irving and L. Fusi

    King’s College London, School of Basic and Medical Biosciences, Randall Centre, UK

    The contractility of cardiac muscle is regulated by thick filament-based mechanisms in addition to the classical calcium/thin-filament mediated mechanisms. Here we tested the hypothesis that titin-based passive tension induces regulatory structural changes in the thick filament which mediate length-dependent activation (LDA) in cardiac muscle. We used fluorescence polarization from bifunctional sulphorhodamine probes on the N- and C-lobes of the myosin regulatory light chain (RLC) to monitor changes in the orientation of the myosin motors induced by increasing sarcomere length in relaxed and partially calcium-activated demembranated trabeculae. Under relaxing conditions at near physiological temperature and lattice spacing the myosin motors are roughly parallel to the filament axis, consistent with the OFF structure of the thick filament. Cooling of the relaxed trabecula induced a more perpendicular orientation of the myosin motors indicating disruption of the OFF state. Application of a staircase stretch protocol in the sarcomere length range 2.0–2.3 μm (25 °C, 3% dextran) at pCa 9.0 also induced a more perpendicular orientation of the myosin motors, but despite an increase in titin-based passive tension to ~ 30% of the maximum active force (T0), the orientation change was only ~ 4% of that associated with full calcium activation. Larger stretch-induced orientation changes (~ 20%) were observed at pCa 6.6 accompanied by an increase in active force of ~ 0.05 T0. RLC phosphorylation further increased the stretch-induced orientation changes at pCa 6.6 to ~ 50% and the active force response to ~ 0.2 T0. The stretch-induced activation at low calcium concentration was abolished in the presence of 25 μM Blebbistatin. Our results indicate that titin-stress applied on the filament backbone during a diastolic stretch induces only small changes in the structure of the thick filament. However at higher calcium concentrations stretch and partial RLC phosphorylation significantly activate the thick filament. (Supported by Wellcome Trust, BHF, UK).

    Does the amount of stretching affect residual force enhancement in cardiac myofibrils?

    S.W. Han 1, V. Joumaa1, A. Jinha1 and W. Herzog1

    1University of Calgary, Calgary, Canada

    Residual force enhancement (RFE) is defined as the increase in the steady-state, isometric force of a muscle following an active stretch compared to the corresponding (same length, same activation) force of a purely isometric contraction. It is well established that the amount of active stretching associated with a stiffening of titin. However, titin isoforms differ substantially between skeletal and cardiac muscles to the extent where reports have indicated that there is no RFE in cardiac muscles. The purpose of this study was to determine if cardiac myofibrils exhibit RFE, and if RFE depends on the amount of stretching.

    RFE was measured following active stretching of rabbit cardiac myofibrils across two sarcomere length (SL) ranges: (i) 1.8–2.0 μm (n = 8), and (ii) 1.8–2.2 μm (n = 2, under data collection). Myofibrils were set at an average SL of 1.8 μm for both length ranges tested, passively stretched to an average SL of 2.0 μm or 2.2 μm and then activated, in order to measure the purely isometric reference force. Myofibrils were then quickly shortened to an average SL of 1.8 μm, paused for 30 s, and actively stretched back to an average SL of 2.0 μm or 2.2 μm, to induce RFE.

    RFE was observed in all myofibrils and for both stretched magnitudes. RFE was 16.8 ± 3.2% (mean ± 1 SD), and 15.1 ± 1.4% for the 0.2 μm and 0.4 μm stretch magnitudes, respectively. Based on these preliminary results, it appears that cardiac myofibrils exhibit RFE that is independent of the stretch magnitude, suggesting that the structural elements of titin that contribute to RFE are retained in cardiac muscle.

    Effects of inorganic phosphate on muscle contraction: a case where either model or experiments may be right

    A. Månsson

    Linnaeus University, Kalmar, Sweden

    It is well-known that the acto-myosin force-generating transition in muscle is intimately associated with release of inorganic phosphate (Pi) from the active site of myosin. However, the exact sequence of events in relation to the actual Pi release step is controversial. Details of this process are reflected in the relationships between [Pi] and the developed force and movement. In order to account for these relationships, models have proposed branched kinetic pathways or loose coupling between biochemical and force-generating transitions. Here it is tested whether a recent model (Rahman, M.A. et al. 2018, Biophysical Journal, 115, 386–397) without such complexities can account for the effects of altered [Pi] on the force–velocity relationship. The tested model accounts for a range of other contractile phenomena both in the presence and absence of the small molecular compound blebbistatin. The model faithfully reproduces the relationship between [Pi] and isometric force. However, in apparent disagreement with experimental findings in the literature it produces an anomalous force–velocity relationship at elevated [Pi] with more than one possible velocity for a given constant load. This seems to suggest that the model does not capture central features of the force-generating cross-bridge cycle. However, an alternative explanation is that the experiments do not capture the anomalous force–velocity relationship at high [Pi] due to non-uniformities in the force-generating capacity between sarcomeres. Such non-uniformities are likely to exist even within muscle fibre segments whose length or load is feed-back controlled. Acknowledgements of support: EU Horizon2020 FET programme (#732482; Bio4comp) and Swedish Research Council (#2015-05290).

    Dynamics of cardiac thin filaments upon Ca2+ activation and heavy meromyosin binding revealed by high-speed atomic force microscopy

    O.S. Matusovsky and D. E. Rassier

    Department of Kinesiology and Physical Education, McGill University, Montreal, Canada

    High-speed atomic force microscopy (HS-AFM) is a powerful tool to study biological processes with real time imaging and characterization of molecules with 1–5 nm resolution. We used HS-AFM to evaluate the dynamics of regulated cardiac thin filaments (cTFs) isolated as a complex of actin filaments, tropomyosin (Tpm) and troponin (Tn) in the absence or presence of Ca2+, and with weakly or strongly bound myosin heads. Images of cTFs deposited on mica-supported lipid bilayer were visualized by using an ultra-fast detector and a tapping mode cantilever with a low spring constant (0.1–0.2 N/m) and a high-resonant frequency (~ 1 MHz), without fixation or freezing of the samples. The dynamical displacement of individual Tm strands in the center of a regulatory unit of cTF (between two Tn complexes) was observed to expose isolated myosin binding sites at the relaxed and activated conditions. The mean topographical peak heights of cTFs, representing the highest points of filament crossover, visualized in the absence of Ca2+ (B-state) showed similar mean values compared to that of bare actin filaments. The distributions of the topographical peak heights in cTFs from pCa 9.0 to pCa 4.5 were used as an index of changes in the filament structure accompanied by changes in Tpm configuration; low peak heights are characteristic of non-activated cTFs (pCa 9.0) while high peaks represent activated cTFs in the C-state (pCa 4.5) and open M-state (presence of HMM). The ability of HMM to move Tpm does not depend on the activation level of cTFs (the absence or presence of Ca2+ and ATP). Our results support a three-state model of TF activation and advance understanding of cTFs activation by Tpm switching from different states that includes weakly and strongly bound myosin. Supported: NSERC, CFI and partly by Bio-SPM project

    Does Ca2+ sensitivity increase during staircase with intermittent submaximal tetanic contractions?

    B.R. MacIntosh 1, L. Glass1, and A. Cheng2

    1Faculty of Kinesiology, University of Calgary, University of Calgary, Calgary, Alberta, Canada; 2School of Kinesiology and Health Sciences, York University, Toronto, Ontario, Canada

    Activity dependent potentiation is thought to result from phosphorylation of the regulatory light chains of myosin, increasing Ca2+ sensitivity. Yet, Ca2+ sensitivity decreases early in a period of intermittent contractions. The purpose of this study was to investigate the early change in Ca2+ sensitivity during intermittent submaximal tetanic contractions. Mouse flexor digitorum brevis muscle fibres were dissected from mice after cervical disarticulation. Fibres were superfused with Tyrode solution at 3 °C. Length was set to yield maximal tetanic force. Indo-1 was microinjected into fibres and allowed to dissipate for 30 min. Fluorescence was measured at 405 and 495 nm wavelength and the ratio was used to estimate [Ca2+]. A control force-Ca2+ relationship was determined with stimulation over a range of frequencies, yielding constants for slope, max force and half-maximal [Ca2+] (pCa2+50). Data were collected for sequential contractions at 40 Hz at 2 s intervals. Active force decreased over the first 1–4 contractions then increased. A force-pCa2+ curve was fit to each contraction, using the control value for the Hill slope and max force by adjusting pCa2+50 until the curve passed through the target contraction. Data are presented for three contractions for each fibre: first, maximum shift to the right and last contraction. There was a significant shift to the right for pCa2+50 (decreased Ca2+ sensitivity), usually early in the series of intermittent contractions, then pCa2+50 shifted to the right, but remained significantly different from the control value. Although potentiation is associated with increased Ca2+ sensitivity, this increase begins only after Ca2+ sensitivity has decreased and in most cases Ca2+ sensitivity does not increase above the control level.

    Low temperature traps myosin motors of mammalian muscle in a state that prevents activation

    M. Caremani1, E. Brunello1, M. Linari1, L. Fusi2, T. Irving3, D. Gore3, T. Narayanan4, G. Piazzesi1, M. Irving2, V. Lombardi1 and M. Reconditi 1

    1University of Florence, Florence, Italy; 2King’s College London, London, UK; 3BioCAT, Illinois Institute of Technology, Chicago IL, USA; 4ESRF, Grenoble, France

    The structural changes induced in the thick filament of mammalian muscle by changes in temperature are investigated by collecting X-ray diffraction patterns from the fast skeletal muscle of the mouse (EDL, Extensor Digitorum Longus) in the temperature range from physiological (30–35 °C) to 10 °C. In resting muscle the X-ray reflections that signal the OFF state of the thick filament, in which the myosin motors are packed in helical tracks on the surface of the thick filament, indicate that lowering the temperature produces a progressive disruption of the OFF state, with motors moving away from the thick filament. In the range of temperature explored, we find that the number of myosin motors in the OFF state at 10 °C is half of that at 35 °C. In the same range of temperatures, the force at the plateau of isometric contraction is decreased by a factor of 3 by lowering temperature. The corresponding changes in the X-ray signals that report the fraction and the conformation of the actin-attached motors can be explained if the threefold decrease in force is due not only to a decrease in the force-generating transition in the attached motors but also to a twofold reduction in their number. Thus, lowering the temperature reduces the fraction of motors in the OFF state at rest and the fraction of motors attached to actin during isometric contraction to the same extent, suggesting that motors that leave the OFF state accumulate in a refractory disordered state that makes them unavailable for interaction with actin upon activation. This regulatory effect of temperature on the thick filament of mammalian skeletal muscle could represent an energetically convenient mechanism for hibernating animals.

    Motivating myosin: 2-deoxy-ATP induced structural alterations that increase myosin activity

    W. Ma1, M. Childers2, J. Powers2, C. Yuan2, K. McCabe3, M. Geeves4, T. Irving5 and M. Regnier 2

    1Argonne National Laboratory; 2University of Washington; 3University of California at Santa Barbara; 4University of Kent; 5Illinois Institute of Technology

    The naturally occurring nucleotide 2-deoxy-ATP (dATP) can be used by cardiac myosin and increases binding to actin and cross-bridge cycling. To understand the structural basis of this we used experimental and computational modeling approaches. Molecular Dynamics (MD) simulations of pre-powerstroke myosin suggest that dADP.Pi (vs. ADP.Pi) induces changes in contact pairs within the nucleotide binding pocket and these local structural changes translate to exposure of more polar and positive charge on the actin binding surface. Brownian Dynamics simulations with these pre-powerstroke structures suggest that M.dADP.Pi binds more rapidly to actin than M.ADP.Pi. X-ray diffraction analysis of resting cardiac muscle indicates a large increase in the I1,1/I1,0 intensity ratio for M.dADP.Pi vs. M.ADP.Pi, suggesting myosin movement towards thin filaments. This difference is eliminated when ionic strength is reduced to 100 mM (exposing protein surface charge), suggesting greater myosin-actin electrostatic interaction is responsible. The SM3 meridional reflection (indicative of myosin crown spacing) is increased for M.dADP.Pi and similar to activated myosin, without a change in the degree of axial ordering (IM3). Relaxation was studied with time-resolved X-ray diffraction of soleus muscle from a transgenic (Tg) mouse with elevated dATP. Force decay from tetanic contraction is slightly faster for WT soleus, but the rate of recovery of the first-order myosin layer line (MLL1) is significantly faster for Tg-dATP soleus. The radii to the center of mass of myosin heads (Rm) and equatorial intensity ratio (I1,1/I1,0) are larger in resting Tg soleus, indicating myosin is extended closer to actin. MD simulations of post-powerstroke myosin show dADP is more mobile in the nucleotide binding pocket, and alters conformation of residues on the actin binding surface that directly interact with actin. These results suggest dATP increases myosin-actin electrostatic interactions that activate myosin in resting muscle and help maintain an activated state following contraction. EU777204.

    The myosin mesa and hypertrophic cardiomyopathy: mutations to mechanisms to therapies

    J. Spudich 1, K. Ruppel1 and D. Trivedi1

    1Stanford University, Stanford, CA, USA

    After 40 years of developing and utilizing assays to understand the molecular basis of energy transduction by the myosin family of molecular motors, all members of our laboratory are now focused on understanding the underlying biochemical and biophysical bases of human hypertrophic (HCM) and dilated (DCM) cardiomyopathies. Our primary focus is on HCM since these mutations cause the heart to be hypercontractile, and we hope to understand the molecular basis of this increased power output. HCM is most often a result of single missense mutations in one of several sarcomeric proteins, the sarcomere being the fundamental contractile unit of the cardiomyocyte. More than 35% of all HCM mutations occur in the motor domain of human beta-cardiac myosin, while another ~ 35% occur in myosin binding protein-C. Associated with HCM worldwide are heart failure, arrhythmias, and sudden cardiac death at any age. We are using in vitro molecular studies of biochemically reconstituted human sarcomeric protein complexes to elucidate the molecular basis of HCM-induced hypercontractility. We postulated in 2015 that a majority of HCM mutations are likely to be shifting beta-cardiac myosin heads from a sequestered off-state to an active on-state for interaction with actin, resulting in the hypercontractility seen clinically in HCM patients. This hypothesis is different from earlier prevailing views, and this ‘viewing an old disease in a new light’ is the basis of all of our current research. With a detailed molecular understanding of the resultant increase in power output caused by HCM mutations, one should be able to design appropriate small molecule therapies, which are desperately needed for treatment of these diseases.

    The T1 curve of Huxley and Simmons (1971) reassessed

    C. Knupp1 and J.M. Squire 2,3

    1School of Optometry and Vision Sciences, Cardiff University, Cardiff CF10 3NB, UK; 2Muscle Contraction Group, School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol BS8 1TD, UK; 3Faculty of Medicine, Imperial College, London SW7 2BZ, UK

    Myosin cross-bridge stiffness is a key factor in analysing possible scenarios to explain myosin head changes during force generation in active muscles. The seminal study of Huxley and Simmons (1971: Nature 233: 533) suggested that most of the observed half-sarcomere instantaneous compliance (= 1/stiffness) resides in the myosin heads. Their T1 plot showed that, after a very fast release, the half-sarcomere tension reduced to zero after a step of about 6 nm (with improved experiments reduced to 4 nm). However, later X-ray diffraction studies showed that myosin and actin filaments themselves stretch under tension, which means that most (at least two-thirds) of the half sarcomere compliance comes from the filaments and not from cross-bridges. We have now modelled the compliances in a virtual half sarcomere in silico using a modified MusLabel program. We show that the T1 curve comes almost entirely from length changes in the myosin and actin filaments, because the calculated cross-bridge stiffness (greater than 4 pN/nm) is higher than previous studies have suggested.

    In the light of this, we present a plausible modified scenario to describe aspects of the myosin cross-bridge cycle in active muscle. In particular, we suggest that, apart from the filament compliances, most of the cross-bridge contribution to the instantaneous T1 response comes from weakly-bound myosin heads, not myosin heads in strongly attached states. The strongly attached heads would still contribute to the T1 curve, but only in a very minor way. We postulate that their stiffness could be around 1 pN/nm. This would generate a working stroke close to 100 Å from the hydrolysis of one ATP molecule. The new MusLabel program can serve as a tool to calculate sarcomere elastic properties for any vertebrate striated muscle once various parameters have been determined (e.g. tension, T1 intercept, temperature, X-ray diffraction spacing results).

    Time resolved depression of isometric force by Mavacamten in single myofibrils from rabbit psoas and human cardiac muscle

    B. Scellini1, N. Piroddi1, M. Dente1, C. Ferrantini1, R. Coppini2, C.Poggesi1 and C. Tesi 1

    1Dept. Experimental and Clinical Medicine- University of Florence- Italy; 2Neurofarba - University of Florence- Italy

    Mavacamtem (MYK-641, Axon Medchem BV) is a promising small molecule designed to act as allosteric inhibitor of sarcomeric myosins and presently used in preclinical/clinical trials for HCM treatment (Anderson et al., 2018)). Studies of the effects of ligands on the force generation mechanism in intact or skinned striated muscle fibres are complicated by diffusional barriers. These limitations can be overcome by the use of single myofibrils submitted to perturbations of the contractile environment by sudden solution changes (Tesi et al., 2000). Here, single myofibrils or thin bundles of myofibrils from rabbit fast skeletal muscle (psoas) and human donor ventricle (frozen biopsies) have been used to study the effects of μmolar Mavacamtem on maximal isometric force. Both myofibril types were mounted in relaxing solution (pCa 9; [Pi] ~ 200 μM, 15 °C,) and then fully activated (pCa 3.5) by fast alternation of perfusing fluxes. Myofibrils were then suddenly moved to and from a second flux of activating solution containing selected concentrations of Mavacamtem (“jumps”; solution change ~ 10 ms). Relaxation of force was achieved by returning myofibrils to the relaxing solution. When submitted to Mavacamten jumps, both myofibril types responded with a rapid, relaxation-like force drop. The effect was fully reversible but with significantly slower kinetics than that of force development. Dose–response curves confirmed a higher sensitivity of cardiac muscle (IC50 ~ 0.5 μM) compared to fast skeletal (IC50 ~ 5 μM), as previously reported for pCa50-activated ATPase of the same myofibrillar systems (Kawas et al., 2017). Interestingly, Mavacamtem also decreased the rate of force development, in agreement with the reported inhibition of Pi release rate but also with a possible effect on the regulation state mediated by the availability of strongly actin binding heads. SilicoFCM grant agreement n. 777204.

    Session II: Muscle: close-up

    Locating titin on the muscle thick filament

    P. Bennett 1, M. Rees1, A. Fukuzawa1 and M. Gautel1

    1King’s College London, London, UK

    The complex sequence of titin reflects features of the structure and function of the vertebrate striated muscle sarcomere. The C-terminal ~ 200 immunoglobulin (Ig) and fibronectin (Fn) domains have been associated with the thick filament from the tip to the centre, however the precise axial register of titin on the thick filament has not been determined. To do this we have characterised several titin antibodies and determined their positions on the thick filament using super resolution microscopies, STORM and STED. These data plotted together with published information show that the antibody positions are linearly related to domain number in the crossbridge region of the A-band, where the data is in excellent agreement with the regression line (R2 = 0.999). The slope of the line is 4 nm per domain, the expected span of an Ig or Fn domain. This is also in agreement with the assumption that 11 titin super repeats (CSR) of 11 domains (Ig-Fn-Fn-Ig-Fn-Fn-Fn-Ig-Fn-Fn-Fn) are related to the disposition of the accessory protein, myosin binding protein C (MyBP-C), on 9 stripes at ~ 43 nm intervals in each half of the thick filament. Using the known position of MyBP-C stripes establishes that the furthest MyBP-C position from the M-band coincides with the end of the second CSR (domains 8–10). The 9 MyBP-C stripes are therefore related to the ends of CSRs 2–10. This is consistent with a recent publication in which the first two CSRs were deleted with the loss of only one MyBP-C stripe. Proximal to the MyBP-C positions are two stripes of unknown origin, one of which is located at the edge of the bare zone. We find the last Fn domain close to this position, refining the start of the titin bare zone sequence. Furthermore, titin kinase neighbours this Fn domain, suggesting that it may contribute to the stripe 1 density.

    Localisation of individual nebulin molecules in sarcomeres of Nebulin-Dendra2-KI mice

    S. Bogaards 1, M. Yuen1, R. van der Pijl1,2, S. Shen2, P. Tonino2, C. Gregorio2, R. Mayfield2, K. Jalink3, J. Kole1, H. Granzier2 and C. Ottenheijm1,2

    Nebulin spans the length of the thin filament, with its C-terminus located in the z-disc and its N-terminus near the thin filament pointed-end. Genetic mutations in nebulin, as well as in proteins that bind to nebulin cause myopathy. The pathophysiological mechanisms are incompletely understood, in part because of a lack of tools to precisely localize nebulin on the thin filament. We localized nebulin N-termini using a mouse with photoconvertable Dendra2 inserted at the nebulin N-terminus (Dendra2-KI).

    Individual nebulin molecules were visualized by photoactivated localization microscopy (PALM). In PALM, Dendra2 switches between fluorescent states so that in every snapshot, only a small, optically resolvable fraction of Dendra2 is detected. Single FDB fibers of Dendra2-KI mice were isolated and imaged with PALM in TIRF mode. The Dendra2 blinks were background subtracted and Gaussian fitted with a reconstruction resolution of 10 nm. Dendra2 blinks were fitted with a precision of 10–15 nm. Individual nebulin N-termini were normally distributed within the sarcomere, with a width at half-maximum of ~ 120 nm. The average position of the nebulin N-terminus molecules was 1.1 um from the Z-disk. Based on 908,078 localizations, nearly 40% of the theoretical number of nebulin molecules in the sarcomere was detected, assuming a lattice spacing of 35 nm and a hexagonal distribution of thin filaments with 2 nebulin molecules per thin filament. Leiomodin3 (Lmod3) is a thin filament associated molecule, and mutations in LMOD3 cause myopathy. Pilot experiments in Lmod3-KO x nebulin-Dendra2-KI mice (Lmod3-KO Dendra2-KI) show less nebulin molecules, with a wider distribution of nebulin N-termini, and with the average position closer to the z-disc. Thus, the nebulin-Dendra2 mouse model is a useful tool to localize individual nebulin molecules in the muscle’s sarcomere.

    Super-resolution 3D mapping of site-specific phosphorylation signatures of ryanodine receptors in the healthy and failing heart

    T.M.D. Sheard1, M.E. Hurley1, J. Colyer1, E. White1, Y. Hou2, C. Soeller3, A.H. Clowsley3, M.A. Colman1 and I. Jayasinghe 1

    1Faculty of Biological Sciences, University of Leeds LS2 9JT, UK; 2Institute of Experimental Medical Research, Oslo University Hospital, Oslo, Norway; 3Living Systems Institute, University of Exeter, Devon, EX4 4QL, UK.

    The primary intracellular calcium release mechanism in cardiac muscle is dependent on the tightly clustered ryanodine receptors (RyRs), found in compact signalling domains called couplons. Classical super-resolution microscopy methods (e.g. dSTORM, PALM) have previously been used to visualise RyR clusters, however individual receptors remained unresolved due to resolution which was limited to ~ 30 nm. In this abstract we present how the new generation of super-resolution techniques, DNA-PAINT and expansion microscopy (ExM), have allowed individual RyRs to be resolved in both planar sub-sarcolemmal couplons as well as the curved couplons located deeper within cardiomyocytes. Both techniques exploit optical resolution of 10–15 nm and allow three-dimensional (3D) immuno-localisation of not only the position of each RyR, but also their individual phosphorylation for the residue Ser2808, in situ. With a 3D imaging protocol, we observed disturbances to the RyR arrays in the nanometre scale, which accompanied right-heart failure caused by acute pulmonary hypertension. Coinciding with the disease, was a distinct nano-scale gradient of RyR phosphorylation from the edge of the couplon towards the centre, not seen in healthy cells. This spatial pro le appeared to contrast distinctly from that sustained by the cells during acute, physiological hyperphosphorylation when they were stimulated with a α-adrenergic agonist. We simulated the couplons’ calcium release properties using experimentally-mapped RyR cluster geometries and individual phosphorylation signatures. This computational modelling visualised how the nanoscale dispersal of the RyRs during pathology diminishes its intrinsic likelihood to ignite a cytoplasmic calcium signal. It also revealed that the natural topography of RyR phosphorylation could offset potential heterogeneity in couplon excitability, which may arise such RyR re-organisation. Our approach showcases how super-resolution microscopy can be combined with spatial models of protein-ion interactions to characterise nanoscale signalling patterns which are still beyond the detection capabilities of contemporary microscopy techniques.

    Molecular dynamics simulations of cardiac troponin show that phosphorylation stabilizes the ‘open’state and that mutations disrupt this relationship

    J. Eiros-Zamora, I. Gould and S. Marston

    Imperial College London, NHLI, ICTEM, Du Cane Road London, W12 0NN, United Kingdom

    For Molecular Dynamics simulations we used a new force field, termed ff14SB. This improved secondary structure predictions and NMR scalar couplings for proteins in solution as seen by the reduced RMSF. We also produced and analysed a substantially larger dataset: over 50 μs simulation for both P and unP troponin, > 50-fold the previous simulation.

    Despite the long simulations we did not achieve significant convergence, confirming the intrinsic disorder of parts of troponin and the absence of measurable changes in protein structure or flexibility due to phosphorylation. Therefore we have used Markov State Models to understand the dynamic transitions between the structural ensembles of cTn. This revealed a significant difference between phosphorylated and unphosphorylated troponin

    The simulation data was interpreted in terms of the current models for the Ca2+ -switch. The cTnC helix A-B interhelical angle was calculated as a function of time. Transitions to the closed conformation were observed to be more frequent in the WT apo unphosphorylated state and the energy barrier associated for the open-to-closed conformation of the hydrophobic patch of cTnC is lower for the unphosphorylated state than for the phosphorylated state. Helicity was investigated throughout the troponin sequence. The I36-V72 pair were of particular interest since they are not sequential but they are spatially close in the loops connecting helices A–B and C–D, and can interact through backbone hydrogen bonds in the open state. Phosphorylation triggers an increase of about 10% in the cluster population corresponding to having both hydrogen bonds present and also affects timescales associated with the creation and dissociation of the bonds. Thus phosphorylation stabilises the ‘open ‘state

    Further simulations show that the DCM-causing mutation G159D in TnC destabilises the open state leading to an uncoupling effect and that small molecules like SilybinB restore the dynamics of the mutation to normal.

    Session III: Muscle: Cytoskeleton

    The pathological contribution of detyrosinated microtubules to human myocardial mechanics

    M.A. Caporizzo 1, C.Y. Chen1, K. Bedi2, K.B. Margulies1,2 and B.L. Prosser1

    1Department of Physiology, University of Pennsylvania, Perelman School of Medicine; 2Department of Medicine, University of Pennsylvania, Perelman School of Medicine

    A proliferation of stable microtubules (MTs) and their post-translational detyrosination (dTyr) occurs in animal models of heart failure and in end-stage human heart failure. A direct observation of dTyr MTs buckling during myocyte contraction implicates microtubules as mechanical resistors to contractility but the pathological consequences on cardiac function remain to be assessed. By utilizing a combination of genetic and pharmacological approaches to reduce MT detyrosination or depolymerize MTs, we demonstrate that dTyr MTs contribute passive viscoelasticity to myocytes, and that reducing MT dTyr improves the speed and amplitude of myocyte shortening and relaxation in primary murine myocytes. Taken together, these results are consistent with MTs providing viscoelastic resistance to myocyte motion and predictive of MTs reducing contractile performance in pathological conditions where they are stabilized. To determine whether dTyr MTs impact myocyte viscoelasticity and contractile dynamics proportional to the severity of disease, we suppress MT dTyr in freshly isolated human cardiomyocytes and measure myocyte shortening mechanical properties. Myocytes isolated from failing human hearts are found to be more viscoelastic, and suppression of dTyr MTs reduces viscoelasticity and increases unloaded shortening more in failing myocytes than in myocytes isolated from non-failing hearts. Genetic manipulation of dTyr MT levels does not change excitation–contraction coupling and mathematical modelling supports attributing increased shortening to the experimentally measured viscoelastic reduction. However, for microtubules to be tractable targets to restore cardiac performance, the improvements in myocytes must scale to the myocardial level. To this end, we excised trabecula from failing donor hearts and assessed passive mechanics before and after MT depolymerization. At strains less than or equal to that observed in diastolic filling, MT depolymerization reduced myocardial viscoelasticity. Thus, we find that microtubules contribute to the viscoelasticity of failing human myocardium and may provide a target to restore some function in heart failure.

    Titin A178D causes cardiomyopathy in mice through loss of telethonin

    H. Jiang1, C. Hooper1, M. Kelly1, V. Steeples1, G. Patone2, N. Huebner2, J. Simon1, B. Davies3, H. Watkins1 and K. Gehmlich 1

    1Division of Cardiovascular Medicine, University of Oxford, Oxford, UK; 2Max Delbrueck Centre for Molecular Medicine, Berlin, Germany; 3Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK

    In recent years titin has gained attention as a disease gene for Dilated Cardiomyopathy: truncating variants in titin are found in up to 20% of affected individuals. However, very little is known about the contribution of missense variants in titin to cardiomyopathies.

    We identified a titin missense variant (A178D) in the Z-disc portion of the protein in a family characterised by a unique cardiomyopathy phenotype and genetic analyses suggested that this variant is causative for inherited cardiac disease in the family. To understand the underlying disease pathways better, we have now created a mouse model carrying the A178D variant in titin. Mice heterozygous for the variant showed no phenotype, in line with the lack of phenotypes in other heterozygous mouse models for human cardiomyopathy pathogenic variants. Homozygous titin A178D mice displayed mild features of Dilated Cardiomyopathy on echocardiography. Furthermore, they had a stronger induction of heart failure markers upon chronic adrenergic stimulation compared to wild-type littermates. At the molecular level, the expression level and localisation of titin in the homozygous mice were similar to wild-type titin, however, reduced sarcomere length was observed in cardiomyocytes isolated from homozygous animals. Moreover, hearts of homozygous mice showed an increased expression of transcripts related to heart failure, in agreement with a Dilated Cardiomyopathy phenotype.

    A combination of transcriptomics and proteomics identified the underlying mechanism responsible for the phenotype: in the presence of the titin A178D variant, telethonin is unable to bind to Z-disc titin. It is subsequently degraded by the ubiquitin-proteasomal system.

    Taken together, our mouse models highlight that the titin A178D missense variant is indeed responsible for cardiomyopathy. Hence, titin missense variants should not be disregarded completely in genetic testing. Novel tools and approaches will be required for the risk stratification of titin missense variants.

    Obscurin/OBSL1 double Knockouts: a new genetic model for HFpEF?

    P. Desmond1, V. Marrocco1, E. Esteve1, A. Velmurugan1, J. Blondelle1, M. Rajan2, Y. Chan1, M. Wright1, S. Myers1, Y. Gu1, N. Dalton1, M. Ghassemian1, M. Klos1, K. Peterson1, E. Borgeson2, and S. Lange 1,2

    1University of California, San Diego, La Jolla, CA-92101, USA; 2University of Gothenburg, Gothenburg, Sweden

    The obscurin protein family consists of three members: the giant obscurin and its splice variants, obscurin-like 1 (Obsl1) as well as striated muscle preferentially expressed gene (SPEG). Skeletal muscle functions for obscurin and Obsl1 have been described, with single and/or double knockout (dKO) mice showing altered sarcoplasmic reticulum (SR) architecture, impaired sarcolemmal integrity and exercise intolerance. However, little is known about the roles of obscurin and/or Obsl1 in the heart.

    We generated and analyzed cardiac-specific obscurin and/or Obsl1 single knockout and dKO mice. While obscurin and Obsl1 knockouts have life expectancies comparable to wildtype controls, dKO mice display shortened median survival rates. Our data from traditional and Doppler echocardiography, as well as hemodynamics studies indicate that obscurin/Obsl1 dKO mice develop severe diastolic dysfunction, while displaying normal fractional shortening, features reminiscent of heart failure with preserved ejection fraction (HFpEF). Intriguingly, the diastolic dysfunction is not accompanied by hypertrophy or increased fibrosis.

    Our data also demonstrate that dKO mice show distinct changes to mitochondria, metabolic enzymes and SR structure. Alterations to SR-structure are reflected in drastically reduced SR-volume, altered protein content and calcium cycling. Isolated dKO cardiomyocytes displayed reduced calcium amplitude (calcium release) and prolonged calcium re-uptake times (tau-values).

    Taken together, our data show that obscurin and Obsl1 are crucial for proper SR-architecture, calcium storage and re-uptake, and their loss results in a profound cardiac relaxation problem.

    We propose that obscurin/Obsl1 dKO mice may serve as a new genetic model to investigate age-dependent diastolic dysfunction and HFpEF.

    Re-evaluation of the contribution from titin to cardiomyocyte viscoelasticity using a mouse model that allows specific cleavage of the titin springs

    J.K. Freundt1, J. Recker1,I. Liashkovich1, C. Loescher1, Y. Li1, J.M. Fernandez2 and W.A. Linke 1

    1University of Münster, Münster, Germany; 2Columbia University, New York, USA

    Background The giant sarcomere protein titin bears passive load in cardiomyocytes and increased titin-based stiffness is frequently found in heart failure (HF). However, recent work suggests that a major contribution to cardiomyocyte stiffness comes from the microtubular network and that microtubule-based stiffening occurs in HF (Robison et al., Science 2016;352:aaf0659; Chen et al., Nat Med 2018;24:1225). These findings call for a re-evaluation of the contribution from titin to cardiomyocyte stiffness.

    Objective To quantify the contribution of titin to total cardiomyocyte viscoelasticity through specific cleavage of the titin springs in situ.

    Methods & Results We developed a knock-in (KI) mouse model carrying a tobacco etch virus (TEV) protease-recognition site and a HaloTag in titin’s elastic region. This HaloTag-TEV cassette allows for specific titin cleavage during mechanical measurements of cardiomyocytes and visualization of successful cleavage. TEV-protease induced a complete, rapid, and specific cleavage of cardiac titin in skinned homozygous KI heart samples. Confocal and atomic force microscopy were combined to quantify the transversal stiffness of cardiomyocytes by nanoindentation. The Young’s modulus of homozygous KI cells at a pre-set force of 3 nN was significantly reduced with titin-cleavage, by 26 ± 4% (n = 26 cells), indicating cell softening with titin-cleavage. The amplitude of this decrease was similar to that found in nanoindentation measurements on human cardiomyocytes upon microtubule depolymerisation (Chen et al., Nat Med 2018;24:1225–1233). Skinned cardiomyocytes were also stretched at their ends and the resulting force recorded before/after TEV-treatment. TEV-protease reduced steady-state passive force by 53 ± 8% and viscous force by 56 ± 7% (n = 9 cells), suggesting that titin is the main contributor to tensile force.

    Conclusions The HaloTag-TEV mouse allows, for the first time, the direct and reliable quantitation of the titin contribution to cardiomyocyte stiffness. Our findings show that intact titin springs are responsible for most of the elastic force of the mouse cardiomyocyte.

    Unique properties of brain-specific tropomyosin isoforms

    V.V. Nefedova1, M.A. Marchenko1,2, Y.G. Ermakova3, D.V. Shchepkin4, G.V. Kopylova4, S.R. Nabiev4, S.Y. Bershitsky4 and A.M Matyushenko 1

    1Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia; 2Department of Biochemistry, School of Biology, Moscow State University, Moscow, 119234, Russia; 3European Molecular Biology Laboratory, 69117 Heidelberg, Germany; 4Institute of Immunology and Physiology, Ural Branch of the Russian Academy of Sciences, Yekaterinburg, Russia;

    The actin cytoskeleton plays a crucial role in cell functioning and viability. The leading role in the discrimination of actin functions is assigned to tropomyosin (Tpm). Sixteen isoforms of this protein are expressed in the brain. However, still not clear how different tropomyosin isoforms cause morphological or functional changes in neurons. In our work, we investigate the structural and functional properties of different brain-specific Tpm isoforms. We revealed that many properties of these isoforms could depend on their molecular weight. Using differential scanning calorimetry, we showed that the structural properties of Tpm molecules are highly dependent on the amino acid sequence encoded by alternatively spliced exons. We determined that the thermal stability of the N-terminal part of Tpm HMW isoforms was lower than for LMW isoforms. It was found that the affinity of HMW tropomyosin isoforms to F-actin is much higher than the affinity of LMW isoforms. The denaturation of Tpm-actin complexes occurs primarily due to the melting of the C-terminal part of the Tpm molecule. We also obtained data on the bending stiffness of the reconstructed thin filaments decorated with the different Tpm brain-specific isoforms. These results have a good correspondence with the thermal stability of the Tpm-actin complexes. Expressive data were obtained on MIN-6 and 1.1B4 cell cultures. The expression of various tropomyosin isoforms led to changes in the shape of cells and the formation of lamellopodia and different protrusions. We have also carried out experiments on the colocalization of various Tpm isoforms with myosin Ic. Some tropomyosin isoforms can be located near to this motor, and some are not. Summarizing the results of all our experiments, we can conclude that Tpm was able to determine the architecture of cells by regulating the properties of actin filaments and myosin motors.

    Supported by RSF No18-74-10099.

    Mechanical properties of nonmuscle myosin 2

    Y. Takagi, S. Heissler, L. Melli, N. Billington, F. Zhang, R. Liu, A. Nagy, E. Homsher and J.R. Sellers

    National Heart, Lung and Blood Institute, NIH, Building 50, Room 3523 Bethesda, MD, 20892 United States

    Three nonmuscle myosin-2 (NM2) paralogs participate in many mammalian cellular phenomena. Here we compare the mechanical properties of NM2A and NM2B. Each form 310 nm bipolar filaments containing 30 myosins. The two paralogs can also co-assemble into the same filament. Both are slow enzymatically compared to most other myosins, but NM2A moves actin filaments 2 to 3 times faster than NM2B in motility assays and NM2B has a higher duty ratio. Neither NM2A nor NM2B demonstrate processive movements as single molecules. We assayed the ability of filaments of these two myosins to move processively on actin filaments bound to a coverslip surface. NM2B filaments move processively and experiments show that when co-polymerized with headless tail fragments about 6 motors per half filament are required for processive movements. NM2A filaments require the presence of methylcellulose to increase viscosity in order to move processively. When assayed at low loads by optical trapping NM2A and NM2B give single attachment events with lifetimes of about 1 and 3 s, respectively. Both paralogs show load dependence of their attachment lifetimes. When actin-attached NM2A is subject to loads of 4–6 pN the lifetimes increase to ~ 10–20 s. Strikingly, when NM2B molecules are subject to similar loads the attachment lifetimes are 80–300 s. This differential load dependence may explain the role of NM2B molecules in stress fibers.

    Myosin-18b regulates sarcomeric myosin dynamics and actin thin filament organization during cardiac myofibril assembly

    S.L. Latham1,3, K. Schwanke2, N. Weiß1, C. Thiel1, R. Zweigerdt2, D.J. Manstein1 and M.H. Taft 1

    1Institute for Biophysical Chemistry, Hannover Medical School, OE 4350, Carl-Neuberg-Str. 1, 30625 Hannover, Germany; 2Department of Cardiac, Thoracic, Transplantation and Vascular Surgery (HTTG), Hannover Medical School, OE 6217, Carl-Neuberg-Str. 1, 30625 Hannover, Germany; 3present address: The Kinghorn Cancer Centre, Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW, 2010, Australia

    MYO18B loss-of-function mutations and depletion prevent cardiac and skeletal muscle sarcomere assembly via the misalignment of actin thin filaments and myosin thick filaments. Currently, the molecular function of the encoded protein, myosin-18B (M18B), is entirely unknown. Here, we delineate the biochemical function of M18B with recombinantly produced protein constructs and utilize a human embryonic stem cell model to define the spatial and temporal organization of M18B during cardiac myogenic differentiation. Our results demonstrate that unlike myosins of other classes, M18B lacks motor ATPase activity and instead functions as an actin filament cross-linker via its four actin-binding domains. This interaction does not inhibit the binding of key sarcomeric proteins to actin. At select physiological ratios, M18B opposes contractile forces exerted by both nonmuscle myosin-2B (NM2B) and beta-cardiac myosin (bCM), two myosins implicated in myofibrillogenesis. However, elevated bCM levels, such as those observed at the onset of synchronous beating in cardiomyocytes, are able to overcome this molecular brake. Within the cell, we show that the nuclear-to-cytoplasmic relocalization of M18B accompanies myogenic maturation and that the incorporation of M18B into NM2B filaments coincides with the onset of sarcomeric striation. Further, we show that M18B remains present within the mature sarcomere, incorporated into bCM thick filaments in the A-band. Together, these data demonstrate that M18B functions as a structural myosin, which tightly regulates the spatial and temporal assembly of the sarcomere through its actin binding and motor modulating capabilities.

    Session IV: Muscle: Mechanobiology

    In silico observation of mechanically-induced binding pockets in muscle proteins

    M. Tiberti1, B.-D. Lechner1 and A. Fornili 1

    1Queen Mary University of London, London, UK

    The binding of small molecules to proteins usually occurs at specific clefts on the protein surface known as binding pockets. In the past years it has become increasingly clear that binding pockets are dynamical entities, whose shape and size can change due to the motions of the protein. Considering the fact that muscle proteins can be often subjected to mechanical stress while performing their function, in this work we aimed at understanding if a mechanical force can induce the opening of new binding pockets on their molecular surface.

    Our Steered Molecular Dynamics simulations show for the first time that the application of forces mimicking a mechanical stress can lead to the formation of a pocket in a Ig-like domain from cardiac Myosin Binding Protein C [1]. This mechanically-induced pocket is energetically stabilised by the external forces and can bind probe molecules in in silico druggability tests. Moreover, preliminary investigations indicate that similar mechano-pockets could be found in other domains from the same fold family.

    Our findings thus uncover in atomistic detail the first example of a potential whole new class of binding pockets, which we believe could lead to the identification of new sets of drug targets, in particular for the treatment of cardiac and skeletal muscle diseases. This research has been supported by the British Heart Foundation and it made use of time on ARCHER granted via the UK High-End Computing Consortium for Biomolecular Simulation, HECBioSim.

    [1] Tiberti M, Lechner B-D, Fornili A. Binding Pockets in Proteins Induced by Mechanical Stress. J Chem Theory Comput. 2019;15: 1–6.

    Cardiomyocyte integrin mechanosignalling: responses to a dynamic extra cellular matrix in development and disease

    W. Hawkes 1,2,3, P. Reynolds4, M. Ward3, P. Pandey1,3, N. Gadegaard4, M. Palma2 and T. Iskratsch3

    1Randall Division of Cell and Molecular Biophysics, King’s College London; 2School of Biological and Chemical Sciences, Queen Mary University of London; 3School of Engineering and Materials Science, Queen Mary University of London; 4School of Engineering, University of Glasgow.

    During development and disease, the cardiac extracellular matrix (ECM) undergoes dramatic changes in stiffness and composition. Integrins bind cells to the ECM and play a key role in the response to the dynamic mechanical and chemical stimuli around cardiomyocytes. Work from our lab has identified unique integrin mediated rigidity sensing behaviours (Pandey et al., Dev Cell, 2018), but as cardiomyocytes respond to changes in ECM composition, the role of different integrin subtypes remains unclear. Utilising a multi-platform approach, we are beginning to elucidate these signalling mechanisms at the single cell (PDMS substrates), single adhesion (nanopillar arrays and ligand micropatterning) and single molecule (DNA origami mediated ligand nanopatterning) level. Our data shows that cardiomyocytes cultured on fibronectin or laminin coated substrates, of various stiffnesses, have significantly different cytoskeletal morphology, traction forces and focal adhesion composition. Using a DNA Origami nanopatterning platform (Hawkes et al., Faraday Discuss, 2019) we are analysing the underpinning molecular mechanisms behind these observations. Thereby, we find that the clustering and signalling of cardiomyocyte integrins differ significantly. Our data provides new insights into the molecular mechanisms of cardiomyocyte integrin signalling and further our understanding of their role in development and disease.

    Do titins rule sarcomeres of insect muscles?

    V. Loreau1, W. Koolhaas2 and F. Schnorrer 1,2

    1Aix Marseille University, CNRS, IBDM, Marseille, France; 2Max Planck Institute of Biochemistry, Martinsried, Germany

    Sarcomeres are the force producing molecular machines of muscles in all higher animals. Each sarcomere consists of a quasi-crystalline assembly of cross-linked parallel actin filaments with bipolar myosin filaments, both of which are linked by gigantic titin molecules. Members of the titin family are essential for sarcomere formation across evolution and were shown to determine sarcomere length by spanning from the sarcomeric Z-disc to the M-band in mammals. While this titin ruler model is well supported for mammalian sarcomeres, titin isoforms in insects are shorter and thus may not simply determine sarcomere length. Here, we are investigating the two Drosophila titin homologs Sallimus and Projectin, both of which are essential for the formation of sarcomeres in Drosophila. Using CRISPR-based genome engineering we modified the various parts of both proteins and quantified the consequences for myofibrillogenesis, sarcomere length and muscle function.

    Sarcomere growth is controlled by a finely tuned mechanism of protein aggregation

    N. González-Morales, Y. S. Xiao, M. A. Schilling, O. Marescal, K. A. Liao and F. Schöck

    Department of Biology, McGill University, Canada

    Myofibrils are aggregates of cytoskeletal proteins forming an array of sarcomeres that are embedded in the cytosol of myotubes and mediate contractility. EM studies showed that they form initially from small aggregates called Z-bodies that develop eventually into Z-discs to which thin filaments are anchored. The size of the Z-disc and potentially the M-line to which thick filaments are anchored therefore sets the diameter of the myofibril. While mechanisms have been proposed that set the length of sarcomeres and initial myofibril assembly, Z-disc growth and growth termination is poorly understood. Here we show that multivalency-driven aggregation of Zasp proteins causes Z-disc growth, whereas upregulation of multivalency-blocking isoforms at later developmental time points terminates Z-disc growth. Zasp proteins are Alp/Enigma family members, which contain PDZ, ZM, and LIM domains and are scaffold proteins localizing to the Z-disc. An imbalance of growing and blocking Zasp isoforms results either in aggregate formation or reduced Z-disc size. We propose that this mechanism has wide implications for diseases caused by aggregate formation.

    Sarcomere dissasembly after unloading is regulated by ubiquitination and acetylation of CapZ and alpha-actinin

    C. Solís and B. Russell

    University of Illinois at Chicago, Chicago, IL, USA

    Assembly and disassembly of sarcomeres occurs to adjust muscle mass to altered mechanical demand. In the heart, hypertrophic cardiomyopathy results from myofibrillar assembly controlled by post-translational modification of proteins directed by signaling pathways. More is known about assembly on loading than disassembly on unloading. Here, the hypothesis tested is that unloading of mechanical forces affects acetylation (Ac) and ubiquitination (Ub) of the actin-binding proteins, CapZ and alpha-actinin. Blebbistatin (1 micromolar) decreased myocyte contractility in rat ventricular myocytes (NRVMs) and caused significant sarcomere disassembly by 6 h and ~ 70% atrophy by 24 h. Ac and Ub levels in alpha-actinin over the 24 h unloading time period were determined by α-actinin immunoprecipitation (IP) western blots using K48 oligo-Ub and acetyl-lysine antibodies, respectively. Ac decreased by 24 h blebbistatin treatment compared to untreated NRVMs. Ac and Ub in the Z-discs were quantified on immunofluorescent images. The Z-discs colocalized oligo-Ub (K-48 oligo-Ub linkage) and Ac in untreated samples; this Z-disc localization of Ub and Ac was diminished with blebbistatin. Fluorescence Recovery after Photobleaching (FRAP) measured the dynamics of alpha-actinin after reduced cell tension. FRAP assays showed that the dynamics of alpha-actinin-YFP localized in the Z-discs decreased with blebbistatin. Similar findings with CapZ FRAP were found with unloading, and IP experiments are ongoing. Overall, results suggest sarcomere assembly is regulated by mechanical forces and signaling pathways involving Ac and Ub of myofibrillar proteins. It is likely that Ac is responsible for reducing the rate of Ub and subsequent degradation in atrophy. These findings could have consequences for cardiac heart disease with abnormal sarcomeric proteostasis. Funded by NIH HL62426.

    Session V: Muscle: Outside In

    Muscles from calsequestrin-1 knockout mice contain pre-assembled calcium entry units that provide constitutively active store-operated Ca2+ entry

    A. Michelucci 1,2, S. Boncompagni2, L. Pietrangelo2, F. Protasi2* and R.T. Dirksen1*

    1Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA; 2CeSI-MeT, Center for Research on Ageing and Translational Medicine, University G. d’Annunzio, Chieti, Italy. *contributed equally to this work

    Store-operated Ca2+ entry (SOCE) is a ubiquitous Ca2+ influx mechanism triggered by depletion of intracellular stores. In wild type (WT) mice, acute treadmill exercise of drives the formation of new intracellular junctions between the sarcoplasmic reticulum (SR) and transverse-tubules (T-tubules). These junctions were named Calcium Entry Units (CEUs), as they contain STIM1 and Orai1, the two proteins that mediate SOCE. Skeletal muscles from mice lacking calsequetrin-1 (CASQ1-null), the primary Ca2+-bindingprotein in the lumen of SR terminal cisternae, exhibit reduced SR Ca2+ content and massive SR depletion during high-frequency stimulation. Here we report that CEUs are constitutively assembled in extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of adult CASQ1-null mice (4 months of age), even in the absence of acute exercise. The higher incidence of CEUs in CASQ1-null muscles directly correlated with: (i) enhanced rate of Mn2+ quench in single FDB fibers, both in the absence of and after store depletion; (ii) higher ability to maintain peak Ca2+ transient amplitude in FDB fibers during repetitive, high-frequency stimulation (40 successive 500 ms 50 Hz stimuli delivered every 2.5 s); and (iii) increased capability of EDL muscles to maintain contractile force, which is abolished by experimental interventions that inhibit Ca2+ influx via SOCE (Ca2+-free extracellular solution or presence of SOCE inhibitor BTP-2). Taken together, these data indicate that muscles from CASQ1-null micecompensate for the lack of CASQ1 and reduced SR Ca2+ content by constitutively assembling CEUs to promote Ca2+ entry via SOCE even in the absence of store depletion.

    Assembly of Ca2+ entry units provides constitutively active store-operated Ca2+ entry

    A. Michelucci1,2, S. Boncompagni1, L. Pietrangelo1, R. T. Dirksen2 and F.Protasi 1

    CeSI-MeT, Center for Research on Ageing and Translational Medicine, University G. d’Annunzio, Chieti, Italy; 2 Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, NY

    Store-operated Ca2+ entry (SOCE) is a ubiquitous cellular Ca2+ influx mechanism triggered by depletion of intracellular Ca2+ stores (endoplasmic and sarcoplasmic reticulum, ER and SR) and mediated by STIM1, the Ca2+ sensor in the ER/SR, and Orai1, a Ca2+ permeable channel in external membranes. SOCE is in skeletal muscle seems important to limits muscle fatigue during repetitive fatiguing stimulation. The precise subcellular location of STIM1-Orai1 SOCE complexes in skeletal muscle is still debated.

    We discovered that exercise in extensor digitorum longus (EDL) of mice drives formation of new junctions between stacks of SR cisternae and transverse-tubules (TTs) containing STIM1 and Orai1.

    These junctions disassemble in the first hours of recovery thanks to the retraction of TTs away for the SR stacks. We also noticed that identical SR/TT junctions are constitutively assembled in extensor digitorum longus (EDL) and flexor digitorum brevis (FDB) muscles of CASQ1-null mice, even in the absence of acute exercise.

    The higher incidence of these junctions in muscle following exercise, and in CASQ1-null muscles, directly correlated with: (i) enhanced rate of Mn2+ quench in single FDB fibers, both in the absence of and after store depletion; (ii) increased capability of EDL muscles to maintain contractile force during high-frequency stimulation in presence of extracellular Ca2+, a fatigue resistance that is abolished in conditions in which Ca2+ entry in prevented (i.e. 0 Ca2+ or presence of SOCE inhibitors).

    Our data strongly suggest that these previously unidentified SR-TT junctions function as Ca2+ Entry Units (CEUs), providing a preferential pathway for constitutively active SOCE. As altered SOCE activity contributes to muscle dysfunction in ageing and various myopathies, our findings may also have implications for the understanding of mechanisms involved in muscular dysfunction.

    Nexilin is essential for cardiac T-tubules development

    S. Spinozzi 1*, C. Liu1*,J.Y. Chen1, X. Fang1, W. Feng1, G. Perkins1, P. Cattaneo2, N. Guimarães-Camboa3, N.D. Dalton1, K.L. Peterson1, T. Wu1, K. Ouyang4, X.D. Fu1, S.M. Evans1 and J. Chen1

    1University of California San Diego, La Jolla, CA, USA; 2Humanitas Clinical and Research Center, Rozzano, Italy; 3Goethe University Frankfurt, Frankfurt, Germany; 4Peking University Shenzhen Graduate School, Shenzhen, China

    *These authors equally contributed to the project

    Membrane contact sites are fundamental for transmission and translation of signals in multicellular organisms. A prime example is the junctional membrane complex (JMC), a protein complex located in the cardiac dyads where the T-tubules are juxtaposed to the sarcoplasmic reticulum (SR). JMCs are fundamental for the calcium signal amplification that enables optimal cardiac contraction, with alterations in JMCs leading to cardiac disease and heart failure. Nexilin (NEXN) has been identified as an actin-binding protein and multiple mutations in the NEXN gene are associated with cardiac diseases. We revealed, unexpectedly, that NEXN is a pivotal component of the JMC. Cardiomyocyte specific loss of Nexn in mice resulted in a rapidly progressive dilated cardiomyopathy. In vivo and in vitro analyses demonstrated that NEXN interacts with junctional SR proteins, is essential for optimal calcium transients, and is required for the initiation of T-tubule invagination seminal for their formation. These results identify NEXN as an essential component for T-tubules maturation and give insight into mechanisms underlying cardiomyopathy in patients with NEXN mutations.

    Homo- and heteromeric interactions determine junctophilins retention at adult skeletal muscle triads

    D. Rossi, A.M. Scarcella, E. Pierantozzi and V. Sorrentino

    Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy

    In adult skeletal muscle fibers, two junctophilin isoforms (JPH1 and JPH2) tether the sarcoplasmic reticulum to the T-tubule membrane, generating stable Membrane Contact Sites known as triads. JPH1 and JPH2 are anchored to the membrane of the sarcoplasmic reticulum by a C-terminal transmembrane domain (TMD) and to the T-tubule membrane by eight lipid-binding (MORN) motifs in the N-terminal region. Expression in FDB fibers of different JPH1mutants, lacking either MORN motifs or the TMD, revealed that a protein containing only the TMD of JPH1 or JPH2 is capable to localize to triads. Bimolecular Complementation experiments showed that the TMD of JPH1 is capable to form homodimers. Additional biochemical experiments identified a second domain in the cytosolic regions of JPH1 and JPH2 that is capable of mediating homo and heterodimeric interactions between these two proteins. FRAP experiments revealed that removal of one or the other of these two domains in JPH1 decreases the association of the resulting proteins with triads. Altogether, these results indicate that localization and stabilization of newly synthesized JPHs is mediated by the ability to establish homo- and heterodimeric interactions with resident triadic JPHs in adult skeletal muscle fibers. Furthermore, based on these results and on already published data, we propose a unifying model where JPHs’ capability to form homo- and heterodimers, and to interact with other e–c coupling proteins, represents the nucleation system that, starting at the early stages of junctional membrane domains assembling, mediates clustering of additional JPHs and of other e–c coupling proteins, including DHPR and RyR1, at these sites in embryonal skeletal muscle fibers. The same function is then carried on when, following alignment with the sarcomere, peripheral coupling and dyads progress to form triads in adult skeletal muscle fibers.

    Session VI: Muscle: Skeletal

    The reduction of selenoprotein S impairs muscle performance independent of dysregulated cellular or inflammatory stress

    A.B. Addinsall 1, C.R. Wright2, T.L. Kotsiakos1, Z.M. Smith3, T.R. Cook3, S. Andrikopoulos4, C. van der Poel5 and N. Stupka1

    1Centre for Molecular and Medical Research, School of Medicine, Deakin University, Geelong, Victoria, Australia; 2Institute for Physical Activity and Nutrition (IPAN), School of Exercise and Nutrition Sciences, Deakin University, Geelong, Victoria, Australia; 3School of Life and Environmental Sciences, Deakin University, Geelong, Victoria, Australia; 4Department of Medicine, University of Melbourne, Parkville Victoria, Australia; 5Department of Physiology, Anatomy & Microbiology, School of Life Sciences, La Trobe University, Bundoora, Victoria, Australia.

    Selenoprotein S (Seps1) in an endoplasmic reticulum resident antioxidant highly expressed in skeletal muscle and microvasculature. Seps1 is thought to be protective against metabolic, cellular and inflammatory stress. Global deletion of Seps1 reduces spontaneous physical activity, compromises fast twitch extensor digitorum longus muscle strength ex vivo and, depending on context, exacerbates inflammation in mice. In the current study, Seps1 global knockout (KO), heterozygous (HT) and wildtype (WT) mice were randomised to a 3-day incremental, high intensity treadmill running protocol to assess (1) if a reduction of Seps1 is detrimental to exercise performance and (2) if the regulation of cellular and inflammatory response previously observed, are associated with the physiological stress of exercise. On day 4, in situ contractile function, where nerve and blood supply remain intact, was determined in fast tibialis anterior (TA) muscles. Here, the genetic reduction or deletion of Seps1 compromised exercise capacity, as defined by a diminished distance run. TA specific force was reduced in Seps1 HT and KO mice, as determined by force frequency curve. The rate of fatigue was increased in TA of sedentary Seps1 KO mice. However, following 3 days of exercise, the rate of fatigue was exacerbated in the TA of Seps1 HT, but not KO mice, indicating a biological interaction between Seps1 protein expression, exercise and endurance of the TA. Impairments in exercise capacity and TA function were not associated with increased muscle or circulating inflammation or dysregulation of muscle redox state (GSH:GSSG ratio). However, Seps1 reduction and deletion decreased Nitrous oxide 1 and Vascular endothelial growth factor A mRNA transcripts in TA muscles, while capillary density remained unchanged. Given the high expression of Seps1 in blood vessels, Seps1 may affect muscle performance via regulation of perfusion, which forms the basis for ongoing research investigating the role of Seps1 in skeletal muscle.

    Advanced PPMO combinations drive multi-exon skipping and dystrophin expression in dystrophic dogs

    M.K. Tsoumpra1, L. Roux2, G. McClorey3, M. Kuraoka1,4, S. Arima1, Y. Mizobe1, Y. Hara1, A.A. Arzumanov3,5, M.J. Gait3,5, K. Kimura6, M. Imamura1, S. Takeda1, T. Yokota7, C. Godfrey2, M.A. Wood3, and Y. Aoki 1

    1Department of Molecular Therapy, National Institute of Neuroscience, National Centre of Neurology and Psychiatry, Kodaira-shi, Tokyo, Japan; 2PepGen Limited, Bioescalator, Innovative Building, Oxford, United Kingdom; 3Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom; 4Laboratory of Experimental Animal Science, Nippon Veterinary and Life Science University, Musashino-shi, Tokyo, Japan; 5Medical Research Council, Laboratory of Molecular Biology, Cambridge Biomedical Campus, Cambridge, United Kingdom; 6Department of General Medicine, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; 7Department of Medical Genetics, School of Human Development, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Canada

    Duchenne muscular dystrophy (DMD) is an X-linked disorder characterized by progressive muscle degeneration caused by the lack of functional dystrophin. There is currently no cure and therefore, an urgent need for new therapies to extend lifespan and improve quality-of-life for DMD patients. The current state-of-the-art is a first generation oligonucleotide gene therapy that restores dystrophin protein and has been developed by US/UK and Japanese investigators (e.g. NS-065/NCNP-01: Viltolarsen) is a phosphorodiamidate morpholino PMO-based oligonucleotide drug). However, this current therapeutic approach has limited efficacy. The use of peptides as delivery agents to boost the levels of exon skipping induced by peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs), which have been extensively developed by Wood and Gait laboratories, is considered a revolutionary therapeutic option for DMD patients. Herein we tested the multi-exon skipping efficacy of novel PPMO combinations in our beagle-based canine X-linked muscular dystrophy (CXMDJ) model. We first screened 3-PPMO and 2-PPMO-based cocktails using CXMDJ-derived primary myoblasts and confirmed dose-dependent 6–8 exon skipping via RT-PCR. We then administered our three most potent cocktails intramuscularly in tibialis cranialis (TC) of CXMDJ dogs and collected samples 14 days later. Notably, even the 2-PPMO-based cocktails were able to drive highly efficient multi-exon skipping and to restore dystrophin expression as proved by Western blot and immunohistochemistry analysis. We assume that the advanced PPMO combinations have comparably potent exon skipping activity to previous experimental compounds but with negligible renal toxicity and is thus highly suitable for clinical development.

    The role of the microcirculation in muscle function and plasticity

    P. Hendrickse1,2 and H. Degens 1,2,3

    1Research Centre for Musculoskeletal Science & Sports Medicine, School of Healthcare Science, Manchester Metropolitan University; 2Lithuanian Sports University, Kaunas; 3University of Medicine and Pharmacy of Targu Mures, Rumania

    It is widely acknowledged that maintenance of muscle, size, strength and endurance is necessary for quality of life and the role that skeletal muscle microcirculation plays in muscle health is becoming increasingly clear. Here we discuss the role that skeletal muscle microcirculation plays in muscle function and plasticity. Besides the density of the capillary network, also the distribution of capillaries is crucial for adequate muscle oxygenation. While capillaries are important for oxygen delivery, the capillary supply to a fibre is related to fibre size rather than oxidative capacity. This link between fibre size and capillary supply is also reflected by the similar time course of hypertrophy and angiogenesis, and the cross-talk between capillaries and satellite cells. A dense vascular network may in fact be more important for a swift repair of muscle damage than the abundance of satellite cells and a lower capillary density may also attenuate the hypertrophic response. Capillary rarefaction does not only occur during ageing, but also during conditions as chronic heart failure, where endothelial apoptosis has been reported to precede muscle atrophy. It has been suggested that capillary rarefaction precedes sarcopaenia. If so, stimulation of angiogenesis by for instance endurance training before a hypertrophic stimulus may enhance the hypertrophic response. The microcirculation may thus well be a little-explored target to improve muscle function and the success of rehabilitation programmes during ageing and chronic diseases.

    Deletion of the cation channel TRPV4 attenuates fibrosis in dystrophic MDX mouse muscle

    R.S. Eller 1, M. Krautwald1 and H. Brinkmeier1

    1University Medicine Greifswald, Institute of Pathophysiology, Greifswald, Germany

    The cation channel TRPV4 is expressed in skeletal muscle fibres and in activated fibroblasts, so called myofibroblasts. In muscle fibres, TRPV4 is a calcium conducting, mechanosensitive channel and a candidate to account for the increased Ca2+ influx and abnormal Ca2+ homeostasis in dystrophin-deficient mdx mouse muscle. To test the hypothesis that deletion of TRPV4 is protective for mdx muscle architecture and performance, we bred a mdx/trpv4−/− double mutant mouse model and investigated its muscles histologically after 100 days of age. Analyses included H&E staining, Sirius red staining and immunofluorescence staining of fibre types on cryosections of soleus muscle, extensor digitorum longus muscle, tibialis anterior muscle and the most severely affected diaphragm. As expected, wildtype muscles and those from TRPV4-deficient mice were characterized by peripheral nuclei, a balanced collagen content (4.20 ± 0.74%) and a comparable fibre type composition. In contrast, mdx muscles showed the typical signs of dystrophic muscle, i.e., centralized nuclei in up to 80.91 ± 3.98% of fibres, extensive Sirius red staining indicating fibrosis and a fast to slow shift of fibre types. Skeletal muscles from mdx/trpv4−/− double mutant mice widely resemble the histological appearance of mdx muscles. However, the area on cryosections consisting of connective tissue was reduced to an average of 5.24 ± 1.13% (n = 6 independent male animals tested for each strain) compared to 9.74 ± 1.45% in the mdx mouse. We conclude that the TRPV4 channel is not substantially involved in the process of muscle fibre degeneration and regeneration in dystrophic mdx muscle. However, the lack of TRPV4 in fibroblasts seems to inhibit fibroblast proliferation and fibrosis in mdx muscles. TRPV4 blockers may be useful for the symptomatic treatment of human muscular dystrophies characterized by replacement of muscle tissue by fibro-fatty tissue.

    The study was supported by the Deutsche Duchenne-Stiftung, Bochum.

    Bench to bedside research on critical illness myopathy and ventilator induced diaphragm muscle dysfunction

    L. Larsson 1, N. Cacciani1, M. Li1, H. Salah1, A. Addinsall1 and Y. Hedström1

    1Karolinska Institutet, Stockholm, Sweden

    Severe muscle wasting and impaired muscle function are frequently observed in immobilized and mechanically ventilated intensive care unit (ICU) patients. Approximately 30% of mechanically ventilated and immobilized ICU patients for durations > 5 days develop generalized muscle paralysis of all limb and trunk muscles, a condition known as critical illness myopathy (CIM). Mechanical ventilation is a lifesaving treatment in critically ill ICU patients; however, the being on a ventilator creates dependence, and the weaning process occupies as much as 40% of the total time of mechanical ventilation. Common components of ICU treatment per se, unrelated to underlying disease, are directly involved in the progressive impairment of muscle function and muscle wasting during long-term ICU treatment. The specific mechanisms underlying the muscle wasting and impaired muscle function associated with the ICU intervention are poorly understood in the clinical setting. There is, accordingly, compelling need for experimental animal models closely mimicking the ICU condition, including long-term exposure to mechanical ventilation and immobilization. In this project, the muscle dysfunction, which by far exceeds the loss in muscle mass in limb and respiratory muscles in patients with CIM and VIDD have been investigated in detail at the cellular and molecular levels in rodent and porcine experimental ICU models, allowing detailed studies in immobilized and mechanically ventilated animals for long durations. Results demonstrate that the motor protein myosin is highly involved in the pathogenesis of both CIM and VIDD, but mechanisms are different. In CIM there is a preferential loss of myosin due to transcriptional down-regulation and enhanced degradation while post-translational modifications of myosin play a significant role for the diaphragm muscle dysfunction in VIDD. Specific intervention strategies targeting the mechanisms underlying CIM and VIDD will be presented and the translation of these interventions to the clinic.

    The effect of heat shock on myogenic differentiation of human skeletal mesenchymal stem cells

    R. Miksiunas 1, G. Juskaite1 and D. Bironaite1

    1State Research Institute for Innovative Medicine, Department of Regenerative Medicine, Vilnius, Lithuania

    Background Muscle injuries, degenerative diseases and other lesions strongly influent the proper functioning of human skeletal system and thus quality of human life. The application of somatic MSC for skeletal muscle regeneration show promising results, however the identification of populations of muscle-derived mesenchymal stem cells (mMSC) responding to external myogenic stimuli could significantly improve regeneration of human skeletal muscle. Therefore, in this study we investigated the impact of heat shock stimulus on the myogenic differentiation of CD56 positive and negative mMSC.

    Materials and Methods mMSC were isolated from post plastic surgery muscle material. mMSC cell were sorted according to CD56 biomarker by flow cytometry. mMSC-derived CD56+ and CD56− populations have been stimulated by heat stress at 42C for 1 h, expression levels of Hsp70, HSF1, VIM and other proteins have been estimated by Western blotting. Myogenic differentiation after heat shock have been evaluated by qPCR and activation of creatine kinase.

    Results Results showed that CD56 + cells displayed four times higher expression of multiple surface markers, such as CD349 and CD318, CD146 and CD292. Additionally, CD56+ cells reacted more rapidly to heat shock compared to CD56− cells: increased HSF1 expression was observed 30 min. after heat shock in CD56+, however response in CD56-cells was detectable only after 2 h. Finally, heat shock marginally increased myogenic differentiation of CD56+ cells increasing expression of late myogenic gene MYOG by 3 times and creatine kinase by 1,5 times compared to untreated control.

    Conclusions Data show that general human mMSC population is heterogeneous and therefore not equally differentiated to myogenic direction. CD56+ mMSCs significantly better differentiated to myogenic direction and responded to external stimuli. External stimuli might purposefully activate hMSC populations and be applied as a promising therapeutic mean stimulating skeletal muscle regeneration.

    Myogenic effects of FN14 targeting antibodies in a mouse skeletal muscle notexin injury model

    A. Pascoe 1, A.J. Johnston1, L. Jenkinson1, C. van der Poel2 and R.M. Murphy1

    1La Trobe Institute for Molecular Sciences, La Trobe University, VIC 3086, Australia; 2School of Human Biosciences, La Trobe University, VIC 3086, Australia

    Muscle wasting is a devastating comorbidity associated with an array of chronic and acute conditions. Maintenance of muscle mass and function is essential for improving health outcomes and quality of life in these individuals. TNFα-like weak inducer of apoptosis (TWEAK) is an emerging cytokine regulator of muscle homeostasis. TWEAK has been shown to regulate muscle growth, repair, and remodelling via interaction with its receptor, fibroblast growth factor-inducible 14 (Fn14). The current study investigates the effects of two anti-Fn14 antibody variants on degeneration and recovery of mouse tibialis anterior (TA) muscle following a snake venom (Notexin) induced injury. Notexin solution (40 μl, 10 μg/ml) was injected intramuscularly in the right TA and 40 μl saline injected in the left TA under isoflurane anaesthesia. Mice received 20 mg/kg intraperitoneal injections of either anti-Fn14 A, anti-Fn14 B, or no antibody treatment, 3–4 h and 7 days post-injury. Mice were culled at days 3, 7, and 14 post injury and both TA muscles collected (n = 4 for each treatment and time point). Tissue architecture was assessed with H+E staining and key myogenic regulatory factors (myogenin, MRF4, Myf5, and MyoD), catabolic markers (calpain-1, atrogin-1, and MuRF1), and structural proteins (actin, desmin, and myosin) were measured with qPCR or western blotting to establish progression of muscle recovery. Fn14 and TWEAK were also assessed using qPCR. Upregulation of Fn14 mRNA following antibody-mediated Fn14 targeting was significant at 14 days post-injury, and positively correlated with changes in MyoD mRNA. These results indicate a potential positive feedback loop, which may positively regulate muscle myogenesis following acute Notexin injury. While preliminary results indicate that Fn14 may contribute to mouse skeletal muscle regeneration following Notexin injury, further work is needed to clarify the in vivo mechanistic actions of the antibody treatments employed and direct future studies.

    Regulation of actin filament dynamics by cofilin-2 and tropomyosin isoforMS TPM1.1 AND TPM3.12

    K. Robaszkiewicz, M. Śliwinska and J. Moraczewska

    Department of Biochemistry and Cell Biology, Faculty of Natural Sciences, Kazimierz Wielki University in Bydgoszcz, Poland

    The length of actin filaments determines the extent of the overlap with myosin filaments, therefore it contributes to the amount of force developed by muscle fibers. Tpm1.1 and Tpm3.12 are tropomyosin isoforms expressed in fast and slow muscle fibers, respectively. We hypothesized that these isoforms have an impact on the dynamics of actin filaments by affecting length, polymerization and fragmentation caused by muscle cofilin-2. The study was done in vitro using actin isolated from skeletal muscle, recombinant tropomyosin and cofilin-2.

    In the absence of cofilin-2 both Tpm isoforms strongly bound to F-actin. When the filament was decorated with cofilin-2, Tpm1.1 bound to F-actin decorated with cofilin-2 non-cooperatively and with 2.2-fold reduced affinity, whereas Tpm3.12 bound cooperatively and with 1.3-fold lower affinity. At 1.5 cofilin per actin, 50% of Tpm1.1 and 25% of Tpm3.12 was removed from the filament. The process was non-cooperative for Tpm.1 and highly cooperative for Tpm3.12. Binding of cofilin-2 to tropomyosin-coated F-actin was cooperative, but the affinity was reduced 1.5–2-fold. Polymerization of G-actin was followed by turbidimetry and showed that the initial rate was reduced 2.2-fold by Tpm1.1 and 3.6-fold by Tpm3.12. Cofilin-2 alone reduced the rate of polymerization 3.2-fold and it was not affected significantly by tropomyosins. Microscopic observations of the actin filaments fluorescently labeled at Gln-41 revealed that Tpm1.1 and Tpm3.12 increased the length of the filaments in the presence and absence of cofilin-2 by about 20% and 40%. Surprisingly, both tropomyosins increased the number of breaks introduced by cofilin-2.

    In conclusion, the regulation of actin dynamics by the fast and slow isoforms of tropomyosin is quantitatively different, which may results in variations in the length of filaments present in sarcomeres of the fast and slow muscle fibers.

    The project was supported by National Science Center, grant 2014/15/B/NZ1/01017.

    Structure of α-actinin-2/Fatz-1 fuzzy complex and implications in Z-disk assembly

    A. Sponga 1, J.L. Arolas1, A. Rodriguez-Chamorro1, E. de Almeida Ribeiro1, T. Peterbauer1, L. Geist1, F. Drepper2, G. Mlynek1, B. Warscheid2, R. Konrat1 and K. Djinovic-Carugo1

    1Max F. Perutz Laboratories, University of Vienna, Austria; 2University of Freiburg, Germany

    α-Actinin-2 plays a pivotal role in Z-disk assembly and stability as it crosslinks actin filaments from adjacent sarcomeres and acts as a binding platform for a number of Z-disk proteins. Among them is FATZ-1, which appears when Z-bodies, the precursors of Z-disks, are formed and is believed to have a central role during these initial stages of myofibrillogenesis by serving as focal point for interactions with Z-disk proteins.

    Here, we first generated a soluble construct (D91 FATZ-1) and used bioinformatics, CD and SAXS to show that FATZ-1 is intrinsically disordered. We next studied the affinity and binding stoichiometry of α-actinin-2/FATZ-1 complex by ITC and SEC-MALS, and further mapped down a shortest construct (mini FATZ-1) using XL/MS, NMR and LP/MS, as the disordered nature of this protein was a challenge for our crystallographic studies. We then managed to solve the structure of α-actinin-2 rod/mini FATZ-1 complex at 2.7Å and that of α-actinin-2 half dimer/D91 FATZ-1 complex at 3.2Å using MR and Se-Met variants of FATZ-1. In addition, we modelled the flexible regions of D91 FATZ-1 in complex with α-actinin-2 by SAXS to have a complete molecular model of the complex. Finally, we studied FATZ-1 capacity to phase separate and form liquid droplets in the presence of α-actinin-2. Together, our results provide a complete and detailed structural model of the α-actinin-2/FATZ-1 fuzzy complex as well as insights into its role in Z-disk assembly.

    Molecular regulation of longitudinal muscle hypertrophy, titin mechanosensing in action

    R. van der Pijl 1,2, S. Shen1, J. Gohlke1, J. Strom1, H. Sorimachi3, S. Lange4, J. Chen4, J. Graumann5, S. Labeit6, H. Granzier1 and C. Ottenheijm1,2

    1University of Arizona, Tucson, United States; 2Amsterdam University medical center, Amsterdam, the Netherlands; 3Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan; 4University California, San Diego, United States; 5Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany; 6Medical Faculty Mannheim, Mannheim, Germany

    Mechanosensing is the ability to perceive and respond to mechanical cues. Muscles rely on mechanosensors to deal with increased strain and initiate hypertrophic remodeling. One of these mechanosensors is titin, a myofilament protein that forms a contiguous filament along the muscle fibril and thus ideally positioned to sense strain. We previously demonstrated that titin-based stiffness modulates diaphragm hypertrophy. In this study we explored the mechanisms by which titin exerts its mechanosensing function.

    We used unilateral diaphragm denervation (UDD), a surgical model for cyclic stretching of the arrested hemidiaphragm. UDD induces a rapid, transient hypertrophy of the denervated hemi-diaphragm. UDD was applied to wildtype mice for 1, 3, 6, 12 or 35 days to characterize the transient hypertrophy, showing increased muscle mass that peaked at 6 days (50%). Focusing on the 3 day UDD group, a phase at which hypertrophic signaling is most active, we pursued the underlying molecular mechanisms. We performed RNAseq and quantitative proteomics using mass spectrometry. RNAseq revealed an extensive transcriptional program, with 5929 up and 5876 down regulated genes, matching 1423 differentially regulated proteins. Subsequently, we performed UDD studies, to pharmacologically inhibit activity of calcineurin and mTOR. These studies revealed that mTOR is involved in the regulation of longitudinal hypertrophy, with a 9% reduction in both tissue mass and the number of serial sarcomeres in the diaphragm of inhibitor-treated compared to untreated mice. Furthermore, we applied UDD in Capn3 inactive-, as well as Fhl1-, Murf1- and muscle ankyrin repeat protein (MARP; Ankrd1, Ankrd2 and Ankrd23) deficient mice (known titin-interacting proteins). Of these models, the MARP knock-out mouse revealed a mass gain of only 36.4% versus 46.5% in wildtype.

    Thus, longitudinal hypertrophy in the diaphragm is in part regulated through titin-mechanosensing. We propose a central role for MARPs, potentially through affecting protein synthesis via the Akt-mTOR pathway.

    Session VII: Muscle: Cardiac

    Structural dynamics of the myosin filament during activation and relaxation of heart muscle cells

    E. Brunello 1, L. Fusi1, A. Ghisleni1, S.-J. Park-Holohan1, J. Garcia Ovejero1, T. Narayanan2 and M. Irving1

    1Randall Centre, King’s College London, UK; 2European Synchrotron Radiation Facility, Grenoble, France

    The efficiency of the heart as a pump implies a precise control in the contraction and relaxation of its constituent muscle cells. We have recently shown that activation of skeletal muscle is regulated by structural changes in both the actin-containing thin filament, triggered by the calcium transient in the muscle cell, and the myosin-containing thick filament, triggered by a stress-dependent structural switch in the thick filament backbone that is independent of intracellular calcium concentration (Linari et al. 2015, Nature 528:276–279; Fusi et al. 2016, Nat Commun 7:13281).

    This dual-filament mechanism of regulation may also operate in heart muscle, but in a modified form adapted for its different regulatory requirements, including the graded control of both the strength and kinetics of contraction and relaxation. To better understand those adaptations, we measured the kinetics of the structural changes in the thick filaments in intact, electrically paced, trabeculae dissected from the right ventricle of rat hearts using X-ray diffraction and sarcomere-level mechanics at beamline ID02 of the European Synchrotron Radiation Facility. We also characterised thick filament structure in demembranated trabeculae in relaxing solution in the presence of 3% dextran T500 and during maximal calcium activation; these states were used as references for the OFF and ON states of the thick filament, respectively.

    The results showed that the change in thick filament backbone periodicity is roughly proportional to force, consistent with the hypothesis of a stress sensor in the thick filament. The helical arrangement of the myosin motors in the OFF state is partially lost during force development and is recovered at the end of mechanical relaxation. The distinct time courses of force, sarcomere length and the X-ray reflections associated with thick filament structure during activation and relaxation provide new insights into thick filament-based regulation of cardiac contractility.

    Supported by British Heart Foundation, UK.

    The evaluation of influence of mechanical deformation on immediate and delayed myocardial contractile responses

    X. Butova 1,2 and O. Lookin1,2

    1Institute of Immunology and Physiology of the Ural Branch of the Russian Academy of Sciences, Yekaterinburg, Russia; 2 Ural Federal University, Yekaterinburg, Russia

    Myocardial force in the given twitch is greatly influenced by the mechanical conditions imposed to the preceding contractions. The effects of deformation can also be delayed, altering myocardial contractility in subsequent contraction cycles. Consequently, immediate and delayed changes in myocardial contractility raise the need to quantify adequately the contractile variability taking into account various parameters of mechanical deformation. To achieve this aim, special biomechanical tests were designed and tried out on trabeculae isolated from right ventricle of healthy rats. Each test was based on alternating contraction modes: isometric contractions (without changes in length during whole twitch) and series of single half-sinus shortening-relengthening cycles with variable deformation parameters. Additionally, the tests were performed at non-physiological and quasi-physiological pacing rates to reveal whether the effects of changes in myocardial contractility observed under conditions of maximal maintenance of vitality in vitro may differ from those occurred under physiological conditions.

    In this study, we tested how muscle pre-stretch, amplitude and duration of single shortening-relengthening cycle and number of repetitive cycles may alter the immediate and prolonged effects in myocardial contractility. We found that inactivation of contractile responses was minimal (and most typical for immediate changes) as opposed to significant activation in subsequent twitches. Moreover, the activation was increased at physiological condition. We hypothesized that the immediate changes in contractility following the mechanical intervention are due to the acto–myosin interaction and cooperative effects on the level of regulatory units while the delayed effects may arise from the changes in the level of intracellular activating Ca2+. Further research is needed to discover the role of the components of Ca2+ homeostasis (e.g. sarcoplasmic reticulum) in this phenomenon.

    The study was carried out within the framework of the IIF UrB RAS and supported by RFBR (grants #18-04-00572).

    Deamidation of ASPARAGINE14 prevents phosphorylation of human cardiac myosin light chain 2

    P.H. Goldspink, E. Levi-D’Ancona, C.M. Warren, J.L. Martin, and P.P. de Tombe

    Department of Physiology and Biophysics, Center for Cardiovascular Research, University of Illinois Chicago, Chicago, IL 60612, USA

    Phosphorylation of cardiac myosin light chain 2 (cMLC2; Myl2) appears to modulate myosin’s function and its interaction with actin. While cMLC2 across species is rather conserved, a difference exists in the critical phosphorylation motif between species. Small rodents carry two phosphorylation sites (S14, S15), whereas human cMLC2 has a single phosphorylation site (S15). In humans, S14 is substituted for Asn (N14) that can undergo deamidation to form Asp (a charged residue), but the influence of N14D transformation on S15 phosphorylation is unknown. To investigate, we expressed human recombinant wild type (Wt), and mutant S15A and N14D/S15 cMLC2 proteins and phosphorylated with known kinases in vitro. Following phosphorylation with cardiac specific myosin light chain kinase (cMLCK; Mylk3), zipper interacting protein kinase (ZIPK), and myosin light chain kinase 4 (Mylk4), proteins were subjected to 1D and 2D SDS-PAGE. Staining 1D gels with Pro-Q diamond, showed that Wt cMLC2 was phosphorylated by all 3 kinases while mutant S15A served as a negative control, and not phosphorylated. Interestingly, N14D/S15 cMLC2 also showed no indication of phosphorylation. To investigate further, proteins were transferred following 2D-PAGE and probed with a phospho-MYL2 S15 Ab (Invitrogen). Analysis of Wt cMLC2 following cMLCK phosphorylation revealed 2 spots both positive for phospho-S15 compared to unphosphorylated Wt cMLC2. A similar phospho-species profile was obtained with Wt cMLC2 following ZIPK treatment, whereas the profile following MLCK4 treatment contained 3 spots. Analysis of the N14D/S15 cMLC2 phospho-species profile with cMLCK treatment was negative, validating our initial Pro-Q staining result.

    While the implications of N14D transformation on the modulation of contraction remain unknown, these data indicate deamidation of Asn 14 prevents access to critical regulatory phosphorylation motifs (S15) and additional sites in human cMLC2. Thus, N14D transformation may play a role in the diminished levels of cMLC2 phosphorylation reported in heart failure.

    The newly detected myosin binding protein C/troponin interaction, function and thin filaments integrity affected by cTn mutants

    H. Budde1*, R. Hassoun 1*, S. Fujita-Becker2, A. Kostareva3,4, H. Milting5, M. Nowaczyk6, P. Reusch7, A. Mügge1, R. Schröder2, H.G. Mannherz8, K. Jaquet1 and D. Cimiotti1

    1Research Laboratory Molecular Cardiology, Cardiology, Bergmannsheil and St. Josef Hospital, Clinics of the Ruhr-University Bochum, Germany; 2Cryoelectron microscopy, Netcell, Bioquant, Medical Faculty, University of Heidelberg, Germany; 3Department of Molecular Biology and Genetics, Almazov Federal Medical Research Center, St. Petersburg, Russia; 4Department of Women’s and Children’s Health and Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden; 5Erich and Hanna Klessmann Institute, Heart and Diabetes Center NRW, University Hospital of the Ruhr-University Bochum, Bad Oeynhausen, Germany; 6Plant Biochemistry, Faculty of Biology and Biotechnology, Ruhr-University Bochum, Germany; 7Department of Clinical Pharmacology, Ruhr-University Bochum, Germany; 8Department of Anatomy and Embryology, Medical Faculty, Ruhr-University Bochum, Germany. *These authors contributed equally to this work

    Mutations in cardiac Troponin (cTn) subunits are common cause for the development of cardiomyopathies (CM). Newly discovered point mutations leading to mutant cTnI (D127Y, R170G/W), associated with restrictive cardiomyopathy, were identified in infants. Additionally, a novel point mutation leading to cTnC-G34S has been linked to the development of an end stage of heart failure in a newborn.

    Our aim is to investigate the functional consequences of these mutations and the effects on interactions between thin and thick filament proteins. Hereby we detected a direct interaction between cTn and Myosin binding protein C (cMyBP-C fragment C0-C2).

    In order to study the mutation-induced changes in binding affinities between cMyBP-C and Tn, microscale thermophoresis (MST) has been applied. The affinity of cTn mutant cTnI-R170G but not cTnI-R170W towards cMyBP-C was significantly increased compared to wildtype cTn. MST measurements using troponin subunits have proven that cTn attaches to cMyBP-C via cTnT and cTnI, but not cTnC. The binding affinities of the mutant subunits alone towards MyBP-C are unchanged compared to wildtype.

    Effects of troponin mutations on thin filament activation were studied by recording the Ca+2 dependent excimer fluorescence of pyrenemaleimide labeled tropomyosin (TM-PM). cTnI R170G/W mutations show, that only the cooperativity but not pCa50 was decreased in the presence of cMyBP-C compared to wildtype. The addition of cMyBP-C and Myosin S1 resulted in decreased pCa50 values of the thin filaments containing either cTnC-G34S or cTnI D127Y. EM analysis of the reconstituted thin filaments revealed significant fragmentation in case of CM mutations.

    The obtained data demonstrate an impaired thin filament activation due to the mutations. Moreover, we have shown that MyBP-C affects the mutant filaments differently and proven for the first time that cTnI and cTnT are its binding partners on cTn.

    Phosphorylation and binding of CA2+ to RLC, counters the effect of Mavacamten by destabilizing the myosin super-relaxed staTE

    N. Sa1, S. Gollapudi1, I. Tomasic1 and S. Nag 1 *

    Dept. of Biology, MyoKardia Inc, South San Francisco, California. *Correspondence:

    Myosin regulatory light chain (RLC) is a regulatory subunit of the myosin molecule, which plays a major role in stabilizing the Interacting Head Motif (IHM) of myosin in non-striated muscle. However, in striated muscle, its purpose is less well defined, and it is only in the past decade that researchers have started unraveling the function of RLC and its phosphorylation in muscle contraction. In this study, using synthetically formed myosin thick filaments, we show that the presence of RLC is critical in stabilizing the Super-Relaxed State (SRX) of myosin, which is an indirect biochemical measure of the auto-inhibited or ‘OFF’ state of myosin. Phosphorylation of the RLC with Myosin Light Chain Kinase (MLCK) destabilizes the SRX state, which renders more myosin to the ‘ON’ state as confirmed by its higher ATPase activity. Naturally occurring cardiomyopathy-causing RLC mutants K104E and D94A and not R58Q abolished this phosphorylation-mediated regulation.

    Interestingly we also found that Ca2+ and not Mg2+ binding to synthetic myosin filaments had modulating effects on the population of the myosin SRX state. At systolic high Ca2+ myosin exhibits lower SRX state than at low diastolic Ca2+. This effect is obliterated in RLC-lacking myosin constructs such as sS1, or RLC stripped myosin filaments suggesting that Ca2+ binding to RLC can activate the myosin from the ‘OFF’ state to a more active ‘ON’ state. Altogether, these observations demonstrate that either RLC phosphorylation or binding to Ca2+ can reduce the number of accessible myosin heads for contraction and could form the basis of the Ca2+-mediated activation of the sarcomeric thick filament—a long-standing hypothesis! Mavacamten, a phase-III clinical myosin inhibitor, stabilizes the SRX population of myosin more at low Ca2+ than at high Ca2+ further bolstering the Ca2+ induced thick filament activation hypothesis.

    Molecular mechanisms and therapeutic approaches to myofilament glycation as a result of diabetes

    M. Papadaki 1, J. Theerachat1 and J.A. Kirk1

    Loyola University Chicago, Maywood, IL, USA

    We previously discovered that methylglyoxal (MG), a glycolysis byproduct, modifies arginine and lysine residues on myofilament proteins to a greater extent in patients with diabetes and heart failure (dbHF) than non-failing controls. These modifications reduced myofilament calcium sensitivity (pCa50) and maximal force (Fmax), and despite being primarily on actin and myosin, did not directly affect their direct binding. Here we aimed to elucidate MG’s molecular mechanisms and identify effective therapeutic options for dbHF patients. We utilized a custom loaded fiber system to simultaneously measure isometric force and ATPase activity. Mouse fibers treated with 100 μM MG for 40 min exhibited a decrease in force production with a concomitant decrease in ATPase activity such that tension cost was unaffected. In skinned myocytes, we also found that treatment with MG decreased ktr, indicating a negative impact on crossbridge cycling kinetics. Together, these data along with our previous findings suggest that MG doesn’t alter myosin function directly but rather crossbridge formation through actin/myosin interactions with tropomyosin/troponin.

    MG modifications are irreversible, so therapeutic approaches should compensate for the functional defects induced. Aminoguanidine (AG) is an MG scavenger that has been efficacious in basic science studies but failed in clinical trials. Agreeing with the clinical trials, 1 mM AG could not reverse MG’s effects. We next used Omecamtiv mecarbil (OM), a myosin activator currently in clinical trials. As reported previously, we found that OM increased pCa50 and decreased Fmax in normal myocytes. However, in myocytes pretreated with MG, OM had no effect of pCa50 but still decreased Fmax. These data suggest OM may have less, or detrimental, effects in diabetic patients. Here we further elucidated the mechanism of MG’s myofilament impact and that these modifications reduce the efficacy of OM, a small molecule currently in clinical trials.

    Measurement of myofilament-localised calcium dynamics in adult cardiomyocytes to assess the effect of small molecule treatments for HCM

    A. J. Sparrow1, K. Sievert1, H. Watkins1, M.A. Geeves2, C. S. Redwood1, M. J. Daniels1 and P. Robinson 1

    1Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford, UK; 2Department of Biosciences, University of Kent, Canterbury, UK

    Hypertrophic cardiomyopathy (HCM) is principally caused by mutations in genes encoding sarcomeric proteins, which alter contractility, Ca2+-buffering, Ca2+-cycling, and Ca2+-dependent signalling via Ca2+/calmodulin dependent protein kinase II, calcineurin and extracellular regulated kinase. In order to investigate small molecule modulators of myofilament Ca2+/contractility and their potential to treat HCM, we have conjugated the genetically encoded calcium indicator RGECO to either troponin T (cTnT) or troponin I (cTnI) (myoRGECO) to report micro-domain calcium-flux. Importantly, myoRGECO conjugation does not alter the intrinsic function of RGECO or troponin complex in vitro. Using adenoviral transduction of myoRGECO, we investigated the changes in myofilament-localised Ca2+ in adult guinea pig left ventricular cardiomyocytes. We find that HCM mutations (TnI R145G and TnT R92Q) introduced by co-adenoviral transduction, increase systolic Ca2+ at the myofilament whereas cytoplasmic Ca2+ transients (measured using unconjugated RGECO) give mutation specific alterations to Ca2+ transient amplitude. Furthermore, these data contrast with previous findings using chemical dyes, which also show no alteration of Ca2+ transient amplitude. We subsequently tested the application of the small molecule modulators of contractility, on control cardiomyocytes to screen for the most suitable drug to rescue the effects of HCM Ca2+ dysregulation. We found that levosimendan increased myofilament systolic Ca2+ and omecamtiv mecarbil did not alter Ca2+ flux, whereas both diltiazem and mavacamten reduced systolic Ca2+ at the myofilament and were considered the most viable candidates to prevent pathological Ca2+ signalling in HCM. Finally, myoRGECO revealed a partial rescue of both myofilament and cytoplasmic Ca2+ transients by mavacamten in co-transduced HCM cardiomyocytes. Of note, there was a reversal in sensor Ca2+ binding rates not detected by a cytoplasmic protein sensor. myoRGECO provides additional mechanistic insights into small molecule modulators of contractility and HCM-causing myofilament mutations and provides a useful screening tool for potential drug treatments for HCM and heart failure.

    Effect of gradual cold acclimation on beta-adrenergic signalling in rat heart

    V. Tibenska 1, A. Benesova1, P. Vebr1, S. Vybiral1, L. Hejnová1, D. Hornikova1, B. Elsnicova1, J. Novotny1, O. Novakova1,2 and J.M. Zurmanova1

    1Department of Physiology, Faculty of Science, Charles University, Prague, Czech Republic; 2 Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic

    Acute cold was considered as a cardiovascular risk factor and a tight correlation between acute cold exposure and winter mortality from heart disease was repeatedly reported. Contrary to that cold acclimation (CA) increases organismal resistance to different pathological stimuli and we have shown that it may improve myocardial ischemic tolerance in rat. However, the molecular mechanism underlying this phenomena is not fully understood. Aim of the study was to assess metabolic response to adrenergic stimulation in rat and to analyse changes in β-AR signalling in the left ventricle (LV) after cold acclimation and their durability. CA of male Wistar rats was initially carried out for 8 h/day at 8 °C for a week, followed by four weeks at 8 °C for 24 h/day. The recovery group (CAR) was kept at 24 °C for another two weeks, while control animals were kept at 24 °C throughout the whole experiment. Metabolic rate was assessed after infusion of noradrenaline (1.8 μg/ml/100 g) by respirometer. Protein levels of β-ARs were analysed by western blotting in the crude membrane fractions and their subcellular distribution by quantitative immunofluorescence of the cryosections. Activation of selected downstream signalling pathways were also analysed. The data showed that CA increased basal metabolic rate and it returned to control level after CAR. Noradrenaline stimulated metabolic rate in both control and CA groups, but not after CAR. In parallel, all β1, β2 and β3-ARs were increased by CA and only β2 and β3-ARs remained increased after CAR. Importantly, the pPKA/PKA ratio was not affected in acclimated rats and PKB was activated only after CAR. In conclusion, the CA- and CAR-elicited upregulation of β2- and β3-ARs suggests a progressive enhancement of coupling of β-adrenergic signalling to the inhibitory Gi-protein/PKB pathway, which may play a role in the persisting cytoprotective effect of CA.

    GAUK No. 64126; GACR 17-07748S

    POSTERS (And TALKS from Young Investigator Session)

    Quantum dots mediated thermometric measure of enzyme features of myosin extracted from single muscle fibre

    M. Li1, L. Coppo2, A.B. Addinsal 1, H. Akkad1, Y. Hedström1 and L. Larsson1

    1Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden; 2Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

    The quantum dots (QDs) are zero-dimensional semiconductors with enhanced photo-stability and brightness. Featured by altered emitted fluorescence intensity and spectral wavelength, QDs have been applied for probing temperature changes in biological systems. This thermometry has been recently introduced to evaluate myosin efficiency during ATP hydrolysis, which is essential to convert chemical energy to mechanical work. The heat loss during ATP hydrolysis increases temperature, which causes decreased QDs fluorescence intensity. In this study, QDs mediated thermometric measure has been adapted to the single muscle fibre approach, which aims to evaluate enzyme kinetics of myosin ATPase extracted from single muscle fibres. Methodologically, the standard cure of linear regression is firstly established between the different concentrations of commercially available myosin and the enzyme reaction velocity. The velocity equals the slope calculated from linear regression between initial section of time course and the relative change of QDs fluorescence intensity, while the applied ATP concentration is optimized. Secondly, for myosin extracted from single muscle fibres, the respective slopes are determined from the linear regression between initial section of time and the relative change of QDs fluorescence intensity while a ladder of ATP concentrations is applied. The concentration of the extracted myosin is calculated by normalizing to the standard curve with known myosin concentrations and common ATP concentration. Thirdly, Michaelis constant (Km) and the turnover number (Kcat) are calculated from non-linear enzyme kinetics regression between different ATP concentrations and the reaction velocity where the extracted myosin concentration is known. This in turn reflects the affinity and efficiency of myosin ATPase. The QDs mediated thermometric measure of enzyme features of myosin extracted from single fibre provides a nanoscale sensitive approach to evaluate myosin function, which will be beneficial to the research targeting myosin protein or the isoform dependent properties and even be complementary to biophysical investigation.

    Matrix rigidity determines smooth muscle cell function

    S. Ahmed and D. Warren

    University of East Anglia, Norwich Research Park Norwich, NR5 8NX United Kingdom

    Decreased aortic compliance is associated with ageing and vascular disease, including atherosclerosis and hypertension. Changes in aortic compliance are driven by altered ECM composition. Vascular smooth muscle cells (VSMCs) line the blood vessel wall and VSMC contraction regulates vascular tone. Mechanical cues, such as stretch, are known to influence VSMC function. Whether changes in ECM rigidity alter VSMC function remains poorly defined. In this study, we investigate how matrix rigidity affects VSMC contractile response. Isolated human aortic VSMCs were grown on 12 kPa (pliable) and 72 kPa (rigid) polyacrylamide hydrogels. Quiescent VSMCs were stimulated with AngII for 30 min to initiate contraction. Analysis revealed that on pliable hydrogels, VSMCs contract, cell area is reduced but cell volume remains unchanged. In contrast, on rigid hydrogels, VSMCs fail to contract and cell area/volume is enhanced. Traction force microscopy (TFM) revealed that VSMCs generate increased traction force (TF) magnitudes on rigid hydrogels. The rigid hydrogel response was abolished by addition of the stretch activated ion channel antagonist GsMTx4 or by depleting Ca2+ from the medium. Finally, live cell imaging revealed that VSMCs contract on pliable hydrogels whereas migration is induced on rigid hydrogels. These data suggest that ECM rigidity alters VSMC function and serves as a VSMC migrational cue.

    Septin 7 has an essential role in differentiation of C2C12 cells

    Á. Angyal, M. Gönczi, Z. Ráduly, L. Szabó, N. Dobrosi, B. Dienes and L. Csernoch

    University of Debrecen, Faculty of Medicine, Department of Physiology, Debrecen, Hungary

    Cytoskeletal components septins have important roles in various cellular processes, e.g. cell mobility, apoptosis, endocytosis and determining cell shape within a wild range of organisms, including yeast, drosophila and mammals. 13 human septins have been described and classified into 4 subgroups, where septin 7 is the only member of its group and it has a crucial role in the formation of higher-order structures of septins.

    As the role of septin 7 in skeletal muscle is not fully understood yet, we wanted to examine the effect of septin 7 expression changes on C2C12 cultured cells. We were able to show cytoplasmic distribution of septin 7 in cultured myoblast and myotubes and its co-localization with actin filaments. To modify the expression of septin 7 in the cultured cells shRNA technique was applied. Three different targeting shRNA-sequences were designed which decreased the protein level while the cells remained alive. However, decreased septin 7 expression generated remarkable changes in cell morphology and other physiological processes. In septin 7 knockdown cells (KD) the average cell area increased compared to both the absolute control and scrambled shRNA transfected cells. The perimeter of septin 7 KD cells did not show any significant alteration, however cells loosed their processes and became more circular than the controls. Cell proliferation and the myotube differentiation were also significantly reduced in KD cells. During the differentiation multinucleated myotubes were hardly detected, the fusion index was markedly reduced, and differentiation marker desmin protein expression was significantly lower in septin 7 modified cultures. These finding in the skeletal muscle model system C2C12 cells indicate that septin 7 has essential role in the proper development and differentiation process of myotubes. The research was supported by Hungarian Research Founds (NKFIH K-115461 and GINOP-2.3.2-15-2016-00044).

    Myofibrillar function differs markedly between denervated and dexamethasone-treated rat skeletal muscles

    Y. Ashida 1, K. Himori1, D. Tatebayashi1 and T. Yamada1

    1Graduate School of Health Sciences, Sapporo Medical University, Sapporo, Japan

    To investigate the role of mechanical load in myofibrillar function, we compared the skinned fiber force in denervated (DEN) or dexamethasone-treated (DEX) rat skeletal muscles with or without neuromuscular electrical stimulation (ES). DEN and DEX were induced by cutting the sciatic nerve and daily injection of dexamethasone (5 mg/kg/day) for 7 days, respectively. For ES training, plantarflexor muscles were electrically stimulated to produce four sets of five isometric contractions each day. In situ maximum torque was markedly depressed in the DEN muscles compared to the DEX muscles (− 74% vs. − 10%), whereas there was not much difference in the degree of atrophy in gastrocnemius muscles between DEN and DEX groups (− 24% vs. − 17%). Similar results were obtained in the skinned fiber preparation, with a greater reduction in maximum Ca2+-activated force in DEN than in DEX group (− 53% vs. − 16%). Moreover, there was a parallel decline in myosin heavy chain and actin content per muscle volume in DEN, but not in DEX, muscles, and augmentation of mRNA levels of muscle RING finger protein 1 and atrogin-1. DEN resulted in upregulation of NADPH oxidase 2, neuronal and endothelial nitric oxide synthase expression. Importantly, mechanical load evoked by ES protects against DEN- and DEX-induced myofibrillar dysfunction and these molecular alterations. Our findings provide novel insights regarding the difference in intrinsic contractile properties between DEN and DEX and suggest an important role of mechanical load in preserving myofibrillar function in skeletal muscle.

    The role of PIEZO1 expression and its activation in the calcification of arterial smooth muscle cells

    N. Balogh 1; Á. Angyal1; C. Tóth2; B. Dienes1; G. Czifra1 and L. Csernoch1

    1Department of Physiology, Faculty of Medicine, University of Debrecen; 2Clinic of Surgery, Clinical Center, University of Debrecen

    Mechanically activated, non-selective Piezo1 cation channels are evolutionarily conserved proteins the presence of which is crucial in many physiology processes. Their activation has already been shown to play a predominant role in the early development of vascular architecture and the lack of function of the channel is embryonically lethal due to a disorganized vascular structure. However, the role of Piezo1 in arterial smooth muscle cells in the adult is not fully clarified. Nevertheless, being a mechanosensitive channel, it presumably plays a role in the development of atherosclerotic plaques and the calcification of the vessels, which likely results from the increased shear forces due to dysregulation of circulation. Here we investigated the expression of Piezo1 channels in human atherosclerotic blood vessel samples derived from superficial femoral artery (SFA) and internal carotid artery (ICA) as well as examining the expression and function of Piezo1 on human aortic cell line (HAOSMC cells). To prove the expression of Piezo1 on human arterial samples immunohistochemistry was used and the labeling was optimized for both fluorescent and chromogenic labelling. Our results suggest that Piezo1 is only weakly expressed in SFAs, while the expression of the channel was high in ICA samples. Expression of the Piezo1 was confirmed by mRNA (qPCR) and protein level (Western blot). The functional measurements were carried out on HAOSMC cells, a widely accepted model of the vascular smooth muscle, after the presence of Piezo1 channels was proven the on these cells, too. Using confocal microscopy the intracellular Ca2+ concentration upon Piezo-1 activation was compared for control and calcified HAOSMC cells. Our preliminary results suggest that Piezo1 channels could indeed be involved in the development of certain arthropathies by influencing the calcification of the vascular smooth muscle cells. The research was financed by EFOP-3.6.2-16-2017-00006; Hungarian Research Found (NKFIH K-115461).

    Effects of manipulating levels of aggregating chaperone and ubiquitination proteins in a drosophila model of myosin-based inclusion body myopathy 3

    K. Manalo1, J.A. Suggs1, G.C. Melkani1, A. Melkani1, D.B. Foster2, S.I. Bernstein 1

    1Department of Biology, San Diego State University, San Diego, California, USA; 2Division of Cardiology, School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA

    We produced a Drosophila model of inclusion body myopathy 3 (IBM-3), a rare, dominant skeletal muscle disease with an E706K substitution in the SH1 helix of myosin heavy chain IIa. As in the human disease, progressive degradation occurs in IBM-3 Drosophila indirect flight muscles (IFM). Homozygotes accumulate thermally unstable myosin and autophagic vesicles. We identified aggregated proteins within inclusions by extracting insoluble proteins from IFM of young and old homozygotes. Quantitative iTRAQ proteomics defined 18 proteins with > 1.5-fold difference in relative abundance at both ages in mutants vs. controls. Two of 6 proteins over-represented in IBM-3 have small heat shock protein domains (Hsp20, CG7409), while the 12 under-represented proteins include Hsp23, Hsp60, and Trim32 (abba/thin), which has ubiquitin–protein transferase activity. Using the GAL4-UAS system for over-expression or knock down (RNAi), we tested the role of these proteins in wild-type muscle and determined how altering expression affects the IBM-3 heterozygote phenotype. While knockdown of Hsp20, Hsp23 or CG7409 yielded normal flight in wild type, Hsp23 or CG7409 over-expression resulted in age-dependent reduction in flight ability. While knockdown or over-expression of these chaperones typically exacerbated the IBM-3/+ phenotype, preliminary results suggest a slight improvement in flight ability when hsp23 is over-expressed. For Trim32, knockdown or over-expression eliminated flight in wild-type or IBM-3/+ flies. These phenotypes correlate with less organized, thinner myofibrils in the knockdown. Over-expression yielded deteriorating myofibrils that hypercontract. Remarkably, electron microscopy shows that Trim32 over-expression in the IBM-3/+ background produces a phenotype that resembles the dramatic destruction of myofibrils observed in IBM-3 homozygotes. Overall, each of the proteins assessed is key to function of wild-type muscle and manipulating their levels typically has detrimental effects upon the IBM-3/+ phenotype. This suggests that levels of these proteins are well regulated to yield optimal proteostasis in both health and disease.

    Changes in α-tropomyosin induced by posttranslational modifications and their effect on the actin-myosin interaction in the myocardium

    G.V. Kopylova1, A.M. Matyushenko2, V.Y. Berg1, S.R. Nabiev1, L.V. Nikitina1, D.I. Levitsky2, D.V. Shchepkin1, S.Y. Bershitsky 1

    1Institute of Immunology and Physiology, Russian Academy of Sciences, Yekaterinburg, 620049, Russia; 2A.N. Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, 119071, Russia.

    In cardiac pathologies like myocardium ischemia or infarction, the two most prevalent posttranslational modifications in the molecule of α-tropomyosin (Tpm), carbonylation and phosphorylation, can occur. Tpm phosphorylation is considered to be one of the regulatory mechanisms of its function, whereas carbonylation is purely pathological modification being a result of protein oxidation. We aimed to investigate the difference in their effects on the Tpm properties and the regulation of the actin-myosin interaction in the cardiac muscle at the molecular level.

    For this, human recombinant WT α-Tpm and the Tpm S283D mutant mimicking its phosphorylated form were expressed in E. coli. Carbonylation of Tpm was attained by oxidation the protein by FeCl3/ascorbic acid solution, according to Maisonneuve et al. (2009). The extent of protein carbonylation was determined with MALDI mass spectrometry. Myosin and troponin from left pig ventricle and rabbit skeletal actin were obtained by standard methods.

    It was found that Tpm phosphorylation slightly increased the thermal stability of Tpm and worsened inhibition of the sliding velocity of thin filaments at low [Ca2+]. At neutral pH, it reduced the dissociation temperature of the Tpm–F-actin complex and substantially increased the viscosity of Tpm solution. Acidulation diminished these effects and increased the Ca2+ sensitivity of the sliding velocity of thin filaments.

    Carbonylation disrupted the cooperative melting of the molecule, increased the bending stiffness of thin filaments due to interchain cross-linking in Tpm molecule by C190 residues, made impossible interaction of Tpm with F-actin, and entirely killed Ca2+ regulation of the sliding velocity of thin filaments.

    From the results obtained, we conclude that phosphorylation of Tpm may serve as a fine-tuning of its functioning, while carbonylation destructs Tpm molecule and so the formation of the Tpm–F-actin complex. Supported by RFBR grants 17-00-00070 and 18-34-20085, Program AAAA-A18-118020590135-3.

    Redistribution of focal adhesion kinase in rat neonatal cardiomyocytes in culture

    N. Bildyug

    Institute of Cytology RAS, St. Petersburg, Russia

    Rat neonatal cardiomyocytes in culture undergo reversible rearrangement of their myofibrillar apparatus with conversion of myofibrils into stress fiber-like structures. Previously we have shown that this rearrangement is accompanied by transient replacement of cardiac actin by smooth muscle isoform and accumulation of extracellular matrix. The concomitant changes in integrins, which provide connection between cells and matrix, suggested that dynamics of contractile apparatus in cardiomyocytes may be regulated by integrin-transmitted extracellular signals. Integrins are known to transfer mechanical stimuli from extracellular matrix into the intracellular signaling via associated kinases, such as focal adhesion kinase (FAK) that is activated upon binding of integrins with extracellular matrix proteins.

    This study was aimed to reveal the distribution of FAK at different stages of contractile apparatus rearrangement in rat neonatal cardiomyocytes and to correlate these data with actin isoform switching. In freshly isolated cardiomyocytes, immunofluorescent staining revealed only traces of FAK, probably due to the initial lack of extracellular matrix. At the stage of total rearrangement of contractile apparatus with the appearance of smooth muscle actin and intensive accumulation of extracellular matrix the distribution of FAK generally corresponded to stress fiber-like structures, which may be explained by the involvement of both components in the formation of focal adhesions. The recovery of myofibrillar apparatus in cardiomyocytes with the re-expression of cardiac actin isoform was accompanied by redistribution of FAK to the newly formed myofibrils. This may be related to the recruitment of FAK to the costamere complexes linking myofibrills with cell membrane and extracellular matrix. In general, the obtained results suggest that FAK may be involved in regulation of contractile apparatus dynamics in cardiomyocytes. Supported by the Russian Science Foundation (Grant 18-74-00129).

    The impact of histone deacetylase inhibitors on cardiomyogenic differentiation of human myocardium-derived mesenchymal stem cells

    R. Miksiunas1, R. Gradauskaite1, J. Buivydaviciute1, K. Rukšaite1, K. Rucinskas2, V. Janusauskas2, S. Labeit3 and D. Bironaite 1

    1State Research Institute for Innovative Medicine, Department of Regenerative Medicine, Vilnius, Lithuania; 2Centre of Cardiothoracic Surgery of Vilnius University Hospital Santariskiu Klinikos, Vilnius, Lithuania; 3Universitätsmedizin Mannenheim, Department of Integrative Pathophysiology, Mannenheim, Germany.

    Background Dilated cardiomyopathy (DCM) is characterized by dilated and systolic dysfunction of left (or both) ventricular subsequently decreasing ventricular ejection fraction. Treatment of DCM is mainly directed towards the reduction of heart failure symptoms and restoration of heart function. Therefore, promotion of cardiomyogenic differentiation of human myocardium-derived primary cells is very important step in restoration of impaired cardiac tissue. In this work, the effect of histone deacetylase inhibitors on functioning and cardiomyogenic differentiation of healthy and dilated human myocardium-derived mesenchymal stem cells (hmMSC) has been investigated. Materials and Methods hmMSC were isolated from post cardiac surgery muscle biopsies by explant outgrowth or enzymatic method. The MSC origin has been proved by the positive and negative expression of specific MSC surface markers (CD73, CD90, CD105, CD45, CD34, CD14) and differentiation potential. The histone deacetylase activity was measured using fluorescent substrate Boc-Lys(Ac) and inhibitors Vorinostat, MC1568 and Mocetinostat. Expression of cardiomyogenic gene (Nkx2,5, cardio alpha-actin, cTnT, HOPX, GATA4 and other) have been identified by qPCR. The protein expression has been proved by the western blotting and immunocytochemistry.

    Results Data show that human healthy and dilated myocardium-derived MSC express similar cell surface markers compared to the other types of somatic MSC. Dilated myocardium-derived hmMSC showed almost two folds’ higher level of histone deacetylase activity which was significantly inhibited by used inhibitors with subsequent inhibition of intracellular calcium, strong activation of mitochondrial activity and cardiomyogenic genes such as cTnT, alpha-actin, Nkx2.5 and other. Used histone deacetylase inhibitors more effectively stimulated cardiomyogenic genes of pathological hmMSC. Conclusions Data show that human dilated myocardium-derived hmMSC still have remaining regenerative potential which possible to activate by the epigenetic regulation of histone acetylation. The compounds suppressing histone deacetylase activity of dilated myocardium-derived hmMSC can be used as promising therapeutic mean stimulating heart muscle regeneration.

    Functional smooth muscle cell lines as in vitro test systems for the drug development

    A. Bleisch 1, S. Dehmel2 and T. May1

    1InSCREENeX GmbH, Braunschweig, Germany; 2Fraunhofer ITEM, Hannover, Germany

    Airway smooth muscle (ASM) is the main effector of airway hyperresponsiveness, a hallmark of asthma and mild-to-moderate chronic obstructive pulmonary disease (COPD). Asthma and COPD are highly prevalent and represent a major global health and economic burden. Currently-available drugs give partial benefit by relieving disease symptoms. For the development of more efficacious drugs, in vitro test systems mimicking in vivo airways are highly desirable. For this purpose smooth muscle cells are indispensable. This study aimed to generate authentic smooth muscle cell lines that can be used to build up functional 3D muscle tissues to improve the drug testing in vitro. Therefore, in a first step bronchial smooth muscle cell lines were established by the CI-SCREEN technology [1]. In this technology, a lentiviral gene library is introduced into the respective primary cell and induces expanded cell proliferation as well as maintenance of the primary phenotype. Resulting cell lines of a healthy donor and a COPD patient showed unlimited proliferation and reached 30 cumulative population doublings within 70–120 days. In contrast primary cells stopped proliferation completely after reaching 2–10 population doublings. Characterization of the novel cell lines showed homogenous expression of smooth muscle specific markers as alpha smooth muscle actin and calponin. Furthermore, the release of calcium and thus the presence of relevant receptors was displayed in response to histamine and methacholine. Moreover smooth muscle constriction was shown after receptor stimulation. These robustly proliferating and highly functional smooth muscle cell lines are an excellent tool to create novel in vivo-like assay systems to forward the development of new and more effective drugs.

    1. Lipps, C., Klein, F., Wahlicht, T., Seiffert, V., Butueva, M., Zauers, J., & May, T. (2018). Expansion of functional personalized cells with specific transgene combinations. Nature Communications, 9,

    Mobility of nebulin molecules in sarcomeres of Nebulin-dendra2-KI mice

    S. Bogaards 1, M. Yuen1, N. Klingberg1, J. Kole1, R. van der Pijl1,2, S. Shen2, P. Tonino2, H. Granzier2 and C. Ottenheijm1,2

    1Amsterdam UMC, Department of Physiology, Amsterdam, the Netherlands; 2University of Arizona, Cellular and Molecular Medicine, Tucson, USA

    Nebulin spans the length of the thin filament, with its C-terminus located in the z-disc and its N-terminus near the thin filament pointed-end. Genetic mutations in nebulin, as well as in proteins that bind to nebulin cause myopathy. The pathophysiological mechanisms are incompletely understood, in part because of a lack of knowledge of the kinetics of nebulin molecules. We studied the kinetics of nebulin molecules using a mouse with photoconvertable Dendra2 inserted at the nebulin N-terminus (Dendra2-KI) using fluorescence recovery after photoconversion (FRAP) microscopy. With FRAP, a fraction of Dendra2 in the fiber is converted from the green to the red fluorescence state. Recovery of the green fluorescence is measured in time. Adult single FDB fibers of Dendra2-KI mice were isolated and imaged with FRAP and recovery was measured to determine the mobile fraction of nebulin. 67% of the green fluorescence was converted to the red state. Only 26% of this loss in fluorescence was recovered during the following 9 days, indicating very slow nebulin kinetics in FDB fibers. Pilot experiments were done with electrically stimulated fibers. The fluorescence recovery, however, was similar to unstimulated fibers, indicating that contractile activity does not affect the kinetics of nebulin mobility.

    Protein synthesis was blocked using cyclohexamide. Fibers survived up to 2 days. Fibers treated with cyclohexamide showed fluorescence recovery comparable to untreated fibers, indicating that de novo protein synthesis did not affect nebulin kinetics within this timespan.

    Myotubes were differentiated from FDB cultures and FRAP was determined. Myotubes allowed us to study nebulin kinetics in a system with higher protein turnover. In myotubes the conversion level of Dendra2 was 55%. In 2 h, 61% of fluorescence was recovered, indicating much higher nebulin kinetics in differentiating myotubes.

    These results indicate that nebulin is a highly immobile protein in mature muscle fibers.

    Exercise prevents formation of tubular aggregates in ageing skeletal muscle fibers

    C. Pecorai1, A. Michelucci1, L. Pietrangelo1, F. Protasi1, and S. Boncompagni 1

    1CeSI-MeT, Center for Research on Ageing and Translational Medicine, Univ. G. d’Annunzio, I-66100 Chieti.

    Tubular aggregates (TAs) are unusual accumulation of sarcoplasmic reticulum (SR) tubes found in skeletal muscle fibers of Tubular Aggregate Myopathy (TAM) patients that have been recently linked to mutations in STIM1 and Orai1. These two proteins are the two main molecular players in store-operated Ca2+ entry (SOCE), a ubiquitous mechanism that allows recovery of extracellular Ca2+ during repetitive muscle activity. We have recently shown that: (i) exercise triggers the formation of unique intracellular junctions between SR and TTs (Ca2+ Entry Units, CEUs) that promotes co-localization of Orai1 with STIM1 and improves muscle function in presence of external Ca2+; (ii) TAs also form in muscle of male mice during ageing starting from the remodeling of SR tubes/vesicles at the I-band. Using a combination of electron and confocal microscopy (EM and CM), western blotting (WB), and functional studies (ex vivo stimulation protocols in presence of absence of external Ca2+), here we analyzed ultra-structure, STIM1-Orai1 localization/expression, and fatigue resistance of EDL muscles dissected from adult and aged mice, the latter divided in 2 sub-groups: control and exercised in wheel cages for 15 months. The results collected using CM and EM indicated that: (i) ageing causes STIM1 and Orai1 to accumulate in TAs; and (ii) exercise significantly reduced formation of TAs and promoted maintenance of CEUs. Parallel functional studies revealed that: (iii) aged EDLs exhibit a faster decay of contractile force than adult muscles, likely caused by their inability to recruit extracellular Ca2+; and (iv) exercise restored the lost capability of aged EDL muscles to use external Ca2+. Our results suggest that exercise prevents improper accumulation of STIM1 and Orai1 in TAs during ageing restoring the capability of aged muscle to use external Ca2+ via SOCE.

    How two tropomyosins activate insect flight muscle

    K. Drousiotis1, D. Koutalianos1, D. Sanfelice2, A. Pastore2 and B. Bullard 1

    1University of York, Department of Biology, York, UK; 2The Wohl Institute, King’s College London, 5 Cutcombe Road, London, SE5 9RT, UK

    The rapid contractions of insect flight muscle (IFM) are activated by periodic stretches at a constant concentration of calcium. We have investigated a possible stretch sensor in flight muscle. Bridges between thick and thin filaments have been observed in electron micrographs of relaxed muscle. The bridges bind to the thin filaments every 38.7 nm, at the position of troponin, and they alternate with target sites for force-producing crossbridges on actin. These troponin bridges may transmit stress between thick and thin filaments during the stretching phase of oscillating contractions. The interactions of the subunits in Lethocerus flight muscle troponin are described; models of the complex, using the structures of skeletal and cardiac troponin as templates, show an overall similarity of vertebrate and flight muscle complexes. The tropomyosin-troponin (Tm-Tn) complex isolated from Lethocerus flight muscle contains two isoforms of tropomyosin (Tm1 and Tm2). Tm-Tn and Tm1 alone bind to thick filaments and to both myosin and myosin-S1, whereas Tm2 does not bind to myosin. Crosslinking the Tms isolated together from IFM, showed that Tm1 and Tm2 are homodimers in situ, and could therefore act independently. Tm1 may be part of a troponin bridge and Tm2 may act as a conventional Tm. Electron microscopy of Tm-Tn or Tm1 associated with thick filaments showed regular binding to the filaments every ~ 40 nm. In some cases, thick filaments were crosslinked. If the non-force producing myosin crossbridges bind to Tm1 at the position of troponin, the whole length of Tm on thin filaments could be pulled from an inhibitory position on actin after a stretch; at the same time, stress on the thick filaments would convert myosin heads to the ON state. This could occur reversibly at every cycle of oscillatory contraction, without any changes in calcium concentration.

    Allelic imbalance of TNNI3 through burst-like transcription may lead to contractile imbalance in hypertrophic cardiomyopathy patients

    V. Burkart 1, J.Beck1, K. Kowalski1, J. van der Velden2, C. dos Remedios3, J. Montag1 and T. Kraft1

    1Hanover Medical School, Hannover, Germany; 2VU University Medical Centre, Amsterdam, Netherland; 3University of Sydney, Sydney, Australia

    Hypertrophic Cardiomyopathy (HCM) is the most common inherited cardiac disease with an incidence of 1:500. About half of the genotyped patients carry heterozygous mutations in genes encoding for sarcomeric proteins affecting their functionality and thereby mechanisms of force generation in cardiomyocytes. We found larger variability in force generation between individual cardiomyocytes from HCM patients with mutations in the myosin heavy chain (beta-MyHC) gene (MYH7) compared to donor cardiomyocytes. In the myocardium, such contractile imbalance among cardiomyocytes most likely contributes to development of HCM features like fibrosis and cardiomyocyte disarray. We provided evidence that functional heterogeneity among individual cardiomyocytes arises from unequal expression of mutated and wildtype beta-MyHC from cell to cell as result of burst-like, stochastic and independent transcription of the two alleles.

    Because mutations in different sarcomeric proteins induce HCM the question was whether other sarcomeric genes are also transcribed burst-like provoking contractile imbalance. We analyzed relative expression of mutant and wildtype cardiac Troponin I (TNNI3) mRNA and measured force generation at different calcium concentrations of individual cardiomyocytes from myocardial tissue of HCM-patients with TNNI3-mutation R145 W (c.433C > T). RNA-fluorescence in situ hybridization revealed that most nuclei contain no active transcription sites (81% in donor and 95% in patient tissue), while others show active transcription of one or both alleles. This indicates independent burst-like transcription of the two TNNI3 alleles. Single cell allele-specific RT-PCR revealed cardiomyocytes with essentially only wildtype, only mutant, or with mixtures of both, mutant and wildtype TNNI3 mRNA, indicating cell-to-cell allelic imbalance. Force measurements of chemically permeabilized cardiomyocytes of the same cardiac tissue samples showed larger variability in force generation at submaximal calcium-concentrations compared to donor cardiomyocytes.

    These results indicate, that TNNI3 is transcribed burst-like, which may induce cell-to-cell allelic imbalance of wildtype and mutated mRNA and contractile imbalance in the myocardium as a trigger for HCM.

    A study of heterogeneity in mechanical activity of single cardiomyocytes and contractile proteins from rat atria and ventricles

    X. Butova 1,2, T. Myachina1,2, V. Berg1, D. Shchepkin1, G. Kopylova1 and A. Khokhlova1,2

    1Institute of Immunology and Physiology of the Ural Branch of the Russian Academy of Sciences, Yekaterinburg, Russia; 2Ural Federal University, Yekaterinburg, Russia

    Uniform cardiac pump function requires heterogeneity at the cellular and molecular level. While electrical heterogeneity of cardiac cells from different chambers has been well-characterized, studies on the regional variation in mechanical properties are sparse. We investigated mechanical function of single cardiomyocytes and contractile proteins from atria, the left (LV) and right ventricle (RV).

    The study followed the Directive 2010/63/EU. Single cardiomyocytes from Wistar rats were isolated using the standard Langendorff-perfusion technique. The measurements of sarcomere shortening and cytosolic calcium with Fluo-8 AM (AAT Bioquest) were performed using laser confocal scanning microscopy. The interaction of myosin with native thin filaments (NTF) extracted from rat atria, the LV, and the RV was studied in an in vitro motility assay. Phosphorylation proteins was analysed by Pro-Q Diamond Phosphoprotein Gel Stain (Thermo Fisher Scientific).

    We found that the end-diastolic sarcomere length, the amplitudes of sarcomere shortening, and calcium transient were smaller in atrial cells compared with LV and RV cardiomyocytes. The negative inotropic effect with increasing frequency of stimulation (0.5–1–2–3 Hz) was observed for heart chambers, but less extent was found for atrial cells as compared with ventricular cardiomyocytes. The time-to-peak of contraction and time of relaxation were smaller for atrial cells compared with ventricular cells, but no significant differences were found between the LV and RV. In the in vitro motility assay, the sliding velocity of F-actin was higher over atrial myosin than over ventricular one. The sliding velocity of NTF over LV and RV myosin did not differ significantly. Phosphorylation degree of thin filament proteins from the LV and the RV was different. Thus, the heart manifests the heterogeneity in contraction at cellular and molecular levels with pronounced differences between the atria and ventricles. Supported by the President grant of the RF #MК-949.2019.4 and IIF UrB RAS theme.

    Mutations in the TNT1 tropomyosin-binding element of troponin T depress its inhibitory properties and stimulate myocardial dysfunction

    A Madan1, M.C. Viswanathan1, G. Vogler2, K.C. Woulfe3, W. Schmidt1, B. Trinh2, S. Madathil4, C. Wilson3, L.S. Tobacman4 and A. Cammarato 1

    1Johns Hopkins University, School of Medicine, Baltimore, MD, USA; 2Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA; 3University of Colorado, School of Medicine, Aurora, CO, USA; 4University of Illinois at Chicago, College of Medicine, Chicago, IL, USA

    In 2002, two separate studies revealed that the N-terminal fragment of Troponin T (TnT), i.e., TnT1, contributes directly to the inhibition of muscle contraction, independent of other troponin subunits. Impaired relaxation is a hallmark of several muscular disorders including hypertrophic (HCM) and restrictive (RCM) cardiomyopathies. Disease-causing cardiac TnT1 mutations may help explain the molecular basis of the enigmatic inhibitory property of TnT. We tested the hypothesis that specific HCM and RCM mutations within the highly-conserved TnT1 tropomyosin (Tpm)-binding element weaken its ability to help confine Tpm to an inhibitory position along F-actin, where the regulatory strand blocks myosin binding during diastole. In vitro, in situ and in vivo experiments were conducted to measure the effects of human TnT1 peptides on myosin-driven F-actin-Tpm sliding, and TnT variants on Drosophila cardiac performance. Motility results confirmed earlier findings that TnT1 substantially enhances Tpm’s native inhibitory properties. However, compared to control, the mutant peptides were significantly less effective at preventing force-generating myosin interactions with actin-Tpm-TnT1 filaments. Likewise, fly hearts over-expressing mutant TnTs displayed impaired relaxation and restrictive physiology that was predominantly due to excessive, unimpeded myosin cycling under low intracellular Ca2+ levels. Individual mutant myofibrils displayed elevated resting tension, also because of Tpm mispositioning and strong actomyosin binding. In light of previous and current findings we present a model that explains the mechanistic basis of TnT’s inhibitory role and of cardiomyopathy. Electrostatic contacts mediate F-actin-Tpm binding and place Tpm in a default location where it impedes actomyosin-dependent force generation. We propose that TnT1’s association with Tpm optimizes the formation of F-actin-Tpm contacts, and thereby promotes thin filament inhibition. Thus, the cardiomyopathy mutations may disrupt TnT’s normal contribution to myocardial relaxation by weakening TnT1-Tpm binding and destabilizing Tpm’s inhibitory positioning, potentially serving as the most proximal cause of pathology in our fly models and humans.

    In vivo structural dynamics of myosin binding protein-C

    J. Chandler 1, M. Irving1, and T. Kampourakis1

    1Randall Division for Cell & Molecular Biophysics, King’s College London, SE1 1UL

    The coordinated activation and de-activation of both the actin-containing thin and myosin-containing thick filaments of the sarcomere, the basic contractile unit of the heart, is fundamental for normal heart muscle function. Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated regulatory protein localized to the inner two thirds of the half-sarcomere A-band, called the ‘C-zone’, via interactions of its C-terminal domains with titin and myosin tail domains. Recent in vitro experiments have highlighted the functional significance of the N-terminal region of cMyBP-C for heart muscle contraction, reporting regulatory interactions with both thick and thin filaments. These interactions, in turn, are believed to control filament activation states, cross-bridge cycling kinetics, and the rate of myofilament activation and relaxation. Moreover, mutations in the gene encoding for cMyBP-C are the second-most common cause of heritable hypertrophic cardiomyopathy (HCM), further underlining its functional significance. However, filament-binding states of the regulatory N-terminal domains of cMyBP-C and the structural dynamics of these interactions during the cardiac cycle are not well understood. To answer these questions we are developing an in situ FRET binding assay between the N-terminus of full-length cMyBP-C, and thick (myosin regulatory light chain, RLC) and thin filament components (cardiac troponin T, cTnT) in living cardiomyocytes. Initial development and validation studies have been performed, confirming the expression of our fluorescently labelled proteins in both mammalian and bacterial cell systems. Moreover, fluorescent protein-tagged cMyBP-C and RLC constructs show correct localization to the sarcomeric C-zone and A-band, respectively, when expressed in isolated neonatal rat cardiomyocytes. Our results demonstrate the feasibility of the developed fluorescent protein-tagged constructs for use in an FRET binding assay in living cardiomyocytes. The results from these experiments will likely lead to a new model for the physiological function of cMyBP-C.

    Cardiomyocyte nuclei in knock-in mice with cardiomyopathy mutation cardiac troponin C A8 V are smaller but do not have lower ploidy than WT

    J.R. Johnston1, F. Ogunfuwa2, C.L. Tougas2, J. Le Patourel2, K. Dieseldorff Jones1, A.S. Martins1, K.M. Crotty2, K.M. Ward2, R. Didier1, J.R. Pinto1 and P.B. Chase 2

    1Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, FL USA; 2Department of Biological Science, Florida State University, Tallahassee, FL USA

    De novo mutation A8V in human cardiac troponin C (cTnC) results in cardiomyopathy (Landstrom et al. 2008 JMCC 45:281), an outcome that is recapitulated in both heterozygous and homozygous knock-in mice (Martins et al. 2015 Circ Cardiovasc Genet 8:653). The mutation alters cardiomyocyte function by markedly increasing Ca2+-sensitivity of steady-state MgATPase activity (Pinto et al. 2009 JBC 284:1990) and isometric force, and the kinetics of tension redevelopment (kTR) at all levels of Ca2+-activation (Gonzalez-Martinez et al. 2018 JMCC 123:26). The cTnC, A8V mutation also alters the structure of cardiomyocyte nuclei, which are significantly smaller in heterozygous and homozygous mice (Tougas et al., 2018 JMRCM 39:90). Because normal adult mouse cardiomyocytes often exhibit ploidy greater than 2, we hypothesized that smaller nuclei in cTnC, A8V cardiomyocytes would be associated with lower ploidy compared with WT. To test this hypothesis, cardiomyocyte nuclei were isolated from WT and cTnT, A8V homozygous adult mouse hearts using a procedure modified from Bergmann and Jovinge (2012, J Vis Exper). DNA content (NucBlue, Life Technologies) was quantified in PCM-1 positive nuclei (~ 30% of nuclei from both WT and cTnC, A8V hearts) using fluorescence-activated nuclear sorting (FANS) performed with a FACSAria Special Order System (Becton–Dickinson). While the majority of nuclei were diploid in both, the percentage of diploid nuclei was lower and that of tetraploid nuclei was higher in cTnC,A8 V nuclei, contrary to expectation. cTnC, A8V cardiomyocytes were also more likely to be multinucleated, although the majority of cardiomyocytes were binucleated in both WT and cTnC, A8V. Thus factors other than ploidy must be responsible for smaller nuclei in cTnC, A8V cardiomyocytes such as a possible direct role of cTnC in the actin component of the nucleoskeleton, or an indirect role such as increased expression of desmin (Sheng et al. 2016 JMCC 99:2018). Support: US NIH NHLBI HL128683 and NIH and AHA predoctoral fellowships.

    O-GlcNAcylation/phosphorylation interplay on desmin fate: focus on its partition and its interaction with alphab-crystallin, its molecular chaperone

    C. Claeyssen1, B. Bastide1 and C. Cieniewski-Bernard 1

    1URePSSS – EA7369 Physical Activity, Muscle, Health, University of Lille, France

    O-GlcNAcylation is an atypical glycosylation akin to phosphorylation. Dynamic and reversible, it modifies a plethora of structural and contractile proteins. We have previously demonstrated that O-GlcNAcylation regulates sarcomeric cytoskeleton since the sarcomere morphometry is modified consecutively to O-GlcNAcylation changes, in correlation with variation of O-GlcNAcylation level of myofibrillar proteins. Desmin, a key protein of striated muscle cells cytoarchitecture and its molecular chaperone alphaB-crystallin seems to be involved in sarcomere morphometry changes through the modification of their interaction. Importantly, their interaction could be modulated through O-GlcNAcylation since the glycosylated sites are localized into the C-term domain of these proteins, known as interaction domain between desmin and its chaperone. Our objective was to determine the impact of O-GlcNAcylation changes on partition and interaction of the desmin and alphaB-crystallin in C2C12 myotubes; we also focused on desmin and alphaB-crystallin expression, so as their O-GlcNAcylation and phosphorylation changes, and analysed the desmin filaments organization by confocal microscopy. We have demonstrated that the global O-GlcNAcylation modulation was associated to desmin O-GlcNAcylation and phosphorylation changes, so as its location toward the cytoskeleton. In parallel, we also showed that alphaB-crystallin partition toward soluble and insoluble fractions was also changed. These data suggest that the interplay between O-GlcNAcylation and phosphorylation could be involved in these partition changes. Furthermore, we also showed that O-GlcNAcylation changes led to the remodelling of desmin filaments. All together, our data confirm the key role of O-GlcNAcylation in organization and reorganization of the sarcomeric cytoskeleton in skeletal muscle cells, and also highlight the influence of O-GlcNAcylation in the modulation of interaction between desmin and its molecular chaperone. At terms, this study will permit to better understand mechanisms involved in the physiological and physiopathological remodelling of sarcomeric cytoskeleton, in particular in some neuromuscular pathologies such as myofibrillar myopathies.

    USP1 deubiquitinates protein kinase akt to inhibit PI3K-AKT-FOXO signaling in muscle atrophy

    D. Goldbraikh, D. Neufeld, Y. Mutlak-Eid, I. Lasry, A. Parnis, and S. Cohen

    Faculty of Biology, Technion Institute of Technology, Haifa, Israel

    PI3K-Akt-FoxO-mTOR signaling is the central pathway controlling growth and metabolism of muscle and all cells. Activation of this pathway requires ubiquitination of Akt prior to its activation by phosphorylation. Here, we found that the deubiquitinating (DUB) enzyme USP1 removes K63-linked polyubiquitin chains on Akt to sustain PI3K-Akt-FoxO signaling low during prolonged starvation. DUB screening platform identified USP1 as a direct DUB for Akt, and USP1 depletion in atrophying muscle increased Akt ubiquitination, PI3K-Akt-FoxO signaling, and glucose uptake in mouse muscle during fasting. Co-immunoprecipitation and mass spectrometry identified Disabled-2 (Dab2) and the tuberous sclerosis complex TSC1/TSC2 as USP1 bound proteins. During starvation, Dab2 was essential for Akt recruitment to USP1/UAF1 complex, and for PI3K-Akt-FoxO inhibition. Additionally, to maintain its own protein levels high, USP1 limits TSC1 levels to sustain mTOR-mediated basal protein synthesis rates. This USP1-mediated suppression of PI3K-Akt-FoxO signaling probably contributes to insulin resistance in catabolic diseases and perhaps to malignancies seen with USP1 mutations.

    Mutation in KBTBD13 causes stiffening of thin filaments in skeletal muscle

    S. Conijn 1, M. van de Locht1, R. van der Pijl2, W. Ma3, T. Irving3, B. Kiss2, H. Granzier2, J. de Winter1 and C. Ottenheijm1

    1Department of Physiology, Amsterdam UMC, Amsterdam, The Netherlands; 2Cellular and Molecular Medicine, University of Arizona, Tucson, USA; 3CSRRI and Dept. BCHS, Illinois Institute of Technology, Chicago, Illinois

    Nemaline myopathy (NEM) is caused by mutations in genes encoding thin filament proteins. NEM6 is caused by mutations in KBTBD13. NEM6 patients display muscle weakness and slow kinetics of muscle relaxation. Whether KBTBD13 plays a role in thin filament structure/function, and whether such role can explain the clinical phenotype of NEM6 patients, is unknown.

    We developed a KBTBD13R408C-knockin (KI) mouse model. In line with the phenotype of NEM6 patients, KI mice exhibit nemaline rods and slow kinetics of muscle relaxation. Here, we used low-angle X-ray diffraction to study whether structural changes in the myofilaments can explain the contractile phenotype of KI mice. Low angle X-ray diffraction experiments were conducted at the BioCAT beamline, Advanced Photon Source, Argonne National Laboratories. Soleus muscles were mounted between a force transducer and a fixed hook, exposed to the X-ray beam during relaxed and maximal tetanus conditions. Images were recorded using a Pilatus 3x M1 detector.

    Results show that the ALL6 [i.e. 59A(ngstrom)] spacing is reduced in KI mice, a finding that is line with previous findings in mucle fibers of NEM6 patients. Furthermore, in relaxed state the actin monomer spacing (27A) and myosin backbone spacing (28A) are reduced in KI compared to wt muscles (2.7329 vs. 2.7343 nm; 2.8764 nm vs. 2.8797 nm). Upon force generation during maximal tetanic activation, which was comparable between KI and wt muscles, the 27A spacing increased less in KI compared to wt muscles (0.00099%/kPa vs. 0.00127%/kPa, respectively). Upon maximal tetanic activation, the 28A spacing increased in KI muscles, but this increase was comparable to that in wt mice (0.00256%/kPa vs. 0.00281%/kPa, respectively).

    These findings indicate that thin filament stiffness, but not thick filament stiffness, is increased in muscles of KI mice. The increased stiffness of thin filaments might contribute to the slow muscle relaxation.

    The role of electrical stimulus in the hiPSC-CMS differentiation and maturation

    T. Crestani 1,2, C. Steichen2, M. Rodrigues2, M.E. Zenteno2, E. Neri2, B. Ormrod1,3, M.R. Holt1,3, J.E. Krieger2 and E. Ehler1,3

    1School of Cardiovascular Medicine and Sciences, BHF Research Excellence Centre, King’s College London; 2Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of São Paulo Medical School, São Paulo, Brazil; 3Randall Centre for Cell and Molecular Biophysics (School of Basic and Medical Biosciences, King’s College London)

    Fibrous tissue replaces cardiomyocyte following an acute myocardial infarction contributing to cardiac remodeling and compromising the organ’s contractile capacity. To counteract these effects several gene/cell therapy approaches have been explored in the last decade with poor results. Recently, the use of human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) appears as a promising strategy to restore cardiac function. Although preliminary evidence indicates that hiPSC-CMs can engraft and improve cardiac function, this response is accompanied by arrhythmia, which can be a life-threatening condition. The arrhythmia can be explained by poor coupling between the cells or excessive specialized conductive cardiomyocytes indicating that we need to better understand the signals driving the maturation of cardiac cells to specialized conductive tissue or working cardiomyocytes. The objective of this work is to investigate the role of electrical stimulus (ES) during the differentiation and maturation process of hiPSC-CMs. All hiPSCs clones presented expression of pluripotent genes and protein markers and normal karyotype. Then iPSCs were differentiated into cardiomyocytes into culture plates (control group) or with ES. hiPSC-CMs with ES displayed higher expression of NKX2-5, IRX3, IRX5, TNNI1, TNNI3, HCN4 and MYL2 compared to the control at day 15. These preliminary results showed that ES is able to increase maturation of iPSC-CMs. We also want to characterize the morphological and functional parameters during the differentiation of hiPSCs-CM according their morphology (shape; myofibrillar maturity; intercalated disc composition) and functional parameters (intracellular Ca2+ transients and action potential). Understanding of the electrical influence may impact the differentiation process to obtain predominantly iPSC-CM of the specialized conductive system, which is essential to increase the efficiency to derive cells for therapeutic purposes or disease modeling.

    Diet induced obesity leads to impaired mitochondrial dynamics in the heart

    H. Daghistani, S. Saxton, S. Prehar, M. Zi, E. Cartwright and A. Kitmitto

    The University of Manchester

    Obesity is a common pre-cursor to type 2 diabetes (T2DM); both are risk factors for developing cardiovascular diseases. Cardiac mitochondrial dysfunction occurs in the early stages of T2DM. However, the mechanisms underpinning changes to mitochondrial function, particularly dynamics, as a result of obesity and/or T2DM remain unclear. Here we have (i) investigated the effects of a high fat diet upon cardiac function and mitochondrial fission/fusion mechanisms and (ii) determined whether introducing an exercise regimen can improve cardiac and mitochondrial function.

    Eight week old C57BL/6J male mice were fed with either a 60% high fat (HF) diet or chow (10% fat) (n = 6–8) for 12 weeks. A second HFD group incorporated exercise training (a daily swimming regimen) at week 13 for 5 weeks to determine the impact upon cardiac and mitochondrial function.

    HF feeding leads to weight gain, hyperglycaemia and insulin resistance with echocardiography revealing increased right wall thickness (p ≤ 0.0001) and decreased stroke volume (p ≤ 0.05). Western blotting and RT-qPCR showed increased expression of the inner mitochondrial fusion protein Opa1, fission proteins Drp1 and Fis1 (p ≤ 0.05) and changes to proteins associated with mitophagy and mitochondrial biogenesis. Exercise training led to weight loss (8.3% ± 1.8) but the mice remained insulin resistant with alterations to the fission–fusion protein axis.

    Our model of obesity exhibits early LV dysfunction in line with symptoms identified in obese patients. Upregulation of Opa1 may be an adaptive response to stabilise cristae structure (as outer mitochondrial membrane fusion proteins are unchanged) with elevation of Drp1 and Fis1 levels indicative of mitochondrial fragmentation; 3D electron microscopy studies are underway to correlate molecular changes to morphology. The exercise animals continue to exhibit insulin resistant and impaired mitochondrial function. Studies are currently on-going to delineate the perturbed mitochondrial pathways and links to loss of cardiac function.

    Neural control and biomechanics of octopus arm muscular hydrostat

    A. Di Clemente 1,3, F. Maiole1,3 and L. Zullo1,2

    1Center for Synaptic Neuroscience and Technology, Istituto Italiano di Tecnologia, Largo Rosanna Benzi, 10, Torre D1, 16132, Genoa, Italy; 2IRCCS Ospedale Policlinico San Martino, Genova, Italy; 3University of Genova, Viale Benedetto XV, 3, 16132, Genova, Italy

    Soft-bodied animals manifest, during movements, considerable variations in their body shape alongside with reversible modification of their stiffness/softness ratio. Accordingly, their body-wall musculature is fitted to generate forces over a wide range of lengths. These properties are especially interesting in the emerging field of soft robotics for their translational aspects.

    Octopus arms represent a ‘one of a kind’ organ where softness is coupled with an extraordinary precision of movements. These properties are built upon the antagonistic action of two main muscle groups, namely the Longitudinal (L) and Transverse (T) muscles, occupying respectively inner and outer layers, and oriented perpendicular to each other.

    In this study, we investigated the physical constraints imposed to them inside the arm showing that, in resting conditions, both muscle groups are held under different degree of compression in the three dimensions.

    We next performed an extensive mechanical characterization of muscle strips excised from the arm. Muscle activation properties showed significant differences in the two groups while length-tension relationships were comparable. Interestingly, they manifested differences in their frequency dependent length-tension curve thus suggesting a distinct sensibility to stimulus frequency. Although showing a similar length-tension curve, due to their different degree of compression inside the arm, muscles will work on different regions of their length-tension curve even when simultaneously activated. This imply that, at any given length of the arm, the two muscles will have a very different outcome in term of force developed Given the above, we suggest that arm muscles are adapted to work in relation to their use in motion and that these differences arise more from extrinsic properties, such as their physical constraints and activation patterns, than intrinsic ones. This information may provide a benchwork for designing new soft materials with muscle-like properties and controllable properties employed in soft robotics.

    Septin 7 has no role in ec-coupling but severly modifies skeletal muscle architecture

    N. Dobrosi 1, L. Szabó1, Z. Ráduly1, M. Gönczi1, G. Kis2, K. Cseri1, B. Dienes1, L. Csernoch1

    1University of Debrecen, Faculty of Medicine, Department of Physiology, Debrecen, Hungary; 2University of Debrecen, Faculty of Medicine, Department of Anatomy, Histology and Embryology, Debrecen, Hungary

    Our goal was to identify the role of septin 7 in the ECC, in the muscle regeneration and in the formation of the skeletal muscle. Knocking out septin 7 is lethal in embryonic state, so a tissue specific, tamoxifen induced Cre-Lox system was used to modify the expression of septin 7 in skeletal muscle. The voltage activation of calcium transients was examined on enzymatically isolated single FDB fibres under whole cell voltage clamp. These results indicate that the reduced expression of septin 7 does not alter the release channel activation significantly and the ECC machinery remains unaltered and fully operational in the mutant. Tibialis posterior muscles were prepared for electron microscopic analysis and images from horizontally and transversally orientated samples were taken to study the occurring structural malformations. Decreased septin 7 expression had a high impact on the architecture of skeletal muscle. The individual myofibrils became smaller but their number increased compared to control. The number of mitochondria also elevated in septin 7 KD mice and they formed large mitochondrial networks distorting the already deformed structure. We induced skeletal muscle damage in control and septin 7 KO mice with BaCl2 injection and septin 7 expression was monitored through the tissue repair. In control mice septin 7 expression significantly increased after the muscle injury and by the end of the regeneration its expression returned to normal. However, in the case of septin 7 KO mice the protein expression was not altered and this modified tissue repair resulted in inflammation lasting longer. These novel insights suggest that septin 7 has a crucial role in skeletal muscle formation and regeneration but does not alter EC-coupling. The research was supported by Hungarian Research Founds (NKFIH K-115461 and GINOP-2.3.2-15-2016-00044).

    Pharmacological modulators of inosine monophosphate metabolism promote activation of AMPK in skeletal muscle cells

    K. Dolinar 1, K. Miš1, T. Hropot1, M. Kolar1, K. Šopar1, A. V. Chibalin2,3 and S. Pirkmajer1

    1University of Ljubljana, Faculty of Medicine, Institute of Pathophysiology, Ljubljana, Slovenia; 2Department of Molecular Medicine and Surgery, Integrative Physiology, Karolinska Institutet, Stockholm, Sweden; 3National Research Tomsk State University, Tomsk, Russia

    Skeletal muscles are one of the main sites for insulin-mediated glucose uptake and therefore important for maintaining whole-body glucose homeostasis. Insulin resistance in skeletal muscle can lead to hyperglycemia and type 2 diabetes (T2D). Activation of AMP-activated protein kinase (AMPK), cellular energy sensor and regulator of cellular metabolism, is a very promising strategy for alleviating insulin resistance in T2D. However, existing experimental AMPK activators often display low bioavailability, serious adverse effects, or preferential activation of AMPK in non-muscle tissues, highlighting the need for new approaches to activate skeletal muscle AMPK. We previously showed that inhibition of de novo purine synthesis pathway with methotrexate promotes AMPK activation in skeletal muscle. Methotrexate inhibits metabolism of ZMP to inosine monophosphate (IMP) and therefore increases level of ZMP, which is a direct activator of AMPK. ZMP is also the active form of the most widely used experimental AMPK activator AICAR. In current study, we investigated whether inhibition of metabolism of IMP to AMP and GMP can activate AMPK and/or enhance AICAR-induced AMPK activation in skeletal muscle.

    We examined the effect of alanosine (inhibitor of adenylosuccinate synthetase), mycophenolate mofetil (MMF, inhibitor of IMP dehydrogenase) and mercaptopurine (inhibitor of IMP dehydrogenase, adenylosuccinate synthetase and adenylosuccinate lyase) on AMPK in primary human and rat L6 skeletal muscle cells. AMPK activation was analysed by measuring phosphorylation of AMPK (Thr172) and phosphorylation of a direct AMPK substrate acetyl-CoA carboxylase (Ser79) with western blotting. Alanosine did not activate AMPK or enhance AICAR-induced AMPK activation, MMF enhanced AICAR-induced AMPK activation, while mercaptopurine stimulated both basal and AICAR-induced AMPK activation. Collectively, our results indicate that MMF and mercaptopurine promote AMPK activation in skeletal muscle cells, suggesting inhibition of enzymes in the final steps of synthesis of AMP and GMP may represent a new strategy to activate skeletal muscle AMPK.

    Role of unconventional myosin VI in the heart development

    AI. Grabowska 1, V. Chumak1,2 and M. J. Redowicz1

    1Nencki Institute of Experimental Biology Polish Academy of Sciences 3 Pasteur St., Warsaw, Poland; 2Center for Translational Research and Molecular Biology of Cancer - Oncology Center 15 Wybrzeze Armii Krajowej St., Gliwice, Poland

    Myosin VI (MVI) is the motor protein involved in a variety of cellular processes through its interactions with actin and tissue/cell specific partners. Mutations within mammalian MVI gene (Myo6) causing sensorineural deafness, mild brain, intestine and kidney abnormalities, as well as dysfunction of the heart were recently reported. We used Snell’s waltzer (SV) mice as the MVI knockout model (MVI-KO) with heterozygotes mice with unaffected phenotype as the controls. Our macroscopic and microscopic analysis performed on P0 nurslings and 3- and 12-moth mice revealed that hearts were hypertrophic in MVI-KO animals. They exhibited abnormal morphology, characteristic for cardiomyopathy such as the ventricular hypertrophy and fibrosis. We observed significant differences in heart/body mass index in all the examined animal groups. Interestingly, in normally developing mice the level of MVI was changing during the lifetime. We detected highest levels of MVI in embryos and P0 mice, a drop in 3-month animals, and a raise in 12-month old individuals. MVI-associated cardiomyopathy seems to affect expression of genes and/or proteins involved in cardiovascular disease and cardiac function similar to MVI expression pattern. Hearts of the SV mice with respect to hearts of control animals also display differences in the cardiac genes/proteins profile. The data gathered so far can indicate involvement of MVI in the cardiac gene expression and thus its importance for the cardiac development and function.

    This work was supported by National Science Centre, Poland grant no 2015/17/D/NZ4/02308.

    Expression of truncated obscurins leads to maladaptive responses in the heart

    A. Grogan 1, L-Y.R. Hu1, H. Joca1, A. Coleman1, C. Ward1, and A. Kontrogianni-Konstantopoulos1

    1University of Maryland Baltimore, Baltimore, USA

    Giant obscurin (720–870 kDa) is a modular protein that surrounds sarcomeres at the level of M-bands and Z-disks where it plays key structural and regulatory roles in striated muscles. Immunoglobulin domains 58/59 (Ig58/59) of obscurin mediate binding to several important regulators of muscle structure and function, including canonical titin, a smaller splice variant of titin, termed novex-3, and phospholamban. Importantly, missense mutations present within obscurin Ig58/59 that are known to alter binding to titins and/or phospholamban are linked to the development of cardiac and skeletal myopathies in humans. Elucidating the impact of the obscurin Ig58/59 module therefore has important implications for cardiac health and disease.

    To this end, we generated a constitutive deletion mouse model, Obscn-ΔIg58/59, that expresses truncated obscurin lacking Ig58/59. Morphometric analysis and transthoracic echocardiography revealed that homozygous males develop compensatory left ventricular (LV) hypertrophy by 6-months that progresses to LV dilation, contractile impairment, and atrial enlargement by 12-months under sedentary conditions. In contrast, females do not exhibit significant structural or functional alterations compared to controls. Moreover, electrocardiography analysis indicated that Obscn-ΔIg58/59 mice of both genders develop spontaneous arrhythmia at 6- and 12-months of age characterized by sinus rhythm variation, junctional escape and/or atrial fibrillation with males exhibiting more severe manifestations. Furthermore, analysis of calcium transients and contractility kinetics in isolated ventricular and atrial cardiomyocytes revealed tissue-specific alterations in calcium release and decay in male Obscn-ΔIg58/59 animals compared to wild-type as a function of aging. Taken together, my studies demonstrate that deletion of obscurin Ig58/59 leads to the development of ventricular and atrial pathologies that manifest to different extents between genders. Given these important findings, and that mutations within Ig58/59 are linked to myopathy in humans, it is apparent that obscurin Ig58/59 is essential for normal muscle function, and its deletion is associated with pathogenicity and disease development.

    Long-term spontaneously contracting 3D cardiac model for in vitro drug testing

    B. Grunow

    Leibniz Institute for Farm Animal Biology, Dummerstorf, Germany

    Cardiovascular diseases are the leading cause of death in humans. This is mirrored by the prevalence of in vitro models for studying heart physiology. During the last 10 years, researchers did prove the high potential of model systems derived from fish for pharmacological testing as their pharmacological properties are very similar to humans. Our here presented model can be gained from fish larvae (like e.g. zebrafish and trout) and shows properties, which are unique in their sum. The model system called spontaneously contracting cell aggregate (SCC) is based on a 3D in vitro model which contracts spontaneously up to 6 month with a human analogue beating frequency of 60 to 80 beats per min. Cell characterization via PCR, immunochemistry, electron microscopy and mass spectrometry exhibited the existence of fully developed cardiomyocytes that were mechanically and electrically coupled. Additionally, immunochemical analysis revealed the existence of the proteins HCN4 and connexin 45 which are specific proteins of the pacemaker center. For pharmacological studies intra- and extracellular electrophysiological recordings were performed. The existence and functionality of L-type calcium channels was proven by isoproterenol application, the occurrence of ATP-sensitive potassium channels via rilmakalim. Furthermore, it could be shown that the SCCs exhibit the ERG-channel, tested with Terfenadine, which is a drug withdrawn from the market and listed on the QT-drug list. After the addition of the ion channel blocker, a significant change in contraction frequency, field respectively action potential duration could be detected. These results show the high potential of the SCCs as an efficient in vitro model for the early preclinical phase of drug development. Moreover, this in vitro model could reduce the amount of mouse models due to its lack of the ERG channel supporting the 3R aim (reduce, refine, replace of animal testing) in research.

    MPH-220 is a pharmacologically safe skeletal muscle myosin inhibitor with superior effect-profile over current muscle relaxants

    M. Gyimesi 1, Á.I. Horváth2, K. Oravecz3, G. Hegyi4, S. Kumar Suthar5, M. Kovács6, A. Málnási-Csizmadia7

    1,2,3,4,6,7Eötvös Loránd University, Dept. of Biochemistry, Hungary; 5Printnet Ltd., Hungary

    MPH-220 is a highly specific fast skeletal muscle myosin-2 inhibitor, which decreases muscle force in rat hindleg by 50% after oral administration in an FDA-approved pharmacological formulation. The residual uninhibited myosin-2 fraction is of high importance when considering that one major drawback of current muscle relaxants is the total loss of muscle tone if the extremely narrow effective range is overdosed. Furthermore, current muscle relaxants act on different target points of the nervous system leading to severe side-effect profles, instead of directly inhibiting the effector molecule of muscle contraction. MPH-220 is a first-in-class inhibitor that directly relaxes skeletal muscles while avoiding neurological, cardiac and respiratory symptoms. We directly compared MPH-220 and the most commonly used muscle relaxant baclofen, and found that even when baclofen was used in a lethal dose the effect size of MPH-220 on muscle force was two-fold higher than that of baclofen. Moreover, a robust accumulation of MPH-220 could be observed in skeletal muscle samples including hindleg, foreleg and trunk muscles, but it was negligible in heart, esophageal and diaphragm samples. The accumulation was in line with slow degradation of MPH-220 by hepatocytes resulting in high bioavailability that is suitable for a daily oral medication. These effects together with the lack of mutagenicity in Ames tests make our molecule an optimal lead compound for further drug development as a highly specific muscle relaxant.

    High-intensity eccentric training improves skeletal muscle dysfunction in a mouse model of polymyositis

    K. Himori 1, Y. Ashida1, D. Tatebayashi1, Y. Saito2, T. Chikenji3 and T. Yamada1

    1Graduate School of Health Sciences, Sapporo Medical University, Japan; 2Department of Anatomy, Sapporo Medical University, Japan; 3Faculty of Health Sciences, Hokkaido University, Japan

    It is common to exclude resistance exercise from rehabilitation program of patients with polymyositis (PM), for fear of increased muscle inflammation. Here, we investigated whether high-intensity eccentric contraction (ECC) training can be a safe and effective intervention counteracting the muscle weakness in experimental autoimmune myositis (EAM) mice, a widely used animal model for PM. Control (CNT) and EAM mice were exposed to an acute bout of damaging ECC exercise (100 contractions at 150 deg/s) or 4 weeks of ECC training (20 contractions at 20 deg/s every other day). EAM was induced in Balb/c mice by immunization with three injections of myosin emulsified in complete Freund’s adjuvant. To induce ECCs, planter flexors were electrically stimulated via surface electrodes (45 V, 100 Hz) while the ankle was forcibly dorsiflexed by servomotor. There was a significant reduction in in situ plantar flexor torque in EAM mice, which was accompanied by reduction in in vitro maximum Ca2+-activated force and decreased myosin heavy chain expression in the gastrocnemius muscles from EAM mice. Moreover, EAM muscles showed an increase in the expression levels of inflammation-redox stress-related proteins including high mobility group box 1, NADPH oxidase-2, and inducible nitric oxide synthase. Evans blue dye uptake into muscle fibers was not higher in EAM muscles compared to CNT muscles after an acute bout of damaging ECC exercise. Importantly, EAM-induced muscle dysfunction was improved by ECC training. Although ECC training did not normalize the expression levels of contractile protein and inflammation-redox stress-related proteins, it increased the expression of catalase, αB-crystallin, and HSP25 in EAM muscles. In conclusion, the susceptibility to ECC-induced damage is not increased in EAM muscles. High-intensity ECC training improves muscle dysfunction in EAM muscles without any detrimental effects, which may be attributed to the enhanced antioxidative defence system against inflammation-oxidative stress insults.

    Non-invasive measurement of contraction, strain and cell–cell coupling in cardiomyocytes

    M.R. Holt 1, W. de Boer2, B. Ormrod1, A.L. Kho1, E. Ehler1, and M. Gautel1

    1King’s BHF Centre of Research Excellence, King’s College London, London. 2University of Amsterdam, The Netherlands.

    We present a simple assay that uses phase contrast microscopy (PCM) coupled with simple image processing routines, written in Wolfram Mathematica, that allow measurement of contractility in cultured cardiomyocytes. Other methods have been developed that use either calcium imaging, edge detection or motion detection to determine contractility. The former approach uses fluorescent chemicals that could alter the behaviour of cells by their requirement to interact with calcium, the key ion driving contraction. There is also the untested possibility for these chemicals to interact with different drugs. The second approach, whilst similar in its use of PCM/image analysis, is mostly limited to single cell behaviour or uses block-based analyses that have low spatial resolution. Furthermore, these approaches fail to differentiate satisfactorily between contraction and relaxation events. Our approach allows us to detect pertinent contractile parameters of cardiomyocytes at the subcellular level. Cardiomyocytes observed using PCM show small, but detectable and repeatable, changes in the amount of light that is transmitted through to a digital CCD camera imaging at > 10 Hz. By analysing these changes on a pixel-by-pixel basis we can determine specific contraction-based parameters such as beating rate, rate of onset of contraction and relaxation, variability in beating rate, i.e., arrhythmia, at the cellular level and rate of transmission of signal across a whole field of cells as an indicator of electrical coupling. Additional details enable simultaneous tracking of intracellular cell movement, which enable estimates of magnitude and direction of strains produced during cellular contraction.

    Regulation of muscle growth by antagonistic control of protein quality and quantity by the ubiquitin ligase ubr4

    L.C. Hunt 1, J. Stover1, B. Schadeberg1, B. Haugen1, T. Shaw2, Y. Li2, V. Pagala2, D. Finkelstein3, Y. Fan3, M. Labelle4, J. Peng2 and F. Demontis1

    1Division of Developmental Biology, Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA, 2Department of Structural Biology and Mass-spectrometry Facility, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA, 3Department of Computational Biology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA, 4Solid Tumor Program, Comprehensive Cancer Center, Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA

    The Ubiquitin proteasome system (UPS) is crucial for protein turnover; many efforts to therapeutically target muscle wasting or promote growth result in altered protein metabolism and inhibition of the UPS. RNAi screening tested the function of all conserved UPS components during Drosophila larval muscle growth and identified genes that acted as both positive and negative regulators of muscle mass, indicating that inhibition of the UPS can both promote and retard muscle growth depending on the gene. Subsequent testing in mouse muscle cell models suggested many of the negative regulators of muscle mass have evolutionary conserved functions, and UBR4, an E3 ubiquitin ligase, was identified as a target to promote muscle growth and attenuate wasting. RNAi mediated loss of UBR4 promoted muscle growth not only in flies, but also in cultured mouse muscle cells. Furthermore, UBR4 siRNA knockdown and muscle specific knockout (tamoxifen inducible ACTA-Cre with UBR4 flox) induced muscle hypertrophy in the tibialis anterior muscle. Increased muscle mass was associated with increased size of myofibers but not number. Loss of UBR4 could not prevent atrophy caused by cachexia but still maintained hypertrophic muscle compared to wild-type. In old mice with sarcopenia, loss of UBR4 also induced hypertrophy. However, chronic loss of UBR4 lead to decreased muscle function in mice and flies that was associated with aberrant protein quality control and led to decreased lifespan. This study identifies UBR4 as a novel regulator of muscle mass and highlights that muscle UPS components have antagonistic but also necessary functions in growth and cell homeostasis respectively via protein quality control.

    Investigating cardiomyocyte mechanosensing with nanopillars and nanopattern

    T. Iskratsch 1

    1School of Engineering and Material Science, Queen Mary University of London

    The composition and the stiffness of cardiac microenvironment change during development and/or in heart disease. Cardiomyocytes (CMs) and their progenitors sense these changes, which decides over the cell fate and can trigger CM (progenitor) proliferation, differentiation, de-differentiation or death. The field of mechanobiology has seen a constant increase in output that also includes a wealth of new studies specific to cardiac or cardiomyocyte mechanosensing. As a result, mechanosensing and transduction in the heart is increasingly being recognized as a main driver of regulating the heart formation and function. However, the molecular mechanism of cardiomyocyte rigidity sensing is still elusive. To study the regulation of cardiomyocyte rigidity sensing on a molecular level we combine nanopillar arrays, PDMS gels with defined stiffness and FRET molecular tension sensors (Pandey et, Dev Cell, 2018). Moreover, because not only the stiffness but also the molecular composition of the adhesions change in pathological conditions (Ward et al., BBAMCR, 2019) we further want to study the implication of the changing adhesion structure and mechanics in detail. To this aim, we adapted a surface functionalization approach using DNA origami with conjugated receptor ligands (uni- or multivalent) that are placed onto nanopatterns fabricated with electron beam lithography (Hawkes et al., Faraday Discussions, 2019). Together our approach indicates a specific cardiomyocyte rigidity sensing mechanism and gives new insights into the nanoscale organisation of cardiomyocyte integrins.

    Rapid modulation of titin-based cardiomyocyte stiffness by ischemic preconditioning in pig hearts

    C. Jahns 1, S. Kötter1, S. Bongardt1, A. Skyschally2, G. Heusch2, P. Kleinbongard2 and M. Krüger2

    1Institute for Cardiovascular Physiology, Medical Faculty, University Düsseldorf, Düsseldorf, Germany; 2Institute for Pathophysiology, West German Heart and Vascular Center, University of Essen, Medical School, Essen, Germany

    Titin largely defines cardiomyocyte passive tension and plays an important role for the structural integrity of the sarcomeres, particularly in situations of increased mechanical stress, e.g. after myocardial injury. We previously demonstrated that myocardial ischemia and reperfusion (I/R) in adult wildtype mice caused rapid PKCalpha-dependent phosphorylation of titin, which increased cardiomyocyte stiffness and contributed to early adaptive remodeling of the non-ischemic (remote) myocardium within the first days after the ischemic event. We speculate that the increased myocyte stiffness adds further stability to the remote myocardium and is beneficial to maintain myocardial function until scar formation is completed. Here, we aimed to translate our findings to larger animals and tested how I/R with or without myocardial ischemic preconditioning affects titin modification and cardiomyocyte stiffness in left ventricular biopsies from mini pig hearts. Pigs underwent 60 min myocardial ischemia by ligation of the left anterior descending artery and 10–120 min reperfusion, with or without 15 min of ischemic preconditioning (n = 5 + 5). Biopsies from non-infarcted left ventricular tissue were taken before and after 55 min ischemia, at the end of ischemic preconditioning and after 10–120 min reperfusion. Titin phosphorylation was analyzed using Western blot, and passive tension was determined by stepwise stretching of permeabilized cardiomyocytes. Ischemia significantly increased phosphorylation of PKCalpha to 130% and of titin S11878 to 160%, and increased passive tension at sarcomere lengths of 2–2.4 μm. Ischemic preconditioning caused similar increases of PKCalpha phosphorylation to 170% and of titin S11878 phosphorylation to 150%, which persisted during the following ischemia. In the reperfusion phase only preconditioned hearts showed an additional increase in relative titin S11878 phosphorylation to 200% and cardiomyocyte passive tension remained significantly elevated. Our data indicate rapid signal transduction from ischemic to non-ischemic tissue and suggest that titin stiffening may contribute to the process of cardioprotection after ischemic preconditioning.

    Sulfasalazine and diflunisal activate AMPK, but exert divergent effects on glucose uptake in skeletal muscle cells

    V. Jan 1*, K. Miš1*, K. Dolinar1, A. Vidović1, N. Kikelj1, R. Komel1, S. Ivanova1, M. Podbregar1,2, T. Marš1, A.V. Chibalin3,4 and S. Pirkmajer1

    1University of Ljubljana, Faculty of Medicine, Institute of Pathophysiology, Ljubljana, Slovenia; 2Celje General Hospital, Department of Internal Intensive Medicine, Celje, Slovenia; 3Department of Molecular Medicine and Surgery, Integrative Physiology, Karolinska Institutet, Stockholm, Sweden; 4National Research Tomsk State University, Tomsk, Russia

    *These authors have contributed equally to this work.

    AMP-activated protein kinase (AMPK), a cellular energy sensor and a major regulator of energy homeostasis, is one of the most promising pharmacological targets in skeletal muscle for treatment of type 2 diabetes. AMPK activation in skeletal muscle increases glucose uptake and lipid oxidation, which improves systemic glucose homeostasis and reduces insulin resistance. Few of the available pharmacological agents activate AMPK in skeletal muscle effectively, which underscores the importance of discovering new AMPK-activating compounds. Salicylic acid and some of its derivatives exert their beneficial metabolic effects, at least in part, via activation of AMPK. Sulfasalazine (SSZ), a widely used anti-rheumatic drug, is an aminosalicylate and sulfonamide conjugate. It increases intracellular concentrations of ZMP, an endogenous purine precursor, in murine splenocytes, possibly due to inhibition of AICAR transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC), an enzyme that converts ZMP into IMP. ZMP is a direct AMPK activator and the active form of the most commonly used experimental AMPK activator AICAR. We examined whether SSZ promotes AMPK activation in cultured skeletal muscle cells and compared its effects with diflunisal, a fluorine-containing salicylate. We found that SSZ activates AMPK in rat L6 skeletal muscle cells. However, although previous studies had indicated that SSZ increases ZMP concentration, it did not enhance AICAR-stimulated AMPK activation in our experiments. This suggests that inhibition of ATIC does not underlie SSZ-stimulated AMPK activation in skeletal muscle cells. Despite activation of AMPK, SSZ failed to increase or even decreased basal and insulin-stimulated glucose uptake. Consistent with this effect, phosphorylation of AS160 (TBC1D4), which stimulates translocation of GLUT4 to plasma membrane, was reduced by SSZ. In contrast to SSZ, therapeutic concentrations of diflunisal potently activated AMPK and increased glucose uptake. In conclusion, SSZ and diflunisal both activate AMPK, but exert divergent effects on glucose uptake in skeletal muscle cells.

    Making a human myosin behave like a rat

    C.A. Johnson 1, J.E. McGreig1, C. Vera2, D. Mulvihill1, M. Ridout1, L. Leinwand2, M.N. Wass1, M.A. Geeves1

    1University of Kent, Canterbury, UK; 2University of Colorado Boulder, Boulder, USA

    Muscle contraction velocity is a property of the myosin isoform expressed and the velocity, at least for isofom1 (or β-cardiac myosin, MyHC-7) is controlled by the rate constant for the release of ADP at the end of the working stroke. The velocity of contraction also varies with size of the species; therefore there are expected to be variations in myosin sequence between orthologues of MyHC-7 from different species. Examining the amino acid sequence of the motor domain of rat and human MyHC-7 (residues 1–800) reveals ~ 35 differences spread throughout the motor domain. These are too numerous to attempt to define which residues are responsible for the changes in myosin properties using an experimental approach. Instead we have examined the sequences of 67 mammalian MyHC-7 sequences (from bat to whale) to identify which residues have a high correlation with the size of the mammal and therefore the velocity of contraction. 57 sites in the 800 amino acid sequence of the motor vary in more than 6 species. Of these, 22 have a high probability of a link to the size of the mammal (P < 0.01) and not the clade (P > 0.05). We identified 12 residues of interest that occur in 3 clusters and of these 9 differ between rat and human MyHC-7. We have taken our Human MyHC-7 construct and replaced all nine residues with the equivalent residue found in rat and predict that the ADP release rate constant should be accelerated by 2–3 fold compared to the WT. The active protein is expressed and the data support our hypothesis.

    Effect of uraemic toxicity on viability of muscle cells

    G. I. Mitrou1, A. G. Wynne2, C. Affourtit2 and C. Karatzaferi 1,3

    1Experimental Myology Cluster, School of Sport, Health and Wellbeing, Plymouth Marjon University, UK; 2School of Biomedical Sciences, University of Plymouth, UK; 3Experimental Physiology Lab, School of PE and Sport Science, University of Thessaly, GR

    Background: Renal disease is associated with muscle atrophy and other indicators of muscle dysfunction collectively termed uraemic myopathy. Uraemic toxicity has been implicated, but mechanistic information is lacking. We thus set out to establish an in vitro model to study the effects of uraemic toxicity factors on growth and differentiation of muscle cells. Here we present results on the effects of a selected uraemic toxin (indoxyl sulfate, IS) on the viability of L6 myoblasts. Methods: Myoblasts were obtained from the European Collection of Cell Culture and were maintained at 37 °C under a humidified carbogen atmosphere in Dulbecco’s Modified Eagle Medium–low glucose (DMEM). Cells between passages 35 and 39 were used for experimentation, cultured in 96-well plates and treated with 0.01, 0.1, 0.5, 1 and 2 mM IS for 48 h [and 72 h (one plate)]. Since IS was diluted in Dimethyl sulfoxide (DMSO), control cells were also treated in DMEM containing respective percentages of DMSO (≤ 5% DMSO) to account for possible effects of DMSO. Cell nuclei were determined by staining with the fluorescent stain 4′,6-diamidino-2-phenylindole (DAPI). A microplate reader was used to determine the relative fluorescent units (RFU). Results: The percent cell loss after 48 h incubation with 0.01, 0.1, 0.5, 1 and 2 mM IS was (MEAN ± SD): 12.4 ± 0.0, 8.4 ± 7.8, 14.9 ± 6.4, 35.1 ± 20.6 and 60.4 ± 20.6, respectively. After 72 h incubation at 0.1, 0.5, 1 and 2 mM IS, the percent cell loss was 1.7, 16.7, 48.1 and 103.5, respectively. Conclusions: Indoxyl sulfate had a potent negative effect on cell viability. Further work will examine synergistic effects of IS with other toxicity factors and/or intracellular environmental conditions affected in uraemic disease.

    GM and CK acknowledge support from the European Union’s Horizon 2020 Research and Innovation Programme MSCAS-RISE “Muscle Stress Relief” under grant agreement no. 645648.

    Different skeletal myopathies caused by tropomyosin mutations: distinctive features of the primary disturbances

    O.E. Karpicheva 1, V.V. Sirenko1, A.O. Simonyan1, C.S. Redwood2 and Y.S. Borovikov1

    1Institute of Cytology RAS, Tikhoretsky Av., 4, St. Petersburg, 194064, Russia; 2University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK

    A number of mutations in genes encoding skeletal muscle tropomyosins is associated with several muscle tissue pathologies—nemaline (NM) and cap (Cap) myopathies, congenital fibre-type disproportion (CFTD), distal arthrogryposis (DA) etc. Identifying the functional changes caused by the various tropomyosin mutations is an important step towards the early and accurate classification of skeletal myopathies and the correct choice of pharmacological agents for the muscle function normalization. This work aims to study the impact of several Tpm2.2 and Tpm3.12 substitutions associated with different myopathies on the tropomyosin position in muscle fibre thin filaments and actomyosin conformational changes during the ATPase cycle using polarization fluorimetry. We have identified hallmark features of contractile dysregulation that are typical for the different diseases at the molecular level. One of the criteria allowing an accurate identification of the disease is the nature of the changes in the number of strong-binding myosin cross-bridges in ATPase cycle. For CFTD and DA the relative number of the strong-binding cross-bridges is typically increased both at high and low Ca2+. Whereas for Cap, and especially for NM, at high Ca2+ we observed fixation of tropomyosin near the blocking position and decrease in the ratio of the essential for force generation strong-binding cross-bridges. For effective work of the myosin cross-bridges, not only the strong binding of actomyosin is important, but also relaxation stage. The appearance of the rigor cross-bridges at relaxation can cause disruption of the contractile apparatus, initiate contracture of the muscle tissue, and contribute to the appearance of sarcomere destruction typical for DA, Cap and NM. Irrespective of whether the number of strong-binding myosin cross-bridges increases or decreases, depending on the mutation in tropomyosin, the result of these disorders is the same—disturbance in the muscle tissue contractility and muscle weakness. Supported by the Russian Science Foundation (17-14-01224).

    Effect of myosin regulatory light chain (RLC) E22K mutation on contraction of cardiac muscle fibers of mice

    J. Zhang1, D. Szczesna-Cordary2, L.Wang1, and M. Kawai 3

    1College of Nursing, Soochow University, Suzhou 215006, China; 2Department of Molecular and Cellular Pharmacology, University of Miami, FL 33136, USA; 3Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA 52242, USA

    Myosin RLC Glu-22 (E22) is located at the N-terminal region between phosphorylation site (S15) and Ca2+-binding loop (residues 37–48). It has been known that this mutation results in an alteration in Ca2+ sensitivity and the phosphorylation of RLC in ventricular muscles to cause hypertrophic cardiomyopathy (HCM) in humans and mice. The purpose of our present study is to investigate the effect of RLC E22 K mutation on cardiac muscle contraction in fibers of E22 K transgenic (Tg-E22 K) mice. The B6SJL mouse line (Ntg), and wild-type (Tg-WT) mice expressing human RLC isoform, were used as controls. Papillary muscles from both ventricles were dissected, activated by Ca2+, rigor was induced, and sinusoidal analysis was performed. The effects of the mutation on active isometric tension (Tact), stiffness during activation (Y) and rigor, Ca2+ sensitivity (pCa50), and cross-bridge kinetics were investigated. Our results demonstrate that there are no significant differences between Ntg and Tg-WT in parameters measured. Our results further demonstrate that active tension and stiffness are significantly attenuated (40–55%) by E22K mutation with a small decrease in Ca2+ sensitivity (0.1 pCa unit) compared with Tg-WT, but the cross-bridge kinetics and the rigor stiffness are not much affected. These results imply that the number of actively cycling cross-bridges decreases in mutant fibers without a change in the force/cross-bridge. The rigor result indicates that there is no change in the total number of cross-bridges available for actomyosin interaction. We conclude that RLC E22K mutation imposes a significant alteration in the cross-bridge function, causing reduced ability of myosin to interact with actin and generate force. This reduction of active tension may result in systolic dysfunction and reduced ejection fraction, which eventually leads to HCM. It is conceivable that the N-terminal region of RLC is critical for the interaction of myosin with the thin filament and force generation.

    Contribution of hydrogen-bonding and hydrophobic interactions to the mechanical stability of titin domains

    G.G. Ferenczy and M. Kellermayer

    Department of Biophysics and Radiation Biology, Semmelweis University, Budapest Hungary

    The mechanical stability of titin’s globular (Ig or FN) domains depends on the free energy of the intradomain interactions and the shape of the domains’ conformational space with respect to the physiologically relevant direction of force. In the present work we explored the role of hydrogen bonding and hydrophobic interactions in the mechanical stability of Ig (I91) and FN (A77 and A78) domains of titin by using steered molecular dynamics (SMD) simulations, which provide force-extension and hydrophobic surface-extension curves. SMD force peaks originate from all interactions, while force peaks calculated from hydrophobic surface unravelling upon extension is attributed to hydrophobic interactions. A comparison of the positions of the SMD and hydrophobic force peaks showed that the latter are smaller and are shifted to greater molecular extensions. This finding can be interpreted based on the different free energy dependence on atomic positions. The steep dependence of the free energy of H-bonds on the positions of the interacting partners leads to high forces when the geometry distorts owing to force-induced extension. Free energy of hydrophobic interactions changes less steeply, resulting in smaller force peaks, which are shifted towards greater extensions. The estimated magnitude of hydrophobic forces does not exceed 50 pN, which is only a fraction of the largest force peak observed for I91 at experimental pulling speeds (~ 200 pN). Moreover, since hydrophobic force peaks are shifted to greater extensions, their contributions to SMD force peaks are smaller than their maximal value. Consequently, force peaks observed in constant-velocity pulling experiments and SMD simulations are dominated by H-bond breaking, while hydrophobic interactions contribute to the high-extension tail of the peaks. Our findings suggest that both H-bonds and hydrophobic interactions contribute to the mechanical stability of most protein domains, but increased stability is achieved in folds where simultaneous H-bond breaking is required for unfolding.

    H9C2 cells as an alternative for cardiomyocytes?

    T. Poppenga1*, N. Klein 1*, A. Alhaj1, H.-G. Mannherz2, P. Reusch3, A. Mügge1, D. Cimiotti1 and K. Jaquet1

    1Research Laboratory Molecular Cardiology, Cardiology, Bergmannsheil and St. Josef Hospital, Clinics of the Ruhr-University Bochum, Germany; 2Department of Anatomy and Embryology, Medical Faculty, Ruhr-University Bochum, Germany; 3Department of Clinical Pharmacology, Ruhr-University Bochum, Germany

    *These authors contributed equally to this work

    Primary cardiomyocytes are difficult to isolate and keep in culture for a long period of time. To reduce the dispatch of animals, an alternative had to be found. In recent years, there has been an increasing interest in H9C2 myoblast cell line, initially isolated from embryonic ventricular rat heart tissue, and which is used as an in vitro cardiac-like cell model for skeletal and cardiac muscle. In the presence of retinoic acid (RA) undifferentiated H9C2 cells should differentiate into cardiac-like cells. Our aim is to verify if H9C2 cells could be used for our research on the role of soluble adenylyl cyclase (sAC) in cardiac hypertrophy.

    In our study H9C2 cells were cultured in media with low serum level and with 1 μM RA for a maximum period of 31 days. For comparison, cardiomyocytes from adult Wistar–Kyoto rats were isolated. Data to identify selected muscle and signaling proteins were collected. Muscle proteins, which are relevant for our research for e.g. were cardiac myosin binding protein C (MyBPC) and cardiac troponin T (cTnT). The presence of proteins was investigated by western blot analysis and immunohistochemistry. However, MyBPC could not be observed in H9C2 cells neither with nor without the presence of RA. Similar results are shown for cTnT. Its expression in H9C2 cells was lower compared to the expression in cardiomyocytes and was independent on the presence of RA. In addition, sarcomeric structures were only observed occasionally. Hypertrophy using isoprenaline could not be generated and furthermore sAC was not detected in H9C2 cells.

    Based on our results we have shown that H9C2 cells cannot be used to study stress induced cardiac hypertrophy, and in addition we verified that H9C2 cells are not a suitable replacement for cardiomyocytes.

    The effect of ADP and omecamtiv mecarbil on the actin–myosin interaction in ventricles and atria using the in vitro motility assay

    D.V. Shchepkin1, V.Y. Berg1, A.M. Kochurova1,2 and G.V. Kopylova 1

    1Institute of Immunology and Physiology, Russian Academy of Sciences, Yekaterinburg, Russia; 2Ural Federal University, Yekaterinburg, Russia

    In heart failure (HF), the ATP regeneration from ADP in cardiomyocytes is disrupted. The accumulation of ADP in the cytosol of cardiomyocytes has been shown to stimulate the formation of myosin cross-bridges in the ventricles, which leads to impaired relaxation and diastolic dysfunction [Sequeira et al., 2015]. To compensate for the decrease in the heart contractility in HF, omecamtiv mecarbil (OM) as an myosin activator was developed [Malik et al., 2011]. Here, we studied the effect of ADP alone and in combination with OM on the actin-myosin interaction in the ventricle and atrium using in an in vitro motility assay. Ventricular and atrial myosin were obtained from pig heart. Calcium dependence of the sliding velocity of thin filaments reconstructed from F-actin, troponin and tropomyosin over myosin in the in vitro motility assay was analysed. The force-generating ability of myosin was assessed using NEM-myosin as a load. We found that ADP in concentration from 50 μM to 5 mM reduces the sliding velocity of F-actin and thin filaments in a dose-dependent manner. The inhibition constant (ADP concentration that decreases the velocity two-times), was higher for atrial myosin (0.83 ± 0.29 mM for F-actin and 2.85 ± 0.40 mM for the thin filament), as compared to ventricular myosin (0.22 ± 0.03 mM and 1.18 ± 0.21 mM, correspondingly). A 100 μM ADP increased the calcium sensitivity of the pCa-velocity relationship for ventricular myosin by 0.2 pCa but did not affect it for atrial myosin. Addition of 100 μM ADP and 0.1 μM OM increased the calcium sensitivity of the pCa-velocity relationship of ventricular myosin by ~ 0.2 pCa. The difference in the ADP effects on the actin-myosin interaction in the ventricle and atrium might have meaning for functioning the heart chambers. Supported by the RFBR grant 18-015-00252 and State Program AAAA-A18-118020590135-3.

    Effects of diet and hypothermia on skeletal muscle contractile function in hibernating animals

    J.K.S. Krishnan 1, S. Rice2, M. Mikes2, M. Hunstiger1, K. Drew2 and S.R. Oliver1*

    Department of Chemistry and Biochemistry, University of Alaska Fairbanks, Fairbanks, Alaska, USA; 2 Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, Alaska, USA.

    Arctic ground squirrels during the hibernating season are physically inactive during torpor and undergo extreme temperature fluctuations during interbout arousals (body temperature, Tb, − 3 °C to 37 °C). During interbout arousals, once Tb reach 16 °C, hibernating mammals recruit skeletal muscle (SkM) for shivering thermogenesis to attain Tb of ~ 37 °C. SkM function is dependent on dietary fatty acids like polyunsaturated fatty acids (PUFA) for tissue maintenance. A recent study in hibernators showed that increased ratio of ω-6:ω-3 PUFA in the diet can increase activity of calcium handling proteins (CHPs) like sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2a) in cardiac muscle, lower Tb, and minimize energy expenditure enabling increased torpor length/frequency during hibernation. CHPs like dihydropyridine receptors and ryanodine receptor 1 are directly involved in muscle contraction via Ca2+ release from sarcoplasmic reticulum (SR). The SERCA pump facilitates Ca2+ re-uptake into the SR completing the Ca2+ cycle and eliciting muscle relaxation. We hypothesized that increased dietary PUFA ω-6:ω-3 ratios enable SERCA 1a enhancement during hypothermic conditions in hibernators, which in turn enhance SkM contractility during hibernation. An Ex-vivo functional assay using a tissue organ bath system was used to characterize alterations in SkM (diaphragm) contractility during hypothermic temperature stress (4 °C; 15 °C; 25 °C and 37 °C) and diet change (ω-6:ω-3, 3:1 (control diet) versus ω-6:ω-3, 1:1 (test diet).To our knowledge this is the first study showing the effects of diet and temperature on muscle contractile function in hibernators within a torpor bout. Preliminary data suggest (1) torpor influences contractile function and calcium re-absorption, (2) diet alters force conservation at lower temperatures, and (3) both ω-6:ω-3 diet variation and temperature exposure affects the ability to produce force, especially functional recovery after hypothermic treatment. This evidence supports the interpretation that functional properties of SkM can be altered by membrane lipid composition and ambient temperature.

    Diabetes impairs adaptive titin modification in response to myocardial ischemia/reperfusion

    M. Isić1, D. Monteiro Barbosa1, S. Kötter1, A. Kronenbitter2, D. Semmler2, J.P. Schmitt2 and M. Krüger 1

    1Institute of Cardiovascular Physiology, Medical Faculty, University Düsseldorf, Düsseldorf, Germany; 2Institute of Pharmacology and Clinical Pharmacology, Medical Faculty, University Düsseldorf, Düsseldorf, Germany

    Myocardial infarction is a leading cause of mortality worldwide, especially in patients with diabetes mellitus. Diabetic patients show greater mortality during the acute phase after myocardial infarction and a higher morbidity in the post-infarction period. Myocardial ischemia and reperfusion (I/R) impair contractile function in the infarcted area, but also affect the non-infarcted regions, i.e. the remote myocardium. We previously demonstrated that I/R caused rapid PKCalpha-dependent phosphorylation of titin, which increased cardiomyocyte passive tension and contributed to early adaptive remodeling of the remote myocardium. Here, we hypothesized that type 2 diabetes mellitus (T2DM) impairs sarcomere function and thereby impedes the adaptive remodeling processes after I/R. Obese leptin receptor deficient mice (db/db) were used as T2DM model and non-diabetic heterozygous littermates (db/+) served as controls. I/R was induced by 60 min ligation of the left anterior descending artery and remote cardiac tissue was collected 24 h after onset of reperfusion. Western blot analysis was performed using phospho-specific antibodies, and passive tension measurements were performed by stretching single permeabilized cardiomyocytes. In db/db mice baseline cardiomyocyte passive tension at sarcomere lengths 2.0–2.4 μm was significantly higher than in heterozygous littermates (db/+). The increase in passive tension was largely caused by phosphorylation of Ca2+-dependent PKCalpha (1.5-fold) and subsequently increased titin PEVK phosphorylation at residues S11878 and S12022 1.4-fold and 2-fold, respectively. In response to I/R, PKCalpha-dependent titin PEVK phosphorylation and cardiomyocyte passive tension were further increased only in non-diabetic db/+ mice, but not in diabetic db/db mice. Moreover, in db/db hearts the already increased passive tension levels of cardiomyocytes from remote myocardium were not further elevated by I/R. We speculate that these changes may alter the adaptive response of the sarcomeres to myocardial injury and possibly contribute to the worsened outcome of type 2 diabetes patients after I/R.

    Skeletal muscle roles for obscurin and OBSL1

    J. Blondelle1, V. Marrocco1, M. Clark1, P. Desmond1, S. Myers1, J. Nguyen1, M. Wright1, S. Bremner1, E. Pierantozzi2, E. Esteve1, S. Ward1, V. Sorrentino2, M. Ghassemian1 and S. Lange 1,2

    1University of California, San Diego, La Jolla, CA-92101, USA; 2University of Gothenburg, Gothenburg, Sweden; 2Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy

    Biological functions of the obscurin protein family members obscurin, Obsl1 (obscurin-like 1) and SPEG (striated muscle preferentially expressed) have been enigmatic. While obscurin and SPEG are expressed exclusively in striated muscles, Obsl1 is found ubiquitously. Accordingly, mutations or loss of SPEG and obscurin have been linked to cardiac and skeletal muscle myopathies, while Obsl1 mutations lead to 3 M growth syndrome.

    We set out to comprehensively investigate the biological roles for obscurin and Obsl1 in skeletal muscles, with a specific emphasis on functional redundancies between both proteins.

    Global knockouts for Obsl1 display an early embryonic lethality phenotype. In contrast, skeletal muscle specific knockouts for Obsl1 show a benign phenotype similar to the mild myopathy observed for obscurin knockouts. Only deletion of both proteins in double knockouts (dKO) and removal of their functional redundancy in skeletal muscles revealed their biological roles. Loss of both proteins negatively impacts the dystrophin-sarcoglycan complex, leading to increased sarcolemmal membrane fragility even at baseline. We also found that both proteins serve important functions for sarcolemmal and sarcoplasmic reticulum (SR) based ion channels, pumps and calcium binding proteins.

    Unbiased analysis of the skeletal muscle proteome reflects our phenotypical analyses of alterations to the sarcolemma, SR and its associated proteins, but also hinted at significant differences in the muscle metabolism, ROS scavenging and cellular protein trafficking.

    Taken together, our analyses suggest that Obsl1 and obscurin play important functions for muscle membrane systems, metabolism and calcium storage/cycling.

    Dietary supplemental linoleic acid attenuates skeletal muscle contractile dysfunction in a rodent model of Barth syndrome

    M. Elkes1, S. Silvera1, J. Wilkinson1, B. Shields1 and P.J. LeBlanc 1

    Center for Bone and Muscle Health, Faculty of Applied Health Sciences, Brock University, St. Catharines, Ontario, Canada

    Cardiolipin (CL) is a unique phospholipid critical to maintaining mitochondrial structure and proper function. Tafazzin (Taz) is an acyltransferase responsible for converting immature monolysocardiolipin (MLCL) to mature CL. Mutations to Taz result in impaired CL maturation, increased MLCL:CL ratio, altered mitochondrial form and function, resulting in skeletal muscle contractile dysfunction. Previous in vitro cell culture studies using cardiomyocytes have examined the influence of exogenous linoleic acid (LA) and reported increased CL, decreased MLCL:CL, and improved mitochondrial respiration and in vitro contractile function. To our knowledge, no study has examined the influence of supplemental LA in vivo on skeletal muscle structure and function mediated through Taz. Using a doxycycline (625 mg/kg) inducible Taz knockdown (TazKD) mouse model, we sought to examine the influence of dietary LA supplementation on skeletal muscle contractile function. We hypothesize that LA supplemented at 70% of total lipids and 25% of total calories will increase CL, reduce MLCL:CL, and improve in vitro skeletal muscle contractile function. At the end of the dietary intervention (8 weeks of a normal (Con) or supplemental LA (LA) diet in TazKD or wildtype littermates (WT)), CL content decreased ~ 70% and MLCL:CL increased ~ 2.5 fold in TazKD Con compared to WT Con and this effect was attenuated in TazKD LA for CL content only. When examining in vitro soleus contractile properties, time to peak twitch, rates of force development, and rates of force relaxation were impaired 2–3-fold in TazKD Con compared to WT Con and attenuated in TazKD LA. These results suggest that supplemental LA may have therapeutic properties to reverse Taz deficient skeletal muscle contractile dysfunction. Future research aims to elucidate the association between CL biosynthesis and skeletal muscle contractile function and examine the influence of supplemental LA on mitochondrial form and function.

    Myosin VI AND PKA signaling in muscle and myogenic cells

    L. Lehka, M. Topolewska, D. Wojton and M. J. Redowicz

    Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093, Warsaw, Poland

    Myosin VI (VI) is a unique unconventional myosin ubiquitously expressed in metazoans. Its diverse cellular functions are mediated by interactions with a number of binding partners present in multi-protein complexes. Our recent data revealed that MVI is present in striated muscles and myogenic cells, and is believed to play important roles in muscle contraction and myogenesis.

    It is well established that cAMP-PKA signaling pathway plays important role in cell metabolism and is crucial for muscles that are high-energy consumers. In addition, activation of endogenous and exogenous muscle-specific genes by Myf-5 and MyoD can be specifically regulated by PKA. We demonstrated that in vitro the MVI cargo domain is phosphorylated by PKA. We noted that myoblasts derived from SV mice differentiate faster than WT littermates and a large amount of myotubes has a myosac-like morphology. We also observed an increase in PKA kinase level in hindlimb muscles of mice not synthesizing MVI (Snell’s waltzer, SV), serving as natural knockouts, with respect to heterozygous littermates (WT). However, the level of phosphorylated (active) form of PKA was significantly reduced. This observation was accompanied by a decrease in the levels of cAMP and adenylate cyclase. Moreover, the amount of CREB (and its active phospho form), transcriptional factor, which is activated by PKA phosphorylation on Ser133, was also lower in MVI-KO mice and myogenic cells. All these observations indicate that MVI is involved in processes essential for muscle and myogenic cells functioning.

    This work was supported by the grant no. 2017/27/B/N23/01984 from National Science Centre, Poland.

    Docking troponin-T onto tropomyosin of thin filaments

    E. Pavadai1, M.J. Rynkiewicz1 and W. Lehman 1

    1Boston University School of Medicine, Boston, U.S.A.

    Complete description of thin filament transitions accompanying muscle regulation requires ready access to atomic structures of actin-bound tropomyosin-troponin. Currently, molecular-docking protocols have been employed to identify troponin interactions on actin-tropomyosin since no experimentally determined structures of filament-based troponin are available at high-resolution. All-atom filament models generated by Manning et al. (2011) and Gangadharan et al. (2017) incorporated tropomyosin-TnT crystal structures of Murakami et al. (2008). However, the Murakami solution, relying on weak electron densities and poor B-factors, is unconvincing. Indeed, our own molecular dynamics simulation of this structure resulted in chain separation and stark corruption of TnT and the tropomyosin N-terminus. The Williams et al. (2017) atomistic model of the thin filament, using actin-tropomyosin coordinates (Li et al. 2011; Orzechowski et al. 2014) and full-length troponin, is more plausible. However, the corresponding paucity of salt bridges linking the TnT-tail (TnT1) to tropomyosin is difficult to reconcile with the high, 20 nM Kd affinity for TnT-tropomyosin interactions (Tobacman et al., 2002; Jin and Chong, 2010). In fact, molecular dynamics simulations show Williams-modeled TnT1 dissociating from tropomyosin in under 100 ns, while actin-tropomyosin remains intact.

    Our own unbiased docking methodology scores binding of approximately 70,000 trial configurations of well-characterized helical domains of TnT1 on tropomyosin. Here the programs PIPER and ClusPro (Orzechowski et al. 2015; Kozakov et al., 2017) are used, followed by flexible-fitting optimization and extensive molecular dynamics. TnT docks along both sides of isolated tropomyosin, but one side is preferred on actin-bound tropomyosin (cf. Flicker et al., 1982). Moreover, overlapping TnT1-segments fit with strong anti-parallel relationship onto tropomyosin, yielding abundant salt bridges and intimately integrated hydrophobic patches between TnT1 and the tropomyosin N-/C-terminal overlap consistent with the expected high affinity interaction. Interaction energy measurements strongly favor this TnT1-tropomyosin design over previous models.

    Equal contributions were made by first two authors.

    The microscale thermophoresis method for the analysis of nebulin-tropomyosin interaction

    J. Lehtonen 1,2, A. Khattab 3, J. Laitila1, M. Grönholm4, C. Wallgren-Pettersson1, V-L. Lehtokari1 and K. Pelin1,4

    1The Folkhälsan Research Center and Medicum, University of Helsinki, Helsinki, Finland; 2Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland; 3Haartman Institute, Translational Immunology Research Program, University of Helsinki, Helsinki, Finland; 4Faculty of Biological and Environmental Sciences, Molecular and Integrative Biosciences Research Programme, University of Helsinki, Helsinki, Finland

    The microscale thermophoresis (MST) method is based on the movement of molecules in a microscopic temperature gradient and it can be used to study biomolecular interactions. For a long-time it has been hypothesized that nebulin and tropomyosin bind to each other, but firm experimental evidence is missing. Our aim is to elucidate whether all nebulin super repeats (SR) bind to tropomyosin and whether there are differences in binding affinities between the SRs. We are currently exploring the utility of the MST method for this purpose.

    Nebulin is an enormous (600–900 kDa) actin-binding protein in the thin filament of the skeletal muscle sarcomere. More than 90% of nebulin consists of SRs. Each SR has seven actin-binding sites and one predicted binding site for tropomyosin. Variants in the nebulin gene (NEB) are known to cause five different forms of myopathy. Alterations in interactions of nebulin between its binding partners might play a role in a pathogenetic mechanism leading to muscle disease.

    We have previously constructed a nebulin SR panel covering all 26 SRs. The panel enables us to produce the SR protein fragments separately and test their binding to different proteins individually. Our previous nebulin-actin binding studies using an in vitro co-sedimentation assay revealed an interesting pattern in nebulin-actin binding: the ends of the nebulin SR region bound F-actin stronger whereas the central part showed weaker binding.

    The project is currently in the optimizing phase. In our preliminary studies, we have observed binding between nebulin and tropomyosin. Further experiments are needed in order to confirm this binding, and to explore the utility of MST for assessing nebulin-tropomyosin binding and the effects of NEB variants on the interaction between the two proteins.

    Effects of myopathy-causing mutations M9R, E151A and K169E in TPM3 gene on structural and functional properties of slow skeletal muscle tropomyosin

    A.M. Matyushenko1, D.V. Shchepkin2, G.V. Kopylova2, S.Y. Kleymenov3, S.Y. Bershitsky2 and D.I. Levitsky 1,4

    1Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia; 2Institute of Immunology and Physiology, Ural Branch of the Russian Academy of Sciences, Yekaterinburg, Russia; 3Koltzov Institute of Developmental Biology of the Russian Academy of Sciences, Moscow, Russia; 4Belozersky Institute of Physico-Chemical Biology, Moscow State University, Russia

    The tropomyosin (Tpm) isoforms γ (Tpm 3.12) and β (Tpm 2.2) are expressed in human slow skeletal muscles leading to formation of γγ-homodimers and γβ-heterodimers of dimeric Tpm molecules. We applied various methods to investigate how myopathy-causing mutations M9R, E151A and K169E in the Tpm γ-chain affect the structure and functional properties of γγ-Tpm homodimers and γβ-Tpm heterodimers. The results showed that M9R mutation significantly decreased the affinity of γγ-Tpm and γβ-Tpm dimers for actin; however, the presence of this mutation in only one of two γ-chains partly restored an ability of Tpm γγ-homodimers to bind actin filament. Using differential scanning calorimetry, we showed that mutation K169E significantly decreased the thermal stability of C-terminal part of γγ-Tpm molecule, while mutation E151A had no appreciable effect on the thermal unfolding of γγ-Tpm. Moreover, mutation K169E strongly decreased the stability of Tpm-F-actin complexes measured from temperature-dependent decrease in their light scattering; however, this effect of K169E mutation was only observed for the complexes containing γγ-Tpm homodimers, but not for the complexes with γβ-Tpm heterodimers. The experiments performed in an in vitro motility assay showed that sliding velocity of regulated thin filaments containing γγ-Tpm with K169E and E151A mutations was dramatically reduced in comparison with the filaments containing γγ-Tpm WT, while M9R mutation significantly increased the velocity. We conclude that myopathy-causing mutations in the Tpm γ-chain may have dramatic effects on the properties of γγ-homodimers and γβ-heterodimers. However, the properties of the Tpm γβ-heterodimers with these mutations in the γ-chain can be substantially differ from those of the Tpm γγ-homodimers with the same substitutions in both chains. Supported by Russian Science Foundation (grant 16-14-10199 to D.I.L).

    Preferential oxidation of elastic titin domains occurs in volume-overloaded hearts and stabilizes the unfolded state of the domains as a prerequisite

    C. Loescher 1, M. Beitkreuz2, Y. Li1, K. Toischer3, L. Leichert2, N. Hamdani2 and W. A. Linke1

    1Universitätsklinikum Münster, Münster, Germany; 2Ruhr University Bochum, Bochum, Germany; 3Universitätsmedizin Göttingen, Göttingen, Germany

    Background The mechanical function of titin is modulated by oxidation and phosphorylation. In vitro, titin oxidation is promoted by unfolding of titin immunoglobulin-like (Ig) domains, which in turn cannot properly refold when S-glutathionylated. However, it is unknown whether titin oxidation occurs in vivo and if there is differential oxidation of sarcomeric I-band (extensible) and A-band (inextensible) titin. Therefore, we measured in vivo titin oxidation as a function of stretch and oxidative stress. Further, interactions between titin oxidation and phosphorylation have not previously been considered, and therefore were investigated in vitro.

    Methods and Results Titin oxidation was studied in the aorto-caval shunt mouse heart, which is under chronic volume overload (preload-increase) and develops oxidative stress. Titin oxidation was quantified by isotope-coded affinity tag labelling followed by mass spectrometry. Hundreds of cysteines in titin became more oxidized under preload-increase conditions and increased the proportion of cysteines oxidized in I-band titin compared to A-band titin. Several preferentially oxidized Ig domains from elastic titin were then recombinantly expressed. Thermal unfolding of these domains, followed by S-glutathionylation, resulted in increased aggregation. Mutant constructs of 82Ig83 (formerly I27) showed that the substitution of one of two cysteines with an alanine prevented the enhanced oxidation-induced aggregation. Unfolding of 82Ig83 was also required for CaMKIIδ-mediated phosphorylation to occur. The unfolding of 82Ig83, together with S-glutathionylation, enhanced phosphorylation by CaMKIIδ further. Force measurements on skinned human cardiomyocytes showed that S-glutathionylation during stretch reduced passive tension.

    Conclusion Titin oxidation occurs in vivo and elastic titin becomes more oxidized than A-band titin after stretch. This effect is due to increased Ig-domain unfolding and results in a reduction of passive tension. Increased S-glutathionylation of titin Ig domains stabilizes their unfolded state and promotes their CaMKIIδ-mediated phosphorylation. Understanding the mechanisms of oxidative stress-induced titin modifications may help design strategies for heart failure treatment.

    Smoothelin-like protein 1: a potential regulator of insulin sensitivity in skeletal muscle

    I.Tamas1, E, Major1, D. Tari1, A. Sipos1, J MacDonald2, T. Haystead3 and B. Lontay 1

    1University of Debrecen, Department of Medical Chemistry, Debrecen, Hungary; 2University of Calgary, Department of Biochemistry and Molecular Biology, Calgary, AB, Canada; 3Duke University Medical Center, Department of Pharmacology, Durham, NC, USA

    Insulin resistance occurs as a variety of disorders including type 2 diabetes, hyperinsulinemia and obesity. Pregnancy can also diminished sensitivity to insulin by the action of progesterone. We hypothesized that the smoothelin-like 1 protein (SMTNL1) as a highly selective progesterone receptor-B (PR-B) corepressor can regulate the gene expression of metabolic enzymes and the elements of insulin signaling. The phenotype of the skeletal muscle (SKM) by virtue of its mass determines the metabolic state of the body and its transformation is regulated by progesterone specifically through PR-B. We demonstrated that pregnancy promotes fiber-type changes from an oxidative to glycolytic isoform in SKM. Smtnl1−/− mice were metabolically less efficient and showed impaired glucose tolerance. Pregnancy antagonized these effects by inducing metabolic activity and increasing glucose tolerance. We also studied the role of SMTNL1 in the insulin signaling in differentiated C2C12 cells in an insulin resistance model. The Ser-phosphorylation of insulin receptor substrate 1 (the marker of insulin resistance) was decreased upon SMTNL1-overexpression and the PKC, Akt and mTOR-related signaling pathways were involved in the regulatory mechanism of SMTNL1 by Proteome Profiler analysis. The metabolic proterties of WT and SMTNL1 overexpressed differntiated C2C12 cells were investigated by measuring the bioenergetic parameters of cell metabolism such as oxidative phosphorylation and glycolysis by Seahorse XF Analyser. The overexpression of SMTNL1 in the presence of R5020 progestin seemed to increase the metabolic properties of C2C12 cells. Finally, we developed PR-B selective membrane permeable inhibitory peptides which can mimick the coregulatory action of SMTNL1 resulting in a change of gene expression and promoted tissue-specific insulin sensitivity in the SKM. We suggest the potential therapeutic application of the SMTNL1-mimick peptides that might improve on the metabolic condition of patients with insulin resistance.

    Development, synthesis and sar analysis of more than 150 different myosin-2 inhibitors reveal structural determinants toward isoform specificity and inhibitory efficiency

    M. Gyimesi1, S. Kumar Suthar2, C. Kurdi3, A. Szabó4, M. Kovács5, and A. Málnási-Csizmadia 6

    1,3,5,6Eötvös Loránd University, Dept. of Biochemistry, Hungary; 2Printnet Ltd., Hungary; 4SONEAS Ltd., Hungary

    We have synthesized over 150 blebbistatin derivatives and tested their physico-chemical and inhibitory properties on pharmacologically relevant myosin 2 isoforms (non-muscle myosin-2 (NM2) A, B and C, skeletal, cardiac and smooth muscle myosins-2). We found that the novel tricyclic cores with modified A-ring structures containing S or N heteroatoms increased the solubility of the compounds 20–50-fold. Besides these improved properties we found a series of derivatives that differently fine-tuned the ATPase activity of myosin 2 isoforms by exhibiting various levels of maximal inhibition in a narrow IC50 range. This enables the development of inhibitors with specifically defined extents of inhibition of certain myosin 2 isoforms. We could also define modifications that resulted in 100-fold lower IC50 values for fast skeletal muscle myosin-2 than the original blebbistatin molecule. Moreover, these findings enabled the development of pharmacologically safe, isoform-specific inhibitors to directly target myosin 2 isoforms playing essential roles in muscle contraction, neuronal plasticity, cell division and migration.

    Characterisation of cardiac actin mutants causing hypertrophic (A295S) and dilative cardiomyopathy (R312H AND E361G)

    C. Erdmann1, S. Schmitt2, R. Hassoun3, A. Rinne4, K. Jaquet3, S. Fuijta-Becker5, R.R. Schröder5, M. Geyer2, and H. G. Mannherz 1

    1Cytoskelettal Dynamics Group, Department of Anatomy and Molecular Embryology, Ruhr-University, Bochum, Germany; 2Institute of Structural Biology, University of Bonn, Germany; 3Research Group Molecular Cardiology, University Hospital Bergmannsheil and St. Josef Hospital, c/o Clinical Pharmacology; 4Institut of Cellular Physiology, Ruhr-University, Bochum, Germany; 5Cryo-Electron Microscopy, BioQuant, University Hospital, Heidelberg, Germany

    Inherited cardiomyopathies are diseases of the cardiac sarcomere and caused by point mutations in genes encoding for contractile proteins. The most frequent forms are hypertrophic and dilated cardiomyopathy (HCM and DCM). One of the affected proteins causing these diseases is α-cardiac actin. We investigated the role of the α-cardiac actin mutation A295S, which is correlated to HCM, and the two mutations R312K and E361G, which have been identified in cases of DCM. For this study we isolated cardiac actins (wildtype and mutants) after untagged expression in Sf21 insect cells by the baculovirus system using gelsolin G4–6 as affinity matrix (1). Subsequently we verified their native state by the DNase I inhibition assay and their ability to polymerize to filamentous actin using the pyrene-assay and electron microscopy after negative staining. The data obtained indicated a reduced rate of polymerisation of the A295S mutant. No differences were, however, detected in their ability to stimulate the ATPase activity of cardiac myosin subfragment 1 in the absence of tropomyosin and troponin. The calcium sensitivity of these actins was determined after decoration with cardiac tropomyosin and troponin by measuring the myosin-S1-ATPase or the movement, i.e. increase of fluorescence intensity of pyrene-labelled tropomyosin (2). The data obtained indicated a decreased calcium sensitivity of the R312H and E361G mutants. Furthermore, the incorporation of the cardiac actin variants into microfilaments of MDCK cells or into the sarcomeric structures of neonatal rat cardiomyocytes after generation of adenoviral constructs was analysed by immunostaining using confocal microscopy.

    (1) Müller M. et al. (2012) Cell Mol Life Sci. 69; 3457–3479. (2) Ishii Y., Lehrer S.S. (1993) Arch Biochem Biophys. 15; 305(1):193–196

    New insights into the interpretation of fluorescence based single molecule atpase assays: effects of actin and drugs

    M. Ušaj, L. Moretto, A. Salhotra, V. Vemula and A. Månsson

    Linnaeus University, Kalmar, Sweden.

    Recent single molecule myosin MgATP turnover studies, using fluorescent ATP analogues and surface immobilized motor fragments report two exponential processes with rates > tenfold faster than the basal myosin MgATPase activity in solution (0.05–0.1 s−1 at 20–30 °C). Here, we use TIRF microscopy, Alexa-647-ATP (Alexa-ATP) and myosin motor fragments (heavy meromyosin [HMM] and subfragment 1 [S1] from fast rabbit skeletal muscle) to reinvestigate this issue. Initial experiments in standard in vitro motility assay solutions (60–130 mM ionic strength, with glucose/glucose oxidase/catalase and 10 mM DTT, 23 °C) gave a double exponential cumulative frequency distributions of Alexa-nucleotide dwell-time events (n = 1273) with rate constants of 5.3 ± 0.14 s−1 (mean ± SE; 60%) and 0.55 ± 0.009 s−1 (40%). After optimizing assay conditions to eliminate artifacts due to non-specific surface fluorescence or dye photophysics (e.g. introduction of Cyclooctatetraene, 4-Nitrobenzyl alcohol and trolox/trolox-quinone) the rate constants were reduced to 0.28 ± 0.002 s−1 (45%) and 0.06 ± 0.0001 s−1 (55%) (n = 1964). Here the slow phase is attributed to MgATP turnover on the catalytic site of myosin whereas the fast phase is most likely due to remaining non-specific artifacts. We next investigated the actomyosin MgATPase at 5–10 nM Alexa-ATP and isometric conditions (HMM:actin = 1:456). The double exponential dwell-time distribution (n = 949) exhibited rate constants of 1.42 ± 0.007 s−1 (77%) and 0.09 ± 0.0005 s−1 (23%) where the fast rate constant reflects the isometric MgATP turnover rate by actomyosin and the slow rate constant the MgATP turnover by myosin. Finally, we describe effects of the small molecular compound paraaminoblebbistatin at varied ionic strength (20–130 mM) and compare the effects to those on HMM induced actin filament sliding velocity in the in vitro motility assay.

    Acknowledgements of support: EU Horizon2020 FET programme (#732482; Bio4comp) and Swedish Research Council (#2015-05290).

    Allosteric communication in actomyosin complexes

    P. Reinke, T. Reindl, S. Giese, C. Thiel, MH Taft and DJ Manstein

    Institute for Biophysical Chemistry, Medizinische Hochschule Hannover, 30623 Hannover, Germany

    Metazoan actomyosin complexes support a wide range of contractile and transport processes. Recent studies have shown how the dynamic association with specific myosin isoforms and other actin binding proteins generates actin filament populations with distinct properties. Critical details of the associated molecular interactions remain unclear. We are reconstructing human actin-dependent contractile complexes from the purified proteins to investigate structure–function relationships, regulatory mechanisms and the impact of allosteric perturbations. Our results show the extent to which changes in myosin and tropomyosin isoform, disease-associated mutations, small molecule effectors, and post-translational modifications affect the frequency, duration, and efficiency of actomyosin interactions. Structures and functional data that are representative for different types of allosteric perturbations are presented.

    Modulation of cardiomyocyte mechanosensing through shape, size and contraction rate

    E. Marhuenda 1 and T. Iskratsch1

    1School of Engineering and Materials Science, Queen Mary University of London

    Cardiac diseases are associated with myocardial fibrosis, which also changes the mechanical environment of the cardiomyocytes, including a stiffening of the extracellular matrix. Cardiomyocytes sense the mechanical properties of their environment and respond by changing their phenotype and function. However, the molecular mechanism is still elusive. Previously our lab identified a cardiomyocyte specific rigidity sensing mechanism through talin that can be stretched either cyclically or continuously depending on the stiffness. This leads to various changes in downstream signalling that we are currently investigating in further detail. Additionally, we are investigating the cross-talk with other mechanical signals, including shape, size or contraction rate using micro and nanopatterning strategies in combination with primary and IPSC derived cardiomycoytes.

    Small molecules can restore the lusitropic response in animal models of hcm and dcm that are uncoupled by mutation

    S.Marston, M.Papadaki, S. Yang and A. Sheehan

    Imperial College London, NHLI, ICTEM, Du Cane Road London, W12 0NN United Kingdom

    Mutations in contractile proteins that cause familial hypertrophic cardiomyopathy (HCM) or familial dilated cardiomyopathy (DCM) often abolish the coupled relationship between Ca2+-sensitivity and troponin I (TnI) phosphorylation by PKA (uncoupling). In normal heart, phosphorylation of Ser22 and 23 of TnI by PKA leads to a twofold decrease in Ca2+-sensitivity and a corresponding increase in the rate of Ca2+ release from TnC and is essential for the lusitropic response to adrenergic stimulation. Therefore uncoupling results in a blunted response to β1-adrenergic activation that has been demonstrated in animal models with HCM or DCM mutations at cell, tissue and whole animal levels. Furthermore, it has been demonstrated in a DCM mouse model that this blunting is sufficient to induce symptoms of heart failure under chronic stress.

    We have identified compounds that can specifically reverse these abnormalities in vitro and therefore have potential for treatment. Recoupling was complete and independent of the causative mutation and the nature of the compound. We have established a biological assay platform for screening EGCG and related analogues in intact cardiomyocytes to study their effects on contractile regulation in vivo, using an E99K ACTC heterozygous-mutant HCM mouse and the ACTC E361G DCM transgenic mouse models. In the mutant mouse the lusitropic response to dobutamine (∆t90rel) is blunted whilst the increased shortening (inotropy) is normal. Addition of the recoupling compounds resveratrol and Silybin B restores the dobutamine response to wild-type values (re-couples). The steroisomer, Silybin A is ineffective as a recoupler, which was also found in the in vitro assay. EGCG and quercetin may recouple but have additional off-target effects.

    We recently repeated these studies using the Cytocypher high throughput system. Data collection and analysis was 20x faster and allowed a great reduction of animal usage. Current results on a wide range of compounds will be reported.

    Myosin-10 is a fast processive molecular motor that operates as single molecule

    G.I. Mashanov 1, T.A. Nenasheva2, Th. G. Baboolal3, M. Peckham3, and J.E. Molloy1

    1The Francis Crick Institute, London, UK; 2Koltzov Institute of Developmental Biology, Moscow, Russia; 3University of Leeds, Leeds, UK

    Myosin-10 is an unconventional myosin critically important for the formation and functioning of filopodia in mammalian cells. It is usually found in multi-molecular puncta at the tips of filopodia, for example in the growth cone at the tip of the axon. Whereas myosin-10 puncta move towards the plus end of actin filaments at a velocity of ~ 0.2 µm s−1 @37 °C (Berg & Cheney, 2002), single-molecule imaging reveals that individual myosin-10’s move towards the tip at much higher velocity.

    We used high-speed, single molecule imaging and tracking to study the fast dynamics of GFP-tagged myosin-10 in live mammalian cells. We have found that, single molecules of myosin-10 use a combination of 3-D and 2-D diffusion within the cell body and at the plasma membrane to navigate towards the cell periphery. After arriving at the base of a filopodium, myosin-10 switches to a highly directed and persistent form of motion towards the plus-ends of the fascin-bundled actin filaments comprising the filopodial core. Single molecules moved from the base to the tip of filopodia (a distance of 5–10 µm) with a velocity of ~ 1.4 µm s−1 @37 °C, much faster than that reported previously. Some molecules exhibited brief intervals of random, back-and-forth motion but soon reverted to the smooth forward motion. We were able to track the path of individual molecules with a precision of ~ 20 nm in the x,y-plane and ~ 40 nm along the z-axis. By plotting the path taken by many individual molecules we mapped the, 100 nm diameter, fascin-bunded actin core. High speed imaging (100fps) allowed us to see, for the first time, the individual ATP-driven steps taken by an individual myosin-10 as it stepped along the actin filament lattice. The fast velocity can be explained by the large step size (36 nm) and fast ATP turnover (14 s−1 @25 °C, Ca. 35 s−1 @37 °C).

    Adaptation of mammalian myosin ii sequences to body mass

    J.E. McGreig 1, S.T. Jeanfavre1,3, C. Henson1,4, M.P. Coghlan1,5, J. Walklate1, M. Ridout2, A.J. Baines1, M.A. Geeves1 and M.N. Wass1

    1School of Biosciences and 2School of Mathematics, Statistics and Actuarial Science, University of Kent, Canterbury, UK; 3Current address: Broad Institute, 415 Main Street, 7029-K, Cambridge MA 02142; 4Current address: Division of Infection & Pathway Medicine, University of Edinburgh Medical School, The Chancellor’s Building, 49 Little France Crescent, Edinburgh, EH16 4SB; 5Current address: London School of Hygiene and Tropical Medicine, University of London, Bloomsbury, London, WC1E 7HT

    The speed of muscle contraction is related to body size; muscles in larger species contract at a slower rate. We investigated the evolution of twelve myosin II isoforms to identify any adapted to increasing body mass. We identified a correlation between body mass and sequence divergence for the motor domain of three adult myosin II isoforms (β, 2A, 2B) suggesting that these isoforms have adapted to increasing body mass. In contrast the non-muscle and developmental isoforms show no correlation of sequence divergence with body mass, while the sarcomeric myosin 7b, extraocular and 2X isoforms showed a divergence intermediate between these two groups. The β-myosin motor domain showed the greatest rate of sequence divergence [− 0.69%/log(kg)]. β-myosin is abundant in cardiac ventricle and slow skeletal muscle. We propose that β-myosin has adapted to enable slower heart beating and contraction of slow skeletal muscle as body mass increased.

    Interventricular differences in contractile function in experimental type 1 diabetes

    T. Myachina 1,2, K. Butova1,2, V. Berg1, K. Sokolova1,2, I. Gette1,2, D. Shchepkin1, G. Kopylova1 and A. Khokhlova1,2

    1Institute of Immunology and Physiology, Ural Brunch of Russian Academy of Science, Yekaterinburg, Russia, 2Ural Federal University, Yekaterinburg, Russia

    Type 1 diabetes (T1D) leads to diabetic cardiomyopathy. There is still a lack of data on the cellular and molecular changes of different heart chambers in diabetic cardiomyopathy. The aim of this work was to study the changes of contractile function of single cardiomyocytes and contractile proteins from the left (LV) and right ventricle (RV) induced by T1D.

    The study was conducted in accordance with the Directive 2010/63/EU. Alloxan-induced T1D in Wistar rats was confirmed by a significant increase in the glucose concentration and glycosylated hemoglobin level. Single cardiomyocytes were isolated using the Langendorff heart perfusion technique. Sarcomere shortening and cytosolic calcium were examined using Fluo-8 (AAT Bioquest). The interaction of myosin with native thin filaments (NTF) extracted from the LV and RV of healthy rat was studied with an in vitro motility assay.

    We found that the end-diastolic sarcomere length was smaller in a T1D group compared with the control rats. The amplitudes of sarcomere shortening and calcium transient of single cardiomyocytes were smaller in animals with T1D compared with control rats. T1D significantly prolonged the time to peak of sarcomere shortening and the time of relaxation in both the LV and the RV but this effect was more pronounced in LV cardiomyocytes. The maximal sliding velocity of NTF over myosins of T1D rats decreased by 30% compared with myosin of the control group. Calcium sensitivity of the pCa-velocity relationship of myosin of T1D rats was lower by 0.2 pCa. A shift of expression of the myosin heavy chain isoforms to β-isoform was found in both ventricles. Thus, T1D impaired and delayed single cardiomyocyte contraction manifesting itself mostly in the LV. This cellular dysfunction may be partly explained by a shift of the myosin heavy chain isoforms and posttranslational modifications of sarcomere proteins. Supported by the RSF grant 18-74-10059.

    Functional role of core gaps in tropomyosin structure

    V.V. Nefedova 1,2, M.M. Marchenko1,2, D.V. Shchepkin3, G.V. Kopylova3, S.R. Nabiev3, S.Y. Bershitsky3, A.M. Matyushenko1, D.I. Levitskiy1

    1Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia; 2Department of Biochemistry, School of Biology, Moscow State University, Moscow, Russia; 3Institute of Immunology and Physiology, Ural Branch of the Russian Academy of Sciences, Yekaterinburg, Russia

    Many proteins take a part in the regulation of muscle contraction. One of them is tropomyosin (Tpm). Tpm 1.1 is the main isoform of Tpms predominantly found in the striated and cardiac muscles. In Tpm structure there are two gaps between α-helixes in central and C-terminal part of the molecule. In order to understand role of these structural rearrangements for Tpm functioning, we made two mutants A134L and E218L that close the gaps between the two helixes. Based on the results of limited trypsinolysis, A134L substitution affects the local structure in this Tpm region and prevents proteolysis after R133. In order to understand the effects of A134L and E218L mutations on the structure of the whole molecule of Tpm, we used differential scanning calorimetry (DSC). There were no significant changes in Tpm caused by A134L substitution but E218L mutation stabilizes C-terminal part of tropomyosin. Surprising results were obtained in experiments with F-actin. All of these mutations had no effects on Tpm affinity to actin and bending stiffness of actin filaments decorated by Tpm. The mutations slightly stabilize complexes formed by actin and Tpm according to experiments of thermally induced dissociation measured by light scattering. In vitro motility assay studies showed that actin filaments with A134L Tpm have higher maximum velocity but lower Ca2+-sensitivity than WT Tpm. This mutation also decreased force generating capacity of myosin molecules with regulated thin filaments. For comparison, the E218L mutation caused both increasing of maximum velocity and Ca2+− sensitivity. Our results indicate that gaps in Tpm structure don’t play crucial role in its binding to actin filament but are important for regulation of myosin-actin interaction. As a result, we can conclude that gaps in the tropomyosin molecules need for fine tuning of regulation of muscle contraction. Supported by RSF No 16-14-10199.

    Effect of tropomyosin phosphorylation on the bending stiffness of thin filaments with cardiomyopathy associated tropomyosin mutants

    L.V. Nikitina 1, S.R. Nabiev1, A.M. Matyushenko2 and S.Y. Bershitsky1

    1Institute of Immunology and Physiology, Russian Academy of Sciences, Yekaterinburg, 620049, Russia; 2A.N. Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, 119071, Russia

    Some mutations in the TPM 1.1 gene induce amino acid substitutions in the tropomyosin molecule (Tpm) associated with cardiomyopathies. Recently, Tpm phosphorylation was shown to affect the phenotype manifestation of these mutations (Schulz ea., 2013). With molecular dynamics modelling, it was demonstrated that Tpm phosphorylation might increase the stiffness of the Tpm head-to-tail overlap domain (Lehman ea., 2015).

    We aimed to investigate the effect of phosphorylation (pseudo-phosphorylation by Ser283D residue) of Tpm with cardiomyopathy associated mutations located in the overlap zone of the N- and C-termini of neighbouring Tpm molecules on the bending stiffness of thin filaments using a two-beam optical trap.

    Human recombinant WT α-Tpm used as a control, the Tpm S283D mutant mimicking its phosphorylated form, and all Tpm mutants associated with cardiomyopathies (K15N and M8R in the N-terminal and M281T, I284V, and A277V in the C-terminal) were expressed in E. coli. Troponin from left pig ventricle and rabbit skeletal actin were obtained by standard methods. Measurement and analysis of the bending stiffness of the thin filament were carried out as described (Nabiev et al., 2015).

    We found that phosphorylation of Tpm with associated with cardiomyopathies the mutations K15N and M8R decreased the bending stiffness of the thin filament from 7.7 ± 0.8 to 4.1 ± 0.4 10–26 N m2 for K15N and from 7.7 ± 0.6 to 3.9 ± 0.3 10–26 N m2 for M8R (Mean ± SEM). The stiffness of the thin filament with Tpm mutants M281T and A277 V was 5.1 ± 0.4 and 6.6 ± 0.5, respectively, and was not affected by phosphorylation. Phosphorylation increased the stiffness for the mutation I284 V from 3.5 ± 0.3 to 5.4 ± 0.4 10–26 N m2. The results showed that Tpm phosphorylation makes the bending stiffness of thin filaments with all mutants tested statistically indistinguishable from that of the WT α-Tpm (5.4 ± 1.1 10–26 N m2).

    Supported by RFBR grant 17-00-00070, Program AAAA-A18-118020590135-3.

    Atypical inhibitor of kappa beta is associated with denervation-induced muscle atrophy in mice

    Y. Ogura 1, C. Kakehashi1 and T. Funabashi1

    1St. Marianna University School of Medicine, Kawasaki, Japan

    Skeletal muscle atrophy is a medical problem in modern society, because it is associated with many pathological conditions such as frailty and metabolic disorders. Nuclear factor kappa-beta (NF-kB) signaling is known to be involved in muscle atrophy; however, the regulatory mechanisms of NF-kB signaling in muscle atrophy is not completely elucidated. Recent studies have reported that the NF-kB transcriptional activity is eventually modulated by intranuclear type of inhibitor of kB (IkB) or atypical IkB, suggesting that the atypical IkB is also associated with muscle atrophy. The aim of this study was to examine the change in atypical IkB including IkB-NS, IkB-Z, IkB-eta, and Bcl3 in denervation-induced muscle atrophy using mice. Male C57BL6/J (8 weeks old) were assigned to either denervation (DEN, n = 5) or control (CON, n = 5) groups. In DEN animals, a part of sciatic nerve in both legs was removed under anesthesia to induce muscle atrophy in lower limb. CON animals were received with the same operation without nerve removal. After 7 days of the surgery, gastrocnemius muscle (GA) of both groups was harvested to examine muscle mass and the expression of genes and proteins. As compared to CON, GA muscle mass in DEN was significantly decreased (P < 0.05). Western blotting analysis showed that phosphorylation of IkB-alpha and conversion from p100 to p52 were significantly increased in DEN (P < 0.05). In qRT-PCR analysis, mRNA level of MuRF1, Atrogin1, and Lc3b was significantly increased in DEN (P < 0.05). Finally, mRNA of IkB-NS, IkB-Z, IkB-eta, and Bcl3, was significantly increased in DEN (P < 0.05). Accordingly, the results of this study suggest that atypical IkB is implicated in the muscle atrophy induced by denervation in mice.

    Effects of acute bout of high-intensity interval exercise on sarcoplasmic reticulum Ca2+ regulatory protein

    N. Okada 1, C. Aibara1 and M. Wada1

    1Graduate School of Integrated Arts and Sciences, Hiroshima Univ, Hiroshima, Japan

    [Background] Fatigue resistance of skeletal muscle depends on the content of mitochondria present in the cell. Recently, high-intensity interval exercise (HIIE) has been shown to bring about greater increases in the mitochondrial content, compared to moderate-intensity continuous exercise (MICE). However, it is equivocal what changes specifically occur in muscles undergoing HIIE. [Purpose] The present study aimed to examine the effects of acute bout of HIIE on Ca2+ regulatory proteins of the sarcoplasmic reticulum (SR). [Methods] Male Wistar rats were used in this study. Under anesthesia, gastrocnemius muscles of the left legs were electrically stimulated in vivo via the sciatic nerve. The muscles of contralateral (right) legs were used as controls. HIIE-mimicking electrical stimulation consisted of six 30 s stimulations (100 Hz, 0.25 s train, every 0.5 s), each followed by a 4 min rest period. MICE-mimicking electrical stimulation consisted of 30 min stimulation (100 Hz, 0.25 s, every 5 s). The number of muscle contractions was the same for MICE and HIIE. Three hours after the end of stimulation, the muscles were excised and used for later analyses. [Results] Force output at 100 Hz returned to resting levels in both MICE and HIIE muscles. On the other hand, the force at 20 Hz was fully restored in MICE but not in HIIE muscles. Decreases in the contents of junctophilin 1 and reduced glutathione were observed only in HIIE muscles. Either HIIE nor MICE affected the ryanodine receptor content, SR Ca2+ release and uptake rates and total calpain activity. [Conclusion] The present results indicate that prolonged low-frequency force depression persists for longer periods in HIIE than in MICE muscles and suggest that muscles subjected to HIIE undergo higher oxidative stress.

    Cellular phenotyping of ipsc-derived cardiomyocytes

    B. Ormrod 1,2,3, Y. Hinits1,2, K. Streckfuss-Bömeke3 and E. Ehler1,2

    1Randall Centre for Cell and Molecular Biophysics, School of Basic and Medical Biosciences, King’s College London, UK; 2School of Cardiovascular Medicine & Sciences, BHF Research Excellence Centre, King’s College London, UK; 3University Medical Centre, Georg-August University Göttingen, Germany

    Cardiomyocytes derived from induced pluripotent stem cells (iPSCs) have become a popular model system to investigate human cardiomyocytes in culture. We are investigating the cytoarchitecture and well-established markers for cardiomyopathy in cardiomyocytes that were differentiated from iPSCs (iPSC-CM) edited to carry hypertrophic and dilated cardiomyopathy (HCM, DCM) causing mutations using CRISPR/Cas9 technology. We will determine whether cellular changes such as myofibril disarray for HCM and alterations in intercalated disc composition for DCM can also be visualized in iPSC-derived cardiomyocytes. This work focuses on validating induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) as a model system for alterations at the cellular level that are seen in cardiomyopathy in situ. One potential problem of this approach is the rather immature status that characterises iPSC-derived cardiomyocytes. Therefore, it is unlikely that the well-known switch to expression of fetal isoforms of myosin (HCM) or myomesin (DCM) that occurs in patients’ hearts can be analysed, since the cells are still expressing these isoforms. In 2D, we are able to show that the cultures are 100% positive for expression of the embryonic EH-myomesin isoform and do not express any M-protein, which characterises the M-bands of mature myofibrils. Using 3D cultures that better mimic the environment of mature cardiomyocytes, we detect M-protein in a subset of myofibrils as well as a decrease in smooth muscle actin and EH-myomesin expression. Further studies using 3D cultures such as engineered heart tissues may help to improve the maturation status.

    X-ray diffraction study of the thick filament off/on transition in isolated cardiac trabeculae

    J. G. Ovejero 1, L. Fusi1, S.-J. Park-Holohan1, A. Ghisleni1, T. Narayanan2, M. Irving1 and E. Brunello1

    1Randall Centre for Cell and Molecular Biophysics, King’s College London, UK, 2European Synchrotron Radiation Facility, Grenoble, France

    The emerging concept of a dual-filament mechanism of contractile regulation has focused attention on the factors including mechano-sensing and RLC phosphorylation that can alter the regulatory state of the thick filament. However, the structural basis of thick filament regulation is still poorly understood.

    Here we characterised the structural basis of the regulatory transition in the cardiac thick filament induced by cooling from 37 °C to 9 °C, using X-ray diffraction from intact and demembranated trabeculae isolated from rat hearts. Thick filament structure in skinned trabeculae in relaxing solution in the presence of 3% dextran T500 at 27–37 °C was similar to that in intact quiescent trabeculae. Maximal calcium activation at 27 °C was used as a reference for the ON state of the thick filament.

    Cooling of intact quiescent trabeculae induces a decrease in the intensity of the meridional myosin-based reflections and of the first myosin layer line indicating disruption of the OFF state of the thick filament. This was associated with an increase in the spacing of the M6 reflection (SM6) associated with the axial periodicity of the thick filament backbone, a biphasic change in that of the M3 reflection (SM3) associated with the myosin motors, and an increase in the equatorial intensity ratio (I11/I10) associated with the movement of myosin motors towards the thin filaments. Cooling of demembranated trabeculae in relaxing solution showed larger changes in the intensity and spacing of the myosin-based reflections, and SM3, SM6 and I11/I10 in relaxing solution at 9 °C were close to their respective ON values.

    The results show that cooling intact quiescent or relaxed skinned trabeculae induces a transition in the structure of the thick filament from the OFF towards the ON state in the absence of calcium activation and filament stress. The structural changes are smaller in intact than skinned trabeculae.

    Supported by BHF, UK.

    Engineered heart tissue for cardiac regeneration

    P. Pandey 1, R. Jabbour1, T. Owen1, M. Reinsch2, C. Terracciano1, F. Weinberger2, T. Eschenhagen2 and S. Harding1

    1Imperial College London, London, United Kingdom; 2University of Hamburg, Hamburg, Germany

    Tissue engineering strategies such as human engineered heart tissues (EHT) have been successfully applied in preclinical models of cardiac injury. However, data regarding EHT patches in clinically relevant models is lacking. We have developed a rabbit model of myocardial infarction and an upscaled EHT patch 1.5 × 2.5 cm has been optimised in a 6 well format, which could contain up to 50 million human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM).

    Whilst in culture, EHTs have demonstrated a higher degree of cardiomyocyte alignment compared with early (2 weeks) versus late (4 weeks). Electrophysiological analysis of the EHTs have demonstrated a better contraction kinetics and faster calcium transients.

    When applied in vivo in an immunosuppressed rabbit model, a sustained retention of cardiomyocytes was seen with 23.3% at 25.2 ± 1.7 days relative to day 0. Staining with human specific marker KU80 confirmed that the cells were of human origin. Preliminary staining for macrophages has revealed that sham patches without cells were non-immunogenic. Evidence of neovascularisation was seen as early as 1-week post implantation and were confirmed to have originated from the host heart. When grafted onto infarcted hearts, preliminary experiments have indicated an improvement in the left ventricular function. Ex-vivo optical mapping revealed evidence of electrical coupling between the graft and host. Correspondingly, in vivo telemetry recordings and ex vivo arrhythmia provocation protocols have indicated no association of the patch with arrhythmogenicity.

    Furthermore, an allogenic model of EHTs is currently being developed with differentiation of rabbit iPS into cardiomyocytes, which could potentially eliminate the use of high dose immunosuppression.

    With the results so far, upscaling of hiPSC-CM derived EHT has been attained to a clinically relevant size. A great insight into the efficacy of tissue engineered cardiomyocyte grafts has been demonstrated with feasibility of integration within an intermediate sized animal model.

    Exploring the heart oF MyBP-C

    A. Pearce 1,2, T. Kampourakis1 and E. Ehler1,2

    1Randall Centre for Cell and Molecular Biophysics, School of Basic and Medical Biosciences and 2School of Cardiovascular Medicine and Sciences, King’s College London, BHF Research Excellence Centre, London, UK

    Mutations in MyBP-C are among the most common causes for hereditary HCM. While the majority lead to frame shifts that are expected to result in a truncated protein that is unstable, a considerable number are point mutations. The N-terminus of MyBP-C seems to play a role in the regulation of contraction whilst the C-terminus appears to target MyBP-C to the thick filaments. However, the function of the middle part of the molecule is unknown. The high occurrence of disease causing point mutations in the central domains of the protein in both man and cat suggests that this region might act as more than a spacer. We are investigating the “heart” of MyBP-C by searching for novel interaction partners using techniques such as the yeast two-hybrid screen. In addition, we are expressing wild type (WT) and mutant MyBP-C constructs encompassing domains C3–C6 via transient transfection of GFP-tagged versions in primary cultures of NRC. Both conventional and high-resolution (STED) confocal microscopy has then been utilised to determine localisation of the WT and mutant constructs. The WT C3–C6 construct shows a diffuse localisation in NRC. Mutant C3–C6 constructs were also seen to express in NRC, showing a similar localisation pattern to WT, suggesting that these single point mutations lead to full-length proteins, which are capable of incorporating into the sarcomere. However, some mutant constructs appear to have increased nuclear expression, indicating that these mutations could be causing mislocalisation of the protein.

    A synthetic nanomachine (myomac) used to define the functional diversity of muscle myosin isoforms

    I Pertici, G. Bianchi, L. Bongini, V. Lombardi and P. Bianco

    PhysioLab, University of Florence, Italy

    The emergent properties of the array arrangement of the molecular motor myosin II in the striated muscle are studied with a synthetic nanomachine (Myomac, Pertici et al., Nat Commun 9:3532, 2018), made by an ensemble of HMM fragments, carried on a piezoelectric nanopositioner and brought to interact with an actin filament attached with the correct polarity to a bead (Bead Tailed Actin, BTA) trapped into the focus of a Dual Laser Optical Tweezers (Bianco et al., Biophys J. 101:866–874, 2011).

    In solution with [ATP] 2 mM, following the attainment of isometric plateau force (F0), up to five different isotonic force–velocity points for each interaction are recorded by switching the control from position to force feedback, allowing to completely define the load dependence of the shortening velocity and the power. Here, we implemented the Myomac by assembling the BTA with a Ca2+-insensitive gelsolin fragment, to allow the [Ca2+] to be an independent parameter. In this way we could test the intrinsic sensitivity of the Myomac to Ca2+ in the absence of the regulatory proteins on the actin filament. We found that, while the Myomac based on rabbit psoas HMM is insensitive to [Ca2+], that based on frog leg muscle HMM is modulated by [Ca2+], showing a 50% increase in F0, a 20% increase in maximum power and a 25% reduction in the maximum shortening velocity (V0) with the increase of [Ca2+]. The results are interpreted with a kinetic model based on mechanics and energetics of the fast skeletal muscle of origin, to define the minimal conditions that recapitulate the performance of the muscle at the level of the simplest actomyosin system in vitro, without the confounding contributions of the cytoskeletal and regulatory proteins, which can then be re-added one at a time.

    Supported by IIT-SEED, Genova and Fondazione CR Firenze, Italy.

    AMPK regulates phosphorylation of TYR10 in NA+, K+-Atpase in skeletal muscle

    M. Petrič 1, K. Miš1, V. Jan1, A. Chibalin2,3 and S. Pirkmajer1

    Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; 2 Department of Molecular Medicine and Surgery, Integrative Physiology, Karolinska Institutet, Stockholm, Sweden; 3 National Research Tomsk State University, Tomsk, Russia

    Na+,K+-ATPase (NKA), a heterodimeric (α/β) ion pump, transports Na+ and K+ across plasmalemma, thus maintaining ion homeostasis, excitability and contractility of skeletal muscle. NKA is regulated by various signalling pathways that modulate its activity, subcellular distribution and abundance. Phosphorylation of the α-subunit is a major downstream mechanism by which signalling pathways exert their effects on NKA. Among the NKA α-subunit phosphosites the role and regulation of Try10 phosphorylation in skeletal muscle has not been elucidated. Here, we explored whether AMP-activated protein kinase (AMPK), a cellular energy sensor, modulates phosphorylation of Try10.

    Using cultured human primary skeletal muscle cells we found that AICAR, the most widely used exogenous AMPK activator, induced a time-dependent decrease in Tyr10 phosphorylation. To explore whether suppression of Tyr10 phosphorylation might be an AICAR-specific effect, we tested several pharmacological AMPK activators, such as A769662 (a direct AMPK activator), FCCP (a mitochondrial uncoupler), ionomycin (a Ca2+-ionophore), and 2-deoxyglucose (an inhibitor of glycolysis). Treatment with each of these AMPK activators decreased phosphorylation of Tyr10 in skeletal muscle cells, strongly suggesting dephosphorylation of Tyr10 is triggered by activation of AMPK and not via AMPK-independent effects of AICAR. In line with this notion, AICAR as well as A769662 suppressed phosphorylation of Try10 also in human proximal tubular cells HK2. In contrast to AMPK activators, fetal bovine serum increased Tyr10 phosphorylation in HK2 cells. Finally, we found that Try10 and the surrounding amino acid sequence are well-conserved in jawed vertebrates (Gnathostomata), suggesting functional importance of Tyr10 in the vertebrate lineage.

    We demonstrated that phosphorylation of Tyr10 of the NKA α1-subunit in cultured cells is modulated by AMPK activators and serum. Taken together, our results suggest that Tyr10 may provide a link between regulation of energy metabolism and NKA in skeletal muscle.

    Antirheumatic drugs modulate energy metabolism in skeletal muscle

    K. Dolinar1, V. Jan1, K. Miš1, U. Matkovič1, M. Zupanc1, A. Vidovič1, K. Šopar1, T. Hropot1, M. Kolar1, K. Bezjak1, K. Perdan-Pirkmajer3, M. Tomšič3, M. Podbregar1, 2, T. Marš1, A. V. Chibalin4,5 and S. Pirkmajer 1

    1University of Ljubljana, Faculty of Medicine, Institute of Pathophysiology, Ljubljana, Slovenia; 2Department for Internal Intensive Care, General and Teaching Hospital Celje, Celje, Slovenia; 3Department of Rheumatology, University Medical Centre Ljubljana, Ljubljana, Slovenia; 4Department of Molecular Medicine and Surgery, Integrative Physiology, Karolinska Institutet, Stockholm, Sweden; 5National Research Tomsk State University, Tomsk, Russia

    Skeletal muscle is a major site of insulin resistance in type 2 diabetes. Activation of AMP-activated protein kinase (AMPK) reduces insulin resistance in skeletal muscle and improves systemic glucose homeostasis. Pharmacological AMPK activation is therefore a promising strategy to develop new treatments for type 2 diabetes.

    Inflammatory rheumatic diseases, such as rheumatoid and psoriatic arthritis, lead to insulin resistance and increase the risk of developing type 2 diabetes. Conversely, the risk is reduced by treatment of these diseases with antirheumatic drugs. Interestingly, some antirheumatic drugs prevent metabolic dysregulation more effectively than others, indicating that they may exert direct metabolic effects in addition to suppressing inflammation. Consistent with this notion, we previously showed that methotrexate, a frequently used antirheumatic drug, promotes glucose uptake and lipid oxidation in skeletal muscle via AMPK activation.

    We extended our methotrexate investigations by testing effects of other antirheumatic drugs on AMPK in skeletal muscle cells. We determined that sulfasalazine, an aminosalicylate and sulfapyridine conjugate, diflunisal, a fluorine-containing salicylate, mycophenolate mofetil, an inhibitor of inosine monophosphate dehydrogenase, and mercaptopurine, a purine antimetabolite and active form of antirheumatic drug azathioprine, all promote AMPK activation in skeletal muscle cells. However, while diflunisal and sulfasalazine both activated AMPK, only diflunisal increased glucose uptake, indicating that AMPK activators may exert distinct metabolic effects. Finally, we found that suppression of the IL-6/JAK/STAT pathway by tocilizumab, an antibody against the IL-6 receptor, and inhibitors of JAK kinase, which are used for treatment of rheumatoid arthritis, modulates insulin signalling in skeletal muscle cells, again highlighting the link between antirheumatic drugs and energy metabolism in skeletal muscle.

    Collectively, our results indicate that several antirheumatic drugs promote AMPK activation, modulate insulin action and/or exert direct metabolic effects in skeletal muscle. These effects might be exploited to treat metabolic dysregulation in rheumatic and metabolic diseases.

    Expression and dynamics of cell–cell contact proteins

    M. Pruna, M. R. Holt and E. Ehler

    1Randall Centre for Cell and Molecular Biophysics, King’s College London, London

    The main aim is to characterise the dynamics of cell–cell contact proteins (beta-catenin, plakoglobin, desmoglein2, desmocollin2) and signalling proteins (FHOD1, CARP1) in Madine-Darby Canine Kidney cells (MDCK) and neonataral rat cardiomyocytes (NRCs) by fluorescent recovery after photobleaching (FRAP). Fluorescently tagged constructs were characterised in MDCK cells by immunoblotting and immunofluorescence. Preliminary FRAP experiments indicated different dynamics for distinct proteins in MDCK cells. Experiments will be repeated in NRCs in control and stressed cardiomyocytes. The effect of beta-adrenergic stress on cell–cell contacts proteins was also assessed by immunofluorescence and will be confirmed by immunoblotting.

    Probing the interaction between CARP1 and PLCβ1

    T. Randall 1,2, K. Dorner1, M. Pfuhl1,2 and E. Ehler1,2

    1School of Cardiovascular Medicine and Sciences, BHF Research Excellence Centre, King’s College London, UK 2Randall Centre for Cell and Molecular Biophysics, School of Basic and Medical Biosciences, King’s College London, UK

    Dilated cardiomyopathy (DCM) is a condition which causes the walls of the left ventricle to thin and the heart chamber to enlarge, this in turn leads to a decreased ability of the heart to pump blood. Protein Kinase C α (PKCα) is a serine and threonine specific kinase which is known to be upregulated at the intercalated disk (ID) of cardiomyocytes in end stage DCM. Stabilisation of PKCα at the ID is thought to be controlled by a complex consisting of PKCα, Phospholipase C β 1 (PLCβ1) and CARP1. It has been shown that genetically removing CARP1, in the MLP knockout mouse model, prevents the development of the DCM phenotype. The focus of this study has been on the interaction of PLCβ1 and CARP1 and how this can be manipulated in order to prevent DCM. PLCβ1 and CARP1 are thought to interact via their coiled coil domains. Using a recombination protein expression approach in E.coli we have expressed and purified several variants of PLCβ1 and CARP1 and their interaction has been assessed via biochemical and biophysical methods. In the future we will further explore this interaction using structural techniques to gain a clearer understanding.

    Physiological studies of katp channels in langendorf rat heart. The impact of ischemia reperfusion

    C R Revnic 1, C Ginghina1, F Revnic2 and S Voinea1

    1UMF”CarolDavila”,Bucharest, Romania; 2 NIGG”Ana Aslan”, Bucharest, Romania

    Background K ATP channels are implicated in myocardium recovery following an ischemic aggression. It has been suggested that phosphorylation of K ATP following activation of protein kinase C (PKC) via diacylglycerol (DOG), leads to the closing of KATP channels. The aim of study was concerned with conjugate investigation of these two cellular components in Langendorf retrograde perfused rat heart system,.with a 45 min. ischemia followed by 120 min reperfusion. The following chemicals for treatment were initially disolved in dimethylsulfoxide (DMSO), 0.04%: (a).1,2-dioctanoil-sn-glycerol (DOG)—activator of PKC—[30 μM]; (b). cheliretrin (CHE)—inhibitor of PKC—[30 μM]; (c) Cromakalim (Kr)-opener of KATP [30 μM]; (d).Glibenclamide (GLY)—blocking of KATP—[1 μM]. Animals: 35 adult Wistar rats were divided into 7 groups of 5 rats each which received in perfusion after 20’ stabilization the following substances alone or in combination: (1). Control, 0.9% saline for 15’, (2). Kr 7 uM for 10’, (3). Gly 1 uM for 15’, (4). DOG 30 uM for 10’, (5). CHE 30 uM for 15’, (6). DOG + Gly for 15’, (7) CHE + Kr for 15’. After 15’ reperfusion a significant decrease in LVDP in all studied cases was recorded.The decrease in cardiac contractility by blocking KATP channels with GLY was intensified with (CHE). In GLY + DOG, the reduction of contractile power is less. Blocking KATP channels with GLY constantly decrease cardiac frequency (CF), while activation of PCK with DOG or opening KATP channels with (Kr) after a significant reduction of (CF), mentain the same values along the experiment. PKC activation abolishes blocking KATP channels effect by GLY in such a way that at the beginning of reperfusion (CF) increases above Control values, with a decrease in time.

    Conclusion Protective mechanisms of KATP channels openers seem to be the result of their capacity to activate KATP channels in ischemic myocardium The hypothesis is sustained by observation that pretreatment of dogs with selective antagonist of KATP—channels GLY—reduces the beneficial effect of Kr.

    1H NMR proton transverse relaxation studies of water state during contraction-relaxation cycles in hyperthyroid rat heart

    F. Revnic 1, C. R. Revnic2 and S Voinea2

    NIGG”Ana Aslan”, Bucharest, Romania; 2 UMF”Carol Davila”, Bucharest, Romania

    Aim To investigate in hyperthyroid rat(HT) heart modifications in myocyte water state during cardiac cycles of contraction–relaxation.We used 16 male Wistar rats aged 12 months old: 8 controls (C) and 8 treated with Thyroxin (i.p.) injections 4.5 mg/kgb.w.for 21 days. Contraction (CO)—relaxations (RE) sequences, of fresh left ventricle samples from C and HT, were monitored at 37C and at different [Ca2+] in extra cellular environment between 0.5 and 2 mM. Samples of left ventricle from C and (HT) were kept in (CO) solution for 20–80 s and in (RE) solution for 100 s each time. From recording the relaxation curves of sample magnetization were calculated myocyte water proton transverse relaxing times (T2). T2 measurements during cardiac cycles were performed with 1H NMR Aremi Spectrometer in impulses at a frequency of 25 MHz using the standard sequence Carr–Purcell–Meiboom–Gill with an interval between impulses of 8 ms. The increase in T2 in HT in (RE) is approximately 5 times lower, than in C. At 1 mM [Ca2+] in extracellular environment, in both C and in HT the highest speed of (RE) was recorded. In C and in HT there is an increase in T2 in (CO) associated with ampliflication of [Ca2+] inductor of (CO), in both cases being present an exception: in C the lengthening times of (CO) is at (80 s) and in HT is at (40 s). The dynamics of (CO) state described by T2 parameter is also deteriorated in HT. T2 parameter may be considered as a marker of myocyte physiology state, which brings information about the proton exchange through myocyte membrane, related with permeability for water. High contractility in HT is associated with a decrease in permeability for water in myocytes induced by Ca2+ and this may constitute a risk factor for installing diffuse ischemia.

    Physiological and biochemical studies of rat heart hypertrophy experimentally induced with isoproterenol. Associated with hormone treatment

    F. Revnic 1, C. R. Revnic2 and S. Voinea2

    1NIGG”Ana Aslan” Bucharest, Romania; 2U.M.F.”Carol Davila”, Bucharest, Romania

    Aim To investigate cardiac hypertrophy (CH) induced with Isoproterenol (Is) associated or not with hormone treatment (Ht): Testosteron (T), Hydrocortisone (Hy), and Estradiol (Es) in rats of different gender and redox potential during ischemia and cardiac hypertrophy in (Is) treated rats and the effect of (Ht) upon myocardial weight/b.w. and left ventricle weight/b.w. ratio. Materials and methods 40 adult Wistar rats: 20 male and 20 female were divided into 4 groups of 10 rats each (5male and 5 female). (A) Controls 0.9% saline, (B) treated for 20 days with Is (0.5 mg/kg b.w. (i.m.). (C). treated with (Hy)(1,8 mg/kg.), (D). male rats treated with (T) 6,75 mg/kg, and female rats treated with (Es) 0,45 mg/kg. Since the 8th day of treatment, (C) and (D) received also treatment with (Is) (0.5 mg/kg. After (Ht) the rats were sacrificed with anesthesia, some hearts were excised and left ventricle homogenized for assays of: GGT, GST and Thyols groups (SH). The rest of hearts were placed in Langendorf retrograde perfusion system for measurement of coronary flow (CF), heart rate (HR) and left ventricle systolic pressure (LVSP) from all groups. Results (RP) of myocardium was intensified in female with (Is) alone or in association (Is + Es). Synthesis and utilization of. GSH was limited in male rats (Is) or in association (Is + T). After (Ht) there was a decrease in GGT in male versus Controls. GTS was not modified in male rats treated with (Is) while the (Ht) in association resulted in a decrease in GTS activity. (Hy) has a modulator effect upon Is increasing cardiac hypertrophy. LVSP was kept almost at constant values in Controls while in (Ht) at the beginning was diminished then the values got higher and higher. Excepting (Is + HY) in male rats, (Ht) led to increase in SH groups content. Conclusion Myocardial insufficiency has biochemical particularities related to genderand etiological factors. Our results may have a clinical significance.

    Actin-myosin function and longevity in nano devices

    A. Salhotra 1, F.W. Lindberg2, R. Lyttleton2, J. Zhu2, M. Usaj1, M. Norrby1, H. Linke2 and A. Månsson1

    1Department of Chemistry and Biomedical Sciences, Linnaeus University, Kalmar SE-391 82, Sweden; 2NanoLund and Solid State Physics, Lund University, Box 118, Lund SE-221 00, Sweden

    The in vitro motility assay (IVMA) is an effective method for functional studies of molecular motors but it is also useful as a basis for nanodevices e.g. in biocomputation and biosensing. In this assay, surface-adsorbed heavy meromyosin (HMM) propels fluorescently labelled actin filaments. In a recent study, we compared methods for the removal of non-functional myosin heads to improve the fraction of motile filaments (FMF) in the IVMA [Rahman et al. J Muscle Res Cell Motil. (2018) 39: 175]. Due to the advantageous effect of blocking actin, where non-fluorescent actin filaments are added at high density to block “dead” myosin heads prior to addition of fluorescent actin filaments, we here study blocking actin concentrations below the standard (1000 nM). We found progressive increase in the FMF upon reduced concentration of blocking actin down to 250 nM. Further reduction in concentration tended to give lower FMF. The data suggest similar trends for the sliding velocity as for the FMF. With expectation to produce improvements in function, we also tested a higher concentration (100 mM) of DTT than usually employed (1–10 mM) in the HMM storage and incubation solutions. The presence of 100 mM DTT slightly, but not dramatically, enhanced the actomyosin function with increased sliding velocity early after incubation. In addition to the mentioned approaches for improving the function in the short term, it is important with actomyosin longevity for effective use in nanodevices. In this regard, we came across several factors of importance, e.g. interaction with atmospheric oxygen, size of flow cell, types of flow cell spacers and effects of the nanofabrication procedure. Approaches to circumvent the problems are also described, resulting in conditions with motile function ranging between less than 20 min to more than 4 h.

    Acknowledgements of support: EU Horizon2020 FET programme (#732482; Bio4comp)

    Screening of 100 plant extracts for the development of a herbal product effective against muscle atrophy

    L. Salvadori 1, M. Mandrone2, T. Manenti3, C. Ercolani3, L. Corgnoli3, M. Lianza2, P. Tomasi2, F. Poli2, G. Sorci1 and F. Riuzzi1

    1Dept. Experimental Medicine, University of Perugia, 06132 Perugia, Italy; 2Dept. Pharmacy and Biotechnology, University of Bologna, 40126 Bologna, Italy; 3Biokyma srl Laboratory, 52031 Anghiari, Italy

    Skeletal muscle atrophy is a loss of muscle mass/strength associated with aging (sarcopenia) or several pathologies including inflammatory diseases, nutrient deprivation, and prolonged treatment with glucocorticoids (GCs) [1]. Despite it represents an enormous medical problem that accelerates the disease progression, increases the hospitalization, and worsens quality of patients’ life, an effective therapy is still lacking. Regardless of etiology, the activation of myofibrillary protein breakdown, especially myosin heavy chain (MyHC), and the reduction of protein synthesis are recognized as the main processes promoting muscle atrophy [1]. The potential of plant metabolites as protective agents against muscle atrophy has been reported [2]. Thus, this work was aimed to individuate promising plants to contrast muscular atrophy, and suitable to develop a herbal product to be marketed. One hundred hydroalcoholic extracts from medical plants were tested in well-characterized in vitro experimental models mimicking muscle atrophy, i.e., treatment of myotubes from C2C12 myoblasts with proinflammatory cytokines (TNFα/IFNγ), nutrient deprivation (starvation) or excess GCs (dexamethasone) [1]. Ten extracts showed extremely potent protective effects against MyHC degradation, as indicated by measurements of myotube diameters, even if at various extent in different atrophic conditions. In particular, four out of them were capable to completely counteract the catabolic effect of TNFα/IFNγ and dexamethasone, and five showed a strong activity against nutrient deprivation. Panax ginseng Meyer emerged as the most potent anti-atrophic agent, together with Withania somnifera Dunal and Silybum marianum Gaertner. Twenty combinations of six extracts selected for their NMR-based metabolic profiles are under investigation, with particular regard to the molecular mechanisms used to counteract muscle atrophy. Our results might lead to the formulation of a low-cost, non-toxic phytotherapy product able to prevent/counteract muscular atrophy associated with aging and pathological conditions. 1 Bonaldo and Sandri 2013 Dis Model Mech 2 Jeong et al. 2018 Evid Based Complement Alternat Med

    C-terminal titin fragments in mouse muscles

    J. Sarparanta 1, A. Vihola1, R. Singh2, I. Richard3 and B. Udd1

    1Folkhälsan Research Center and University of Helsinki, Helsinki, Finland; 2Albert Einstein College of Medicine, Bronx, NY, USA; 3Généthon, Evry, France

    We have previously observed that normal human and murine muscle extracts show reproducible patterns of small (10–50 kDa) fragments derived from the C-terminal (M-band) end of titin. Coexpression studies indicated that the fragments can be generated by the muscle-specific protease calpain 3 (CAPN3), which binds the alternatively spliced is7 region near titin C-terminus. Also conventional calpains may cleave titin in vitro at the same locations. Immunofluorescence microscopy demonstrated the presence of cleavage products in the M-band in situ, suggesting that calpain-mediated proteolytic processing of titin takes place in physiological conditions.

    To characterize titin processing in vivo, we now analyzed C-terminal titin fragments in mouse hindlimb muscles (tibialis anterior, gastrocnemius, soleus, and plantaris) by immunoblotting using antibodies against different epitopes in C-terminal titin. Further, to understand the biological context of titin processing, we compared titin fragments in baseline conditions and after eccentric exercise (downhill running)—a condition previously shown to cause a prolonged increase in intracellular calcium concentration and subsequent CAPN3 activation. Different muscles showed distinct fragment pattens, presumably reflecting their fibre type and titin isoform composition. We observed a correlation between titin fragment levels and CAPN3 activation status, and preliminary results suggest increased titin processing in exercised muscle.

    Mutations in extreme C-terminal titin underlie tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy 2J (LGMD2J, aka LGMD R10 titin-related), whereas CAPN3 mutations cause LGMD2A (aka LGMD R1 calpain 3-related). Understanding C-terminal titin processing will be important for elucidating the relationship of titin and CAPN3 in the M-band, and for unraveling the molecular pathomechanisms of these diseases.

    Do hypertrophic cardiomyopathy mutations affect beta-cardiac myosin interacting-heads motif formation and stability?

    C.A. Scarff 1, B. Barua2, D. Winkelmann2 and J. Trinick1

    1Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK; 2Department of Pathology and Laboratory Medicine, Robert Wood Johnson Medical School, Rutgers University, Piscataway, New Jersey 08854, USA

    Hypertrophic cardiomyopathy (HCM) affects more than 1 in 500 people and is the most common cause of heart failure in the young. The majority of mutations resulting in disease are found in beta-cardiac myosin heavy chain and cardiac myosin-binding protein c (c-MyBP-C) yet the link between mutation and disease outcome is poorly characterised. During relaxation, pairs of myosin molecules form a shutdown energy-saving state named the interacting-heads motif (IHM), in which one the actin-binding region of one myosin head (denoted as ‘blocked’) binds to the converter region of the partner ‘free’ head. Recently, it has been proposed that mutations affect the number of heads trapped in this ‘off state’, affecting energy consumption, relaxation and ultimately force production and thus fully explaining HCM pathology. Based on a human beta-cardiac myosin IHM quasi-atomic model, Alamo et al. (eLife 2017) examined HCM mutation locations and predicted that they would impair IHM formation and stability. Here, we test this hypothesis by examining the effect of mutations on IHM formation and stability. We examine the percentage of myosin molecules exhibiting the IHM in the absence and presence of c-MyBP-C by negative-stain electron microscopy. Our results provide experimental evidence for the link between IHM formation and stability and HCM pathogenesis.

    Structural and functional properties of κ-TPM

    A.M. Matyushenko1, V.V. Nefedova1, L.V. Nikitina2, S.R. Nabiev2, V.Y. Berg2, G.V. Kopylova2 and D.V. Shchepkin 2

    1A.N. Bach Institute of Biochemistry, Research Center of Biotechnology, Russian Academy of Sciences, Moscow, Russia; 2Institute of Immunology and Physiology, Russian Academy of Sciences, Yekaterinburg, Russia

    In cardiac muscle of mammalians, tropomyosin (Tpm) isoforms Tpm 1.1 and minor Tpm 1.2, or κ-Tpm (products of TPM1 gene, α-chain), are expressed. In the myocardium there is up to 7% of κ-Tpm and its distribution regionally is not homogeneous [Peng et al., 2013]. An increase in the expression of κ-Tpm in mice myocardium led to dilated cardiomyopathy [Rajan et al., 2010; Karam et al., 2011]. We studied the properties of κ-Tpm with a complex of structural and functional methods. DSC experiments showed two highly-cooperative calorimetric domains with half-transition temperature of ~ 37 °C and ~ 41 °C, and one low-cooperative transition at 51 °C. The structure of κ-Tpm corresponded to the typical structure of the α-helix, as follows from the CD spectra. The affnity of Tpm 1.2 and Tpm 1.1 for F-actin is similar. The thermal stability of κ-Tpm complex with F-actin was lower than that of Tpm 1.1, its melting temperature was 44.5 °C against 47 °C of Tpm 1.1. Characteristics of the calcium regulation of the interaction of cardiac myosin with thin filaments containing Tpm 1.2 and Tpm1.1 did not differ. Therefore, the structural properties of κ-Tpm differ from Tpm 1.1, and κ-Tpm forms less stable complex with F-actin as compared to Tpm 1.1. Supported by the RFBR grant 18-34-20085 and State Program AAAA-A18-118020590135-3.

    Cofilin in mechanosensitive podosomes

    H.M. Sit 1,2,3, C.H. Yu2 and T. Iskratsch1

    1The School of Engineering and Materials Science, Queen Mary University of London, London, United Kingdom; 2School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong, HKSAR, Hong Kong; 3Randall Centre for Cell and Molecular Biophysics, Faculty of Life Sciences & Medicine, King’s College London, London, United Kingdom

    Podosomes are actinbased membrane protrusions that serve as extracellular matrix anchors, mechanosensors, and force transducer. However, it is still unclear whether the behaviour and dynamics of podosomes are influenced by substrate rigidity. The molecular pathway transducing the mechanical signals to podosomal actin regulation also remains elusive. Here we show the podosome lifetimes in PDBuinduced A7r5 vascular smooth muscle cells increase with extracellular rigidity. Besides, the activity of the actin remodelling factor, cofilin, also changes with the stiffness of the culturing substratum. The abundances of cofilin at podosomes measured by fluorescence intensities from immunostaining also follow the same trend. We functionally tested the matrix degradation abilities on stiffnesses with iPSC derived vascular smooth muscle cells and have observed a higher degradation rate on soft stiffnesses. Altogether our results demonstrate the behavioural and functional changes of vascular smooth muscle cells on stiffnesses, the presence of active cofilins at the mechanosensitive podosomes, and the presumptive roles of cofilin in regulating podosomal actin dynamics in response to extracellular stiffness. Future work will focus on mapping out the upstream mechanosensitive molecular pathway. We anticipate our studies to be a starting point to study the possibility to suppress podosome activity on pathological stiffnesses by targeting this cofilin-associated pathway.

    Novel synthesised analogues of epigallocatechin-3-gallate for the treatment of hypertrophic cardiomyopathy

    AJ. Sparrow 1, P. Robinson1, H. Shi2, C. Lindsay1, H. Watkins1, D. Dixon2 and C. S. Redwood1

    1Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, UK; 2Department of Chemistry, University of Oxford, UK

    Hypertrophic cardiomyopathy (HCM) is principally caused by mutations in genes encoding sarcomeric proteins. We modelled HCM in an isogenic isolated guinea pig cardiomyocyte model by adenoviral transduction of human wild type cardiac troponin T and cardiac troponin T R92Q. Thin filament HCM mutations lead to altered contractility and increased myofilament Ca2+ buffering, causing altered Ca2+ handling and activation of Ca2+ dependent signalling pathways that contribute to HCM pathophysiology. A therapeutic strategy for reversing the increase in myofilament calcium sensitivity is direct modulation of troponin Ca2+ affinity. The green tea catechin epigallocatechin-3-gallate (EGCg) has been shown to reduce myofilament Ca2+ binding via interaction with troponin C. We tested EGCg on in vitro actomyosin ATPase regulation and on cardiomyocyte contractility and found that ECGg has low efficacy, requiring 30–100 micromolar to desensitise the myofilament. EGCg also has significant off-target effects at concentrations under 1 micromolar when applied to isolated cardiomyocytes. We have now screened a series of novel analogues of EGCg and identified several with greater efficacy and specificity than the parent compound. These compounds desensitise the myofilament to the same or greater extent than EGCg and at up to 100 fold lower concentrations. Similar concentrations applied to isolated cardiomyocytes expressing the HCM troponin T mutant R92Q were sufficient to restore normal Ca2+ handling and contractility. Novel analogues of EGCg have improved specificity and efficacy in reversing the increase in myofilament Ca2+ buffering compared with EGCg. This could provide a promising avenue for the therapeutic treatment of HCM.

    Skeletal muscle responses to short term disuse: molecular markers and functional characteristics after dry immersion on humans

    L. Stevens, V. Montel, L. Cochon and B. Bastide

    Lab Physical Activity, Muscle and Health, University of Lille, France

    Muscle disuse is a well-described phenomenon accompanied by a loss of strength mainly due to a loss of muscle mass. This study aimed to identify the early functional and molecular markers of muscle disuse after a 5-day dry immersion (DI) period on humans. The DI model accurately induced muscle deconditioning. In addition to its potential uses in the space field, the dry immersion model could have potentially valuable uses in studying the physiological effects of support muscle unloading and hypokinesia after short term duration. Twenty healthy volunteers completed 5 days of DI by remaining strictly immersed in a supine position, in a controlled thermo-neutral water bath. Muscle biopsies were taken off from vastus lateralis before (Pre) and after (Post) deconditioning conditions and treated either for skinned fiber measurements or muscle proteome western blot analyses. Muscle phenotype was identified using myosin heavy chain (MHC) isoform expression. Muscle contraction was registered on more than 400 isolated skinned muscle fibers and calcium-tension relationships were established for all muscles identified by their MHC expressions. In parallel of the muscle fiber atrophy in most of the subjects, a good correlation between the functional data and the muscle contractile proteome changes were seen after DI. We suggested that the short-term DI period was sufficient to activate muscular variations and that this study could contribute to the importance of characterizing early changes and biomarkers of skeletal muscle disuse, mainly to implement strategies or countermeasures to limit muscle alterations. This study was funded by the French spatial agency “Centre National d’Etudes Spatiales” (CNES).

    Establishing new role(s) for the nuclear envelope in the mammalian heart

    N. Arcos1, J. Ross1, L. Gerace2 and M.J. Stroud 1$

    1BHF Centre of Excellence, King’s College London. London. SE5 9NU. UK; 2Scripps Research Institute, La Jolla, CA. 92037. USA. $Corresponding author

    One of the least understood areas of cardiovascular disease is the role of the nuclear envelope (NE), which is due to a lack of relevant animal models that are able to recapitulate the condition. This is remarkable considering that mutations in NE-encoding genes are the second highest cause of familial dilated cardiomyopathy. NE components play structural and mechanotransduction roles, which are crucial for heart development, physiology and pathophysiology. One such NE protein that is an excellent candidate for disease modelling is NET 25. Mutations in NET 25 lead to sudden cardiac death and cardiomyopathy in humans. However, little is known regarding the underlying molecular pathways that drive disease pathogenesis.

    Here, we show for the first time that NET 25 is essential for normal heart development. Specifically, removal of NET 25 in cardiomyocytes led to late embryonic lethality after E16.5. High Resolution Episcopic Microscopy revealed cKO hearts were severely underdeveloped prior to lethality, with abnormally thin walls. Furthermore, high levels of apoptotic cardiomyocytes were observed throughout development, likely accounting for fewer cells and thinner compact myocardium observed. Because the cardiomyocyte NE is physically coupled to the cytoskeleton, it is under constant mechanical stress, and therefore requires continual maintenance. Previous work highlighted NET 25’s function in NE repair in cell lines, we therefore reasoned that NET 25 might play a similar role in cardiomyocytes. Indeed, NET 25-deficient cardiomyocytes displayed increased incidences of nuclear blebbing and elevated DNA damage levels.

    Our data fit the working hypothesis whereby increased hemodynamic load during heart development or stress augments the force exerted on the NE. In the case of NET 25 mutant cardiomyocytes, this reaches a critical point at which the NE is unable to undergo proper repair, leading to rupture, apoptosis, remodeling, and death.

    Spatio-temporal features of elementary calcium release events of striated muscle from insects and vertebrates

    P. Szentesi 1, C. Collet2, M. Takacs3, L. Szabo4 and L. Csernoch1

    1Department of Physiology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary; 2Department Bees and Environment—Environmental toxicology, National Institute for Agricultural Research, Avignon, France; 3Department of Animal Science, Faculty of Agricultural and Food Sciences and Environmental Management, University of Debrecen, Debrecen; 4Department of Electrical Engineering, Sapientia Hungarian University of Transylvania, Tîrgu Mureş, Romania

    Elementary calcium release events (ECREs) underlie global calcium signaling, they are regulated by local, subcellular signaling processes. In vitro, in isolated striated muscle fibers, calcium release from the sarcoplasmic reticulum (SR) can occur spontaneously or after cell stimulation (e.g. depolarization, mechanical stimulation). Species and tissue-specific features in the occurrence of ECREs have been identified in vertebrate cells under physiological conditions. Increased probability in ECREs seems to correlate with some pathological cases. Our recent characterization of ECREs in an insect species may help understanding their origin and regulation.

    We compared spontaneous ECREs recorded from skeletal muscle fibers isolated from the honey bee (Apis mellifera), the frog (Rana pipiens) and the mouse (Mus musculus). Although measurement conditions were slightly different between species the common confocal 2D/line-scan imaging method was successfully applied using calcium sensitive fluorescence dyes of similar affinity and Ca-binding kinetics. Spontaneous ECREs were detected and analyzed with a self-developed program, which automatically extracts their characteristic spatio-temporal parameters.

    In bee leg muscle fibers showing spontaneous activity, ECRE’s frequency was calculated to be 2.20 ± 0.47 kHz/mm2 (n = 15 fibers), while it was 1.47 ± 0.30 kHz/mm2 (n = 106 fibers) in frog skeletal muscle. In contrast, ECREs are 100 fold less frequent in mouse skeletal muscle (0.0119 ± 0.005 kHz/mm2, n = 43 fibers). While the average spatial spread at half maximum is around 2–2.5 μm in both frogs and mice, it is wider (around 3.5 μm) in bees. The mean amplitude of the events seems to be species independent (approx. 0.2 μF/F0). Our results suggest that the importance of ECREs is decreasing evolutionary in skeletal muscle. The fact that they show up more frequently in myopathies highlight their importance in pathological conditions, which has to be studied further. The research was supported by the French National Research Agency (ANR-13-BSV7-0010), Hungarian Research Fund (NKFIH K-115461, EFOP-3.6.2-16-2017-0006) and Balaton grant (2018-2.1.13-TÉT-FR).

    Drosophila model of myosin myopathy rescued by overexpression of a trim-protein family member

    M. Dahl-Halvarsson1,2, M. Olive3,4, M. Pokrzywa1, K. Ejeskär5, R.H. Palmer2, A.E. Uv2, and H. Tajsharghi 5,1

    1Department of Pathology, Institute of Biomedicine, University of Gothenburg, 405 30 Gothenburg, Sweden; 2Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, University of Gothenburg, 405 30 Gothenburg, Sweden; 3Institute of Neuropathology, Department of Pathology, Institut Investigació Biomèdica de Bellvitge–Hospital de Bellvitge, Hospitalet de Llobregat, 08908 Barcelona, Spain; 4Neuromuscular Unit, Department of Neurology, Institut Investigació Biomèdica de Bellvitge–Hospital de Bellvitge, Hospitalet de Llobregat, 08908 Barcelona, Spain; 5Translational Medicine, School of Health and Education, University of Skövde, SE-541 28, Skövde, Sweden

    Myosin is a molecular motor indispensable for body movement and heart contractility. Apart from pure cardiomyopathy, mutations in MYH7 encoding slow/β-cardiac myosin heavy chain also cause skeletal muscle disease with or without cardiac involvement. Mutations within the α-helical rod domain of MYH7 are mainly associated with Laing distal myopathy. To investigate the mechanisms underlying the pathology of the recurrent causative MYH7 mutation (K1729del), we have developed a Drosophila melanogaster model of Laing distal myopathy by genomic engineering of the Drosophila Mhc locus. Homozygous MhcK1728del animals die during larval/pupal stages, and both homozygous and heterozygous larvae display reduced muscle function. Flies expressing only MhcK1728del in indirect flight and jump muscles, and heterozygous MhcK1728del animals, were flightless, with reduced movement and decreased lifespan. Sarcomeres of MhcK1728del mutant indirect flight muscles and larval body wall muscles were disrupted with clearly disorganized muscle filaments. Homozygous MhcK1728del larvae also demonstrated structural and functional impairments in heart muscle, which were not observed in heterozygous animals, indicating a dose-dependent effect of the mutated allele. The impaired jump and flight ability and the myopathy of indirect flight and leg muscles associated with MhcK1728del were fully suppressed by expression of Abba/Thin, an E3-ligase that is essential for maintaining sarcomere integrity. This model of Laing distal myopathy in Drosophila recapitulates certain morphological phenotypic features seen in Laing distal myopathy patients with the recurrent K1729del mutation. Our observations that Abba/Thin modulates these phenotypes suggest that manipulation of Abba/Thin activity levels may be beneficial in Laing distal myopathy.

    Cellular uptake of blood plasma exosomes by endothelial cells and adult rat cardiomyocytes

    M. Tangos 1, K. Jaquet1 and D. Kolijn1

    Ruhr University Bochum, Bochum, Germany

    Introduction/Aim Exosomes are extracellular vesicles in a size range of 40–100 nm and are known for their role in cell-to-cell communication. They play an important role in diseases by affecting cell proliferation, cellular differentiation, inflammation, aging, angiogenesis, stress response, and cardiovascular remodelling. My focus is the examination of exosomes from patients with coronary heart disease (CHD) and atrial fibrillation (AF) as common examples of cardiovascular complications. As a first approach I investigated the cellular uptake of blood plasma exosomes after incubation with endothelial cells (HUVEC) and adult rat cardiomyocytes. Methods 1 ml of blood plasma was used to isolate exosomes via size exclusion chromatography. Fractions containing exosomes were selected for investigation. Commonly used methods for visualization of cellular processes are fluorescence microscopy and live cell imaging. For this purpose, blood plasma exosomes and cell membrane were dyed with PKH, a fluorescent general membrane marker. For exosomal RNA tracking SYTOTM RNASelectTM dye was used. Hoechst staining was chosen to counterstain the cell nucleus. Results The results of HUVEC live cell imaging indicate that blood plasma exosomes are grabbed by filopodias before cellular uptake takes place. Further investigation showed an accumulation of exosomes and exosome-associated RNA in the perinuclear region of endothelial cells, with RNA being partially present inside the nucleus, too. These findings could be observed within a few hours after exosome incubation. First results showed a completely different behaviour concerning cardiomyocytes: exosomes seem not to reach the nucleus. Conclusion In this study I was able to prove that blood plasma exosomes communicate with endothelial recipient cells in vitro via RNA cargo. With the knowledge of altered exosomal cargo during disease state, further investigations with HUVEC and cardiomyocytes will be done to get a better understanding of the cellular effects during CHD and AF.

    Supplementation with bcaas plus glutamine prevent skeletal muscle atrophy induced by cast-immobilization. The cross effect of physical exercise

    P. Tavares 1,2, E. Ribeiro1,2, V. H. Pinheiro3, J. Martins2, S. Simões2, A. F. Ambrósio2 and C.A. Fontes Ribeiro1,2

    1Faculty of Sport Sciences and Physical Education, University of Coimbra; 2IBILI – Faculty of Medicine, University of Coimbra; 3Orthopaedic Department of CHUC, Coimbra

    It’s important to look for a strategy that can prevent and/or treat skeletal muscle atrophy by immobilization. Therefore, we aimed to study the response of muscle and satellite cells (SC) with and without Branched Chain Amino Acids (BCAAs) plus glutamine.

    Adult male Wistar rats has been used. The animals were divided into control group [A]; a group that performed 4 weeks of aerobic exercise [B]; a group in which the right hindlimb was immobilized with a cast for 1 week [C]; a group with exercise, as described, rest for 1 week and then was immobilized [D]; a group that received daily oral BCAAs and was immobilized [E]; a group as [E] but with rest for a week and exercise for 4 weeks (F); a group that was immobilized for 1 week, rest another week and then performed 4 weeks of exercise [G].

    The aerobic exercise was performed in a treadmill starting at low velocity and increased until the end of four weeks. After the above protocols, animals were sacrificed by anaesthetic overdose and the gastrocnemius and soleus muscles, from both hindlimbs, were taken. The samples were heighted and prepared for histological analysis and for immunohistochemistry. To evaluate SC, we used antibodies against Pax-7, Myf-5 and c-met, identification by fluorescence which allows to count immunoreactive cells. Immobilization caused atrophy and muscle damage. SC were activated to regenerate the skeletal muscle, as seen by the increase in Pax-7 and Myf-5 expression with a decrease of c-met, suggesting mobilization of SC. Visible cell niches in the muscle reinforced these data.

    The supplement had a partial protective effect when it was taken during immobilization, but it seems to hamper the muscle regeneration, when exercise is performed after immobilization. Thus, our results suggested that BCAAs prevent skeletal muscle atrophy by acting over SC.

    FCT: UID/NEU/04539/2013.

    Differences between cardiac and skeletal musculature: eccentric force generation and muscle structure

    A. Tomalka 1, O. Roehrle2,3, J.-C. Han4, T. Pham5, A.J. Taberner4,6 and T. Siebert1

    1Department of Motion and Exercise Science, University of Stuttgart, Stuttgart, Germany; 2Institute of Applied Mechanics (Civil Engineering), University of Stuttgart, Stuttgart, Germany; 3Cluster of Excellence for Simulation Technology (SimTech), Stuttgart, Germany; 4Auckland Bioengineering Institute, The University of Auckland, Auckland, New Zealand; 5Department of Physiology, The University of Auckland, Auckland, New Zealand; 6Department of Engineering Science, The University of Auckland, Auckland, New Zealand

    In skeletal muscle, experimentally observed progressive forces during active stretching (force enhancement) and enhanced isometric forces following active stretching (residual force enhancement), can not be explained by classic theories of muscle contraction. Non-cross-bridge structures may account for this striking behaviour in skeletal muscle. So far it is unknown whether differences between non-cross-bridge structures of heart and skeletal muscle yield to deviating contractile behaviour of cardiac muscle during and after long eccentric contractions. Here, for the first time, we investigated the force response of intact cardiac trabeculae during and after isokinetic eccentric muscle contractions with extensive magnitudes of stretch. Separation of cross-bridge and non-cross-bridge structures contributing to total muscle force was achieved by using an actomyosin inhibitor.

    We found that in cardiac trabeculae the force length response during long stretch was similar to the active isometric force–length relation. This implies that no residual force enhancement exists in cardiac muscle under these experimental conditions. This result is in contrast with that obtained in skeletal muscle where residual force enhancement is present following eccentric muscle contractions with extensive magnitudes of stretch. The current findings allow for the hypothesis that titin stiffness does not increase with activation in cardiac muscle. This contributes to an improved understanding of heart functioning.

    Myosin VI in skeletal muscle: a new player in muscle metabolism and myofiber organization

    M. Topolewska 1, A. M. Kaminska2, L. Lehka1 and M. J. Redowicz1

    1Nencki Institute of Experimental Biology Polish Academy of Sciences, Pasteura 3 Warsaw, 02-093 Poland

    Myosin VI (MVI), one of the unconventional myosins, is involved in numerous cellular processes associated with the actin cytoskeleton such as cell migration, adhesion, maintenance of the Golgi apparatus, endocytosis, autophagy, and also in gene transcription. We showed that it is also expressed in skeletal muscle where it localizes to the sarcoplasmic reticulum (SR), postsynaptic region of the neuromuscular junction (NMJ) and muscle nuclei. Since the knowledge of MVI involvement in muscle functions is still very limited, we aimed at examination of the role of MVI in skeletal muscle. In order to achieve this goal, we used Snell’s waltzer mice serving as MVI knockout animals (MVI-KO). We investigated hindlimb muscles of P0, 3-month, and 12-month old mice. We observed symptoms of hypertrophy and fibrosis in gastrocnemius muscle (GM), composed mainly from slow-type myofibers. This result led us to investigate myofiber structure and metabolism in GM of MVI-KO mice with respect to wild type animals (WT). Immunofluorescence staining and electron microscopy revealed aberrations in the neuromuscular junction, nucleus, sarcoplasmic reticulum and mitochondria as well as appearance of tubular aggregates. To examine whether these changes could affect muscle metabolism, we analyzed the levels of ATP and cAMP. We revealed significant changes in the level of these nucleotides, so crucial for muscle metabolism and contraction. Thus our data indicate that MVI could indeed be involved in the maintenance of myofiber compartments and regulation of muscle metabolism.

    This work was supported by the grant no. 2017/27/B/NZ3/01984 from National Science Centre, Poland.

    Effects of dietary restriction on muscle aging in caenorhabditis elegans

    S. Tumbapo 1, A. Strudwick1, J. Stastna1, S. Harvey1 and M. Bloemink1*

    1Biomolecular Research Group, School of Human and Life Sciences, Canterbury Christ Church University, Canterbury CT1 1QU, United Kingdom

    *Corresponding author

    A key feature of the ageing process is a progressive decline in muscle mass and function, known as sarcopenia. Sarcopenia leads to loss of functional mobility and independence in elderly people and is also a major component of frailty. With a growing aging population, it is essential to understand the molecular mechanisms behind sarcopenia in order to develop effective therapies.

    In this study, GFP-labelled unc-54 (e1092) strain of C. elegans were used to test the effect of three different dietary restriction (mild, medium and severe) on its lifespan and sarcopenia. This strain has one of its myosin heavy chain (MHC) isoform labelled with GFP so the fluorescence intensity can be used as an indicator of myosin density. Lifespan under mild and medium dietary restriction (DR) was significantly extended with a mean lifespan of 17 days in comparison to the control group (14 days). In contrast, the mean lifespan of worms under severe DR decreased to 11 days in comparison to the control group. Worms under mild and medium DR also maintained significantly higher motility rates compared to severe DR and control group. Fluorescence measurements of the C.elegans body-wall muscle displayed an age-related decrease in fluorescence in all groups, consistent with reduced unc-54 myosin levels and indicating the development of sarcopenia. The fluorescence intensity in worms under mild and medium DR decreased at a slower rate compared to the severe DR and control group. These findings suggest that moderately restricted diet is most effective in inducing the beneficial effects of DR on C. elegans’ lifespan and sarcopenia while severely restricted diet leads to detrimental effects.

    Titin cleavage in striated muscles from a knock-in Halotag-Tev mouse model visualized by electron microscopy

    A. Unger 1, Y. Li1 and W.A. Linke1

    1Institute of Physiology II, University of Munster, D-48149 Munster, Germany

    The giant protein titin is a fundamental component of sarcomeres, where it is continuously subject to stretching forces. Spanning half the length of a sarcomere, titin determines the passive stiffness of myocytes and enables force transduction essential for the mechanical activity of striated muscle. We used a genetically engineered knock-in mouse model carrying a HaloTag-TEV insertion located at the end of the I-band of titin (between Ig domains I86 and I87). By specifically digesting native HaloTag-TEV titin with TEV protease, we quantified the contribution of titin to the overall stiffness of muscle fibers. Treatment of HaloTag-TEV psoas myofibers with TEV resulted in a time-dependent drop in passive force of more than 50%. Electron microscopical observations of TEV-treated fibers after stretching demonstrated structural alterations and showed the important role of titin for sarcomeric A-band and Z-disc integrity. In addition, isolated cardiac trabecular myofibers were subjected to selective removal of actin filaments by a calcium-independent gelsolin fragment. In these fibers, we could visualize titin by immuno-gold EM before and after TEV protease treatment. Taken together, the HaloTag-TEV model can be used to explore the mechanical function of native titin in situ and demonstrate the precise ultrastructural localization of titin domains within striated muscle cells.

    Mutations in fast skeletal troponin C (TNNC2) cause contractile dysfunction

    M. van de Locht 1, J.M. de Winter1, S.H. Conijn1, W. Ma2, M.H.B. Helmes1,3, T.C. Irving2, S. Donkervoort4, P. Mohassel4, L. Medne5, C. Quinn6, O.L.A. Neto4, S. Moore4, A.R. Foley4, J.R. Pinto7, N.C. Voermans8, C.G. Bönnemann4 and C.A.C. Ottenheijm1

    1Department of Physiology, Amsterdam UMC (location VUMC), Amsterdam, The Netherlands; 2Biophysics Collaborative Access Team, Center for Synchrotron Radiation Research and Instrumentation, and Department of Biological Sciences, Illinois Institute of Technology, Chicago, Illinois, USA; 3IonOptix Llc.,Milton, MA, USA; 4Neuromuscular and Neurogenetics Disorders of Childhood Section, Neurogenetics Branch, National Institutes of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; 5Roberts Individualized Medical Genetics Center, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; 6Department of Neurology, University of Pennsylvania, Philadelphia, PA, USA; 7Department of Biomedical Sciences, The Florida State University College of Medicine, Tallahassee, FL, USA; 8Department of Neurology, Radboud University Medical Centre, Nijmegen, Netherlands.

    Nemaline myopathy (NEM) is a group of rare muscle diseases caused by mutations in genes encoding proteins associated with the thin filament. To date, fast skeletal (fs)TnC has not been implicated in disease. Here, we investigate muscle biopsies of two patients with heterozygous, predicted to be pathogenic mutations (Patient 1 (P1), 27 yrs: p.Asp34Tyr & patient 2 (P2), 19 yrs: p.Met79Ile) in the gene encoding fsTnC (TNNC2). P1 has congenital weakness and vocal cord paralysis requiring tracheostomy, with ptosis, opthalmoplegia, osteopenia and clinical improvement over time. Also, a brother, mother and maternal grandmother, presenting with similar symptoms were found to carry the TNNC2 mutation. P2 has a milder phenotype, with early respiratory weakness, dysphagia and generalized hypotonia, which improved with age, resulting in normal ambulance with mild proximal weakness at age 19. To investigate the mechanism underlying muscle weakness, we performed contractility measurements in single muscle fibers isolated from patient (N = 2) and control (N = 5) biopsies. Permeabilized fibers were activated by exogenous calcium. P1 showed atrophied type II fibers and decreased absolute and specific force. A decreased calcium-sensitivity of force was observed in type II myofibers in both patients. The more severe clinical phenotype of P1 compared to P2 is reflected in these experimental results. To determine whether the decrease in calcium-sensitivity of force is a direct result of the mutations in TNNC2, we performed experiments in which endogenous (i.e. mutated) fsTnC was removed from type II fibers of P1 and replaced by WT-fsTnC. Introducing WT-fsTnC in myofibers of P1 restored the calcium-sensitivity of force, while introducing WT-fsTnC in control myofibers did not affect the calcium-sensitivity of force. These results show that the muscle weakness in these patients is a direct consequence of the mutated protein, and that restoration of troponin function is a therapeutic target.

    Excision of titin’s C-terminal PEVK exons alters skeletal muscle contractility and induces hypertrophy in slow twitch skeletal muscles

    R. van der Pijl 1,2, B. Hudson1, T. Granzier-Nakajima1, F. Li1, J. Smith 3rd1, C. Chung1,3, M. Gotthardt4, H. Granzier1 and C. Ottenheijm1,2

    1University of Arizona, Tucson, United States; 2Amsterdam University Medical Center, Amsterdam, the Netherlands; 3Wayne State University, Detroit, United States; 4Max-Delbruck-Center for Molecular Medicine, Berlin, Germany.

    We previously published a titin PEVK deletion mouse in which all exons that constitute the PEVK region of the cardiac N2B isoform (exons 219–225) had been excised, and reported cardiac hypertrophy and increased passive stiffness. Here we investigated the phenotype in skeletal muscles, focusing on EDL (fast twitch) and soleus (slow twitch) muscles.

    Gel electrophoresis revealed, in both EDL and soleus, co-expression of two titin isoforms in TtnΔ219–225, versus a single isoform in wildtype mice. The larger isoform represents the TtnΔ219–225 N2A titin. The smaller isoform, referred to as N2A2, represents a splicing adaption, as indicated by titin exon array. Passive stiffness was significantly increased in soleus and EDL muscles. This increase was observed at the whole muscle level (SL 3.0 μm: 23 ± 6% and 60 ± 15%, respectively) and at the skinned fiber level (SL 3.0 μm: 48 ± 5% and 41 ± 5%, respectively), in the absence of changes of extracellular matrix. Active force was increased in TtnΔ219–225 soleus (max. specific force: WT 240 ± 9; TtnΔ219–225 276 ± 17 mN/mm2), while EDL showed reduced force (max. specific force: WT 315 ± 9; TtnΔ219–225 280 ± 14 mN/mm2). The change in force coincided with a slow fiber type switch and, curiously, faster kinetics of muscle activation and relaxation in both muscles. TtnΔ219–225 mice display a sensitivity to hypertrophic remodeling as they developed more hypertrophy in response to muscle overload (ablation), and increased muscle sparing in response to unloading (hindlimb suspension).

    Thus, excision of titin’s c-terminal PEVK exons prompts aberrant titin splicing, resulting in increased titin-based passive stiffness that alter the mechanics of the myofilament activation and relaxation and sensitize slow twitch skeletal muscle to hypertrophic stimuli.

    Early-onset and adult-onset hcm mutations in the myosin motor domain have similar properties

    C.D. Vera1, C.A. Johnson2, J. Walklate 2, A. Adhikari3, Z. Ulfalusi4, M. Svicevic5, S.M. Mijailovich6, A. Combs1, S.J. Langer1, K.M. Ruppel7, J.A. Spudich7, L.A. Leinwand1 and M.A. Geeves2

    1BioFrontiers Institute and Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder CO 80309, USA; 2School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK; 3Rumi Scientific, San Francisco CA 94107, USA; 4Department of Biophysics, Univeristy of Pécs, Medical School, Szigeti Street 12, H-7624 Pécs, Hungary; 5Faculty of Science, University of Kagujevac, Serbia; 6Department of Biology, Illinois Institute of Technology, Chicago IL 60616, USA; 7Stanford University School of Medicine, Department of Biochemistry, Stanford CA 94305, USA

    Hypertrophic Cardiomyopathy (HCM) is a common genetic disorder affecting 1 in 500 people that leads to left ventricular hypertrophy and abnormal contractions of the cardic muscle. One of the major causes of HCM are mutations in the beta cardiac myosin heavy chain (beta-MyHC) isoform. These mutations are typically characterised by cardiac hypercontractility with the mechanism of how the change in myosin function leads to disease not fully understood. Due to the nature of the samples required a detailed molecular characterisation is difficult to achieve. In addition to this, the time course over which the disorder manifests (20–40 years), the changes must have subtle affects. With the identification of cardiomyopathy causing mutations in children, we hypothesised that these would bring about significant changes in the molecular function when compared to the adult-onset mutations. In this work using recombinantly expressed beta-MyHC subfragment 1 (S1) and a combination of steady-state and transient kinetics, 8 missense mutations have been analysed, 5 of which have been identified as early-onset cardiomyopathies. Using these derived parameters the cross-bridge cycle was modelled to show the cycle occupancy. Contrary to our hypothesis, there was no clear difference between the early and adult-onset cardiomyopathy mutations. Despite this the predicted actomyosin.ADP occupancy for [actin] = 3Kapp along with the duty ratio and measured ATPase rates all change in parallel to the wild type values. Six of the eight HCM mutations presented here are clearly distinct from the set of dilated cardiomyopathy (DCM) mutations previously characterised.

    Matrix stiffness dependent nuclear shuttling of MLP and PDLIM5 in cultured cardiomyocytes

    M. Ward 1 and T. Iskratsch1

    1School of Engineering and Materials Science, Queen Mary University of London

    The extracellular matrix of the myocardium can change its mechanical properties throughout life, both as a result of normal maturation and in disease states. These changes include alterations to the rigidity of the matrix. Cardiomyocytes have been demonstrated to be sensitive to changes in matrix rigidity, for example increasing matrix rigidity can result in the activation of the hypertrophic response.

    The mechanisms by which these changes in matrix stiffness lead to this response are poorly understood. At the costamere the proteins such talin has been shown to have altered stretching dynamics depending on matrix stiffness and thought to be implicated in mechanotransduction on cardiomyocytes, however, the mechanisms by which this information is relayed to the nucleus to alter transcriptional programs is unclear. Several LIM domain containing proteins have been implicated as potential mediators of this information.

    Here, using neonatal rat cardiomyocytes (NRCs) cultured on polydimethylsiloxane (PDMS) substrates of varying stiffness, followed by analysis with immunofluorescent imaging, western blotting and immunoprecipitation we find that various LIM domain proteins show different stiffness dependent dynamics, which we hypothesize will have overlapping effects. The mechanisms by which these proteins translocate to the nucleus and their role within remains to be investigated.

    Pluripotent stem cell-derived cardiomyocytes from a patient with ARG723GLY β-myosin heavy chain mutation as model for hypertrophic cardiomyopathy?

    N. Weber 1, T. Holler1, N. Peschel1, K. Schwanke2, B. Piep1, U. Martin2, R. Zweigerdt2 and T. Kraft1

    1Institute of Molecular & Cell Physiology, Hannover Medical School, Hannover, Germany; 2Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Hannover Medical School, Hannover, Germany.

    Hypertrophic cardiomyopathy (HCM) is the most frequent inherited cardiac disease. In mutation-positive patients one third of the mutations are located in the β-cardiac myosin heavy chain protein (β-MyHC). Yet, availability of human heart tissue samples from HCM patients is limited and in primary cardiomyocytes functional effects directly linked to the mutations are often masked by adaptational changes. Recently developed in vitro cultures of cardiomyocytes differentiated from induced pluripotent stem cells (hiPSC-CMs) appear attractive to investigate the direct effects of HCM mutations. Here we developed an in vitro disease model using HCM-patient-derived hiPSC-CMs carrying Arg723Gly mutation in β-MyHC. We characterized the impact of the mutation on twitch contractions, intracellular calcium transients and morphology of Arg723Gly-β-MyHC vs wildtype (WT) CMs. After differentiation, patient-derived hiPSC-CMs were cultivated on glass coverslips for ~ 35 days. Using our novel single cell mapping technique, we were able to identify the MyHC protein composition (α- vs. β-MyHC) of all functionally characterized cardiomyocytes and to compare functional and morphological parameters only for pure β-MyHC protein expressing CMs. Analysis of electrically evoked twitch contractions showed slightly larger contraction amplitude and significantly longer time to peak and half relaxation time of twitch for Arg723Gly-β-MyHC in comparison to WT-CMs. Analysis of intracellular calcium transients using Fura-2 AM, showed longer time courses of calcium transients for Arg723Gly-β-MyHC vs. hiPSC-WT CMs and notably for some batches of Arg723Gly-β-MyHC arrhythmic calcium transients in up to ~ 20% of CMs.

    Morphologically, Arg723Gly-β-MyHC-CMs exhibited a significant increase in cellular width and length with consecutive increase in cell area under basal conditions and under stimulation with hypertrophic agonists (isoprenaline and endotheline-1) versus WT CMs. Taken together Arg723Gly-β-MyHC CMs showed alterations in twitch contractions and intracellular calcium transients as well as of morphology. The question is how these alterations are linked to a missense mutation in β-MyHC.

    DLC1 expression and localisation is stiffness dependent in cardiomyocytes

    I. Xanthis, M. Ward and T. Iskratsch

    School of Engineering and Materials Science, Queen Mary University of London

    The cardiac microenvironment constantly changes during development and disease and the mechanical properties of the extracellular environment affect cellular responses, such as migration or differentiation. Cardiomyocytes have a network of mechanosensitive molecules in order to decode mechanical signals originated from the extracellular matrix. Talin’s head region binds to the cytoplasmic tails of integrins and is connected to a large flexible rod, which includes multiple binding sites for other proteins, such as DLC1—a molecule with unknown function in cardiomyocytes so far. Several studies have highlighted the importance of talin stretching dynamics, especially in relation to extracellular stiffness. However, the expression profiles and function of molecules binding to talin remain unclear.

    We hypothesised that DLC1 has distinct expression and localisation profiles in cardiomyocytes seeded on different stiffness substrates. To address this question, neonatal rat cardiomyocytes (NRMCs) were isolated and seeded on PDMS-coated coverslips, with increasing substrate stiffness. To investigate the role of DLC1 in cardiomyocyte mechanosensing, we combined here immunofluorescence, Western blot and FRET/FLIM microscopy with various inhibitor drug treatments in NRCMs. Thereby, we find that DLC-1 expression and localisation changes in relation to substrate stiffness, affecting downstream signalling pathways.

    However, the role of DLC-1 and its interactions downstream of talin in cardiomyocytes remain to be elucidated.

    A physiological characterization of heart failure progression and the role of phosphorylated regulatory light chain in cardiac function recovery

    H. Yu 1, W. Song1,2, K. D/O Markandran1 and M.A. Ferenczi1

    1Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore; 2National Heart Research Institute Singapore, Singapore

    In Singapore, a mortality rate of 68% is due to heart failure (HF). In addition, hypertrophic cardiomyopathy (HCM) arising from spontaneous or inherited mutations is among the most common cardiac disorders, with a prevalence of 1/500 in young adults around the world. In this study, a detailed physiological characterization of heart failure progression in mouse is performed to investigate the progression from compensation to decompensation. In particular, we aim to characterize the role of phosphorylation of the myosin regulatory light chain (RLC) as an indicator and modulator of heart failure progression and to test its potential as a therapeutic intervention. Mouse myocardial infraction model was created by left anterior descending (LAD) artery ligation surgery. The isometric contraction force of isolated cardiac papillary muscle was measured at six time points from day 2 to day 28 after LAD surgery. The phosphorylation level of native RLC was characterized at some time points. Recombinant RLC with a high phosphorylation level was expressed, modified, and exchanged into cardiac papillary muscle to study its potential to recover heart function. Based on the result of the pattern of cardiac muscle contraction force during these 28 days, the compensation happened in the early days but was lost from day 10. Hypertrophy also happened in the early stages but in the late stage, dilated cardiomyopathy was dominant with a much lower muscle contraction force. Exchange of phosphorylated RLC could recover at least some of cardiac muscle function and could be a potential treatment for heart failure disease.

    The scaffold protein Nesprin-2 is a novel binding partner for telethonin in cardiomyocytes

    Q.P. Zhang 1, C. Li1, 2, C. Zhou1, A.J. Candasamy1, D.T. Warren1,3, M. Garcia4, M. Wheeler5, M. Avkiran1 and C.M. Shanahan1

    1King’s College London British Heart Foundation Centre of Research Excellence, Cardiovascular Division, London, UK; 2Department of Cardiology, West China Hospital of Sichuan University, Chengdu, PR China; 3School of Pharmacy, University of East Anglia, UK; 4The Randall Centre for Cell and Molecular Biophysics, King’s College London, UK; 5Dept. of Cardiac Development and Remodelling, Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany

    Nesprins comprise a family of multi-isomeric scaffolding proteins that bind to lamin A/C, emerin and SUN1/2 at the nuclear envelope (NE) to form the linker of nucleoskeleton and cytoskeleton (LINC) complex. Mutations in nesprin-1 and -2 have been found in patients with the laminopathic disorders Emery–Dreifuss muscular dystrophy (EDMD) and dilated cardiomyopathy (DCM). We have identified that specific nesprin-2 isoforms are highly expressed in cardiac muscle cells and localise to the Z-disc and I band of the sarcomere. Importantly, expression of a GFP-tagged nesprin-2-TM construct, which lacks the NE targeting KASH domain, localised to the sarcomere of neonatal rat cardiomyocytes. Furthermore, yeast two-hybrid screening of a cardiac muscle library identified telethonin as a potential nesprin-2 binding partner. GST pull-down, in vivo IP and Native Page Novex gel all confirmed interactions between nesprin-2 and telethonin. Moreover, the binding affinity between nesprin-2/telethonin was tightly regulated by telethonin phosphorylation, suggesting that nesprin-2 may form a complex with telethonin at the Z-disc in a phosphorylation-dependent manner. In addition, their interaction was also impaired by both nesprin-2 and telethonin mutations identified in EDMD-DCM and HCM patients. These mutations were close to nesprin-2/telethonin binding or phosphorylation sites, further suggesting that appropriate binding affinity between these proteins is of great physiological importance. Taken together, these data suggest that nesprin-2 is a novel scaffold protein that may potentially participate in maintenance of sarcomeric organization. These findings provide essential information for further investigating the cardiac functions of nesprin-2.

    Deficiency of the actin binding protein CAP2 causes abnormal myofibril architecture without obvious effects on muscle performance

    Y. Zhang 1, L.-J. Kepser3, J. Brenmoehl2, A. Hoefiich2, M. Rust3 and H. Brinkmeier1

    1University Medicine Greifswald, Institute of Pathophysiology, Greifswald, Germany; 2Leibniz Institute for Farm Animal Biology (FBN), Dummerstorf, Germany; 3University of Marburg, Institute of Physiological Chemistry, Marburg, Germany

    Cyclase-associated proteins (CAP) are actin regulatory molecules with widely unknown functions. Recently it was shown that CAP2 deficient mice exhibit delayed muscle fibre maturation and an abnormal myofibril architecture, including a high frequency of type IIb ring fibers. To investigate physiological consequences of CAP2 deficiency we studied muscle forcesof isolated EDL and soleus (SOL) muscles of wildtype (WT) and CAP2 knockout (KO) mice. Muscles were electrically stimulated to produce twitches and tetani under isometric conditions. EDL muscles werefurther subjected of eccentric contractions. For this, they were stretched to 110% of their optimal length during 120-Hz tetani. For SOL a fatigue protocol of 400 s duration was performed (SOL: n = 4–5; EDL: n = 8–10 independent animals, each genotype). CAP2 deficient mice at an age of 3 months were slightly smaller than wildtype animals. Also weight and cross-sectional area (CSA) of their EDL muscles were significantly lower (muscle mass, WT/mutant: 8.5 ± 1.2/6.8 ± 1.1 mg; CSA: 1.6 ± 0.2/1.3 ± 0.1 mm2). Absolute forces of twitches and responses to 10 Hz, 50 Hz and 120-Hzstimulation were lower for KO EDL muscles. However, when normalized to CSA maximal isometric forces of WT and mutant were identical. Eccentric contractionscaused decreases of EDL muscle forces, both for WT and KO mice. After four eccentric contractions relative force had dropped to 0.78 ± 0.08 (WT) and to 0.81 ± 0.08 (KO), respectively. Kinetics of twitches and tetani of EDL and SOL muscles were not different in CAP2 deficient mice compared to WT. We conclude that CAP2 deficiency causes a developmental delay and abnormal myofibril architecture of mouse muscles. However, the force parameters studied here were surprisingly unaffected. More detailed analyses will be necessary to characterize the physiological muscle phenotype of CAP2 deficiency.

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    Talks. J Muscle Res Cell Motil 40, 227–274 (2019).

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