Abstract
D166V point mutation in the ventricular myosin regulatory light chain (RLC) is one of the causes of familial hypertrophic cardiomyopathy (FHC). We show here that the rates of cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from right ventricle of WT and Tg-D166V mice. To avoid averaging over ensembles of molecules composing muscle fibers, the data was collected from a single molecule. Kinetics were derived by tracking the orientation of a single actin molecule by fluorescence anisotropy. Orientation oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The cross-bridge in a wild-type (healthy) heart stayed attached and detached from thin filament on average for 0.7 and 2.7 s, respectively. In FHC heart, these numbers increased to 2.5 and 5.8 s, respectively. These findings suggest that alterations in myosin cross-bridge kinetics associated with D166V mutation of RLC ultimately affect the ability of a heart to efficiently pump the blood.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Abbreviations
- t ON :
-
The time cross-bridge is strongly attached to actin
- t OFF :
-
The time cross-bridge is detached from actin
- Ψ :
-
Duty cycle of the cross-bridge
- AP:
-
Alexa488-phalloidin
- RP:
-
Rhodamine-phalloidin
- UP:
-
Unlabeled-phalloidin
- DA:
-
Detection area
- EDC:
-
1-Ethyl-3-(3′-dimethylaminopropyl) carbodiimide
- ROI:
-
Region-of-interest
- DTT:
-
Cleland’s reagent
- APD:
-
Avalanche photodiode
- FCS:
-
Fluorescence correlation spectroscopy
- SMD:
-
Single molecule detection
References
Nihei T, Mendelson RA, Botts J (1974) Use of fluorescence polarization to observe changes in attitude of S1 moieties in muscle fibers. Biophys J 14:236–242
Borejdo J, Assulin O, Ando T, Putnam S (1982) Cross-bridge orientation in skeletal muscle measured by linear dichroism of an extrinsic chromophore. J Mol Biol 158:391–414
Thomas DD, Cooke R (1980) Orientation of spin-labeled myosin heads in glycerinated muscle fibers. Biophys J 32:891–905
Huxley AF, Simmons RM (1971) Proposed mechanism of force generation in striated muscle. Nature 233:533–538
Lymn RW, Taylor EW (1971) Mechanism of adenosine triphosphate hydrolysis by actomyosin. Biochemistry 10:4617–4624
Siemankowski RF, Wiseman MO, White HD (1985) ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle. Proc Natl Acad Sci U S A 82:658–662
Geeves MA, Holmes KC, Bodis E et al (2005) The molecular mechanism of muscle contraction. Adv Protein Chem 71:161–193
Magde D, Elson EL, Webb WW (1974) Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers 13:29–61
Qian H, Saffarian S, Elson EL (2002) Concentration fluctuations in a mesoscopic oscillating chemical reaction system. Proc Natl Acad Sci U S A 99:10376–10381
Bagshaw CR (1982) Muscle contraction. Chapman & Hall, London
Enderlein J, Ambrose WP (1997) Optical collection efficiency function in single-molecule detection experiments. Appl Opt 36:5298–5302
Willets KA, Ostroverkhova O, He M, Twieg RJ, Moerner WE (2003) Novel fluorophores for single-molecule imaging. J Am Chem Soc 125:1174–1175
Wang Y, Qin H, Kudaravalli RD et al (2007) Single-molecule structural dynamics of EF-G-ribosome interaction during translocation. Biochemistry 46:10767–10775
Taniguchi Y, Karagiannis P, Nishiyama M, Ishii Y, Yanagida T (2007) Single molecule thermodynamics in biological motors. Biosystems 88:283–292
Warshaw DM, Hayes E, Gaffney D et al (1998) Myosin conformational states determined by single fluorophore polarization. Proc Natl Acad Sci U S A 95:8034–8039
Quinlan ME, Forkey JN, Goldman YE (2005) Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy. Biophys J 89:1132–1142
Forkey JN, Quinlan ME, Shaw MA, Corrie JE, Goldman YE (2003) Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization. Nature 422:399–404
Yildiz A, Forkey JN, McKinney SA, Ha T, Goldman YE, Selvin PR (2003) Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300:2061–2065
Toprak E, Enderlein J, Syed S et al (2006) Defocused orientation and position imaging (DOPI) of myosin V. Proc Natl Acad Sci U S A 103:6495–6499
Minton AP, Wilf J (1981) Effect of macromolecular crowding upon the structure and function of an enzyme: glyceraldehyde-3-phosphate dehydrogenase. Biochemistry 20:4821–4826
Minton AP (1981) Excluded volume as a determinant of macromolecular structure and reactivity. Biopolymers 20:2093–2120
Eigen M, Rigler R (1994) Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc Natl Acad Sci U S A 91:5740–5747
Borejdo J, Shepard AA, Akopova I, Grudzinski W, Malicka J (2004) Rotation of the lever-arm of myosin in contracting skeletal muscle fiber measured by two-photon anisotropy. Biophys J 87:3912–3921
Levene MJ, Korlach J, Turner SW, Foquet M, Craighead HG, Webb WW (2003) Zero-mode waveguides for single-molecule analysis at high concentrations. Science 299:682–686
Lewis A (1991) The optical near-field and cell biology. Semin Cell Biol 2:187–192
de Lange F, Cambi A, Huijbens R et al (2001) Cell biology beyond the diffraction limit: near-field scanning optical microscopy. J Cell Sci 114:4153–4160
Borejdo J, Talent J, Akopova I, Burghardt TP (2006) Rotations of a few cross-bridges in muscle by confocal total internal reflection microscopy. Biochim Biophys Acta 1763:137–140
Vecer J, Kowalczyk AA, Davenport L, Dale RE (1993) Reconvolution analysis in time-resolved fluorescence experiments, an alternative approach: reference-to-excitation-to-fluorescence reconvolution. Rev Sci Instrum 64:3413–3424
Bukatina AE, Fuchs F, Watkins SC (1996) A study on the mechanism of phalloidin-induced tension changes in skinned rabbit psoas muscle fibres. J Muscle Res Cell Motil 17:365–371
Prochniewicz-Nakayama E, Yanagida T, Oosawa F (1983) Studies on conformation of F-actin in muscle fibers in the relaxed state, rigor, and during contraction using fluorescent phalloidin. J Cell Biol 97:1663–1667
Shepard A, Borejdo J (2004) Correlation between mechanical and enzymatic events in contracting skeletal muscle fiber. Biochemistry 43:2804–2811
Szczesna D, Lehrer SS (1993) The binding of fluorescent phallotoxins to actin in myofibrils. J Muscle Res Cell Motil 14:594–597
Yanagida T, Oosawa F (1980) Conformational changes of F-actin-epsilon-ADP in thin filaments in myosin-free muscle fibers induced by Ca2+. J Mol Biol 140:313–320
Yanagida T, Oosawa F (1998) Polarized fluorescence from epsilon-ADP incorporated into F-actin in a myosin-free single fiber: conformation of F-actin and changes induced in it by heavy meromyosin. J Mol Biol 126:507–524
Borovikov YS, Kuleva NV, Khoroshev MI (1991) Polarization microfluorimetry study of interaction between myosin head and F-actin in muscle fibers. Gen Physiol Biophys 10:441–459
Borejdo J, Shepard A, Dumka D et al (2004) Changes in orientation of actin during contraction of muscle. Biophys J 86:2308–2317
Dos Remedios CG, Millikan RG, Morales MF (1972) Polarization of tryptophan fluorescence from single striated muscle fibers. A molecular probe of contractile state. J Gen Physiol 59:103–120
Dos Remedios CG, Yount RG, Morales MF (1972) Individual states in the cycle of muscle contraction. Proc Natl Acad Sci U S A 69:2542–2546
Tregear RT, Mendelson RA (1975) Polarization from a helix of fluorophores and its relation to that obtained from muscle. Biophys J 15:455–467
Morales MF (1984) Calculation of the polarized fluorescence from a labeled muscle fiber. Proc Nat Acad Sci U S A 81:145–149
Maron BJ, Olivotto I, Spirito P et al (2000) Epidemiology of hypertrophic cardiomyopathy-related death: revisited in a large non-referral-based patient population. Circulation 102:858–864
Elson EL (2008) Introduction to FCS. Course Cell Mol Fluorescence 2:8
Huxley AF (1957) A hypothesis for the mechanism of contraction of muscle. Prog Biophys Biophys Chem 7:255–318
Cooke R, Crowder MS, Thomas DD (1982) Orientation of spin labels attached to cross-bridges in contracting muscle fibres. Nature 300:776–778
Thomas DD, Ramachandran S, Roopnarine O, Hayden DW, Ostap EM (1995) The mechanism of force generation in myosin: a disorder-to-order transition, coupled to internal structural changes. Biophys J 68:135S–141S
Reedy MC (2000) Visualizing myosin’s power stroke in muscle contraction. J Cell Sci 113:3551–3562
Sweeney HL, Houdusse A (2004) The motor mechanism of myosin V: insights for muscle contraction. Philos Trans R Soc Lond B Biol Sci 359:1829–1841
Takagi Y, Shuman H, Goldman YE (2004) Coupling between phosphate release and force generation in muscle actomyosin. Philos Trans R Soc Lond B Biol Sci 359:1913–1920
Poetter K, Jiang H, Hassanzadeh S et al (1996) Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle. Nat Genet 13:63–69
Flavigny J, Richard P, Isnard R et al (1998) Identification of two novel mutations in the ventricular regulatory myosin light chain gene (MYL2) associated with familial and classical forms of hypertrophic cardiomyopathy. J Mol Med 76:208–214
Andersen PS, Havndrup O, Bundgaard H et al (2001) Myosin light chain mutations in familial hypertrophic cardiomyopathy: phenotypic presentation and frequency in Danish and South African populations. J Med Genet 38:E43
Kabaeva ZT, Perrot A, Wolter B et al (2002) Systematic analysis of the regulatory and essential myosin light chain genes: genetic variants and mutations in hypertrophic cardiomyopathy. Eur J Hum Genet 10:741–748
Richard P, Charron P, Carrier L et al (2003) Hypertrophic cardiomyopathy: distribution of disease genes. Spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation 107:2227–2232
Morner S, Richard P, Kazzam E et al (2003) Identification of the genotypes causing hypertrophic cardiomyopathy in northern Sweden. J Mol Cell Cardiol 35:841–849
Hougs L, Havndrup O, Bundgaard H et al (2005) One third of Danish hypertrophic cardiomyopathy patients have mutations in MYH7 rod region. Eur J Hum Genet 13:161–165
Maron BJ (2002) The young competitive athlete with cardiovascular abnormalities: causes of sudden death, detection by preparticipation screening, and standards for disqualification. Card Electrophysiol Rev 6:100–103
Ao X, Lehrer SS (1995) Phalloidin unzips nebulin from thin filaments in skeletal myofibrils. J Cell Sci 108:3397–3403
Borovikov YS, Chernogriadskaia NA (1979) Studies on conformational changes in F-actin of glycerinated muscle fibers during relaxation by means of polarized ultraviolet fluorescence microscopy. Microsc Acta 81:383–392
Houdusse A, Sweeney HL (2001) Myosin motors: missing structures and hidden springs. Curr Opin Struct Biol 11:182–194
Pesce AJ, Rosen CG, Pasby TL (1971) Fluorescence spectroscopy. Marcel Dekker, New York
Mettikolla P, Luchowski R, Gryczynski I, Gryczynski Z, Szczesna-Cordary D, Borejdo J (2009) Fluorescence lifetime of actin in the FHC transgenic heart. Biochemistry 48(6):1264–1271
Cooper WC, Chrin LR, Berger CL (2000) Detection of fluorescently labeled actin-bound cross-bridges in actively contracting myofibrils. Biophys J 78:1449–1457
Duong AM, Reisler E (1989) Binding of myosin to actin in myofibrils during ATP hydrolysis. Biochemistry 28:1307–1313
Hilber K, Sun YB, Irving M (2001) Effects of sarcomere length and temperature on the rate of ATP utilisation by rabbit psoas muscle fibres. J Physiol 531:771–780
Kerrick WG, Kazmierczak K, Xu Y, Wang Y, Szczesna-Cordary D (2009) Malignant familial hypertrophic cardiomyopathy D166V mutation in the ventricular myosin regulatory light chain causes profound effects in skinned and intact papillary muscle fibers from transgenic mice. FASEB J 23:855–865
Ando T (1987) Propagation of Acto-S-1 ATPase reaction-coupled conformational change in actin along the filament. J Biochem (Tokyo) 105:818–822
Acknowledgments
Supported by NIH grant R01AR048622 to J.B. and by Texas ETF grant (CCFT). R.L. is the recipient of the Research Mobility program from the Polish Ministry of Science and Higher Education.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Borejdo, J. et al. (2012). Single Molecule Detection Approach to Muscle Study: Kinetics of a Single Cross-Bridge During Contraction of Muscle. In: Bujalowski, W. (eds) Spectroscopic Methods of Analysis. Methods in Molecular Biology, vol 875. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-806-1_17
Download citation
DOI: https://doi.org/10.1007/978-1-61779-806-1_17
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-805-4
Online ISBN: 978-1-61779-806-1
eBook Packages: Springer Protocols