A study of tropomyosin’s role in cardiac function and disease using thin-filament reconstituted myocardium
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- Bai, F., Wang, L. & Kawai, M. J Muscle Res Cell Motil (2013) 34: 295. doi:10.1007/s10974-013-9343-z
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Tropomyosin (Tm) is the key regulatory component of the thin-filament and plays a central role in the cardiac muscle’s cooperative activation mechanism. Many mutations of cardiac Tm are related to hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and left ventricular noncompaction (LVNC). Using the thin-filament extraction/reconstitution technique, we are able to incorporate various Tm mutants and protein isoforms into a muscle fiber environment to study their roles in Ca2+ regulation, cross-bridge kinetics, and force generation. The thin-filament reconstitution technique poses several advantages compared to other in vitro and in vivo methods: (1) Tm mutants and isoforms are placed into the real muscle fiber environment to exhibit their effect on a level much higher than simple protein complexes; (2) only the primary and immediate effects of Tm mutants are studied in the thin-filament reconstituted myocardium; (3) lethal mutants of Tm can be studied without causing a problem; and (4) inexpensive. In transgenic models, various secondary effects (myocyte disarray, ECM fibrosis, altered protein phosphorylation levels, etc.) also affect the performance of the myocardium, making it very difficult to isolate the primary effect of the mutation. Our studies on Tm have demonstrated that: (1) Tm positively enhances the hydrophobic interaction between actin and myosin in the “closed state”, which in turn enhances the isometric tension; (2) Tm’s seven periodical repeats carry distinct functions, with the 3rd period being essential for the tension enhancement; (3) Tm mutants lead to HCM by impairing the relaxation on one hand, and lead to DCM by over inhibition of the AM interaction on the other hand. Ca2+ sensitivity is affected by inorganic phosphate, ionic strength, and phosphorylation of constituent proteins; hence it may not be the primary cause of the pathogenesis. Here, we review our current knowledge regarding Tm’s effect on the actomyosin interaction and the early molecular pathogenesis of Tm mutation related to HCM, DCM, and LVNC.
KeywordsHCMDCMLVNCHypertrophic cardiomyopathyDilated cardiomyopathyLeft ventricular noncompactionCross-bridge kineticsElementary stepsSinusoidal analysis
Tropomyosin (Tm) is a regulatory protein of contraction in striated muscles. Together with troponin (Tn) complex, Tm interacts with actin and regulates the actomyosin (AM) interaction. The cooperative Ca2+ activation process in cardiomyocytes involves sequential conformational changes of sarcomeric proteins, which is an extremely complex process. The precise mechanism through which Tm modulates the conformational changes has been one of the vigorously researched subjects for the past 20 years, and numerous proposals have been made to account for the role of Tm in normal cardiac functions as well as in the diseased states. Here, we review our current knowledge on cardiac Tm in three aspects: (1) the mechanism relating to how Tm promotes the AM interaction and enhances force generation; (2) the role of each period of Tm in the thin-filament activation mechanism; and (3) the mechanism how Tm’s mutations change the cardiac function and lead to the disease phenotype. There have been several review articles written in these areas recently (Wieczorek et al. 2008; Tardiff 2011; Jagatheesan et al. 2010; Nevzorov and Levitsky 2011; Lehman and Craig 2008; Hitchcock-DeGregori 2008; Kobayashi et al. 2008), hence we try to avoid unnecessary duplications.
Using thin-filament extraction/reconstitution technique and sinusoidal analysis to investigate Tm functions
In this analysis method, small amplitude sinusoidal length changes are applied to the reconstituted myocardium at 18 different frequencies (f). The tension transients resulting from the length changes are analyzed and the quantity called complex modulus Y(f) is obtained. Y(f) is the ratio of the stress change to the strain change represented in the frequency domain.
In the cross-bridge scheme (Fig. 4), K1 is the ATP association constant, K0 is the ADP association constant, k2 is the forward rate constant of the cross-bridge detachment step 2, and k-2 is its reversal step. k4 is the forward rate constant of the force generation (isomerization) step 4, and k-4 is its reversal step. K5 is the Pi association constant. σ in Eq.  is calculated from K1 and K2 obtained from the MgATP study and S = 5 mM, the condition of the Pi study.
The number of actively cycling cross bridges and the tension supported by each cross bridge can be deduced with the kinetic constants and S, P, and D dependence of isometric tension (Kawai et al. 1993). Tension per cross bridge are used as a direct indicator of the AM binding. When combined with temperature studies, the ionic and hydrophobic surface area changes of the AM interaction can be deduced (Kawai 2003). A part of the interacting surface area change comes from Tm’s allosteric effect on actin. These thermodynamic parameters can provide information on dynamic structural changes among Tm, actin, and myosin during isometric contraction.
Tm’s allosteric effect on the AM interaction
For more than 40 years, Tm has been known to enhance the AM interaction at the saturating Ca2+ concentration (Bremel and Weber 1972; Lehrer and Morris 1982; Eaton 1976). This is called the positive allosteric effect of Tm on the AM interaction. It has been shown that Tm increases AM binding affinity by three-sevenfold in the presence of Tn and Ca2+ (Williams and Greene 1983). Fujita and his colleagues demonstrated, in skinned fibers, that the isometric tension increased by ~50 % after reconstitution with Tm and Tn, compared to tension generated by unregulated actin filament and the thick filament (Fujita et al. 2002). In vitro motility assay also demonstrated a similar increase in tension and sliding velocity when Tm and Tn were reconstituted (Oguchi et al. 2011; Bing et al. 2000a; Kawai et al. 2006; Gordon et al. 1998; VanBuren et al. 1999). From a structural point of view, this allosteric effect may happen because Tm binding to the AM complex recruits more hydrophobic residues to the AM interface, leading to an enhanced AM binding (Holmes 1995). At the low Ca2+ concentration in the presence of Tn, Tm sterically blocks the myosin binding site of actin (Lehman et al. 2009; Huxley 1973). At saturating Ca2+ concentration, Tm moves away from the blocked position and allows the initial binding between actin and myosin to take place (Lehman et al. 2009). This binding changes the actin conformation, which in turn allows a further azimuthal shift of Tm’s position; this shift promotes the stronger AM binding and may recruit more hydrophoic residues into the AM interface. AM consequently achieves the strong binding state, in which cross-bridges are fully active (Lehman et al. 2009; Holmes 1995). Structural studies have shown that most amino acid residues in the AM interface are hydrophobic (Behrmann et al. 2012; Holmes et al. 2004; Rayment et al. 1993) and there is thermodynamic evidence to this (Zhao and Kawai 1994; Kawai 2003). It has been thought that Tm directly interacts with myosin in the rigor state and stabilize the AM interaction (Behrmann et al. 2012).
The initial binding between actin and myosin involves ionic amino acid residues and it is presumably a “weak” interaction. This is convenient because the ionic interaction takes place over a long distance. It has been known for some time that actin’s N-terminus has four negative charges (Asp-1, Glu-2, Asp-3, Glu-4) (Sutoh et al. 1991), which interact with myosin’s loop 2 (residues 633–642) with 5 Lys residues (Furch et al. 1998). Another ionic interaction between next actin’s Glu-99 and myosin’s positively charged segment Lys-567 through His-578 is possible (Bookwalter and Trybus 2006; Holmes et al. 2004). The actin sequence is based on rabbit skeletal actin (virtually the same as human cardiac actin), and the myosin sequence on chicken skeletal myosin. The ionic interaction is followed by the hydrophobic and stereospecific interaction, which is only effective at a short distance, and corresponds to the “strong” interaction. The primary binding site on actin consists of helix-turn-helix (residues 337–351) in subdomain 1, and that on myosin is helix-turn-helix of the lower 50 K domain (residues 526–559); The secondary binding site on actin consists of segments Ala-26 through Ala-29 and Pro-333 through Tyr-337, and that on myosin is in its upper 50 K domain called “cardiomyopathy loop” (around residues 405–414) (Holmes et al. 2004). The weak interaction between N-terminus of actin and the loop 2 of myosin is known to induce the strong interaction subsequently, because a decrease in the number of N-terminal negative charges of actin results in a decrease in isometric tension and stiffness (Lu et al. 2005). The strong interaction is detected as isometric tension or stiffness measured by low frequency (<1 kHz). Unfortunately, how Tm affects these interactions, or at which point the “closed” to “open” transition occurs, is not yet known, because the AM interaction is based on observations without Tm, except for the recent study of the rigor complex between actin, myosin, and Tm (Behrmann et al. 2012).
Physiological and mechanical evidence for the enhanced hydrophobic interaction by Tm comes from temperature studies of skinned cardiac muscle fibers. Ca2+ activatable tension increases with an increase in temperature (called endothermic force generation) (Coupland et al. 2001; Zhao and Kawai 1994; Ranatunga 1996), because of an increase in the hydrophobic interaction which requires heat to be absorbed (Kawai 2003). When unregulated and regulated cardiac muscle fibers are compared, we reported that the slope of the temperature-tension plot increased by ~3 times upon reconstitution of Tm and Tn, indicating increased hydrophobic interaction of AM (Fujita and Kawai 2002). Sinusoidal analysis also demonstrated that tension supported by each cross-bridge was enhanced to ~2 times upon the reconstitution of Tm/Tn to the actin-filament reconstituted muscle fibers (Fujita et al. 2002). This increase may come in part from the direct Tm-myosin interaction that is known to occur during the rigor state (Behrmann et al. 2012). Further thermodynamic analysis under different temperatures provided evidence that in the presence of Tm/Tn, the activation energy of 2πc (apparent rate constant of fast tension recovery step) decreased significantly compared to that in the absence of Tm/Tn (Fujita and Kawai 2002), indicating that the elementary step of the cross-bridge cycle associated with 2πc (cross-bridge detachment step 2, Fig. 1) becomes easier to occur in the presence of Tm/Tn than in their absence.
Tm’s positive allosteric effect on AM binding can also be induced without Tn. In the unregulated cardiac muscle fibers (no Tm/Tn), a reconstitution with Tm increases the isometric tension by ~20 % (Fujita et al. 2004). Further reconstitution with Tn increases the tension by another ~25 % (Fujita et al. 2004, 2002). In the absence of Tm/Tn, cross bridge detachment rate was decreased and the equilibrium constant of force generation step (K4, Fig. 1) was increased, leading to ~30 % more strongly binding cross-bridges compared to that in the presence of Tm/Tn (Fujita et al. 2002). However, adding Tm alone to unregulated cardiac muscle fibers shifts the number of active cycling cross bridges to the same as that in the presence of both Tm and Tn (Fujita et al. 2004). These results indicate that Tm is the key protein in the positive allosteric effect, and an addition of the Tn complex further enhances this effect as an amplifier.
Isoforms and post-translational modification of Tm in cardiac muscle fibers
In striated muscles, there are four major isoforms of Tm: α-Tm, β-Tm, γ-Tm, and κ-Tm. However, γ-Tm is not expressed in cardiomyocytes. While β-Tm is expressed during the development of the heart, α-Tm is the predominant isoform in adult cardiac muscles (Muthuchamy et al. 1993). A recent study showed that in adult human heart, there are 90–94 % α-Tm, 3–5 % β-Tm, and 3–5 % κ-Tm. κ-Tm is a novel Tm isoform which is generated from alternative splicing of TPM1 gene (Rajan et al. 2010). Different isoforms of Tm bear functional differences despite of the fact that they are highly homologous in their amino acid sequences. κ-Tm isoform showed almost no actin binding affinity, which indicates that the presence and expression level of this isoform may be important for regulating the overall Tm function (Rajan et al. 2010). This isoform also decreased Ca2+ sensitivity and caused cardiac dysfunction in transgenic mice expressing 90 % κ-Tm (Rajan et al. 2010). Replacing α-Tm with β-Tm in skinned cardiac muscle fibers resulted in an increased Ca2+ sensitivity and a decreased cooperativity (Lu et al. 2010). Transgenic mice expressing 55 % β-Tm showed no change in cardiac morphology and function, but also caused an increase in myofilament Ca2+ sensitivity (Muthuchamy et al. 1995). Transgenic mice expressing 50 % γ-Tm and 50 % α-Tm caused both systolic and diastolic dysfunction and a decrease in Ca2+ sensitivity (Pieples et al. 2002). Structural analysis showed that different Tm isoform composition shifted the position and movement of Tm’s coiled-coil on the actin filament (Lehman et al. 2009, 2000). However, except for the recent report that κ-Tm expression is increased in DCM patients’ heart (Karam et al. 2011), it is still not clear how the ratio among different isoform of Tm in the human heart affects normal cardiac functions or development of diseases.
Phosphorylation of sarcomeric proteins has been shown to be an important mechanism to modulate contraction, relaxation, and Ca2+ activation processes (Solaro and Kobayashi 2011). Both α-Tm and β-Tm have a potential phosphorylation site at Ser-283, a region critical for the Tm-TnT interaction, and the head-to-tail overlap between adjacent Tm’s coiled-coil on the actin-filament (Mak et al. 1978; Heeley 1994). α-Tm phosphorylation may be essential in heart development as majority of α-Tm is phosphorylated during the fetal stage, whereas only 10–30 % of α-Tm is phosphorylated in adult cardiomyocytes (Heeley et al. 1982; Yuan et al. 2008). β-Tm, although carrying 86 % sequence identity with α-Tm, is not shown to be phosphorylated in either fetal stage or adult cardiomyocytes (Heeley et al. 1982). The functional effect of α-Tm phosphorylation was studied using different approaches and the results varied accordingly. In vitro studies showed that phosphorylation of α-Tm had no effect on Tm-actin binding (Heeley et al. 1989), but promoted the head-to-tail interaction between two neighboring Tms (Rao et al. 2009). Pseudo-phosphorylation of α-Tm is shown to decrease the relaxation rate in myofibrils (Nixon et al. 2012). Skinned fiber study showed that 100 % phosphorylated α-Tm increased the Ca2+ sensitivity without changing the contractility compared to the cardiac muscle fiber containing 100 % unphosphorylated α-Tm (Lu et al. 2010). Sinusoidal analysis showed that the cross-bridge kinetics during the steady state remained largely unchanged by the phosphorylation of α-Tm (Lu et al. 2010). Tg mice bearing α-Tm S283A mutation (mimic unphosphorylated state) showed mild hypertrophy without a change in cardiac function, Ca2+ sensitivity, or cooperativity compared to non Tg mice (Schulz et al. 2012). From our thin-filament reconstitution experiments, we conclude that the Tm phosphorylation is a fast method of increasing the Ca2+ sensitivity, whereas isoform switch from α-Tm to β-Tm is a slow method of increasing the Ca2+ sensitivity (Lu et al. 2010).
Function of different periods of Tm on cooperative activation and disease
Inherited cardiomyopathy causing mutations in α-Tm
(Chang et al. 2011)
(Olson et al. 2001)
(Olson et al. 2001)
(Jongbloed et al. 2003)
(Yamauchi-Takihara et al. 1996)
(van de Meerakker et al. 2012)
(Karibe et al. 2001)
(Van Driest et al. 2003)
TnT Interactiion; actin-Tm-myosin interface
TnT Interaction; actin-Tm-myosin interface
(Thierfelder et al. 1994)
TnT Interaction; actin-Tm-myosin interface
(Regitz-Zagrosek et al. 2000)
(Probst et al. 2011)
(Morita et al. 2008)
(Lakdawala et al. 2010)
(Probst et al. 2011)
Tm head-to-tail binding
(Van Driest et al. 2003)
Molecular pathogenesis of Tm mutations leading to inherited cardiomyopathies
So far, 18 Tm mutations are known to cause HCM, DCM, or LVNC in humans. These mutations scatter in different periods of Tm and interact with different Tm binding proteins (Table 1; Fig. 5). However, despite extensive investigations, the mechanism of how the mutations within Tm lead to various disease phenotypes remains unknown.
HCM Tm mutations
Hypertrophic cardiomyopathy (HCM) is a disease of the heart muscle characterized by the abnormally thickened left ventricular wall and interventricular septum. Familial forms of HCM are the most common inherited heart disease caused by sarcomeric protein mutations (Elliott and McKenna 2004; Ho and Seidman 2006). HCM prevalence is ∼0.2 % in the total population and is the leading cause of sudden death in young adults (Ommen and Gersh 2009; Maron et al. 2003). The most notable clinical manifestation of HCM is left ventricular hypertrophy. However, individuals carrying HCM causing genes can remain asymptomatic, or can experience severe heart failure and sudden cardiac death (Marian and Roberts 2001; Barron 1999). So far, more than 400 mutations within the sarcomeric proteins have been identified to be associated with HCM, 11 of which occur in Tm. In different countries, Tm mutation accounts for 5–11 % of all familial HCM cases (Tardiff 2005; Nakajima-Taniguchi et al. 1995; Yamauchi-Takihara et al. 1996; Jaaskelainen et al. 1998). This difference in prevalence is likely due to the “founder” effect in the specific population (Wieczorek et al. 2008). The phenotype of the Tm mutation related HCM is generally benign, but also it can be severe depending on the specific mutation (Tardiff 2005; Nakajima-Taniguchi et al. 1995; Yamauchi-Takihara et al. 1996; Jaaskelainen et al. 1998).
Of the 11 HCM Tm mutations, six of them (E62Q, K70T, D175N, E180G, E180V, and L185R) cause charge changes. Because Tm-actin interaction is largely electrostatic, the charge changes are considered critical to cause abnormal Tm-actin interaction (Brown and Cohen 2005; Lorenz et al. 1995). Four of the mutations (D175N, E180G, E180V, and L185R) are located in or close proximity to the region involved in the actin-Tm-myosin interface and possibly interfere with the electrostatic interaction (Behrmann et al. 2012). This region of Tm has also been shown to interact with TnT and plays a key role in the electrostatic interaction between Tm and actin (Barua et al. 2011). Other five HCM mutations which do not cause a charge change in Tm (A63V, V95A, I172T, S215L and M281T) all cause changes in the hydrophobicity. Hydrophobic interaction is critical for stabilizing the Tm’s coiled-coil structure. It has been shown that Ala clusters in Tm molecule is critical for maintaining its flexibility and replacing Ala residues in the a and d position of the heptad repeat with more hydrophobic residues significantly improve the stability of Tm’s coiled-coil (Singh and Hitchcock-DeGregori 2003). Hydrophobic interaction is also involved in Tm (period 5 and 6) and actin binding (Brown et al. 2005; Miki et al. 2011).
The underlying molecular mechanism how these Tm mutants lead to HCM phenotype has been extensively investigated. The most studied mutants include V95A, D175N and E180G. Structural analysis has shown that A63V, K70T, D175N and E180G decreased molecular stability (Ly and Lehrer 2012; Heller et al. 2003). Fluorescence polarization measurement showed that D175N and E180G shifted the Tm’s position in the thin-filament towards the open position (Borovikav et al. 2011). Solution studies indicated that V95A decreased ATPase activity (Mathur et al. 2011). D175N and E180G reduced the Tm-actin and Tm-TnT affinities, but they exhibited no effect on the ATP hydrolysis rate (Mathur et al. 2011). In vitro motility assay also showed that D175N and E180G increased the Ca2+ sensitivity in the fraction of motile filaments (Bing et al. 2000b).
Tg mouse models have been created for D175N and E180G (Prabhakar et al. 2001, 2003 Muthuchamy et al. 1999). D175N mutation caused only mild hypertrophic phenotype: increased end diastolic pressure and increased Ca2+ sensitivity in skinned fiber preparations (Muthuchamy et al. 1999). E180G caused more severe and progressive hypertrophy and fibrosis, leading to cardiac death in 4.5–6 months (Prabhakar et al. 2001). E180G mice also showed increased end diastolic pressure and Ca2+ sensitivity (Prabhakar et al. 2001). Both mutants caused a significant decrease in contraction and relaxation rate (Muthuchamy et al. 1999; Prabhakar et al. 2001). A human patient study also indicated D175N to be one of the benign HCM-causing mutations based on the prognosis and symptom (Van Driest et al. 2002).
HCM Tm mutant proteins V95A, D175N, and E180G were incorporated into skinned cardiac muscle fibers by using the thin-filament removal and reconstitution protocol to investigate the underlying molecular pathogenesis at the earliest stage (Bai et al. 2011). Our results demonstrated that all these three Tm mutants exhibit abnormal increase in the number of active cycling cross-bridges at pCa 8, leading to the increased tension at the relaxed state, which causes diastolic dysfunction (Bai et al. 2011). Diastolic pressure overload in the affected myocardium may stimulate cardiac growth, causing abnormal release of angiotensin II and aldosterone (Sadoshima et al. 1993), and eventually lead to the HCM phenotype: left ventricular hypertrophy (Koide et al. 1999) and abnormal extracellular matrix formation (Sadoshima et al. 1993). V95A and D175N also cause decreased Ca2+ activatable tension, which contributes to a systolic problem. V95A and E180G significantly increase the Ca2+ sensitivity compared to that of WT and D175N. Our results also demonstrated that these Tm mutations altered the steady-state cross-bridge kinetics, with V95A showed the most severe effect (Bai et al. 2011). Interestingly, V95A also exhibited severe symptom and poor prognosis in human patients (Karibe et al. 2001). By comparing the observed parameters (Table 1 in (Bai et al. 2011)) and results from human patient studies (Karibe et al. 2001; Coviello et al. 1997), we conclude that both impaired relaxation and elevated Ca2+ sensitivity cause diastolic problem leading to the HCM phenotype. Other changes in contractility and cross-bridge kinetics may also contribute to the severity of HCM symptoms and disease prognosis.
DCM Tm mutations
Dilated cardiomyopathy (DCM) is characterized by dilated left ventricle and systolic dysfunction (Fuster et al. 1981). DCM affects ~36.5 people in 100,000 and causes ~10,000 death annually (Gillum 1986; Codd et al. 1989). It is estimated that 25–30 % of DCM cases are caused by genetic defects (Grunig et al. 1998; Zimmerman et al. 2010; Michels et al. 1992; Marston 2011). Mutations in more than 20 proteins, including β-myosin heavy chain, cardiac myosin binding protein-C (cMyBP-C), α-actin, troponin, titin, desmin, vinculin, and Tm, have been shown to cause DCM in humans (Hershberger et al. 2009). However, the number of DCM causing mutations is small compared to that causing HCM, hence there are fewer studies on DCM related mutations than its HCM counterpart. So far, there are only four Tm mutations which have been identified to cause DCM: E40K, E54K, D84N and D230N (Table 1; Fig. 5). All four DCM related Tm mutations are located on the surface of Tm coiled-coil and carry a charge change. Both E40 and E54 are located at the e position of the heptad repeat and respectively interact with R35 (g) and K49 (g) on the other Tm chain to stabilize the Tm’s coiled-coil structure (Brown et al. 2001). D84 is located at the g position of the heptad repeat and interacts with E75 (e) of the other Tm chain, which increases the flexibility of Tm’s coiled-coil (Whitby and Phillips 2000). D230 is located in the outer surface of Tm (f position) and possibly involved in the cTnT interaction (Tardiff 2011). E40K, E54K and D84N are also located in the actin-binding motif of the Tm (McLachlan and Stewart 1976). Furthermore, E40K is located in Tm period 1b, which interacts with actin in the presence of Ca2+, whereas E54K is located in Tm period 2a, which interacts with actin in the absence of Ca2+ (Holthauzen et al. 2004). Previous studies showed that E40K and E54K decreased the Ca2+ sensitivity of myocardium (Chang et al. 2005), and the decreased Ca2+ sensitivity was thought to be critical to DCM pathogenesis (Willott et al. 2010; Mirza et al. 2007). In a different study, E40K caused a decreased maximum ATPase activity, but E54K showed only diminished Tm-actin affinity in the absence of Tn and myosin (other parameters remained unchanged) (Mirza et al. 2007). Co-sedimentation assay showed that D84N decreased the Tm’s affinity to F-actin by 25 % (van de Meerakker et al. 2012). ATPase assay showed that D230N led to a significant decrease in both maximal ATPase activity and Ca2+ sensitivity (Lakdawala et al. 2010).
To investigate the in vivo effects of these DCM causing Tm mutants, Tg mouse models have been generated for E54K and D230N mutations (Rajan et al. 2007; Tal et al. 2012). E54K mice showed typical DCM phenotype with both systolic and diastolic dysfunction. Isolated cardiac myofibers from E54K mice showed significantly decreased tension generation and Ca2+ sensitivity (Rajan et al. 2007). D230N mice showed early dilation and systolic failure without any histological changes (Tal et al. 2012). Secondary effect of these mutations on the transgenic models was also studied. Real-time RT-PCR proved that the expression level of β-myosin heavy chain and skeletal actin was increased in E54K mice; however, the expression level of two Ca2+ handling proteins, sarcoplasmic reticulum Ca2+ ATPase and ryanodine receptor, was decreased in E54K mice (Rajan et al. 2007). Isolated myocytes from D230N mice showed no changes in Ca2+ transients, indicating this mutation has no effect on the Ca2+ handling proteins (Tal et al. 2012).
We have also studied Tm mutation E40K and E54K using thin-filament reconstituted myocardium (Bai et al. 2012). Our results demonstrated that E40K and E54K didn’t change the Ca2+ sensitivity compared to that of the WT, which was mainly dues to 8 mM phosphate (Pi) included in the activating solution (see below). However, E40K and E54K caused an over-inhibition effect on force generation and decreased the maximal tension (pCa 4.66) by ~20 % compared to that of the WT. This over-inhibition effect also decreased tension at pCa 7.0 by ~50 % compared to that of the WT, from which we conclude that the over-inhibition occurs regardless of [Ca2+]. Sinusoidal analysis demonstrated that under pCa 4.66, E40K decreased tension by decreasing the number of actively cycling cross-bridges without changing force/cross-bridge, whereas E54K decreased tension both by decreasing the number of actively cycling cross-bridges and force/cross-bridge. At pCa 7.0, both E40K and E54K decreased tension by decreasing the number of actively cycling cross-bridges. These results demonstrated that E40K and E54K can directly decrease the isometric tension of the myocardium, leading to the systolic dysfunction. The decreased tension under pCa 7.0 may contribute to the altered filling pattern of the left ventricle in DCM patients. Kinetic analysis showed that both E40K and E54K changed the steady state cross-bridge kinetics, with E40K had more severe effect than E54K.
Our results demonstrated that E40K and E54K caused no change in Ca2+ sensitivity. This result is at variance with the generally accepted concept that DCM causing sarcomeric protein mutations lead to a decreased Ca2+ sensitivity in the affected myocardium. This inconsistency can be resolved in part by different activating solutions used in different laboratories. 8 mM phosphate (Pi) was used in our activating solutions, while other investigators did not use Pi in their solutions (Chang et al. 2005; Mirza et al. 2005, 2007; Rajan et al. 2007). The Pi concentration under physiological conditions in cardiomyoctes has been reported to be ~6 mM (Opie et al. 1971; Roth et al. 1989). Therefore, adding Pi in the activation solution better mimics the physiological condition in the heart, as described (Bai et al. 2012). The inconsistency can also be caused by different approaches used. Actin-Tm-myosin S1 complex used for the ATPase assay in solution may be too simple to account for the complex interactions which take place in cardiomyocytes. Isolated muscle fibers from Tg models may be a better system to study the effect of Tm mutations on cardiac performance. At the same time, the Tg models generally involves many secondary effects caused by the mutation, including altered protein expression levels (Rajan et al. 2007), and altered post-translational modifications (Sfichi-Duke et al. 2010). These secondary effects have been shown to significantly alter muscle regulation and performances (Jacques et al. 2008; Layland et al. 2005; Sadayappan et al. 2005), making it difficult to identify the primary effect of the mutation. In fact, Tardiff (2011) states in her recent review said that “the predicted result of focusing on the earliest stages of cardiac remodeling (before the activation of potentially complex signaling cascades) would be that the observed molecular and biophysical effects of thin filament mutations would provide a more robust mechanistic link between genotype and phenotype.” Using thin-filament reconstituted myocardium, we can assure that only Tm is replaced with mutants, and all the other components in the myocardium remain unchanged (Kawai and Ishiwata 2006). Therefore, direct and primary effects of the mutation on regulation and contraction can be studied without complications. Altogether, we conclude that the over inhibition of the AM interaction is the immediate and primary cause of DCM in E40K and E54K Tm, that causes systolic (and perhaps diastolic) problems. A decreased Ca2+ sensitivity in transgenic models may not be the major cause of DCM in these mutations, because this phenomenon was observed in the absence of Pi, which is unphysiological.
Tm mutation in left ventricular noncompaction (LVNC)
LVNC occurs with both DCM and HCM and is classified as the primary cardiomyopathy by The American Heart Association and The European Society of Cardiology. The major symptom of LVNC involves numerous and prominent ventricular trabeculations and deep intertrabecular recesses (Sarma et al. 2010; Chin et al. 1990; Oechslin et al. 2000). LVNC can be caused by genetic mutations of proteins including taffazin, β-dystrobrevin, lamin A/C, myosin, and cMyBP-C (Zaragoza et al. 2007). Recently, 3 mutations of Tm (E37K, E192K, and K248E) have been identified to cause LVNC in humans (Chang et al. 2011; Probst et al. 2011). All three mutants are located at the outer surface of Tm’s coiled-coil (E37K is at the b position of the heptad repeat, whereas E192K and K248E are located at the c position) and bear an opposite charge change. The functional consequence of these Tm mutants at the molecular level has not been worked out yet. It would be very interesting to investigate the molecular mechanism of how these Tm mutations lead to a specific phenotype, which is neither DCM nor HCM.
Tm mutations lead to DCM or HCM through different molecular mechanisms
The genotype-phenotype connection between a Tm mutation and familial cardiomyopathy (DCM or HCM) has been established by patient screening and Tg models. However, how these distinctively different phenotypes originate from mutations within the same protein or even the same region of the protein remains unknown. Mechanical stress caused by these mutations has been generally considered the key to the cardiac remodeling in HCM and DCM pathogenesis (Bernardo et al. 2010). Tm plays the key role in the mechanical performance of the heart, therefore, it is natural to assume different mutations in Tm leads to different mechanical changes in myocardium and finally lead to different disease phenotypes.
It has been suggested that mutations that increase the Ca2+ sensitivity lead to HCM, and mutations that decrease the Ca2+ sensitivity lead to DCM (Willott et al. 2010). In the case of Tm, both Tg mice models and in vitro studies seem to support this hypothesis as D175N and E180G increased Ca2+ sensitivity and E40K, E54K and D230N decreased Ca2+ sensitivity (Muthuchamy et al. 1999; Prabhakar et al. 2003; Rajan et al. 2007; Bing et al. 2000b; Mirza et al. 2007; Chang et al. 2005). However, studies using human DCM myocardium showed an increased Ca2+ sensitivity (Wolff et al. 1996; Lee et al. 2010). A recent Tg study provided solid counter evidence for this hypothesis: Tg mice bearing one TnT mutation, which caused significant increased Ca2+ sensitivity in skinned fibers, showed no change in phenotype or survival rate compared to the non-Tg controls (Gollapudi et al. 2012). Our study also showed that Tm mutants E40K (DCM), E54K (DCM), D175N (HCM) and cardiac TnT mutant ΔK210 (DCM) had no effect on Ca2+ sensitivity under the physiological condition (Bai et al. 2011, 2012, 2013). Furthermore, it has been demonstrated that Ca2+ sensitivity can be regulated by many other factors, such as the mutant protein expression level (McConnell et al. 1999), altered phosphorylation status of sarcomeric proteins (Sadayappan et al. 2005), cardiac remodeling (Wang et al. 1994), and even exercise (Wisloff et al. 2002). Although most disease-related sacomeric-protein mutations have been found to associate with Ca2+ sensitivity changes, it is still too early to assume that increasing or decreasing Ca2+ sensitivity will lead to a specific disease phenotype. A most recent study also proved that E54K didn’t change the Ca2+ sensitivity and D230N even increased the Ca2+ sensitivity (Memo et al. 2013). Therefore, we conclude that in disease related Tm mutations, Ca2+ sensitivity change may not be the key factor in the pathogenesis.
Both DCM and HCM Tm mutants changed the kinetic constants of elementary steps of the cross-bridge cycle. However, this effect largely depended on individual mutants, and not on a specific disease phenotype (Table 1 in Ref (Bai et al. 2012)). Among HCM mutants, V95A disturbed the cross-bridge kinetics by promoting ADP dissociation, weakening ATP binding, diminishing cross-bridge detachment, and accelerating Pi release (decreased K0, K1, K2, and K5); In D175N, K0 and K2 did not change; In E180G, K0 increased, but K2 did not change (Bai et al. 2011). Among DCM mutants, E40K changed all equilibrium constants (decrease in K0, K4, and K5; increase in K1 and K2); but E54K only promoted ATP binding to myosin (increased K1) (Bai et al. 2012). In the case of V95A, the severely disturbed cross-bridge kinetics coincided with severe disease symptoms and poor prognosis (Karibe et al. 2001). Our results demonstrate that the cross-bridge kinetics are affected differently among the same disease phenotype.
The thin-filament removal and reconstitution technique has been shown to be a powerful method to investigate the immediate and early effect of mutation on cardiac muscle contraction and regulation before the complex signaling cascades start. We found that, in Tm mutants, inadequate inhibition of the AM interaction by regulatory proteins during diastole causes HCM, and over-inhibition of the AM interaction during systole causes DCM. Ca2+ sensitivity may not be the primary cause of the pathogenesis, because this is affected by the amount of inorganic phosphate in the myocyte, ionic strength, and phosphorylation status of myofilament proteins. The sinusoidal analysis technique is a good method to characterize the cross-bridge kinetics and the elementary steps of the cross-bridge cycle. Both methods are valuable tools to investigate the molecular mechanisms of contraction.
This work was supported by grant from the NIH (HL70041) to MK. The content of this study is solely the responsibility of the authors, and does not necessarily represent the official view of the awarding organization.