Force-generating Cross-bridges Research Articles

Dissecting the mechanisms by which a pathogenic variant in cardiac TnT tail domain leads to disturbed myofilament performance.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and high rate of sudden cardiac arrest at juvenile age. cTnT-I79N is located in the cTnT N-terminal (TNT1) loop region and is well-known for its pathological and prognostic relevance. Recent structural study revealed that I79 is part of hydrophobic interface between TNT1 loop and actin which stabilizes the OFF state of the cardiac thin filament. Given the importance of understanding: i) the role of TNT1 loop region on the regulation of the cardiac thin filament, and ii) the underlying mechanisms of cTnT-I79N-linked pathogenesis; we sought to investigate the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased passive force and passive sinusoidal stiffness, which could be due in part to an increase in force-generating cross-bridges during diastole. Tg-I79N also displayed increased myofilament Ca2+-sensitivity, shorter myofilament lattice spacing, and slower cross-bridge kinetics. These findings can be attributed to the destabilization of cardiac thin filament's inhibitory property, and the increase in the overall number of cross-bridges during Ca2+ activation. Finally, we show that cTnT-I79N dysregulates SRX↔DRX equilibrium, which can be explained by the excessive binding of myosin heads to the dysregulated thin filament that causes the SRX↔DRX equilibrium to shift towards the ON state. Altogether, biochemical and functional measurements, and structural data points to the following mechanism: cTnT-I79N weakens the interaction of the TNT1 loop with the actin filament, which in turn destabilizes the OFF state of the cardiac thin filament.

Shortening the thick filament by partial deletion of titin's C-zone alters cardiac function by reducing the operating sarcomere length range

Titin's C-zone is an inextensible segment in titin, comprised of 11 super-repeats and located in the cMyBP-C-containing region of the thick filament. Previously we showed that deletion of titin's super-repeats C1 and C2 (TtnΔC1–2 model) results in shorter thick filaments and contractile dysfunction of the left ventricular (LV) chamber but that unexpectedly LV diastolic stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the LV and intact cardiomyocyte, cellular work loops of intact cardiomyocytes, Ca2+ transients, cross-bridge kinetics, and myofilament Ca2+ sensitivity. Intact cardiomyocytes of TtnΔC1–2 mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance at both LV and single-cell levels showed that activation kinetics are normal in TtnΔC1–2 mice, but that relaxation is slower. The slowed relaxation is, in part, attributable to an increased myofilament Ca2+ sensitivity and slower early Ca2+ reuptake. Cross-bridge dynamics showed that cross-bridge kinetics are normal but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements revealed that in TtnΔC1–2 mice the operating SL range of the LV is shifted towards shorter lengths. This normalizes the apparent cell and LV diastolic stiffness but further reduces systolic force as systole occurs further down on the ascending limb of the force-SL relation. We propose that the reduced working SLs reflect titin's role in regulating diastolic stiffness by altering the number of sarcomeres in series. Overall, our study reveals that thick filament length regulation by titin's C-zone is critical for normal cardiac function.

Abstract P315: Shortening The Thick Filament By Partial Deletion Of Titin’S C-zone Alters Cardiac Function By Reducing The Operating Sarcomere Length Range Of The Heart.

Titin’s C-zone is the inextensible part of titin that binds along the thick filament at its cMyBP-C -containing region. Previously it was shown that deletion of titin’s super-repeats C1 and C2 ( Ttn ΔC1-2 mouse model) results in shorter thick filaments and contractile dysfunction, but LV chamber stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the left ventricle (LV) and from cellular work loops of intact loaded cardiac myocytes. Ca 2+ transients were also measured as well as crossbridge cycling kinetics and Ca 2+ sensitivity of force. It was found that intact cardiomyocytes of Ttn ΔC1-2 mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance of the LV chamber showed that the kinetics of LV activation are normal but that relaxation is slower in Ttn ΔC1-2 mice. The slowed relaxation was, in part, attributable to an increased myofilament Ca 2+ sensitivity and slower early Ca 2+ reuptake. Dynamic stiffness at the myofilament level showed that cross-bridge kinetics are normal, but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements in the mid-wall region of the LV revealed that the operating SL range is shifted in Ttn ΔC1-2 mice towards shorter lengths. This normalizes the apparent cell and LV chamber stiffness but reduces the number of force generating cross-bridges due to suboptimal thin and thick filament overlap. Thus the contractile dysfunction in Ttn ΔC1-2 mice is not only due to shorter thick filaments but also to a reduced operating sarcomere length range. Overall these results reveal that for normal cardiac function, thick filament length regulation by titin’s C-zone is critical.

Omecamtiv mecarbil activates ryanodine receptors from canine cardiac but not skeletal muscle

Due to the limited results achieved in the clinical treatment of heart failure, a new inotropic strategy of myosin motor activation has been developed. The lead molecule of myosin activator agents is omecamtiv mecarbil, which binds directly to the heavy chain of the cardiac β-myosin and enhances cardiac contractility by lengthening the lifetime of the acto-myosin complex and increasing the number of the active force-generating cross-bridges. In the absence of relevant data, the effect of omecamtiv mecarbil on canine cardiac ryanodine receptors (RyR 2) has been investigated in the present study by measuring the electrical activity of single RyR 2 channels incorporated into planar lipid bilayer. When applying 100nM Ca2+ concentration on the cis side ([Ca2+]cis) omecamtiv mecarbil (1–10µM) significantly increased the open probability and opening frequency of RyR 2, while the mean closed time was reduced. Mean open time was increased moderately by 10µM omecamtiv mecarbil. When [Ca2+]cis was elevated to 322 and 735nM, the effect of omecamtiv mecarbil on open probability was evident only at higher (3–10µM) concentrations. All effects of omecamtiv mecarbil were fully reversible upon washout. Omecamtiv mecarbil (up to 10µM) had no effect on the open probability of RyR 1, isolated from either canine or rabbit skeletal muscles. It is concluded that the direct stimulatory action of omecamtiv mecarbil on RyR 2 has to be taken into account when discussing the mechanism of action or the potential side effects of the compound.

The myosin activator omecamtiv mecarbil: a promising new inotropic agent

Heart failure became a leading cause of mortality in the past few decades with a progressively increasing prevalence. Its current therapy is restricted largely to the suppression of the sympathetic activity and the renin-angiotensin system in combination with diuretics. This restrictive strategy is due to the potential long-term adverse effects of inotropic agents despite their effective influence on cardiac function when employed for short durations. Positive inotropes include inhibitors of the Na+/K+ pump, β-receptor agonists, and phosphodiesterase inhibitors. Theoretically, Ca2+ sensitizers may also increase cardiac contractility without resulting in Ca2+ overload; nevertheless, their mechanism of action is frequently complicated by other pleiotropic effects. Recently, a new positive inotropic agent, the myosin activator omecamtiv mecarbil, has been developed. Omecamtiv mecarbil binds directly to β-myosin heavy chain and enhances cardiac contractility by increasing the number of the active force-generating cross-bridges, presumably without major off-target effects. This review focuses on recent in vivo and in vitro results obtained with omecamtiv mecarbil, and discusses its mechanism of action at a molecular level. Based on clinical data, omecamtiv mecarbil is a promising new tool in the treatment of systolic heart failure.

Nebulin Deficiency in Adult Muscle Causes Sarcomeric Defects and Muscle-Type Dependent Changes in Trophicity -- Novel Insights in Nemaline Myopathy

Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin's functional roles in adult muscle we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscles but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survive to adulthood with low nebulin levels (<5% of control), contain nemaline rods, and undergo fiber-type switching towards oxidative types. Nebulin deficiency causes a large deficit in specific-force and mechanistic studies provide evidence that a reduced fraction of force-generating crossbridges and shortened thin filaments contribute to the force deficit. Muscles rich in glycolytic fibers upregulate proteolysis pathways (MuRF-1, Fbxo30/MUSA1, Gadd45a) and undergo hypotrophy with smaller cross-sectional areas, worsening their force deficit. Muscles rich in oxidative fibers do not have smaller weights and can even hypertrophy, offsetting their specific-force deficit. These studies reveal nebulin as critically important for force development and trophicity in adult muscle. The Neb cKO phenocopies important aspects of nemaline myopathy (muscle weakness, oxidative fiber type predominance, variable trophicity effects, nemaline rods) and will be highly useful to test therapeutic approaches to ameliorate muscle weakness.

Ca2+-Sensitivity and Elementary Steps of the Cross-Bridge Cycle in Papillary Muscle Fibers from the Troponin C (TnC)-A8V Knock-In Mouse, which Exhibits Hypertrophic Cardiomyopathy (HCM)

Mutations of TnC alter its function by two mechanisms: changing Ca2+-affinity, and/or changing the TnC-TnI interaction. A mutation, A8V, exhibits HCM and diastolic dysfunction in both mice and humans as demonstrated by echocardiography. Here we have used homozygous knock-in TnC mutant A8V mice (7-8 months-old) in which the myofilaments carry only mutant protein. These mice develop HCM at 3 months (Circ Cardiovasc Genetic 2015, in press). This mutation is located in the N-helix of TnC, a region known to affect Ca2+-affinity. Here we studied the effects of TnC-A8V mutation in small fiber bundles (90-110µm diameter) from skinned papillary muscles. These were activated at 25°C under the intracellular ionic conditions of working cardiomyocytes (5mM ATP, 1mM Mg2+, 8mM phosphate, 200mM ionic strength, and pH 7.0). With the pCa-tension study, Ca2+-sensitivity increased (3x) and cooperativity decreased (0.8x) compared to the wild-type. The maximum active tension decreased slightly (0.95x). The elementary steps of the cross-bridge cycle were investigated by small-amplitude sinusoidal analysis. With the ATP study, there was no significant change in the ATP affinity (K1) to the myosin head, but cross-bridge detachment rate (k2) and its equilibrium constant (K2) increased to 1.5x, which causes slightly fewer force-generating cross-bridges. With the phosphate study, no effect of the mutation on the rate constants of the force-generation step (k4, k-4) or Pi-release step (1/K5) was observed. When the rigor state was induced, there was no effect of the mutation on stiffness, implying that force/cross-bridge is not altered. We infer from these observations that TnC-A8V mutation recruits more cycling cross-bridges during systole as a result of increased myofilament Ca2+-sensitivity, but unable to relax adequately resulting in diastolic dysfunction, followed by HCM development.

Nebulin deficiency in adult muscle causes sarcomere defects and muscle-type-dependent changes in trophicity: novel insights in nemaline myopathy.

Nebulin is a giant filamentous protein that is coextensive with the actin filaments of the skeletal muscle sarcomere. Nebulin mutations are the main cause of nemaline myopathy (NEM), with typical adult patients having low expression of nebulin, yet the roles of nebulin in adult muscle remain poorly understood. To establish nebulin's functional roles in adult muscle, we studied a novel conditional nebulin KO (Neb cKO) mouse model in which nebulin deletion was driven by the muscle creatine kinase (MCK) promotor. Neb cKO mice are born with high nebulin levels in their skeletal muscles, but within weeks after birth nebulin expression rapidly falls to barely detectable levels Surprisingly, a large fraction of the mice survive to adulthood with low nebulin levels (<5% of control), contain nemaline rods and undergo fiber-type switching toward oxidative types. Nebulin deficiency causes a large deficit in specific force, and mechanistic studies provide evidence that a reduced fraction of force-generating cross-bridges and shortened thin filaments contribute to the force deficit. Muscles rich in glycolytic fibers upregulate proteolysis pathways (MuRF-1, Fbxo30/MUSA1, Gadd45a) and undergo hypotrophy with smaller cross-sectional areas (CSAs), worsening their force deficit. Muscles rich in oxidative fibers do not have smaller weights and can even have hypertrophy, offsetting their specific-force deficit. These studies reveal nebulin as critically important for force development and trophicity in adult muscle. The Neb cKO phenocopies important aspects of NEM (muscle weakness, oxidative fiber-type predominance, variable trophicity effects, nemaline rods) and will be highly useful to test therapeutic approaches to ameliorate muscle weakness.

High ionic strength depresses muscle contractility by decreasing both force per cross-bridge and the number of strongly attached cross-bridges.

An increase in ionic strength (IS) lowers Ca(2+) activated tension in muscle fibres, however, its molecular mechanism is not well understood. In this study, we used single rabbit psoas fibres to perform sinusoidal analyses. During Ca(2+) activation, the effects of ligands (ATP, Pi, and ADP) at IS ranging 150-300 mM were studied on three rate constants to characterize elementary steps of the cross-bridge cycle. The IS effects were studied because a change in IS modifies the inter- and intra-molecular interactions, hence they may shed light on the molecular mechanisms of force generation. Both the ATP binding affinity (K1) and the ADP binding affinity (K 0) increased to 2-3x, and the Pi binding affinity (K5) decreased to 1/2, when IS was raised from 150 to 300 mM. The effect on ATP/ADP can be explained by stereospecific and hydrophobic interaction, and the effect on Pi can be explained by the electrostatic interaction with myosin. The increase in IS increased cross-bridge detachment steps (k2 and k-4), indicating that electrostatic repulsion promotes these steps. However, IS did not affect attachment steps (k-2 and k4). Consequently, the equilibrium constant of the detachment step (K2) increased by ~100%, and the force generation step (K4) decreased by ~30%. These effects together diminished the number of force-generating cross-bridges by 11%. Force/cross-bridge (T56) decreased by 26%, which correlates well with a decrease in the Debye length that limits the ionic atmosphere where ionic interactions take place. We conclude that the major effect of IS is a decrease in force/cross-bridge, but a decrease in the number of force generating cross-bridge also takes place. The stiffness during rigor induction did not change with IS, demonstrating that in-series compliance is not much affected by IS.

Elevated Ionic Strength Diminishes Force/Cross-Bridge and the Number of Force-Generating Cross-Bridges

Ionic strength (IS) is an important parameter to govern the inter- and intra-molecular interactions. In muscle, it has been known that an increase in IS lowers Ca2+ activated tension, however, its molecular mechanism is not well understood. Our aim was to determine whether force/cross-bridge or the number of force-generating cross-bridges changes with IS. Stiffness during rigor was studied on single fibers from rabbit psoas, which showed that there was no effect of IS, demonstrating that in-series compliance is not affected by IS. This observation indicates that stiffness of thick filament, thin filament, myosin head, and actomyosin interface are not affected by IS. Sinusoidal analyses were performed during Ca2+ activation, and the effects of ATP, phosphate (Pi), and ADP on three rate constants were studied at IS ranging 150mM-300mM to characterize elementary steps of the cross-bridge cycle. Both ATP binding (K1) and ADP binding (K0) increased to 2x, and the Pi binding (K5) decreased to 1/2 when IS was increased from 150mM to 300mM. The effect of Pi can be explained by the electrostatic interaction with the Pi binding site on myosin. The effect on ATP/ADP can be attributed to improved stereoscopic and hydrophobic interaction with the nucleotide binding site. The increase in IS increased cross-bridge detachment steps (k2 and k(-4)), indicating that electrostatic force counteracts these steps. However, IS did not affect attachment steps (k(-2) and k4). Consequently, the equilibrium constant of the detachment step (K2) increased to 2x, and the force generation step (K4) decreased to 0.7x. These effects together diminished the number of force-generating cross-bridges by ∼10%. Because associated decrease of tension was ∼40%, the major effect of IS is a decrease in force/cross-bridge, but also a decrease in the number of force generating cross-bridge occurs.

Deletion of the titin N2B region accelerates myofibrillar force development but does not alter relaxation kinetics

Cardiac titin is the main determinant of sarcomere stiffness during diastolic relaxation. To explore whether titin stiffness affects the kinetics of cardiac myofibrillar contraction and relaxation, we used subcellular myofibrils from the left ventricles of homozygous and heterozygous N2B-knockout mice which express truncated cardiac titins lacking the unique elastic N2B region. Compared with myofibrils from wild-type mice, myofibrils from knockout and heterozygous mice exhibit increased passive myofibrillar stiffness. To determine the kinetics of Ca(2+)-induced force development (rate constant kACT), myofibrils from knockout, heterozygous and wild-type mice were stretched to the same sarcomere length (2.3 µm) and rapidly activated with Ca(2+). Additionally, mechanically induced force-redevelopment kinetics (rate constant kTR) were determined by slackening and re-stretching myofibrils during Ca(2+)-mediated activation. Myofibrils from knockout mice exhibited significantly higher kACT, kTR and maximum Ca(2+)-activated tension than myofibrils from wild-type mice. By contrast, the kinetic parameters of biphasic force relaxation induced by rapidly reducing [Ca(2+)] were not significantly different among the three genotypes. These results indicate that increased titin stiffness promotes myocardial contraction by accelerating the formation of force-generating cross-bridges without decelerating relaxation.

Titin-mediated control of cardiac myofibrillar function

According to the Frank–Starling relationship, ventricular pressure or stroke volume increases with end-diastolic volume. This is regulated, in large part, by the sarcomere length (SL) dependent changes in cardiac myofibrillar force, loaded shortening, and power. Consistent with this, both cardiac myofibrillar force and absolute power fall at shorter SL. However, when Ca2+ activated force levels are matched between short and long SL (by increasing the activator [Ca2+]), short SL actually yields faster loaded shortening and greater peak normalized power output (PNPO). A potential mechanism for faster loaded shortening at short SL is that, at short SL, titin becomes less taut, which increases the flexibility of the cross-bridges, a process that may be mediated by titin’s interactions with thick filament proteins. We propose a more slackened titin yields greater myosin head radial and azimuthal mobility and these flexible cross-bridges are more likely to maintain thin filament activation, which would allow more force-generating cross-bridges to work against a fixed load resulting in faster loaded shortening. We tested this idea by measuring SL-dependence of power at matched forces in rat skinned cardiac myocytes containing either N2B titin or a longer, more compliant N2BA titin. We predicted that, in N2BA titin containing cardiac myocytes, power-load curves would not be shifted upward at short SL compared to long SL (when force is matched). Consistent with this, peak normalized power was actually less at short SL versus long SL (at matched force) in N2BA-containing myocytes (N2BA titin: ΔPNPO (Short SL peak power minus long SL peak power)=−0.057±0.049 (n=5) versus N2B titin: ΔPNPO=+0.012±0.012 (n=5). These findings support a model whereby SL per se controls mechanical properties of cross-bridges and this process is mediated by titin. This myofibrillar mechanism may help sustain ventricular power during periods of low preloads, and perhaps a breakdown of this mechanism is involved in impaired function of failing hearts.

The troponin I: inhibitory peptide uncouples force generation and the cooperativity of contractile activation in mammalian skeletal muscle

Hodges and his colleagues identified a 12 amino acid fragment of troponin I (TnI-ip) that inhibits Ca(2+)-activated force and reduces the effectiveness Ca(2+) as an activator. To understand the role of troponin C (TnC) in the extended cooperative interactions of thin filament activation, we compared the effect of TnI-ip with that of partial troponin TnC extraction. Both methods reduce maximal Ca(2+)-activated force and increase [Ca(2+)] required for activation. In contrast to TnC extraction, TnI-ip does not reduce the extended cooperative interactions between adjacent thin filament regulatory units as assessed by the slope of the pCa/force relationship. Additional evidence that TnI-ip does not interfere with extended cooperativity comes from studies that activate muscle by rigor crossbridges (RXBs). TnI-ip increases both the cooperativity of activation and the concentration of RXBs needed for maximal force. This shows that TnI-ip binding to TnC increases the stability of the relaxed state of the thin filament. TnI-ip, therefore, uncouples force generation from extended cooperativity in both Ca(2+) and RXB activated muscle contraction. Because maximum force can be reduced with no change-or even an increase-in cooperativity, force-generating crossbridges do not appear to be the primary activators of cooperativity between thin filament regulatory units of skeletal muscle.

Titin Mediates Sarcomere Length Dependence of Myocyte Power

According to the Frank-Starling relationship, ventricular pressure increases with end-diastolic volume. This is controlled, in large part, by sarcomere length (SL) dependence of cardiac myofibrillar force, loaded shortening, and power. Consistent with this, both myofibrillar force and power fall at shorter SL, however, when Ca2+ activated force and presumably thin filament activation levels are matched between short and long SL (by increasing the activator [Ca2+]) short SL actually yields faster loaded shortening and greater peak normalized power output (PNPO). A potential mechanism for faster loaded shortening at short SL is that as SL decreases titin becomes less taut which reduces the impedance of cross-bridges, a process that may be mediated by titin's interactions with the thick filament. We propose a more slackened titin yields greater myosin head radial and azimuthal mobility and these flexible cross-bridges are more likely to maintain thin filament activation, which would allow more force-generating cross-bridges to work against a fixed load resulting in faster loaded shortening. We tested this idea by measuring SL dependence of power at matched forces in rat skinned cardiac myocytes containing either N2B titin or a longer, more compliant N2BA titin. We predicted less overshoot in power by short SL in N2BA titin myocytes. Consistent with this, peak power did not overshoot but was actually less at short versus long SL at matched thin filament activation levels in N2BA-containing myocytes (Wt: ΔPNPO = +0.0121 ± 0.0115 (n=5), N2BA titin: ΔPNPO = −0.0566 ± 0.0493 (n=5)). These findings are consistent with SL per se modulating the mechanical properties of cross-bridges with this modulation being mediated by titin. This myofibrillar mechanism may help sustain ventricular power during periods of low preloads, and perhaps a breakdown of this mechanism is involved in impaired function of failing hearts.

Kinetic Mechanism of Ca2+-controlled Changes of Skeletal Troponin I in Psoas Myofibrils

Conformational changes in the skeletal troponin complex (sTn) induced by rapidly increasing or decreasing the [Ca2+] were probed by 5-iodoacetamidofluorescein covalently bound to Cys-133 of skeletal troponin I (sTnI). Kinetics of conformational changes was determined for the isolated complex and after incorporating the complex into rabbit psoas myofibrils. Isolated and incorporated sTn exhibited biphasic Ca2+-activation kinetics. Whereas the fast phase (kobs∼1000 s−1) is only observed in this study, where kinetics were induced by Ca2+, the slower phase resembles the monophasic kinetics of sTnI switching observed in another study (Brenner and Chalovich. 1999. Biophys. J. 77:2692–2708) that investigated the sTnI switching induced by releasing the feedback of force-generating cross-bridges on thin filament activation. Therefore, the slower conformational change likely reflects the sTnI switch that regulates force development. Modeling reveals that the fast conformational change can occur after the first Ca2+ ion binds to skeletal troponin C (sTnC), whereas the slower change requires Ca2+ binding to both regulatory sites of sTnC. Incorporating sTn into myofibrils increased the off-rate and lowered the Ca2+ sensitivity of sTnI switching. Comparison of switch-off kinetics with myofibril force relaxation kinetics measured in a mechanical setup indicates that sTnI switching might limit the rate of fast skeletal muscle relaxation.

Enhanced Active Cross-Bridges during Diastole: Molecular Pathogenesis of Tropomyosin's HCM Mutations

Three HCM-causing tropomyosin (Tm) mutants (V95A, D175N, and E180G) were examined using the thin-filament extraction and reconstitution technique. The effects of Ca 2+, ATP, phosphate, and ADP concentrations on cross-bridge kinetics in myocardium reconstituted with each of these mutants were studied at 25°C, and compared to wild-type (WT) Tm at physiological ionic strength (200 mM). All three mutants showed significantly higher (2–3.5 fold) low Ca 2+ tension ( T LC ) and stiffness than WT at pCa 8.0. High Ca 2+ tension ( T HC ) was significantly higher for E180G than that for WT, whereas T HC of V95A and D175N was similar to WT; high Ca 2+ stiffness ( Y HC ) had the same trend. The Ca 2+ sensitivity of isometric force was significantly greater for V95A and E180G than for WT, whereas that of D175N remained the same as for WT; for all mutants, cooperativity was lower than for WT. Nine kinetic constants and the cross-bridge distribution were deduced using sinusoidal analysis. The number of force-generating cross bridges was similar among the D175N, E180G, and WT Tm forms, but it was significantly larger in the case of V95A than WT. We conclude that the increased number of actively cycling cross bridges at pCa 8 is the major cause of Tm mutation-related HCM pathogenesis, which may result in diastolic dysfunction. Decreased contractility ( T act ) in V95A and D175N may further contribute to the severity of myocyte hypertrophy and related prognosis of the disease.

The FHC Mutation D166V in the Cardiac Muscle Regulatory Light-Chain Causes Both a Diastolic and Systolic Dysfunction in Mouse Cardiac Muscle Performance

Previously we showed that the D166V (aspartic acid to valine) mutation in the myosin regulatory light-chain (RLC), implicated in familial hypertrophic cardiomyopathy, was associated with a decrease in the rate that force-generating cross-bridges dissociate (g) and an increased Ca2+ sensitivity of Ca2+ activated skinned fiber force and ATPase (Kerrick et al. FASEB J. 2009, 23:855-65). Here, we tested the hypothesis that the previous findings most likely manifested a diastolic dysfunction of the mutated myocardium. Transgenic intact papillary muscle fibers from D166V and WT mice were stimulated to contract at different frequencies, and force and intracellular Ca2+ were measured simultaneously. Results showed that as frequency of stimulation increased, the end-twitch force increased greatly in transgenic D166V papillary muscles compared to WT, demonstrating diastolic dysfunction. The end-twitch intracellular Ca2+ increased similarly with increasing frequency in both D166V and WT mice, as expected. However, it was found that the twitch force in D166V muscles initially increased and then decreased with increasing frequency of stimulation as did the amplitude of the intracellular Ca2+ transient, a possible explanation for why force amplitude decreased at higher frequencies. Another explanation for a decrease in force at high contraction frequencies is that RLC phosphorylation, activated by the increasing levels of diastolic Ca2+, may affect force differently in D166V (lowers force) than in WT muscles. In contrast to D166V, WT papillary muscle twitch force increased linearly over the same range of frequencies studied reaching a maximum force far exceeding that of D166V muscles. In summary the FHC D166V-RLC mutation of cardiac muscle would be expected to cause both a diastolic and systolic dysfunction in cardiac muscle performance. Supported by AHA 10GRNT4450042(WGK) and 10POST3420009(PM), and NIH-HL090786(DSC).

Kinetic Mechanism of Ca 2+-Induced Conformational Changes of Skeletal Muscle Troponin I in Rabbit Psoas Myofibrils

The kinetics of Ca2+-controlled conformational changes of the inhibitory subunit (sTnI) of the heterotrimeric skeletal troponin complex were determined by rapid stopped-flow at 10 °C. Conformational changes were probed by fluorescent labelling of sTnI at Cys134 located in between the second actin binding site of sTnI that interacts with actin-tropomyosin at low [Ca2+] and the switch peptide of sTnI that interacts with sTnC at high [Ca2+]. The kinetics was analysed for the sTn-complex in isolation and after its incorporation into rabbit psoas myofibrils. The rapid increase of [Ca2+] to pCa 4.5 induced biphasic fluorescence transients with similar rate constants of k+Ca1.phase ∼800 s−1 and k+Ca2.phase ∼100 s−1 in both preparations. Incorporation changed the polarity of the faster phase but not the polarity of the slower phase. Rapid reduction of [Ca2+] resulted in a monophasic fluorescence change whose rate constant k-Ca was 1.5 s−1 for isolated and 12 s−1 for incorporated sTn-complex. Thus, incorporation of the sTn-complex into the sarcomere increases the off-rate ∼8-fold while leaving the on-rate unaffected. The values of k+Ca2.phase and k-Ca determined in our myofibrils experiments are very similar to the values of kON and kOFF reported by Brenner & Chalovich (Biophys J. 1999 77:2692-708). They observed monophasic kinetics for sTnI labelled at Cys134 incorporated into skinned rabbit psoas fibers. In contrast to our experiments, they triggered sTnI-kinetics mechanically, i.e. by rapidly changing the number of force-generating cross-bridges. The faster conformational change in sTnI that is only observed in our Ca2+-triggered experiments is therefore likely associated with the Ca2+-binding process while the slower phase reports the conformational change of sTnI involved in force regulation. Supported by the Center of Molecular Medicine Cologne (CMMC-A6) and the DFG (SFB612-A2).

Reply from Vincenzo Lombardi

Reply from Vincenzo Lombardi

Twitchin of mollusc smooth muscles can induce “catch”-like properties in human skeletal muscle: support for the assumption that the “catch” state involves twitchin linkages between myofilaments

Molluscan catch muscles can maintain tension with low or even no energy utilization, and therefore, they represent ideal models for studying energy-saving holding states. For many decades it was assumed that catch is due to a simple slowing of the force-generating myosin head cross-bridge cycles. However, recently evidences increased suggesting that catch is rather caused by passive structures linking the myofilaments in a phosphorylation-dependent manner. One possible linkage structure is the titin-like thick filament protein twitchin, which could form bridges to the thin filaments. Twitchin is known to regulate the catch state depending on its phosphorylation state. Here, we found that twitchin induces a catch-like stiffness in skinned human skeletal muscle fibres, when these fibres are exposed to this protein. Subsequent phosphorylation of twitchin reduces the stiffness. These findings support the assumption that catch of molluscan smooth muscle involves twitchin linkages between thick and thin filaments.