Cooperative mechanisms underlie differences in myocardial contractile dynamics between large and small mammals.

Ca2+ binding to troponin C (TnC) and myosin cross-bridge binding to actin act in a synergistic cooperative manner to modulate myocardial contraction and relaxation. The responsiveness of the myocardial thin filament to the activating effects of Ca2+ and myosin cross-bridge binding has been well-characterized in small mammals (e.g., mice). Given the nearly 10-fold difference in resting heart rates and twitch kinetics between small and large mammals, it is unlikely that the cooperative mechanisms underlying thin filament activation are identical in these two species. To test this idea, we measured the Ca2+ dependencies of steady-state force and the rate constant of force redevelopment (ktr) in murine and porcine permeabilized ventricular myocardium. While murine myocardium exhibited a steep activation-dependence of ktr, the activation-dependent profile of ktr was significantly reduced in porcine ventricular myocardium. Further insight was attained by examining force-pCa and ktr-pCa relationships. In the murine myocardium, the pCa50 for ktr was right-shifted compared with the pCa50 for force, meaning that increases in steady-state force occurred well before increases in the rate of force redevelopment were observed. In the porcine myocardium, we observed a tighter coupling of the force-pCa and ktr-pCa relationships, as evidenced by near-maximal rates of force redevelopment at low levels of Ca2+ activation. These results demonstrate that the molecular mechanisms underlying the cooperative activation of force are a dynamic property of the mammalian heart, involving, at least in part, the species- and tissue-specific expression of cardiac myosin heavy chain isoforms.

RLC phosphorylation amplifies Ca2+ sensitivity of force in myocardium from cMyBP-C knockout mice.

Hypertrophic cardiomyopathy (HCM) is the leading genetic cause of heart disease. The heart comprises several proteins that work together to properly facilitate force production and pump blood throughout the body. Cardiac myosin binding protein-C (cMyBP-C) is a thick-filament protein, and mutations in cMyBP-C are frequently linked with clinical cases of HCM. Within the sarcomere, the N-terminus of cMyBP-C likely interacts with the myosin regulatory light chain (RLC); RLC is a subunit of myosin located within the myosin neck region that modulates contractile dynamics via its phosphorylation state. Phosphorylation of RLC is thought to influence myosin head position along the thick-filament backbone, making it more favorable to bind the thin filament of actin and facilitate force production. However, little is known about how these two proteins interact. We tested the effects of RLC phosphorylation on Ca2+-regulated contractility using biomechanical assays on skinned papillary muscle strips isolated from cMyBP-C KO mice and WT mice. RLC phosphorylation increased Ca2+ sensitivity of contraction (i.e., pCa50) from 5.80 ± 0.02 to 5.95 ± 0.03 in WT strips, whereas RLC phosphorylation increased Ca2+ sensitivity of contraction from 5.86 ± 0.02 to 6.15 ± 0.03 in cMyBP-C KO strips. These data suggest that the effects of RLC phosphorylation on Ca2+ sensitivity of contraction are amplified when cMyBP-C is absent from the sarcomere. This implies that cMyBP-C and RLC act in concert to regulate contractility in healthy hearts, and mutations to these proteins that lead to HCM (or a loss of phosphorylation with disease progression) may disrupt important interactions between these thick-filament regulatory proteins.

Cardiac MyBP-C phosphorylation regulates the Frank-Starling relationship in murine hearts.

The Frank-Starling relationship establishes that elevated end-diastolic volume progressively increases ventricular pressure and stroke volume in healthy hearts. The relationship is modulated by a number of physiological inputs and is often depressed in human heart failure. Emerging evidence suggests that cardiac myosin-binding protein-C (cMyBP-C) contributes to the Frank-Starling relationship. We measured contractile properties at multiple levels of structural organization to determine the role of cMyBP-C and its phosphorylation in regulating (1) the sarcomere length dependence of power in cardiac myofilaments and (2) the Frank-Starling relationship in vivo. We compared transgenic mice expressing wild-type cMyBP-C on the null background, which have ∼50% phosphorylated cMyBP-C (Controls), to transgenic mice lacking cMyBP-C (KO) and to mice expressing cMyBP-C that have serine-273, -282, and -302 mutated to aspartate (cMyBP-C t3SD) or alanine (cMyBP-C t3SA) on the null background to mimic either constitutive PKA phosphorylation or nonphosphorylated cMyBP-C, respectively. We observed a continuum of length dependence of power output in myocyte preparations. Sarcomere length dependence of power progressively increased with a rank ordering of cMyBP-C KO = cMyBP-C t3SA < Control < cMyBP-C t3SD. Length dependence of myofilament power translated, at least in part, to hearts, whereby Frank-Starling relationships were steepest in cMyBP-C t3SD mice. The results support the hypothesis that cMyBP-C and its phosphorylation state tune sarcomere length dependence of myofibrillar power, and these regulatory processes translate across spatial levels of myocardial organization to control beat-to-beat ventricular performance.

cMyBP-C phosphorylation modulates the time-dependent slowing of unloaded shortening in murine skinned myocardium.

In myocardium, phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) is thought to modulate the cooperative activation of the thin filament by binding to myosin and/or actin, thereby regulating the probability of cross-bridge binding to actin. At low levels of Ca2+ activation, unloaded shortening velocity (Vo) in permeabilized cardiac muscle is comprised of an initial high-velocity phase and a subsequent low-velocity phase. The velocities in these phases scale with the level of activation, culminating in a single high-velocity phase (Vmax) at saturating Ca2+. To test the idea that cMyBP-C phosphorylation contributes to the activation dependence of Vo, we measured Vo before and following treatment with protein kinase A (PKA) in skinned trabecula isolated from mice expressing either wild-type cMyBP-C (tWT), nonphosphorylatable cMyBP-C (t3SA), or phosphomimetic cMyBP-C (t3SD). During maximal Ca2+ activation, Vmax was monophasic and not significantly different between the three groups. Although biphasic shortening was observed in all three groups at half-maximal activation under control conditions, the high- and low-velocity phases were faster in the t3SD myocardium compared with values obtained in either tWT or t3SA myocardium. Treatment with PKA significantly accelerated both the high- and low-velocity phases in tWT myocardium but had no effect on Vo in either the t3SD or t3SA myocardium. These results can be explained in terms of a model in which the level of cMyBP-C phosphorylation modulates the extent and rate of cooperative spread of myosin binding to actin.

Deletion of Enigma Homologue from the Z-disc slows tension development kinetics in mouse myocardium.

Enigma Homologue (ENH) is a component of the Z-disc, a structure that anchors actin filaments in the contractile unit of muscle, the sarcomere. Cardiac-specific ablation of ENH protein expression causes contractile dysfunction that ultimately culminates in dilated cardiomyopathy. However, whether ENH is involved in the regulation of myocardial contractility is unknown. To determine if ENH is required for the mechanical activity of cardiac muscle, we analyze muscle mechanics of isolated trabeculae from the hearts of ENH +/+ and ENH -/- mice. We detected no differences in steady-state mechanical properties but show that when muscle fibers are allowed to relax and then are restretched, the rate at which tension redevelops is depressed in ENH -/- mouse myocardium relative to that in ENH +/+ myocardium. SDS-PAGE analysis demonstrated that the expression of ß-myosin heavy chain is increased in ENH -/- mouse myocardium, which could partially, but not completely, account for the depression in tension redevelopment kinetics. Using top-down proteomics analysis, we found that the expression of other thin/thick filament regulatory proteins is unaltered, although the phosphorylation of a cardiac troponin T isoform, cardiac troponin I, and myosin regulatory light chain is decreased in ENH -/- mouse myocardium. Nevertheless, these alterations are very small and thus insufficient to explain slowed tension redevelopment kinetics in ENH -/- mouse myocardium. These data suggest that the ENH protein influences tension redevelopment kinetics in mouse myocardium, possibly by affecting cross-bridge cycling kinetics. Previous studies also indicate that ablation of specific Z-disc proteins in myocardium slows contraction kinetics, which could also be a contributing factor in this study.

Phosphorylation of cardiac Myosin-binding protein-C is a critical mediator of diastolic function.

BACKGROUND: Heart failure (HF) with preserved ejection fraction (HFpEF) accounts for ≈50% of all cases of HF and currently has no effective treatment. Diastolic dysfunction underlies HFpEF; therefore, elucidation of the mechanisms that mediate relaxation can provide new potential targets for treatment. Cardiac myosin-binding protein-C (cMyBP-C) is a thick filament protein that modulates cross-bridge cycling rates via alterations in its phosphorylation status. Thus, we hypothesize that phosphorylated cMyBP-C accelerates the rate of cross-bridge detachment, thereby enhancing relaxation to mediate diastolic function. METHODS AND RESULTS: We compared mouse models expressing phosphorylation-deficient cMyBP-C(S273A/S282A/S302A)-cMyBP-C(t3SA), phosphomimetic cMyBP-C(S273D/S282D/S302D)-cMyBP-C(t3SD), and wild-type-control cMyBP-C(tWT) to elucidate the functional effects of cMyBP-C phosphorylation. Decreased voluntary running distances, increased lung/body weight ratios, and increased brain natriuretic peptide levels in cMyBP-C(t3SA) mice demonstrate that phosphorylation deficiency is associated with signs of HF. Echocardiography (ejection fraction and myocardial relaxation velocity) and pressure/volume measurements (-dP/dtmin, pressure decay time constant τ-Glantz, and passive filling stiffness) show that cMyBP-C phosphorylation enhances myocardial relaxation in cMyBP-C(t3SD) mice, whereas deficient cMyBP-C phosphorylation causes diastolic dysfunction with HFpEF in cMyBP-C(t3SA) mice. Simultaneous force and [Ca(2+)]i measurements on intact papillary muscles show that enhancement of relaxation in cMyBP-C(t3SD) mice and impairment of relaxation in cMyBP-C(t3SA) mice are not because of altered [Ca(2+)]i handling, implicating that altered cross-bridge detachment rates mediate these changes in relaxation rates. CONCLUSIONS: cMyBP-C phosphorylation enhances relaxation, whereas deficient phosphorylation causes diastolic dysfunction and phenotypes resembling HFpEF. Thus, cMyBP-C is a potential target for treatment of HFpEF.

Cardiac MyBP-C regulates the rate and force of contraction in mammalian myocardium.

Cardiac myosin-binding protein-C (cMyBP-C) is a thick filament-associated protein that seems to contribute to the regulation of cardiac contraction through interactions with either myosin or actin or both. Several studies over the past several years have suggested that the interactions of cardiac myosin-binding protein-C with its binding partners vary with its phosphorylation state, binding predominantly to myosin when dephosphorylated and to actin when it is phosphorylated by protein kinase A or other kinases. Here, we summarize evidence suggesting that phosphorylation of cardiac myosin binding protein-C is a key regulator of the kinetics and amplitude of cardiac contraction during ß-adrenergic stimulation and increased stimulus frequency. We propose a model for these effects via a phosphorylation-dependent regulation of the kinetics and extent of cooperative recruitment of cross bridges to the thin filament: phosphorylation of cardiac myosin binding protein-C accelerates cross bridge binding to actin, thereby accelerating recruitment and increasing the amplitude of the cardiac twitch. In contrast, enhanced lusitropy as a result of phosphorylation seems to be caused by a direct effect of phosphorylation to accelerate cross-bridge detachment rate. Depression or elimination of one or both of these processes in a disease, such as end-stage heart failure, seems to contribute to the systolic and diastolic dysfunction that characterizes the disease.

Myosin binding protein-C phosphorylation is the principal mediator of protein kinase A effects on thick filament structure in myocardium.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a regulator of pump function in healthy hearts. However, the mechanisms of regulation by cAMP-dependent protein kinase (PKA)-mediated cMyBP-C phosphorylation have not been completely dissociated from other myofilament substrates for PKA, especially cardiac troponin I (cTnI). We have used synchrotron X-ray diffraction in skinned trabeculae to elucidate the roles of cMyBP-C and cTnI phosphorylation in myocardial inotropy and lusitropy. Myocardium in this study was isolated from four transgenic mouse lines in which the phosphorylation state of either cMyBP-C or cTnI was constitutively altered by site-specific mutagenesis. Analysis of peak intensities in X-ray diffraction patterns from trabeculae showed that cross-bridges are displaced similarly from the thick filament and toward actin (1) when both cMyBP-C and cTnI are phosphorylated, (2) when only cMyBP-C is phosphorylated, and (3) when cMyBP-C phosphorylation is mimicked by replacement with negative charge in its PKA sites. These findings suggest that phosphorylation of cMyBP-C relieves a constraint on cross-bridges, thereby increasing the proximity of myosin to binding sites on actin. Measurements of Ca(2+)-activated force in myocardium defined distinct molecular effects due to phosphorylation of cMyBP-C or co-phosphorylation with cTnI. Echocardiography revealed that mimicking the charge of cMyBP-C phosphorylation protects hearts from hypertrophy and systolic dysfunction that develops with constitutive dephosphorylation or genetic ablation, underscoring the importance of cMyBP-C phosphorylation for proper pump function.

Dissociation of structural and functional phenotypes in cardiac myosin-binding protein C conditional knockout mice.

BACKGROUND: Cardiac myosin-binding protein C (cMyBP-C) is a sarcomeric protein that dynamically regulates thick-filament structure and function. In constitutive cMyBP-C knockout (cMyBP-C(-/-)) mice, loss of cMyBP-C has been linked to left ventricular dilation, cardiac hypertrophy, and systolic and diastolic dysfunction, although the pathogenesis of these phenotypes remains unclear. METHODS AND RESULTS: We generated cMyBP-C conditional knockout (cMyBP-C-cKO) mice expressing floxed cMyBP-C alleles and a tamoxifen-inducible Cre-recombinase fused to 2 mutated estrogen receptors to study the onset and progression of structural and functional phenotypes caused by the loss of cMyBP-C. In adult cMyBP-C-cKO mice, knockdown of cMyBP-C over a 2-month period resulted in a corresponding impairment of diastolic function and a concomitant abbreviation of systolic ejection, although contractile function was largely preserved. No significant changes in cardiac structure or morphology were immediately evident; however, mild hypertrophy developed after near-complete knockdown of cMyBP-C. In response to pressure overload induced by transaortic constriction, cMyBP-C-cKO mice treated with tamoxifen also developed greater cardiac hypertrophy, left ventricular dilation, and reduced contractile function. CONCLUSIONS: These results indicate that myocardial dysfunction is largely caused by the removal of cMyBP-C and occurs before the onset of cytoarchitectural remodeling in tamoxifen-treated cMyBP-C-cKO myocardium. Moreover, near ablation of cMyBP-C in adult myocardium primarily leads to the development of hypertrophic cardiomyopathy in contrast to the dilated phenotype evident in cMyBP-C(-/-) mice, which highlights the importance of additional factors such as loading stress in determining the expression and progression of cMyBP-C-associated cardiomyopathy.

Transgenic overexpression of γ-cytoplasmic actin protects against eccentric contraction-induced force loss in mdx mice.

BACKGROUND: γ-cytoplasmic (γ-cyto) actin levels are elevated in dystrophin-deficient mdx mouse skeletal muscle. The purpose of this study was to determine whether further elevation of γ-cyto actin levels improve or exacerbate the dystrophic phenotype of mdx mice. METHODS: We transgenically overexpressed γ-cyto actin, specifically in skeletal muscle of mdx mice (mdx-TG), and compared skeletal muscle pathology and force-generating capacity between mdx and mdx-TG mice at different ages. We investigated the mechanism by which γ-cyto actin provides protection from force loss by studying the role of calcium channels and stretch-activated channels in isolated skeletal muscles and muscle fibers. Analysis of variance or independent t-tests were used to detect statistical differences between groups. RESULTS: Levels of γ-cyto actin in mdx-TG skeletal muscle were elevated 200-fold compared to mdx skeletal muscle and incorporated into thin filaments. Overexpression of γ-cyto actin had little effect on most parameters of mdx muscle pathology. However, γ-cyto actin provided statistically significant protection against force loss during eccentric contractions. Store-operated calcium entry across the sarcolemma did not differ between mdx fibers compared to wild-type fibers. Additionally, the omission of extracellular calcium or the addition of streptomycin to block stretch-activated channels did not improve the force-generating capacity of isolated extensor digitorum longus muscles from mdx mice during eccentric contractions. CONCLUSIONS: The data presented in this study indicate that upregulation of γ-cyto actin in dystrophic skeletal muscle can attenuate force loss during eccentric contractions and that the mechanism is independent of activation of stretch-activated channels and the accumulation of extracellular calcium.

Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development.

Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein-C (cMyBP-C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca(2+) sensitivity of force (pCa(50)), PKA treatment has been shown to decrease pCa(50), presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca(2+)-independent force and maximum Ca(2+)-activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase-specific differential effects on steady-state force, we used synchrotron low-angle X-ray diffraction to compare equatorial intensity ratios (I(1,1)/I(1,0)) to assess the proximity of myosin cross-bridge mass relative to actin and to compare lattice spacings (d(1,0)) to assess the inter-thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP-C increases I(1,1)/I(1,0) and, as hypothesized, treatment with MLCK also increased I(1,1)/I(1,0), which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by 2 nm (3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP-C phosphorylation increases the proximity of cross-bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP-C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca(2+) sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

Context-dependent functional substitution of alpha-skeletal actin by gamma-cytoplasmic actin.

We generated transgenic mice that overexpressed gamma-(cyto) actin 2000-fold above wild-type levels in skeletal muscle. gamma-(cyto) actin comprised 40% of total actin in transgenic skeletal muscle, with a concomitant 40% decrease in alpha-actin. Surprisingly, transgenic muscle was histologically and ultrastructurally identical to wild-type muscle despite near-stoichiometric incorporation of gamma-(cyto) actin into sarcomeric thin filaments. Furthermore, several parameters of muscle physiological performance in the transgenic animals were not different from wild type. Given these surprising results, we tested whether overexpression of gamma-(cyto) actin could rescue the early postnatal lethality in alpha-(sk) actin-null mice (Acta1(-/-)). By quantitative Western blot analysis, we found total actin levels were decreased by 35% in Acta1(-/-) muscle. Although transgenic overexpression of gamma-(cyto) actin on the Acta1(-/-) background restored total actin levels to wild type, resulting in thin filaments composed of 60% gamma-(cyto) actin and a 40% mixture of cardiac and vascular actin, the life span of transgenic Acta1(-/-) mice was not extended. These results indicate that sarcomeric thin filaments can accommodate substantial incorporation of gamma-(cyto) actin without functional consequences, yet gamma-(cyto) actin cannot fully substitute for alpha-(sk) actin.

Overexpression of TEAD-1 in transgenic mouse striated muscles produces a slower skeletal muscle contractile phenotype.

TEA domain (TEAD) transcription factors serve important functional roles during embryonic development and in striated muscle gene expression. Our previous work has implicated a role for TEAD-1 in the fast-to-slow fiber-type transition in response to mechanical overload. To investigate whether TEAD-1 is a modulator of slow muscle gene expression in vivo, we developed transgenic mice expressing hemagglutinin (HA)-tagged TEAD-1 under the control of the muscle creatine kinase promoter. We show that striated muscle-restricted HA-TEAD-1 expression induced a transition toward a slow muscle contractile protein phenotype, slower shortening velocity (Vmax), and longer contraction and relaxation times in adult fast twitch extensor digitalis longus muscle. Notably, HA-TEAD-1 overexpression resulted in an unexpected activation of GSK-3alpha/beta and decreased nuclear beta-catenin and NFATc1/c3 protein. These effects could be reversed in vivo by mechanical overload, which decreased muscle creatine kinase-driven TEAD-1 transgene expression, and in cultured satellite cells by TEAD-1-specific small interfering RNA. These novel in vivo data support a role for TEAD-1 in modulating slow muscle gene expression.

Destabilization of the dystrophin-glycoprotein complex without functional deficits in alpha-dystrobrevin null muscle.

Alpha-dystrobrevin is a component of the dystrophin-glycoprotein complex (DGC) and is thought to have both structural and signaling roles in skeletal muscle. Mice deficient for alpha-dystrobrevin (adbn(-/-)) exhibit extensive myofiber degeneration and neuromuscular junction abnormalities. However, the biochemical stability of the DGC and the functional performance of adbn(-/-) muscle have not been characterized. Here we show that the biochemical association between dystrophin and beta-dystroglycan is compromised in adbn(-/-) skeletal muscle, suggesting that alpha-dystrobrevin plays a structural role in stabilizing the DGC. However, despite muscle cell death and DGC destabilization, costamere organization and physiological performance is normal in adbn(-/-) skeletal muscle. Our results demonstrate that myofiber degeneration alone does not cause functional deficits and suggests that more complex pathological factors contribute to the development of muscle weakness in muscular dystrophy.

Protein kinase A-mediated phosphorylation of cMyBP-C increases proximity of myosin heads to actin in resting myocardium.

Protein kinase A-mediated (PKA) phosphorylation of cardiac myosin binding protein C (cMyBP-C) accelerates the kinetics of cross-bridge cycling and may relieve the tether-like constraint of myosin heads imposed by cMyBP-C. We favor a mechanism in which cMyBP-C modulates cross-bridge cycling kinetics by regulating the proximity and interaction of myosin and actin. To test this idea, we used synchrotron low-angle x-ray diffraction to measure interthick filament lattice spacing and the equatorial intensity ratio, I(11)/I(10), in skinned trabeculae isolated from wild-type and cMyBP-C null (cMyBP-C(-/-)) mice. In wild-type myocardium, PKA treatment appeared to result in radial or azimuthal displacement of cross-bridges away from the thick filaments as indicated by an increase (approximately 50%) in I(11)/I(10) (0.22+/-0.03 versus 0.33+/-0.03). Conversely, PKA treatment did not affect cross-bridge disposition in mice lacking cMyBP-C, because there was no difference in I(11)/I(10) between untreated and PKA-treated cMyBP-C(-/-) myocardium (0.40+/-0.06 versus 0.42+/-0.05). Although lattice spacing did not change after treatment in wild-type (45.68+/-0.84 nm versus 45.64+/-0.64 nm), treatment of cMyBP-C(-/-) myocardium increased lattice spacing (46.80+/-0.92 nm versus 49.61+/-0.59 nm). This result is consistent with the idea that the myofilament lattice expands after PKA phosphorylation of cardiac troponin I, and when present, cMyBP-C, may stabilize the lattice. These data support our hypothesis that tethering of cross-bridges by cMyBP-C is relieved by phosphorylation of PKA sites in cMyBP-C, thereby increasing the proximity of cross-bridges to actin and increasing the probability of interaction with actin on contraction.

Mutation that dramatically alters rat titin isoform expression and cardiomyocyte passive tension.

Titin is a very large alternatively spliced protein that performs multiple functions in heart and skeletal muscles. A rat strain is described with an autosomal dominant mutation that alters the isoform expression of titin. While wild type animals go through a developmental program where the 3.0 MDa N2B becomes the major isoform expressed by two to three weeks after birth (approximately 85%), the appearance of the N2B is markedly delayed in heterozygotes and never reaches more than 50% of the titin in the adult. Homozygote mutants express a giant titin of the N2BA isoform type (3.9 MDa) that persists as the primary titin species through ages of more than one and a half years. The mutation does not affect the isoform switching of troponin T, a protein that is also alternatively spliced with developmental changes. The basis for the apparently greater size of the giant titin in homozygous mutants was not determined, but the additional length was not due to inclusion of sequence from larger numbers of PEVK exons or the Novex III exon. Passive tension measurements using isolated cardiomyocytes from homozygous mutants showed that cells could be stretched to sarcomere lengths greater than 4 mum without breakage. This novel rat model should be useful for exploring the potential role of titin in the Frank-Starling relationship and mechano-sensing/signaling mechanisms.

Radial displacement of myosin cross-bridges in mouse myocardium due to ablation of myosin binding protein-C.

Myosin binding protein-C (cMyBP-C) is a thick filament accessory protein, which in cardiac muscle functions to regulate the kinetics of cross-bridge interaction with actin; however, the underlying mechanism is not yet understood. To explore the structural basis for cMyBP-C function, we used synchrotron low-angle X-ray diffraction to measure interfilament lattice spacing and the equatorial intensity ratio, I(11)/I(10), in skinned myocardial preparations isolated from wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)). In relaxed myocardium, ablation of cMyBP-C appeared to result in radial displacement of cross-bridges away from the thick filaments, as there was a significant increase ( approximately 30%) in the I(11)/I(10) ratio for cMyBP-C(-/-) (0.37+/-0.03) myocardium as compared to WT (0.28+/-0.01). While lattice spacing tended to be greater in cMyBP-C(-/-) myocardium (44.18+/-0.68 nm) when compared to WT (42.95+/-0.43 nm), the difference was not statistically significant. Furthermore, liquid-like disorder in the myofilament lattice was significantly greater ( approximately 40% greater) in cMyBP-C(-/-) myocardium as compared to WT. These results are consistent with our working hypothesis that cMyBP-C normally acts to tether myosin cross-bridges nearer to the thick filament backbone, thereby reducing the likelihood of cross-bridge binding to actin and limiting cooperative activation of the thin filament.