Myosin Binding protein-C Research Articles

Thick Filament Protein Network, Functions, and Disease Association.

Sarcomeres consist of highly ordered arrays of thick myosin and thin actin filaments along with accessory proteins. Thick filaments occupy the center of sarcomeres where they partially overlap with thin filaments. The sliding of thick filaments past thin filaments is a highly regulated process that occurs in an ATP-dependent manner driving muscle contraction. In addition to myosin that makes up the backbone of the thick filament, four other proteins which are intimately bound to the thick filament, myosin binding protein-C, titin, myomesin, and obscurin play important structural and regulatory roles. Consistent with this, mutations in the respective genes have been associated with idiopathic and congenital forms of skeletal and cardiac myopathies. In this review, we aim to summarize our current knowledge on the molecular structure, subcellular localization, interacting partners, function, modulation via posttranslational modifications, and disease involvement of these five major proteins that comprise the thick filament of striated muscle cells. © 2018 American Physiological Society. Compr Physiol 8:631-709, 2018.

Contractile responses to endothelin-1 are regulated by PKC phosphorylation of cardiac myosin binding protein-C in rat ventricular myocytes

The shortening of sarcomeres that co-ordinates the pump function of the heart is stimulated by electrically-mediated increases in [Ca2+]. This process of excitation-contraction coupling (ECC) is subject to modulation by neurohormonal mediators that tune the output of the heart to meet the needs of the organism. Endothelin-1 (ET-1) is a potent modulator of cardiac function with effects on contraction amplitude, chronotropy and automaticity. The actions of ET-1 are evident during normal adaptive physiological responses and increased under pathophysiological conditions, such as following myocardial infarction and during heart failure, where ET-1 levels are elevated. In myocytes, ET-1 acts through ETA- or ETB-G protein-coupled receptors (GPCRs). Although well studied in atrial myocytes, the influence and mechanisms of action of ET-1 upon ECC in ventricular myocytes are not fully resolved. We show in rat ventricular myocytes that ET-1 elicits a biphasic effect on fractional shortening (initial transient negative and sustained positive inotropy) and increases the peak amplitude of systolic Ca2+ transients in adult rat ventricular myocytes. The negative inotropic phase was ETB receptor-dependent, whereas the positive inotropic response and increase in peak amplitude of systolic Ca2+ transients required ETA receptor engagement. Both effects of ET-1 required phospholipase C (PLC)-activity, although distinct signalling pathways downstream of PLC elicited the effects of each ET receptor. The negative inotropic response involved inositol 1,4,5-trisphosphate (InsP3) signalling and protein kinase C epsilon (PKCε). The positive inotropic action and the enhancement in Ca2+ transient amplitude induced by ET-1 were independent of InsP3 signalling, but suppressed by PKCε. Serine 302 in cardiac myosin binding protein-C was identified as a PKCε substrate that when phosphorylated contributed to the suppression of contraction and Ca2+ transients by PKCε following ET-1 stimulation. Thus, our data provide a new role and mechanism of action for InsP3 and PKCε in mediating the negative inotropic response and in restraining the positive inotropy and enhancement in Ca2+ transients following ET-1 stimulation.

Skeletal myosin binding protein-C isoforms regulate thin filament activity in a Ca2+-dependent manner

Muscle contraction, which is initiated by Ca2+, results in precise sliding of myosin-based thick and actin-based thin filament contractile proteins. The interactions between myosin and actin are finely tuned by three isoforms of myosin binding protein-C (MyBP-C): slow-skeletal, fast-skeletal, and cardiac (ssMyBP-C, fsMyBP-C and cMyBP-C, respectively), each with distinct N-terminal regulatory regions. The skeletal MyBP-C isoforms are conditionally coexpressed in cardiac muscle, but little is known about their function. Therefore, to characterize the functional differences and regulatory mechanisms among these three isoforms, we expressed recombinant N-terminal fragments and examined their effect on contractile properties in biophysical assays. Addition of the fragments to in vitro motility assays demonstrated that ssMyBP-C and cMyBP-C activate thin filament sliding at low Ca2+. Corresponding 3D electron microscopy reconstructions of native thin filaments suggest that graded shifts of tropomyosin on actin are responsible for this activation (cardiac > slow-skeletal > fast-skeletal). Conversely, at higher Ca2+, addition of fsMyBP-C and cMyBP-C fragments reduced sliding velocities in the in vitro motility assays and increased force production in cardiac muscle fibers. We conclude that due to the high frequency of Ca2+ cycling in cardiac muscle, cardiac MyBP-C may play dual roles at both low and high Ca2+. However, skeletal MyBP-C isoforms may be tuned to meet the needs of specific skeletal muscles.

MYBP-C Phosphorylation Accelerates Sarcomere Shortening as Thin Filaments Slide through the C-Zone

Myosin Binding Protein-C (MyBP-C) is a 140-150 kDa thick filament protein located in the middle third of each sarcomere, i.e., the C-zone, and is likely a key regulator of contraction. Phosphorylation of MyBP-C occurs at 4 residues within its N-terminus and PKA phosphorylation of MyBP-C in hearts is associated with faster cardiac muscle twitch dynamics (Tong et. al. Circ. Res. 2008). Here we investigated potential mechanisms by which MyBP-C phosphorylation regulates myofilament function. We utilized slow-twitch skeletal muscle fibers since PKA phosphorylates slow skeletal (ss)MyBP-C but not (ss)troponin I. Permeabilized rat slow-twitch fibers were mounted between a force transducer and motor, and calcium activated to elicit ∼30% maximal force. We measured rates of force development and transient force overshoots following a slack re-stretch maneuver and monitored sarcomere shortening during load-clamps. Following PKA, force development rates increased ∼40% and there was a near-doubling in the magnitude of transient force overshoot. We also observed faster shortening velocities at all loads following PKA treatment; this may arise from (i) faster cross-bridge cycling rates, (ii) greater number of cross-bridges during load clamps, and/or (iii) attenuation of internal drag imposed by binding of MyBP-C to actin. To address these mechanisms, we quantified sarcomere length shortening as thin filaments traversed into the C-zone. Before PKA, there tended to be a slowdown as sarcomeres shortened from ∼3.05 to 2.90 µm. After PKA, sarcomeres shortened a considerably greater distance during similar load clamps and, at times, shortening even accelerated as thin filaments slid through the C-zone. Overall these results suggest MyBP-C phosphorylation speeds sarcomere shortening and power output by a combination of faster cross-bridge cycling kinetics, recruitment of cross-bridges, and reduction in drag forces as thin filaments slide through the C-zone.

Impact of Hypertrophic Cardiomyopathy Mutations and the Role of Myosin Binding Protein-C on the Sequestered State of Myosin

The heart contracts by the virtue of multiple ATP-dependent interactions between the actin-containing thin filaments and myosin-containing thick filaments. This sliding of the thin and thick filaments generates force necessary to pump blood throughout the body. It is imperative for the heart to modulate this force-generating mechanism thus fine-tuning its power output. An abrogation of this fine-tuning may lead to hypercontractility of the cardiac tissue which in-turn leads to diseases like Hypertrophic Cardiomyopathy (HCM). For the first time, we report biochemical evidence outlining the molecular basis of this fine-tuning which involves an intramolecular interaction between the myosin head and tail region. This interaction holds myosin heads in reserve, only to be released under dynamic needs of the heart. Another protein of the thick filament called Myosin Binding Protein-C also interacts with myosin thus additionally reigning more myosin heads on the thick filament. We demonstrate that many HCM-associated mutations abrogate these interactions thus bringing more myosin heads into play leading to the observed clinical hypercontractility.

Association of Cardiac Myosin Binding Protein-C with the Ryanodine Receptor-Ca2+ Release Channel: Putative Retrograde Regulation?

Cardiac ryanodine receptor (RyR2) mediates the sarcoplasmic reticulum (SR) Ca2+ efflux that induces cardiomyocyte contraction, whereas sarcomeric myosin binding protein-C (cMyBP-C) regulates actomyosin cross-bridge cycling. We report the first evidence for direct interaction between these two essential proteins using several complementary methods, suggesting existence of a RyR2:cMyBP-C complex. Our studies indicate the C-terminus of cMyBP-C binds to the RyR2 N-terminus in mammalian cells with minimal requirement of a fibronectin-like type 3 domain. Importantly, we observe that specific binding between both recombinant cMyBP-C and RyR2 also occurs with the native proteins extracted from cardiac tissue. Co-expression of cMyBP-C and RyR2 in HEK293 cells alters cellular Ca2+ dynamics, with reduced frequency of RyR2-mediated spontaneous Ca2+ oscillations, suggesting a potential inhibitory effect of cMyBP-C on RyR2 Ca2+ release. Our discovery of direct, functional cMyBP-C association with RyR2 provides novel evidence for putative linkage between sarcomeric cMyBP-C regulation of myofilament contraction and SR-mediated Ca2+ release via RyR2. A distinct mechanism for retrograde sarcomere regulation of SR RyR2 channels may exist, with significant relevance to cMyBP-C and RyR2 dysfunction observed in inherited cardiac disorders.

C1 IG-Domain of Myosin Binding Protein-C Activates Cardiac Thin Filament by Means of Thethering Tropomyosin to the Subdomain-1 of Actin

Cardiac muscle contraction is based on the interaction between two filamentous systems: the thick filament, which is comprised of myosin and its accessory protein, cardiac myosin binding protein C (cMyBP-C) and the thin filament (TF), which is comprised of troponin (Tn), tropomyosin (Tpm), and filamentous actin (F-actin). Mutations in the gene encoding cMyBP-C are one of the most common causes of hypertrophic cardiomyopathy (HCM), a disease that affects approximately 1 in 500 people. The interaction between thick and thin filaments is regulated through translocation of Tpm cable by the Tn-complex in response to Ca2+ followed by additional Tpm azimuthal movement upon binding of rigor myosin cross-bridges to F-actin. Therefore, activation of the TF is a two-step process which depends on both Ca2+-induced translocation of the Tpm and subsequent binding of rigor myosin heads. The N-terminal domain of cMyBP-C is comprised of three Ig-domains (C0, C1 and C2) and a regulatory linker (M-domain). We show that C1 Ig-domain is the only Ig-domain that can activate the TFs at low Ca2+. 3D-reconstruction of frozen hydrated cardiac TFs decorated with C1 Ig-domain shows that the C1 Ig-domain on its own can translocate the Tpm cable on the surface of F-actin to the same extent as the combination of Ca2+ and rigor myosin by tethering the Tpm cable to the subdomain-1 (SD1) of actin. Disruption of the interaction of the C1 Ig-domain interaction with either the SD1 of actin or Tpm cable by point mutations in C1 Ig-domain results in a complete loss of the C1-induced TF activation at low Ca2+. These data suggest how cMyBP-C can modulate the activation of the TF in heart muscle.

Time-Resolved FRET Detection of the Myosin Super-Relaxed off State in Cardiac Thick Filament

We developed time-resolved fluorescence resonance energy transfer (FRET) sensors to detect the structural state of myosin in cardiac thick filaments. Cardiac myosin thick filaments are hypothesized to isomerize between an active state, termed RX (relaxed) that interacts with actin and generates force in the sarcomere, and an inactive state, termed SRX (super-relaxed) that does not bind actin and exhibits inhibited ATP hydrolysis compared to soluble myosin without actin. Regulation of the transition between these states is hypothesized to underlie the Frank-Starling law of the heart, and dysregulation of the transition to lead to genetically-driven hypertrophic cardiomyopathy; however, exactly how remains unclear, because the structural basis of the SRX is poorly understood. To investigate the structural transition between RX and SRX, we developed a time-resolved FRET assay that measures the intermolecular distance between two adjacent myosin regulatory light chains. Based on proposed structural models, formation of the SRX increases FRET while disruption decreases FRET. We tested this model with known modulators of the SRX biochemical kinetics, including temperature and ionic strength, and with two physiologically relevant SRX modulators, RLC phosphorylation and myosin-binding protein-C. This work was supported by National Institute of Health grants AR032961 and AR057220 (DDT), and American Heart Association Scientist Development Grant (JMM).

Altered myofilament structure and function in dogs with Duchenne muscular dystrophy cardiomyopathy

AimDuchenne Muscular Dystrophy (DMD) is associated with progressive depressed left ventricular (LV) function. However, DMD effects on myofilament structure and function are poorly understood. Golden Retriever Muscular Dystrophy (GRMD) is a dog model of DMD recapitulating the human form of DMD. ObjectiveThe objective of this study is to evaluate myofilament structure and function alterations in GRMD model with spontaneous cardiac failure. Methods and resultsWe have employed synchrotron X-rays diffraction to evaluate myofilament lattice spacing at various sarcomere lengths (SL) on permeabilized LV myocardium. We found a negative correlation between SL and lattice spacing in both sub-epicardium (EPI) and sub-endocardium (ENDO) LV layers in control dog hearts. In the ENDO of GRMD hearts this correlation is steeper due to higher lattice spacing at short SL (1.9μm). Furthermore, cross-bridge cycling indexed by the kinetics of tension redevelopment (ktr) was faster in ENDO GRMD myofilaments at short SL. We measured post-translational modifications of key regulatory contractile proteins. S-glutathionylation of cardiac Myosin Binding Protein-C (cMyBP-C) was unchanged and PKA dependent phosphorylation of the cMyBP-C was significantly reduced in GRMD ENDO tissue and more modestly in EPI tissue. ConclusionsWe found a gradient of contractility in control dogs' myocardium that spreads across the LV wall, negatively correlated with myofilament lattice spacing. Chronic stress induced by dystrophin deficiency leads to heart failure that is tightly associated with regional structural changes indexed by increased myofilament lattice spacing, reduced phosphorylation of regulatory proteins and altered myofilament contractile properties in GRMD dogs.

Calcium-Dependent Interaction Occurs between Slow Skeletal Myosin Binding Protein C and Calmodulin

Myosin binding protein C (MyBP-C) is a multi-domain protein that participates in the regulation of muscle contraction through dynamic interactions with actin and myosin. Three primary isoforms of MyBP-C exist: cardiac (cMyBP-C), fast skeletal (fsMyBP-C), and slow skeletal (ssMyBP-C). The N-terminal region of cMyBP-C contains the M-motif, a three-helix bundle that binds Ca2+-loaded calmodulin (CaM), but less is known about N-terminal ssMyBP-C and fsMyBP-C. Here, we characterized the conformation of a recombinant N-terminal fragment of ssMyBP-C (ssC1C2) using differential scanning fluorimetry, nuclear magnetic resonance, and molecular modeling. Our studies revealed that ssC1C2 has altered thermal stability in the presence and absence of CaM. We observed that site-specific interaction between CaM and the M-motif of ssC1C2 occurs in a Ca2+-dependent manner. Molecular modeling supported that the M-motif of ssC1C2 likely adopts a three-helix bundle fold comparable to cMyBP-C. Our study provides evidence that ssMyBP-C has overlapping structural determinants, in common with the cardiac isoform, which are important in controlling protein–protein interactions. We shed light on the differential molecular regulation of contractility that exists between skeletal and cardiac muscle.

Whole length myosin binding protein C stabilizes myosin S2 as measured by gravitational force spectroscopy

The mechanical stability of the myosin subfragment-2 (S2) was tested with simulated force spectroscopy (SFS) and gravitational force spectroscopy (GFS). Experiments examined unzipping S2, since it required less force than stretching parallel to the coiled coil. Both GFS and SFS demonstrated that the force required to destabilize the light meromyosin (LMM) was greater than the force required to destabilize the coiled coil at each of three different locations along S2. GFS data also conveyed that the mechanical stability of the S2 region is independent from its association with the myosin thick filament using cofilaments of myosin tail and a single intact myosin. The C-terminal end of myosin binding protein C (MyBPC) binds to LMM and the N-terminal end can bind either S2 or actin. The force required to destabilize the myosin coiled coil molecule was 3 times greater in the presence of MyBPC than in its absence. Furthermore, the in vitro motility assay with full length slow skeletal MyBPC slowed down the actin filament sliding over myosin thick filaments. This study demonstrates that skeletal MyBPC both enhanced the mechanical stability of the S2 coiled coil and reduced the sliding velocity of actin filaments over polymerized myosin filaments.

Abstract 21304: Calmodulin Modulates the Functional Effects of cMyBP-C on the Thin Filament

Introduction: Cardiac myosin binding protein-C (cMyBP-C) is an essential regulator of heart muscle function that is necessary for both normal contraction and for increased contractility in response to inotropic signals. Effects of cMyBP-C on contraction are complex and attributable to dynamic interactions with both actin and myosin. For instance, cMyBP-C binding to actin can exert either activating or inhibitory effects on contraction by shifting tropomyosin on the thin filament or by blocking myosin heads, respectively. Recently, calmodulin (Ca 2+ /CaM) was also shown to bind cMyBP-C at a unique sequence within the regulatory M-domain of cMyBP-C (Lu et al, JBC 287:31596, 2012). Importantly, the binding site for CaM overlaps with a conserved actin binding site that we found was important for mediating the activating and inhibitory effects of cMyBP-C on the thin filament (Bezold et al. JBC 288: 21496, 2013). Hypothesis: Here we hypothesized that CaM competes with actin for cMyBP-C binding and thus influences the functional effects of cMyBP-C that are mediated through the thin filament. Methods: Recombinant N’-terminal domains of cMyBP-C were used in co-sedimentation binding assays, pull-down assays, and actomyosin ATPase assays to determine effects of CaM binding on cMyBP-C function. Results: We found that CaM binds to N’-terminal domains of cMyBP-C in the presence of Ca 2+ (Ca 2+ /CaM) with an affinity comparable to cMyBP-C binding to actin (3-10 UM). We further show that Ca 2+ /CaM reduces apparent binding affinity of cMyBP-C for actin, consistent with Ca 2+ /CaM competing with actin for binding to cMyBP-C. Also consistent with reduced binding of cMyBP-C to actin, Ca 2+ /CaM reversed the inhibitory effects of cMyBP-C N’-terminal domains on actin activated myosin ATPase rates. However, apo-CaM showed reduced binding affinity for cMyBP-C, did not compete with actin, and did not reverse the activating effects of cMyBP-C on the thin filament at low Ca 2+ . Conclusions: These data suggest that Ca 2+ /CaM is a novel modulator of cMyBP-C function that can temporally tune the complex effects of cMyBP-C on contraction, potentially as Ca 2+ rises and falls during a single heartbeat.

Hypertrophic cardiomyopathy clinical phenotype is independent of gene mutation and mutation dosage

Over 1,500 gene mutations are known to cause hypertrophic cardiomyopathy (HCM). Previous studies suggest that cardiac β-myosin heavy chain (MYH7) gene mutations are commonly associated with a more severe phenotype, compared to cardiac myosin binding protein-C (MYBPC3) gene mutations with milder phenotype, incomplete penetrance and later age of onset. Compound mutations can worsen the phenotype. This study aimed to validate these comparative differences in a large cohort of individuals and families with HCM. We performed genome-phenome correlation among 80 symptomatic HCM patients, 35 asymptomatic carriers and 35 non-carriers, using an 18-gene clinical diagnostic HCM panel. A total of 125 mutations were identified in 14 genes. MYBPC3 and MYH7 mutations contributed to 50.0% and 24.4% of the HCM patients, respectively, suggesting that MYBPC3 mutations were the most frequent cause of HCM in our cohort. Double mutations were found in only nine HCM patients (7.8%) who were phenotypically indistinguishable from single-mutation carriers. Comparisons of clinical parameters of MYBPC3 and MYH7 mutants were not statistically significant, but asymptomatic carriers had high left ventricular ejection fraction and diastolic dysfunction when compared to non-carriers. The presence of double mutations increases the risk for symptomatic HCM with no change in severity, as determined in this study subset. The pathologic effects of MYBPC3 and MYH7 were found to be independent of gene mutation location. Furthermore, HCM pathology is independent of protein domain disruption in both MYBPC3 and MYH7. These data provide evidence that MYBPC3 mutations constitute the preeminent cause of HCM and that they are phenotypically indistinguishable from HCM caused by MYH7 mutations.

Reversible expression of cardiac MyBP-C using an inducible tet-off system

Loss of cMyBP-C has been linked to left ventricular dilation, cardiac hypertrophy and impaired ventricular function in both cMyBP-C constitutive and conditional knockout mice. At present, it remains unclear whether the structural and functional phenotypes associated with the MYBPC3 null mouse are reversible. To test this idea, we generated a cardiac-specific transgenic mouse model using a Tet-Off inducible system to permit the controlled expression of wild-type (WT) cardiac myosin binding protein-C (cMyBP-C) on the MYBPC3 null background. Functional cMyBP-C Tet-off mice were generated by crossing tetracycline transactivator mice with responder mice carrying the WT cMyBP-C transgene. Prior to dietary DOX administration, cMyBP-C expression and left ventricular function in cMyBP-C Tet-off mice were not statistically different from WT mice. Introduction of dietary doxycycline (DOX) for four weeks resulted in a partial knock-down of cMyBP-C expression and commensurate impairment of systolic and diastolic function to levels approaching that observed in MYBPC3 null mice. Subsequent withdrawal of DOX from the diet resulted in the re-expression of cMyBP-C to levels comparable to that observed in WT mice, along with the near complete recovery of in vivo cardiac function. These results indicate that the cardiac phenotypes associated with the MYBPC3 null mouse are reversible, at least in part, and furthermore, validate the use of the Tet-Off inducible system as a means to study the mechanisms underlying hypertrophic cardiomyopathy.

The Sphingosine-1-Phosphate Receptor Modulator, FTY720, Reverses Diastolic Dysfunction and Hypertrophy in Hypertrophic Cardiomyopathy

Background: Hypertrophic cardiomyopathy (HCM) patients present with progressively worsening hypertrophy, diastolic dysfunction and fibrosis, ending in heart failure with preserved ejection fraction. At the molecular level, a common pathology found is an increase in myofilament-calcium (Ca) sensitivity. Previously, we have shown that oxidative modification of myofilament proteins, as a result of increased oxidative stress in HCM, may be a direct mediator of this increase. FTY720 is an immunomodulating drug, clinically used in the treatment of multiple sclerosis, which we have previously identified to have anti-fibrotic and anti-hypertrophic effects in a model of pressure-overload-induced hypertrophy. We sought to understand whether agonism of the sphingosine-1-phosphate receptor by FTY720 would be of therapeutic benefit in HCM, and its effects on oxidative modification. Methods: We treated an HCM mouse model linked to a mutation in tropomyosin (Tm-E180G) and non-transgenic (NTG) littermates with FTY720 or vehicle for six weeks. We assessed cardiac function and morphology using echocardiography and pressure-volume (PV) relations and the myofilament-Ca-response using detergent-treated (skinned) fibers. We also investigated if signaling pathways were altered. Results: We found that FTY720-treated Tm-E180G mice had a significant reduction in left atrial size, E/A and E/Em ratios compared to significantly increased baseline measurements as assessed by echocardiography. PV relations revealed significant improvements in the end- diastolic pressure volume relationship, preload recruitable stroke work, and relaxation constant (Tau). FTY720-treated NTG mice displayed a significant decrease in ejection fraction and rate of maximum pressure development in early systole compared to vehicle-treated NTG mice assessed by PV relations. Skinned fiber bundlesrevealed a significant decrease in myofilament- Ca-responsiveness in FTY720-treated Tm-E180G mice. We attributed these functional improvements to a down-regulation of S-glutathionylation of cardiac myosin binding protein-C in FTY720-treated Tm-E180G mice. The increases in oxidative modification in vehicle treated Tm- E180G mice were due to an up-regulation of NADPH oxidase 2 enzyme expression (NOX2). We attributed the and fibrosis and hypertrophy to increased periostin, TGF-beta expression, and GATA4 phosphorylation respectively. These maladaptive changes were reversed by FTY720 treatment. Conclusions: In Tm-E180G mice, FTY720 improves diastolic function and morphology by reversing oxidative modification of myofilament proteins, resulting in a decrease in myofilament- Ca-responsiveness, via down-regulation of NOX2 and other maladaptive remodeling signals.

The W792R mutation in cardiac myosin binding protein-C reduces the C6 FnIII domain stability and causes hypertrophic cardiomyopathy through haploinsufficiency

The W792R mutation in cardiac myosin binding protein-C reduces the C6 FnIII domain stability and causes hypertrophic cardiomyopathy through haploinsufficiency

Ablation of cMyBP-C eliminates the activation-dependence of unloaded shortening velocity at low levels of Ca2 + activation

At low levels of Ca2+ activation, the time-course of unloaded shortening velocity (Vo) in striated muscle is biphasic, i.e., shortening is comprised of an initial high-velocity phase and a subsequent low-velocity phase. The respective velocities of both the fast and slow phases are known to scale with the level of activation, culminating in a single high-velocity phase at saturating Ca2+ (i.e., Vmax). The molecular mechanism underlying the activation-dependence of Vo most likely involves a shortening-induced cooperative inactivation of the thin filament that results in reduced numbers of strongly-bound cross-bridges, giving rise to the slow phase of unloaded shortening. Cardiac myosin binding protein-C (cMyBP-C) is believed to modulate the cooperative activation of the thin filament by binding to myosin and/or actin and thereby regulating the probability of cross-bridge binding to actin. To test the idea that cMyBP-C contributes, at least in part, to the activation-dependence of unloaded shortening velocity, we measured Vo in skinned trabeculae isolated from wild-type (WT), cTnI-Ala2 (i.e., Ser23,24Ala) and cTnI-Ala2 x cMyBP-C-/- mice. During maximal activation, Vmax was monophasic and not significantly different among the three groups. However, at low levels of activation, biphasic shortening was observed in both the WT and cTnI-Ala2 myocardium, but only the high-velocity phase was observed in the cTnI-Ala2 x cMyBP-C-/- myocardium. Treatment of WT and cTnI-Ala2 trabeculae with PKA accelerated both the low- and high-velocity phases of shortening during submaximal activation, but did not eliminate the low-velocity phase. Thus, phosphorylation of cMyBP-C but not cTnI was required to evoke these effects. The results can be explained in terms of a model in which cMyBP-C regulates the cooperative binding of myosin cross-bridges to actin with the degree of cooperative propagation dependent on the phosphorylation state of cMyBP-C.

A missense mutation within the C6-domain of cMyBP-C results in cardiac enlargement and depressed ventricular function

Cardiac myosin-binding protein-C (cMyBP-C) plays a key role in determining the force and kinetics of myocardial contraction, primarily by binding to either myosin subfragment-2 or actin or both. By modulating the probability of myosin cross-bridge binding to actin, cMyBP-C normally acts to govern the kinetics of force development and relaxation. To determine the effects on cardiac morphology and function due to a novel cMyBP-C W792R missense mutation associated with clinical hypertrophic cardiomyopathy in humans. We generated cMyBP-C-W792R heterozygous and homozygous knock-in mice to investigate the structural and functional phenotypes associated with a missense mutation within the C6-domain of cMyBP-C. Compared to wild-type, homozygous W792R knock-in hearts exhibited significant ventricular and atrial dilation. While the W792R mutant protein was expressed at equivalent levels as in wild-type mice, the extent of phosphorylation of the mutant cMyBP-C protein was markedly reduced. Measurements of steady-state force and cross-bridge cycling kinetics showed that homozygous cMyBP-C W792R skinned myocardium exhibited a significant increase in the Ca2+-sensitivity of force and accelerated cross-bridge cycling kinetics (ktr) at maximal levels of Ca2+-activation, compared to WT myocardium. Cardiac function, as assessed by transthoracic echocardiography, revealed depressed indices of systolic and diastolic ventricular function in the homozygous W792R KI mice. These results demonstrate that a mutation within the mid-region of cMyBP-C can disrupt the dynamic regulation between myosin and actin that is normally governed by cMyBP-C, thereby leading to aberrant cardiac enlargement and commensurate diminution of in vivo ventricular function.

Muscle Lim Protein and myosin binding protein C form a complex regulating muscle differentiation

Muscle Lim Protein (MLP) is a protein with multiple functional roles in striated muscle physiology and pathophysiology. Herein, we demonstrate that MLP directly binds to slow, fast, and cardiac myosin-binding protein C (MyBP-C) during myogenesis, as shown by yeast two-hybrid and a range of protein-protein interaction assays. The minimal interacting domains involve MLP inter-LIM and MyBP-C [C4]. The interaction is sensitive to cytosolic Ca2+ concentrations changes and to MyBP-C phosphorylation by PKA or CaMKII. Confocal microscopy of differentiating myoblasts showed MLP and MyBP-C colocalization during myoblast differentiation. Suppression of the complex formation with recombinant MyBP-C [C4] peptide overexpression, inhibited myoblast differentiation by 65%. Suppression of both MLP and MyBP-C expression in myoblasts by siRNA revealed negative synergistic effects on differentiation. The MLP/MyBP-C complex modulates the actin activated myosin II ATPase activity in vitro, which could interfere with sarcomerogenesis and myofilaments assembly during differentiation. Our data demonstrate a critical role of the MLP/MyBP-C complex during early myoblast differentiation. Its absence in muscles with mutations or aberrant expression of MLP or MyBP-C could be directly implicated in the development of cardiac and skeletal myopathies.

The naked mole-rat exhibits an unusual cardiac myofilament protein profile providing new insights into heart function of this naturally subterranean rodent.

The long-lived, hypoxic-tolerant naked mole-rat well-maintains cardiac function over its three-decade-long lifespan and exhibits many cardiac features atypical of similar-sized laboratory rodents. For example, they exhibit low heart rates and resting cardiac contractility, yet have a large cardiac reserve. These traits are considered ecophysiological adaptations to their dank subterranean atmosphere of low oxygen and high carbon dioxide levels and may also contribute to negligible declines in cardiac function during aging. We asked if naked mole-rats had a different myofilament protein signature to that of similar-sized mice that commonly show both high heart rates and high basal cardiac contractility. Adult mouse ventricles predominantly expressed α-myosin heavy chain (97.9±0.4%). In contrast, and more in keeping with humans, β myosin heavy chain was the dominant isoform (79.0± 2.0%) in naked mole-rat ventricles. Naked mole-rat ventricles diverged from those of both humans and mice, as they expressed both cardiac and slow skeletal isoforms of troponin I. This myofilament protein profile is more commonly observed in mice in utero and during cardiomyopathies. There were no species differences in phosphorylation of cardiac myosin binding protein-C or troponin I. Phosphorylation of both ventricular myosin light chain 2 and cardiac troponin T in naked mole-rats was approximately half that observed in mice. Myofilament function was also compared between the two species using permeabilized cardiomyocytes. Together, these data suggest a cardiac myofilament protein signature that may contribute to the naked mole-rat's suite of adaptations to its natural subterranean habitat.