Cardiac Myosin Binding protein-C Research Articles

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.

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.

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.

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.

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.

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

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.

Usefulness of Released Cardiac Myosin Binding Protein-C as a Predictor of Cardiovascular Events

Cardiac myosin binding protein-C (cMyBP-C) is a heart muscle-specific thick filament protein. Elevated level of serum cMyBP-C is an indicator of early myocardial infarction (MI), but its value as a predictor of future cardiovascular disease is unknown. Based on the presence of significant amount of cMyBP-C in the serum of previous study subjects independent of MI, we hypothesized that circulating cMyBP-C is a sensitive indicator of ongoing cardiovascular stress and disease. To test this hypothesis, 75 men and 83 women of similar ages were recruited for a prospective study. They underwent exercise stress echocardiography to provide pre- and poststress blood samples for subsequent determination of serum cMyBP-C levels. The subjects were followed for 1 to 1.5 years. Exercise stress increased serum cMyBP-C in all subjects. Twenty-seven primary events (such as death, MI, revascularization, invasive cardiovascular procedure, or cardiovascular-related hospitalization) and 7 critical events (CE; such as death, MI, stroke, or pulmonary embolism) occurred. After adjusting for sex and cardiovascular risk factors with multivariate Cox regression, a 96% sensitive prestress cMyBP-C threshold carried a hazard ratio of 8.1 with p = 0.041 for primary events. Most subjects (6 of 7) who had CE showed normal ejection fraction on echocardiography. Pre-stress cMyBP-C demonstrated area under receiver operating curve of 0.91 and multivariate Cox regression hazard ratio of 13.8 (p = 0.000472) for CE. Thus, basal cMyBP-C levels reflected susceptibility for a variety of cardiovascular diseases. Together with its high sensitivity, cMyBP-C holds potential as a screening biomarker for the existence of severe cardiovascular diseases.

The Sphingosine‐1‐Phosphate Analog, FTY720, Reverses Diastolic Dysfunction and Hypertrophy in Familial Hypertrophic Cardiomyopathy

BackgroundHypertrophic 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.MethodsWe 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.ResultsWe 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 bundles revealed a significant decrease in myofilament‐Ca‐responsiveness. 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), and fibrosis and hypertrophy to increased periostin, TGF‐beta expression, and GATA4 phosphorylation. These maladaptive changes were reversed by FTY720 treatment.ConclusionsIn 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.Support or Funding InformationR01 HL128468 (BMW, RJS) and F31 HL127996 (DMR)

Myofilaments: Movers and Rulers of the Sarcomere.

Striated cardiac and skeletal muscles play very different roles in the body, but they are similar at the molecular level. In particular, contraction, regardless of the type of muscle, is a precise and complex process involving the integral protein myofilaments and their associated regulatory components. The smallest functional unit of muscle contraction is the sarcomere. Within the sarcomere can be found a sophisticated ensemble of proteins associated with the thick filaments (myosin, myosin binding protein-C, titin, and obscurin) and thin myofilaments (actin, troponin, tropomyosin, nebulin, and nebulette). These parallel thick and thin filaments slide across one another, pulling the two ends of the sarcomere together to regulate contraction. More specifically, the regulation of both timing and force of contraction is accomplished through an intricate network of intra- and interfilament interactions belonging to each myofilament. This review introduces the sarcomere proteins involved in striated muscle contraction and places greater emphasis on the more recently identified and less well-characterized myofilaments: cardiac myosin binding protein-C, titin, nebulin, and obscurin. © 2017 American Physiological Society. Compr Physiol 7:675-692, 2017.

Cardiac Myosin Binding Protein-C Autoantibodies Are Potential Early Indicators of Cardiac Dysfunction and Patient Outcome in Acute Coronary Syndrome

The degradation and release of cardiac myosin binding protein-C (cMyBP-C) upon cardiac damage may stimulate an inflammatory response and autoantibody (AAb) production. We determined whether the presence of cMyBP-C-AAbs associated with adverse cardiac function in CVD patients. Importantly, cMyBP-C-AAbs were significantly detected in ACS patient sera upon arrival to the emergency department, particularly in STEMI patients. Patients positive for cMyBP-C-AAbs had a reduced LVEF and elevated levels of clinical biomarkers of MI. We conclude that cMyBP-C-AAbs may serve as early predictive indicators of deteriorating cardiac function and patient outcome in ACS patients prior to the infarction.

C0 Domain of Cardiac Myosin Binding Protein-C Modulates Interaction of the Neighboring C1 Domain with Tropomyosin Through the Allosteric Interaction with F-Actin

Mutations in cardiac myosin binding protein C (cMyBP-C) are the most common cause of hypertrophic cardiomyopathy affecting millions of people worldwide. Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown. Recently, we demonstrated that despite being structural homologs, C0 and C1 NTDs exhibit different patterns of binding on the surface of F-actin. Moreover, we showed that C1 but not C0 can activate the TF by means of shifting the tropomyosin (Tm) cable from the “closed” to the “open” structural state via direct interaction with Tm. Here we used cryo electron microscopy and image analysis to reveal how C0 and C1 Ig-domains connected by the Proline/Alanine-linker communicate with each other when interacting with the TF in tandem. Our results show that C0 binds to the front of actin subdomain-1 in two distinct modes, and that in these two modes C0 has different effect on the interaction of C1 domain with both actin filament and Tm cable. In one mode C0 enhances the activating effect of C1 domain so that the Tm cable is shifted towards the subdomain-4 of actin to an even larger extent than found in the “open” state of Tm. In the second mode C0 reverses the activation effect of C1 by means of disrupting the interaction of C1 with Tm. Importantly, we demonstrate that the two domains communicate with each other allosterically through the actin filament. Our data suggest that the internal dynamics of the actin molecule is essential for the regulation of the actomyosin interaction in cardiac muscle. We propose a mechanism by which cMyBP-C may modulate actomyosin interactions in cardiac muscle.

Activation and Inhibition of Cardiac Thin Filaments by Single and Multiple Domains Constructs of Human Cardiac Myosin Binding Protein-C (cMyBP-C) at Low Calcium

We have previously shown that the C1 domain of human cardiac myosin binding protein-C (c-MyBP-C) produces a biphasic regulation of cardiac thin filaments at low calcium concentrations. Increasing [C1] first produces activation followed by inhibition of myosin-S1 ATP hydrolysis by cardiac thin filaments at higher [C1]. In contrast, domains C0 and C2 only inhibit and do not activate ATP hydrolysis at any concentration. N’-terminal proteins containing the C1 domain along with C0 or C2 (e.g., C0-PA-C1, C1-M-C2 and C0-PA-C1-M-C2) show a similar pattern of activation and inhibition of ATP hydrolysis at low calcium concentration as the C1 domain alone, but the concentrations required for maximum activation and inhibition are ∼10 fold lower than required using C1 alone.

Investigation of the Molecular Interactions Regulating the Function of Human Cardiac Myosin

Cardiac myosin interacts cyclically with actin in the presence of ATP in the sarcomere to drive heart contraction. Previous fiber studies suggested that the structure and function of myosin in the thick filaments are regulated by its own regulatory light chain (RLC) phosphorylation and through its interaction with myosin binding protein-C (MyBP-C). Interestingly, ∼80% of mutations that cause hypertrophic cardiomyopathy (HCM), a relatively common genetic cardiac disorder characterized by hypercontractility, have been found in the genes encoding human β-cardiac myosin and human cardiac MyBP-C. It has been hypothesized that the heads of the two-headed motor can adopt a sequestered structure where the heads interact asymmetrically with their own tail as Ser-15 residue of RLC remains dephosphorylated, and the N-terminal domains of MyBP-C binds to myosin to stabilize the structure. We designed two different two-headed human β-cardiac myosin motor constructs differing in the length of their S2 tail domains and co-expressed them with both of the human light chains to test the hypothesis that the number of myosin heads available for interaction with actin is regulated by phosphorylation of the RLC and MyBP-C. The shorter construct (2-hep HMM) is devoid of the tail region needed for the interaction of S2 with its S1 heads whereas the longer one (25-hep HMM) has the required length of S2 tail region to allow such interactions. We found that the maximal actin-activated ATPase rate for 2-hep HMM and the single-headed myosin construct S1 are essentially the same, and are only slightly changed by RLC phosphorylation. On the other hand, de-phosphorylated 25-hep HMM has a significantly lower maximal actin-activated ATPase than the 2-hep HMM, consistent with the former having fewer actin-accessible S1 heads than the latter. Additionally, binding experiments of 2-hep and 25-hep HMM with the N-terminal C0-C2 domain of human cardiac MyBP-C showed that the affinity of complex formation is weakened by phosphorylation of either RLC or MyBP-C.

Peroxisome proliferator-activated receptor-α expression induces alterations in cardiac myofilaments in a pressure-overload model of hypertrophy.

Although alterations in fatty acid (FA) metabolism have been shown to have a negative impact on contractility of the hypertrophied heart, the targets of action remain elusive. In this study we compared the function of skinned fiber bundles from transgenic (Tg) mice that overexpress a relatively low level of the peroxisome proliferator-activated receptor α (PPARα), and nontransgenic (NTg) littermates. The mice (NTg-T and Tg-T) were stressed by transverse aortic constriction (TAC) and compared with shams (NTg-S and Tg-S). There was an approximate 4-fold increase in PPARα expression in Tg-S compared with NTg-S, but Tg-T hearts showed the same PPARα expression as NTg-T. Expression of PPARα did not alter the hypertrophic response to TAC but did reduce ejection fraction (EF) in Tg-T hearts compared with other groups. The rate of actomyosin ATP hydrolysis was significantly higher in Tg-S skinned fiber bundles compared with all other groups. Tg-T hearts showed an increase in phosphorylation of specific sites on cardiac myosin binding protein-C (cMyBP-C) and β-myosin heavy chain isoform. These results advance our understanding of potential signaling to the myofilaments induced by altered FA metabolism under normal and pathological states. We demonstrate that chronic and transient PPARα activation during pathological stress alters myofilament response to Ca2+ through a mechanism that is possibly mediated by MyBP-C phosphorylation and myosin heavy chain isoforms.NEW & NOTEWORTHY Data presented here demonstrate novel signaling to sarcomeric proteins by chronic alterations in fatty acid metabolism induced by PPARα. The mechanism involves modifications of key myofilament regulatory proteins modifying cross-bridge dynamics with differential effects in controls and hearts stressed by pressure overload.

Diagnostic and Prognostic Value of Plasma Levels of Cardiac Myosin Binding Protein-C as a Novel Biomarker in Heart Failure.

Heart failure (HF) has high morbidity and mortality in children. This study aimed to investigate the value of cardiac myosin binding protein-C (cMyBP-C) as a diagnostic and prognostic biomarker in children with heart failure. This study was a prospective case-control study that involved 50 children with acute HF and 25 healthy children of matched age and sex as a control group. cMyBP-C plasma levels were measured in patients with HF at the time of admission and 1month after treatment. Echocardiographic assessment was done for all children. All patients were followed up for a period of 3months. There was a significant increase in plasma levels of cMyBP-C (ng/ml) in patients with HF at admission (122.44±41.01) as compared to patients after treatment (71.38±49.68) and to control group (24.40±9.83). This increase was associated with increased severity of HF according to pediatric Ross classification of HF. Significant increase in plasma levels of cMyBP-C at admission and its persistent increase after treatment were associated with adverse outcome of mortality and readmission. Plasma levels of cMyBP-C were significantly correlated with echocardiographic and clinical assessment of heart failure. Plasma levels of cMyBP-C were a good biomarker for diagnosis of HF with sensitivity 100% and specificity 96% at cutoff point of 45ng/ml. Its value in predicting adverse outcome in HF patients was obtained by ROC curve with sensitivity of 90% and specificity 93% at a cutoff point of 152ng/ml cMyBP-C at admission. cMyBP-C may be a novel useful diagnostic and prognostic biomarker in children with heart failure and determination of severity of HF in these patients.