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Heart muscle has the unique property that it can never rest; all cardiomyocytes contract with each heartbeat which requires a complex control mechanism to regulate cardiac output to physiological requirements. Changes in calcium concentration regulate the thin filament activation. A separate but linked mechanism regulates the thick filament activation, which frees sufficient myosin heads to bind the thin filament, thereby producing the required force. Thick filaments contain additional nonmyosin proteins, myosin-binding protein C and titin, the latter being the protein that transmits applied tension to the thick filament. How these three proteins interact to control thick filament activation is poorly understood. Here, we show using 3-D image reconstruction of frozen-hydrated human cardiac muscle myofibrils lacking exogenous drugs that the thick filament is structured to provide three levels of myosin activation corresponding to the three crowns of myosin heads in each 429Å repeat. In one crown, the myosin heads are almost completely activated and disordered. In another crown, many myosin heads are inactive, ordered into a structure called the interacting heads motif. At the third crown, the myosin heads are ordered into the interacting heads motif, but the stability of that motif is affected by myosin-binding protein C. We think that this hierarchy of control explains many of the effects of length-dependent activation as well as stretch activation in cardiac muscle control. [ABSTRACT FROM AUTHOR]

Key points: The Anrep effect represents the alteration of left ventricular (LV) contractility to acutely enhanced afterload in a few seconds, thereby preserving stroke volume (SV) at constant preload. As a result of the missing preload stretch in our model, the Anrep effect differs from the slow force response and has a different mechanism.The Anrep effect demonstrated two different phases. First, the sudden increased afterload was momentary equilibrated by the enhanced LV contractility as a result of higher power strokes of strongly‐bound myosin cross‐bridges. Second, the slightly delayed recovery of SV is perhaps dependent on Ca2+/calmodulin‐dependent protein kinase II activation caused by oxidation and myofilament phosphorylation (cardiac myosin‐binding protein‐C, myosin light chain 2), maximizing the recruitment of available strongly‐bound myosin cross‐bridges.Short‐lived oxidative stress might present a new facet of subcellular signalling with respect to cardiovascular regulation.Relevance for human physiology was demonstrated by echocardiography disclosing the Anrep effect in humans during handgrip exercise. The present study investigated whether oxidative stress and Ca2+/calmodulin‐dependent protein kinase II (CaMKII) activity are involved in triggering the Anrep effect. LV pressure–volume (PV) analyses of isolated, preload controlled working hearts were performed at two afterload levels (60 and 100 mmHg) in C57BL/6N wild‐type (WT) and CaMKII‐double knockout mice (DKOCaMKII). In snap‐frozen WT hearts, force–pCa relationship, H2O2 generation, CaMKII oxidation and phosphorylation of myofilament and Ca2+ handling proteins were assessed. Acutely raised afterload showed significantly increased wall stress, H2O2 generation and LV contractility in the PV diagram with an initial decrease and recovery of stroke volume, whereas end‐diastolic pressure and volume, as well as heart rate, remained constant. Afterload induced increase in LV contractility was blunted in DKOCaMKII‐hearts. Force development of single WT cardiomyocytes was greater with elevated afterload at submaximal Ca2+ concentration and associated with increases in CaMKII oxidation and phosphorylation of cardiac‐myosin binding protein‐C, myosin light chain and Ca2+ handling proteins. CaMKII activity is involved in the regulation of the Anrep effect and associates with stimulation of oxidative stress, presumably starting a cascade of CaMKII oxidation with downstream phosphorylation of myofilament and Ca2+ handling proteins. These mechanisms improve LV inotropy and preserve stroke volume within few seconds. Key points: The Anrep effect represents the alteration of left ventricular (LV) contractility to acutely enhanced afterload in a few seconds, thereby preserving stroke volume (SV) at constant preload. As a result of the missing preload stretch in our model, the Anrep effect differs from the slow force response and has a different mechanism.The Anrep effect demonstrated two different phases. First, the sudden increased afterload was momentary equilibrated by the enhanced LV contractility as a result of higher power strokes of strongly‐bound myosin cross‐bridges. Second, the slightly delayed recovery of SV is perhaps dependent on Ca2+/calmodulin‐dependent protein kinase II activation caused by oxidation and myofilament phosphorylation (cardiac myosin‐binding protein‐C, myosin light chain 2), maximizing the recruitment of available strongly‐bound myosin cross‐bridges.Short‐lived oxidative stress might present a new facet of subcellular signalling with respect to cardiovascular regulation.Relevance for human physiology was demonstrated by echocardiography disclosing the Anrep effect in humans during handgrip exercise. [ABSTRACT FROM AUTHOR]

The heart is an extraordinarily versatile pump, finely tuned to respond to a multitude of demands. Given the heart pumps without rest for decades its efficiency is particularly relevant. Although many proteins in the heart are essential for viability, the non-essential components can attract numerous mutations which can cause disease, possibly through alterations in pumping efficiency. Of these, myosin binding protein C is strongly over-represented with ~ 40% of all known mutations in hypertrophic cardiomyopathy. Therefore, a complete understanding of its molecular function in the cardiac sarcomere is warranted. In this review, we revisit contemporary and classical literature to clarify both the current standing of this fast-moving field and frame future unresolved questions. To date, much effort has been directed at understanding MyBP-C function on either thick or thin filaments. Here we aim to focus questions on how MyBP-C functions at a molecular level in the context of both the thick and thin filaments together. A concept that emerges is MyBP-C acts to govern interactions on two levels; controlling myosin access to the thin filament by sequestration on the thick filament, and controlling the activation state and access of myosin to its binding sites on the thin filament. Such affects are achieved through directed interactions mediated by phosphorylation (of MyBP-C and other sarcomeric components) and calcium. [ABSTRACT FROM AUTHOR]

In patients with (ACS ), it is necessary to identify a sensitive biomarker for early diagnosis to avoid misdiagnosis of this serious condition and despite the high sensitivity and specificity of troponin in this context, the diagnosis of ACS represents a challenge. cMYBP-C might improve the ability of early diagnosis of ACS and the aim of the current study is to evaluate its role in the diagnosis depending on troponin as a reference biomarker. A total number of 150 patients with ACS were randomly selected to participate in the current study. They were equally divided into ST-elevation myocardial infarction (STTEMI), patients with non-ST elevation myocardial infarction (NSTEMI) and unstable angina (UA )in addition to50 individuals as a control healthy group. All participants were investigated for both troponin and c-MYBP-Cand the results were statistically evaluated. The current study has shown a significant increase in serum level of c-MYBP-C in patients with ACS apart from those with unstable angina when compared to the control group. This increase was so comparable to troponin increase that both biomarkers were even significantly higher in STEMI compared to NSTEMI. In those with unstable angina, there was no significant difference in both biomarkers compared to the control group. c-MYBP-Cis a useful biomarker for early diagnosis of ACS and in differentiating myocardial infarction (whether STEMI or NSTEMI) from unstable angina. [ABSTRACT FROM AUTHOR]

Mutations in cardiac myosin-binding protein C (cMyBP-C) are a major cause of familial hypertrophic cardiomyopathy. Although cMyBP-C has been considered to regulate the cardiac function via cross-bridge arrangement at the C-zone of the myosin-containing A-band, the mechanism by which cMyBP-C functions remains unclear. We identified formin Fhod3, an actin organizer essential for the formation and maintenance of cardiac sarcomeres, as a cMyBP-C-binding protein. The cardiac-specific N-terminal Ig-like domain of cMyBP-C directly interacts with the cardiac-specific N-terminal region of Fhod3. The interaction seems to direct the localization of Fhod3 to the C-zone, since a noncardiac Fhod3 variant lacking the cMyBP-C-binding region failed to localize to the C-zone. Conversely, the cardiac variant of Fhod3 failed to localize to the C-zone in the cMyBP-C-null mice, which display a phenotype of hypertrophic cardiomyopathy. The cardiomyopathic phenotype of cMyBP-C-null mice was further exacerbated by Fhod3 overexpression with a defect of sarcomere integrity, whereas that was partially ameliorated by a reduction in the Fhod3 protein levels, suggesting that Fhod3 has a deleterious effect on cardiac function under cMyBP-C-null conditions where Fhod3 is aberrantly mislocalized. Together, these findings suggest the possibility that Fhod3 contributes to the pathogenesis of cMyBP-C-related cardiomyopathy and that Fhod3 is critically involved in cMyBP-C-mediated regulation of cardiac function via direct interaction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cardiac myosin binding protein C (cMyBP-C) has a key regulatory role in cardiac contraction, but the mechanism by which changes in phosphorylation of cMyBP-C accelerate cross-bridge kinetics remains unknown. In this study, we isolated thick filaments from the hearts of mice in which the three serine residues (Ser273, Ser282, and Ser302) that are phosphorylated by protein kinase A in the m-domain of cMyBP-C were replaced by either alanine or aspartic acid, mimicking the fully nonphosphorylated and the fully phosphorylated state of cMyBP-C, respectively. We found that thick filaments from the cMyBP-C phospho-deficient hearts had highly ordered cross-bridge arrays, whereas the filaments from the cMyBP-C phospho-mimetic hearts showed a strong tendency toward disorder. Our results support the hypothesis that dephosphorylation of cMyBP-C promotes or stabilizes the relaxed/superrelaxed quasi-helical ordering of the myosin heads on the filament surface, whereas phosphorylation weakens this stabilization and binding of the heads to the backbone. Such structural changes would modulate the probability of myosin binding to actin and could help explain the acceleration of crossbridge interactions with actin when cMyBP-C is phosphorylated because of, for example, activation of β1-adrenergic receptors in myocardium. [ABSTRACT FROM AUTHOR]

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 1 month after treatment. Echocardiographic assessment was done for all children. All patients were followed up for a period of 3 months. 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 45 ng/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 152 ng/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. [ABSTRACT FROM AUTHOR]

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During each heartbeat, cardiac contractility results from calcium-activated sliding of actin thin filaments toward the centers of myosin thick filaments to shorten cellular length. Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filament that appears to tune these mechanochemical interactions by its N-terminal domains transiently interacting with actin and/or the myosin S2 domain, sensitizing thin filaments to calcium and governing maximal sliding velocity. Both functional mechanisms are potentially further tunable by phosphorylation of an intrinsically disordered, extensible region of cMyBP-C's N terminus, the M-domain. Using atomic force spectroscopy, electron microscopy, and mutant protein expression, we demonstrate that phosphorylation reduced the M-domain's extensibility and shifted the conformation of the N-terminal domain from an extended structure to a compact configuration. In combination with motility assay data, these structural effects of M-domain phosphorylation suggest a mechanism for diminishing the functional potency of individual cMyBP-C molecules. Interestingly, we found that calcium levels necessary to maximally activate the thin filament mitigated the structural effects of phosphorylation by increasing M-domain extensibility and shifting the phosphorylated N-terminal fragments back to the extended state, as if unphosphorylated. Functionally, the addition of calcium to the motility assays ablated the impact of phosphorylation on maximal sliding velocities, fully restoring cMyBP-C's inhibitory capacity. We conclude that M-domain phosphorylation may have its greatest effect on tuning cMyBP-C's calcium-sensitization of thin filaments at the low calcium levels between contractions. Importantly, calcium levels at the peak of contraction would allow cMyBP-C to remain a potent contractile modulator, regardless of cMyBP-C's phosphorylation state. [ABSTRACT FROM AUTHOR]

Enhanced cardiac contractile function with increased sarcomere length (SL) is, in part, mediated by a decrease in the radial distance between myosin heads and actin. The radial disposition of myosin heads relative to actin is modulated by cardiac myosin binding protein-C (cMyBP-C), suggesting that cMyBP-C contributes to the length-dependent activation (LDA) in the myocardium. However, the precise roles of cMyBP-C in modulating cardiac LDA are unclear. To determine the impact of cMyBP-C on LDA, we measured isometric force, myofilament Ca2+-sensitivity (pCa50) and length-dependent changes in kinetic parameters of cross-bridge (XB) relaxation (krel), and recruitment (kdf) due to rapid stretch, as well as the rate of force redevelopment (ktr) in response to a large slack-restretch maneuver in skinned ventricular multicellular preparations isolated from the hearts of wild-type (WT) and cMyBP-C knockout (KO) mice, at SL's 1.9μm or 2.1μm. Our results show that maximal force was not significantly different between KO and WT preparations but length-dependent increase in pCa50 was attenuated in the KO preparations. pCa50 was not significantly different between WT and KO preparations at long SL (5.82 ± 0.02 in WT vs. 5.87 ± 0.02 in KO), whereas pCa50 was significantly different between WT and KO preparations at short SL (5.71 2 ± 0.02 in WT vs. 5.80 ± 0.01 in KO; p < 0.05). The ktr, measured at half-maximal Ca2+-activation, was significantly accelerated at short SL in WT preparations (8.74 ± 0.56 s-1 at 1.9μm vs. 5.71 ± 0.40 s-1 at 2.1μm, p < 0.05). Furthermore, krel and kdf were accelerated by 32% and 50%, respectively at short SL in WT preparations. In contrast, ktr was not altered by changes in SL in KO preparations (8.03 ± 0.54 s-1 at 1.9μm vs. 8.90 ± 0.37 s-1 at 2.1μm). Similarly, KO preparations did not exhibit length-dependent changes in krel and kdf. Collectively, our data implicate cMyBP-C as an important regulator of LDA via its impact on dynamic XB behavior due to changes in SL. [ABSTRACT FROM AUTHOR]

Cardiac myosin binding protein-C (cMyBP-C) is a modular protein anchored to the thick filament through interactions mediated by its C-terminal region. The N-terminal region of cMyBPC-C regulates myocardial contractility by modifying actin-myosin association. Phosphorylation of the N-terminal region diminishes cMyBP-C's capacity to regulate actin-myosin function. Despite a substantial body of literature, many issues remain unclear regarding the structural and functional roles of cMyBP-C. While no high-resolution structures of the intact protein exist, crystallographic and nuclear magnetic resonance (NMR) structures of isolated N-terminal domains provide important molecular details regarding cMyBP-C's role in controlling contractility. In this review, we summarize the emerging structural understanding of cMyBP-C with a particular emphasis placed on describing how its dynamic molecular interactions with both thin and thick filament proteins likely contribute to contractile regulation. Furthermore, we discuss the future directions and strategies by which we may improve the mechanistic understanding of its role in modulating cardiac muscle contraction. [ABSTRACT FROM AUTHOR]

Myosin-binding protein-C (MyBP-C) is an accessory protein of the myosin filaments of vertebrate striated muscle. In the heart, it plays a key role in modulating contractility in response to β-adrenergic stimulation. Mutations in the cardiac isoform (cMyBP-C) are a leading cause of inherited hypertrophic cardiomyopathy. Understanding cMyBP-C function and its role in disease requires knowledge of the structure of the molecule, its organization in the sarcomere, and its interactions with other sarcomeric proteins. Here we review the main structural features of this modular, elongated molecule and the properties of some of its key domains. We describe observations suggesting that the bulk of the molecule extends perpendicular to the thick filament, enabling it to reach neighboring thin filaments in the sarcomere. We review structural and functional evidence for interaction of its N-terminal domains with actin and how this may modulate thin filament activation. We also discuss the effects that phosphorylation of cMyBP-C has on some of these structural features and how this might relate to cMyBP-C function in the beating heart. [ABSTRACT FROM AUTHOR]

Diagnosis of myocardial infarction (MI) is based on ST-segment elevation on electrocardiographic evaluation and/or elevated plasma cardiac troponin (cTn) levels. However, troponins lack the sensitivity required to detect the onset of MI at its earliest stages. Therefore, to confirm its viability as an ultra-early biomarker of MI, this study investigates the release kinetics of cardiac myosin binding protein-C (cMyBP-C) in a porcine model of MI and in two human cohorts. Release kinetics of cMyBP-C were determined in a porcine model of MI (n = 6, pigs, either sex) by measuring plasma cMyBP-C level serially from 30 min to 14 days after coronary occlusion, with use of a custom-made immunoassay. cMyBP-C plasma levels were increased from baseline (76 ± 68 ng/l) at 3 h (767 ± 211 ng/l) and peaked at 6 h (2,418 ± 780 ng/l) after coronary ligation. Plasma cTnI, cTnT, and myosin light chain-3 levels were all increased 6 h after ligation. In a cohort of patients (n = 12) with hypertrophic obstructive cardiomyopathy undergoing transcoronary ablation of septal hypertrophy, cMyBP-C was significantly increased from baseline (49 ± 23 ng/l) in a time-dependent manner, peaking at 4 h (560 ± 273 ng/l). In a cohort of patients with non-ST segment elevation MI (n = 176) from the SYNERGY trial, cMyBP-C serum levels were significantly higher (7,615 ± 4,514 ng/l) than those in a control cohort (416 ± 104 ng/l; n = 153). cMyBP-C is released in the blood rapidly after cardiac damage and therefore has the potential to positively mark the onset of MI. [ABSTRACT FROM AUTHOR]

Cardiac myosin binding protein-C (cMyBP-C) is a cardiac-specific thick filament assembly, accessory, and regulatory protein. Physiologically, it is a key regulator of cardiac contractility. With more than 200 mutations in the cMyBP-C gene directly linked to the development of cardiomyopathy and heart failure, cMyBP-C clearly plays a critical role in heart function. At baseline, cMyBP-C is highly phosphorylated, a condition required for normal cardiac function. However, the level of cMyBP-C phosphorylation is significantly decreased during heart failure, indicating that the level of cMyBP-C phosphorylation is directly linked to signaling and cardiac function. Early studies indicated that cMyBP-C interacts with myosin and titin, whereas studies now show that it also interacts with thin filament proteins. However, the exact role(s) of cMyBP-C in the heart remain(s) to be elucidated. As such, we invited experts in the field of cardiac muscle to identify and address key issues related to cMyBP-C by contributing a mini review on such topics as structure, function, regulation, cardiomyopathy, proteolysis, and gene therapy. Starting from this issue, Pflügers Archiv European Journal of Physiology will publish two invited mini review articles each month to discuss the most recent advances in the study of cMyBP-C. [ABSTRACT FROM AUTHOR]

Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric septal hypertrophy and is often caused by mutations in MYBPC3 gene encoding cardiac myosin-binding protein C. In contrast to humans, who are already affected at the heterozygous state, mouse models develop the phenotype mainly at the homozygous state. Evidence from cell culture work suggested that altered proteasome function contributes to the pathogenesis of HCM. Here we tested in two heterozygous Mybpc3-targeted mouse models whether adrenergic stress unmasks a specific cardiac phenotype and proteasome dysfunction. The first model carries a human Mybpc3 mutation (Het-KI), the second is a heterozygous Mybpc3 knock-out (Het-KO). Both models were compared to wild-type (WT) mice. Mice were treated with a combination of isoprenaline and phenylephrine (ISO/PE) or NaCl for 1 week. Whereas ISO/PE induced left ventricular hypertrophy (LVH) with increased posterior wall thickness to a similar extent in all groups, it increased septum thickness only in Het-KI and Het-KO. ISO/PE did not affect the proteasomal chymotrypsin-like activity or β5-subunit protein level in Het-KO or wild-type mice (WT). In contrast, both parameters were markedly lower in Het-KI and negatively correlated with the degree of LVH in Het-KI only. In conclusion, adrenergic stress revealed septal hypertrophy in both heterozygous mouse models of HCM, but proteasome dysfunction only in Het-KI mice, which carry a mutant allele and closely mimic human HCM. This supports the hypothesis that proteasome impairment contributes to the pathophysiology of HCM. [ABSTRACT FROM AUTHOR]

It is now generally accepted that phosphorylation of cMyBP-C is critically important in maintaining normal cardiac function. Although much of the work to date on phospho-regulation of cMyBP-C has focused on the role of protein kinase A (PKA, also known as cAMP-dependent protein kinase), recent evidence suggests that a number of non-PKA serine/threonine kinases, such as Ca/calmodulin-dependent protein kinase II, protein kinase C, protein kinase D and the 90-kDa ribosomal S6 kinase are also capable of targeting this key regulatory sarcomeric protein. This article reviews such evidence and proposes a hypothetical role for some of the pertinent signalling pathways in phospho-regulation of cMyBP-C in the setting of heart failure. [ABSTRACT FROM AUTHOR]

Myocardial injury is associated with inflammation and fibrosis. Cardiac myosin-binding protein-C (cMyBP-C) is cleaved by µ-calpain upon myocardial injury, releasing C0-C1f, an N-terminal peptide of cMyBP-C. Previously, we reported that the presence of C0-C1f is pathogenic within cardiac tissue and is able to activate macrophages. Fibroblasts also play a crucial role in cardiac remodeling arising from ischemic events, as they contribute to both inflammation and scar formation. To understand whether C0-C1f directly modulates fibroblast phenotype, we analyzed the impact of C0-C1f on a human fibroblast cell line in vitro by performing mRNA microarray screening, immunofluorescence staining, and quantitative real-time PCR. The underlying signaling pathways were investigated by KEGG analysis and determined more precisely by targeted inhibition of the potential signaling cascades in vitro. C0-C1f induced pro-inflammatory responses that might delay TGFβ-mediated myofibroblast conversion. TGFβ also counteracted C0-C1f-mediated fibroblast activation. Inhibition of TLR4 or NFκB as well as the delivery of miR-146 significantly reduced C0-C1f-mediated effects. In conclusion, C0-C1f induces inflammatory responses in human fibroblasts that are mediated via TRL4 signaling, which is decreased in the presence of TGFβ. Specific targeting of TLR4 signaling could be an innovative strategy to modulate C0-C1f-mediated inflammation. [ABSTRACT FROM AUTHOR]