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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.

Hypertrophic cardiomyopathy (HCM) is an inherited disorder characterized by left ventricular hypertrophy and diastolic dysfunction, and increases the risk of arrhythmias and heart failure. Some patients with HCM develop a dilated phase of hypertrophic cardiomyopathy (D-HCM) and have poor prognosis; however, its pathogenesis is unclear and few pathological models exist. This study established disease-specific human induced pluripotent stem cells (iPSCs) from a patient with D-HCM harboring a mutation in MYBPC3 (c.1377delC), a common causative gene of HCM, and investigated the associated pathophysiological mechanisms using disease-specific iPSC-derived cardiomyocytes (iPSC-CMs). We confirmed the expression of pluripotent markers and the ability to differentiate into three germ layers in D-HCM patient-derived iPSCs (D-HCM iPSCs). D-HCM iPSC-CMs exhibited disrupted myocardial sarcomere structures and an increased number of damaged mitochondria. Ca 2+ imaging showed increased abnormal Ca 2+ signaling and prolonged decay time in D-HCM iPSC-CMs. Cell metabolic analysis revealed increased basal respiration, maximal respiration, and spare-respiratory capacity in D-HCM iPSC-CMs. RNA sequencing also showed an increased expression of mitochondrial electron transport system-related genes. D-HCM iPSC-CMs showed abnormal Ca 2+ handling and hypermetabolic state, similar to that previously reported for HCM patient-derived iPSC-CMs. Although further studies are required, this is expected to be a useful pathological model for D-HCM.

Chest pain is responsible for 6–10% of all presentations to acute healthcare providers. Triage is inherently difficult and heavily reliant on the quantification of cardiac Troponin (cTn), as a minority of patients with an ultimate diagnosis of acute myocardial infarction (AMI) present with clear diagnostic features such as ST-elevation on the electrocardiogram. Owing to slow release and disappearance of cTn, many patients require repeat blood testing or present with stable but elevated concentrations of the best available biomarker and are thus caught at the interplay of sensitivity and specificity.We identified cardiac myosin-binding protein C (cMyC) in coronary venous effluent and developed a high-sensitivity assay by producing an array of monoclonal antibodies and choosing an ideal pair based on affinity and epitope maps. Compared to high-sensitivity cardiac Troponin (hs-cTn), we demonstrated that cMyC appears earlier and rises faster following myocardial necrosis. In this review, we discuss discovery and structure of cMyC, as well as the migration from a comparably insensitive to a high-sensitivity assay facilitating first clinical studies. This assay was subsequently used to describe relative abundance of the protein, compare sensitivity to two high-sensitivity cTn assays and test diagnostic performance in over 1900 patients presenting with chest pain and suspected AMI. A standout feature was cMyC’s ability to more effectively triage patients. This distinction is likely related to the documented greater abundance and more rapid release profile, which could significantly improve the early triage of patients with suspected AMI.

Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral‐mediated AON transfer in a Mybpc3 ‐targeted knock‐in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var‐4) deleted of exons 5–6 in wild‐type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON‐5 and AON‐6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno‐associated virus (AAV‐U7‐AON‐5+6). Transduction of cardiac myocytes or systemic administration of AAV‐U7‐AON‐5+6 increased Var‐4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof‐of‐concept study paves the way towards a causal therapy of HCM. Graphical Abstract Exon skipping is a promising therapy for selected genetic diseases. Here, the authors show as a proof‐of‐principle that MYBPC3 mutation‐induced cardiomyopathy can be rescued by AAV‐U7‐antisense oligoribonucleotides in the heart of neonatal mice.

Cardiomyopathies caused by double gene mutations are rare but conferred a remarkably increased risk of end‐stage progression, arrhythmias, and poor outcome. Compound genetic mutations leading to complex phenotype in the setting of cardiomyopathies represent an important challenge in clinical practice, and genetic tests allow risk stratification and personalized clinical management of patients. We report a case of a 50‐year‐old woman with congestive heart failure characterized by dilated cardiomyopathy, diffuse coronary disease, complete atrioventricular block, and missense mutations in cardiac myosin‐binding protein C (MYBPC3) and myopalladin (MYPN). We discuss the plausible role of genetic profile in phenotype determination.

[...]triage has become reliant on detection of elevated levels of the biomarker cardiac troponin (cTn) in the blood. [...]cMyC is a cardiac-restricted protein which rapidly enters the systemic circulation after myocardial injury and is relatively more abundant than troponin. Financial and competing interests disclosure The authors are supported by grants from the Medical Research Council (UK; G1000737), Guy's and St Thomas’ Charity (R060701, R100404), British Heart Foundation (TG/15/1/31518, FS/15/13/31320) and the UK Department of Health through the NIH Research Biomedical Research Centre award to Guy's and St Thomas’ NHS Foundation Trust.M Marber is named as an inventor on a patent held by King's College London for the detection of cMyC as a biomarker of myocardial injury. [...]universal definition of myocardial infarction.

Pumping of the heart is powered by filaments of the motor protein myosin that pull on actin filaments to generate cardiac contraction. In addition to myosin, the filaments contain cardiac myosin-binding protein C (cMyBP-C), which modulates contractility in response to physiological stimuli, and titin, which functions as a scaffold for filament assembly 1 . Myosin, cMyBP-C and titin are all subject to mutation, which can lead to heart failure. Despite the central importance of cardiac myosin filaments to life, their molecular structure has remained a mystery for 60 years 2 . Here we solve the structure of the main (cMyBP-C-containing) region of the human cardiac filament using cryo-electron microscopy. The reconstruction reveals the architecture of titin and cMyBP-C and shows how myosin’s motor domains (heads) form three different types of motif (providing functional flexibility), which interact with each other and with titin and cMyBP-C to dictate filament architecture and function. The packing of myosin tails in the filament backbone is also resolved. The structure suggests how cMyBP-C helps to generate the cardiac super-relaxed state 3 ; how titin and cMyBP-C may contribute to length-dependent activation 4 ; and how mutations in myosin and cMyBP-C might disturb interactions, causing disease 5 , 6 . The reconstruction resolves past uncertainties and integrates previous data on cardiac muscle structure and function. It provides a new paradigm for interpreting structural, physiological and clinical observations, and for the design of potential therapeutic drugs. The intricate molecular architecture and interactions of the human cardiac myosin filament offer insights into cardiac physiology, disease and drug therapy.

BackgroundCardiac myosin-binding protein C (cMyC) is a novel biomarker of myocardial injury. We hypothesise that calpain-mediated cleavage of cMyC differs between different types of myocardial injury. In order to test our hypothesis we optimised two existing in-house assays capable of detecting intact and total forms of cMyC and assessed their analytic performance.AimsOptimise our existing in-house assay for the quantification of total concentration of cMyC to improve its sensitivity.Optimise our existing in-house assay for the selective detection and quantification of fuller/intact forms of cMyC.MethodsTwo in-house electrochemiluminescence sandwich ELISA platforms for the detection and quantification of total (all species) and intact (fuller length) cMyC in the circulation were optimised. Both protocols were rigorously and iteratively refined to optimise sensitivity. This included trials of multiple buffers, incubation periods, antibodies and antibody concentrations. The detection antibodies of both assays are tagged with Ruthenium.The total cMyC assay uses high affinity mouse monoclonal N-terminal antibodies targeting epitopes which are in close proximity within the N-terminus part of cMyC. This has been designed to detect and quantify all circulating species of cMyC containing the N-terminus.Our intact assay uses a N-terminal, and a C-terminal mouse monoclonal antibody which are widely separated to straddle the calpain cleavage site within cMyC. This assay will only detect the intact (fuller-length) cMyC. ResultsUsing different combinations of antibodies targeting separate portions of the protein we have previously created two independent assays measuring different forms of cMyC.The sensitivity and quantification capacity of both assays was optimised.ConclusionsWe have successfully optimised two sensitive assays capable of detecting two different forms of cMyC at low concentrations. Both assays are sensitive enough to detect cMyC released by minimal myocardial injury seen in clinical scenarios since their LoDs are approximately 10% of the concentration reported as the 99th centile.Abstract BS40 Figure 1Depicts the Sandwich ELISA using N-terminal mAb (3H8) for capture and another (1A4-ruthenium) for detection. The cMyC C0C2 fragment recombinant protein is used to create the standards starting at 80,000ng/L and diluting by a factor of 4. Each sample is run in duplicate. This demonstrates a sensitive assay for total cMyC with low variability (%CV). The limit of detection (LoD) for this assay is 5.6ng/L with all %CV less than 10%Abstract BS40 Figure 2Depicts the Sandwich ELISA using N-terminal mAb (3H8) and a C-terminal anti-C5 mAb for detection. Full complex cMyC recombinant protein is used to create the standards starting at 80,000ng/L and diluting by a factor of four. Each sample is ran in duplicate. This demonstrates a sensitive assay for intact cMyC with low variability. The LoD for this assay is 6.05ng/L with all %CV less than 5%. The control used is recombinant C0C2, which is not detected by this assayConflict of Interestnil

The thick filament is a key component of sarcomeres, the basic units of striated muscle 1 . Alterations in thick filament proteins are associated with familial hypertrophic cardiomyopathy and other heart and muscle diseases 2 . Despite the central importance of the thick filament, its molecular organization remains unclear. Here we present the molecular architecture of native cardiac sarcomeres in the relaxed state, determined by cryo-electron tomography. Our reconstruction of the thick filament reveals the three-dimensional organization of myosin, titin and myosin-binding protein C (MyBP-C). The arrangement of myosin molecules is dependent on their position along the filament, suggesting specialized capacities in terms of strain susceptibility and force generation. Three pairs of titin-α and titin-β chains run axially along the filament, intertwining with myosin tails and probably orchestrating the length-dependent activation of the sarcomere. Notably, whereas the three titin-α chains run along the entire length of the thick filament, titin-β chains do not. The structure also demonstrates that MyBP-C bridges thin and thick filaments, with its carboxy-terminal region binding to the myosin tails and directly stabilizing the OFF state of the myosin heads in an unforeseen manner. These results provide a foundation for future research investigating muscle disorders involving sarcomeric components. A cryo-electron tomography study reports the structure of thick myosin filaments of mouse cardiac muscle in the relaxed state in situ and the MyBP-C links that connect them with the surrounding thin actin filaments.

Myosin-based mechanisms are increasingly recognized as supplementing their better-known actin-based counterparts to control the strength and time course of contraction in both skeletal and heart muscle. Here we use synchrotron small-angle X-ray diffraction to determine the structural dynamics of local domains of the myosin filament during contraction of heart muscle. We show that, although myosin motors throughout the filament contribute to force development, only about 10% of the motors in each filament bear the peak force, and these are confined to the filament domain containing myosin binding protein-C, the “C-zone.” Myosin motors in domains further from the filament midpoint are likely to be activated and inactivated first in each contraction. Inactivated myosin motors are folded against the filament core, and a subset of folded motors lie on the helical tracks described previously. These helically ordered motors are also likely to be confined to the C-zone, and the associated motor conformation reforms only slowly during relaxation. Myosin filament stress-sensing determines the strength and time course of contraction in conjunction with actin-based regulation. These results establish the fundamental roles of myosin filament domains and the associated motor conformations in controlling the strength and dynamics of contraction in heart muscle, enabling those structures to be targeted to develop new therapies for heart disease.