The mechanisms by which truncating mutations in MYBPC3 (encoding cardiac myosin-binding protein C; cMyBPC) or myosin missense mutations cause hypercontractility and poor relaxation in hypertrophic cardiomyopathy (HCM) are incompletely understood. Using genetic and biochemical approaches, we explored how depletion of cMyBPC altered sarcomere function. We demonstrated that stepwise loss of cMyBPC resulted in reciprocal augmentation of myosin contractility. Direct attenuation of myosin function, via a damaging missense variant (F764L) that causes dilated cardiomyopathy (DCM), normalized the increased contractility from cMyBPC depletion. Depletion of cMyBPC also altered dynamic myosin conformations during relaxation, enhancing the myosin state that enables ATP hydrolysis and thin filament interactions while reducing the super relaxed conformation associated with energy conservation. MYK-461, a pharmacologic inhibitor of myosin ATPase, rescued relaxation deficits and restored normal contractility in mouse and human cardiomyocytes with MYBPC3 mutations. These data define dosage-dependent effects of cMyBPC on myosin that occur across the cardiac cycle as the pathophysiologic mechanisms by which MYBPC3 truncations cause HCM. Therapeutic strategies to attenuate cMyBPC activity may rescue depressed cardiac contractility in patients with DCM, whereas inhibiting myosin by MYK-461 should benefit the substantial proportion of patients with HCM with MYBPC3 mutations.

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Cardiomyopathies are a leading cause of heart failure and are often caused by mutations in sarcomeric genes, resulting in contractile dysfunction and cellular damage. This may stimulate the production of a robust proinflammatory response. To determine whether myocardial inflammation is associated with cardiac dysfunction in dilated cardiomyopathy (DCM) caused by MYBPC3 mutation, we used the well-characterized cMyBP-C(t/t) mouse model of DCM at 3 months of age. Compared to wild type (WT) mice, DCM mice exhibited significantly decreased fractional shortening (36.4±2% vs. 15.5±1.0%, p < 0.0001) and significantly increased spleen weight (5.3±0.3% vs. 7.2±0.4 mm/mg, p = 0.002). Intriguingly, flow cytometry analysis revealed a significant increase in total (CD45+CD11b+Ly6C−MHCII+F480+) macrophages (6.5±1.4% vs. 14.8±1.4%, p = 0.002) and classically activated (CD45+CD11b+Ly6C−MHCII+F480+CD206−) proinflammatory (M1) macrophages (3.4±0.8% vs. 10.3±1.2%, p = 0.0009) in DCM hearts as compared with WT hearts. These results were further confirmed by immunofluorescence analysis of heart tissue sections. Splenic red pulp (CD11b+Ly6C+MHCIIlowF480hi) macrophages were significantly elevated (2.4±0.1% vs. 1.3±0.1%, p = 0.0001) in DCM compared to WT animals. Serum cytokine analysis in DCM animals exhibited a significant increase (0.65±0.2 vs. 2.175±0.5 pg/mL, p = 0.02) in interleukin (IL)-6 compared to WT animals. Furthermore, RNAseq analysis revealed the upregulation of inflammatory pathways in the DCM hearts. Together, these data indicate a robust proinflammatory response in DCM hearts, likely in response to cellular damage triggered by MYBPC3 mutation and resultant contractile dysfunction.

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Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N ′ )-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C ∆C0-C1f ), displayed dilated cardiomyopathy, underscoring the importance of the N ′ -region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, Abbreviations: cMyBP-C FL , full-length cardiac myosin binding protein C; cMyBP-C ∆C0-C1f , cMyBP-C lacking the C0-C1f region; TG, transgenic; WT, wild type; S2, subfragment 2; RLC, regulatory light chain; non-transgenic, NTG; C0-C1f, the first 271 residues of N ′ -region of cMyBP-C; I-R, ischemia-reperfusion; N ′ , amino terminal; t/t, cMyBP-C null homozygous mice; HF, heart failure; C0-C2, the first 448 residues of N ′ -region of cMyBP-C.

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Myocardial infarction (MI) leads to loss and degradation of contractile cardiac tissue followed by sterile inflammation of the myocardium through activation and recruitment of innate and adaptive cells of the immune system. Recently, it was shown that cardiac myosin binding protein-C (cMyBP-C), a protein of the cardiac sarcomere, is degraded following MI, releasing a predominant N-terminal 40-kDa fragment (C0C1f) into myocardial tissue and the systemic circulation. We hypothesized that early release of C0C1f contributes to the initiation of inflammation and plays a key role in recruitment and activation of immune cells. Therefore, we investigated the role of C0C1f on macrophage / monocyte activation using both mouse bone marrow-derived macrophages and human monocytes. Here we demonstrate that C0C1f leads to macrophage / monocyte activation in vitro. Furthermore, C0C1f induces strong upregulation of pro-inflammatory cytokines (interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and interleukin-1β (IL-1β)) in cultured murine macrophages and human monocytes, resulting in a pro-inflammatory phenotype. We identified the toll-like receptor 4 (TLR4), toll-like receptor 2 (TLR2), and Advanced Glycosylation End Product-Specific Receptor (RAGE) as potential receptors for C0C1f whose activation leads to mobilization of the NFκB signaling pathway, a central mediator of the pro-inflammatory signaling cascade. Thus, C0C1f appears to be a key player in the initiation of inflammatory processes and might also play an important role upon MI.

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