Background: About 45% of identified HCM-causing mutations are in cardiac myosin binding protein C (cMyBP-C). One prevalent missense mutation is R495Q, which is in a positively charged pocket and alters the predicted electrostatic properties of the C3 domain. Many known HCM-causing mutations cause hypercontractility, likely through promoting the disordered relaxed state (DRX), in which myosin heads are more likely to bind to actin to generate contractile force, relative to the super relaxed state (SRX) in which myosin heads fold back on myosin tails. Mavacamten (MAVA), an FDA-approved drug for HCM patients with left ventricular outflow obstruction, is proposed to reduce DRX:SRX toward normal by inhibiting the myosin ATPase. It is unclear if all HCM-causing mutations act via this mechanism. Question: What is the contractile phenotypic timeline of MYBPC3 R495Q in human iPSC-engineered heart tissue (EHT) and can chronic MAVA ameliorate this phenotype? Aim: Define the phenotype progression in EHTs homozygous for the R495Q mutation (QQ), compared to isogenic controls (RR). We will use this timeline, combined with pharmacologic intervention to gain mechanistic insight into the pathogenesis of the R495Q mutation. Methods: 3D EHTs were generated from CRISPR/Cas-9 edited QQ and RR human iPSC cardiomyocytes. We used capillary immunoassay to quantify cMyBP-C expression and immunocytochemistry to determine protein localization. Contractile function of EHTs was assessed weekly (weeks 1-6) with chronic treatment of MAVA (100nM) starting at day 8 of EHT culture. Results: QQ EHTs express normal cMyBP-C protein levels with appropriate incorporation into the sarcomere, yet at day 8 show increased twitch force (TF) and fast relaxation (Fig A-B). At day 36, QQs have normalized TF and slow relaxation. Importantly, MAVA normalizes relaxation time (Fig C,D). Conclusion: These data suggest that R495Q presents with an early hypercontractile phenotype. With prolonged time in culture, TF is reduced, and relaxation time slows. Chronic administration of MAVA quickened relaxation, but reduced total force compared to untreated normal EHT, suggesting that caution may be warranted to ensure that systolic function isn’t impaired.

Hypercontractility, caused by mutations that increase the number of myosin heads in the disordered relaxed (DRX) state, is a prevalent cause of hypertrophic cardiomyopathy (HCM). A subgroup of HCM mutations cause primary hypocontractility, by stabilizing super-relaxed crossbridges (SRX). Mavacamten (mava) directly targets hypercontractility by reducing the number of DRX heads and improves outcomes in ~70% patients with obstructive HCM. We here assessed the efficacy of chronic mava treatment at preventing phenotype development in human iPSC engineered heart tissue (EHT) in a cMyBP-C null model (-/-), that increases the number of DRX heads, and EHT carrying the W792R (RR) HCM mutation. While the primary contractile defect caused by the W792R mutation is unknown, we postulated that it differed from the cMyBP-C -/- model, as RR mice die prior to weaning while -/- mice do not. Twitch force (TF) measurements performed on day 7 on control (+/+), -/- and RR EHT show that -/- EHT were hypercontractile while RR EHT were hypocontractile (Fig. 1). Following measurement, EHT from each genotype were either treated with vehicle or 100nM mava and cultured for 21 days. By day 28, TF production was similar between +/+ and -/- vehicle treated EHT while RR EHT remained hypocontractile (Fig. 2). Chronic mava treatment significantly reduced TF production in +/+ EHT but not in -/- or RR EHT (Fig. 2). Consistent with diastolic dysfunction, a hallmark of HCM in humans, day 28 -/- and RR EHT displayed slow relaxation, though the effect was more pronounced in RR EHT (Fig. 3). While chronic mava treatment accelerated relaxation in -/- EHT to levels that were similar to that observed in vehicle treated +/+ EHT, it failed to do so in RR EHT (Fig. 3). cMyBP-C -/- EHTs initially present with a hypercontractile phenotype, consistent with an increase in DRX crossbridges, while W792R RR EHT present with a hypocontractile phenotype. By 4 weeks in EHT culture, both genotypes present with impaired relaxation, that is more severe in RR EHT. While chronic mava treatment prevents development of slow relaxation in -/- EHT, it failed to do so in RR EHT. These findings suggest that the primary disease mechanism of the W792R mutation is not an increase in DRX crossbridges and that mavacamten may not be an effective treatment for patients carrying this mutation, further highlighting the need for studying the pathomechansims of HCM mutations and the development of novel therapies.

Missense mutations in cardiac myosin binding protein C (cMyBP-C) are known to cause hypertrophic cardiomyopathy (HCM). The W792R mutation in the C6 domain of cMyBP-C causes severe, early onset HCM in humans, yet its impact on the function of cMyBP-C and the mechanism through which it causes disease remain unknown. To fully characterize the effect of the W792R mutation on cardiac morphology and function in vivo, we generated a murine knock-in model. We crossed heterozygous W792RWR mice to produce homozygous mutant W792RRR, heterozygous W792RWR, and control W792RWW mice. W792RRR mice present with cardiac hypertrophy, myofibrillar disarray and fibrosis by postnatal day 10 (PND10), and do not survive past PND21. Full-length cMyBP-C is present at similar levels in W792RWW, W792RWR and W792RRR mice and is properly incorporated into the sarcomere. Heterozygous W792RWR mice displayed normal heart morphology and contractility. Permeabilized myocardium from PND10 W792RRR mice showed increased Ca2+ sensitivity, accelerated cross-bridge cycling kinetics, decreased cooperativity in the activation of force, and increased expression of hypertrophy-related genes. In silico modeling suggests that the W792R mutation destabilizes the fold of the C6 domain and increases torsion in the C5-C7 region, possibly impacting regulatory interactions of cMyBP-C with myosin and actin. Based on the data presented here, we propose a model in which mutant W792R cMyBP-C preferentially forms Ca2+ sensitizing interactions with actin, rather than inhibitory interactions with myosin. The W792R-cMyBP-C mouse model provides mechanistic insights into the pathology of this mutation and may provide a mechanism by which other central domain missense mutations in cMyBP-C may alter contractility, leading to HCM.

Many hypertrophic cardiomyopathy (HCM) causing mutations result in myocardial hypercontractility by increasing the number of myosin crossbridges in the disordered relaxed (DRX) state, or by sensitizing the sarcomere to the effects of Ca 2+ . Mavacamten (Mava) recently became the first FDA-approved treatment of obstructive HCM that directly targets hypercontractility by reducing the number of DRX cross bridges. Mava improves outcomes in patients with obstructive HCM but has not been approved in children or adults without obstructive HCM. The efficacy of chronic Mava treatment at preventing phenotype development in asymptomatic HCM mutation carriers remains largely unexplored. We recently showed that ablation of cardiac myosin binding protein-C (cMyBP-C -/-) in engineered heart tissue (EHT) causes rapid phenotype development in a dish. Initial hypercontractility progresses to hypocontractility with impaired relaxation, mediated by Ca 2+ mishandling. Here we investigate whether chronic Mava administration prevents phenotype progression. cMyBP-C +/+, +/- and -/- EHT were treated with 100nM Mava starting on day 7 of EHT culture. Serial twitch force (TF) measurements were performed weekly for 5 weeks, using the Mantarray platform. At day 7, prior to Mava treatment, +/- and -/- produced significantly more TF than +/+ EHT (392 ± 9 μN vs. 390 ± 15 μN vs. 199 ± 13 μN). At day 14, Mava treatment reduced TF by 31% (p < 0.001) in +/+ EHT, 30% (p < 0.001) in +/- EHT and 12% (p = 0.115) in -/- EHT. After an additional 4 weeks, chronic Mava treatment reduced TF by 54% (p < 0.001) in +/+ EHT, 65% (p < 0.001) in +/- EHT but increased TF by 23% (p = 0.034) in -/- EHT.These data show that chronic Mava treatment prevents/slows maladaptive remodeling in -/- EHT, but depresses contractility in +/+ and +/- ECT, suggesting that chronic Mava treatment may be detrimental to patients with mild HCM. Our ongoing studies are assessing the effect of chronic Mava treatment on other EHT models of HCM and on Ca 2+ -handling.