Abstract 4141331: The HCM-causing Mutation R495Q in Cardiac Myosin Binding Protein C Causes Contractile Changes in Human Engineered Heart Tissue that is Ameliorated by Chronic Mavacamten Treatment

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.