Abstract 915: Dysregulation of the Myosin and Myosin Binding Protein-C interaction in Hypertrophic Cardiomyopathy

Rationale: Affecting 1 in 300 individuals, hypertrophic cardiomyopathy (HCM) is a genetic heart disease characterized by left ventricular hypertrophy, myocardial disarray, and sudden cardiac death. Often, HCM is associated with myocardial hypercontractility. The subfragment-2 (S2) of beta-myosin heavy chain contains a cluster of missense and deletion mutations associated with severe HCM. Interestingly, myosin S2 interacts with the C0-C2 region of cardiac myosin binding protein C (cMyBP-C) in a phosphorylation-dependent manner to regulate sarcomere contractility. However, the nature of myosin S2 and cMyBP-C interactions and the mechanism(s) by which mutations in myosin S2 cause HCM remain to be elucidated. Objective: To determine whether mutations in myosin S2 weaken its interaction with cMyBP-C, resulting in enhanced myofilament contractility. Methods and Results: Myosin S2 proteins (126 amino acids) containing three clinically relevant mutations (R870H, E924K, E930del, or wild type), and recombinant C0-C2 region of cMyBP-C were produced and purified by metal affinity chromatography. Solid-phase binding assays and isothermal calorimetry experiments revealed a significantly dampened binding to C0-C2 for these three mutants in myosin S2 (30% lower than wild type, p<0.002), suggesting that mutations in S2 regions reduce their bindings to cMyBP-C. Conversely, upon protein kinase A phosphorylation of C0C2, these S2 mutants displayed an increased affinity to cMyBP-C, an effect opposite that of wild-type S2. Structural analyses of these mutations within myosin S2 are predicted to reduce the alpha-helical content, thereby reducing stability of the critical coiled-coil structure. Conclusions: Mutations in myosin S2 result in reduced binding to cMyBP-C. Functionally, this would result in a greater attachment of cross-bridges, and thus enhance myofilament contractility. Strikingly, these mutations increase their affinity to cMyBP-C upon phosphorylation, demonstrating fundamental changes to the regulation of contractile function.