Dynamics of the β-cardiac myosin auto-inhibited state explain cardiomyopathy pathogenesis

Abstract Cardiac contractility requires precise regulation. A recently discovered form of regulation of cardiac contractility involves a β-cardiac myosin off-state, the Interacting-Heads Motif ( Car IHM). Despite its central role in cardiac physiology and disease, Car IHM structural dynamics remain poorly understood. Here, we integrate near-atomic resolution cryo-EM with all-atom molecular dynamics to characterize Car IHM in solution and in the context of the filament. We describe its conformational ensembles maintained by dynamic interfaces, and the stabilizing effect of the dilated cardiomyopathy mutation E525K, which limits S2 coiled-coil flexibility and impairs myosin activation. Intrinsically disordered regions of Car IHM and MyBP-C further modulate these dynamics. Our findings provide evidence for how Car IHM ensembles balance off/on states and anchor myosin heads in distinct thick filament environments. This integrated structural and dynamic approach enhances the understanding of thick filament regulation and facilitates predictions of the effects of genetic variants in inherited cardiomyopathies.