Impact of Hypertrophic Cardiomyopathy Mutations on the Cardiac Myosin Super-Relaxed State

Abstract

We aim to examine the effects of hypertrophic cardiomyopathy (HCM) associated mutations on the myosin super-relaxed state (SRX) through functional ATPase assays and time-resolved FRET structural measurements. Cardiac myosin isomerizes between the relaxed state (RX), which is available for activation and subsequent force generation with actin, and the kinetically inactive SRX, in which myosin is hypothesized to be structurally occluded from the force-generation cycle by the interacting-heads motif (IHM). We hypothesize that myosin regulatory light chain (RLC) mutations associated with HCM, specifically E22K and R58Q, cause hypercontractility through disruption of the myosin SRX. Based on proposed models of the IHM, these residues form stabilizing salt-bridge interactions with the opposite myosin. We hypothesize that mutations in the RLC associated with HCM increase myosin's basal ATPase activity due to disruption of SRX. To observe the effect of these mutations, we have performed ATPase assays to measure changes in myosin function, and time-resolved FRET to directly detect the changes in head-head interactions, due to changes in the population of SRX myosin. These measurements were performed on bovine cardiac myosin containing human cardiac RLC. This work was supported by NIH grants AR032961 and AR057220 (to DDT), and an American Heart Association Scientist Development Grant (to JMM).