β-Cardiac myosin is the primary motor protein in the human heart responsible for force generation by converting chemical energy from ATP hydrolysis to mechanical work. It binds actin, produces force through its powerstroke, and releases actin, and must complete this cycle several times a second within each heartbeat. Like other muscle myosins, cardiac myosin has a low duty ratio of ∼5%, the fraction of time in the actin-bound force-producing state. Here, we present a hypertrophic cardiomyopathy (HCM) causing mutation, G768R, that increases cardiac myosin’s duty ratio to >60%, an unprecedented >10× increase unmatched by any previously studied muscle myosin mutation. In recombinantly expressed human β-cardiac myosin subfragment-1 (sS1), G768R dramatically decreases the load-sensitive actin-detachment rate and step size of single molecules measured by optical tweezers. The ∼15× longer actin-bound time combined with only a modest change in overall ATPase rate predicts a >60% duty ratio. Motility velocity is also severely slowed, as expected given the long bound time and decreased step size. All-atom molecular dynamic simulations of the pre-powerstroke and post-rigor states predict that the mutation may stabilize the lever arm in a high-angle primed state, hinder the subsequent powerstroke, and reduce ADP pocket opening, providing a possible structural mechanism to corroborate the experimental observations. Finally, ATPase and single-turnover experiments on 2-headed heavy meromyosin (HMM) constructs suggest that G768R destabilizes the super-relaxed state (SRX). Our findings of high duty ratio and reduced SRX provide molecular mechanisms of hypercontractility. A muscle myosin in the heart that stays bound to actin for over half its period is bound to have devastating functional and clinical consequences.
Pathak et al. (2026) studied this question.