RationaleMavacamten is a novel, FDA-approved, small molecule therapeutic designed to regulate cardiac function at the sarcomere level by selectively but reversibly inhibiting the enzymatic activity of myosin. It shifts myosin towards orderedoffstates close to the thick filament backbone.ObjectiveThe purpose of this study is to investigate whether mavacamten permanently sequester these myosin heads in theoffstate(s) and can these heads be recruited in response to physiological stimuli when required to boost cardiac output.Methods and ResultsWe show that cardiac myosins stabilized in theseoffstate(s) by mavacamten are recruitable by 1) Ca2+, 2) increased chronotropy (heart rate), 3) stretch, and 4) β-adrenergic (β-AR) stimulation, all known physiological inotropic effectors. At the molecular level, we show that Ca2+increases myosin ATPase activity by shifting mavacamten-stabilized myosin heads from the inactive super-relaxed (SRX) state to the active disordered relaxed (DRX) state. At the myofilament level, both Ca2+and passive lengthening can shift mavacamten-orderedoffmyosin heads from positions close to the thick filament backbone to disorderedonstates closer to the thin filaments. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmedin vivoin telemetered rats, where left-ventricular dP/dtmax,an index of inotropy, increased with heart rate in mavacamten treated animals. Finally, we show that β-AR stimulationin vivoincreases left-ventricular function and stroke volume in the setting of mavacamten.ConclusionsOur data demonstrate that the mavacamten-promotedoffstates of myosin in the thick filament are activable, thus leading to the preservation of the cardiac reserve. These results provide a potential mechanistic explanation, beyond mere LV outflow tract obstruction removal, for the clinical observation of increased peak oxygen uptake (pVO2) with exercise in HCM patients receiving mavacamten.