Myosin in autoinhibited off state(s), stabilized by mavacamten, can be recruited via inotropic effectors

Rationale Mavacamten 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 ordered off states close to the thick filament backbone. Objective The purpose of this study is to investigate whether mavacamten permanently sequester these myosin heads in the off state(s) and can these heads be recruited in response to physiological stimuli when required to boost cardiac output. Methods and Results We show that cardiac myosins stabilized in these off state(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-ordered off myosin heads from positions close to the thick filament backbone to disordered on states closer to the thin filaments. In isolated rat cardiomyocytes, increased stimulation rates enhanced shortening fraction in mavacamten-treated cells. This observation was confirmed in vivo in 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 stimulation in vivo increases left-ventricular function and stroke volume in the setting of mavacamten. Conclusions Our data demonstrate that the mavacamten-promoted off states 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. ### Competing Interest Statement C.Z, and R.S, are employees and own shares in Bristol Myers Squibb (formerly MyoKardia). S.N and S.G are former employees of Bristol Myers Squibb (formerly MyoKardia). C.dR. has a consulting relationship with Bristol Myers Squibb. S.M.M and M.P are partners in FilamenTech, Inc. and they provided the simulations at no cost. T.C.I. consults for Edgewise Therapeutics, but this activity has no relation to the current work.