Mavacamten, formerly known as MYK-461 is a recently discovered novel small-molecule modulator of cardiac myosin that targets the underlying sarcomere hypercontractility of hypertrophic cardiomyopathy, one of the most prevalent heritable cardiovascular disorders. Studies on isolated cells and muscle fibers as well as intact animals have shown that mavacamten inhibits sarcomere force production, thereby reducing cardiac contractility. Initial mechanistic studies have suggested that mavacamten primarily reduces the steady-state ATPase activity by inhibiting the rate of phosphate release of β-cardiac myosin-S1, but the molecular mechanism of action of mavacamten has not been described. Here we used steady-state and presteady-state kinetic analyses to investigate the mechanism of action of mavacamten. Transient kinetic analyses revealed that mavacamten modulates multiple steps of the myosin chemomechanical cycle. In addition to decreasing the rate-limiting step of the cycle (phosphate release), mavacamten reduced the number of myosin-S1 heads that can interact with the actin thin filament during transition from the weakly to the strongly bound state without affecting the intrinsic rate. Mavacamten also decreased the rate of myosin binding to actin in the ADP-bound state and the ADP-release rate from myosin-S1 alone. We, therefore, conclude that mavacamten acts on multiple stages of the myosin chemomechanical cycle. Although the primary mechanism of mavacamten-mediated inhibition of cardiac myosin is the decrease of phosphate release from β-cardiac myosin-S1, a secondary mechanism decreases the number of actin-binding heads transitioning from the weakly to the strongly bound state, which occurs before phosphate release and may provide an additional method to modulate myosin function.

We have identified a small molecule inhibitor, MYK0000461, of the cardiac myosin ATPase. This agent was characterized in steady state and transient kinetic assays to understand its mechanism of action. MYK0000461 decreases the steady-state rate of the ATPase activity of purified bovine β-cardiac myosin subfragment-1 (S1) as well as that of bovine cardiac myofibrils, wild type and the mutant R453C of recombinant human β-cardiac myosin S1. We also find that MYK0000461 inhibits cardiac myosin selectively as compared to systems containing rabbit skeletal or chicken smooth muscle myosins Analysis of the individual steps of the chemo-mechanical cycle of cardiac myosin suggests that MYK0000461 exerts its effect by inhibiting the actin-stimulated release of phosphate, presumably by stabilizing the detached state of cardiac myosin prior to the release of phosphate. We find no evidence to suggest that MYK000461 inhibits cardiac myosin in a strongly bound state and no other steps in the chemo-mechanical cycle are affected by MYK0000461. Thus, the enzymatic step governing the weak to strong transition of S1 binding to actin is inhibited without affecting the release from the strongly bound states. This decrease in the rate of transition from the weak to strongly bound state should decrease force production and may underlie its ability to decrease cardiac contractility in cellular and in vivo models of cardiac function. An agent such as MYK0000461 could potentially be used to treat cardiac disorders that stem from hyper contractility such as the genetic hypertrophic cardiomyopathies (HCM). By decreasing the net force of contraction and restoring it back to normal level could potentially be useful in treating patients that suffer from this disease.

following ischemia-reperfusion, indicating mg53-/mice are more susceptible to CS injury. rhMG53 protein (6 mg/kg) applied intravenously to the wild type mice prior to the onset of CS could protect skeletal muscle injury, as demonstrated by the reduction of CK in serum and Evans Blue positive (injured) muscle fibers. Histochemical studies revealed that rhMG53 treatment could ameliorate pathological changes in mouse skeletal muscle associated with CS. When rhMG53 was examined in a rat model of CS-induced muscle injury, we saw a lesser degree of muscle injury and minimum effect for the exogenous rhMG53 in protection against the development of CS. The lack of effect of rhMG53 in the rat model was likely due to the fact that serum level of endogenous MG53 protein in the rat is more than 20-fold higher than that in the mouse and other animal models. Taken together, our data suggest that rhMG53 can protect ischemia-reperfusion induced muscle injury as a potential therapy for protection against compartment syndrome.

Genetic hypertrophic cardiomyopathy (HCM) results from mutations in the cardiac sarcomere, including β-cardiac myosin, with HCM afflicting about 1 out of every 500 people in the United States. HCM is characterized by hyper-contractility and myocyte hypertrophy. Current agents used to treat HCM include β-blockers and Ca2+ channel blockers to decrease the hyper-contractility and improve cardiac relaxation. A novel and more direct approach to decreasing hyper-contractility and improving diastolic relaxation in HCM patients is by modulating β-cardiac myosin to produce less force. We believe modulation at the sarcomere level provides a focused strategy while lowering the potential for adverse drug events. In this study we examined the effects of MYK0000461, a cardiac myosin selective inhibitor, on adult rat cardiomyocytes to fully understand the mechanism of action on excitation-contraction (E-C) coupling. Cellular contractility was assessed using edge detection and the calcium transient was measured using fura-2 loaded myocytes. MYK0000461 decreased contractility in a dose dependent manner with an IC50 of 250nM without altering the calcium transient. This inhibitory effect can be reversed by stimulation of the β-adrenergic pathway with the known agonist isoproterenol. We hypothesize that modulation of mutant β-cardiac myosin activity with a small molecule agent such as MYK0000461 could potentially treat disorders resulting from hyper-contractility such as HCM.