Mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin

Abstract

We used transient biochemical and structural kinetics to elucidate the molecular mechanism of mavacamten, an allosteric cardiac myosin inhibitor and a prospective treatment for hypertrophic cardiomyopathy. We find that mavacamten stabilizes an autoinhibited state of two-headed cardiac myosin not found in the single-headed S1 myosin motor fragment. We determined this by measuring cardiac myosin actin-activated and actin-independent ATPase and single-ATP turnover kinetics. A two-headed myosin fragment exhibits distinct autoinhibited ATP turnover kinetics compared with a single-headed fragment. Mavacamten enhanced this autoinhibition. It also enhanced autoinhibition of ADP release. Furthermore, actin changes the structure of the autoinhibited state by forcing myosin lever-arm rotation. Mavacamten slows this rotation in two-headed myosin but does not prevent it. We conclude that cardiac myosin is regulated in solution by an interaction between its two heads and propose that mavacamten stabilizes this state.

Impact of the Myosin Modulator Mavacamten on Force Generation and Cross‐Bridge Behavior in a Murine Model of Hypercontractility

“…Interestingly, our data also show that Myk461 incubation significantly slowed k rel in the KO myocardium but not in the WT myocardium, effectively normalizing the basal differences in k rel between KO and WT groups (Table 3; Figure 5), suggesting a slowing in the strain‐induced XB detachment from actin in the KO myocardium. Our observation of unaltered k rel in WT myocardium agrees with results from solution‐based transient kinetic studies, which show that Myk461 does not affect the ADP release rate from actomyosin 22, 48. However, our result that Myk461 slows XB detachment in KO indicates a differential impact of Myk461 on XB detachment, suggesting that the impact of Myk461 may vary depending on the presence or absence of cMyBPC in an intact sarcomeric lattice system that is influenced by strain‐dependent mechanisms, a phenomenon that is difficult to replicate using solution‐based transient kinetic studies.…”

“…It has been shown that myocardial samples isolated from HCM patients expressing cMyBPC mutations often display reduced total cMyBPC expression7, 25 and accelerated XB kinetics27; therefore, we sought to investigate the effects of Myk461 on the contractile function of myocardium lacking cMyBPC. In this context, recent data show that Myk461 stabilizes an autoinhibited state of 2‐headed myosin structure48 and also strengthens a folded‐back sequestered super‐relaxed state of myosin heads onto the thick filament backbone49—an effect that contributes to Myk461‐induced force depression by stabilizing the “ off ” state of myosin heads and preventing acto‐myosin formation.…”

“…However, our result that Myk461 slows XB detachment in KO indicates a differential impact of Myk461 on XB detachment, suggesting that the impact of Myk461 may vary depending on the presence or absence of cMyBPC in an intact sarcomeric lattice system that is influenced by strain‐dependent mechanisms, a phenomenon that is difficult to replicate using solution‐based transient kinetic studies. Furthermore, the impact of Myk461 on myosin head kinetics varies depending on the presence of a 2‐headed cardiac heavy meromyosin or a single‐headed S1,48 suggesting that the structural complexity of the experimental preparations may be an important determinant in interpreting the effects of Myk461 on cardiac muscle contractile function.…”

Hypertrophic cardiomyopathy mutations at the folded-back sequestered β-cardiac myosin S1-S2 and S1-S1 interfaces release sequestered heads and increase myosin enzymatic activity

“…Anderson et al showed that the level of SRX measured by mant-ATP single turnover by WT 25hep HMM corresponds well to a folded-back state of the 25-hep WT HMM, as observed by negative staining electron microscopy 24 . To investigate whether the HCM mutations R249Q and H251N release sequestered myosin heads into a functionally active state, we used mant-ATP single turnover kinetics, a technique originally used to probe the level of SRX in cardiac myosin fibers 41,42 which has more recently been applied to purified cardiac myosin 24,44 . When bound to myosin, mant-ATP shows increased fluorescence.…”

The molecular basis of hypercontractility caused by the hypertrophic cardiomyopathy mutations R403Q and R663H

“…The potential importance of a folded-back sequestered state of cardiac myosin has recently been highlighted in diseases like HCM, and also in the mechanism of action of cardiac inhibitors like mavacamten (15,37,38). The cardiac activator omecamtiv mecarbil has also been shown to stabilize the open state of cardiac myosin while also impacting the kinetics of actin-bound myosin motors (39)(40)(41).…”