CONSERVED INTERACTIONS IN CARDIAC SYNTHETIC THICK FILAMENTS DIFFERENTLY AFFECT MYOSIN SUPER-RELAXED STATE IN HEALTHY, DISEASE AND MAVACAMTEN-TREATED MODELS

Abstract

A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low ionic strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filament. While these STFs have been extensively used for structural characterization, their use for functional studies has been very limited. In this report, we show that the ultra-low ATP-consuming super-relaxed (SRX) state of myosin is electrostatically more stable in STFs as compared with shorter myosin sub-fragments that lack the distal tail required for thick filament assembly. However, this electrostatic stability of the SRX state is weakened by phosphorylation of myosin light chains or the hypertrophic cardiomyopathy-causing myosin R403Q mutation. We also show that ADP binding to myosin depopulates the SRX population in STFs made of wild-type (WT) myosin, but not in S1, HMM, or STFs made of mutant R403Q myosin. Collectively, these findings emphasize that a critical network of inter- and intra-molecular interactions that underlie the SRX state of myosin are mostly preserved in STFs, establishing it as a native-like tool to interrogate myosin regulation. Next, using STFs, we show that a clinical-stage small molecule inhibitor, mavacamten, is more effective in promoting the myosin SRX state in STFs than in S1 or HMM and that it is equally potent in STFs made of atrial-WT, ventricular-WT, and mutant-R403Q myosin. Also, we found that mavacamten-bound heads are not permanently protected in the SRX state but can be recruited in response to physiological perturbations, thus providing new insights into its inhibitory mechanism.