Synthetic thick filaments: A new avenue for better understanding the myosin super-relaxed state in healthy, diseased, and mavacamten-treated cardiac systems

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 functional evaluation has been limited. In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin sub-fragments, heavy meromyosin (HMM) and myosin subfragment-1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and increased ADP:ATP ratio destabilize the SRX population. Furthermore, wild-type myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small molecule inhibitor, mavacamten, showed that it is not only more effective in increasing myosin SRX population in STFs than in S1 or HMM , but it also increases myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations such as elevated ADP concentration weakens the mavacamten’s ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, disease, and therapeutic conditions.

Cardiac myosin super relaxation (SRX): a perspective on fundamental biology, human disease and therapeutics

“…This result inferred that the mechanism of action of RLC phosphorylation is the increase in Huxley's N a parameter, which is consistent with a reduction of SRX and increase in DRX myosin states (Craig et al, 1987;Nag et al, 2017;Toepfer et al, 2016b). This has recently been validated in a synthetic thick filament system (Gollapudi et al, 2020). In addition phosphorylation of cRLC leads to a higher power production and V max in trabecular muscle (Toepfer et al, 2013), inferring that cRLC phosphorylation is central to adapting sarcomeric power, which is at least partly mediated by altering IHM interactions.…”

TWO CLASSES OF MYOSIN INHIBITORS, BLEBBISTATIN AND MAVACAMTEN, STABILIZE Β-Cardiac MYOSIN IN DIFFERENT STRUCTURAL AND FUNCTIONAL STATES

“…1C and Table 1). IC50 values reported for mavacamten, and blebbistatin in this study are consistent with those reported in the literature (21,25,26,28,29,38).…”

“…3A. The relative SRX population increased gradually with increasing mavacamten concentration, reaching 100% at 3.25 μM, suggesting that mavacamten shifted the myosin DRX-SRX equilibrium more towards the SRX state, consistent with what has been observed earlier (23,25,26). The concentration of mavacamten required to attain a half-maximal increase of myosin population in the SRX state is 1.21±0.35 μM, close to the IC50 of ATPase data measured in various systems (Table 1).…”

“…The ability of mavacamten to counter enhanced contractility has also been investigated in vitro using hypertrophic cardiomyopathy-causing mutations in myosin, myosinbinding protein C, troponin T, and troponin I (21)(22)(23)(24). Many elegant mechanistic studies showed that, functionally, mavacamten populates myosin in the low-energy consuming SRX state (23,25,26). Using electron microscopy and muscle fiber X-ray diffraction studies, it has also been shown that mavacamten structurally stabilizes a closed state reminiscent of the IHM state (23), thereby suggesting a link between the structurally closed state and the SRX functionality.…”

To lie or not to lie: Super-relaxing with myosins

“…This report is discussed further under section ‘Diseased state and therapeutic modulators of myosin SRX state ’. In reconstituted full-length porcine cardiac myosin thick filaments, it has also been recently reported that either phosphorylation of the RLC with MLCK or depletion of RLC decreases the myosin population in the SRX state ( Gollapudi et al, 2020a ). All of these observations together suggest an RLC phosphorylation-mediated shift in DRX↔SRX state equilibrium toward the DRX state of myosin.…”