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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 filaments. Although 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 subfragments, 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 an increased ADP:ATP ratio, destabilize the SRX population. Furthermore, WT 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 more effective in not only increasing myosin SRX population in STFs than in S1 or HMM but also in increasing 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 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, diseased, and therapeutic conditions.
Highlights
Vertebrate striated muscle contraction is a result of cyclic interactions between myosin heads on the thick filaments and actin monomers on the thin filament
Steady-state Measurements of Basal Myosin ATPase Activity in S1, heavy meromyosin (HMM), and synthetic thick filaments (STFs) To evaluate whether bovine cardiac (Bc) S1, HMM, and STF differ in their biochemical properties, we first measured the basal ATPase activity in these myosin systems
The basal ATPase measured in other myosin models such as porcine cardiac (Pc) STFs made of WT and R403Q myosin in this study was in the range of 0.010-0.025 s-1
Summary
Vertebrate striated muscle contraction is a result of cyclic interactions between myosin heads on the thick filaments and actin monomers on the thin filament. In the muscle thick filaments, it has been hypothesized that, in addition to myosin interactions with titin and myosinbinding protein C (MyBPC), the folded-back myosin IHM state is stabilized by several intramolecular interactions among various sub-domains of myosin such as the regulatory light chain (RLC), heavy meromyosin (HMM), light meromyosin (LMM), subfragment-1 (S1) and subfragment-2 (S2), that give rise to S1-S1, S1-S2, RLC-RLC, S1-LMM and S2-LMM interactions (reviewed in [1,2,3]) If these interactions underlying the IHM state give rise to the functional SRX state, it is essential to build an experimental model that can capture most of these features.
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