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.

中文翻译：

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合成粗丝：更好地了解健康、疾病和经 mavacamten 治疗的心脏系统中肌球蛋白超松弛状态的新途径


肌球蛋白-II 的一个标志性特征是它可以在低离子强度缓冲液中自发自组装成双极性合成粗丝 (STF)，从而作为肌肉粗丝的体外重建模型。虽然这些 STF 已广泛用于结构表征，但它们的功能评估受到限制。在本报告中，我们表明 STF 中的肌球蛋白反映了节能超松弛 (SRX) 状态下更多的静电和协作相互作用，而使用较短的肌球蛋白子片段、重的肌球蛋白 (HMM) 和肌球蛋白子片段则看不到这种相互作用。 -1（S1）。使用这些 STF，我们显示了肥厚型心肌病中的几种病理生理学损伤，包括 R403Q 肌球蛋白突变、肌球蛋白轻链磷酸化以及 ADP:ATP 比率增加导致 SRX 群体不稳定。此外，含有STF的野生型肌球蛋白，但不包括S1、HMM或含有STF的R403Q肌球蛋白，重现了ADP诱导的SRX状态的不稳定。涉及临床阶段小分子抑制剂 mavacamten 的研究表明，它不仅在增加 STF 中的肌球蛋白 SRX 数量方面比 S1 或 HMM 更有效，而且在健康和疾病制成的 STF 中也能同样有效地增加肌球蛋白 SRX 数量。引起 R403Q 肌球蛋白。重要的是，我们还发现病理生理学扰动（例如 ADP 浓度升高）会削弱 mavacamten 增加肌球蛋白 SRX 数量的能力，这表明与 mavacamten 结合的肌球蛋白头在 SRX 状态下不会受到永久保护，但可以被招募来发挥作用。这些发现共同强调，STF 作为一种有价值的工具，可以为健康、疾病和治疗条件下肌球蛋白 SRX 状态提供新的见解。