Striated muscle contraction is the result of sarcomeres, the basic contractile unit, shortening because of hydrolysis of adenosine triphosphate (ATP) by myosin molecular motors. In noncontracting, "relaxed" muscle, myosin still hydrolyzes ATP slowly, contributing to the muscle's overall resting metabolic rate. Furthermore, when relaxed, myosin partition into two kinetically distinct subpopulations: a faster-hydrolyzing "relaxed" population, and a slower-hydrolyzing "super relaxed" (SRX) population. How these two myosin subpopulations are spatially arranged in the sarcomere is unclear, although it has been proposed that myosin-binding protein C (MyBP-C) may stabilize the SRX state. Because MyBP-C is found only in a distinct region of the sarcomere, i.e., the C-zone, are SRX myosin similarly colocalized in the C-zone? Here, we imaged the binding lifetime and location (38-nm resolution) of single, fluorescently labeled boron-dipyrromethene-labeled ATP molecules in relaxed skeletal muscle sarcomeres from rat soleus. The lifetime distribution of fluorescent ATP-binding events was well fitted as an admixture of two subpopulations with time constants of 26 ± 2 and 146 ± 16 s, with the longer-lived population being 28 ± 4% of the total. These values agree with reported kinetics from bulk studies of skeletal muscle for the relaxed and SRX subpopulations, respectively. Subsarcomeric localization of these events revealed that SRX-nucleotide-binding events are fivefold more frequent in the C-zone (where MyBP-C exists) than in flanking regions devoid of MyBP-C. Treatment with the small molecule myosin inhibitor, mavacamten, caused no change in SRX event frequency in the C-zone but increased their frequency fivefold outside the C-zone, indicating that all myosin are in a dynamic equilibrium between the relaxed and SRX states. With SRX myosin found predominantly in the C-zone, these data suggest that MyBP-C may stabilize and possibly regulate the SRX state.

中文翻译：

---

静息骨骼肌中的 ATP 消耗成像：一次一个分子

横纹肌收缩是肌节（基本收缩单位）由于肌球蛋白分子马达水解三磷酸腺苷 (ATP) 而缩短的结果。在非收缩、“放松”的肌肉中，肌球蛋白仍然缓慢地水解 ATP，有助于肌肉的整体静息代谢率。此外，当放松时，肌球蛋白分为两个动力学不同的亚群：水解速度更快的“放松”群体和水解速度较慢的“超级放松”（SRX）群体。这两个肌球蛋白亚群如何在肌节中空间排列尚不清楚，尽管有人提出肌球蛋白结合蛋白 C (MyBP-C) 可能稳定 SRX 状态。因为 MyBP-C 仅存在于肌节的不同区域，即 C 区，SRX 肌球蛋白是否同样位于 C 区？在这里，我们对来自大鼠比目鱼肌的松弛骨骼肌肌节中单个荧光标记的硼-二吡咯亚甲基标记的 ATP 分子的结合寿命和位置（38 nm 分辨率）进行了成像。荧光 ATP 结合事件的寿命分布非常适合作为两个亚群的混合物，时间常数为 26 ± 2 和 146 ± 16 s，寿命较长的种群占总数的 28 ± 4%。这些值分别与松弛和 SRX 亚群的骨骼肌批量研究报告的动力学一致。这些事件的亚肌节定位表明，与没有 MyBP-C 的侧翼区域相比，C 区（存在 MyBP-C 的地方）中 SRX 核苷酸结合事件的频率高五倍。用小分子肌球蛋白抑制剂治疗，mavacamten，在 C 区没有引起 SRX 事件频率的变化，但在 C 区外将它们的频率增加了五倍，表明所有肌球蛋白都处于松弛状态和 SRX 状态之间的动态平衡。由于 SRX 肌球蛋白主要位于 C 区，这些数据表明 MyBP-C 可能稳定并可能调节 SRX 状态。