Muscle Biophysics Laboratory, Department of Kinesiology, University of Massachusetts, Amherst, Massachusetts.
Am J Physiol Cell Physiol. 2019 Oct 1;317(4):C714-C718. doi: 10.1152/ajpcell.00196.2019. Epub 2019 Jul 24.
Intracellular acidosis is a putative agent of skeletal muscle fatigue, in part, because it depresses the calcium (Ca) sensitivity of the myofilaments. However, the molecular mechanism behind this depression in Ca sensitivity is unknown, providing a significant challenge to a complete understanding of the fatigue process. To elucidate this mechanism, we directly determined the effect of acidosis on the ability of a single myosin molecule to bind to a regulated actin filament in a laser trap assay. Decreasing pH from 7.4 to 6.5 significantly ( < 0.05) reduced the frequency of single actomyosin-binding events at submaximal (pCa 8-pCa 6) but not at maximal Ca concentration (pCa 5-pCa 4). To delineate whether this was due to a direct effect on myosin versus an indirect effect on the regulatory proteins troponin (Tn) and tropomyosin (Tm), binding frequency was also quantified in the absence of Tn and Tm. This revealed that acidosis did not significantly alter the frequency of actomyosin binding events in the absence of regulatory proteins (1.4 ± 0.15 vs. 1.4 ± 0.15 events/s for pH 7.4 and 6.5, respectively). Acidosis also did not significantly affect the size of myosin's powerstroke or the duration of binding events in the presence of regulatory proteins, at every [Ca]. These data suggest acidosis impedes activation of the thin filament by competitively inhibiting Ca binding to TnC. This slows the rate at which myosin initially attaches to actin; therefore, less cross bridges will be bound and generating force at any given submaximal [Ca]. These data provide a molecular explanation for the acidosis-induced decrease in force observed at the submaximal Ca concentrations that might contribute to the loss of force during muscle fatigue.
细胞内酸中毒是骨骼肌疲劳的一个潜在因素，部分原因是它降低了肌球蛋白丝对钙 (Ca) 的敏感性。然而，这种 Ca 敏感性降低的分子机制尚不清楚，这对全面了解疲劳过程构成了重大挑战。为了阐明这一机制，我们在激光阱测定中直接测定了酸中毒对单个肌球蛋白分子与调节肌动蛋白丝结合能力的影响。将 pH 值从 7.4 降低到 6.5 显著 ( < 0.05) 降低了亚最大 Ca 浓度 (pCa 8-pCa 6) 而非最大 Ca 浓度 (pCa 5-pCa 4) 下单个肌球蛋白-肌动球蛋白结合事件的频率。为了确定这是由于对肌球蛋白的直接作用还是对调节蛋白肌钙蛋白 (Tn) 和原肌球蛋白 (Tm) 的间接作用，还在没有 Tn 和 Tm 的情况下量化了结合频率。这表明酸中毒并没有显著改变在没有调节蛋白的情况下肌球蛋白-肌动球蛋白结合事件的频率 (pH 7.4 和 6.5 时分别为 1.4±0.15 和 1.4±0.15 个事件/s)。酸中毒也没有显著影响在有调节蛋白存在的情况下，每个 [Ca] 时肌球蛋白的功率冲程大小或结合事件的持续时间。这些数据表明酸中毒通过竞争性抑制 Ca 与 TnC 的结合来阻碍细肌丝的激活。这会降低肌球蛋白最初与肌动蛋白结合的速度；因此，在任何给定的亚最大 [Ca] 下，将结合更少的横桥并产生力。这些数据为酸中毒导致的亚最大 Ca 浓度下力的降低提供了分子解释，这可能导致肌肉疲劳时力的丧失。
