MECHANISMS OF EXERCISE INDUCED MUSCLE FIBER INJURY

这项研究的目的是了解 运动中短暂的肌膜撕裂可能会导致长期的 肌肉细胞内的损伤。杜兴氏肌营养不良症的肌肉 其中最初的损害是较高水平的一过性肌膜 撕裂，表现出类似于偏心运动的超微结构损伤。 Duchenne营养不良肌肉发生更大的钙专一性活动 漏水通道反过来又负责升高休止点 细胞内游离钙([Ca~(2+)]i)和更高的钙速率- 依赖蛋白水解酶。我们假设，在锻炼过程中，局部的 通过瞬间泪水的钙内流导致局部激活 通过蛋白分解的钙泄漏通道。最终，积累的 激活的渗漏通道会增加钙内流，静息[钙]i， 激活钙依赖的降解途径。瞬变 收缩活动中的肌膜撕裂，尤其是营养不良 肌肉，可能代表了过程激活的第一步 最终导致肌肉细胞死亡。 本研究将通过以下问题来检验这一假设： (1)[Ca~(2+)]_i的时空变化 严格定义、可重现的伤人事件？(2)伤人是否会导致 局部激活泄漏通道，如果是这样的话，(钙]i有什么变化 激活频道所需的？是创伤性渗漏通道 蛋白分解和对细胞骨架的影响所介导的激活？ (3)泄漏通道的活性是如何被蛋白质分解或 在切除的斑块中操纵细胞骨架？(4)是激活 收缩活动后钙依赖的降解过程 依赖于钙离子通过更活跃的渗漏渠道内流？(问题 1)在培养的小鼠骨骼肌管上进行精确的创伤观察 通过测量[Ca~(2+)]i的空间和时间变化(问题2) 然后将使用[Ca~(2+)]i变化的知识来确定 应用贴片观察钙离子内流与通道激活的关系 钳制方法。(问题3)监管渠道活动 蛋白质分解和细胞骨架的调节将使用 切开由内向外的膜片钳配置。(问题4)肌管将 处于长时间的收缩活动或静止状态 泄漏通道的二氢吡啶抑制剂与相关抑制 钙依赖酶降低的渗漏通道 流程。

The goal of this research is to understand the mechanisms by which transient tears at the sarcolemma during exercise might lead to long-term damage within a muscle cell. Muscles in Duchenne muscular dystrophy, in which the initial lesion is greater levels of transient sarcolemmal tearing, exhibit similar ultrastructural damage as eccentric exercise. Duchenne dystrophic muscles develop greater activity of calcium-specific leak channels which in turn are responsible for elevated resting intracellular free calcium ([Ca2+]i) and higher rates of calcium- dependent proteolysis. We hypothesize that, during exercise, local calcium influx through transient tears leads to local activation of calcium leak channels via proteolysis. Eventually, accumulation of activated leak channels will increase calcium influx, resting [Ca2+]i, and activation of calcium-dependent degradative pathways. Transient sarcolemmal tears during contractile activity, especially in dystrophic muscle, may represent the initial step in the activation of processes which eventually mediate muscle cell death. This study will examine this hypothesis through the following questions: (1) What are the spatial and temporal changes in [Ca2+]i near a rigorously defined, reproducible wounding event? (2) Does wounding lead to local activation of leak channels, and, if so, what changes in (Ca2+]i are required to activate channels? Is wound-induced leak channel activation mediated by proteolysis and by effects on the cytoskeleton? (3) How is activity of the leak channel modulated by proteolysis or manipulation of the cytoskeleton in excised patches? (4) Is activation of calcium-dependent degradative processes after contractile activity dependent on calcium influx through more active leak channels? (Question 1) Precise wounds on cultured mouse skeletal myotubes will be followed by measurement of spatial and temporal changes in [Ca2+]i. (Question 2) Knowledge of [Ca2+]i changes will then be employed to determine the relationship between calcium influx and channel activation using patch clamp methods. (Question 3) The regulation of channel activity by proteolysis and modulation of the cytoskeleton will be examined using the excised inside-out patch clamp configuration. (Question 4) Myotubes will be subject to long periods of contractile activity or quiescence and dihydropyridine inhibitors of the leak channel to correlate inhibition of leak channels with decreased rates of calcium-dependent enzymatic processes.