MECHANISMS OF EXERCISE INDUCED MUSCLE FIBER INJURY

运动引起肌纤维损伤的机制

• 批准号: 2607934
• 负责人: RICHARD A STEINHARDT
• 金额: $ 20.99万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 2000-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2607934
• 关键词: actins; calcium channel; calcium flux; chloride channels; confocal scanning microscopy; endopeptidases; exercise; laboratory mouse; muscle cells; muscle function; muscle metabolism; muscle proteins; muscular dystrophy; myotubes; nitric oxide synthase; phosphodiesterases; protease inhibitor; sarcolemma; striated muscles; tissue /cell culture; voltage /patch clamp; wound healing

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.

这项研究的目的是了解其机制 运动过程中肌膜的短暂撕裂可能会导致长期的 肌肉细胞内的损伤。杜氏肌营养不良症中的肌肉 最初的病变是较高水平的暂时性肌膜 撕裂，表现出与离心运动类似的超微结构损伤。 杜氏营养不良性肌肉产生更大的钙特异性活性 泄漏通道反过来又负责提高休息 细胞内游离钙 ([Ca2+]i) 和较高的钙-比率 依赖性蛋白水解。我们假设，在运动过程中，局部 通过短暂的眼泪钙流入导致局部激活 通过蛋白水解作用的钙泄漏通道。最终积累 激活的渗漏通道将增加钙流入，静息 [Ca2+]i， 和钙依赖性降解途径的激活。瞬态 收缩活动期间肌膜撕裂，尤其是营养不良者 肌肉，可能代表过程激活的初始步骤 最终介导肌肉细胞死亡。 本研究将通过以下问题检验这一假设： (1) a附近[Ca2+]i的时空变化是怎样的？ 严格定义的、可重复的伤害事件？ (2) 伤人是否导致 泄漏通道的局部激活，如果是这样，(Ca2+]i 发生了什么变化 需要激活通道吗？是否是伤口引起的渗漏通道 由蛋白水解和对细胞骨架的影响介导的激活？ (3) 渗漏通道的活性如何通过蛋白水解或 操纵切除斑块中的细胞骨架？ (4) 是否激活 收缩活动后钙依赖性降解过程的影响 依赖于通过更活跃的渗漏通道的钙流入？ （问题 1) 培养小鼠骨骼肌管上的精确伤口将被跟踪 通过测量 [Ca2+]i 的空间和时间变化。 （问题2） 然后将利用 [Ca2+]i 变化的知识来确定 使用贴片钙流入和通道激活之间的关系 夹紧方法。 （问题3）渠道活动的监管 将使用以下方法检查细胞骨架的蛋白水解和调节 切除的内向外膜片钳配置。 （问题 4）Myotubes 将 受到长时间的收缩活动或静止，并且 泄漏通道的二氢吡啶抑制剂与抑制相关 泄漏通道的钙依赖性酶促速率降低 流程。