EXERCISE INDUCED MUSCLE FIBER INJURY

本研究的长期目标是确定机制（S） 潜在的离心收缩引起的骨骼肌损伤， 强调病因学中的始动因素和自生因素。 的 本建议中研究的具体目标是回答以下问题 问题. (1)游离胞浆[Ca 2 +]（[Ca 2 +]i）是否升高， 体外离心收缩的性能，如果是这样， 降解的（即，蛋白水解和磷脂水解）过程平行 随着时间的推移[Ca 2 +]i的升高？ (2)[Ca 2 +]i升高 导致降解过程的增加？ (3)有哪些 总肌肉[Ca 2 +]和肌肉中[Ca 2 +]i升高的机制 因偏心性宫缩而受伤 它是（a）通过电压激活的 通道、钠/钙交换器或Ca 2+动员受体 机制，或（B）它是由肌膜破坏引起的， 允许Ca 2+沿其电化学梯度被动扩散 纤维里吗 (4)[Ca 2 +]i是否在执行以下操作后升高 离心收缩偏向体内运动，如果有，做变化 降解过程平行升高[Ca 2 +]i随着时间的推移？ 为了回答前三个问题， 长肌（EDL）肌肉会受到高力量离心的伤害 收缩使用肌肉杠杆系统，并与对照组相比 不收缩的肌肉和等长的肌肉 宫缩 为了确定离心收缩对 [Ca2+]i（问题#1），共聚焦激光扫描显微镜（CLSM）将 用于使用Ca 2 +-浓度曲线跟踪[Ca 2 +]i随时间的局灶性变化。 敏感染料Fluo-3;将使用电子探针X射线微量分析 测量肌肉细胞器中[Ca 2 +]的变化。 蛋白质变化 并将磷脂降解与[Ca 2 +]i的变化进行比较 通过测量酪氨酸和3-甲基组氨酸的释放以及 前列腺素E2和白三烯B4。 伤后休息 将测量VO 2以评估与Ca 2+相关的代谢率 超载。 为了回答问题#2，细胞外[Ca 2 +]将变化 （0.5-5.0 mM），以检验高细胞外[Ca 2 +] 增加[Ca 2 +]i，进而增加蛋白水解和磷脂分解 受伤的肌肉。 回答第三个问题，伤害会 在存在特异性药理学阻滞剂的情况下， 通道、钠/钙交换器和Ca 2+动员受体; 将使用放射性示踪剂和荧光方法来评估破坏情况 的肌膜。 为了解决第四个问题，[Ca 2 +]i的变化 蛋白水解和磷脂分解标记物将在 离心偏置运动后大鼠比目鱼肌 （下坡行走）。

The long term goal of this research is to determine the mechanism(s) underlying eccentric contraction-induced skeletal muscle injury, with emphasis on the initiating and autogenetic factors in the etiology. The specific aims of the studies in this proposal are to answer the following questions. (1) Is free cytosolic [Ca2+] ([Ca2+]i) elevated following performance of eccentric contractions in vitro, and if so, do changes in degradative (i.e., proteolytic and phospholipolytic) processes parallel the elevations in [Ca2+]i over time? (2) Do the elevations in [Ca2+]i cause the increases in degradative processes? (3) What are the mechanisms for the rise in total muscle [Ca2+] and [Ca2+]i in muscles injured by eccentric contractions? Is it (a) through voltage-activated channels, the sodium/calcium exchanger, or Ca2+-mobilizing receptor mechanisms, or (b) does it result from disruption of sarcolemma, permitting passive diffusion of Ca2+ down it electrochemical gradient into the fibers? (4) Is [Ca2+]i elevated following performance of eccentric contraction-biased exercise in vivo, and if so, do changes in degradative processes parallel the elevation in [Ca2+]i over time? To answer the first three questions, isolated mouse extensor digitorum longus (EDL) muscles will be injured with high force eccentric contractions using a muscle lever system and compared with control muscles that perform no contractions and muscles that perform isometric contractions. To determine the effect of eccentric contractions on [Ca2+]i (Question #1), confocal laser scanning microscopy (CLSM) will be used to follow focal changes in [Ca2+]i over time using the Ca2+- sensitive dye, Fluo-3; electron probe x-ray microanalysis will be used to measure changes in [Ca2+] in muscle organelles. Changes in protein and phospholipid degradation will be compared with the changes in [Ca2+]i by measuring release of tyrosine and 3-methylhistidine and production of prostaglandin E2 and leukotriene B4, respectively. Post-injury resting VO2 will be measured to assess the metabolic rate associated with Ca2+ overload. To answer Question #2, extracellular [Ca2+] will be varied (0.5-5.0 mM) to test the hypothesis that high extracellular [Ca2+] will increase [Ca2+]i and in turn increase proteolytic and phospholipolytic rates in the injured muscles. To answer the third question, injury will be induced in the presence of specific pharmacological blockers of slow channels, the sodium/calcium exchanger, and Ca2+-mobilizing receptors; radio-tracer and fluorescent methods will be used to assess disruption of the sarcolemma. To address the fourth question, changes in [Ca2+]i and the proteolytic and phospholipolytic markers will be followed in the soleus muscles of rats following a bout of eccentric-biased exercise (downhill walking).