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).
