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Exercise-induced muscle damage : role of the calpain-calpastatin system in skeletal muscle myofibrillar protein composition Ball, Chad Geoffrey

Abstract

The purpose of this study was to exainine the relationship between the activation of the calcium stimulated cysteine protease, calpain, its endogenous inhibitor, calpastatin, and myofibrillar protein composition (troponin I (Tnl), troponin T (TnT) and tropomyosin (TM)) in an exercise-induced muscle damage model. It was hypothesized that this protease system initiated skeletal myofibrillar protein loss (and perhaps subsequent peptide release) from the contractile apparatus. In addition, lowering calpain activity (by the use of an exogenous inhibitor) was hypothesized to attenuate the composition of myofibrillar protein substrates for calpain (i.e. Tnl, TnT, TM). To test these hypotheses, male Wistar rats (~315g) were randomly assigned to one of six groups: 1) control sedentary (n=8), 2) control + cysteine protease inhibitor (E64c)(n=8), 3) running (25 meters/minute (-16°) for 45 min.)(n=8), 4) running + E64c (n=8), 5) running + 6 nr. recovery (n=8), or 6) running + 6 nr. recovery + E64c (n=8). Calpain I and II isoforms were isolated from rat hind-limb skeletal muscles, purified via phenyl-sepharose chromatography and their activities quantified using a casein-release assay. Calpastatin was isolated by a heat-release procedure and assayed based on its ability to inhibit calpain. Finally, myofibrillar proteins were resolved from a muscle homogenate via SDS-PAGE and their concentrations quantified using computer densitometry. Calpain I and II activities increased by 36.1% and 37.5% respectively, immediately following exercise, and at 6 hours of recovery were 16.4% and 15.9% compared to controls (p0.05). Calpastatin activity did not change with exercise, however at the 6 hour recovery, activity was elevated by 74.8% (p0.05). With E64c injection, any exercise-induced loss of sTnl (108.0% of control), sTnT (105.5% of control) or TM (110.7% of control) following running was arrested (p>0.05). This trend was maintained into the recovery (104.2%, 101.8% and 113.8% respectively)(p>0.05). The proportion of cytosolic protein bands corresponding to sTnl, sTnT and TM increased to 363.8%, 343.5% and 430.1% of control respectively, immediately after exercise (p

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