Exercise-induced muscle damage : role of the calpain-calpastatin system in skeletal muscle myofibrillar protein composition
Creators: name: Ball, Chad Geoffrey
Publisher: name: University of British Columbia
Year: 2009

description: 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 (p&lt;0.05). With E64c administration, the run-induced activation of calpain I (3.7%) and II (8.2%) following exercise was much less, which was maintained into the recovery (5.3% and 9.7% respectively)(p&gt;0.05). Calpastatin activity did not change with exercise, however at the 6 hour recovery, activity was elevated by 74.8% (p&lt;0.05). With E64c injection, this activity increase remained elevated over control levels (97.4%)(p&lt;0.05). The proportion of bands corresponding to myofibrillar sTnl, sTnT and T M decreased to 75.1%, 75.6% and 90.1% of control values respectively, immediately following exercise (p&gt;0.05) and at 6 hours of recovery were 73.2%, 75.4% and 80.3% (p&gt;0.05). 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&gt;0.05). This trend was maintained into the recovery (104.2%, 101.8% and 113.8% respectively)(p&gt;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&lt;0.05) and at 6 hours of recovery were 386.4%, 372.7% and 473.2% (p&lt;0.05). With E64c administration, the exercise-induced increase in sTnl (532.3% of control), sTnT (509.0% of control) and TM (478.6% of control) was enhanced (p&lt;0.05). This change remained into the recovery (537.4%, 530.7% and 514.9% respectively)(p&lt;0.05). It is concluded, based on E64c's ability to attenuate both the exercise-induced activation of calpain and myofibrillar protein loss/breakdown, that calpain is a causative factor for protein composition changes in vivo. Calpastatin also dominates the protease system during recovery, shifting the environment to one of protein maintenance.