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ANIMAL PRODUCTS |
Roman L. Hruska U.S. Meat Animal Research Center, ARS, USDA, Clay Center, NE 68933-0166 and
and
Department of Pediatrics and UCLA Duchenne Muscular Dystrophy Research Center, University of California, Los Angeles 90095-1606
3 Correspondence: P.O. Box 166 (phone: 402-762-4221; fax: 402-762-4149; e-mail: koohmaraie{at}email.marc.usda.gov).
Abstract
Using both in vitro and in vivo approaches, numerous studies have provided evidence that µ-calpain is responsible for postmortem proteolysis. This paper reports the effect of overexpression of calpastatin on postmortem proteolysis in transgenic mice. Transgenic mice (n = 8) with a human calpastatin gene, whose expression was driven by the human skeletal muscle actin promoter, were killed along with control nontransgenic littermates (n = 5). Hind limbs were removed and stored at 4°C, and muscle samples were dissected at 0, 1, 3, and 7 d postmortem and analyzed individually. At time 0, active human calpastatin was expressed in transgenic murine skeletal muscle at a level 370-fold greater (P < 0.001) than calpastatin in control mice. Although the native isoform of this protein was degraded with storage, at 7 d postmortem, approximately 78% of at-death activity remained, indicating that degraded calpastatin retains activity. Calpain (µ- and m-) expression was unaffected (P > 0.05) by the transgene as assessed by immunoreactivity at d 0. Over 7 d, 33% of at-death 80-kDa isoform immunoreactivity of µ-calpain was lost in transgenics compared to an 87% loss in controls, indicating that autolysis of µ-calpain was slowed in transgenic mice. Desmin degradation was also inhibited (P < 0.05) in transgenics when compared to controls. Control mice lost 6, 78, and 91% of at-death native desmin at 1, 3, and 7 d postmortem, respectively; conversely, transgenic mice lost only 1, 3, and 17% at the same times. A similar trend was observed when examining the degradation of troponin-T. Interestingly, m-calpain seemed to undergo autolysis in control mice, which in postmortem tissue is indicative of proteolysis. Further investigation revealed that both µ- and m-calpain are active postmortem in normal murine skeletal muscle. In conclusion, a high level of expression of active calpastatin was achieved, which, by virtue of its inhibitory specificity, was determined to be directly responsible for a decrease in postmortem proteolysis.
Key Words: Calpastatin • Calpain • Postmortem • Proteolysis • Transgenic Mice
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