|Spencer, M - UNIV OF CA, LOS ANGELES|
Submitted to: Journal of Animal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 10, 2003
Publication Date: April 20, 2004
Citation: Kent, M.P., Spencer, M.J., Koohmaraie, M. 2004. Postmortem proteolysis is reduced in transgenic mice overexpressing calpastatin. Journal of Animal Science. 82:794-801. Interpretive Summary: Current evidence shows that u-calpain activity is responsible for the breakdown or degradation of structural proteins in postmortem skeletal muscle, and that this degradation is responsible for improvements in meat tenderness. However, research relating calpain activity to postmortem proteolysis will benefit from additional supporting evidence. To address this, we have examined postmortem protein degradation in genetically altered mice which over-produce calpastatin; the inhibitor of calpain. This paper reports the impact of over-production of calpastatin on postmortem protein degradation. Calpastatin was over-produced in experimental mice at a level 370-fold greater than in unaltered, normal mice. Postmortem, in experimental mice, the activity of u-calpain was reduced, as was the degradation of desmin and Troponin T (two important structural proteins). Interestingly, our investigations also revealed that m-calpain, which is inactive in postmortem beef, lamb, or pigs, is active in postmortem mouse muscle. This is the first evidence of m-calpain being active postmortem in normal, whole muscle. In conclusion, a high level of over-production of calpastatin was achieved which, by virtue of its inhibitory action over u-calpain, was determined to be directly responsible for a reduction in postmortem protein degradation.
Technical Abstract: Using both in vitro and in vivo approaches, numerous studies have provided evidence that calpain is responsible for postmortem proteolysis. This paper reports the impact of over-expression of calpastatin on postmortem proteolysis in transgenic mice. Transgenic mice (n = 8), containing a human calpastatin gene whose expression was driven by the human skeletal muscle actin promoter, were sacrificed along with control non-transgenic littermates (n = 5). The hind limbs were removed and stored at 4 deg C; muscle samples were dissected at 0, 1, 3 and 7 days postmortem and analyzed individually. At time 0, active human calpastatin was expressed in transgenic murine skeletal muscle at a level 370-fold greater than calpastatin in control mice. Although the native isoform of this protein was degraded with storage, at 7 days postmortem approximately 78% of at-death activity remained, indicating that degraded calpastatin retains activity. Calpain (u- and m-) expression was unaffected by the transgene as assessed by immunoreactivity at day 0. Over 7 days, 33% of at-death 80kDa isoform immunoreactivity of u-calpain was lost in transgenics, compared to an 87% loss in controls, indicating that autolysis of u-calpain was slowed in transgenic mice. Desmin degradation was also reduced in transgenics when compared to controls. Control mice lost 6, 78 and 91% of at-death native desmin at 1, 3, and 7 days postmortem respectively; in contrast, 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 appeared to undergo autolysis in control mice, which in postmortem tissue is indicative of proteolysis. Further investigation revealed that both u- 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 reduction in postmortem proteolysis.