Postmortem proteolysis is reduced in transgenic mice overexpressing calpastatin.
Using both in vitro and in vivo approaches, numerous studies have provided evidence that mu-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 degrees 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 (mu- 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 mu-calpain was lost in transgenics compared to an 87% loss in controls, indicating that autolysis of mu-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 mu- 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.
Kent
M P
MP
Roman L. Hruska U.S. Meat Animal Research Center, ARS, USDA, Clay Center, NE 68933-0166, USA.
Spencer
M J
MJ
Koohmaraie
M
M
D000818:Animals; D015153:Blotting, Western / Q000662:veterinary; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism; D004247:DNA / Q000032:analysis; D003893:Desmin / Q000037:antagonists & inhibitors*; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D005260:Female; D008297:Male; D008460:Meat / Q000592:standards*; D051379:Mice; D008822:Mice, Transgenic; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D009210:Myofibrils / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000662:veterinary; D011180:Postmortem Changes; D020107:Troponin T / Q000032:analysis; D016596:Vinculin / Q000032:analysis
D002135:Calcium-Binding Proteins; D003893:Desmin; D020107:Troponin T; D016596:Vinculin; C033668:calpastatin; D004247:DNA; D002154:Calpain