DOWN-REGULATION OF CALPASTATIN IN RAT-HEART AFTER BRIEF ISCHEMIA AND REPERFUSION

The activities of calpain and its endogenous inhibitor, calpastatin, w ere measured in the soluble fraction of perfused rat heart after ische mia for 5-20 min and reperfusion for up to 30 min, The method for m-ca lpain measurement was modified: washing of the DEAE-cellulose column w ith 0.18 M NaCl instead of 0.15 M NaCl increased the m-calpain activit y 12.5-fold, Ischemia for 20 min followed by reperfusion for 30 min di d not affect the m-calpain activity but decreased the calpastatin acti vity, m-Calpain was enriched in the nucleus-myofibril fraction but was not further translocated on ischemia-reperfusion. mu-Calpain was belo w the limit of detection on immunoblotting or casein zymography, but i ts mRNA was substantially expressed, as detected on Northern blotting, Casein zymography also revealed a novel Ca2+-dependent protease witho ut the typical characteristics of mu- or m-calpain, The immunoblotting of myocardial fractions showed that calpastatin was proteolyzed on is chemia-reperfusion. The calpastatin proteolysis was suppressed by a ca lpain inhibitor, Ac-Leu-Leu-norleucinal. Calpastatin may sequester cal pain from its substrates in the normal myocardium, but may be proteoly zed by calpain in the presence of an unidentified activator in the ear ly phase of calpain activation during ischemia-reperfusion, resulting in the proteolysis of calpastatin and then other calpain substrates.