Downregulation of calpastatin in rat heart after brief ischemia and reperfusion.
The activities of calpain and its endogenous inhibitor, calpastatin, were measured in the soluble fraction of perfused rat heart after ischemia for 5-20 min and reperfusion for up to 30 min. The method for m-calpain measurement was modified: washing of the DEAE-cellulose column with 0.18 M NaCl instead of 0.15 M NaCl increased the m-calpain activity 12.5-fold. Ischemia for 20 min followed by reperfusion for 30 min did not affect the m-calpain activity but decreased the calpastatin activity. m-Calpain was enriched in the nucleus-myofibril fraction but was not further translocated on ischemia-reperfusion. Mu-calpain was below the limit of detection on immunoblotting or casein zymography, but its mRNA was substantially expressed, as detected on Northern blotting. Casein zymography also revealed a novel Ca2+-dependent protease without the typical characteristics of mu- or m-calpain. The immunoblotting of myocardial fractions showed that calpastatin was proteolyzed on ischemia-reperfusion. The calpastatin proteolysis was suppressed by a calpain inhibitor, Ac-Leu-Leu-norleucinal. Calpastatin may sequester calpain from its substrates in the normal myocardium, but may be proteolyzed by calpain in the presence of an unidentified activator in the early phase of calpain activation during ischemia-reperfusion, resulting in the proteolysis of calpastatin and then other calpain substrates.
Sorimachi
Y
Y
Department of Legal Medicine, Yamaguchi University School of Medicine, Ube.
Harada
K
K
Saido
T C
TC
Ono
T
T
Kawashima
S
S
Yoshida
K
K
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification; D002848:Chromatography, DEAE-Cellulose; D015853:Cysteine Proteinase Inhibitors / Q000302:isolation & purification / Q000378:metabolism; D015536:Down-Regulation; D006868:Hydrolysis; D008297:Male; D017202:Myocardial Ischemia / Q000378:metabolism*; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D012333:RNA, Messenger; C033668:calpastatin; D002154:Calpain