Altered expression of high-molecular-weight calmodulin-binding protein in human ischaemic myocardium.
A high-molecular-weight calmodulin-binding protein (HMWCaMBP) was previously identified and purified from the cytosolic fraction of bovine heart. Based on the sequence homology, amino acid analysis, antibody reactivity, and calpain inhibition, HMWCaMBP has been identified as a homologue of the calpain inhibitor calpastatin. In the present study the expression of HMWCaMBP was investigated in normal and ischaemic human myocardium. Western blot analysis of normal human cardiac muscle extract with the polyclonal antibody raised against bovine HMWCaMBP indicated a prominent immunoreactive band with a molecular mass of 140 kD. HMWCaMBP was localized in the cytoplasm and myofilaments of cardiac myocytes. Furthermore, Western blot analysis of normal and ischaemic cardiac tissues indicated a decrease in the expression of HMWCaMBP in ischaemic tissues. These studies were further substantiated by immunohistochemical studies, indicating strong to moderate HMWCaMBP immunoreactivity in normal cardiac muscle and poor to negative immunoreactivity in ischaemic muscle. The results obtained from the rat ischaemic model suggested that the expression of cardiac HMWCaMBP was significantly decreased during ischaemia/reperfusion. In addition, micro-calpain and m-calpain expression was higher in ischaemic cardiac tissue samples than in normal controls. The calpain inhibitory activity of ischaemic cardiac tissues was significantly lower than normal cardiac tissue samples. In some cases of cardiac ischaemia, HMWCaMBP highlighted the contraction band necrosis seen at the margins of a myocardial infarct. In vitro, HMWCaMBP was proteolysed by micro-calpain and m-calpain. These results indicate that HMWCaMBP could be susceptible to proteolysis by calpains during ischaemia or reperfusion and may play a contributory role in myocardial injury.
Kakkar
R
R
Department of Pathology and Saskatoon Cancer Centre, College of Medicine, Royal University Hospital, University of Saskatchewan, Canada.
Radhi
J M
JM
Rajala
R V
RV
Sharma
R K
RK
D008841:Actin Cytoskeleton / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D016022:Case-Control Studies; D002417:Cattle; D003593:Cytoplasm / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D066298:In Vitro Techniques; D008297:Male; D008970:Molecular Weight; D017202:Myocardial Ischemia / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000378:metabolism
D002148:Calmodulin-Binding Proteins