Decreased expression of high-molecular-weight calmodulin-binding protein and its correlation with apoptosis in ischemia-reperfused rat heart.
A cardiac high-molecular-weight calmodulin-binding protein (HMWCaMBP) was previously identified as a homologue of the calpain inhibitor, calpastatin. In the present study, we investigated the expression of HMWCaMBP and calpains in rat heart after ischemia and reperfusion. Western blot analysis of normal rat heart extract with a polyclonal antibody raised against bovine HMWCaMBP indicated a prominent immunoreactive band of 140kDa. Both the expression and the activity of HMWCaMBP were decreased by ischemia reperfusion. Immunohistochemical studies showed strong-to-moderate HMWCaMBP immunoreactivity in normal heart and poor immunoreactivity in ischemia-reperfused heart muscle. However, the expression of micro-calpain and m-calpain in ischemia-reperfused heart was increased as compared to normal heart. The calpain inhibitory activity of ischemia-reperfused heart tissues was significantly lower as compared to normal heart tissues. The pre-ischemic and post-ischemic perfusion of hearts with a cell-permeable calpain inhibitor suppressed the increase in calpain expression but increased the HMWCaMBP expression. In-vitro HMWCaMBP was proteolyzed by micro-calpain and m-calpain. We also measured apoptosis in normal and ischemia-reperfused tissues. An increase in the number of apoptotic bodies was observed with increased duration of ischemia and reperfusion. Bcl-2 expression did not change in any of the groups, whereas Bax expression increased with ischemia-reperfusion and correlated well with the degree of apoptosis. Our findings suggest that HMWCaMBP may sequester calpains from its substrates in the normal myocardium, but it is susceptible to proteolysis by calpains during ischemia-reperfusion. Thus, decreased expression of HMWCaMBP may play an important role in myocardial injury.
Kakkar
R
R
Department of Pathology, College of Medicine, University of Saskatchewan, Saskatoon, Canada.
Wang
X
X
Radhi
J M
JM
Rajala
R V
RV
Wang
R
R
Sharma
R K
RK
D000818:Animals; D000906:Antibodies; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D002148:Calmodulin-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism; D006023:Glycoproteins / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D008297:Male; D008970:Molecular Weight; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D009206:Myocardium / Q000201:enzymology* / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley
D000906:Antibodies; D002148:Calmodulin-Binding Proteins; D006023:Glycoproteins; C039304:calpain inhibitors; D002154:Calpain