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 p reviously identified as a homologue of the calpain inhibitor, calpastatin. In the present study, we investigated the expression of HMWCaMBP and calpai ns in rat heart after ischemia and reperfusion. Western blot analysis of no rmal rat heart extract with a polyclonal antibody raised against bovine HMW CaMBP indicated a prominent immunoreactive band of 140 kDa. Both the expres sion and the activity of HMWCaMBP were decreased by ischemia reperfusion. I mmunohistochemical studies showed strong-to-moderate HMWCaMBP immunoreactiv ity in normal heart and poor immunoreactivity in ischemia-reperfused heart muscle. However, the expression of mu -calpain and m-calpain in ischemia-re perfused heart was increased as compared to normal heart. The calpain inhib itory activity of ischemia-reperfused heart tissues was significantly lower as compared to normal heart tissues. The pre-ischemic and post-ischemic pe rfusion of hearts with a cell-permeable calpain inhibitor suppressed the in crease in calpain expression but increased the HMWCaMBP expression. In-vitr o HMWCaMBP was proteolyzed by mu -calpain and m-calpain. We also measured a poptosis in normal and ischemia-reperfused tissues. An increase in the numb er 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 wit h the degree of apoptosis. Our findings suggest that HMWCaMBP may sequester calpains from its substrates in the normal myocardium, but it is susceptib le to proteolysis by calpains during ischemia-reperfusion. Thus, decreased expression of HMWCaMBP may play an important role in myocardial injury. (C) 2001 Harcourt Publishers Ltd.