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Published on: January 12, 2016
Decreased expression of high-molecular-weight calmodulin-binding protein and its correlation with apoptosis in
1Department of Pathology, College of Medicine, University of Saskatchewan, Saskatoon, Canada.
Insights
Cardiac high-molecular-weight calmodulin-binding protein (HMWCaMBP) decreases during heart ischemia-reperfusion, leading to increased calpain activity and myocardial injury. This suggests HMWCaMBP plays a protective role against heart damage.
Area of Science:
- Cardiology
- Molecular Biology
- Biochemistry
Background:
- Cardiac high-molecular-weight calmodulin-binding protein (HMWCaMBP) is a calpastatin homologue.
- Calpains are implicated in myocardial injury.
Purpose of the Study:
- Investigate HMWCaMBP and calpain expression and activity in rat hearts post-ischemia-reperfusion.
- Determine the role of HMWCaMBP in myocardial injury during ischemia-reperfusion.
Main Methods:
- Western blot and immunohistochemistry to assess protein expression.
- Assay of calpain inhibitory activity.
- In-vitro proteolysis assays.
- Measurement of apoptosis and Bcl-2/Bax expression.
Main Results:
- Ischemia-reperfusion decreased HMWCaMBP expression and activity.
- Micro-calpain and m-calpain expression increased post-ischemia-reperfusion.
- Calpain inhibitory activity was reduced.
- HMWCaMBP was proteolyzed by calpains in vitro.
- Apoptosis increased with ischemia-reperfusion duration, correlating with Bax expression.
Conclusions:
- HMWCaMBP expression is reduced during myocardial ischemia-reperfusion.
- HMWCaMBP is degraded by calpains, contributing to increased calpain activity.
- Decreased HMWCaMBP levels may promote myocardial injury.
Abstract:
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.
