Abstract 11550: Calpain Mediated Proteolysis of The Cardiac Sodium-Calcium Exchanger 1 (NCX1) in Failing Hearts: Molecular Interactions
Introduction: In cardiac excitation-contraction coupling, the sarcolemmal Na+/Ca2+ exchanger 1 (NCX1) is a central component in maintaining intracellular Ca2+ homeostasis. Upregulation of NCX1 activity is associated with cardiac diseases such as heart failure, hypertrophy or ischemia reperfusion. Moreover, increased calpain activity, a ubiquitous intracellular Ca2+-activated protease, has been implicated in degradation of several cardiac proteins in failing hearts. Previous studies showed that calpain cleaves NCX1 and thereby increases conductance of NCX1, suggesting that calpain is an important regulator of NCX1 activity, but further insights into the underlying molecular mechanisms remain to be addressed. In the present study, we aim to identify NCX1-calpain protein interaction and their binding sites.
Methods and Results: Pull-down assays and peptide overlay assays have been employed and our preliminary results show that calpain binds directly to two distinct sites in the cytoplasmic loop of NCX1. In reciprocal experiments, we also found that NCX1 binds to catalytic region and domain III (membrane binding domain) in calpain. Further investigations on mapping of protein binding sites are ongoing to confirm their interaction sites. Consistent with these observations, NCX1 and calpain-1 are co-immunoprecipitated in the rat left ventricle suggesting NCX1 and calpain-1 physical interact. Interestingly, we also found that both full-length NCX1 and the 75 kDa proteolytic NCX1 fragment were upregulated in the failing left ventricle of rats after chronic pressure-overload induced by aortic banding for six weeks, indicating a role for NCX1 proteolysis during development of heart failure. In addition, we also observed an increased proteolysis of protein kinase Cα (PKCα), one of well-known calpain substrates, providing strong evidence that calpain activity was upregulated in our cardiac disease model.
Conclusion: Our present study shows a direct NCX1-calpain interaction and increased levels of the proteolytic fragment of NCX1 in the pressure-overloaded heart, providing further step to understand how calpain modulates NCX1 activity during development of heart failure.
- © 2012 by American Heart Association, Inc.