Abstract 13025: Modulation of the Sodium-calcium Exchanger 1 (NCX1) by Calpain; Molecular Interactions and Identification of a Calpain Cleavage Site
Introduction: Chronic heart disease due to pressure overload, such as in hypertension and aortic stenosis (AS), is a major cause of morbidity and mortality. In response to pressure overload, altered Ca2+ homeostasis is a key determinant of cardiac remodeling and contractility. Aberrant activation of calpain, a ubiquitous Ca2+ dependent- protease can contribute to pathophysiology associated with loss of Ca2+ control in cardiomyocytes. Calpain cleaves NCX1 and modulates NCX1 exchange rate but underlying molecular mechanisms of NCX1 and calpain interaction remain to be determined. The effect of calpain on modulation of NCX1 was examined in this study.
Results: Investigation of NCX1 protein in left ventricular biopsies from AS patients and in the failing left ventricle of rats following aortic banding (AB) showed that both full-length NCX1 and a 75 kDa proteolytic NCX1 fragment were increased compared to control biopsies and sham-operated rats, respectively. We demonstrated that calpain-1 bound directly to two sites in cytoplasmic part of NCX1. By bioinformatics and mutation analysis we identified M369 as a putative calpain cleavage site residing within the α-catenin-like domain (CLD) in NCX1. Importantly, cleavage of NCX1 at M369 corresponded to a proteolytic fragment of 75 kDa. Competitive in vitro assay of calpain cleavage of NCX1 with a peptide containing M369 abolished the cleavage reaction. In order to investigate the functional role of calpain cleavage at M369 and the 75 kDa proteolytic NCX1 fragment, we mutated the identified site to a Tobacco Etch Virus (TEV) protease cleavage site. Co-transfection of NCX1(TEV) with the TEV protease resulted in specific cleavage of NCX1 at M369 in the membrane fraction.
Conclusion: We have identified a direct NCX1-calpain interaction, a calpain cleavage site at M369 in the α-catenin-like domain (CLD) in NCX1 and increased levels of full length NCX1 and the 75 kDa proteolytic NCX1 fragment in the pressure overloaded rat and AS biopsies. Our findings provide insight into a calpain-dependent mechanism that might be important for regulating Ca2+ homeostasis during heart failure progression.
- © 2013 by American Heart Association, Inc.