Caveolin-3 Directly Interacts and Regulates the Function of Cardiac CaV3.2 (A1H) T-Type Ca2+ Channels

Abstract

Voltage-gated T-type Ca2+ channel (TTCC), Cav3.1 and Cav3.2, are normally expressed during cardiac development but are re-expressed in cardiac hypertrophy and may contribute to the altered intracellular Ca2+ during this disease. However, the mechanism of altered Ca2+ signaling in cardiac hypertrophy is not clearly known. Caveolae containing scaffolding protein caveolin-3 (Cav-3), provide spatiotemporal regulation of intracellular Ca2+ in cardiomyocytes. To define the source of signaling Ca2+ involved and basis of dysregulated contractile function in cardiac hypertrophy and to investigate the role of caveolae and TTCC in the regulation of Ca2+ signaling, we used a transthorasic aortic constriction (TAC) induced mouse model of cardiac hypertrophy. Western blot analysis revealed re-expression of Cav3.1 and Cav3.2 proteins and significant increase in expression of Cav-3 in adult ventricle from TAC mice but not from sham treated mice. Electron microscopy analysis demonstrated significant increase in the number of caveolae and co-localization of Cav3.2 and Cav-3 in the ventricular myocytes in the TAC hearts. Co-immunoprecipitation analysis using anti-Cav-3 antibody revealed that re-expressed Cav3.2 co-IPs with Cav-3 in the TAC hearts, but not in sham hearts. GST pull-down analysis using Cav-3 fusion proteins confirmed that Cav-3 directly associates with Cav3.2 channels. Whole cell patch clamp analysis in HEK293 cells co-expressed with either CaV3.2 and wild-type Cav-3 or GFP revealed that co-expression of Cav3.2 + Cav-3 significantly decreased the peak ICav3.2 (−12 ± 3 pA/pF, n=11) compared to Cav3.2+GFP (−31 ± 4 pA/pF, n=11). Whereas co-expression of Cav-3 had no effect on the ICav3.1. Cav-3 coexpression did affect the voltage dependent activation or inactivation of ICav3.2. We conclude that Cav-3 associates with Cav3.2 channels and regulates its function. Increased Cav-3 expression may play a crucial role in regulation of Ca2+ signaling during hypertrophic cardiomyopathy.