Calcium Sensing Receptor Promotes Cardiac Fibroblast Proliferation and Extracellular Matrix Secretion

Aims: Calcium-sensing receptor (CaR) acts as a G protein coupled receptor that mediates the increase of the intracellular Ca2+ concentration. The expression of CaR has been confirmed in various cell types, including cardiomyocytes, smooth muscle cells, neurons and vascular endothelial cells. However, whether CaR is expressed and functions in cardiac fibroblasts has remained unknown. The present study investigated whether CaR played a role in cardiac fibroblast proliferation and extracellular matrix (ECM) secretion, both in cultured rat neonatal cardiac fibroblasts and in a model of cardiac hypertrophy induced by isoproterenol (ISO). Methods and Results: Immunofluorescence, immunohistochemistry and Western blot analysis revealed the presence of CaR in cardiac fibroblasts. Calcium and calindol, a specific activator of CaR, elevated the intracellular calcium concentration in cardiac fibroblasts. Pretreatment of cardiac fibroblasts with calhex231, a specific inhibitor of CaR, U73122 and 2-APB attenuated the calindol- and extracellular calcium-induced increase in intracellular calcium ([Ca2+]i). Cardiac fibroblast proliferation and migration were assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), cell count and the cell scratch assay. ECM production was detected by expression of matrix metalloproteinase-3 and -9 (MMP-3 and -9). Activation of CaR promoted cardiac fibroblast proliferation and migration and ECM secretion. More importantly, calhex231, suppressed cardiac fibroblast proliferation and migration and MMP-3 and -9 expression. To further investigate the effect of CaR on cardiac fibrosis, a model of ISO-induced cardiac hypertrophy was established. Pretreatment with calhex231 prevented cardiac fibrosis and decreased the expression of MMP-3 and -9 expression. Conclusions: Our results are the first report that CaR plays an important role in Ca2+ signaling involved in cardiac fibrosis through the phospholipase C- inositol 3,4,5 phosphate (PLC-IP3) pathway