Multiple Ca2+ Signaling Pathways Regulate Intracellular Ca2+ Activity in Human Cardiac Fibroblasts

Ca2+ signaling pathways are well studied in cardiac myocytes, but not in cardiac fibroblasts. The aim of the present study is to characterize Ca2+ signaling pathways in cultured human cardiac fibroblasts using confocal scanning microscope and RT-PCR techniques. It was found that spontaneous intracellular Ca2+ (Ca-i(2+)) oscillations were present in about 29% of human cardiac fibroblasts, and the number of cells with Ca-i(2+) oscillations was increased to 57.3% by application of 3% fetal bovine serum. Ca-i(2+). oscillations were dependent on Ca2+ entry. Ca-i(2+) oscillations were abolished by the store-operated Ca2+ (SOC) entry channel blocker La3+, the phospholipase C inhibitor U-73122, and the inositol trisphosphate receptors (IP3Rs) inhibitor 2-aminoethoxydiphenyl borate, but not by ryanodine. The IP3R agonist thimerosal enhanced Ca-i(2+) oscillations. Inhibition of plasma membrane Ca2+ pump (PMCA) and Na+-Ca2+ exchanger (NCX) also suppressed Ca-i(2+) oscillations. In addition, the frequency of Ca-i(2+) oscillations was reduced by nifedipine, and increased by Bay K8644 in cells with spontaneous Ca2+ oscillations. RT-PCR revealed that mRNAs for IP3R1-3, SERCA1-3, Ca(v)1.2, NCX3, PMCA1,3,4, TRPC1,3,4,6, STIM1, and Orai1-3, were readily detectable, but not RyRs. Our results demonstrate for the first time that spontaneous Ca-i(2+) oscillations are present in cultured human cardiac fibroblasts and are regulated by multiple Ca2+ pathways, which are not identical to those of the well-studied contractile cardiomyocytes. This study provides a base for future investigations into how Ca2+ signals regulate biological activity in human cardiac fibroblasts and cardiac remodeling under pathological conditions. J. Cell. Physiol. 223: 68-75, 2010. (C) 2009 Wiley-Liss, Inc.