ABSTRACT

Excessive cardiac fibrosis, characterized by increased collagen-rich extracellular matrix (ECM) deposition, is a major predisposing factor for mechanical and electrical dysfunction in heart failure (HF). The human ventricular fibroblast (hVF) remodeling mechanisms that cause excessive collagen deposition in HF are unclear, although reports suggest a role for intracellular free Ca²⁺ in fibrosis. Therefore, we determined the association of differences in cellular Ca²⁺ dynamics and collagen secretion/deposition between hVFs from failing and normal (control) hearts. Histology of left ventricle sections (Masson trichrome) confirmed excessive fibrosis in HF versus normal. In vitro, hVFs from HF showed increased secretion/deposition of soluble collagen in 48 h of culture compared with control [85.9±7.4 µg/10⁶ cells vs 58.5±8.8 µg/10⁶ cells, P<0.05; (Sircol™ assay)]. However, collagen gene expressions (COL1A1 and COL1A2; RT-PCR) were not different. Ca²⁺ imaging (fluo-3) of isolated hVFs showed no difference in the thapsigargin-induced intracellular Ca²⁺ release capacity (control 16±1.4% vs HF 17±1.1%); however, Ca²⁺ influx via store-operated Ca²⁺ entry/Ca²⁺ release-activated channels (SOCE/CRAC) was significantly (P≤0.05) greater in HF-hVFs (47±3%) compared with non-failing (35±5%). Immunoblotting for I_CRAC channel components showed increased ORAI1 expression in HF-hVFs compared with normal without any difference in STIM1 expression. The Pearson's correlation coefficient for co-localization of STIM1/ORAI1 was significantly (P<0.01) greater in HF (0.5±0.01) than control (0.4±0.01) hVFs. The increase in collagen secretion of HF versus control hVFs was eliminated by incubation of hVFs with YM58483 (10 µM), a selective I_CRAC inhibitor, for 48 h (66.78±5.87 µg/10⁶ cells vs 55.81±7.09 µg/10⁶ cells, P=0.27). In conclusion, hVFs from HF have increased collagen secretion capacity versus non-failing hearts and this is related to increase in Ca²⁺ entry via SOCE and enhanced expression of ORAI, the pore-forming subunit. Therapeutic inhibition of SOCE may reduce the progression of cardiac fibrosis/HF.