Data from: Enhanced store-operated Ca2+ influx and ORAI1 expression in ventricular fibroblasts from human failing heart

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 [Ca2+]i 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 vs normal. In vitro, hVFs from HF showed increased secretion/deposition of soluble collagen in 48 hours of culture compared with control [85.9±7.4 μg/106 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 [SOCE /CRAC (Ca2+ release-activated)] channels was significantly (P≤0.05) greater in HF-hVFs (47±3%) compared with non-failing (35±5%). Immunoblotting for ICRAC 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 vs control hVFs was eliminated by incubation of hVFs with YM58483 (10 μM), a selective ICRAC inhibitor for 48 hours (66.78±5.87μg/106 cells vs 55.81±7.09 μg/106 cells, P=0.27). In conclusion, hVFs from HF have increased collagen secretion capacity vs 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.

以富含胶原蛋白的细胞外基质（extracellular matrix, ECM）沉积增多为特征的过度心肌纤维化，是心力衰竭（heart failure, HF）患者发生机械与电功能障碍的主要易感因素。尽管已有研究提示细胞内钙离子浓度[Ca²⁺]i参与纤维化进程，但心力衰竭状态下导致胶原蛋白过度沉积的人心室成纤维细胞（human ventricular fibroblast, hVF）重塑机制仍未明确。为此，本研究探讨了心力衰竭与正常（对照）心脏来源的人心室成纤维细胞之间，细胞钙动力学及胶原蛋白分泌/沉积的差异关联。

左心室组织切片的马松三色（Masson trichrome）染色组织学分析证实，心力衰竭组较正常对照组存在更为显著的心肌纤维化。体外实验中，与对照组相比，心力衰竭来源的人心室成纤维细胞在培养48小时后，可溶性胶原蛋白的分泌与沉积水平显著升高[85.9±7.4 μg/10⁶ vs 58.5±8.8 μg/10⁶ 细胞，P<0.05；Sircol™ 检测法]。但COL1A1与COL1A2的胶原蛋白基因表达经rt-PCR检测后，未见显著组间差异。

对分离得到的人心室成纤维细胞进行钙离子成像（Fluo-3探针标记）结果显示，毒胡萝卜素诱导的细胞内钙释放能力两组间无显著差异（对照组16±1.4% vs 心力衰竭组17±1.1%）；但通过钙池操纵性钙内流（store-operated Ca²⁺ entry, SOCE）/钙释放激活钙通道（Ca²⁺ release-activated, CRAC）介导的钙离子内流，在心力衰竭来源的成纤维细胞中显著升高（P≤0.05），心力衰竭组为47±3%，非衰竭组为35±5%。针对ICRAC通道组分的免疫印迹实验结果显示，心力衰竭来源的成纤维细胞中ORAI1蛋白表达水平较正常对照组升高，而STIM1蛋白表达无显著组间差异。STIM1与ORAI1共定位的皮尔逊相关系数，在心力衰竭组人心室成纤维细胞中显著高于对照组（P<0.01），心力衰竭组为0.5±0.01，对照组为0.4±0.01。

将心力衰竭组与对照组的人心室成纤维细胞与选择性ICRAC抑制剂YM58483（10 μM）共同孵育48小时后，心力衰竭组较对照组的胶原蛋白分泌升高现象被完全消除（66.78±5.87 μg/10⁶ 细胞 vs 55.81±7.09 μg/10⁶ 细胞，P=0.27）。

综上，心力衰竭来源的人心室成纤维细胞较非衰竭心脏来源的细胞，其胶原蛋白分泌能力显著升高，这一现象与钙池操纵性钙内流介导的钙离子内流增加以及孔形成亚基ORAI的表达上调密切相关。对钙池操纵性钙内流进行治疗性抑制，或可延缓心肌纤维化与心力衰竭的进展。