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）沉积增加为特征的过度心肌纤维化，是引发心力衰竭（heart failure, HF）患者机械与电生理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）在心肌纤维化进程中发挥调控作用，但心力衰竭状态下引发胶原蛋白过度沉积的人心室成纤维细胞（human ventricular fibroblast, hVF）重塑机制仍未阐明。因此，本研究旨在探究衰竭心脏与正常（对照）心脏来源的hVF之间，细胞钙离子动力学差异与胶原蛋白分泌/沉积水平的关联。对左心室组织切片进行Masson三色染色（Masson trichrome）的组织学分析证实，相较于正常心脏，心力衰竭患者的心肌纤维化程度显著更重。体外实验结果显示，与对照组相比，心力衰竭来源的hVF在培养48小时后，可溶性胶原蛋白的分泌与沉积水平更高[85.9±7.4 μg/10⁶ vs 58.5±8.8 μg/10⁶ 细胞，P<0.05；采用Sircol™检测试剂盒（Sircol™ assay）完成检测]。然而，两组的胶原蛋白基因表达（COL1A1与COL1A2；rt-PCR）水平无显著差异。对分离得到的hVF进行钙离子成像（fluo-3标记）结果显示，毒胡萝卜素（thapsigargin）诱导的细胞内钙离子释放能力无显著差异（对照组为16±1.4%，心力衰竭组为17±1.1%）；但相较于非衰竭心脏来源的hVF（35±5%），心力衰竭组hVF通过钙池操纵性钙离子进入（store-operated Ca²+ entry, SOCE）与钙释放激活型Ca²+通道（Ca²+ release-activated, CRAC）介导的钙离子内流显著升高（P≤0.05，47±3%）。针对ICRAC通道组分的免疫印迹分析显示，相较于正常对照组，心力衰竭组hVF的ORAI1表达水平升高，而STIM1表达无显著差异。STIM1与ORAI1共定位的Pearson相关系数在心力衰竭组（0.5±0.01）显著高于对照组（0.4±0.01，P<0.01）。使用选择性ICRAC抑制剂YM58483（10 μM）孵育hVF 48小时后，心力衰竭组相较于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，相较于非衰竭心脏来源的hVF，心力衰竭患者来源的hVF胶原蛋白分泌能力增强，这一现象与SOCE介导的钙离子内流增加以及孔形成亚基ORAI1的表达上调相关。对SOCE进行治疗性抑制或可延缓心肌纤维化与心力衰竭的进展。