Department of Medicine, and Research Centre of Heart, Brain, Hormone and Healthy Aging, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China.
J Mol Cell Cardiol. 2011 Aug;51(2):198-206. doi: 10.1016/j.yjmcc.2011.05.008. Epub 2011 May 18.
Our previous study demonstrated that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts, including a large-conductance Ca(2+)-activated K(+) current (BKCa), a volume-sensitive chloride current (I(Cl.vol)), and voltage-gated sodium currents (I(Na)). The present study was designed to examine the possible involvement of these ion channels in proliferation of cultured human cardiac fibroblasts using approaches of cell proliferation assay, whole-cell patch voltage-clamp, siRNA and Western blot analysis. It was found that the blockade of BKCa with paxilline (1-3μM) or I(Cl.vol) with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium (DIDS, 100-200μM), but not I(Na) with tetrodotoxin (0.1-10μM), remarkably suppressed proliferation in human cardiac fibroblasts. Knockdown of KCa1.1 or Clcn3 with specific siRNAs significantly reduced BKCa or I(Cl.vol) current, mRNA and channel protein levels, and inhibited growth of human cardiac fibroblasts. Flow cytometry analysis showed accumulation of cardiac fibroblasts at G0/G1 phase and reduced cell number in S phase after inhibition of BKCa or I(Cl.vol) with channel blockers or knock down of the corresponding channels with specific siRNAs; these effects were accompanied by a decreased expression of cyclin D1 and cyclin E. The present results demonstrate the novel information that BKCa and I(Cl.vol) channels, but not I(Na) channels, are involved in the regulation of proliferation in cultured human cardiac fibroblasts by promoting cell cycle progression via modulating cyclin D1 and cyclin E expression.
我们之前的研究表明，人类心肌成纤维细胞中存在多种离子通道的异质性表达，包括大电导钙激活钾电流（BKCa）、容积敏感性氯离子电流（I(Cl.vol)）和电压门控钠电流（I(Na)）。本研究旨在通过细胞增殖测定、全细胞膜片钳、siRNA 和 Western blot 分析等方法，研究这些离子通道是否参与培养的人心脏成纤维细胞的增殖。结果发现，BKCa 通道阻断剂 paxilline（1-3μM）或 I(Cl.vol)通道阻断剂 4,4'-二异硫氰基二苯乙烯-2,2'-二磺酸二钠盐（DIDS，100-200μM）而非 I(Na)通道阻断剂河豚毒素（0.1-10μM），可显著抑制人心肌成纤维细胞的增殖。用特异性 siRNA 敲低 KCa1.1 或 Clcn3，可显著减少 BKCa 或 I(Cl.vol)电流、mRNA 和通道蛋白水平，并抑制人心肌成纤维细胞的生长。流式细胞术分析表明，BKCa 或 I(Cl.vol)通道被通道阻断剂抑制或用特异性 siRNA 敲低相应通道后，心肌成纤维细胞在 G0/G1 期积累，S 期细胞数量减少；同时，cyclin D1 和 cyclin E 的表达减少。这些结果表明，BKCa 和 I(Cl.vol)通道而不是 I(Na)通道，通过调节 cyclin D1 和 cyclin E 的表达，促进细胞周期进程，参与调节培养的人心肌成纤维细胞的增殖。
