Rapid Electrical Stimulation Increased Cardiac Apoptosis Through Disturbance of Calcium Homeostasis and Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
Rapid Electrical Stimulation Increased Cardiac Apoptosis Through Disturbance of Calcium Homeostasis and Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes
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快速电刺激通过干扰人诱导多能干细胞来源的心肌细胞中的钙稳态和线粒体功能障碍来增加心脏细胞凋亡
DOI：
10.1159/000490213
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发表时间：
2018-01-01
影响因子：
--
通讯作者：
Chen, Minglong
中科院分区：
文献类型：
--
作者：
Geng, Le;Wang, Zidun;Chen, Minglong
背景/目的：心衰是临床上最常见的心律失常。本研究旨在探讨其潜在机制。方法：采用频率为3hz的快速电刺激（RES）对人诱导多能干细胞来源的心肌细胞（hiPSC-CMs）进行7天，分别设置8 h/天和24 h/天代表短期和长期心动过速。将年龄匹配的无电刺激或慢电刺激（1 Hz）的hiPSC-CMs设为无电刺激（NES）对照和低频电刺激（LES）对照。刺激后进行JC-1染色流式细胞术分析，检测线粒体状况。采用Hoechst染色和膜联蛋白V/碘化丙啶（AV/PI）染色流式细胞术分析hiPSC-CMs细胞的凋亡情况。记录钙瞬态和l型钙电流以评估钙稳态。Western blotting和qPCR检测细胞凋亡相关基因和钙稳态调控基因的蛋白和mRNA表达水平。结果：与对照组相比，RES后hiPSC-CMs出现线粒体功能障碍，凋亡百分比增加。与res相比，hiPSC-CMs中钙瞬态和l型钙电流的振幅显著降低。分子分析显示Caspase3表达上调，Bax/Bcl-2比值升高。res后，钙重序列相关基因下调，而磷酸化Ca2+/钙调素依赖性蛋白激酶II （CaMKII）显著上调，NES对照组与LES对照组在这些方面无显著差异。1µM KN93对CaMKII的抑制作用部分逆转了RES的这些不良反应。结论：RES对hiPSC-CMs的影响扰乱了钙稳态，导致线粒体应激，促进细胞凋亡，并以时间依赖性的方式引起电生理重构。CaMKII在RES引起的损伤中发挥了核心作用，CaMKII活性的药理抑制部分逆转了RES对细胞结构和电生理特性的不利影响。
Background/Aims: Heart failure induced by tachycardia, the most common arrhythmia, is frequently observed in clinical practice. This study was designed to investigate the underlying mechanisms. Methods: Rapid electrical stimulation (RES) at a frequency of 3 Hz was applied on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for 7 days, with 8 h/day and 24 h/day set to represent short-term and long-term tachycardia, respectively. Age-matched hiPSC-CMs without electrical stimulation or with slow electrical stimulation (1 Hz) were set as no electrical stimulation (NES) control or low-frequency electrical stimulation (LES) control. Following stimulation, JC-1 staining flow cytometry analysis was performed to examine mitochondrial conditions. Apoptosis in hiPSC-CMs was evaluated using Hoechst staining and Annexin V/propidium iodide (AV/PI) staining flow cytometry analysis. Calcium transients and L-type calcium currents were recorded to evaluate calcium homeostasis. Western blotting and qPCR were performed to evaluate the protein and mRNA expression levels of apoptosis-related genes and calcium homeostasis-regulated genes. Results: Compared to the controls, hiPSC-CMs following RES presented mitochondrial dysfunction and an increased apoptotic percentage. Amplitudes of calcium transients and L-type calcium currents were significantly decreased in hiPSC-CMs with RES. Molecular analysis demonstrated upregulated expression of Caspase3 and increased Bax/Bcl-2 ratio. Genes related to calcium re-sequence were downregulated, while phosphorylated Ca2+/calmodulin-dependent protein kinase II (CaMKII) was significantly upregulated following RES. There was no significant difference between the NES control and LES control groups in these aspects. Inhibition of CaMKII with 1 µM KN93 partly reversed these adverse effects of RES. Conclusion: RES on hiPSC-CMs disturbed calcium homeostasis, which led to mitochondrial stress, promoted cell apoptosis and caused electrophysiological remodeling in a time-dependent manner. CaMKII played a central role in the damages induced by RES, pharmacological inhibition of CaMKII activity partly reversed the adverse effects of RES on both structural and electrophysiological properties of cells.