Geng Le, Wang Zidun, Cui Chang, Zhu Yue, Shi Jiaojiao, Wang Jiaxian, Chen Minglong
Cell Physiol Biochem. 2018;47(3):1167-1180. doi: 10.1159/000490213. Epub 2018 Jun 15.
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.
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.
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.
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.
背景/目的：心动过速诱发的心力衰竭是临床实践中最常见的心律失常，本研究旨在探讨其潜在机制。
以3 Hz的频率对人诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）进行快速电刺激（RES），持续7天，分别设置每天8小时和24小时以代表短期和长期心动过速。将未进行电刺激或进行缓慢电刺激（1 Hz）的年龄匹配的hiPSC-CMs设置为无电刺激（NES）对照组或低频电刺激（LES）对照组。刺激后，进行JC-1染色流式细胞术分析以检测线粒体状况。使用Hoechst染色和膜联蛋白V/碘化丙啶（AV/PI）染色流式细胞术分析评估hiPSC-CMs中的细胞凋亡。记录钙瞬变和L型钙电流以评估钙稳态。进行蛋白质印迹和qPCR以评估凋亡相关基因和钙稳态调节基因的蛋白质和mRNA表达水平。
与对照组相比，RES处理后的hiPSC-CMs出现线粒体功能障碍且凋亡百分比增加。RES处理后的hiPSC-CMs中钙瞬变和L型钙电流的幅度显著降低。分子分析表明Caspase3表达上调且Bax/Bcl-2比值增加。与钙重排相关的基因下调，而RES处理后磷酸化的Ca2+/钙调蛋白依赖性蛋白激酶II（CaMKII）显著上调。在这些方面，NES对照组和LES对照组之间没有显著差异。用1 μM KN93抑制CaMKII部分逆转了RES的这些不利影响。
对hiPSC-CMs进行RES会扰乱钙稳态，导致线粒体应激，促进细胞凋亡并以时间依赖性方式引起电生理重塑。CaMKII在RES诱导的损伤中起核心作用，药理学抑制CaMKII活性部分逆转了RES对细胞结构和电生理特性的不利影响。
