高频电刺激人源诱导多能干细胞分化心房肌细胞构建心房肌细胞损伤模型

Establishment of injury model using high-frequency electrical stimulation of atrial myocytes differentiated from human induced pluripotent stem cells

目的通过高频电刺激人源诱导多能干细胞分化心房肌细胞构建细胞水平损伤模型,探讨损伤发生的机制。方法利用人源诱导多能干细胞技术并通过视黄酸(RA)诱导分化特异性心房肌细胞,并通过免疫荧光染色以及实时荧光定量聚合酶链式反应(qRT-PCR)验证心房肌特异性核蛋白标志物NR2F2的表达。将分离培养的心房肌细胞分为高频组、对照组。高频组对心房肌细胞以频率7 Hz,持续24 h的高频电刺激。对照组不予电刺激。利用共聚焦显微镜检测两组钙瞬变。利用Annexin V/propidium iodide (AV/PI)分析两组细胞凋亡率的变化。通过qRT-PCR检测凋亡相关基因(bax以及bcl-2)以及钙离子通道RyR2表达量变化。4-苯基丁酸钠(4PBA)处理高频电刺激组细胞后检测细胞钙瞬变。结果利用人源诱导多能干细胞技术能获得较多的心房肌细胞。高频组荧光强度变化值/最低荧光强度(ΔF/F0)较对照组明显下降,同时RYR2基因表达量下降。利用4PBA预处理高频组细胞后,ΔF/F0明显增加。高频组早期凋亡及晚期凋亡的细胞比率之和明显高于对照组,同时促凋亡基因bax表达上调而抑制凋亡基因bcl-2表达下降。结论高频电刺激人源诱导多能干细胞分化的心房肌细胞可致细胞内钙稳态异常以及细胞凋亡增加,构成心房肌细胞的电损伤。

Objective To investigate the mechanism of atrial injury induced by high-frequency electrical stimulation in the atrial myocytes differentiating from human induced pluripotent stem cells. Methods Human induced pluripotent stem cells were differentiated into specific atrial myocytes with retinoic acid(RA)and the specific atrial nucleoprotein marker was verified by immunofluore scence staining and quantitative real time polymerase chain reaction(qRT-PCR).Then,the atrial myocytes were divided into rapid atrial pacing(RAP)group and control group.The RAP group was subjected to high-frequency electrical stimulation with frequency of 7 Hz for 24 h.The control group received no electrical stimulation.Calcium homeostasis was detected by calcium transient.Annexin V/propidium iodide(AV/PI)was used to analyze the apoptosis rate of cells and the gene expression of apoptosis indicators(Bax and Bcl-2)and calcium channel(RYR2)were detected by qRT-PCR.Calcium transient was conducted after the treatment of 4-phenylbutyrate(4 PBA)in tachypacing cells. Results The high purity of atrial myocytes were obtained by differentiation.After high-frequency electrical stimulation,theΔF/F0 was significantly lower in RAP group compared with the control group.Meanwhile,the expression of gene RYR2 was decreased compared with control group.The disordered calcium homeostasis of atrial myocytes was significantly reduced after the treatment of 4 PBA.The sum rate of cell early and late apoptosis in the RAP group was also higher than control group.The expression of bax was up-regulated and the expression of bcl-2 was decreased compared with control group. Conclusion High-frequency electrical stimulation would lead to abnormal intracellular calcium homeostasis and cell apoptosis,which accounting for electrical injury of atrial myocytes.