【摘要】 目的：糖尿病患者发生心律失常的风险增加，但高血糖及胰岛素治疗对人心肌细胞电生理特性的影响尚未完全明确。本研究探讨高糖与胰岛素对人诱导多潜能干细胞来源的心肌细胞(human induced pluripotent stem cells derived cardiomyocytes,hiPSC-CMs)电生理特性及诱发心律失常现象的影响。方法：采用流式细胞术分析健康个体来源的hiPSC-CMs纯度。将hiPSC-CMs分为3组：对照组(NM组，培养基含5 mmol/L葡萄糖)、高糖组(HG组，培养基含15 mmol/L葡萄糖)和高糖+胰岛素组(HG+INS组，培养基含15 mmol/L葡萄糖+100 mg/L胰岛素)，各组干预时间均为4 d。采用微电极阵列技术检测3组hiPSC-CMs在干预前后的电生理改变，包括跳动频率(beating rate,BR)、阈电位时程[(field potential duration,FPD)，类似于心电图QT间期]、FPDc(经BR校正的FPD)、锋电位与传导速率(conduction velocity,CV)；此外，采用索他洛尔干预各组hiPSC-CMs，观察其诱发FPDc延长及心律失常现象发生的情况。结果：hiPSC-CMs中心肌标志物肌钙蛋白T呈高表达，其纯度为99.06%。与NM组相比，HG组hiPSC-CMs的BR增快(9.14±0.8)%(P<0.01)；高糖处理后，HG组hiPSC-CMs的FPD从处理前的(460.4±9.0) ms延长至处理后的(587.6±23.7) ms,NM组FPD从(462.5±14.5) ms延长至(512.6±17.6) ms，与NM组相比，HG组FPD延长了(16.8±1.4)%(P<0.01)；高糖处理后，HG组hiPSC-CMs的FPDc从处理前的(389.1±13.7) ms延长至处理后的(478.3±31.5) ms,NM组从(387.7±21.6) ms延长至(422.6±32.9) ms，与NM组相比，HG组hiPSC-CMs的FPDc延长了(13.9±1.3)%(P<0.05)；但高糖处理后，NM组和HG组锋电位与CV均无明显变化(均P>0.05)。药物诱发实验发现10μmol/L索他洛尔可使HG组各培养孔hiPSC-CMs诱发出心律失常现象。高糖+胰岛素处理后，HG+INS组BR相对HG组增快(8.3±0.5)%(P<0.05);HG+INS组FPD从处理前的(463.4±9.7) ms延长至处理后的(532.6±12.8) ms,HG组从处理前的(460.4±9.0) ms延长至(587.6±23.7) ms，与HG组比较，HG+INS组FPD缩短了(12.7±1.9)%(P<0.01);HG+INS组FPDc从处理前的(387.4±4.1) ms延长至处理后的(422.4±10.0) ms,HG组从处理前的(384.8±4.0) ms延长至(476.3±11.5) ms，与HG组相比，HG+INS组FPDc缩短了(14.7±1.1)%(P<0.01)；胰岛素处理后，hiPSC-CMs锋电位显著增加，HG+INS组锋电位从处理前的(3.12±0.46) mV增加至处理后的(4.35±0.64) mV,HG组从(3.06±0.35) mV增加至(3.33±0.41) mV，与HG组相比，HG+INS组锋电位增加了(30.8±3.7)%(P<0.05)；胰岛素处理后，HG+INS组CV从处理前的(0.23±0.08) mm/ms增加至处理后的(0.32±0.08) mm/ms,HG组CV从处理前的(0.21±0.04) mm/ms增加至处理后的(0.30±0.07) mm/ms，与HG组相比，HG+INS组CV无明显变化(P>0.05)；药物诱导实验发现10μmol/L索他洛尔可使HG+INS组的FPDc延长(78.9±11.6)%，但各孔均未诱发出心律失常现象。结论：高糖可诱导hiPSC-CMs的FPD/FPDc延长，并增加其药物诱发心律失常现象的风险；胰岛素可缩短高糖诱导的FPD/FPDc延长，并能降低高糖作用下药物诱发的心律失常现象。这为糖尿病患者心肌的电生理改变及胰岛素治疗对其电生理的影响提供了实验依据；进一步的机制研究将可能为获得性甚至遗传性长QT综合征的治疗提供新的思路与方法。更多还原

【Abstract】 Objective: The risk of arrhythmia increases in diabetic patients. However, the effects of hyperglycemia and insulin therapy on the electrophysiological properties of human cardiomyocytes remain unclear. This study is to explore the effects of high glucose and insulin on the electrophysiological properties and arrhythmias of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs).Methods: Immunofluorescent staining and flow cytometry were used to analyze the purity of hiPSC-CMs generated from human skin fibroblasts of a healthy donor. The hiPSC-CMs were divided into 3 group (treated with normal medium, high glucose and insulin for 4days): a control group (NM group, containing 5 mmol/L glucose), a high glucose group(HG group, containing 15 mmol/L glucose), and a high glucose combined with insulin(HG+INS group, containing 15 mmol/L glucose+100 mg/L insulin). Electrophysiological changes of hiPSC-CMs were detected by microelectrode array (MEA) before or after treatment with glucose and insulin, including beating rate (BR), field potential duration(FPD) (similar to QT interval in ECG), FPDc (FPD corrected by BR), spike amplitude and conduction velocity (CV). Effects of sotalol on electrophysiological properties and arrhythmias of hiPSC-CMs were also evaluated.Results: The expression of cardiac-specific marker of cardiac troponin T was high in the hiPSC-CMs. The purity of hiPSC-CMs was 99.06%. Compared with the NM group, BR was increased by (9.14±0.8)% in the HG group (P<0.01). After treatment with high glucose, FPD was prolonged from (460.4±9.0) ms to (587.6±23.7) ms in the HG group,while it was prolonged from (462.5±14.5) ms to (512.6±17.6) ms in the NM group.Compared with the NM group, FPD of hiPSC-CMs was prolonged by (16.8±1.4)% in the HG group (P<0.01). The FPDc of hiPSC-CMs was prolonged from (389.1±13.7) ms to(478.3±31.5) ms in the HG group, and that was prolonged from (387.7±21.6) ms to (422.6±32.9) ms in the NM group. Compared with the NM group, the FPDc of hiPSC-CMs was prolonged by (13.9±1.3)% in HG group (P<0.01). The spike amplitude and CV remained unchanged between the HG group and the NM group (P>0.05). Ten μmol/L of sotalol can induce significant arrhythmias from all wells in the HG group. After treatment with insulin and high glucose, compared with the HG group, BR was increased by (8.3±0.5)% in the HG+INS group (P<0.05). The FPD was prolonged from (463.4±9.7) ms to (532.6±12.8) ms in the HG+INS group, while it was prolonged from (460.4±9.0) ms to (587.6±23.7) ms in the HG group. Compared with the HG group, the FPD of hiPSC-CMs was shortened by(12.7±1.9)% in the HG+INS group (P<0.01). The FPDc of hiPSC-CMs was prolonged from (387.4±4.1) ms to (422.4±10.0) ms in the HG+INS group, and that was prolonged from (384.8±4.0) ms to (476.3±11.5) ms in HG group. Compared with the HG group, the FPDc of hiPSC-CMs was shortened by (14.7±1.1)% in HG group (P<0.01). After the insulin treatment, the spike amplitude of hiPSC-CMs was increased from (3.12±0.46) mV to (4.35±0.64) mV in the HG+INS group, while it was enhanced from (3.06±0.35) mV to(3.33±0.41) m V in the HG group. The spike amplitude of hiPSC-CMs was increased by(30.8±3.7)% in the HG+INS group compared with that in the HG group (P<0.05). The CV in the HG+INS group was increased from (0.23±0.08) mm/ms to (0.32±0.08) mm/ms after insulin treatment, which was increased from (0.21±0.04) mm/ms to (0.30±0.07) mm/ms in the HG group, but there was no significant difference in CV between the HG+INS group and the HG group (P>0.05). The induction experiment showed that 10 μmol/L of sotalol could prolong the FPDc of hiPSC-CMs by (78.9±11.6)% in the HG+INS group, but no arrhythmia was induced in each well.Conclusion: High glucose can induce FPD/FPDc of hiPSC-CMs prolongation and increase the risk of arrhythmia induced by drugs. Insulin can reduce the FPD/FPDc prolongation and the risk of induced arrhythmia by high glucose. These results are important to understand the electrophysiological changes of the myocardium in diabetic patients and the impact of insulin therapy on its electrophysiology. Further study on the mechanism may provide new ideas and methods for the treatment of acquired and even inherited long QT syndrome.更多还原