基于实时细胞分析技术验证人诱导多能干细胞分化的心肌细胞毒性评价模型

目的： 利用人诱导多能干细胞分化的心肌细胞（hiPSC-CMs）联合实时细胞分析（RTCA）技术，建立hiPSC-CMs体外心脏毒性评价模型，并选用阳性化合物进行验证。方法： 研究选用商业化来源的hiPSC-CMs，选用不同作用机制的心脏毒性药物：蒽环类抗癌药阿霉素（0.3、1、3 μmol·L^-1）、K⁺通道阻滞剂多非利特（0.03、0.1、0.3 μmol·L^-1）和E-4031（0.1、0.3、1 μmol·L^-1）、Na⁺通道阻滞剂美西律（3、10、30 μmol·L^-1）、多离子通道阻滞剂维拉帕米（0.1、0.3、1 μmol·L^-1）和苄普地尔（0.3、1、3 μmol·L^-1）、抗组胺药物特非那定（0.3、1、3 μmol·L^-1）和西沙必利（3、10、30 μmol·L^-1），分别给予hiPSC-CMs作用48 h，采用RTCA技术连续监测心肌细胞搏动的频率、振幅、细胞指数、搏动图谱等指标在给药前后的变化。结果： 阿霉素对心肌细胞的抑制作用呈现时效和量效依赖性特点；多非利特显著降低心肌细胞搏动幅度及频率；E-4031为1 μmol·L^-1时显著降低心肌细胞搏动频率，缩短振幅，而0.1、0.3 μmol·L^-1时则对心肌细胞无影响；30 μmol·L^-1美西律作用2 h内心肌细胞搏动频率相对空白对照组降低了66.4%，振幅缩短了60.7%，并导致心肌细胞搏动周期延长；苄普地尔浓度低于1 μmol·L^-1时对心肌细胞无影响，3 μmol·L^-1苄普地尔作用2 h导致心肌细胞搏动间歇性停搏，并伴随细胞活力轻微下降；维拉帕米作用2 h内各剂量组心肌细胞搏动频率分别增强了12.2%、13.1%、52.3%，而振幅分别降低了20.0%、61.7%、67.6%。0.3 μmol·L^-1以上浓度的维拉帕米导致心肌细胞搏动骤停（3/9）；特非那定浓度≥ 1 μmol·L^-1时短时间内即导致心肌细胞间歇性搏动骤停；西沙必利短期作用2 h内显著降低心肌细胞搏动频率和幅度，10 μmol·L^-1以上浓度的西沙必利持续抑制心肌细胞搏动频率。结论： hiPSC-CMs结合RTCA技术可用于预测药物的心脏毒性风险。

Objective: To establish an early screening model for cardiotoxicity by using cardiomyocytes derived from human induced pluripotent stem cells(hiPSC-CMs) combined with real time cellular analysis(RTCA),and select reference compounds for validation. Methods: Commercially available hiPSC-CMs were selected and exposed to cardiotoxic compounds with different mechanisms for 48 h. Anthracycline doxorubicin(0.3,1, 3 μmol·L^-1),K⁺ channel blocker dofetilide(0.03,0.1,0.3 μmol·L^-1) and E-4031(0.1,0.3,1 μmol·L^-1),Na⁺ channel blocker mexiletine(3,10,30 μmol·L^-1),Multi-ion channel blocker verapamil(0.1,0.3,1 μmol·L^-1) and bepridil(0.3,1,3 μmol·L^-1),the antihistamine terfenadine(0.3,1,3 μmol·L^-1) and cisapride(3,10,30 μmol·L^-1). Impedance signals from hiPSC-CMs before and after exposure were analyzed for beating rate,contraction amplitude,cell index and beating traces by using RTCA system. Results: Doxorubicin inhibited hiPSC-CMs in time and dose-dependent manner. Dofetilide significantly reduced the amplitude and beat rate in a concentration-dependent manner. E-4031,at concentrations between 0.1 and 0.3 μmol·L^-1,had no effect on the hiPSC-CMs,A significant reduction in total spike amplitude and beat rate was seen at 1 μmol·L^-1. For mexiletine(30 μmol·L^-1),a 66.4% reduction in spike amplitude and 60.7% reduction in beat rate was seen within 2 h,combined with a prolonged period compared with the time-matched vehicle controls. 3 μmol·L^-1 bepridil produced cessation of beating within 2 h and a slight decrease in cell viability,while no effect in hiPSC-CMs was observed at concentrations below 1 μmol·L^-1. Verapamil significantly increased the beat rate of the cells in a concentration-dependent manner,the percentage change from the baseline in beat rates increased 12.2%,13.1%,52.3% and amplitude decreased 20.0%,61.7%,67.6% at 0.1,0.3,1 μmol·L^-1 respectively within 2 h. Concentration over 0.3 μmol·L^-1 verapamil produced cessation of beating in three out of nine wells tested. Terfenadine produced periods of intermittent cessation of beating(quiescence) at concentrations over 1 μmol·L^-1. Cispride produced a significant decrease in spontaneous beat rates and amplitude. Conclusion: hiPSC-CMs combined with RTCA can be used for drug-induced cardiotoxicity risk prediction.

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