应用hiPSC-ACM和hiPSC-VCM进行药物毒性检测的研究

Cardiotoxicity assessment using human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes

目的:建立可用于评价药物心脏毒性的人源性诱导多能干细胞分化的心房、心室肌细胞(human induced pluripotent stem cell derived atrial and ventricular cardiomyocytes,hiPSC-ACM、hiPSC-VCM)模型,并采用此模型评价化合物潜在的心脏毒性。方法:通过调控视黄酸(retinoic acid,RA)通路分化出目标细胞,分为RA处理组(RA)和RA抑制剂处理组(RAi),用流式细胞法以及免疫荧光染色法检测心室特异性标志物(MLC2v)和心肌细胞特异性标志物(α-actinin),荧光定量RT-PCR法检测细胞心房、心室基因(MLC2v、MYH7、NR2F2、KCNA5)表达情况,通过细胞活性实验(CCK8)检测已知致心律失常药物特非那定、索他洛尔、伊布利特在不同浓度(0.1、1.0、100.0、1 000.0μmol/L)下对细胞的活性影响,通过钙敏染料Fluo-3 AM检测特非那定(50、100、500 nmol/L)、索他洛尔(1、10、100μmol/L)、伊布利特(0.1、1.0、10.0μmol/L)对于细胞钙瞬变的影响。结果:流式、免疫荧光染色以及荧光定量RT-PCR均显示,心房肌细胞特异性标志物在RA组高表达而在RAi组低表达,心室肌细胞标志物在RA组低表达而在RAi组高表达。RA可以促进hiPSCs向心房肌(ACM)分化,RAi可以促进hiPSCs向心室肌(VCM)分化。特非那定浓度达到较高浓度(100μmol/L)时,两组细胞的活性显著下降,而两组细胞钙瞬变振幅在较低浓度(50 nmol/L)时即出现显著下降。在高浓度索他洛尔(1 mmol/L)的刺激下两组细胞活性显著下降,同样伊布利特的浓度达到1 mmol/L时两组细胞活性均有显著下降。hiPSC-VCM在不同浓度索他洛尔以及伊布利特的刺激下钙瞬变振幅均无显著变化,然而hiPSC-ACM在特非那定1μmol/L及索他洛尔1μmol/L刺激下即开始出现振幅显著下降。结论:人源性诱导多能干细胞可以通过调控视黄酸通道分化的心房、心室肌细胞建立体外心脏毒性评价模型,通过检测其钙瞬变的改变可以迅速、准确地评价药物毒性。

Objective:To establish human induced pluripotent stem cell derived atrial and ventricular cardiomyocytes for cardiotoxicity assessment. Method:Temporarily manipulating retinoic acid signal to differentiate atrial and ventricular cardiomyocytes.Flow cytometry analysis and immunofluorescent staining were conducted to detect the cardiac-specific marker:α-actinin and ventricle-specific marker:MLC2 v. qRT-PCR was done to detect ventricle-specific marker(MLC2 v,MYH7)and atrial-specific marker(NR2 F2,KCNA5). Cell viability and calcium transient were assessed to evaluate cytotoxicity and cellular electrophysiological alterations caused by different drugs(terfenadine,sotalol and ibutilide). Result:The results of flow cytometry,immunofluorescent staining and qRT-PCR demonstrated that ventricle-specific markers were highly expressed in RAi group while atrial-specific markers were expressed in RA group. By assessing calcium transient and cell viability,we proposed a drug assessment platform using hiPSC-ACM and hiPSC-VCM.After treated with terfenadine,cytotoxic effects occured at a concentration of 100 μmol/L both in hiPSC-ACM and hiPSC-VCM.Meanwhile,both sotalol and ibutilide exhibited cardiotoxicity potential at a concentration of 1 mmol/L. In the following calcium transient assessment,the amplitudes of calcium transient were significantly decreased in hiPSC-ACM after 1 μmol/L sotalol and 1μmol/L ibutilide treatment,while in hiPSC - VCM,the amplitude showed no significant difference in such concentrations. The amplitudes of calcium transient were significantly decreased in both groups when the concentration of terfenadine reached to 1 mmol/L.Conclusion:By modulating retinoic acid signaling during hiPSC differentiation,we generated atrial and ventricular cardiomyocytes.Human induced pluripotent stem cell-derived atrial and ventricular cardiomyocytes were useful models for assessing cardiotoxicity of drugs by detecting calcium transient.