Probing flecainide block of INa using human pluripotent stem cell-derived ventricular cardiomyocytes adapted to automated patch-clamping and 2D monolayers
Probing flecainide block of INa using human pluripotent stem cell-derived ventricular cardiomyocytes adapted to automated patch-clamping and 2D monolayers
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DOI：
10.1016/j.toxlet.2018.05.006
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发表时间：
2018-09-15
影响因子：
3.5
通讯作者：
Li, Ronald A.
中科院分区：
文献类型：
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作者：
Geng, Lin;Kong, Chi-Wing;Li, Ronald A.
人多能干细胞衍生的心肌细胞（hPSC-CM）是用于诸如药物发现和筛选作为药物消耗的主要原因的促血管生成和心脏毒性的应用的新兴工具。了解hPSC-CM的电生理学（EP）是必不可少的，但传统的手动膜片钳非常费力且低通量。在这里，我们适应hPSC-CM来自两个人胚胎干细胞（hESC）系，HES 2和H7，为16通道自动平面记录方法的单细胞EP表征。自动电流和电压钳位，总体成功率为55.0 +/-11.3%，表明90%的hPSC-CM显示心室样动作电位（AP），来自两个hESC系的心室心肌细胞（VCM）表达相似水平的I-Na，I-CaL，I-kr和I-f，同样缺乏I-to和I-K1。这些充分表征的hPSC-VCM也可以容易地适用于促炎药物筛选的自动化测定。作为一个例子，我们表明氟卡尼（FLE）诱导的I-Na阻断，抑制稳态失活偏移，减缓了我们的hPSC-VCM从失活中的恢复。由于单细胞EP测定不足以预测药物诱导的折返性心律失常，因此将hPSC-VCM进一步重新组装成2D人心室心肌单层（hvCML）用于多细胞电生理评估。事实上，FLE显著减慢传导速度，同时导致AP延长。我们的RNA-seq数据表明，细胞-细胞相互作用增强了hPSC-VCM的成熟。总的来说，需要使用单细胞EP和hvCML的组合方法来全面评估药物诱导的致炎性。
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are emerging tools for applications such as drug discovery and screening for pro-arrhythmogenicity and cardiotoxicity as leading causes for drug attrition. Understanding the electrophysiology (EP) of hPSC-CMs is essential but conventional manual patch-clamping is highly laborious and low-throughput. Here we adapted hPSC-CMs derived from two human embryonic stem cell (hESC) lines, HES2 and H7, for a 16-channel automated planar-recording approach for single-cell EP characterization. Automated current- and voltage-clamping, with an overall success rate of 55.0 +/- 11.3%, indicated that 90% of hPSC-CMs displayed ventricular-like action potential (AP) and the ventricular cardiomyocytes (VCMs) derived from the two hESC lines expressed similar levels of I-Na, I-CaL, I-kr and I-f and similarly lacked I-to and I-K1. These well-characterized hPSC-VCMs could also be readily adapted for automated assays of pro-arrhythmic drug screening. As an example, we showed that flecainide (FLE) induced I-Na blockade, leftward steady-state inactivation shift, slowed recovery from inactivation in our hPSC-VCMs. Since single-cell EP assay is insufficient to predict drug-induced reentrant arrhythmias, hPSC-VCMs were further reassembled into 2D human ventricular cardiac monolayers (hvCMLs) for multi-cellular electrophysiological assessments. Indeed, FLE significantly slowed the conduction velocity while causing AP prolongation. Our RNA-seq data suggested that cell-cell interaction enhanced the maturity of hPSC-VCMs. Taken collectively, a combinatorial approach using single-cell EP and hvCMLs is needed to comprehensively assess drug-induced arrhythmogenicity.