Scheel Olaf, Frech Stefanie, Amuzescu Bogdan, Eisfeld Jörg, Lin Kun-Han, Knott Thomas
1 Cytocentrics Bioscience GmbH , Rostock, Germany .
Assay Drug Dev Technol. 2014 Oct;12(8):457-69. doi: 10.1089/adt.2014.601. Epub 2014 Oct 29.
Recent progress in embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) research led to high-purity preparations of human cardiomyocytes (CMs) differentiated from these two sources-suitable for tissue regeneration, in vitro models of disease, and cardiac safety pharmacology screening. We performed a detailed characterization of the effects of nifedipine, cisapride, and tetrodotoxin (TTX) on Cor.4U(®) human iPSC-CM, using automated whole-cell patch-clamp recordings with the CytoPatch™ 2 equipment, within a complex assay combining multiple voltage-clamp and current-clamp protocols in a well-defined sequence, and quantitative analysis of several action potential (AP) parameters. We retrieved three electrical phenotypes based on AP shape: ventricular, atrial/nodal, and S-type (with ventricular-like depolarization and lack of plateau). To suppress spontaneous firing, present in many cells, we injected continuously faint hyperpolarizing currents of -10 or -20 pA. We defined quality criteria (both seal and membrane resistance over 1 GΩ), and focused our study on cells with ventricular-like AP. Nifedipine induced marked decreases in AP duration (APD): APD90 (49.8% and 40.8% of control values at 1 and 10 μM, respectively), APD50 (16.1% and 12%); cisapride 0.1 μM increased APD90 to 176.2%; and tetrodotoxin 10 μM decreased maximum slope of phase to 33.3% of control, peak depolarization potential to 76.3% of control, and shortened APD90 on average to 80.4%. These results prove feasibility of automated voltage- and current-clamp recordings on human iPSC-CM and their potential use for in-depth drug evaluation and proarrhythmic liability assessment, as well as for diagnosis and pharmacology tests for cardiac channelopathy patients.
胚胎干细胞（ESC）和诱导多能干细胞（iPSC）研究的最新进展，使得从这两种来源分化出的高纯度人心肌细胞（CMs）得以制备出来，这些心肌细胞适用于组织再生、疾病体外模型以及心脏安全药理学筛选。我们使用CytoPatch™ 2设备进行自动全细胞膜片钳记录，在一个将多种电压钳和电流钳协议按明确顺序组合的复杂试验中，对硝苯地平、西沙必利和河豚毒素（TTX）对Cor.4U®人iPSC-CM的影响进行了详细表征，并对几个动作电位（AP）参数进行了定量分析。我们根据AP形状检索到三种电生理表型：心室型、心房/结型和S型（具有类似心室的去极化且无平台期）。为了抑制许多细胞中存在的自发放电，我们持续注入-10或-20 pA的微弱超极化电流。我们定义了质量标准（封接和膜电阻均超过1 GΩ），并将研究重点放在具有类似心室AP的细胞上。硝苯地平使AP持续时间（APD）显著缩短：APD90（在1 μM和10 μM时分别为对照值的49.8%和40.8%），APD50（16.1%和12%）；0.1 μM西沙必利使APD90增加到176.2%；10 μM河豚毒素使第3相最大斜率降低到对照的33.3%，峰值去极化电位降低到对照的76.3%，并使APD90平均缩短到80.4%。这些结果证明了在人iPSC-CM上进行自动电压钳和电流钳记录的可行性，以及它们在深入药物评估和致心律失常风险评估中的潜在用途，以及对心脏离子通道病患者的诊断和药理学测试中的潜在用途。
