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To improve proarrhythmic predictability of preclinical models, we assessed whether human ventricular-like embryonic stem cell-derived cardiomyocytes (hESC-CMs) can be selected following a standardized protocol. Also, we quantified their arrhythmogenic response and compared this to a contemporary used rabbit Purkinje fiber (PF) model. Multiple transmembrane action potentials (AP) were recorded from 164 hESC-CM clusters (9 different batches), and 12 isolated PFs from New Zealand White rabbits. AP duration (APD), early afterdepolarizations (EADs), triangulation (T), and short-term variability of repolarization (STV) were determined on application of the I(Kr) blocker E-4031 (0.03/0.1/0.3/1 muM). Isoproterenol (0.1 muM) was used to assess adrenergic response. To validate the phenotype, RNA isolated from atrial- and ventricular-like clusters (n=8) was analyzed using low-density Taqman arrays. Based on initial experiments, slow beating rate (<50 bpm) and long APD (>200 ms) were used to select 31 ventricular-like clusters. E-4031 (1 muM) prolonged APD (31/31) and induced EADs only in clusters with APD90>300 ms (11/16). EADs were associated with increased T (1.6+/-0.2 vs 2.0+/-0.3) and STV (2.7+/-1.5 vs 6.9+/-1.9). Rabbit PF reacted in a similar way with regards to EADs (5/12), increased T (1.3+/-0.1 vs 1.9+/-0.4), and STV (1.2+/-0.9 vs 7.1+/-5.6). According to ROC values, hESC-CMs (STV 0.91) could predict EADs at least equivalent to PF (STV 0.69). Isoproterenol shortened APD and completely suppressed EADs. Gene expression analysis revealed that HCN1/2, KCNA5, and GJA5 were higher in atrial/nodal-like cells, whereas KCNJ2 and SCN1B were higher in ventricular-like cells (P<0.05). Selection of hESC-CM clusters with a ventricular-like phenotype can be standardized. The proarrhythmic results are qualitatively and quantitatively comparable between hESC-CMs and rabbit PF. Our results indicate that additional validation of this new safety pharmacology model is warranted.

The xCELLigence real time cell analyzer Cardio system offers a new system for real-time cell analysis that measures impedance-based signals in a label-free noninvasive manner. The aim of this study was to test whether impedance readings are a useful tool to detect compound effects on beating frequency (beats per minute, bpm) and arrhythmias of human induced pluripotent stem cell-and a mouse embryonic stem cell-derived cardiomyocyte line (hiPSC-CM and mESC-CM, respectively). Baseline values for control wells were 45 -3 and 179 -6 bpm, respectively (n = 6). Correspondingly, isoproterenol increased beating frequency by 77% and 71%, whereas carbachol decreased frequency by 11% and 100% (stopped in 5/6 mESC-CM wells). E-4031 decreased beating rate and caused arrhythmias in both cell types, however, more pronounced in the human iPSC-CMs. Amlodipine inhibited contractions in both models, and T-type calcium channel block strongly reduced beating rate and eventually stopped beating in mESC-CM but caused a smaller effect in hiPSC-CM. The results of this initial study show that, under the right conditions, the beating frequency of a monolayer of cells can be stably recorded over several days. Additionally, the system detects changes in beating frequency and amplitude caused by added reference compounds. This assay system has the potential to enable medium-throughput screening, but for implementation into routine daily work, extended validation, testing of additional batches of cardiomyocytes, and further assay optimization (e.g., frequency of media exchange, growth matrix, seeding density, age of cells after plating, and temperature control) will be needed.

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