Korean J Physiol Pharmacol.  2023 May;27(3):267-275. 10.4196/kjpp.2023.27.3.267.

New in vitro multiple cardiac ion channel screening system for preclinical Torsades de Pointes risk prediction under the Comprehensive in vitro Proarrhythmia Assay concepta

• An JR
• Mun SY
• Jung IK
• Kim KS
• Kwon CH
• Choi SO
• Park WS

• KMID: 2541638
• DOI: http://doi.org/10.4196/kjpp.2023.27.3.267

Abstract

Cardiotoxicity, particularly drug-induced Torsades de Pointes (TdP), is a concern in drug safety assessment. The recent establishment of human induced pluripotent stem cell-derived cardiomyocytes (human iPSC-CMs) has become an attractive human-based platform for predicting cardiotoxicity. Moreover, electrophysiological assessment of multiple cardiac ion channel blocks is emerging as an important parameter to recapitulate proarrhythmic cardiotoxicity. Therefore, we aimed to establish a novel in vitro multiple cardiac ion channel screening-based method using human iPSC-CMs to predict the drug-induced arrhythmogenic risk. To explain the cellular mechanisms underlying the cardiotoxicity of three representative TdP high- (sotalol), intermediate- (chlorpromazine), and low-risk (mexiletine) drugs, and their effects on the cardiac action potential (AP) waveform and voltage-gated ion channels were explored using human iPSC-CMs. In a proof-of-principle experiment, we investigated the effects of cardioactive channel inhibitors on the electrophysiological profile of human iPSC-CMs before evaluating the cardiotoxicity of these drugs. In human iPSC-CMs, sotalol prolonged the AP duration and reduced the total amplitude (TA) via selective inhibition of I _Kr and I _Na currents, which are associated with an increased risk of ventricular tachycardia TdP. In contrast, chlorpromazine did not affect the TA; however, it slightly increased AP duration via balanced inhibition of I _Kr and I _Ca currents. Moreover, mexiletine did not affect the TA, yet slightly reduced the AP duration via dominant inhibition of I _Ca currents, which are associated with a decreased risk of ventricular tachycardia TdP. Based on these results, we suggest that human iPSC-CMs can be extended to other preclinical protocols and can supplement drug safety assessments.

Keyword

Ion channels; Patch-clamp techniques; Safety; Stem cell; Torsades de pointes

Figure

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  Fig. 1 Action potential (AP) characterization of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). AP was elicited by minimal current injection to overcome the threshold in the current-clamp mode. (A) Representative traces of nodal-like (top), atrial-like (middle), and ventricular-like (bottom) APs in human iPSC-CMs. The AP subtypes of human iPSC-CMs are classified based solely on AP morphology, with ventricle-like CMs having longer AP duration (APD90 > 600 ms) than both nodal- and atrial-like CMs (APD90 between 500 and 600 ms). The distinction between nodal- and atrial-like APs is made based on differences in resting membrane potential (RMP), total amplitude (TA), and upstroke velocity (dV/dtmax). (B) The relative proportion of three different AP subtypes in human iPSC-CMs. n = 80. (C) The parameters of the three AP types recorded in human iPSC-CMs. Error bars, SEM. Student’s t-test, *p < 0.05.

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  Fig. 2 Effects of the selective cardiac ion channel inhibitors on action potential (AP) waveforms in human induced pluripotent stem cell-derived cardiomyocytes. Representative ventricular-type AP traces in the control and in the presence of 300 nM E-4031 (A), 1 μM nifedipine (B), and 1 μM tetrodotoxin (TTX) (C).

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  Fig. 3 Effects of the high, intermediate, and low Torsades de Pointes risk drugs on action potential (AP) waveforms in human induced pluripotent stem cell-derived cardiomyocytes. Representative ventricular-type AP traces in the control and in the presence of 14.69 μM sotalol (A), 0.03 μM chlorpromazine (B), and 2.5 μM mexiletine (C).

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  Fig. 4 Effects of the high, intermediate, and low Torsades de Pointes risk drugs on the IKr channel in human induced pluripotent stem cell-derived cardiomyocytes. To elicit the IKr current, a –80 mV holding potential was used, followed by a 2 sec depolarization to +20 mV and a 2 sec repolarization to –40 mV. The IKr currents were isolated by eliminating the ICa currents using 1 μM nifedipine in the external solution. (A) Representative traces demonstrating the effect of sotalol on the IKr channel at 14.69, 44.07, and 146.9 μM. (B) Summary of panel (A) (mean ± SEM, n = 3). (C) Representative traces demonstrating the effect of chlorpromazine on the IKr channel at 0.03, 0.09, and 0.3 μM. (D) Summary of panel (C) (mean ± SEM, n = 4). (E) Representative traces demonstrating the effect of mexiletine on IKr channel at .5, 7.5, and 25 μM. (F) Summary of panel (E) (mean ± SEM, n = 5).

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  Fig. 5 Effects of the high, intermediate, and low Torsades de Pointes risk drugs on ICa channel in human induced pluripotent stem cell-derived cardiomyocytes. To elicit the ICa current, a –50 mV holding potential was used, followed by a 300 ms depolarization to 0 mV. The ICa currents were isolated by eliminating the IKr currents using 2 mM and 4-aminopyridine in the external solution. (A) Representative traces demonstrating the effect of sotalol on the ICa channel at 14.69, 44.07, and 146.9 μM. (B) Summary of panel (A) (mean ± SEM, n = 3). (C) Representative traces demonstrating the effect of chlorpromazine on the ICa channel at 0.03, 0.09, and 0.3 μM. (D) Summary of panel (C) (mean ± SEM, n = 3). (E) Representative traces demonstrating the effect of mexiletine on the ICa channel at 2.5, 7.5, and 25 μM. (F) Summary of panel (E) (mean ± SEM, n = 3).

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  Fig. 6 Effects of the high, intermediate, and low Torsades de Pointes risk drugs on INa channel in human induced pluripotent stem cell-derived cardiomyocytes. To elicit the INa current, a –100 mV holding potential was used, followed by a 50 ms depolarization to –40 mV. The INa currents were isolated by eliminating the ICa currents using 1 μM nifedipine in the external solution. (A) Representative traces demonstrating the effect of sotalol on the INa channel at 14.69, 44.07, and 146.9 μM. (B) Summary of panel (A) (mean ± SEM, n = 3). (C) Representative traces demonstrating the effect of chlorpromazine on the INa channel at 0.03, 0.09, and 0.3 μM. (D) Summary of panel (C) (mean ± SEM, n = 3). (E) Representative traces demonstrating the effect of mexiletine on the INa channel at 2.5, 7.5, and 25 μM. (F) Summary of panel (E) (mean ± SEM, n = 3). TTX, tetrodotoxin.

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