Korean J Physiol Pharmacol 2023; 27(3): 267-275
Published online May 1, 2023 https://doi.org/10.4196/kjpp.2023.27.3.267
Copyright © Korean J Physiol Pharmacol.
Jin Ryeol An1, Seo-Yeong Mun2, In Kyo Jung1, Kwan Soo Kim1, Chan Hyeok Kwon1, Sun Ok Choi1,*, and Won Sun Park2,*
1Pharmacological Research Division, Toxicological Evaluation and Research Department, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Cheongju 28159, 2Department of Physiology, Kangwon National University School of Medicine, Chuncheon 24341, Korea
Correspondence to:Sun Ok Choi
Won Sun Park
Author contributions: J.R.A. conceptualization, writing – original draft, data collection, data analysis. S.Y.M. and I.K.J. data analysis and interpretation. K.S.K. drafting the manuscript. C.H.K. critical revision of the paper. S.O.C. and W.S.P. conceptualization, funding acquisition, and final approval of the completed manuscript.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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 IKr and INa 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 IKr and ICa currents. Moreover, mexiletine did not affect the TA, yet slightly reduced the AP duration via dominant inhibition of ICa 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.
Keywords: Ion channels, Patch-clamp techniques, Safety, Stem cell, Torsades de pointes
In the 1990s to early 2000s, eight non-cardiac drugs were withdrawn from the market because of their association with Torsade de Pointes (TdP), a potentially life-threatening ventricular arrhythmia condition . Up to 90% of new compounds that pass preclinical testing fail at the clinical trial phase, with cardiotoxicity accounting for the failure of 45% of the compounds . Hence, the International Conference for Harmonization (ICH) presented the regulatory guidelines S7B and E14 in 2005, which focuses on two markers to assess TdP risk:
The three preclinical elements of the CiPA initiative include (1)
In this study, we established a modified human iPSC-CM-based
Sotalol, chlorpromazine, mexiletine, E-4031, nifedipine, tetrodotoxin (TTX), and 4-aminopyridine were purchased from Tocris Cookson and dissolved in distilled water. Fibronectin was purchased from Gibco.
Human iPSC-CMs (Cardiosight-S C-001; NEXEL) were cultured for single-cell electrophysiological experiments. Frozen vials of human iPSC-CMs were thawed quickly in a water bath at 37°C and mixed with an ice-cold plating medium (Cardiosight-S Advanced Plating Medium CM-010A). The cells were transferred to 24-well culture plates (1.4 × 105 cells/well) containing 50 µg/ml fibronectin-coated glass coverslips at a low density to yield single uncoupled cells. Thereafter, the cells were maintained in a humidified incubation chamber containing 5% CO2 at 37°C. After 24 h of incubation, the plating medium was replaced with a maintenance medium (Cardiosight-S Advanced Maintenance Medium CM-001A), which was changed every 2 days. The cells were cultured for 1 week and used 7–14 days post-thaw for electrophysiological analysis.
To record whole-cell membrane currents, human iPSC-CMs were placed in an experimental chamber (RC-26G; Warner Instruments) mounted on the stage of an Olympus BX51WI upright microscope. A heated external solution (37°C) was continuously perfused into the chamber. The patch pipettes were pulled from borosilicate glass capillaries (TW100-4; World Precision Instruments) using a vertical puller (PP-830; Narishige Scientific Instrument Laboratory) to establish 1–3 MΩ when filled with the internal solution. We recorded whole-cell membrane voltage or current using a patch clamp amplifier (Axopatch 200 B; Axon Instruments) in voltage-clamp or current-clamp mode and an A/D converter (Axon Digidata 1550B; Axon Instruments) data acquisition system controlled by software (pCLAMP 10; Axon Instruments).
The external solution for APs and IKr channel recordings contained NaCl (150 mM), KCl (5.5 mM), HEPES (10 mM), NaH2PO4 (0.33 mM), CaCl2 (1.8 mM), MgCl2 (1 mM), and dextrose (10 mM), with the pH adjusted to 7.4 using 1 M NaOH. The internal solution for APs and IKr channel recordings contained K-Asp (120 mM), NaCl (5 mM), KCl (20 mM), HEPES (10 mM), EGTA (5 mM), Mg-ATP (5 mM), and CaCl2 (1.8 mM), with the pH adjusted to 7.25 using 1 M KOH. The external solution for ICa channel recordings contained NaCl (135 mM), CsCl (10 mM), HEPES (5 mM), NaH2PO4 (0.33 mM), CaCl2 (1.8 mM), MgCl2 (0.5 mM), and dextrose (16.6 mM), with the pH adjusted to 7.4 using 1 M NaOH. The external solution for INa channel recordings contained NaCl (130 mM), CsCl (15 mM), HEPES (5 mM), NaH2PO4 (0.33 mM), CaCl2 (1.8 mM), MgCl2 (0.5 mM), and dextrose (16.6 mM), with the pH adjusted to 7.4 using 1 M NaOH. The internal solution for ICa and INa channel recordings contained Cs-Asp (120 mM), NaCl (5 mM), CsCl (20 mM), TEA-Cl (10 mM), HEPES (10 mM), EGTA (10 mM), and Mg-ATP (5 mM), with the pH adjusted to 7.25 using 1 M CsOH.
Data acquisition and statistical analyses were performed using pCLAMP (Axon Instruments) and Origin 8.0 (OriginLab Corp.). Data are presented as the mean ± SEM, and
Using the whole-cell patch clamp technique in the current-clamp mode, we first measured the APs in spontaneously beating cells isolated from human iPSC-CMs. Based on the distinct heterogeneity of human iPSC-CMs, three major AP subtypes (nodal, atrial, or ventricular-like) (Fig. 1 and Table 1) were characterized based on a predominant AP morphology. Fig. 1A shows a schematic overview of representative traces of spontaneous APs recorded from human iPSC-CMs. Differentiation of ventricular-like APs (Fig. 1A, bottom) was performed on the relatively more negative resting membrane potential (RMP) and rapid AP upstroke with a long plateau phase. Atrial-like APs showed a lack of a plateau phase and shorter AP duration compared to those shown by ventricular-like APs (Fig. 1A, middle). A prominent phase 4 depolarization with a less negative RMP than those of atrial- and ventricular-like CMs was characteristic of nodal-like APs, resulting in slower AP upstroke (Fig. 1A, top). Most cells (~87.5%) showed ventricular-like APs, whereas nodal-like APs and atrial-like APs were also recorded among all the cells (
Table 1 . Comparison of action potential parameters between human iPSC-CMs and native human VCMs.
|Cell types||RMP (mV)||TA (mV)||dV/dtmax (V/s)||APD90 (ms)|
|Human iPSC-CMs||Nodal-like||–30.5 ± 2.54||42.4 ± 3.25||6.2 ± 3.77||520.5 ± 5.54|
|Atrial-like||–59.6 ± 1.25||96.1 ± 2.54||37.1 ± 2.01||541.6 ± 3.54|
|Ventricular-like||–63.5 ± 1.87||120.6 ± 1.21||46 ± 1.52||730.6 ± 1.01|
|Native human VCMs ||–81.8 ± 3.3||106.7 ± 1.4||215 ± 33||351 ± 14|
Values are expressed as mean ± SEM. iPSC-CMs, induced pluripotent stem cell-derived cardiomyocytes; Native human VCMs, native human ventricular cardiomyocytes; RMP, resting membrane potential; TA, total amplitude; dV/dtmax, maximum upstroke velocity; APD90, action potential duration at 90%.
To identify the utility of human iPSC-CMs as testbeds for drug-induced cardiotoxicity, we investigated whether the APs were actually affected by cardiac ion channel inhibitors, such as E-4031, nifedipine, and TTX specific for IKr, ICa, and INa channels, respectively (Fig. 2). Application of 300 nM E-4031 induced a significant prolongation of the APs; APD90 was increased by 20.6 ± 2.12%. There were no significant changes in the RMP, TA, and dV/dtmax (
In the CiPA initiative, human iPSC-CM-based cardiovascular safety assessment and pharmacology tests were performed using various drugs with clinical torsogenic information . We first tested the effects of high, intermediate, and low TdP risk drugs on the AP waveforms of human iPSC-CMs using whole-cell patch-clamp techniques. Sotalol, a high TdP risk drug, significantly prolonged the APD90 to 26.12 ± 1.2% at 1× Cmax (14.69 µM), with a minor reduction in TA (
Repolarization-related currents (IKr) and depolarization-related currents (ICa and INa) are the major cardiac ion currents most closely related to the cardiac safety of drugs . To understand the cardiotoxic mechanism of TdP risk drug-induced modification of AP waveforms, the effects of these drugs on IKr, ICa, and INa in human iPSC-CMs were analyzed using the whole-cell patch clamp technique. Each ion current was activated using appropriate one-step pulse protocols under pharmacological conditions to selectively isolate the currents (see the Materials and Methods section).
To investigate the effects of the high, intermediate, and low TdP risk drugs on the repolarization-related current, IKr was recorded. To record IKr, human iPSC-CMs were depolarized for 2 sec to +20 mV from a holding potential of –80 mV, followed by repolarization back to –40 mV for 2 sec. Fig. 4 shows the concentration-dependent effects of sotalol, chlorpromazine, and mexiletine on IKr amplitude in human iPSC-CMs. Sotalol at 1×, 3×, and 10× free plasma Cmax reduced the amplitude of IKr by 12.07%, 33.68%, and 71.34%, respectively (
The effects of high, intermediate, and low TdP risk drugs on depolarization-related currents, ICa and INa, were analyzed. The ICa of human iPSC-CMs was elicited using a one-step pulse from a holding voltage of –50 mV to 0 mV for a duration of 300 ms. Fig. 5 shows the representative ICa traces under control conditions and after exposure to sotalol, chlorpromazine, and mexiletine. All drugs were found to reduce the amplitude of ICa in a concentration-dependent manner, with mexiletine having the greatest effect and sotalol having the least effect. Sotalol at 1×, 3×, and 10× free plasma Cmax reduced the amplitude of ICa by 7.91%, 18.91%, and 22.25%, respectively (
The aim of the present study was to establish a new
Cardiotoxicity screening, including
Previous studies have demonstrated that sotalol, chlorpromazine, and mexiletine inhibit hERG currents, which may explain the cardiac toxicity of these drugs [14-16]. Although it is widely accepted that TdP, known as fatal ventricular arrhythmia, is primarily caused by hERG inhibition, the limitations of such a simple interpretation have also been reported . An abnormally prolonged QT is sensitive; however, it is not highly specific for predicting which drugs can cause TdP. Additionally, drug-induced blockade of hERG does not indicate a clear correlation with QT interval prolongation risk or the occurrence of proarrhythmia. Therefore, the integrated effects of the non-hERG cardiac ionic currents should be considered. Inhibition of both INa-late and ICa has been associated with a reduction in QT interval prolongation and TdP, even in the presence of hERG inhibition [18-20]. Therefore, when assessing TdP liabilities, a new alternative
In this study, we investigated the effects of sotalol, chlorpromazine, and mexiletine on AP features and major cardiac ion channels, including IKr, ICa, and INa channels, in human iPSC-CMs. Included in the drugs tested was one representative drug from each of the CiPA risk categories (high-, intermediate-, and low-risk TdP). These categories were based on the risk of drug-associated TdP . We compared the ion channel inhibition against all tested ion currents at 1×, 3×, and 10× free plasma Cmax. Sotalol, which is classified as having a high risk for TdP, inhibited IKr at the clinical free plasma Cmax either exclusively or to a much greater extent than any other currently examined drug. In the intermediate TdP risk category, the extent of blocking IKr by chlorpromazine was, on average, less than that by a drug in the high-risk category, supporting a lower risk of TdP. In the low TdP risk category, mexiletine was associated with greater inhibition of ICa than that of IKr. The extent of APD prolongation was determined from the complex interactions with IKr, ICa, and INa channels, which were differentially altered by tested drugs. This was consistent with the CiPA study, in which high-risk drugs exhibited greater hERG/IKr inhibition than inhibition of other channels, whereas intermediate- and low-risk drugs exhibited greater Cav1.2/ICa inhibition than hERG/IKr inhibition . Our results demonstrated the potential of human iPSC-CMs as valuable tools for predicting drug-induced cardiotoxicities and support their use as part of a tiered testing strategy. Thus, we suggest that human iPSC-CMs could be a comparable or more effective platform than the comprehensive
In conclusion, our findings suggest that, compared to heterologous expression systems, human iPSC-CMs efficiently replicated the effects of drugs on cardiac AP and voltage-gated ion channels. Therefore, human iPSC-CMs can replace the established
This study was supported by a grant (21181MFDS276) from the Ministry of Food and Drug Safety, Republic of Korea from 2021–2022.
The authors declare no conflicts of interest.
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