Human induced pluripotent stem cell-cardiomyocytes for cardiotoxicity assessment: a comparative study of arrhythmiainducing drugs with multi-electrode array analysis

Abstract

Reliable preclinical models for assessing drug-induced cardiotoxicity are essential to reduce the high rate of drug withdrawals during development. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising platform for such assessments due to their expression of cardiacspecific ion channels and electrophysiological properties. In this study, we investigated the effects of eight arrhythmogenic drugs-E4031, nifedipine, mexiletine, JNJ303, flecainide, moxifloxacin, quinidine, and ranolazine-on hiPSC-CMs derived from both healthy individuals and a long QT syndrome (LQTS) patient using multielectrode array systems. The results demonstrated dose-dependent changes in field potential duration and arrhythmogenic risk, with LQTS-derived hiPSC-CMs showing increased sensitivity to hERG channel blockers such as E4031. Furthermore, the study highlights the potential of hiPSC-CMs to model disease-specific cardiac responses, providing insights into genetic predispositions and personalized drug responses. Despite challenges related to the immaturity of hiPSC-CMs, their ability to recapitulate human cardiac electrophysiology makes them a valuable tool for preclinical cardiotoxicity assessments. This study underscores the utility of integrating patientderived hiPSC-CMs with advanced analytical platforms, such as multi-electrode array systems, to evaluate drug-induced electrophysiological changes. These findings reinforce the role of hiPSC-CMs in drug development, facilitating safer and more efficient screening methods while supporting precision medicine applications.

Keywords: Electrophysiology; Arrhythmias, cardiac; Comprehensive in vitro proarrhythmia assay; Human induced pluripotent stem cells; Myocytes, cardiac.

Fig. 1. Gene expression analysis of cardiomyocyte-specific and ion channel genes in control and hiPSC-CM lines by RT-PCR.

(A) Cardiomyocyte-specific markers: The expression levels of TNNT2 and MYH6 were measured in control cells and four hiPSC-CM lines (CMC-006, CMC-011, CMC-016 derived from healthy individuals, and DPHC01i-AR derived from a patient with LQTS). The results are shown as fold changes normalized to the housekeeping gene GAPDH. The CMs of the four cell lines were normalized to their respective hiPS cells, with the hiPS cells set to 1 (upper panel). All hiPSC-CM cell lines exhibited significant upregulation of TNNT2 and MYH6 compared to the control, indicating successful differentiation into cardiomyocytes. (B) Ion channel gene expression: Key ion channel gene (SCN5A, KCNH2, KCND3, KCNQ1, and CACNA1C) expression levels were analyzed in the same cell lines. SCN5A, KCNH2, and KCNQ1 showed comparable expression levels across all cell lines, whereas KCND3 and CACNA1C exhibited variability in expression. Values are presented as mean ± SD. hiPSC-CM, human induced pluripotent stem cell-derived cardiomyocyte; cTnT, cardiac Troponin T; LQTS, long QT syndrome.

Fig. 2. Immunocytochemical analysis of cardiomyocyte markers in hiPSC-CMs.

Immunofluorescence staining was performed to detect cardiomyocyte-specific proteins in the four hiPSC-CM lines (CMC-006, CMC-011, CMC-016, and DPHC01i-AR). Myosin heavy chain α (MHCα) is shown in red, cardiac Troponin T (cTnT) is shown in green, and nuclei are stained with 4’,6-diamidino-2-phenylindole (DAPI) in blue. The merged images display co-localization of these markers, confirming the expression of cardiomyocyte-specific proteins in all cell lines. The structural organization and marker expression exhibit slight variations across the cell lines, indicating successful differentiation with some heterogeneity in morphology. Scale bar: 100 μm. hiPSC-CMs, human induced pluripotent stem cell-derived cardiomyocytes.

Fig. 3. Analysis of action potentials (AP) in hiPSC-CMs.

(A) AP characteristics: A schematic showing the AP parameters measured in hiPSC-CMs, including maximum upstroke velocity (Vmax), amplitude (Amp), maximum diastolic potential (MDP), APD50, and APD80. (B) Representative AP: AP traces recorded from the CMC-006, CMC-011, CMC-016, and DPHC01i-AR cell lines using patch-clamps. Each trace displays the characteristic features of either ventricular-like or nodal-like APs. The DPHC01i-AR cell line exhibits prolonged APD with abnormal repolarization, reflective of its LQT background. (C) Proportions of AP types: The percentage distribution of ventricular-like, atrial-like, and nodal-like APs in each cell line (CMC-006, CMC-011, CMC-016, and DPHC01i-AR). A majority of cells in each cell line exhibit ventricular-like APs. hiPSC-CMs, human induced pluripotent stem cell-derived cardiomyocytes; LQT, long QT.

Fig. 4. Multi-electrode array (MEA) analysis of electrophysiological responses to eight arrhythmogenic drugs in hiPSC-CMs (CMC-006).

MEA analysis showing changes in field potential duration (FPD), corrected FPD (FPDc), beat period, and amplitude (expressed as percentages) in response to varying concentrations of eight drugs: E4031, nifedipine, mexiletine, JNJ303, flecainide, moxifloxacin, quinidine, and ranolazine. The arrhythmic beats observed with E4031 are represented as the number of occurrences relative to the total number of repetitions (observations/repetitions). The blue dot line indicates the 20% change threshold for FPD and FPDc, which is considered biologically relevant for cardiotoxicity. The arrow indicates the FPD20 or FPDc20, with the depth of the red color representing the decrease in FPD20 or FPDc20. hiPSC-CMs, human induced pluripotent stem cell-derived cardiomyocytes.

Fig. 5. Multi-electrode array (MEA) analysis of electrophysiological responses to eight arrhythmogenic drugs in hiPSC-CMs (CMC-011).

MEA evaluation depicting the percentage alterations in field potential duration (FPD), corrected FPD (FPDc), beat period, and amplitude in response to varying concentrations of eight arrhythmogenic drugs: E4031, nifedipine, mexiletine, JNJ303, flecainide, moxifloxacin, quinidine, and ranolazine. The arrhythmic beats observed with mexiletine, JNJ303, moxifloxacin, and ranolazine are represented as the number of occurrences relative to the total number of repetitions (observations/repetitions). The blue dot line indicates the 20% change threshold for FPD and FPDc. The arrow indicates the FPD20 or FPDc20, with the depth of the red color representing the decrease in FPD20 or FPDc20. hiPSC-CMs, human induced pluripotent stem cell-derived cardiomyocytes.

Fig. 6. Multi-electrode array (MEA) study of drug-induced electrophysiological effects in hiPSC-CMs (CMC-016).

MEA study illustrating the percentage changes in electrophysiological parameters, including field potential duration (FPD), corrected FPD (FPDc), beat period, and amplitude, observed after exposure to eight drugs: E4031, nifedipine, mexiletine, JNJ303, flecainide, moxifloxacin, quinidine, and ranolazine. The blue dot line indicates the 20% change threshold for FPD and FPDc. The arrow indicates the FPD20 or FPDc20, with the depth of the red color representing the decrease in FPD20 or FPDc20. hiPSC-CMs, human induced pluripotent stem cell-derived cardiomyocytes.

Fig. 7. Multi-electrode array (MEA) analysis of electrophysiological changes in response to drugs in hiPSC-CMs (DPHC01i-AR).

MEA analysis highlighting the percentage changes in field potential duration (FPD), corrected FPD (FPDc), beat period, and amplitude, triggered by varying concentrations of eight drugs: E4031, nifedipine, mexiletine, JNJ303, flecainide, moxifloxacin, quinidine, and ranolazine. The blue dot line indicates the 20% change threshold for FPD and FPDc. The arrow indicates the FPD20 or FPDc20, with the depth of the red color representing the decrease in FPD20 or FPDc20. hiPSC-CMs, human induced pluripotent stem cell-derived cardiomyocytes.

Fig. 8. Comparison of drug-induced responses in hiPSC-CMs across cell lines.

(A) Heatmap showing the minimum concentrations that induce a change in FPD of more than 20% (FPD20). (B) Heatmap showing the minimum concentrations that induce a change in FPDc of more than 20% (FPDc20). The depth of red color represents FPD20 or FPDc20 concentrations, from the lowest to the highest, respectively. White indicates that no concentration tested induced a change in FPD or FPDc of over 20%. hiPSC-CMs, human induced pluripotent stem cell-derived cardiomyocytes; FPD, field potential duration; FPDc, corrected FPD.