Recapitulation of Clinical Individual Susceptibility to Drug-Induced QT Prolongation in Healthy Subjects Using iPSC-Derived Cardiomyocytes

Abstract

To predict drug-induced serious adverse events (SAE) in clinical trials, a model using a panel of cells derived from human induced pluripotent stem cells (hiPSCs) of individuals with different susceptibilities could facilitate major advancements in translational research in terms of safety and pharmaco-economics. However, it is unclear whether hiPSC-derived cells can recapitulate interindividual differences in drug-induced SAE susceptibility in populations not having genetic disorders such as healthy subjects. Here, we evaluated individual differences in SAE susceptibility based on an in vitro model using hiPSC-derived cardiomyocytes (hiPSC-CMs) as a pilot study. hiPSCs were generated from blood samples of ten healthy volunteers with different susceptibilities to moxifloxacin (Mox)-induced QT prolongation. Different Mox-induced field potential duration (FPD) prolongation values were observed in the hiPSC-CMs from each individual. Interestingly, the QT interval was significantly positively correlated with FPD at clinically relevant concentrations (r > 0.66) in multiple analyses including concentration-QT analysis. Genomic analysis showed no interindividual significant differences in known target-binding sites for Mox and other drugs such as the hERG channel subunit, and baseline QT ranges were normal. The results suggest that hiPSC-CMs from healthy subjects recapitulate susceptibility to Mox-induced QT prolongation and provide proof of concept for in vitro preclinical trials.

Keywords: QT prolongation; cardiomyocytes; clinical trial; drug safety assessment; drug susceptibility; iPSC; inter-individual difference.

Graphical abstract

Figure 1

Study Outline and Profiling of Human iPSCs Derived from Blood Samples of Each Volunteer (A) Schematic representation of the experimental approach. (B) Representative slopes of ΔQTcF with the plasma concentration of moxifloxacin (Mox) and immunostaining for OCT3/4 (red) and NANOG (green) in iPSCs. Nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 50 μm. The bottom panel (scale bars, 200 μm) shows alkaline phosphatase (ALP) staining of iPSC colonies. (C) Immunostaining for BRACHYURY (red), SOX17 (red), and OTX2 (red) in iPSC-derived mesoderm, endoderm, and ectoderm, respectively. Nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 50 μm.

Figure 2

Profiling of hiPSC-Derived Cardiomyocytes from Healthy Participants (A) Immunostaining for cardiac actin (green), cardiac troponin T (cTnT, green), and GATA4 (red) in iPSC-derived cardiomyocytes (iPSC-CMs). Nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 50 μm. (B and C) iPSC-derived cardiomyocytes isolated from beating cell clusters. (B) Detection of cardiac actin-positive cells using an IN Cell Analyzer 1000. Scale bar, 200 μm. (C) Average ratio of cardiac actin-positive to total cells in hiPSC-derived beating cell clusters. A–J show the study subjects. Error bars represent the mean ± SEM of the independent experiments (n = 3–9). (D and E) Hierarchical cluster analysis of gene expression in hiPSC-CMs with undifferentiated iPSCs and adult ventricular tissue. (D) Heatmap of genes (241 genes, 331 probes) categorized as regulating muscle contraction (GO: 6937) and having voltage-gated ion channel activity (GO: 5244). (E) Heatmap of genes related to cardiac contractile function. VE, adult ventricular tissue; A–J_CM, hiPSC-CMs derived from each volunteer.

Figure 3

Prolongation of Mox-Induced Field Potential Duration in iPSC-CMs from Each Participant (A) Phase-contrast image of iPSC-CMs plated onto a multielectrode chamber. (B) Representative waveforms of field potential recorded after dosing with Mox (0, 3, 10, 30, 50, and 100 μM). (C) Changes in field potential duration (FPD) induced by Mox (0, 3, 10, 30, 50, and 100 μM) in iPSC-CMs from all volunteers. Error bars represent the mean ± SEM of the independent experiments (n = 10–16). (D) Slopes of FPD for the indicated concentrations of Mox in iPSC-CMs from each individual. Error bars represent the mean ± SEM of the independent experiments (n = 10–16). (E) Relationship between timing of differentiation (days) and FPD in hiPSC-CMs from all volunteers.

Figure 4

Correlation between In Vivo and In Vitro Data (A) Correlation coefficient between in vitro FPD slope for the indicated Mox concentrations from each participant and ΔQTcF (blue) and ΔΔQTcF (red) slopes. ^∗p < 0.05. (B and C) Correlation between in vitro FPD slope from each participant at 0–10 μM Mox, and ΔQTcF (B) and ΔΔQTcF (C) slopes for the same individual. (D) Correlation coefficient between in vitro FPD slope for the indicated Mox concentrations from each participant and ΔQTcF-C_max (blue) and ΔΔQTcF-C_max (red) slopes. ^∗p < 0.05. (E and F) Correlation between in vitro FPD slope from each participant at 0–10 μM Mox, and ΔQTcF-C_max (E) and ΔΔQTcF-C_max (F) slopes for the same individual.