Delayed Repolarization Caused by hERG Block With Different Drug Modalities Can Be Detected in Stem Cell-Derived Cardiomyocytes: Incubation Time Matters

Abstract

The intrinsic characteristics of oligonucleotides pose a challenge for their assessment in conventional primary in vitro cardiac models, which were designed for the acute application of small molecule agents and are not suitable for transfection and extended culture periods. Conversely, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) offer a viable platform for the evaluation of agents over prolonged application and recording times. Our previous experiments demonstrated that a chronic protocol of 48 h is necessary to discern the functional effects of a siRNA targeting hERG in a stable cell line heterologously expressing hERG. To investigate whether a targeted hERG siRNA induces delayed repolarization in hiPSC-CM, we recorded field potentials (FPs) using a multielectrode array. FP duration (FPD) prolongation was noted as early as 10 min after exposure to moxifloxacin, whereas pentamidine required 24 h to induce FPD prolongation. Transfection with hERG-targeting siRNA reduced mRNA expression at 6 h post-transfection. However, FPD prolongation was only observed after 24 h post-transfection, with significantly larger effects at 48 h, which is indicative of the time needed for turnover of the hERG protein on the plasma membrane. Our findings provide compelling evidence that MEA recordings in hiPSC-CM can accurately detect disruptions in cardiac repolarization due to various mechanisms that impair hERG channel function, including direct channel blockade, inhibition of protein trafficking, and gene silencing via siRNA. The findings also indicate that indirect mechanisms of hERG knockdown, including gene silencing, require assessment at least 48 h following treatment to detect delayed repolarization in the hiPSC-CM model.

Keywords: PCR; hERG; hiPSC‐CM; multielectrode array; oligonucleotide; siRNA.

FIGURE 1

Moxifloxacin increased beat rate corrected field potential duration (FPDc) in hiPSC‐CM. Percent changes from baseline and the application time of 10 min, 6, and 24 h are presented under different conditions as labeled. Compared to vehicle control (DMSO), statistically significant prolongation of FPDc was observed at 10 and 30 μM moxifloxacin at all time points. The number of wells, from the same plate, in each condition are indicated, and statistical significance is indicated as **p < 0.01; 5.**p < 0.001; ****p < 0.0001.

FIGURE 2

Pentamidine increased beat rate corrected field potential duration (FPDc) in hiPSC‐CM. Effects are observed at 24 h after application while having no effect on FPDc at 10 min and 6 h after treatment. Percent changes from baseline are presented under different conditions as labeled. At 24 h after application, compared to vehicle control (DMSO), statistically significant prolongation of FPDc was achieved at 1 and 3 μM of pentamidine. The number of wells, from the same plate, in each condition are indicated, and statistical significance is indicated as *p < 0.05; ****p < 0.0001.

FIGURE 3

Lack of effects on hKCNH2 expression by treatments with moxifloxacin and pentamidine for 24 h. These were the same cells after the MEA recordings. Each measurement was performed with four to six wells pooled together from the same plate. The hKCNH2 expression was normalized to HPRT, a housekeeping gene in hiPSC‐CM.

FIGURE 4

Selection of positive hKCNH2 siRNA, negative siRNA, and the testing concentration. Left panel: Expression level of hKCNH2 relative to HPRT1 following transfection of hiPSC‐CM with negative and positive KCNH2 siRNAs. Relative hKCNH2 expression after 48 and 72 h transfection with negative sRNAs (1 and 2), and KCNH2 siRNA (1, 2, 3, 4, and a mixture of all 4 were shown). Each individual siRNA was tested at 10 nM and the concentration of each siRNA in the mixture was 2.5 nM. Each condition was tested twice, and three wells of cells from the same plate were pooled together. Right panel: Relative expression level of hKCNH2 was decreased by KCNH2 siRNA transfection in a concentration‐dependent manner while there was no effect of Negative KCNH2 siRNA 48 h after transfection in hiPSC‐CM. Each sample was from eight wells pooled together from one plate.

FIGURE 5

Time‐dependent effects of KCNH2 siRNA on beat rate corrected field potential duration (FPDc) in hiPSC‐CM. Percent change from baseline is presented under different conditions as labeled. The tested concentration of siRNA was 30 nM. No effects were observed at 6 h, statistically significant prolongation of FPDc was achieved at 24 and 48 h after transfection with greater effects observed at 48 h. The number of wells in each condition are indicated, one plate was used for each time point. Statistical significance is indicated as ****p < 0.0001.

FIGURE 6

Time‐dependent effects of KCNH2 siRNA on relative hKCNH2 expression levels. The measurement of qPCR was performed with the same cells after MEA recordings. The cells were harvested from five to six wells and pooled from the same plate (three samples at each condition). Each symbol represented one sample and the bar represented the mean value.

FIGURE 7

Gene silencing occurs before functional changes. Overlay plots of percent changes in beat rate corrected field potential duration (FPDc) and KCNH2 mRNA level caused by KCNH2 siRNA transfection in hiPSC‐CM.