Screening drug-induced arrhythmia [corrected] using human induced pluripotent stem cell-derived cardiomyocytes and low-impedance microelectrode arrays

Abstract

Background: Drug-induced arrhythmia is one of the most common causes of drug development failure and withdrawal from market. This study tested whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) combined with a low-impedance microelectrode array (MEA) system could improve on industry-standard preclinical cardiotoxicity screening methods, identify the effects of well-characterized drugs, and elucidate underlying risk factors for drug-induced arrhythmia. hiPSC-CMs may be advantageous over immortalized cell lines because they possess similar functional characteristics as primary human cardiomyocytes and can be generated in unlimited quantities.

Methods and results: Pharmacological responses of beating embryoid bodies exposed to a comprehensive panel of drugs at 65 to 95 days postinduction were determined. Responses of hiPSC-CMs to drugs were qualitatively and quantitatively consistent with the reported drug effects in literature. Torsadogenic hERG blockers, such as sotalol and quinidine, produced statistically and physiologically significant effects, consistent with patch-clamp studies, on human embryonic stem cell-derived cardiomyocytes hESC-CMs. False-negative and false-positive hERG blockers were identified accurately. Consistent with published studies using animal models, early afterdepolarizations and ectopic beats were observed in 33% and 40% of embryoid bodies treated with sotalol and quinidine, respectively, compared with negligible early afterdepolarizations and ectopic beats in untreated controls.

Conclusions: We found that drug-induced arrhythmias can be recapitulated in hiPSC-CMs and documented with low impedance MEA. Our data indicate that the MEA/hiPSC-CM assay is a sensitive, robust, and efficient platform for testing drug effectiveness and for arrhythmia screening. This system may hold great potential for reducing drug development costs and may provide significant advantages over current industry standard assays that use immortalized cell lines or animal models.

Keywords: arrhythmias, cardiac; genomics; myocytes, cardiac; pharmacogenetics; pharmacology; stem cells.

Figure 1. hiPSC-CM expression of cardiac ion channel genes and sarcomeric proteins

A, Heat map showing the gene expression levels of the main adrenoreceptors and ion channels essential for cardiomyocyte function in hiPSCs and human adult heart chambers. Color scale represents delta-delta Ct values relative to the expression levels in hiPSC-CMs. Green indicates upregulation and red downregulation. Strong red corresponds to genes which are not expressed. Average linkage cluster for the samples is presented. LA refers to left atrium, RA to right atrium, LV to left ventricle, and RV to right ventricle (n=4). B, Confocal microscopy image of alpha-actinin (top) and troponin T (middle) immunostaining demonstrates the presence of cardiac specific sarcomeric proteins in a single hiPSC-CM. The inset in the merged image at the bottom shows organized horizontal myofilaments with parallel red striations indicating the sarcomeric Z-lines.

Figure 2. Video contractility analyses demonstrate high percentage of hiPSC-CMs in embryoid bodies

A, Embryoid body plated on a gelatin-coated MEA probe grid. B, Heat map showing the levels of contractility within the EB at a given point in time. C, Ratio of troponin T positive cells (in red) to nuclei (stained for DAPI in blue) was used to determine the percentage of cardiomyocytes in dissociated EBs. D, Graph comparing the contractile area of the EBs studied to the percentage of cells stained positively for TNNT2 (n=10).

Figure 3. QT interval, action potential duration, and field potential duration

A, EKG (lead 4) obtained from the subject illustrating the QT interval. B, Action potential recorded via whole-cell patch-clamping of a representative ventricular hiPSC-CM from a dissociated EB showing the APD. C, Field potentials recorded from a representative EB via MEAs denoting the electrophysiological parameters of interest. Note the similarity between the EKG inset on the top left corresponding to the time course of a typical action potential recorded via conventional patch-clamping on the top right inset.

Figure 4. Norepinephrine causes sustained increases in hiPSC-CM beating frequency

A, Rhythmic spontaneous field potentials at baseline during the last 30 seconds of a 10 minute recording. B, Effect of 1 μM norepinephrine (NE) on beat frequency. C, Graph plotting the timecourse of increase in beat frequency due to 10 μM NE in a representative EB. D, Steady-state concentration-dependent increases on absolute beating frequency. Note the stability of the response during the last 30 seconds of a 10-minute recording. n=8 EBs for the NE testing.

Figure 5. Norepinephrine induces a dose-dependent increase in BPM and consequently a decrease in FPD

A, The dose dependent effect of NE on beat rate could be fitted well with the Hill equation (red line), yielding a half maximal excitatory concentration of 41.66 ± 16.50 nM (n=4). B, Representative traces illustrating the dose-dependent shortening of FPD secondary to NE. Arrow points to the left-shifting peak of the repolarizing wave to indicate shortening of the FPD. C, Graph plotting NE concentration - FPD response and fit to the Hill equation (red line connecting the data points, n=3). The IC₅₀ was 19.38 nM. D, NE concentration vs. FPD corrected according to the Bazett formula and Hill fit (red line). n=8 EBs for the NE testing.

Figure 6. hERG blocker sotalol prolongs FPD despite reducing BPM

A, The effect of sotalol (30 μM) on the FPD of a representative EB begins almost immediately after drug administration and stabilizes within approximately 7 minutes. B, Dose-dependent prolongation of field potentials caused by sotalol in a representative EB. Arrow points to the right-shifting peak of the repolarizing wave to indicate lengthening of the FPD. C, Sotalol also affects BPM, necessitating a normalization procedure because FPD is related to beat frequency (n=4). D, The dose dependent effect of sotalol on FPD (corrected according to the Bazett formula) could be fitted nicely with the Hill equation (red line) (n=4). Asterisks note the FPDc prolongation was statistically and physiologically significant at concentrations of 100 uM and higher.

Figure 7. hERG block induces arrhythmic activity in hiPSC-CMs

A, Typical notched or bifurcated repolarizing wave seen in LQTS2 and sotalol cardiotoxicity (n=6). B, Ectopic beats in a short-long-short rhythm were observed at a sotalol concentration of 500 μM and higher (n=6). C, Early afterdepolarizations (EADs) induced by quinidine (1 μM). Arrows point to the different arrhythmic events (n=5).

Figure 8. MEA/hiPSC-CM platform identifies false-positive and false-negative hERG blockers

A, Dose-dependent shortening of field potentials caused by verapamil in a representative EB. Arrow points to the left-shifting peak of the repolarizing wave to indicate shortening of the FPD. B, The dose-dependent decreases in the normalized mean FPDc could be fitted nicely with the Hill equation (red line), evidencing a half maximal effect at 169.28 ± 24.00 nM (n=4). C, Alfuzosin induced dose-dependent FPD prolongation in our hiPSC-CMs as shown in this graph of a representative EB's responses. Arrow points to the right-shifting peak of the repolarizing wave to indicate lengthening of the FPD. D, FPDc prolongation was statistically and physiologically significant at 300 nM and higher concentrations as noted by the asterisks (n=4).