Model for long QT syndrome type 2 using human iPS cells demonstrates arrhythmogenic characteristics in cell culture

Abstract

Long QT syndrome (LQTS) is caused by functional alterations in cardiac ion channels and is associated with prolonged cardiac repolarization time and increased risk of ventricular arrhythmias. Inherited type 2 LQTS (LQT2) and drug-induced LQTS both result from altered function of the hERG channel. We investigated whether the electrophysiological characteristics of LQT2 can be recapitulated in vitro using induced pluripotent stem cell (iPSC) technology. Spontaneously beating cardiomyocytes were differentiated from two iPSC lines derived from an individual with LQT2 carrying the R176W mutation in the KCNH2 (HERG) gene. The individual had been asymptomatic except for occasional palpitations, but his sister and father had died suddenly at an early age. Electrophysiological properties of LQT2-specific cardiomyocytes were studied using microelectrode array and patch-clamp, and were compared with those of cardiomyocytes derived from control cells. The action potential duration of LQT2-specific cardiomyocytes was significantly longer than that of control cardiomyocytes, and the rapid delayed potassium channel (I(Kr)) density of the LQT2 cardiomyocytes was significantly reduced. Additionally, LQT2-derived cardiac cells were more sensitive than controls to potentially arrhythmogenic drugs, including sotalol, and demonstrated arrhythmogenic electrical activity. Consistent with clinical observations, the LQT2 cardiomyocytes demonstrated a more pronounced inverse correlation between the beating rate and repolarization time compared with control cells. Prolonged action potential is present in LQT2-specific cardiomyocytes derived from a mutation carrier and arrhythmias can be triggered by a commonly used drug. Thus, the iPSC-derived, disease-specific cardiomyocytes could serve as an important platform to study pathophysiological mechanisms and drug sensitivity in LQT2.

Fig. 1.

Mutation and ECG analysis. (A) Mutation analysis confirmed the hERG-FinB mutation in the LQT2 iPSC line, which gave altered DNA cleavage by the SmaI restriction enzyme (lower arrow). (B,C) ECG from leads V1–V3 of the index patient, with a QTc of 437 ms (B), and from the patient’s sister, with the presence of a U-wave following the T-wave; QT(U)c=550 ms.

Fig. 2.

Characterization of iPSCs. (A) Morphology of the iPSC colonies is similar to those of hESCs. The colonies are rather roundish and the edges are well defined and sharp, which is typical for a stem cell colony. (B) Expression of pluripotency markers in LQT2-specific iPSCs is shown by RT-PCR. All the endogenous pluripotency genes studied were turned on in iPSCs by passage 6 (top panel). As a positive control, they were also expressed in hESCs (H7). Expression of Sox2 and very modest expression of Rex1 and Myc was found also in EBs, which were used as a negative control. β-actin served as a loading control. None of the exogenous genes were expressed in iPSCs at passage 11. As a positive control, PCR was also done using the transfected plasmids as templates (bottom panel). RT-PCR results were similar for all the iPSC lines. (C) Immunocytochemical staining of the cells shows that pluripotency markers are expressed also at the protein level. The expression of Nanog, Oct3/4, Sox2, SSEA-4, TRA1-60 and TRA1-81 was similar in all iPSC lines and there were no differences between LQT2-specific and control lines. (D) Karyotypes of all the iPSC lines were analyzed and proved to be normal. (E) Teratomas were made from one LQT2-specific line and two control lines to further confirm the pluripotency of the lines. Tissues from all three germ layers were found in teratomas from every line. (F) EBs were also formed from all the lines to show the pluripotent differentiation capacity. The EB-derived cells from both LQT2-iPSC and all control iPSC lines expressed markers from the three embryonic germ layers.

Fig. 3.

The expression of cardiac markers in iPSC-derived cardiomyocytes and the electrical properties of the cells. (A) Immunocytochemical staining of different cardiac markers: troponin T and α-actinin are shown in red; green indicates connexin-43 and blue represents DAPI-staining for nuclei. The expression was similar in LQT2-specific and control cells, and there were no line-specific differences in the expression of cardiac proteins. (B) The expression of a larger repertoire of cardiac markers was also studied, with RT-PCR showing that the iPSC-derived cardiac cells manifest cardiac properties. TNTT2, MLC2V, MLC2A, Cx-43, MYH7, GATA4 and HERG were present in the cells at the mRNA level. (C) Electrical properties of the cells were studied with MEA, which revealed the differences between LQT2-specific and control cells. FPD was significantly longer in LQT2-specific cardiomyocytes than in control cardiac cells. However, all lines evince the typical electrical properties of cardiomyocytes. (D) LQT2-specific cardiomyocytes showed increased chronotrophy when challenged with isoprenaline, a β-adrenergic agonist.

Fig. 4.

Current-clamp recordings from human iPSC-derived cardiomyocytes. (A) Spontaneous APs from healthy control iPSC-derived (upper APs) and LQT2 patient-derived (lower APs) cardiomyocytes. The dashed line denotes 0 mV. (B) The action potential duration (APD) measured at 50% and 90% repolarization from the AP peak (APD₅₀ and APD₉₀) of spontaneous atrial-like (n=5–6) and ventricular-like APs. For the latter, both the APD₅₀ and APD₉₀ of LQT2 patient-derived cardiomyocytes (n=13) were significantly prolonged compared with those of hESCs (n=7) or control-iPSC origin (n=11). (C) Spontaneous arrhythmogenic activity of an LQT2-iPSC-derived cardiomyocyte.

Fig. 5.

I_Kr recorded from iPSC cardiomyocytes with a ventricular-like AP. (A) Example of the isolation of I_Kr. Whole-cell current, here from a control iPS cardiomyocyte, was recorded in the absence (a) and then presence of 1 μmol/l E-4031 (b), with I_Kr defined as the subtracted current (a–b), i.e. the E-4031-sensitive current. Current was evoked by a 5-second depolarization from a holding potential of −40 mV as shown in the inset. (B) I_Kr of a control iPS (black) and LQT2 iPS (red) cardiomyocyte evoked as in A with the time segment between the arrows expanded to show the peak tail currents on return to −40 mV following a step to +20 mV. (C) The peak tail I_Kr densities of control iPSC (black; n=4) and LQT2 iPSC (red; n=5) cardiomyocytes at membrane potentials from 0 to +40 mV were significantly different (*P<0.01, **P<0.005). (D) I_Kr currents of control iPSC (black) and LQT2 iPSC (red) cardiomyocytes evoked by a voltage protocol of step to +20 mV for 150 ms and ramp of 120 ms back to the −40 mV holding potential.

Fig. 6.

FPD measured on MEA. (A) The effect of the beating rate on FPD in control and LQT2 cardiomyocytes (CMs). Control and LQT2 cardiomyocytes had a negative correlation with moderately high coefficients of determination (R²). The exponential function gave the best fit as determined by R² between different fitting functions. The LQT2 cardiomyocytes had significantly more prolonged FPD compared with controls, especially at low beating frequencies, as determined by nonlinear regression analysis (P=0.0136). (B) At beating rates below 50 beats per minute (bpm), the FPD of LQT2 cardiomyocytes differed significantly from control cardiomyocytes (*P<0.05) as determined by t-test. (C) Drug responses of control and LQT2 cardiomyocytes. Sotalol (19 μmol/l) and E-4031 (500 nmol/l for LQT2-specific cells and 700 nmol/l for control cells) was administered to the cardiomyocyte aggregates derived from control iPSCs and LQT2 iPSCs. For both cell lines, baseline and drug conditions for sotalol and E-4031 are shown. Arrows mark the site of pharmacologically induced EADs. With 500 nmol/l E-4031 there were no EADs observed in control cells.