hiPSC-Derived Cardiomyocyte Model of LQT2 Syndrome Derived from Asymptomatic and Symptomatic Mutation Carriers Reproduces Clinical Differences in Aggregates but Not in Single Cells

Figures

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  Figure 1. Expression ratios of the wild type (WT) and the mutant (MUT) HERG alleles and localization of the HERG protein. (A) Allelic imbalance determination with the standard curve method. Standard data points were plotted as log2 (WT plasmid/MUT plasmid) ratios vs. corresponding mean ΔCt values (Ct(MUT)–Ct(WT)). Standard curve (dashed line) was determined with the least squares fit method. Expression ratios of the HERG alleles in the long QT syndrome type-2 (LQT2) samples were calculated using the mean ΔCt values of the samples and the standard curve equation. Mean ΔCt value for the LQT2 asymptomatic was calculated from the triplicates of the UTA.03412.LQT2 human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) samples and the mean ΔCt value for the symptomatic was calculated from the UTA.03809.LQT2 and the UTA.03810.LQT2 hiPSC-CM samples. Error bars indicate the standard errors of ΔCt means. The locations of the data points indicate that the HERG (KCNH2) alleles are expressed in all three cell lines with the wild type/mutant allele ratio somewhere between 1:2 and 1:1. The allelic imbalance assay was repeated with another set of samples of the three cell lines, yielding…

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  Figure 2. IKr densities: (A) IKr currents were obtained as 1 µM E-4031 sensitive currents with the used protocol. Inset: the voltage protocol used to elicit the IKr current. (B,C) Current-voltage plot of the peak IKr current densities and the tail IKr densities respectively from controls (black), for the LQT2 asymptomatic (red), and symptomatic (blue) hiPSC-CMs. Values inside parentheses indicate the number of hiPSC-CMs analyzed. Data is presented as the mean ± SEM, (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001 between control and asymptomatic and (#) p < 0.05, (##) p < 0.01 and (###) p < 0.001 between control and symptomatic hiPSC-CMs.

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  Figure 3. LQT2 hiPSC-CM shows increased calcium transient abnormalities. (A) Response of the control, the LQT2 asymptomatic and the symptomatic hiPSC-CMs to application of 10 nM adrenaline. (B) Percentage of CMs showing arrhythmia at baseline (BL) and during 10 nM adrenaline (Adr 10nM) perfusion. (C) Comparison of type of arrhythmia observed at baseline and under adrenaline (Adr) in the control, the LQT2 asymptomatic and the symptomatic CMs. (D) Representative arrhythmia observed in the control, the LQT2 asymptomatic and the symptomatic hiPSC-CMs Ca2+ transients. LQT2 asymptomatic CMs have most occurrences of oscillations and low amplitude peaks, while symptomatic CMs have quite equal amounts of all the five kinds of abnormalities but more delay in rise and decay phase than the LQT2 asymptomatic. RG = Regular, OS = oscillations and multiple peaks, LP = low amplitude peaks, PL = plateau abnormality/delay in decay, RD = rise delay, VA = varying amplitude. (E–L) Ca2+ imaging parameters at baseline and in the presence of 10 nM adrenaline. Data is presented for n = 68, 78, 44 for the control, the LQT2 asymptomatic, and the symptomatic respectively, as the mean ± SEM, (*) p < 0.05, (**) p < 0.01…

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  Figure 4. LQT2 hiPSC-CMs from the symptomatic patients presenting with increased arrhythmia on adrenaline application (A) Representative field potential traces from the control, the LQT2 asymptomatic and the symptomatic aggregates at baseline. The red arrows depict the end point of the repolarization duration, i.e., the field potential duration (FPD); the FPD was calculated from the start of the depolarization wave until the repolarization wave reaching the base. (B,C) The scatter-dot plots show the beat rate and the corrected field potential durations (cFPDs) from the control, the LQT2 asymptomatic and the symptomatic (labelled as (C,A and S with black, red and blue colors respectively), the boxes show the mean and SEM, for n = 62, 46, 38, respectively, for both the plots. (D) Percentage of aggregates showing arrhythmia at baseline, vehicle and under 100 nM, 300 nM, 600 nM and 1 µM adrenaline application; n numbers for the control, the LQT2 asymptomatic and the symptomatic; Bl-Veh n = 20, 19, 20, Bl–Adr100nM n = 22, 17, 22, Bl-Adr300nM n= 20, 16, 23, BL-Adr600nM n = 23, 19, 22 and BL-Adr1µM n = 24, 18, 21, respectively. (E) Comparison of the types of arrhythmia observed at baseline and…

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  Figure 5. The effects of the IKr and IKs blockers (A,B) The plots show the responses of the control, the LQT2 asymptomatic and the symptomatic hiPSC-CM aggregates to the IKr blocker E4031 at 300 nM and the occurrence of major arrhythmia for the vehicle (distilled H2O) n = 26, 18, 19 respectively, and for 300 nM E4031 n = 39, 17, 24, respectively. (C,D) Responses of the control, the LQT2 asymptomatic and the symptomatic hiPSC-CM aggregates to the IKs blocker JNJ303 300 nM and the occurrence of major arrhythmia for the vehicle (DMSO) n = 9, 3, 11, respectively, and 300 nM JNJ303 n = 27, 13, 12, respectively. Data is presented as mean ± SEM, (*) p < 0.05 and (***) p < 0.001 compared between Long QT asymptomatic or symptomatic and control and (#) p <0.05, compared on all three lines by ANOVA. (E,F) Representative field potentials and the characteristic arrhythmia observed in the control, the LQT2 asymptomatic and the symptomatic hiPSC-CM aggregates at baseline (black) and after application of E4031 and JNJ303 (red); the prolongation in FPD is marked by orange and blue lines, whereas EAD-like arrhythmias and premature ectopic beats (PEBs) are marked with black arrowheads.

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  Figure 6. Drug responses of healthy controls vs. LQT2 hiPSC-CMs in-silico. (A) Comparison of AP from the healthy control hiPSC (black) and the LQT2 (blue) showing a prolongation of 20.53% in APD90. (B) Effect of p.L552S HERG mutation on the densities of K+ currents, with a reduction in IKr, but an increase in IKs. (C,D) Effect of the IKr blocker E4031 at 0 µM (black), 0.1 µM (yellow), 0.2 µM (orange) and 0.3 µM (red) on AP morphology (left panel), in healthy control hiPSC and LQT2, and the effect of the IKr block 0%–100% on the L-type calcium channel (middle panel) and the sodium channel exchanger NCX current (Sodium calcium exchanger- calcium efflux current) (right). (E,F) Effect of the IKs blocker JNJ303 on the control (top) and the LQT2 (bottom) with baseline as (solid line) 0.3 µM JNJ303 (dotted line) on the AP morphology. Finally, the effect of the IKs block 0%–100% on the densities of IKr and IKs, shown using black solid and red dotted lines for the controls and blue solid and pink dotted lines for the LQT2, respectively.