Flecainide ameliorates arrhythmogenicity through NCX flux in Andersen-Tawil syndrome-iPS cell-derived cardiomyocytes

Abstract

Andersen-Tawil syndrome (ATS) is a rare inherited channelopathy. The cardiac phenotype in ATS is typified by a prominent U wave and ventricular arrhythmia. An effective treatment for this disease remains to be established. We reprogrammed somatic cells from three ATS patients to generate induced pluripotent stem cells (iPSCs). Multi-electrode arrays (MEAs) were used to record extracellular electrograms of iPSC-derived cardiomyocytes, revealing strong arrhythmic events in the ATS-iPSC-derived cardiomyocytes. Ca²⁺ imaging of cells loaded with the Ca²⁺ indicator Fluo-4 enabled us to examine intracellular Ca²⁺ handling properties, and we found a significantly higher incidence of irregular Ca²⁺ release in the ATS-iPSC-derived cardiomyocytes than in control-iPSC-derived cardiomyocytes. Drug testing using ATS-iPSC-derived cardiomyocytes further revealed that antiarrhythmic agent, flecainide, but not the sodium channel blocker, pilsicainide, significantly suppressed these irregular Ca²⁺ release and arrhythmic events, suggesting that flecainide's effect in these cardiac cells was not via sodium channels blocking. A reverse-mode Na^+/Ca²⁺exchanger (NCX) inhibitor, KB-R7943, was also found to suppress the irregular Ca²⁺ release, and whole-cell voltage clamping of isolated guinea-pig cardiac ventricular myocytes confirmed that flecainide could directly affect the NCX current (I_NCX). ATS-iPSC-derived cardiomyocytes recapitulate abnormal electrophysiological phenotypes and flecainide suppresses the arrhythmic events through the modulation of I_NCX.

Keywords: Andersen-Tawil syndrome; Arrhythmia; Cardiomyocyte; iPS cell.

Fig. 1

Electrocardiograms of patients with ATS, Electrocardiograms from the patients with ATS during sinus rhythm.　QT and QTc intervals: QT interval/(RR interval)^1/2 are 480 ms and 462 ms (R218W), 400 ms and 400 ms (R67W), 420 ms and 383 ms (R218Q), respectively. QU and QUc intervals: QU interval/(RR interval)^1/2 are 680 ms and 654 ms (R218W), 680 ms and 680 ms (R67W), 720 ms and 657 ms (R218Q), respectively. The normal QTc interval is <440 ms. The boxes on the right represent the sequence analysis of genomic KCNJ2 from each iPSC.

Fig. 2

Generation of iPSCs from three patients with ATS, a. Immunofluorescence staining for stem cell markers (Tra1-81, NANOG, OCT3/4, and SSEA3) in iPSCs of ATS case (R218W). Nuclei were counterstained with DAPI. b. Microscopic observation of teratoma sections, showing tissue structures resembling gut (endoderm), cartilage (mesoderm), adipose (mesoderm), and neural tissue (ectoderm). c. Immunofluorescence staining for cardiomyocyte markers (α-actinin, ANP, cardiac troponin T (cTnT), and GATA4) in control- and ATS-iPSC-derived cardiomyocytes. Nuclei were counterstained with DAPI.

Fig. 3

Electrophysiological features of ATS-iPSC-derived cardiomyocytes, a. Representative MEA recordings from the control- and ATS-iPSC-derived beating EBs. The bidirectional red arrows indicate FPD. b. Beating rate of the control- (n=14) and ATS-iPSC-derived EBs (n=12–14) at 60 days after differentiation (control, 54.8±3.5; R218W, 58.6±8.1; R67W, 52.1±5.2; R218Q, 47.2±3.7 ms, data are mean±SEM). c. cFPD obtained from the control- (n=14) and ATS-iPSC-derived beating EBs (n=12–14) at 60 days after differentiation (control, 447.1±29.3; R218W, 529.5±27.3; R67W, 492.2±52.0; R218Q, 459.8±26.3 ms, data are mean±SEM). d, e, f, g. Representative recordings of action potential for iPSC-derived ventricular-type cardiomyocytes. h, i, j. Statistical parameters of action potential duration at 50% repolarization (APD₅₀) (control, n=13, 341.1±22.8; R218W, n=10, 343.5±27.2; R67W, n=12, 351.0±29.6; R218Q, n=6, 397.0±28.9 ms), 90% repolarization (APD₉₀) (control, 403.7±26.2; R218W, 409.1±30.4; R67W, 417.2±32.7; R218Q, 490.0±32.8 ms), and maximum diastolic potential (MDP) (control, −58.0±2.0; R218W, −55.9±1.7; R67W, −57.4±1.5; R218Q, −52.3±3.6mv) respectively. Data are mean±SEM.

Fig. 4

Isoproterenol responses of ATS-iPSC-derived cardiomyocytes, a. Representative MEA recordings showing increased beating rates after isoproterenol administration in control-iPSC-derived beating EBs. b. Representative MEA recordings after isoproterenol and flecainide administration in ATS-iPSC-derived beating EBs. c. The rates of EBs with arrhythmic events in MEA analyses (control, n =8; ATS, n=26, *P<0.05 vs. control by Fisher's exact probability test). d. The incidences of EBs with arrhythmic events after isoproterenol (1000 nM) and flecainide (5 μM) administration by MEA analysis (control, n=9; ATS, n=38, **P<0.01 by Fisher's exact probability test).

Fig. 5

Ca²⁺ transients of ATS-iPSC-derived cardiomyocytes, a. Representative line scan images of spontaneous Ca²⁺ transients in control- and ATS-iPSC-derived single cardiomyocytes. Arrowhead indicates the irregular Ca²⁺ release. b. The incidences of cardiomyocytes with irregular Ca²⁺ release at the baseline condition (control, n=58, 37.9%; ATS, R218W, n=68, 60.3%, R67W, n=53, 62.3%, R218Q, n=68, 64.7%, *P<0.05, **P<0.01 vs. control by Chi-square test). c. Representative line scan images of Ca²⁺ transients paced at 1 Hz in control- and ATS-iPSC-derived single cardiomyocytes. d, e, f, g. Statistical parameters of Ca²⁺ transient intensity (ΔF/F0) (d), time to peak (e), time to 90% decay of the Ca²⁺ transient (CaT ₉₀) (f), and of Ca²⁺ transient decline (τ) (g), **P<0.01 vs. control by student's t-test. in control- (n=22) and ATS-iPSC-derived cardiomyocytes (n=54). Data are mean±SEM. h. SR Ca²⁺ content was determined by the caffeine-induced ΔF/F0 (F0 is the baseline fluorescence and ΔF is the baseline subtracted fluorescence.) (control, n =6, 1.46±0.19; ATS, n =7, 1.29±0.15, **P<0.01 vs. control by Student's t-test, Data are mean±SEM.). i. Representative line scan images of spontaneous Ca²⁺ transients at baseline in ATS-iPSC-derived single cardiomyocytes. j. Representative line scan images of spontaneous Ca²⁺ transients after flecainide (500 nM) administration in ATS-iPSC-derived single cardiomyocytes. k. The incidences of cardiomyocytes with irregular Ca²⁺ release after flecainide administration (control, n=46, 8.7% decrease in incidence; ATS, R218W, n=38, R67W, n=57, R218Q, n=52, 23.6%, 28.1%, 23.1% decreases in incidence, respectively, *P<0.05, **P<0.01 vs. baseline by Fisher's exact probability test).

Fig. 6

Therapeutic electrophysiological pathway of ATS-iPSC-derived cardiomyocytes, a. Representative MEA recordings showing the arrhythmic events after isoproterenol and pilsicainide administration in ATS-iPSC-derived cardiomyocytes. b. The incidences of EBs with arrhythmic events after isoproterenol and pilsicainide administration in MEA analysis (n=11). c. The incidences of cardiomyocytes with irregular Ca²⁺ release after KB-R7943 administration (R218W, n =30, R67W, n=31, R218Q, n=34, 40.0%, 32.3%, 26.5% decreases in incidence, respectively. *P<0.05, **P<0.01 vs. baseline by Fisher's exact probability test).

Fig. 7

Effect of flecainide on I_NCX, a. Representative chart recording of membrane current. The horizontal bars above the current indicate when 30 μM flecainide and 5 mM Ni were applied externally. b. I-V curves obtained at the corresponding labels in panel a. a, control; b, in the presence of flecainide; c, in the presence of Ni. c. Summarized data of the augmentation effect of 30 μM and 100 μM flecainide on bi-directional I_NCX. (Ca efflux mode; 33.8±6.8% increase in I_NCX at 30 µM flecainide and 51.0±8.9% increase in I_NCX at 100 µM flecainide, Ca influx mode; 32.4±7.7% increase in I_NCX at 30 µM flecainide and 61.0±8.0% increase in I_NCX at 100 µM flecainide, **P<0.01, †P<0.001 vs. control by paired t-test; values are means±SEM. of 5 cells).