Identification of an INa-dependent and Ito-mediated proarrhythmic mechanism in cardiomyocytes derived from pluripotent stem cells of a Brugada syndrome patient

Figures

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  Figure 1. Characterization of hiPSC line and hiPSC-CMs. Representative data from BrS hiPSCs are presented. (A) The expression of pluripotent stem cell markers Oct-4, SSEA-4, Tra-1–60 and Tra-1–81; (B) Teratoma formation in SCID mice after hiPSCs injection confirmed by identifying three primitive germ layers: intestine (top) for endoderm, cartilage (middle) for mesoderm and neuroepithelium (bottom) for ectoderm. (C) Normal karyotype identified with BrS hiPSCs. (D) markers of cardiomyocytes identified with hiPSC-CMs. Top: α-actinin; Middle: β-MHC; and Bottom: cardiac titin.

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  Figure 2. Expression of SCN5A/Nav1.5 and INa density documented in tsa201 heterologous expression system. tsa201 cells transfected with equal amount of WT-SCN5A, A226V-SCN5A, R1629X-SCN5A and A226V-SCN5A + R1629X-SCN5A (50% each) were assayed for SCN5A/Nav1.5 expression and INa density. (Aa) Representative immunofluorescence staining images of Nav1.5 (purple) in tsa201cells. Arrows indicate the Nav1.5 staining. Levels of Nav1.5 were semi-quantified and plotted in a bar-graph (n = 3). (Ab) Representative Western blotting image of Nav1.5 in tsa201 transfected cells (n = 3). (B) Peak INa density measured in transfected tsa201 cells. (C) and (D) Steady state (SS)-inactivation and activation curves of INa measured in tsa201 expressing WT- and A226V-SCN5A. *p < 0.05; vs. WT-SCN5A.

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  Figure 3. SCN5A and Nav1.5 expressions and INa measured in hiPSC-CMs. (A) Level of SCN5A mRNA in BrS1 (hiPSC-CMs from a BrS patient with compound SCN5A mutations) and CON1 (the sib of BrS1) determined by qPCR. n = 3. (B) Representative traces of sodium currents in the control (CON1) and patient (BrS1). (C) Peak INa density measured in BrS1 and CON1. (D–F) Steady state (SS)-inactivation, activation and recovery from inactivation curves of INa. Values given are mean ± SEM. †p < 0.01; vs. CON1.

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  Figure 4. A basal action potential pattern of BrS identified in BrS1. (A and B) Representative AP waveforms recorded from a CON1 and a BrS1 paced at 1.0 Hz. The solid and dashed arrows indicate APA measured at phase-0 and phase-2, respectively. (C) The main AP parameters of CON1 and BrS1 are plotted and compared in bar-graphs. Values given are mean ± SEM. ||p < 0.00001; vs. CON1 (by unpaired t-test).

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  Figure 5. Effects of low heart rates on APs of BrS1 and CON1 hiPSC-CMs. (A, B1 and B2) (Top): representative superimposed AP waveforms recorded from CON1 and two BrS1 subgroups (non-early repolarization and early repolarization), sequentially paced at 1.0 Hz, 0.5 Hz and 0.1 Hz. (A, B1 and B2) (Bottom): superimposed AP waveforms continually recorded from CON1 and two BrS1 subgroups sequentially paced at 1.0 Hz and 0.1 Hz. (C) Main AP parameters plotted and compared among groups in bar-graphs. Values given are mean ± SEM. Statistics were performed with two-way repeated measures ANOVA followed by the Bonferroni post hoc testing. *p < 0.05; †p < 0.01; ‡P < 0.001. Black symbols: Intra-(sub) group comparisons. 0.1 Hz/0.5 Hz vs. 1.0 Hz. Pink symbols: vs. BrS1 (non-ER subgroup) at the same frequency. Green symbols: vs. CON1 subgroup at 1.0, 0.5 and 0.1 Hz.

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  Figure 6. Validation of the rate-dependent effects on APs in BrS2 and CON2. (A) Representative superimposed AP waveforms of a CON2 cell paced at 1.0 and 0.1 Hz at 24 °C. (B and C) Representative superimposed AP waveforms of BrS2 cells paced at 1 and 0.1 Hz at 24 °C and 34 °C, respectively. (D) Main AP parameters are plotted and compared among groups in bar-graphs. Values given are mean ± SEM. Statistics were performed with two-way repeated measures ANOVA followed by the Bonferroni post hoc testing (by paired t-test). *p < 0.05; ‡P < 0.001.

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  Figure 7. Effects of heart rates on Ito in hiPSC-CMs. (A-left and B-left) Representative traces of Ito currents recorded from CON1 (n = 26) and BrS (n = 22) paced at 1.0 Hz and 0.1 Hz. (A-middle and B-middle) I-V relationships of Ito show the current densities. (A-right and B-right) Graphic demonstrations and comparisons of Ito (recorded at +50 mV) in each cell are presented. Values given are mean ± SEM. §p < 0.0001; ||p < 0.00001; vs. 1.0 Hz (by paired t-test and one-way ANOVA test). ‡P < 0.001, vs. low Ito group.

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  Figure 8. Effects of 4-AP and flecainide on APs. (A) Representative superimposed AP waveforms of a BrS1 cell paced at 0.1 Hz and subsequently exposed to 0.05 mM and 0.5 mM 4-AP. Main AP parameters (showing only the results of 0.5 mM 4-AP) are presented in bar-graphs. (B) Representative superimposed AP waveforms of a V-like BrS1 hiPSC-CM paced at 1.0 Hz followed by flecainide (10 µM) treatment. Main AP parameters are presented in bar-graphs. Values given are mean ± SEM. *p < 0.05; †p < 0.01; §p < 0.0001; ||p < 0.00001 (by paired t-test); vs. 0.1 Hz (for 4-AP) or 0.1 Hz (for flecainide).