Ajmaline blocks I Na and I Kr without eliciting differences between Brugada syndrome patient and control human pluripotent stem cell-derived cardiac clusters

Figures

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  Fig. 1. Reprogramming of hiPSCs and differentiation to cardiac clusters. A) Immunocytochemistry of pluripotency associated factors in iPS-HS1M hiPSCs following reprogramming from HDFs. Bar = 100 μm. B) Immunocytochemistry of lineage markers of mesoderm (ACTA2), neurectoderm (TUBB3) and endoderm (SOX17) plus DAPI staining (blue) following non-directed EB differentiation and outgrowth. Bar = 100 μm. C) G banding of iPS-HS1M, indicating a normal karyotype following reprogramming. D) Schematic with phase contrast images inset of hiPSC differentiation to cardiac clusters. For media formulations see Supplementary table S2. Bar = 500 μm. E) Flow cytometry analysis of TNNT2 expression in iPS-HS1M at differentiation day 21, with IgG isotype control inset. F) Immunocytochemistry of NKX2-5 and TNNT2 plus DAPI at day 28 of differentiation following partial dissociation of iPS-HS1M cardiac clusters. Bar = 100 μm.

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  Fig. 2. Effects of ion channel inhibitors on field potential (FP) of hiPSC cardiac clusters. A) Raw FP signal from a single MEA electrode, showing the effect following 6 mins superfusion of 500 nM isoprenaline (black arrow). B) Schematic of MEA protocol, with phase contrast image inset of cardiac clusters loaded onto an MEA. Bar = 500 μm. C) Representative ensemble FP signals at individual electrodes, showing effect of increasing doses of ajmaline, mexiletine or dofetilide on iPS-HS1M clusters. Arrow heads indicate examples for time points of activation and recovery peaks, to calculate cFPD. Grey dashed line shows 0 μV. D–E) Mean iPS-HS1M cluster activation-recovery interval over increasing drug doses (D), or activation complex downslope gradient dV/dt(minimum) at highest drug dose (E), showing effect of ajmaline (n ≥ 9), mexiletine (n ≥ 9) and dofetilide (n ≥ 4, some clusters ceased beating or were too inconsistent to generate ensemble averages at higher doses) normalised to baseline, error bars show ± s.e.m. One way ANOVA followed by Tukey HSD (D) or paired t-test (E), ** = p ≤ 0.01 or * = p ≤ 0.05 respectively vs baseline before normalisation.

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  Fig. 3. Ajmaline blocks both INa and IKr in hiPSC-CMs. A) Representative INa traces and current density (pA/pF) under control conditions and after application of ajmaline (100 μM), n = 15 cells. B) Representative traces showing the effect of application of ajmaline (100 μM) on IKr tail currents. The voltage protocol and scale are inset. C) Digital subtraction of the current blocked by ajmaline. D) In some cells the IKr tail current partially recovered after removal (washout) of ajmaline (indicated by arrow) and this could then be blocked by the addition of the IKr specific blocker dofetilide (1 mM). E) Current density before and after ajmaline application. F) Peak-tail current density (PTCD) before and after ajmaline application. * = p ≤ 0.05 (paired t-test) compared to baseline value before ajmaline addition, n = 8 cells. Data displayed as mean ± s.e.m.

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  Fig. 4. BrS patient ECGs. A–C) ECGs of patients BR1-P3M (A), BR1-P5M (B) and BR1-P6M (C), with an abnormal ST segment and T wave evident in precordial lead 1 (V1) and/or V2, either spontaneously (B), or after administering 1 mg/kg ajmaline (A, C).

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  Fig. 5. Comparison of BrS patient and control hiPSC-CM differentiation and APs. A–B) qRT-PCR analysis of marker expression during cardiac cluster differentiation of control iPS-HS1M and BrS patient iBR1-P5M-L1 lines. Data displayed from n = 3 (A) or n = 4 (B) independent differentiation experiments ± s.e.m. C–G) APs recorded from isolated iPS-HS1M (n = 21) and iBR1-P5M-L1 CMs (n = 17) at weeks 6–8 of differentiation. C) Representative traces and proportion of AP types (assigned as described in Methods). The arrow heads indicate that the AP was triggered (at 1 Hz). The grey dashed line indicates 0 mV. D–G) Collated AP data displaying the range of APD90 values (D), Resting Membrane Potential (RMP) (E), AP amplitude (F) and Upstroke Velocity (G). Data displayed as mean ± s.e.m. * = p ≤ 0.05 (independent t-test) vs control line iPS-HS1M.

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  Fig. 6. Effect of ajmaline on FP of BrS patient and control hPSC cardiac clusters. A) Representative ensemble FP signals at individual electrodes, showing effect of increasing doses of ajmaline on cardiac clusters from different hPSC lines. Grey dashed line shows 0 μV. B) Mean activation-recovery interval of clusters from different hPSC lines with increasing doses of ajmaline, calculated from cFPD relative to baseline reading. Data averaged from n ≥ 8 electrodes from n ≥ 3 MEA assays (see Supplementary table S1). One way ANOVA followed by Tukey HSD performed before normalisation, * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001 vs. baseline.