Comparing human iPSC-cardiomyocytes versus HEK293T cells unveils disease-causing effects of Brugada mutation A735V of NaV1.5 sodium channels

Figures

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  Figure 1. Inducing CRISPR/Cas9 mediated A735V-NaV1.5 mutation and cardiac differentiation. (a) Scheme of CRISPR/Cas9-mediated introduction of point mutation g.2204C > T in SCN5A. (b) Sequences of SCN5A showing mutation g.2204C > T in two derived MUT hiPSC-CMs compared to the isogenic WT hiPSC-CMs. One mutant clone (MUT2) possesses an additional heterozygote point mutation at position g.2197 T > G resulting in p.F733V and thus heterozygous mutant (the relevant sequence position is indicated by an arrowhead). However, this point mutation has not been reported in any cardiac disease and following Supplementary Fig. S7 mutation p.F733V presumably does not influence the channel properties. (c) Pluripotency markers (SOX2, OCT4) expression in WT and derived MUT hiPSC-CMs. (d) Flow cytometry for the CM-specific markers cardiac Troponin T (cTnT), sarcomeric Actinin (Sarc.Act) and pan-myosin heavy chain (MyHC) showed ~50–70% CMs for WT, MUT1 and MUT2 clones after 14 days of differentiation. Lower bar graphs show qRT-PCR results on MYH6 and SCN5A expression levels for WT, MUT1 and MUT2 clones. (e) IF staining of cardiac aggregates with antibodies against SCN5A (red), sarcomeric actinin (Sarc.Act,…

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  Figure 2. Mutant A735V-NaV1.5 channels reduce the upstroke velocity of APs in hiPSC-CMs. (a) Distribution of cardiomyocyte phenotypes for all hiPSC-CMs (WT + A735V, left), WT hiPSC-CMs (middle) and A735V hiPSC-CMs (right). Cells were classified according to their AP duration as ventricular-like (APD50> 200 ms), atrial-like (APD50: 20-200 ms) or atypical (APD50 <20 ms or no AP). (b,c) Representative traces of spontaneous action potentials from ventricular-like hiPSC-CMs expressing WT (b) or mutant A735V (c) NaV1.5 channels. (d–g) Electrophysiological properties of spontaneous APs (mean values ± s.e.m.) from WT (n = 16) and A735V hiPSC-CMs (n = 41): Maximum diastolic potential MDP (d), AP duration at 50% repolarization level APD50 (e), AP amplitude (f) and upstroke velocity (g). (h,i) Representative traces of evoked action potentials from ventricular-like hiPSC-CMs expressing WT (h) or mutant A735V (i) NaV1.5 channels. For such recordings CMs were hyperpolarized to physiological resting potentials (-80 mV) and action potentials were elicited by intracellular application of short depolarizing current pulses (≤3 nA, 1 ms). (j–m) Electrophysiological properties of evoked APs (mean values ±…

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  Figure 3. Mutation A735V shifts the activation curve of NaV1.5 channels in hiPSC-CMs. (a,b) Voltage protocol for sodium channel activation and representative current traces of cardiac NaV1.5 channels recorded from WT hiPSC-CMs (a) and mutant A735V hiPSC-CMs (b). (c) Voltage dependence of mean NaV1.5 current densities (pA/pF) from WT and mutant A735V hiPSC-CMs. Inset: Comparison of the maximum NaV1.5 current density from WT and mutant A735V hiPSC-CMs. (d,e) Voltage protocol for sodium channel inactivation and representative current traces of cardiac NaV1.5 channels recorded from WT hiPSC-CMs (d) and mutant A735V hiPSC-CMs (e). (f) Sodium channel activation (G/Gmax) and inactivation (I/Imax) curves of NaV1.5 channels from WT and A735V hiPSC-CMs. Solid lines represent Boltzmann functions that were fit to the mean values. (g,h) Scatter plots and mean values (±s.e.m.) of mid voltages (V0.5) of activation (g) and inactivation (h) for WT and A735V-NaV1.5 channels from hiPSC-CMs. - Data volume for sodium channel activation: WT, n = 20; A735V, n = 57; for sodium channel inactivation: WT, n = 17; A735V, n = 56.

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  Figure 4. Effects of A735V mutation are reproducible from clone-to-clone and for different approaches of hiPSC-CMs. (a) Mean values (±s.e.m.) of upstroke velocities of two WT and two A735V mutant clones. There are no differences among WT clones or A735V clones, but differences between WT and A735V clones are highly significant (one way ANOVA with Tukey’s post-hoc comparison test F(3,78) = 26.32, p < 0.001). (b) Inter-experimental variability between different approaches with WT and mutant A735V hiPSC-CMs showing no changes in upstroke velocities among themselves, while WT versus A735V differed considerably from each other (one way ANOVA with Tukey’s post-hoc test F(6,75) = 12.82, p < 0.001). (c) Inter-experimental variability of mean maximal sodium current densities for WT and A735V mutant hiPSC-CMs (one way ANOVA with Tukey’s post-hoc test F(6,70) = 7.467, p < 0.001). (d) An increase in sodium channel density directly correlates to upstroke velocity acceleration of the same cells throughout the different hiPSC-CM approaches corroborating the effect of A735V sodium channel mutation on upstroke velocity (d, linear regression with R² = 0.9937 and F(1,5) = 749.9; n = 7-19). Coloured symbols…

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  Figure 5. Mutation A735V shifts the activation curve of NaV1.5 channels heterologously expressed in HEK293T cells. (a,b) Voltage protocol for sodium channel activation and representative current traces of WT (a) and mutant A735V (b) -NaV1.5 channels heterologously expressed in HEK293T cells. c) Voltage dependence of mean current densities (pA/pF) from HEK293T cells expressing WT or mutant A735V-NaV1.5 channels. Inset: Bar graph on comparison of the maximum current density for WT and mutant A735V NaV1.5 channels. (d,e) Voltage protocol for sodium channel inactivation and representative current traces of WT (d) and mutant A735V (e) NaV1.5 channels heterologously expressed in HEK293T cells. (f) Sodium channel activation (G/Gmax) and inactivation (I/Imax) curves of WT and A735V NaV1.5 channels expressed in HEK293T cells. Solid lines represent fits with Boltzmann functions. (g,h) Scatter plots and mean values (±s.e.m.) of mid-voltages (V0.5) of activation (g) and inactivation (h) for WT and mutant A735V-NaV1.5 channels. (i) Representative western blot analysis for heterologous NaV1.5 channel expression in HEK293T cells, showing similar intensities for WT and mutant A735V-NaV1.5 bands.…

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  Figure 6. Mutation A735V prolongs the time constant of recovery from inactivation for NaV1.5 sodium channels expressed in hiPSC-CM and HEK293T cells. (a) Two pulse protocol to investigate the recovery from inactivation and representative current responses of WT and mutant A735V NaV1.5 channels expressed in HEK293T cells or hiPSC-CMs. Inter-pulse interval (Δt) varied from 1 to 1000 ms (inter-pulse voltage: -120 mV). (b,c) Time dependent increase of relative current amplitudes (INa (pulse2)/INa (pulse1)) fitted by a bi-exponential function reveal two time constants (τ1 and τ2) for recovery from inactivation of WT and mutant A735V NaV1.5 channels expressed in HEK293T cells (b) and hiPSC-CMs (c; data points < 10 ms are not displayed for hiPSC-CMs, according to improper fast voltage clamp conditions with large cardiomyocytes). (d–f) Scatter plot and mean values (±s.e.m.) of fast (d) and slow time constants (e,f) of recovery from inactivation for WT and mutant A735V channels expressed in HEK293T cells (d,e) or hiPSC-CMs (f). - Data volume in (b,d) and (e): WT, n = 18; A735V, n = 16. Data volume in (c) and (f): WT, n = 6; A735V, n = 36.