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2022 Dec 09;2412:2015-2027. doi: 10.1093/europace/euac090.
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The G213D variant in Nav1.5 alters sodium current and causes an arrhythmogenic phenotype resulting in a multifocal ectopic Purkinje-related premature contraction phenotype in human-induced pluripotent stem cell-derived cardiomyocytes.
AIMS: Variants in SCN5A encoding Nav1.5 are associated with cardiac arrhythmias. We aimed to determine the mechanism by which c.638G>A in SCNA5 resulting in p.Gly213Asp (G213D) in Nav1.5 altered Na+ channel function and how flecainide corrected the defect in a family with multifocal ectopic Purkinje-related premature contractions (MEPPC)-like syndrome.
METHODS AND RESULTS: Five patients carrying the G213D variant were treated with flecainide. Gating pore currents were evaluated in Xenopus laevis oocytes. The 638G>A SCN5A variant was introduced to human-induced pluripotent stem cell (hiPSC) by CRISPR-Cas9 gene editing and subsequently differentiated to cardiomyocytes (hiPSC-CM). Action potentials and sodium currents were measured in the absence and presence of flecainide. Ca2+ transients were measured by confocal microscopy. The five patients exhibited premature atrial and ventricular contractions which were suppressed by flecainide treatment. G213D induced gating pore current at potentials negative to -50 mV. Voltage-clamp analysis in hiPSC-CM revealed the activation threshold of INa was shifted in the hyperpolarizing direction resulting in a larger INa window current. The G213D hiPSC-CMs had faster beating rates compared with wild-type and frequently showed Ca2+ waves and alternans. Flecainide applied to G213D hiPSC-CMs decreased window current by shifting the steady-state inactivation curve and slowed the beating rate.
CONCLUSION: The G213D variant in Nav1.5 induced gating pore currents and increased window current. The changes in INa resulted in a faster beating rate and Ca2+ transient dysfunction. Flecainide decreased window current and inhibited INa, which is likely responsible for the therapeutic effectiveness of flecainide in MEPPC patients carrying the G213D variant.
Flecainide treatment reduces MEPPC symptoms in patients with the G213D variant in Nav1.5. G213D shifts Nav1.5 activation to more negative voltages and induces a gating pore current. hIPSC cardiomyocytes have depolarised resting membrane potential, rapid firing and alternans, which can be improved by flecainide. Flecainide decreased INa, which is likely responsible for the therapeutic effectiveness.
ECGs of Patient 3. (A) ECG before initiation of flecainide with PACs and PVCs. (B) ECG during treatment with flecainide with sinus rhythm.
G213D induce gating pore current in Nav1.5 G213D. (A) Alignment of S3 and S4 of the first domain of Nav1.5 from different species and DMNAV, the voltage-gated sodium channel from Drosophila melanogaster. Below is an alignment of the members of the human Nav family. Residues that have been associated with complex arrhythmias and dilated cardiomyopathy4 are underscored. (B) Cartoon showing the central pore (left part of the cartoons) and one voltage-sensing domain. S1–S3 form the gating pore that allows S4 movement in response to voltage changes (right part of the cartoons). The positive charges of the S4 are coordinated by negative (green, marked with "-") and aromatic (turquois empty circle) amino acids in the gating charge transfer centre. The G213D substitution induces a negative charge as well as more stiffness in the S3–S4 linker, which may interfere with the S4 movement through the gating charge centre. (C) Nav1.5 WT and G213D were expressed in Xenopus laevis oocytes and currents measured by two-electrode voltage-clamp. Representative recordings are shown. (D) Mean current amplitude at the end of the 500 ms step plotted as a function of voltage, n = 17 for WT and n = 21 for G213D. Statistical significance was evaluated by a two-way ANOVA followed by a Bonferroni posttest.
Representative whole-cell INa recordings from a single WT (A) and G213D (B) hiPSC-CM. Current recordings were obtained at test potentials between −80 and 30 mV in 5 mV increments from a holding potential of −120 mV. (C) I–V relation showing the threshold for G213D activation was shifted in hyperpolarized direction but reversal was unaffected. (D) Steady-state activation curves for WT and G213D hiPSC-CM. Chord conductance was determined using the ratio of current to the electromotive potential for the cells shown in (C). Data were normalized and plotted against the test potential. n = 18 for WT, n = 22 for G213D 1A and n = 21 for G213D 1C.
Representative action potentials recorded from WT (A) and G213D hiPSC-CMs (B). Triggered activity was occasionally noted in the G213D hiPSC-CM action potentials (top panel) as was the appearance of alternans (bottom panel). (C) Representative line scans recorded from a WT (top traces) and G213D (bottom traces) hiPSC-CM. The hiPSC-CM were spontaneously active with G213D cardiomyocytes showing an inherently faster beating rate. (D) Representative line scans recorded from a G213D hiPSC-CM showing different types of chaotic Ca2+ transients. The corresponding time course of F − Fo/Fo is shown below the line scans. Ca2+ transient alternans and EAD/DAD-like activity was frequently observed in the G213D hiPSC-CMs. Occasionally, Ca2+ waves were observed (bottom panel). Summary of the data is shown in Supplementary material online, Tables S1 and S2.
Representative impedance signals recorded in WT and G213D hiPSC-CMs over a 10 s window. G213D hiPSC-CMs have a faster spontaneous beating rate and occasionally showed chaotic activity compared with WT. Application of flecainide (red traces of right panels) suppressed chaotic activity and slowed the spontaneous beat rate. Results were obtained from a 96-well plate where each well contained about 75 000 cells. Within the 96-well plate, 48 wells were WT myocytes and 48 wells were G213D myocytes. Cells were from two separate differentiations (n = 2 plates).
Steady-state inactivation relation for WT (A, n = 9) and G213D myocytes (B, n = 6) in the absence and presence of flecainide (10 µM). (C) Overlay of activation and steady-state inactivation curves from WT (black traces) and G213D hiPSC-CMs (red traces) taken from Figure 3 and Supplementary material online, Figure S1, respectively. A larger window current occurring at more negative potentials was observed in G213D hiPSC-CM. (D) The shift in steady-state inactivation by flecainide altered the overlay of activation and steady-state inactivation relation resulting in less window current in G213D hiPSC-CMs. Development of use-dependent block of INa in WT (E) and G213D hiPSC-CMs (F) in the absence (black traces) and presence of flecainide (red traces). Sixty test pulses of constant duration were applied to cells at a rate of 1 Hz from a holding potential of −120 mV and every 10th pulse is shown. In the presence of flecainide (10 μM) substantial use-dependent block was observed in both WT and G213D cells. Mean data showing use-dependent block of INa by flecainide in the two cell types, n = 9 for WT and n = 6 for G213D (G).
Supplementary Figure 1: Representative recovery from inactivation traces recorded from a WT hiPSC-CM using a holding potential of either -120 (Panel A) or -100 mV (Panel B). Recovery was measured using two identical voltage-clamp steps to -20 mV from a holding potential of either -120 mV, -100 mV separated by selected time intervals. The recovery time-course of INa recorded from WT and G213D hiPSC-CM was not dramatically altered at the different holding potentials (Panel C and D). HP=-100mV: n = 24 for WT, n = 27 for G213D-1A and n = 13 for G213D-1C. HP=-120mV: n = 25 for WT, n = 30 for G213D-1A and n = 17 for G213D-1C. Representative steady-state inactivation recordings from WT (Panel E) and G213D (Panel F) hiPSC-CM. The voltage-clamp protocol is at the top of figure. Peak current was normalized to the respective maximum values and plotted against the conditioning potential. The steady–state inactivation relation showed no difference in the mid-inactivation potential (Panel G). n = 25 for WT, n = 30 for G213D-1A and n = 16 for G213D-1C