Common Genetic Variants of The Cardiac Sodium Channel Alter Patient Response to Class 1b Antiarrhythmics

Figures

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  Figure 1:. Automated planar patch clamp recordings of the mexiletine block of Nav1.5 currents in transiently transfected HEK293 cells. A) Patchliner schematic. B) Representative INa trace. C) The protocol used to measure UDB consists of a train of 50 test pulses to −20mV at varying frequencies. D) As the frequency of stimulation is increased, the mexiletine dose response becomes increasingly left-shifted. E) Mexiletine tonic block measured at 0Hz frequency. The S1103Y IC50 (44.6±10.2 μM) is significantly left-shifted compared to WT (IC50: 185.1±22.4 μM), p=0.006. The other variants do not exhibit a significant effect. B) Mexiletine use-dependent block measured at 10Hz frequency. The S1103Y IC50 (13.4±2.7 μM) is significantly left-shifted compared to WT (IC50: 31.1±4.4 μM), p=0.014. The other variants do not exhibit a significant effect. Sample size is 8–12 cells per variant.

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  Figure 2:. NaV1.5 localization within iPSC-derived cardiomyocyte nanodomains. NaV1.5 (purple) colocalizes with α-actinin at the z-discs (A-C, blue) and with N-cadherin at the intercalated discs (D-F, yellow) in WT corrected S1103Y−/− (A, D), patient heterozygous S1103Y+/− (B, E), and homozygous S1103Y+/+ (C, F) cardiomyocytes. Single plane images were presented as whole cells and zoomed in views.

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  Figure 3:. Mexiletine block of Nav1.5 currents in iPSC-CMs lines with WTc or MVET-55 background. Top panel: the S1103Y homozygous line was CRISPR-engineered from the WTc line. A) Representative INa traces before and after mexiletine. B) Mexiletine tonic block measured at 0Hz stimulation. The S1103Y+/+ IC50 (7.08±1.8 μM) is significantly left-shifted compared to WTc (IC50: 100.3±22.9 μM), p=0.0005. C) Mexiletine use-dependent block measured at 10Hz frequency. The S1103Y+/+ IC50 (4.2±1.0 μM) is significantly left-shifted compared to WT corrected (IC50: 24.4±2.7μM), p=0.0004. Bottom panel: the WT corrected and S1103Y homozygous lines were CRISPR-engineered from the patient-derived MVET-55 line (S1103Y heterozygous). D) Representative INa traces before and after mexiletine. E) Mexiletine tonic block measured at 0Hz frequency. The S1103Y+/+ IC50 (12.2±3.3 μM) is significantly left-shifted compared to WT corrected (IC50: 37.8±7.8 μM), p=0.018. S1103Y+/− (IC50: 39.7±8.1 μM) exhibits a dose response similar to WT corrected (p=0.98), while it is significantly right-shifted compared to S1103Y+/+ (p=0.018). F) Mexiletine use-dependent block measured at 10Hz frequency. The S1103Y+/+ IC50 (8.2±2.1 μM)…

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  Figure 4:. Action potential duration (APD) and late sodium current measurements in MVET-55 background iPSC-CM lines. A) Representative APD90 traces measured at 1Hz in control and mexiletine-treated cells. 20μM mexiletine was applied 15 minutes before recording. The Savitzky-Golay filter was applied to all representative traces. B) Mexiletine significantly prolongs the APD90 (+175.8ms) in the S1103Y homozygous line (p < 0.001) compared to control. The WT corrected and S1103Y heterozygous lines do not exhibit significant changes in APD90 after mexiletine treatment. C) Late-to-peak sodium current ratio in control and mexiletine-treated cells measured at −20mV. The S1103Y homozygous line shows a significant increase in late-to-peak INa ratio (+0.0147, p = 0.02) after mexiletine treatment. No changes are observed in the WT corrected and S1103Y heterozygous line. D) Representative sodium current traces in control and mexiletine treated cells in WT corrected (top row), S1103Y heterozygous (patient line, middle row), or S1103Y homozygous iPSC-CMs, with recording voltage protocol in inset.

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  Figure 5:. Nav1.5 kinetics in control and mexiletine-treated iPSC-CMs. A) Acute mexiletine (20μM) application does not significantly reduces peak INa in the S1103Y−/−, S1103Y+/− or S1103Y+/+ lines (p = 0.99, 0.55, 0.72 respectively). B) The conductance-voltage and steady-state inactivation curves are not significantly different across the S1103Y−/−, S1103Y+/−, and S1103Y+/+ lines in control vs mexiletine-treated cells (GV V1/2: p > 0.99. SSI V1/2: p > 0.99). C) Mexiletine slows down the fast phase of recovery in S1103Y−/−, S1103Y+/−, and S1103Y+/+ cells. τ10 p-values = 0.0001, 0.04, 0.02, respectively. The slow phase of recovery, although slightly slower with mexiletine, is not significantly different. τ1 p-values > 0.99. D) Voltage protocols for Nav1.5 activation, inactivation and recovery.