Beneficial effects of chronic mexiletine treatment in a human model of SCN5A overlap syndrome

Figures

• 

  Figure 1. Forty-eight hour incubation with 10 µm mexiletine increases peak INa in HEK293A cells transfected with SCN5A-WT or SCN5A-1795insD. (A) Schematic representation of the performed experiments. (B, C) Representative INa traces recorded in HEK293A cells transfected with either SCN5A-WT (B) or SCN5A-1795insD (C) following 48 h incubation with vehicle or mexiletine (10 µm). (D–G) Average peak INa current–voltage (I–V) relationships (D, E) and voltage dependence of activation and inactivation (F, G) in HEK293A cells transfected with SCN5A-WT or SCN5A-1795insD following 48 h incubation with vehicle or mexiletine (10 µm). Insets: voltage protocols; *P < 0.05, two-way RM ANOVA followed by Holm–Sidak test for post hoc analysis. WT, wild-type.

• 

  Figure 2. Acute mexiletine treatment does not affect peak INa in HEK293A cells transfected with SCN5A-WT or SCN5A-1795insD previously incubated with either vehicle or mexiletine. (A) Schematic representation of the performed experiments. (B–E) Average peak INaI–V relationships before (baseline) and after 5 min wash-in of 10 µm mexiletine in HEK293A cells transfected with SCN5A-WT (B, C) or SCN5A-1795insD (D, E), following 48 h incubation with either vehicle or mexiletine. WT, wild-type.

• 

  Figure 3. Forty-eight hour incubation with mexiletine increases late INa in HEK293A cells transfected with SCN5A-1795insD. (A) Schematic representation of the performed experiments and typical late INa recordings obtained using a descending ramp protocol in HEK293A cells transfected with SCN5A-1795insD previously incubated for 48 h with vehicle (left panel) or mexiletine (right panel) before (baseline) and after application of 30 µm TTX. TTX-sensitive current was obtained by subtraction of the current before and after application of TTX. (B) Average I–V relationships for TTX-sensitive current in HEK293A cells transfected with SCN5A-1795insD previously incubated for 48 h with vehicle or mexiletine (left panel); *P < 0.05, two-way RM ANOVA followed by Holm–Sidak test for post hoc analysis. Boxplots (right panel) depicting late INa densities (median: boxes represent interquartile range; whiskers represent 95% interval) determined at −10 mV; *P < 0.05, Mann–Whitney test. TTX, tetrodotoxin.

• 

  Figure 4. Acute mexiletine treatment blocks late INa in HEK293A cells transfected with SCN5A-1795insD. (A) Schematic representation of the performed experiments (top panel). Typical late INa measurements obtained using a descending ramp protocol (see inset) in HEK293A cells transfected with SCN5A-1795insD previously incubated for 48 h with vehicle (middle left panel) and 10 µm mexiletine (middle right panel) prior to drug application (baseline), after 5 min wash-in of mexiletine (10 µm) and TTX (30 µm) in the continued presence of mexiletine. The mexiletine-sensitive currents and TTX-sensitive currents in the presence of mexiletine are shown in the bottom panels. (B) Average I–V relationships for mexiletine-sensitive current and TTX-sensitive current in the presence of mexiletine in HEK293A cells transfected with SCN5A-1795insD previously incubated for 48 h with vehicle (left panel) and mexiletine (right panel). The TTX-sensitive current in the presence of mexiletine is around the 0 pA/pF level, indicating that mexiletine already blocked completely late INa. TTX, tetrodotoxin.

• 

  Figure 5. Chronic and acute effects of mexiletine on action potential properties in SCN5A-1795insD hiPSC-CMs. (A) Schematic representation of the performed experiments and typical examples of APs recorded at 1 Hz in SCN5A-1795insD hiPSC-CMs after 48 h incubation with either vehicle or mexiletine (10 µm). (B) Average data for maximal upstroke velocity (Vmax), AP amplitude, resting membrane potential, AP duration at 20, 50, and 90% repolarization (APD20, APD50, and APD90). *P < 0.05, unpaired Student’s t-test. (C) Schematic representation of the performed experiments and typical examples of APs recorded at 1 Hz in SCN5A-1795insD hiPSC-CMs incubated for 48 h with mexiletine followed by removal of the drug (baseline) and after 5 min acute re-administration (wash-in) of mexiletine. (D) Average data for Vmax, APA, RMP, APD20, APD50, and APD90. Insets: time derivatives of the AP upstrokes. *P < 0.05, paired Student’s t-test. AP, action potential; APA, AP amplitude; RMP, resting membrane potential.

• 

  Figure 6. Forty-eight hour incubation with 10 µm mexiletine increases peak INa in SCN5A-1795insD hiPSC-CMs while acute mexiletine has no effect. (A, B) Schematic representations of the performed experiments (see panels) and average peak INaI–V relationships for SCN5A-1795insD hiPSC-CMs after 48 h incubation with vehicle or mexiletine (10 µm) followed by removal of the drug (A) and after 5 min of re-administration (wash-in) of mexiletine (10 µm) (B). (C, D) Voltage dependence of (in)activation measured in SCN5A-1795insD hiPSC-CMs after 48 h incubation with vehicle or mexiletine (10 µm) followed by removal of the drug (C) and after 5 min re-administration (wash-in) of mexiletine (D). Insets: voltage protocols; *P < 0.05, two-way RM ANOVA followed by Holm–Sidak test for post hoc analysis.