Cardiac-Specific Deletion of Scn8a Mitigates Dravet Syndrome-Associated Sudden Death in Adults

Abstract

Background: Sudden unexpected death in epilepsy (SUDEP) is a fatal complication experienced by otherwise healthy epilepsy patients. Dravet syndrome (DS) is an inherited epileptic disorder resulting from loss of function of the voltage-gated sodium channel, Na_V 1.1, and is associated with particularly high SUDEP risk. Evidence is mounting that Na_Vs abundant in the brain also occur in the heart, suggesting that the very molecular mechanisms underlying epilepsy could also precipitate cardiac arrhythmias and sudden death. Despite marked reduction of Na_V 1.1 functional expression in DS, pathogenic late sodium current (I_Na,L) is paradoxically increased in DS hearts. However, the mechanisms by which DS directly impacts the heart to promote sudden death remain unclear.

Objectives: In this study, the authors sought to provide evidence implicating remodeling of Na⁺ - and Ca²⁺ -handling machinery, including Na_V 1.6 and Na⁺/Ca²⁺exchanger (NCX) within transverse (T)-tubules in DS-associated arrhythmias.

Methods: The authors undertook scanning ion conductance microscopy (SICM)-guided patch clamp, super-resolution microscopy, confocal Ca²⁺ imaging, and in vivo electrocardiography studies in Scn1a haploinsufficient murine model of DS.

Results: DS promotes I_Na,L in T-tubular nanodomains, but not in other subcellular regions. Consistent with increased Na_V activity in these regions, super-resolution microscopy revealed increased Na_V 1.6 density near Ca²⁺release channels, the ryanodine receptors (RyR2) and NCX in DS relative to WT hearts. The resulting I_Na,L in these regions promoted aberrant Ca²⁺ release, leading to ventricular arrhythmias in vivo. Cardiac-specific deletion of Na_V 1.6 protects adult DS mice from increased T-tubular late Na_V activity and the resulting arrhythmias, as well as sudden death.

Conclusions: These data demonstrate that Na_V 1.6 undergoes remodeling within T-tubules of adult DS hearts serving as a substrate for Ca²⁺ -mediated cardiac arrhythmias and may be a druggable target for the prevention of SUDEP in adult DS subjects.

Keywords: Dravet syndrome; Na(V)1.6; sodium channels; sudden cardiac death; sudden unexpected death in epilepsy.

FIGURE 1. DS Is Associated With an Increase in Mortality and Predisposition to Arrhythmia Development, Which Are Mitigated by Reduction in Na_V1.6

(A) Kaplan-Meier survival curves for WT (n = 163), DS (n = 238), DSxNa_V1.6^Het (n = 68), and DSxcNa_V1.6^KO (n = 92) mice. The data from all strains tested for WT and DS were pooled. *P < 0.001 relative to WT, ^&P < 0.001 relative to DS. (B) Representative ECG from WT and DS mice. (C) QT-interval prolongation in DS, mitigated by Na_V1.6 reduction (WT: n = 11; DS: n = 18; DSxNa_V1.6^Het: n = 17; DSxcNa_V1.6^KO: n = 29 mice). (D) Representative ECGs following catecholamine challenge with caffeine and epinephrine illustrate SR followed by VT. (E) Summary data showing significant increase in arrhythmia burden in DS mice (WT: n = 38; DS: n = 59; DSxNa_V1.6^Het: n = 17; DSxcNa_V1.6^KO: n = 40). Arrhythmia scores: 0 = no abnormalities; 1 = premature ventricular complexes observed; 2 = bigeminy; 3 = VT; 4 = ventricular fibrillation. *q < 0.05, ****q < 0.0001 relative to DS. DS = Dravet syndrome; ECG = electrocardiogram; Na_V = voltage-gated sodium channel; cNa_V1.6^KO = cardiac-specific Na_V1.6 deletion; Na_V1.6^Het = Scn8a haploinsufficient; SR = sinus rhythm; VT = ventricular tachycardia; WT = wild type.

FIGURE 2. Whole-Cell Patch Clamp Recordings Reveal Increased Late I_Na in DS Cardiomyocytes, Which Are Mitigated by Reduction in Na_V1.6

(A) Representative late I_Na recordings from cardiomyocytes of each experimental group; increased late I_Na in DS cardiomyocytes is mitigated by Na_V1.6 reduction (WT: n = 10 cells, 4 mice; DS: n = 15 cells, 8 mice; DSxNa_V1.6^Het: n = 13 cells, 6 mice; DSxcNa_V1.6^KO: n = 9 cells, 5 mice). (B) Peak I_Na (WT: n = 5 cells, 3 mice; DS: n = 8 cells, 7 mice; DSxNa_V1.6^Het: n = 9 cells, 5 mice; DSxcNa_V1.6^KO: n = 12 cells, 4 mice). *q < 0.05, **q < 0.01 relative to DS. I_Na,L = voltage-gated sodium current; other abbreviations as in Figure 1.

FIGURE 3. No Changes in Late Na_V Activity Within the Intercalated Disc or the Lateral Membrane Crests of DS Cardiomyocytes

(A, top) Representative immunofluorescence confocal microscopy images of Na_V (Na_V1.5: green; Na_V1.1: purple; Na_V1.6: blue) distributions relative to ID (N-cadherin: orange) and T-tubular (RyR2: red) regions and (bottom) representative scanning ion conductance microscopy images of cardiomyocyte topography (specific regions indicated by markers; left ID, right crest and T-tubule). Scale bars: (left) 20 μm and (right) 2 μm. (B) Cell-attached patch I_Na recordings from the ID. (D) Scanning ion conductance microscopy-guided “smart” patch I_Na recordings from the lateral membrane crest. (C and E) Summary data of late activity at the ID (WT: n = 10 cells, 3 mice; DS: n = 6 cells, 3 mice) and later membrane crest (WT: n = 9 cells, 3 mice; DS: n = 9 cells, 3 mice). ID = intercalate disc; ns = not significant; RyR2 = ryanodine receptors; T-tubule = transverse tubule; other abbreviations as in Figures 1 and 2.

FIGURE 4. Na_V1.6 Contributes to Increased Late Na_V Activity Within the T-Tubules of DS Cardiomyocytes

(A) Representative scanning ion conductance microscopy-guided “smart” patch I_Na recordings from T-tubule openings. (B) Summary data of late activity at the T-tubules (WT: n = 9 cells, 5 mice; DS: n = 13 cells, 7 mice; DSxNa_V1.6^Het: n = 13 cells, 5 mice; DSxcNa_V1.6^KO: n = 7 cells, 4 mice). *q < 0.05, **q < 0.01, ***q < 0.001 relative to DS. Abbreviations as in Figures 1 to 3.

FIGURE 5. Increased Na_V1.6 Expression in DS Hearts

(A) Representative Western blots of Na_V1.6 and GAPDH (loading control) in WT and DS hearts. (B) Summary Western blot data (n = 6). *P < 0.05 relative to DS. GAPDH = glyceraldehyde 3-phosphate dehydrogenase; other abbreviations as in Figures 1 to 3.

FIGURE 6. STORM Reveals Enhanced Na_V1.6 Clustering in Close Proximity to NCX and RyR2 in DS Hearts

Representative stochastic optical reconstruction microscopy (STORM) images from (A and C) WT and (B and D) DS hearts immunolabeled for RyR2 (top, red), Na_V1.6 (top and bottom, blue), and NCX (bottom, yellow). Protein distribution measured as percentage of molecules (E: Na_V1.6 to RyR2; G: Na_V1.6 to NCX) and percentage of clusters (F: Na_V1.6 to RyR2; H: Na_V1.6 to NCX) (n = 5 images/heart from 3 WT hearts and 3 DS hearts). Differences in distributions and medians were tested with the 2-sample Kolmogorov-Smirnov test and Wilcoxon rank-sum test test, respectively (P > 0.05: ns; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). (I) Probability distribution and (J) cumulative distribution plots of Na_V1.6 clusters relative to RyR2 in WT and DS mice. (K) Probability distribution and (L) cumulative distribution plots of Na_V1.6 clusters relative to NCX in WT and DS mice (n = 5 images/heart from 3 WT hearts and 3 DS hearts). Scale bars: (A, left) 7 μm, (A, right) 2 μm, (B, left) 7 μm, (B, right) 2 μm, (C, left) 5 μm, (C, right) 1 μm, (D, left) 6 μm, and (D, right) 1 μm. NCX = sodium/calcium exchanger; other abbreviations as in Figures 1 to 3.

FIGURE 7. Monte Carlo Clustering Simulations Predict That Channel-Channel Interactions Are Sufficient to Explain Enhanced Na_V1.6 Clustering in DS

(A) Representative map showing simulated NCX, Na_V1.6, and Na_V1.1 clustering. (B) Representative map of NCX, Na_V1.6, and Na_V1.1 clustering in DS. Note the smaller Na_V1.1 cluster. (C) Distribution and cumulative distribution plots showing the distribution of Na_V1.6 relative to NCX. Reduction in Na_V1.1 in DS shifts Na_V1.1 distribution to the left (red), without other changes in expression or Na_V1.6-NCX interactions. (D) Distribution and cumulative distribution plots showing the distribution of NCX relative to Na_V1.6. Reduction in Na_V1.1 in DS does not change this distribution. The data shown in C and D are pooled from 5 simulations for each case. Abbreviations as in Figures 1 to 3 and 6.

FIGURE 8. Na_V1.6 Remodeling Within T-Tubules in DS Contributes to Aberrant Ca²⁺ Release

(A) Representative Ca²⁺ line scans of isolated cardiomyocytes (top) and corresponding Ca²⁺ transients (bottom). (B) Summary of frequency and distribution of Ca²⁺ sparks (WT: n = 121 cells, 7 mice; DS: n = 102 cells, 13 mice; DSxNa_V1.6^Het: n = 211 cells, 7 mice; DSxcNa_V1.6^KO: n = 126 cells, 11 mice) and (C) Ca²⁺ waves. WT: n = 123 cells, 7 mice; DS: n = 181 cells, 15 mice; DSxNa_V1.6^Het: n = 103 cells, 4 mice; DSxcNa_V1.6^KO: n = 152 cells, 11 mice. *q < 0.05 relative to DS. Abbreviations as in Figures 1 to 3.

FIGURE 9. Remodeling of Na_Vs Within the Ca²⁺-Handling Machinery–Rich Regions of Cardiac T-Tubules in DS Serves as a Substrate for Arrhythmogenic Ca²⁺ Release

Reduction in Na_V1.1 evidenced in DS facilitates the repopulation of cardiac sarcolemma (SL) sites typically occupied by Na_V1.1 by Na_V1.6. The resulting increase in subsarcolemmal Na⁺ through Na_V1.6 promotes Na⁺/Ca²⁺ exchange and intracellular Ca²⁺ loading, thereby setting a stage for aberrant Ca²⁺ release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR). Abbreviations as in Figures 1 to 3 and 6.

CENTRAL ILLUSTRATION. Targeting the Heart to Prevent Sudden Death in Dravet Syndrome

NA_v = voltage-gated sodium channel; VT = ventricular tachycardia; WT = wild-type.