Tetrodotoxin‐Sensitive Neuronal‐Type Na ⁺ Channels: A Novel and Druggable Target for Prevention of Atrial Fibrillation

Figures

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  Figure 1. Inhibition of tetrodotoxin (TTX)‐sensitive neuronal NaVs (nNavs) is sufficient to prevent induction of aberrant, repetitive Ca2+ oscillations in R33Q atrial myocytes. A, Representative examples of the line scan images and corresponding Ca2+ transients recorded in field‐stimulated R33Q atrial cardiomyocytes paced at 0.5 Hz and loaded with the Ca2+ indicator, Fluo‐3 AM. Cells were treated with isoproterenol (ISO, 100 nmol/L) and subsequently TTX (100 nmol/L; green arrow indicates time when TTX was added) was rapidly applied. β‐Adrenergic stimulation with ISO promoted aberrant, repetitive Ca2+ oscillations (median pacing frequency was 2 Hz with the range of 0.5 to 4 Hz), while TTX (100 nmol/L) significantly decreased their incidence. Ca2+ oscillations were induced after drug washout (red arrow; number of cells tested depicted under the corresponding bars, N=8 animals for ISO and ISO‐TTX, N=6 animals for ISO‐washout, respectively; ***P<0.0001 McNemar test for ISO vs ISO‐TTX, P<0.0001 Fisher exact test for ISO‐TTX vs ISO‐washout). B, Treatment of ISO (100 nmol/L)‐exposed R33Q atrial myocytes with 10 μmol/L riluzole (Ril) significantly reduced the incidence of aberrant, repetitive Ca2+…

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  Figure 2. Effect of neuronal Nav (nNav) blockade with riluzole (Ril) on isoproterenol (ISO)‐promoted inward Na+ currents (INa) in R33Q atrial myocytes. A, Representative INa (top) were elicited by a protocol presented in the inset before (black) or after addition of ISO (100 nmol/L; red) or ISO (100 nmol/L)+Ril (10 μmol/L; purple). (Bottom) Corresponding current‐voltage relationship from control, ISO, and ISO+Ril (n=10, 9, and 7 cells from 5, 6, and 5 mice, respectively). Addition of ISO+Ril reduced INa density relative to ISO alone (peak INa at −40 mV of −37.1±3.2 vs −22.4±3.9 pA/pF for ISO and ISO+Ril, respectively; P=0.0102 ANOVA, P=0.0418 for ISO vs ISO+Ril). B, Representative traces of persistent INa elicited using the protocol shown in the inset were recorded before (black) or after addition of ISO (100 nmol/L; red) or ISO+Ril (10 μmol/L; purple). ISO enhanced persistent INa in R33Q cardiomyocytes, while Ril suppressed β‐adrenergic–mediated increase in persistent INa (n=9, 8, and 8 cells from 3, 3, and 4 mice for R33Q, ISO, and ISO‐Ril, respectively; P<0.0001 ANOVA, ***P=0.0004 for persistent INa integral and ***P<0.0001 for normalized late INa). Summary data are presented as…

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  Figure 3. The extent of late inward Na+ currents (INa) inhibition corresponds to prevention of aberrant, repetitive Ca2+ oscillations in R33Q atrial myocytes. A, (Left) INa obtained by step protocol illustrated in 1‐second intervals. (Right) In isoproterenol (ISO; 100 nmol/L)‐treated R33Q atrial myocytes, addition of ranolazine (Ran; 10 μmol/L, blue trace and bar; n=5 from N=4 animals) or R‐propafenone (R‐prop; 300 nmol/L, orange trace and bar; n=4 from N=3 animals) significantly reduced peak INa (P=0.0016 ANOVA, *P=0.0418 for ISO vs ISO+Ran and *P=0.0246 for ISO vs ISO+R‐prop) relative to ISO alone (n=7 from N=5 animals). Notably, there was no difference in peak INa reduction between the groups. B, (Left) Representative persistent INa elicited using the protocol shown in the inset. (Right) ISO (100 nmol/L) increased persistent INa, while Ran and R‐prop reduced it (n=13, 13, 7, and 7 cells from 6, 6, 6 and 4 mice for R33Q, ISO, ISO+Ran, and ISO+R‐prop, respectively; P<0.0001 ANOVA, ***P<0.0001, **P=0.0082, and *P=0.0480). Notably, R‐prop reduced ISO‐induced persistent INa to a greater extent than Ran (P=0.0480). C, Treatment of ISO (100 nmol/L)‐exposed R33Q atrial myocytes with Ran (10…

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  Figure 4. Neuronal Na+ channel (nNaV) and ryanodine receptor 2 (RyR2) colocalize to the same discrete subcellular regions. Representative confocal micrographs of myocytes isolated from R33Q mice labeled for (A) RyR2 (red) and (B) Na+/Ca2+ exchange (NCX; red) with various Na+ channel (Nav) isoforms (Nav1.x, green). These often resulted in an overlap between the immunofluorescent signals (yellow) when overlaid. (Right) Close‐up views of regions highlighted by dashed white boxes. (Bottom) Representative fluorescent proximity ligation assay signal for RyR2 (A) and NCX (B) with different nNav isoforms (NaV1.x).

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  Figure 5. Augmentation of tetrodotoxin (TTX)‐sensitive neuronal Nav (nNav) is sufficient to initiate aberrant, repetitive Ca2+ oscillations in wild‐type (WT) atrial myocytes. A, Representative traces of persistent inward Na+ currents (INa) recorded in WT atrial cardiomyocytes before (black) and after (red) exposure to β‐pompilidotoxin (β‐PMTX; 40 μmol/L). β‐PMTX increased persistent INa relative to control (n=11 and 10 cells from 3 mice, respectively; ***P<0.0001 Wilcoxon rank sum test). B, (Left) Representative line scan images obtained from WT atrial cardiomyocytes exposed to isoproterenol (ISO; 100 nmol/L) and paced at 1 Hz. (Right) Rapid application of β‐PMTX (40 μmol/L) induced aberrant, repetitive Ca2+ oscillations. (Median pacing frequency was 0.5 Hz with the range of 0.5 to 2 Hz; number of cells tested depicted under the corresponding bars, from N=3 mice; ***P<0.0001 Fisher exact test for ISO vs ISO–β‐PMTX, ***P<0.0001 Fisher exact test for ISO–β‐PMTX vs ISO‐washout).

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  Figure 6. Modulation of tetrodotoxin (TTX)‐sensitive neuronal Nav (nNav) channel correspondingly modulates atrial arrhythmias in mice. A, Simultaneous surface ECG (lead II) and intracardiac atrial electrograms with frequent, rapid P waves and irregular RR intervals suggestive of atrial arrhythmia such as atrial flutter and atrial fibrillation in R33Q mice after burst pacing. Pretreatment with riluzole (Ril; 15 mg/kg IP), targeting plasma concentrations of ~10 μmol/L,33 reduced the atrial arrhythmia inducibility (n=7 mice; **P=0.0160 Wilcoxon signed rank test). B, Representative surface ECG recordings of wild‐type (WT) mice treated (top, red ECG) or untreated (bottom, black ECG) with β‐pompilidotoxin (β‐PMTX; 40 mg/kg IP) and exposed to catecholamine challenge with epinephrine (Epi, 1.5 mg/kg) and caffeine (Caff, 120 mg/kg). Since increased heart rate has been linked to reduced arrhythmia inducibility in calsequestrin null mice, and WT mice show higher heart rate relative to calsequestrin null mice,32 all WT animals were pretreated with ivabradine (3 mg/kg) for 10 minutes before any intervention. Epi+Caff challenge during β‐PMTX exposure precipitated repetitive P waves and irregular RR…

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  Figure 7. Riluzole (Ril) reduces enhanced, persistent inward Na+ currents (INa) and prevents induction of aberrant, repetitive Ca2+ oscillations in canine heart failure (HF) atrial myocytes. A, Representative traces of persistent INa integral elicited using the protocol shown in the inset. Recordings were made in control (top) and failing (bottom) atrial myocytes before (black) and after exposure to isoproterenol (ISO; 100 nmol/L, red) and after treatment with Ril (10 μmol/L, purple). At baseline, atrial cardiomyocytes from failing hearts showed a larger persistent INa integral relative to control. ISO (100 nmol/L) enhanced persistent INa only in control cardiomyocytes. Ril reduced persistent INa integral in both control and failing atrial myocytes. B, Summary data are presented as persistent INa integral Amp‐ms/F (AmsF−1), which was measured by integrating INa between 50 and 450 ms (n=7 and 9 cells from 5 control and 3 failing dogs; P<0.0001 Kruskal–Wallis test; *P=0.0126 for control vs ISO, *P=0.0209 for control vs ISO+Ril, *P=0.0268 for control‐ISO vs HF‐ISO, ***P<0.0001). C, Representative examples of the line‐scan images and corresponding Ca2+ transients recorded in canine HF atrial…

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  Figure 8. Riluzole prevents cardiac arrhythmias in patients with amyotrophic lateral sclerosis (ALS). Two retrospective cohorts of ALS one exposed to riluzole vs no riluzole (controls), were compared by Cox proportional hazard models. The time‐to‐first composite arrhythmic events was analyzed using Kaplan–Meier production limit estimator. A, Overall cohort, (B) US cohort. HR indicates hazard ratio.