Introduction
Atrial arrhythmias, in particular atrial fibrillation (AF), contribute to the morbidity and mortality of western societies [
4]. However, pharmacological therapeutic options are still limited due to moderate potency and severe side effects. Therefore, identification and evaluation of new targets involved in atrial arrhythmogenesis are of clinical interest. The mechanisms for atrial arrhythmogenesis include electrical remodelling and disturbances in ion homeostasis [
16]. Both can cause focal triggered activity, which might evoke atrial arrhythmias or promote re-entry mechanisms. One potent substrate in promoting electrical disturbances and focal triggered activity in the atria is an increased late Na
+ current (
INaL), which is a persistent Na
+ influx throughout the action potential [
3,
16,
22,
33]. By prolonging the duration of the action potential,
INaL increases the probability of early afterdepolarizations (EADs), which constitute a trigger for arrhythmias. Moreover, by increasing cytosolic [Na
+] an enhanced
INaL may lead to Na
+/Ca
2+ exchanger (NCX)-mediated Ca
2+ overload [
26]. Consecutively, this induces arrhythmogenic Ca
2+ release events (Ca
2+ sparks) from the sarcoplasmic reticulum (SR) during diastole [
12,
13]. Increasing diastolic Ca
2+ levels may promote a depolarizing inward current (
Iti), resulting in delayed afterdepolarizations (DADs), which serve as a trigger for irregular action potentials and focal arrhythmias [
32]. However, the mechanisms involved in
INaL generation in the atria are not fully understood.
While SCN5A sodium channels (Na
V1.5) are the predominant isoform in the heart [
14], recent evidence suggested the involvement of SCN10A sodium channels (Na
V1.8) in atrial conduction [
8]. Moreover, genome-wide association studies showed that variants of Na
V1.8 are associated with the development of atrial fibrillation [
17,
21,
25]. Therefore, the aim of our study was to fundamentally investigate the molecular and functional role of Na
V1.8 in the human and murine atria. Moreover, we studied the involvement of Na
V1.8 in atrial arrhythmogenesis and evaluated the channel as a specific target for antiarrhythmic pharmacotherapy.
Discussion
This study comprehensively investigated NaV1.8 in human atrial myocardium and its role in cellular electrophysiology and arrhythmogenesis. We could detect relevant NaV1.8 mRNA and protein levels in the human atrium. While pharmacological NaV1.8 modulation showed no significant effects on action potentials, it depicted a contribution to INaL generation and thereby to diastolic SR Ca2+ leak in human atrial cardiomyocytes. Importantly, selective inhibition of NaV1.8 with two agents potently reduced cellular arrhythmogenic triggers. These findings could be confirmed in mice lacking NaV1.8 (SCN10A−/−). Finally, in vivo studies revealed that SCN10A−/− mice are protected against AF induction.
We not only found that Na
V1.8 is expressed in the human atrium but could show that mRNA and protein expression is higher in atrial compared to ventricular myocardium. The presence of Na
V1.8 in the human atria was indirectly suggested by genome-wide association studies (GWAS) reporting that the SCN10A gene (encoding Na
V1.8) impacts atrial conduction, in particular PR interval and
P wave duration [
8,
18]. Data from mice further support our findings by showing a higher Na
V1.8 expression in the atria compared to the ventricle [
34]. Of note, one previous study reported a generally lower Na
V1.8 mRNA expression in the atria compared to other Na
V isoforms [
19] and other studies described difficulties in the detection of Na
V1.8, which may be due to a high rate of alternate splicing [
6,
9]. Recent genetic studies demonstrated an involvement of SCN10A in atrial cellular electrophysiology and could associate SCN10A variants with AF [
17,
18,
25]. We therefore investigated whether Na
V1.8 compared to Na
V1.5 expression might be differentially regulated in patients with SR or with AF. However, we observed no differences in Na
V1.8 protein or mRNA expression levels between SR and AF myocardium.
We therefore investigated human atrial cardiomyocytes from patients with sinus rhythm to elucidate the cellular role of Na
V1.8 in the human atria. In patch clamp experiments, pharmacological inhibition of Na
V1.8 did not change APA, RMP or
dv/dt in human atrial cardiomyocytes, which could be confirmed in SCN10A
−/− mice. Since
dv/
dt is a surrogate for the fast Na
+ influx and hence peak Na
+ current [
5], these observations suggest that the involvement of Na
V1.8 in the peak Na
+ current is negligible and therefore atrial conduction may not be affected. We observed a trend towards a reduced APD after Na
V1.8 inhibition, which however did not reach statistical significance. Thus, while we could previously show a distinct APD abbreviation upon Na
V1.8 inhibition in ventricular cardiomyocytes [
10], the impact on atrial APD appears minor. However, APD is abbreviated in AF and APD shortening may not be a suitable strategy for the treatment of AF [
16]. A critical issue is that many previous experimental reports on AF treatment strategies investigated permanent AF atria with a very short action potential. However, patients with permanent or long-standing AF are probably not suitable patients for a pharmacological rhythm strategy due to advanced remodeling. Since atrial APD is differentially regulated in different cardiac diseases, i.e., atrial APD is prolonged in patients with left-ventricular dysfunction [
24], further patient-specific studies are needed.
We here demonstrate that pharmacological and genetic Na
V1.8 inhibition markedly reduced
INaL in human and murine atrial cardiomyocytes. Previous studies in animal ventricular cardiomyocytes by Yang et al. and in human ventricular cardiomyocytes by our group described a reduction of
INaL as well as an abbreviation of APD due to Na
V1.8 inhibition [
10,
34]. SCN10A variants associated with AF were also found to modulate
INaL after transfection in ND7/23 cells, which further strengthens findings about the role of Na
V1.8 for
INaL [
23]. Of note, we observed a clear trend towards a further
INaL reduction in SCN10A
−/− cardiomyocytes after exposure to TTX suggesting that other Na
V isoforms still contribute to
INaL generation. Since
INaL directly impacts atrial arrhythmogenesis [
3,
13,
28], we consecutively evaluated whether specific Na
V1.8 inhibition could prevent cellular arrhythmias. We have previously shown that in the human atrium
INaL-mediated Na
+ influx can induce Ca
2+ influx via reverse-mode NCX leading to an increased cytosolic [Ca
2+] and an enhanced incidence of Ca
2+ sparks [
13]. In the present study, selective inhibition or ablation of Na
V1.8 markedly suppressed SR Ca
2+ spark frequency and the total calculated diastolic Ca
2+ leak in atrial cardiomyocytes. Most importantly, the incidence of major diastolic Ca
2+ release events like Ca
2+ waves, which are considered as a proarrhythmic trigger, was significantly blunted after Na
V1.8 inhibition/ablation. Interestingly,
INaL inhibition by TTX showed similar antiarrhythmic effects compared to Na
V1.8 inhibition/ablation. Thus, Na
V1.8-dependent
INaL inhibition alone might be sufficient enough for disrupting the vicious circle of
INaL-dependent SR Ca
2+ leak. The electrogenic exchange of Ca
2+ against Na
+ via NCX can induce a transient inward current (
Iti) leading to depolarization of the cell, which serves as a trigger for spontaneous action potentials [
32]. In human atrial cardiomyocytes, both Na
V1.8 blockers significantly diminished the incidence of EADs and prevented the generation of DADs and spontaneous action potentials during rest. Accordingly, SCN10A
−/− mice and PF-01247324-treated WT cells also showed a lower incidence of triggered activity. Cellular afterdepolarizations as well as irregular action potentials are considered as a potent underlying mechanism for triggered ectopic activity/ectopic firing, which may promote and/or maintain atrial arrhythmias [
16].
To translate our cellular experimental findings in an in vivo model, we here demonstrate that SCN10A
−/− mice were protected against AF induction by rapid pacing and the duration of induced AF was significantly shorter in these mice. Ca
2+ sparks and DAD-related ectopic activity have been shown to trigger ectopic beats, re-entry mechanisms [
7] and may also lead to dispersion of repolarization, which further increases the susceptibility to arrhythmias/AF [
31]. Accordingly, Ca
2+ sparks and DAD-related ectopic activity could previously be linked to pacing induced AF in mice [
20,
27]. Thus, our in vivo data in SCN10A
−/− mice may serve as a translation of our mechanistic findings into an in vivo system.
Using genetic ablation, the proarrhythmic role of Na
V1.8 in the absence of pharmacological approaches and also our findings based on the Na
V1.8 inhibitor PF-01247324 could be confirmed. Interestingly, few association studies in patients with early onset AF also report that SCN10A variants are associated with AF susceptibility [
17,
23]. Of note, as Na
V1.8 was discussed to modulate cardiac conduction [
6,
29] the influence of SCN10A expressed in cardiac neurons/ganglia [
30] may theoretically contribute to our in vivo findings. However, we demonstrate a distinct functional proarrhythmogenic role of Na
V1.8 on human and murine cardiomyocyte level. Notably, Na
V1.8 did not change
dv/dt and amplitude of action potentials in atrial cardiomyocytes in our study as well as in ventricular cardiomyocytes [
1,
10]. In addition, the QRS complex in the ECG was also unchanged in SCN10A
−/− mice. In sharp contrast, Na
V1.5 inhibition (e.g., by flecainide) and reduction of peak Na
+ influx causing changes in cardiac conduction can adversely affect mortality by promoting arrhythmogenic mechanisms [
11].
We propose NaV1.8-dependent selective INaL reduction and prevention of atrial arrhythmogenesis to constitute a novel antiarrhythmic approach in the human, in particular for atrial arrhythmias involving focal and/or ectopic activity. Importantly, the current study investigated atrial cardiomyocytes from patients with sinus rhythm (or murine atrial cardiomyocytes) stressed with isoproterenol. From a clinical point of view, patients with permanent or long-standing AF, which are characterized by advanced structural atrial remodeling, are likely not the optimal patients for a pharmacological rhythm strategy. Therefore, we believe that atrial samples from patients at high risk for triggered/ectopic activity or paroxysmal AF may be more appropriate to investigate from a translational point of view. Nevertheless, NaV1.8 dysregulation might also have functional implications in long-standing AF.
Taken together, the herein presented functional evidence of NaV1.8 in human atrial cardiomyocytes and, most importantly, the potent antiarrhythmic effects of Nav1.8 inhibition and deletion in vitro and in vivo, could lay the foundation development towards a novel therapeutic option for atrial rhythm disorders.
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