Inhibition of NaV1.8 prevents atrial arrhythmogenesis in human and mice

Pharmacologic approaches for the treatment of atrial arrhythmias are limited due to side effects and low efficacy. Thus, the identification of new antiarrhythmic targets is of clinical interest. Recent genome studies suggested an involvement of SCN10A sodium channels (NaV1.8) in atrial electrophysiology. This study investigated the role and involvement of NaV1.8 (SCN10A) in arrhythmia generation in the human atria and in mice lacking NaV1.8. NaV1.8 mRNA and protein were detected in human atrial myocardium at a significant higher level compared to ventricular myocardium. Expression of NaV1.8 and NaV1.5 did not differ between myocardium from patients with atrial fibrillation and sinus rhythm. To determine the electrophysiological role of NaV1.8, we investigated isolated human atrial cardiomyocytes from patients with sinus rhythm stimulated with isoproterenol. Inhibition of NaV1.8 by A-803467 or PF-01247324 showed no effects on the human atrial action potential. However, we found that NaV1.8 significantly contributes to late Na+ current and consequently to an increased proarrhythmogenic diastolic sarcoplasmic reticulum Ca2+ leak in human atrial cardiomyocytes. Selective pharmacological inhibition of NaV1.8 potently reduced late Na+ current, proarrhythmic diastolic Ca2+ release, delayed afterdepolarizations as well as spontaneous action potentials. These findings could be confirmed in murine atrial cardiomyocytes from wild-type mice and also compared to SCN10A−/− mice (genetic ablation of NaV1.8). Pharmacological NaV1.8 inhibition showed no effects in SCN10A−/− mice. Importantly, in vivo experiments in SCN10A−/− mice showed that genetic ablation of NaV1.8 protects against atrial fibrillation induction. This study demonstrates that NaV1.8 is expressed in the murine and human atria and contributes to late Na+ current generation and cellular arrhythmogenesis. Blocking NaV1.8 selectively counteracts this pathomechanism and protects against atrial arrhythmias. Thus, our translational study reveals a new selective therapeutic target for treating atrial arrhythmias. Electronic supplementary material The online version of this article (10.1007/s00395-020-0780-8) contains supplementary material, which is available to authorized users.


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 (I NaL ), which is a persistent Na + influx throughout the action potential [3,16,22,33]. By prolonging the duration of the action potential, I NaL increases the probability of early afterdepolarizations (EADs), which constitute a trigger for arrhythmias. Moreover, by increasing cytosolic [Na + ] an enhanced I NaL 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 (I ti ), resulting in delayed afterdepolarizations (DADs), which serve as a trigger for irregular action potentials and focal arrhythmias [32]. However, the mechanisms involved in I NaL generation in the atria are not fully understood.
While SCN5A sodium channels (Na V 1.5) are the predominant isoform in the heart [14], recent evidence suggested the involvement of SCN10A sodium channels (Na V 1.8) in atrial conduction [8]. Moreover, genome-wide association studies showed that variants of Na V 1.8 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 V 1.8 in the human and murine atria. Moreover, we studied the involvement of Na V 1.8 in atrial arrhythmogenesis and evaluated the channel as a specific target for antiarrhythmic pharmacotherapy.

Human myocardial tissue
All procedures were performed according to the Declaration of Helsinki and were approved by the local ethics committee. Informed consent was obtained from all patients. Human atrial myocardium from patients with sinus rhythm or atrial fibrillation was acquired from atrial resections during open heart surgery (for patient characteristics, see Table 1). For molecular purposes, we utilized left ventricular myocardium from healthy donor hearts that were not transplanted due to technical reasons.

SCN10A −/− and wild-type mice
SCN10A −/− and respective wild-type mice (WT) were studied to reveal the functional consequence of genetic Na V 1.8 ablation [2]. The animal investigations conform to the "Guide for the Care and Use of Laboratory Animals" published by the US National Institutes of Health (publication No. 85-23, revised 1996) and the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Murine atrial cardiomyocytes were isolated as previously reported [13]. Mice used for cardiomyocyte isolation were sacrificed under isoflurane 133 inhalation anesthesia (5%) by cervical dislocation. Mice used for in vivo studies were anesthetized via intraperitoneal injection of medetomidine (0.5 mg/kg), midazolam (5 mg/ kg) and fentanyl (0.05 mg/kg body weight) and killed by cervical dislocation after the procedure.

Human atrial cardiomyocytes isolation
Atrial myocardium from patients with sinus rhythm was used for cellular experiments. Before starting isolation, human atrial tissue was cleared from fat and blood, then cut into very small pieces and rinsed thoroughly. Cardiomyocytes were isolated using collagenase (Worthington type 1, 370 U/ mg) and proteinase (Sigma Type XXIV, 7.0-14.0 U/mg) as described previously [15]. Enzymatic digestion was stopped by adding BCS (2%

Murine atrial cardiomyocytes isolation
Atrial cardiomyocytes from SCN10A −/− and respective WT mouse hearts were isolated as previously described [13]. Cellular experiments were performed at room temperature.

Quantitative real-time PCR (qPCR)
Human atrial tissue or ventricular non-failing tissue were snap-frozen in liquid nitrogen and stored at − 80 °C. RNA was isolated by use of the SV total RNA isolation System (Promega). Primer sequences (forward and four reverse) of SCN10A (Origene, cat No HP209444), SCN5A and GAPDH were used for quantitative RT-PCR.

Patch-clamp experiments I NaL measurements
Ruptured-patch whole-cell voltage-clamp was used to measure I NaL in human atrial cardiomyocytes (HEKA electronics). Cardiomyocytes were held at − 120 mV and I NaL was elicited using a train of pulses to − 35 mV (1 s duration, ten pulses, BCL 2 s). Recordings were initiated 3 min after rupture. The measured current was integrated (between 100 and 500 ms) and normalized to the membrane capacitance (Suppl. Fig. 3).

Action potential recordings
For action potential recordings, the whole-cell patch-clamp technique was used (current clamp configuration, HEKA electronics). Access resistance was typically ~ 5-10 MΩ after patch rupture. Action potentials were continuously elicited by square current pulses of 0.5-1 nA amplitude and 1-5 ms duration at a frequency of 1 Hz. For assessing DADs and spontaneous action potentials, stimulation was paused for 15 s and for 30 s.

Confocal Ca 2+ spark measurements
Isolated atrial cardiomyocytes were loaded with the Ca 2+ indicator Fluo 4-AM (10 µmol/L for 15 min, Molecular Probes) at RT. The solution was substituted and cells were incubated for 15 min with Tyrode's solution and the respective agents. Line scans for Ca 2+ spark measurements were obtained with a laser scanning confocal microscope (Zeiss). Line scans were recorded during rest after loading the sarcoplasmic reticulum with Ca 2+ by continuous field stimulation at 1 Hz. Ca 2+ sparks were analysed with the program SparkMaster for ImageJ.

In vivo arrhythmia studies
For electrophysiological studies, SCN10A −/− and respective wild-type mice [2] were anesthetized and temperature controlled (37 °C). As previously described, a Millar 1.1F octapolar EP catheter (EPR-800; Millar Instruments) was inserted via the right jugular vein [20]. Right atrial pacing was performed using 2 ms current pulses delivered by an external stimulator. Atrial capture was confirmed by atrial pacing prior to the arrhythmia protocol. Inducibility of atrial arrhythmias was tested by decremental burst pacing (5 episodes/mice). AF was defined as the occurrence of rapid and fragmented atrial electrograms with irregular AV nodal conduction and ventricular rhythm for at least 1 s.

Statistics
All data are presented as the mean values ± SEM. For statistical analysis of two groups containing parametric data Student's t test was used, for non-parametric data Mann-Whitney test was used. For analysis of parametric data comparing more than two groups, one-way ANOVA was used. P values were corrected for multiple comparisons by the Tukey method. For analysis of proportions, Fisher's exact test was used. Analysis was performed using GraphPad Prism 8. P values are two-sided and considered statistically significant if P < 0.05.

Expression of Na V 1.8 in human atrial myocardium
To investigate whether Na V 1.8 is expressed in the human atrium, we used myocardium from patients with SR and patients with AF for mRNA and protein analysis. At the protein level, we could confirm the existence of Na V 1.8 in the human atria. Moreover, Na V 1.8 protein expression is significantly higher in the human atria (n = 6) compared to the human ventricle (n = 5 Fig. 1a, b). Using qPCR, we detected the expression of Na V 1.8 mRNA in human atrial tissue, which was 3.0 ± 0.9-fold higher in the human atrium as compared to ventricular non-failing myocardium (ventricle: n = 10 patients, atria: n = 7 patients, Fig. 1c). To evaluate whether Na V 1.8 or the major cardiac sodium channel isoform Na V 1.5 might be differentially regulated in atrial fibrillation (AF) compared to sinus rhythm (SR), we investigated atrial myocardium from patients with SR or with AF. However, neither Na V 1.8 (SR: n = 14 patients, AF: n = 14 patients) nor Na V 1.5 (SR: n = 14 patients, AF: n = 13 patients) protein expression levels were different between myocardium from patients with AF and SR ( Fig. 1d-f). Moreover, while Na V 1.8 mRNA was lower compared to Na V 1.5, we found no changes between SR versus AF for Na V 1.8 (SR: n = 8 patients, AF: n = 8). Na V 1.5 mRNA levels differed between SR (n = 8 patients) and AF (n = 8, Fig. 1g), which however did not translate into protein expression differences. Therefore, Na V 1.8 was confirmed to be present in the human atria without being regulated in patients with AF.

Role of Na V 1.8 for generation of I NaL
The contribution of Na V 1.8 in I NaL generation was studied in human atrial cardiomyocytes from patients with sinus rhythm using ruptured-patch whole-cell voltage-clamp. Patch-clamp recordings of isolated human atrial cardiomyocytes showed that Na V 1.8 inhibition caused a significant reduction of I NaL by 44.9 ± 13.5% after exposure to A-803467 (n = 12 cardiomyocytes/4 patients) and by 53.5 ± 12.7% after PF-01247324 (n = 10/4) compared to control (n = 15/6, Fig. 4a, b). Furthermore, isolated atrial cardiomyocytes from SCN10A −/− mice (n = 11 cells/ 5 mice) showed a significantly lower I NaL compared to WT (n = 8 cells/ 4 mice, Fig. 4c-e). While pharmacological Na V 1.8 inhibition by PF-01247324 (n = 7 cells/ 5 mice) exerted no effect on I NaL in SCN10A −/− , I NaL could be significantly reduced by application of PF-01247324 in atrial WT cardiomyocytes (n = 7 cells/ 4 mice, Fig. 4c-e). To determine the contribution of Na V 1.8 to I NaL generation, we also performed measurements with TTX (2 µmol/L) to globally inhibit I NaL . We observed a trend towards a lower I NaL in TTX-treated SCN10A −/− cardiomyocytes and also compared to PF-01247324-treated WT cardiomyocytes (Fig. 4e) suggesting that other Na V -dependent I NaL is still relevant under these conditions.

Role of Na V 1.8 for sarcoplasmic Ca 2+ -leak generation
It is well known that I NaL can potently induce arrhythmogenic diastolic SR Ca 2+ release events [13]. To investigate whether the reduction of I NaL caused by Na V 1.8 inhibition may lead to a diminished incidence of diastolic SR Ca 2+ release in human atrial cardiomyocytes we used confocal microscopy. The frequency of diastolic SR Ca 2+ sparks (CaSpF) in line scans of human atrial cardiomyocytes was 3.2 ± 0.5 × 100/µm/s (n = 84 cardiomyocytes/13 patients) which could be significantly attenuated to 1.2 ± 0.2 Fig. 1 Expression of Na V 1.8 in the human atrium. Data are presented as scatter plot with mean ± SEM. P values were calculated using unpaired Student's t test. a Original Western blot for Na V 1.8 expression in atrial myocardium from patients with sinus rhythm compared to ventricular myocardium from non-failing donors (NF). b Normalized densitometry data comparing the protein expression of Na V 1.8 in human atria (n = 6) and human ventricle (n = 5). GAPDH was used as an internal loading control in all blots. c Normalized mRNA expression of Na V 1.8/GAPDH in human atrial myocardium (n = 7 patients) compared to ventricular myocardium from healthy subjects (n = 10 patients). d Original Western Blot for Na V 1.8 and Na V 1.5 protein in human atria from patients with sinus rhythm (SR) or atrial fibrillation (AF). e Normalized densitometry data from Western Blots using atrial myocardium from patients with SR or AF showing the protein expression of Na V 1.5 (SR: n = 14 patients, AF: n = 13) and (f) Na V 1.8 (SR: n = 14 patients, AF: n = 14). GAPDH was used as an internal loading control in all blots. g Normalized mRNA expression of Na V 1.5/GAPDH (SR: n = 8 patients, AF: n = 8) and Na V 1.5/GAPDH (SR: n = 8 patients, AF: n = 8) in human atrial myocardium from SR compared to AF × 100/µm/s after addition of A-803467 (n = 73/9) or PF-01247324 to 1.1 ± 0.2 × 100/µm/s (n = 88/11, Fig. 5a,  b). Moreover, the calculated diastolic SR Ca 2+ leak was reduced by 64.3 ± 22.2% after addition of A-803467 and by 80.6 ± 20.6% after PF-01247324 (control: n = 84 cardiomyocytes/13 patients, A-803467: n = 73/9; PF-01247324: n = 88/11, Fig. 5c). In line with that, atrial cardiomyocytes from SCN10A −/− mice (n = 57 cells/ 7 mice, Fig. 5d) showed a lower frequency of diastolic Ca 2+ sparks as well as a lower diastolic Ca 2+ leak compared to WT cells (n = 57 cells/ 7 mice, Fig. 5e-g). While PF-01247324-(n = 62 cells/7 mice) and TTX-(n = 61 cells/ 7 mice) treated cardiomyocytes had a significantly reduced Ca 2+ spark frequency and Ca 2+ leak in WT, both drugs had no further effects in SCN10A −/− cardiomyocytes (n = 57 cells/7 mice and 64/7 respectively). Interestingly, we observed no further antiarrhythmic effect of TTX compared to Na V 1.8 inhibition alone. Thus, the reduction of both I NaL and diastolic SR Ca 2+ release reveal the significant role of Na V 1.8 for cellular arrhythmogenesis in the human atria. Of note, we observed no effects of pharmacological Na V 1.8 inhibition on systolic Ca 2+ transient amplitude and SR Ca 2+ load (Suppl. Figs. 1, 2).

Blocking Na V 1.8 suppresses diastolic Ca 2+ waves
To elucidate potential antiarrhythmic effects of Na V 1.8 inhibition, we have studied the effects of pharmacological inhibition and genetic ablation of Na V 1.8 on the incidence of diastolic Ca 2+ waves, which are major diastolic Ca 2+ release events and constitute proarrhythmic triggers. Indeed, human atrial cardiomyocytes from patients with sinus rhythm treated with A-803467 or PF-01247324 showed a significantly reduced frequency of diastolic Ca 2+ waves (Fig. 6a, b). Also, the percentage of cells developing diastolic Ca 2+ waves, 26.1% under control conditions (n = 116 cardiomyocytes/13 patients), decreased after exposure to either A-803467 (to 8.4%, n = 90/9) or PF-01247324 (to 8.3%, n = 104/11, Fig. 6c). Also, SCN10A −/− mice (n = 67 cells/7 mice) had a reduced Ca 2+ wave frequency as well as a reduced fraction of cardiomyocytes with arrhythmic events compared to WT (n = 80 cells/7 mice, Fig. 6d-g). Pharmacological Na V 1.8 inhibition with PF-01247324 exerted no effects in SCN10A −/− mice (n = 67 cells/7 mice), but significantly decreased Ca 2+ wave frequency and the percentage of cells with arrhythmic events in WT (n = 70 cells/7 mice, Fig. 6f-g). I NaL inhibition by TTX caused no further effects  Fig. 6f-g).

SCN10A −/− mice are protected against AF induction
The role of Na V 1.8 for in vivo arrhythmias was investigated using SCN10A −/− mice and respective WT. After transjugular vein catheterization, five episodes of atrial burst stimulation were performed in anaesthetized mice (Fig. 8a). Electrocardiograms of SCN10A −/− mice showed no changes in cardiac conduction and repolarization compared to WT (Suppl. Table 1). In WT mice undergoing atrial burst stimulation AF was inducible in all animals (n = 5 mice). However, only in two out of eight SCN10A −/− mice AF could be induced indicating a significantly lower susceptibility to AF (n = 8 mice, Fig. 8b). Moreover, the AF duration after respective burst episodes was markedly shorter in SCN10A −/− mice (12.0 ± 3.8 s) compared to WT (33.2 ± 5.5 s, Fig. 8c). These data demonstrate that Na V 1.8 ablation is protective against AF Fig. 6 Effects of Na V 1.8 on diastolic Ca 2+ waves. Data are presented as mean ± SEM. P values were calculated using one-way ANOVA with Tukey's test for multiple comparisons or Fisher's exact test (for c, g). a Confocal line scan images of human atrial cardiomyocytes loaded with the Ca 2+ indicator Fluo-4 representing the occurrence of major diastolic arrhythmogenic Ca 2+ release events. b Mean frequency of Ca 2+ waves in control (n = 108 cardiomyocytes/13 patients) and after treatment with A-803467 (n = 87/9) or PF-01247324 (n = 104/11). c Percentage of cells developing arrhythmic events (Ca 2+ waves or spontaneous transients; n = 24 of 116 cardiomyo-cytes/13 patients) and effects of Na V 1.

Discussion
This study comprehensively investigated Na V 1.8 in human atrial myocardium and its role in cellular electrophysiology and arrhythmogenesis. We could detect relevant Na V 1.8 mRNA and protein levels in the human atrium. While pharmacological Na V 1.8 modulation showed no significant effects on action potentials, it depicted a contribution to I NaL generation and thereby to diastolic SR Ca 2+ leak in human atrial cardiomyocytes. Importantly, selective inhibition of Na V 1.8 with two agents potently reduced cellular arrhythmogenic triggers. These findings could be confirmed in mice lacking Na V 1.8 (SCN10A −/− ). Finally, in vivo studies revealed that SCN10A −/− mice are protected against AF induction.
We not only found that Na V 1.8 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 V 1.8 in the human atria was indirectly suggested by genome-wide association studies (GWAS) reporting that the SCN10A gene (encoding Na V 1.8) 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 V 1.8 expression in the atria compared to the ventricle [34]. Of note, one previous study reported a generally lower Na V 1.8 mRNA expression in the atria compared to other Na V isoforms [19] and other studies described difficulties in the detection of Na V 1.8, 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 V 1.8 compared to Na V 1.5 expression might be differentially regulated in patients with SR or with AF. However, we observed no differences in Na V 1.8 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 V 1.8 in the human atria. In patch clamp experiments, pharmacological inhibition of Na V 1.8 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 V 1.8 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 V 1.8 inhibition, which however did not reach statistical significance. Thus, while we could previously show a distinct APD abbreviation upon Na V 1.8 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 V 1.8 inhibition markedly reduced I NaL 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 I NaL as well as an abbreviation of APD due to Na V 1.8 inhibition [10,34]. SCN10A variants associated with AF were also found to modulate I NaL after transfection in ND7/23 cells, which further strengthens findings about the role of Na V 1.8 for I NaL [23]. Of note, we observed a clear trend towards a further I NaL reduction in SCN10A −/− cardiomyocytes after exposure to TTX suggesting that other Na V isoforms still contribute to I NaL generation. Since I NaL directly impacts atrial arrhythmogenesis [3,13,28], we consecutively evaluated whether specific Na V 1.8 inhibition could prevent cellular arrhythmias. We have previously shown that in the human atrium I NaL -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 V 1.8 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 V 1.8 inhibition/ablation. Interestingly, I NaL inhibition by TTX showed similar antiarrhythmic effects compared to Na V 1.8 inhibition/ablation. Thus, Na V 1.8-dependent I NaL inhibition alone might be sufficient enough for disrupting . P value was calculated using Fisher's exact test. d AF duration after burst stimulation (5 episodes/mice) in WT (n = 5) and SCN10A −/− mice (n = 8). P value was calculated using Mann-Whitney test the vicious circle of I NaL -dependent SR Ca 2+ leak. The electrogenic exchange of Ca 2+ against Na + via NCX can induce a transient inward current (I ti ) leading to depolarization of the cell, which serves as a trigger for spontaneous action potentials [32]. In human atrial cardiomyocytes, both Na V 1.8 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 V 1.8 in the absence of pharmacological approaches and also our findings based on the Na V 1.8 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 V 1.8 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 V 1.8 on human and murine cardiomyocyte level. Notably, Na V 1.8 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 V 1.5 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 Na V 1.8-dependent selective I NaL 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, Na V 1.8 dysregulation might also have functional implications in long-standing AF.
Taken together, the herein presented functional evidence of Na V 1.8 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.