Molecular and Functional Relevance of NaV1.8-Induced Atrial Arrhythmogenic Triggers in a Human SCN10A Knock-Out Stem Cell Model

In heart failure and atrial fibrillation, a persistent Na+ current (INaL) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. We have recently shown that NaV1.8 contributes to arrhythmogenesis by inducing a INaL. Genome-wide association studies indicate that mutations in the SCN10A gene (NaV1.8) are associated with increased risk for arrhythmias, Brugada syndrome, and sudden cardiac death. However, the mediation of these NaV1.8-related effects, whether through cardiac ganglia or cardiomyocytes, is still a subject of controversial discussion. We used CRISPR/Cas9 technology to generate homozygous atrial SCN10A-KO-iPSC-CMs. Ruptured-patch whole-cell patch-clamp was used to measure the INaL and action potential duration. Ca2+ measurements (Fluo 4-AM) were performed to analyze proarrhythmogenic diastolic SR Ca2+ leak. The INaL was significantly reduced in atrial SCN10A KO CMs as well as after specific pharmacological inhibition of NaV1.8. No effects on atrial APD90 were detected in any groups. Both SCN10A KO and specific blockers of NaV1.8 led to decreased Ca2+ spark frequency and a significant reduction of arrhythmogenic Ca2+ waves. Our experiments demonstrate that NaV1.8 contributes to INaL formation in human atrial CMs and that NaV1.8 inhibition modulates proarrhythmogenic triggers in human atrial CMs and therefore NaV1.8 could be a new target for antiarrhythmic strategies.


Introduction
Voltage-gated sodium channels (Na V ) trigger the fast upstroke of the action potential (AP), making them important for the physiological conduction of electrical impulses in the heart. Under physiological conditions, Na V channels (predominantly Na V 1.5) quickly become inactive after activation. However, in some cardiac pathologies such as ischemia and heart failure (HF), Na V channels were described to remain persistently open or reopen, thus creating the late sodium current (I NaL ) as a persistent inward current [1][2][3][4]. It has been demonstrated that this pathologically enhanced I NaL has detrimental effects on cellular electrophysiology and can induce arrhythmias [3,[5][6][7][8]. Previous reports have been published on the existence of non-cardiac Na V isoforms in the heart including Na V 1.8. Na V 1.8 is encoded by the SCN10A gene and described as a voltage-gated sodium channel like the predominant cardiac isoform Na V 1.5. Na V 1.8 has been shown to be predominantly expressed in neuronal tissues with mainly nociception functions and in human and rat spinal cord ganglia and cranial sensory ganglia [9][10][11][12]. Recent work has demonstrated that Na V 1.8 mRNA is expressed in murine and human myocardia [13,14]. In situ hybridization experiments displayed that Na V 1.8 has comparable cellular localizations to Na V 1.5 in murine cardiomyocytes [15]. Genome-wide association studies reported that variants in the SCN10A gene (coding for Na V 1.8) are associated with cardiac arrhythmias such as atrial fibrillation, sudden cardiac death [16,17], impaired conduction in the form of alterations in the PQ and QRS intervals, heart rate and increased arrhythmogenic risk [16], and with J-wave syndromes, specifically Brugada syndrome (BrS) and early repolarization syndrome (ERS) [18]. However, it remains controversial whether these Na V 1.8-associated effects are mechanistically mediated by Na V 1.8 and, if so, if they occur in cardiac ganglia or cardiomyocytes (CMs). Na V 1.8 mRNA and protein were found to be significantly more abundant in human atrial myocardium compared to the ventricular myocardium. The expression levels of Na V 1.8 and Na V 1.5 did not show any differences between myocardial samples obtained from patients with atrial fibrillation and those with sinus rhythm [19][20][21][22]. Functional single-cell experiments of atrial and ventricular human and murine CMs demonstrated direct effects of pharmacological Na V 1.8 inhibition on the I NaL and cellular arrhythmogenesis [19,22]. However, these studies were limited by utilizing respective ion channel blockers that could theoretically have unspecific effects. The currently available drugs, such as amiodarone, have limited efficacy, poor tolerability, and notable adverse side effects, including life-threatening ventricular arrhythmias. Clinical guidelines recommend amiodarone treatment for most patients with severe structural heart disease, especially heart failure (HF). However, chronic use of amiodarone can lead to severe extra-cardiac side effects and organ toxicity despite its relative effectiveness against arrhythmias. There is a demand for new and safer innovative compounds to address this issue. Therefore, we aimed to investigate the electrophysiological contribution of Na V 1.8 using CRISPR/Cas9-generated homozygous atrial SCN10A knock out (KO) induced-pluripotent stem cell CMs (iPSC-CMs). We ultimately describe the influence of the Na V 1.8 channel on the electrophysiological and molecular properties of human atrial CMs and further demonstrate that Na V 1.8 is a potential new target for atrial antiarrhythmic strategies.

Influence of NaV1.8 on INaL in Human Atrial iPSC-Cardiomyocytes
We hypothesized that the KO of SCN10A in atrial iPSC-CMs would reduce the proarrhythmogenic INaL. Therefore, whole-cell voltage clamp experiments were performed to direct measure the INaL integral in human atrial SCN10A KO and control iPSC-CMs.
Since the amplitude of the INaL is relatively small in healthy hiPSC-CMs under physiological conditions [24], we used isoproterenol (Iso, 50 nmol/L) for slight beta-adrenergic stimulation in control and experimental groups during all functional experiments as described previously [20]. To further compare SCN10A KO with pharmacological inhibition of NaV1.8 and test for potential side effects of either KO or pharmacological intervention, we used the specific NaV1.8 blocker PF-01247324 (1 µmol/L) [19,22]. Voltage-clamp experiments demonstrated that the INaL was significantly reduced by genetical KO of NaV1.8 as well as by pharmacological inhibition. The Iso-induce increase in the INaL in control iPSC-CMs (−125.5 ± 8.4 A*ms*F −1 ) was significantly reduced in KO iPSC-CMs (−34.4 ± 4.8 A*ms*F −1 , p < 0.0001, Figure 2). Moreover, the INaL was reduced to the level of KO iPSC-CMs by application of the specific NaV1.8 inhibitor [PF-01247324, 1 µmol/L, atrial control iPSC-CMs vs. PF-01247324 (−44.9 ± 3.8 A*ms*F −1 , p < 0.001, Figure 2)]. Notably, we observed no additional effects on the INaL in KO iPSC-CMs after application of PF-01247324. Nuclei were stained with DAPI. (c) mRNA expression level of atrial marker PITX2 normalized to house-keeping gene HPRT in atrial control and SCN10A KO iPSC-CMs (n = 6/3 differentiations) compared to ventricular control and SCN10A KO iPSC-CMs (n = 7/4 differentiations). Student's t-test was applied for normally distributed data. *: p < 0.05; **: p < 0.01.

Influence of Na V 1.8 on I NaL in Human Atrial iPSC-Cardiomyocytes
We hypothesized that the KO of SCN10A in atrial iPSC-CMs would reduce the proarrhythmogenic I NaL . Therefore, whole-cell voltage clamp experiments were performed to direct measure the I NaL integral in human atrial SCN10A KO and control iPSC-CMs.
Since the amplitude of the I NaL is relatively small in healthy hiPSC-CMs under physiological conditions [24], we used isoproterenol (Iso, 50 nmol/L) for slight beta-adrenergic stimulation in control and experimental groups during all functional experiments as described previously [20]. To further compare SCN10A KO with pharmacological inhibition of Na V 1.8 and test for potential side effects of either KO or pharmacological intervention, we used the specific Na V 1.8 blocker PF-01247324 (1 µmol/L) [19,22]. Voltage-clamp experiments demonstrated that the I NaL was significantly reduced by genetical KO of Na V 1.8 as well as by pharmacological inhibition. The Iso-induce increase in the I NaL in control iPSC-CMs (−125.5 ± 8.4 A*ms*F −1 ) was significantly reduced in KO iPSC-CMs (−34.4 ± 4.8 A*ms*F −1 , p < 0.0001, Figure 2). Moreover, the I NaL was reduced to the level of KO iPSC-CMs by application of the specific Na V 1.8 inhibitor [PF-01247324, 1 µmol/L, atrial control iPSC-CMs vs. PF-01247324 (−44.9 ± 3.8 A*ms*F −1 , p < 0.001, Figure 2)]. Notably, we observed no additional effects on the I NaL in KO iPSC-CMs after application of PF-01247324.

Effects of NaV1.8 on the Atrial Action Potential
To assess the potential influence of KO and pharmacological NaV1.8 inhibitio action potential characteristics in human atrial iPSC-CMs, we performed whole-c rent-clamp experiments. The data presented herein are representative of measur conducted at a frequency of 1 Hz. No effects on atrial action potential duration repolarization (APD90) were observed in KO iPSC-CMs as well as after the additio plication of the specific NaV1.8 blocker PF-01247324 (Figure 3a,b; control at 1.0 Hz 30.5 ms vs. control + PF 229.3 ± 19.0 ms,−5.8%; KO control 204.4 ± 24.2 ms, −16%, v PF 198.9 ± 30.1 ms, −3%). Furthermore, no discernible impacts were observed on th tion of atrial action potential at 20% repolarization (APD20), action potential dur 50% repolarization (APD50), and action potential duration at 70% repolarization ( The available data, including Table S1 and Figure S1, were included in the Suppl Materials. To rule out potential side effects of KO or pharmacological inhibition of we compared the resting membrane potential and action potential amplitud groups. No significant effects of KO or pharmacological inhibition of NaV1.8 on ei amplitude (APA, Figure 3c

Effects of Na V 1.8 on the Atrial Action Potential
To assess the potential influence of KO and pharmacological Na V 1.8 inhibition on the action potential characteristics in human atrial iPSC-CMs, we performed whole-cell current-clamp experiments. The data presented herein are representative of measurements conducted at a frequency of 1 Hz. No effects on atrial action potential duration at 90% repolarization (APD 90 ) were observed in KO iPSC-CMs as well as after the additional application of the specific Na V 1.8 blocker PF-01247324 (Figure 3a,b; control at 1.0 Hz, 243.0 ± 30.5 ms vs. control + PF 229.3 ± 19.0 ms,−5.8%; KO control 204.4 ± 24.2 ms, −16%, vs. KO + PF 198.9 ± 30.1 ms, −3%). Furthermore, no discernible impacts were observed on the duration of atrial action potential at 20% repolarization (APD 20 ), action potential duration at 50% repolarization (APD 50 ), and action potential duration at 70% repolarization (APD 70 ). The available data, including Table S1 and Figure S1, were included in the Supplemental Materials. To rule out potential side effects of KO or pharmacological inhibition of Na V 1.8, we compared the resting membrane potential and action potential amplitude in all groups. No significant effects of KO or pharmacological inhibition of Na V 1.8 on either AP amplitude (APA, Figure 3c, 113.7 ± 4.4 ms vs. control + PF 118.7 ± 3.4 ms, KO control 105.7 ± 5.1 ms, KO + PF 102.1 ± 4.2 ms ), resting membrane potential (RMP, Figure 3d; −76.0 ± 6.2 ms vs. control + PF −72.2 ± 5.7 ms; KO control −66.7 ± 2.6 ms vs. KO + PF −67.3 ± 3.9 ms), or upstroke velocity (Vmax, Figure 3e; 106.2 ± 11.2 vs. control + PF 127.5 ± 11.9 mV/ms; KO control 92.7 ± 13.8 vs. KO + PF 82.8 ± 12.4 mV/ms) could be observed.

Effects of NaV1.8 on Atrial Sarcoplasmic Reticulum Ca 2+ Leak and Arrhythmogenesis
As we previously demonstrated, NaV1.8 exerts its arrhythmogenic potential in the atria via enhancement of the INaL [19]. To investigate the functional cellular effects of the NaV1.8-dependent INaL on Ca 2+ homeostasis and cellular arrhythmogenesis in atrial iPSC-CMs, we recorded line scans in confocal microscopy experiments using Fluo 4-AM in human atrial SCN10A KO and control iPSC-CMs. Diastolic confocal line scans (Fluo 4-AM) showed that KO of SCN10A in atrial iPSC-CMs massively decreased the frequency of

Effects of Na V 1.8 on Atrial Sarcoplasmic Reticulum Ca 2+ Leak and Arrhythmogenesis
As we previously demonstrated, Na V 1.8 exerts its arrhythmogenic potential in the atria via enhancement of the I NaL [19]. To investigate the functional cellular effects of the Na V 1.8-dependent I NaL on Ca 2+ homeostasis and cellular arrhythmogenesis in atrial iPSC-CMs, we recorded line scans in confocal microscopy experiments using Fluo 4-AM in human atrial SCN10A KO and control iPSC-CMs. Diastolic confocal line scans (Fluo 4-AM) showed that KO of SCN10A in atrial iPSC-CMs massively decreased the frequency of spontaneous arrhythmogenic Ca 2+ sparks compared to the respective control cells (KO: 3.25 ± 0.23 sparks/100 µm/s vs. control: 6.34 ± 0.43, p = 0.0142). Similarly, pharmacological inhibition of Na V 1.8 by PF-01247324 led to a significant reduction of diastolic Ca 2+ sparks in atrial control iPSC CMs (control + PF-01247324: 3.96 ± 0.21, p = 0.0469), while having no further effect on SCN10A KO cells (KO + PF-01247324: 3.47 ± 0.34, p = 0.9998) (Figure 4a,b). Furthermore, we investigated the incidence of spontaneous diastolic Ca 2+ waves as major arrhythmogenic events. The proportion of cells exhibiting diastolic Ca 2+ waves was significantly reduced from 24.7% in atrial control -iPSC-CMs to 5.5% in the SCN10A KO group. After pharmacological inhibition of Na V 1.8, we observed a comparable reduction of cells displaying Ca 2+ waves compared to control (9.0%). There was no significant additional effect of Na V 1.8 inhibition in SCN10A KO cells (8.7%) (Figure 4c,d).  (Figure 4a,b). Furthermore, we investigated the incidence of spontaneous diastolic Ca 2+ waves as major arrhythmogenic events. The proportion of cells exhibiting diastolic Ca 2+ waves was significantly reduced from 24.7% in atrial control -iPSC-CMs to 5.5% in the SCN10A KO group. After pharmacological inhibition of NaV1.8, we observed a comparable reduction of cells displaying Ca 2+ waves compared to control (9.0%). There was no significant additional effect of NaV1.8 inhibition in SCN10A KO cells (8.7%) (Figure 4c,d).

Influence of SCN10A KO on Intracellular Ca 2+ Transients
Since KO of SCN10A was shown to reduce the arrhythmogenic potential of the increased I NaL in atrial human iPSC-CMs by reduction of spontaneous SR Ca 2+ release events, we further sought to rule out any potential adverse effects on cellular Ca 2+ handling.
We therefore performed epifluorescence microscopy (Fura 2-AM) in atrial iPSC-CMs with and without KO of SCN10A and/or pharmacological inhibition of Na V 1.8. SCN10A KO did not show any significant effects on Ca 2+ transient amplitude, diastolic Ca 2+ levels, time to peak, or relaxation time (RT 80%), demonstrating intact Ca 2+ handling in both KO and WT cells. Of note, specific inhibition of Na V 1.8 by PF-01247324 also did not exert any additional effects on Ca 2+ transient parameters in either control or KO atrial iPSC-CMs ( Figure 5).

Influence of SCN10A KO on Intracellular Ca 2+ Transients
Since KO of SCN10A was shown to reduce the arrhythmogenic potential of the increased INaL in atrial human iPSC-CMs by reduction of spontaneous SR Ca 2+ release events, we further sought to rule out any potential adverse effects on cellular Ca 2+ handling.
We therefore performed epifluorescence microscopy (Fura 2-AM) in atrial iPSC-CMs with and without KO of SCN10A and/or pharmacological inhibition of NaV1.8. SCN10A KO did not show any significant effects on Ca 2+ transient amplitude, diastolic Ca 2+ levels, time to peak, or relaxation time (RT 80%), demonstrating intact Ca 2+ handling in both KO and WT cells. Of note, specific inhibition of NaV1.8 by PF-01247324 also did not exert any additional effects on Ca 2+ transient parameters in either control or KO atrial iPSC-CMs ( Figure 5).

The Expression of Key Proteins of Excitation-Contraction Coupling Is Not Altered by a SCN10A KO
Since we demonstrated a reduction in the arrhythmogenic potential in atrial human SCN10A KO iPSC-CMs, we wanted to analyze the potential underlying effects on a molecular level. Therefore, we investigated the expression of key proteins of excitation-contraction coupling (voltage-gated sodium channel isoform Na V 1.5; L-type Ca 2+ channel Ca V 1.2; cardiac ryanodine receptor 2, RyR 2 ) using Western blot experiments.
In atrial control iPSC-CMs, we found a lower expression of Na V 1.5 compared to SCN10A KO iPSC-CMs, but it did not reach statistical significance (Figure 6a,d). Furthermore, RyR2 and Ca V 1.2 were not regulated in atrial SCN10A KO iPSC-CMs compared to control atrial iPSC-CMs according to the Western blot results (Figure 6b,c,e,f). Thus, SCN10A KO seems to exert no significant side effects on the expression of the other main proteins relevant to excitation-contraction coupling in atrial iPSC-CMs compared with their respective control cells.

The Expression of Key Proteins of Excitation-Contraction Coupling Is Not Altered by a SCN10A KO
Since we demonstrated a reduction in the arrhythmogenic potential in atrial human SCN10A KO iPSC-CMs, we wanted to analyze the potential underlying effects on a molecular level. Therefore, we investigated the expression of key proteins of excitation-contraction coupling (voltage-gated sodium channel isoform NaV1.5; L-type Ca 2+ channel CaV1.2; cardiac ryanodine receptor 2, RyR2) using Western blot experiments.
In atrial control iPSC-CMs, we found a lower expression of NaV1.5 compared to SCN10A KO iPSC-CMs, but it did not reach statistical significance (Figure 6a,d). Furthermore, RyR2 and CaV1.2 were not regulated in atrial SCN10A KO iPSC-CMs compared to control atrial iPSC-CMs according to the Western blot results (Figure 6b,c,e,f). Thus, SCN10A KO seems to exert no significant side effects on the expression of the other main proteins relevant to excitation-contraction coupling in atrial iPSC-CMs compared with their respective control cells. KO iPSC-CMs. Normalized values of Na V 1.5 (d) (n = 3 control/6 KO differentiations), Ca V 1.2 (e) (n = 5/7 differentiations), and RyR2 (f) (n = 5/7 differentiations) in atrial control and SCN10A KO iPSC-CMs normalized to GAPDH (n = 5/7 differentiations). Student's t-test was used for statistical analysis.

Discussion
Atrial fibrillation (AF) is the most prevalent clinically significant arrhythmia. It represents a major risk factor for embolic stroke and exacerbation of heart failure (HF), consequently contributing to heightened morbidity and mortality rates [25]. The current prevalence of atrial fibrillation (AF) in adults ranges between 2% and 4%, with an anticipated 2.3-fold increase due to the extended longevity of the general population. For patients with atrial fibrillation (AF), first-line therapies for rhythm control include anti-arrhythmic drugs and/or left atrial pulmonary vein ablation [25]. However, pharmacological rhythm control is notably restricted in patients with underlying structural heart disease. The currently available drugs for these patients have limitations, poor tolerability, and adverse side effects [26]. Therefore, there is a demand for new and safer innovative compounds to address the treatment of AF in patients with structural heart disease. Sodium currents are effective therapeutic targets for the treatment of AF. In this context, the I NaL has been increasingly identified as a potential target to inhibit cellular arrhythmogenic triggers in AF and the first hopeful results have been shown in clinical trials [26][27][28][29][30].
However, the mechanism of I NaL regulation with respect to cellular arrhythmogenic triggers is not yet well understood. Besides Na V 1.5, other Na V isoforms have been reported to be present in the heart. We have shown that the expression of the Na + channel Na V 1.8 in left ventricular CMs is upregulated in human HF myocardium [20], and that Na V 1.8 contributes to arrhythmogenesis by inducing the I NaL [19,20,22,31]. Variants in the SCN10A gene (Na V 1.8) were shown to be associated with cardiac arrhythmias such as atrial fibrillation and sudden cardiac death [32]. Whether these Na V 1.8-related effects are mediated by cardiac ganglia or cardiomyocytes is still under debate. In the present study, we used human atrial SCN10A KO iPSC-CMs and demonstrated that Na V 1.8 is responsible for the generation of the I NaL . Both inhibition and KO of Na V 1.8 potently suppressed the I NaL and diastolic SR-Ca 2+ leak as proarrhythmogenic triggers in atrial CMs. These findings suggest that targeting Na V 1.8 constitutes a novel therapeutic antiarrhythmic strategy for the treatment of atrial rhythm disorders.

Na V 1.8 and Atrial I NaL
Under pathological conditions, the enhanced persistent Na + influx, known as enhanced I NaL , has been demonstrated to play an important role throughout the action potential [33]. The prolongation of the action potential duration caused by an I NaL increases the likelihood of early afterdepolarizations (EADs), which serve as triggers for the occurrence of arrhythmias. The specific Na V isoforms involved in the generation of an I NaL , particularly in clinically relevant conditions like atrial fibrillation (AF) and heart failure (HF), remain unclear. This information is of translational relevance because selectively targeting the inhibition of the I NaL would be a desirable antiarrhythmic approach.
Genome-wide association studies have identified SCN10A as a regulator of cardiac conduction. By employing various methodologies in both human and mouse cardiomyocytes, we have demonstrated the significance of Na V 1.8 in the generation of the late sodium current (I NaL ). We found that Na V 1.8 is upregulated under conditions of HF and cardiac hypertrophy [20,22,31]. Recent studies have provided evidence for the involvement of Na V 1.8 in atrial cellular electrophysiology and have successfully linked SCN10A variants to AF [32,34]. However, some of the preliminary studies are limited by the use of appropriate ion channel blockers, which theoretically could have nonspecific effects.
Therefore, in the present study we used homozygous atrial SCN10A-KO iPSC-CMs to show that the Na V 1.8-associated effects are mechanistically mediated by Na V 1.8. Since under healthy conditions the I NaL is very low, we applied isoproterenol in order to enhance the I NaL for a better comparison between the control and KO iPSC CMs. Casini et al. did not detect any Na V 1.8-based I NaL in non-diseased human atrial and rabbit ventricular CMs without beta-adrenergic stimulation [24]. Most importantly, the incidence of an enhanced I NaL depends on pharmacological (beta-adrenergic activation) or pathological stimulation and explain the absence of Na V 1.8 effects in this study. Here, we show that Na V 1.8 contributes to an enhanced I NaL in atrial control iPSC-CMs by reducing the I NaL by simultaneous treatment with isoproterenol and PF-01247324. Moreover, the specific blocker PF-01247324, when used to inhibit Na V 1.8, did not induce any additional effects on the I NaL in Na V 1.8 KO atrial cells compared to untreated Na V 1.8 KO atrial cells. This finding highlights the specificity of the drug in targeting Na V 1.8 [35]. Pabel et al. demonstrated that both pharmacological inhibition and genetic ablation of Na V 1.8 resulted in a reduction of the late sodium current (I NaL ) in human and murine atrial CMs [19]. In line with this, patch-clamp recordings of isolated human atrial CMs obtained from patients in sinus rhythm revealed that following mild beta-adrenergic stimulation with isoproterenol, the inhibition of Na V 1.8 using PF-01247324 and A-803467 led to a significant reduction in the late sodium current (I NaL ) [19]. Isolated atrial CMs from SCN10A-/mice revealed a significantly lower I NaL compared to WT while pharmacological inhibition by PF-01247324 exerted no additional effect on the I NaL in SCN10A-/mice [19]. Therefore, the results of the present study are in line with previous findings in atrial human and mice atrial KO [19] and ventricular KO mice and human iPSC-CMs and isolated CMs [22,36]. Moreover, the impact of SCN10A variants associated with AF on the modulation of the I NaL was demonstrated through transfection experiments in ND7/23 cells. This additional evidence further strengthens the notion that Na V 1.8 plays a significant role in the development of I NaL -related arrhythmias [37].

Na V 1.8 and Atrial Action Potential Duration
Previous studies have provided evidence that the I NaL plays a significant role in determining the APD in both atrial and ventricular CMs [2,3,8,27,29]. Having demonstrated the upregulation of Na V 1.8 expression in human AF and HF, we proceeded to investigate the impact of Na V 1.8-induced I NaL on various action potential parameters. We also used isoproterenol to enhance the I NaL . In line with previous data from our group in atrial human and mice CMs [19], the present study showed that in atrial control or SCN10A KO iPSC CMs, Na V 1.8 has negligible effects on the atrial action potential parameters. In AF, the APD becomes shorter and a further shortening of APD may lead to shorter refractory periods, thereby further facilitating reentry. Therefore, negligible effects on APD point towards a positive therapeutic profile of targeting Na V 1.8 in AF. Since dv/dt is a surrogate for the fast Na + influx and peak Na + current, these data show that there is no involvement of Na V 1.8 in the peak Na + current in atrial iPSC CMs. A negligible effect of Nav1.8 inhibition on cardiac conduction peak Na + current blockade would be desirable in order to treat patients with structural heart disease and AF.

Na V 1.8 and Atrial Ca 2+ Handling
In our previous studies, we demonstrated that the I NaL -mediated Na + influx has the ability to induce Ca 2+ influx through reverse-mode NCX, resulting in elevated cytosolic [Ca 2+ ] levels and an increased occurrence of Ca 2+ sparks in the human atrium [21,28]. Furthermore, the inhibition of the I NaL through specific targeting of Na V 1.8 has the capability to reduce the reverse mode NCX, thereby also mitigating diastolic proarrhythmogenic SR-Ca 2+ leak [20,21,31]. The relationship between enhanced I NaL and an increased risk of arrhythmias is indeed complex. This complexity arises from the fact that the increased leak of Ca 2+ from the SR can induce a transient inward current, which, in turn, leads to arrhythmogenic delayed afterdepolarizations. Additionally, it can also result in significant spontaneous proarrhythmic Ca 2+ release from the SR [8,28].
In the present study, we show a reduction in spontaneous SR Ca 2+ spark frequency as well as a decreased frequency of spontaneous Ca 2+ waves in human atrial SCN10A KO CMs and in control CMs after pharmacological inhibition. As Ca 2+ waves represent a major proarrhythmic trigger, we hereby establish the principle of Na V 1.8-induced I NaL and its triggering role in cellular arrhythmogenesis that is independent of neuronal influence in isolated human atrial CMs. Interestingly, we found no effects on intracellular Ca 2+ transients in either SCN10A KO or following Na V 1.8 inhibition in control CMs. Thus, we propose that the intracellular Ca 2+ handling and likely contractile function of CMs remain mostly unaffected by Na V 1.8. In summary, our results and current evidence indicate that the discussed Na V 1.8-induced I NaL mainly influences arrhythmogenesis on a subcellular level while leaving cellular Ca 2+ release and contractile function unaffected.

Clinical Relevance
The currently available anti-arrhythmic drugs, particularly for patients with structural heart disease, are limited in their effectiveness. Drugs like flecainide or amiodarone, which are commonly used, demonstrate suboptimal efficacy and are associated with significant adverse side effects, including life-threatening ventricular arrhythmias and organ toxicity. Therefore, new, safer, and more precise compounds for the treatment of atrial arrhythmias are highly desirable. Na V 1.8 was detected in atria, and human hypertrophied and failing ventricles [19,22,31]. The results of the present study demonstrate that either genetic ablation of Na V 1.8 using SCN10A KO iPSC-CMs or pharmacological inhibition can reverse cellular proarrhythmic effects in the atria. Both inhibition and KO of Na V 1.8 potently suppressed proarrhythmogenic triggers (e.g., I NaL and diastolic SR-Ca 2+ leak) while leaving the peak Na + current unaffected. These findings suggest targeting Na V 1.8-dependent I NaL constitutes a novel therapeutic anti-arhythmic strategy for the treatment of atrial rhythm disorders.

Generation of Homozygous Knockout iPSCs Using CRISPR/Cas9 and Directed Differentiation into Atrial iPSC-Cardiomyocytes
All procedures conducted in this study adhered to the principles outlined in the Declaration of Helsinki and received approval from the local ethics committee of the University Medicine of Göttingen (Az-10/9/15). Informed consent was signed by all tissue donors. A homozygous SCN10A KO iPSC line was generated from a control iPSC line by CRISPR/Cas9 genome editing as described in detail in previous studies [22,23]. The generated SCN10A KO iPSCs were differentiated into functionally beating, atrial iPSC-derived cardiomyocytes as described in [38].
In order to achieve directed atrial cardiac differentiation of the induced pluripotent stem cells (iPSCs), manipulation of the Wnt signaling pathway was employed, as previously described [38]. The cells were cultured for 60 days and then passaged onto glass-bottom Fluoro Dishes (WPI, 30 K/dish) by subjecting them to trypsinization at 37 • C for 3 min. The cells were allowed to settle for 7 days prior to further measurements, with medium changes performed every 2 days. iPSC-derived cardiac myocytes (iPSC-CMs) were analyzed 8-10 weeks after the initiation of differentiation, unless otherwise specified. The purity of the iPSC-CMs was determined by flow analysis, with a focus on cardiac troponin T positivity (>90% cardiac TNT+), as well as through qPCR and immunofluorescence analysis of atrial-specific markers (PITX2, MLC2a). Four to five differentiation experiments were performed to generate atrial iPSC-CMs from two Na V 1.8 knockout lines and their corresponding healthy isogenic control line.

Pharmacological Intervention
For selective inhibition of Na V 1.8-induced sodium currents, a specific Na V 1.8 blocker PF-01247324 (1 µmol/L, Sigma-Aldrich, Taufkirchen, Germany)) was used. Cellular electrophysiological measurements were performed under slight beta-adrenergic stimulation (isoproterenol (Iso), 50 nmol/L, Sigma-Aldrich, Taufkirchen, Germany)) [20]. Prior to the start of experiments, the CMs were incubated for 15 min with both substances or isoproterenol alone as a control.
Action potential recordings were performed using the whole-cell patch-clamp technique. To elicit action potentials, square current pulses with amplitudes of 0.5-1 nA and durations of 1-5 ms were applied. The stimulation frequency was increased gradually from 0.5 to 2 Hz.
The late sodium current (I NaL ) was measured using the ruptured-patch whole-cell patch-clamp technique. The pipette used had a resistance ranging from 2 to 3 mega-ohms (MΩ). I NaL recordings were performed exclusively in CMs where a seal with a resistance of over 1 giga-ohm (GΩ) was achieved, and the access resistance remained below 7 MΩ. After a stabilization period of 3 min, the iPSC-derived CMs were held at a holding potential of −120 mV and then depolarized to −35 mV for 1000 ms with 10 pulses and a basic cycle length of 2 s. The I NaL was quantified as the integral current amplitude between 100 and 500 ms and was normalized to the membrane capacitance.

Confocal Ca 2+ Imaging
A total of 35.000 atrial iPSC-CMs plated on glass-bottom FluoroDishes were incubated with the Ca 2+ indicator Fluo 4-AM (10 µmol/L, Invitrogen, Darmstadt, Germany) for 15 min at RT for de-esterification of the dye. The solution was substituted with Tyrode's solution (as described in [19]) and the respective pharmacological agents and left to incubate for 15 min. Confocal line scans were obtained with a laser scanning confocal microscope (LSM 5 Pascal, Zeiss, Jena, Germany). Scans were conducted after continuous electrical field stimulation at 1 Hz during pausing of stimulation. Ca 2+ release events were analyzed using the SparkMaster plugin for ImageJ. The mean Ca 2+ spark frequency was calculated from the number of sparks normalized to scan width, duration, and scan rate (100 µm/s). Cells exhibiting major Ca 2+ release events (Ca 2+ wavelets or waves) were excluded from the calculation of Ca 2+ spark frequency and separately classified as proarrhythmic cells as a proportion of all cells.

Epifluorescence Microscopy for Ca 2+ Transient Measurements
A total of 35.000 atrial CMs were dissociated and plated as described above and loaded with the radiometric Ca 2+ indicator Fura 2-AM (5 µmol/L, Invitrogen) for 15 min at RT. Subsequently, the cells were washed with Tyrode's solution for de-esterification and incubated with pharmacological agents as described above. The measurements were performed using a fluorescence detection system (IonOptix, Amsterdam, Netherlands) connected to an inverted microscope with oil immersion lens (40×). Cardiomyocytes were subjected to electrical field stimulation at 1 Hz for the duration of the experiment to ensure steady intracellular Ca 2+ concentrations. Recording of Ca 2+ transients for analysis was performed at 1 Hz at steady state. For each cell, the stimulation was paused for 30 s to detect spontaneous Ca 2+ release events and evaluate the spontaneous beating frequency of the iPSC-CMs. Ca 2+ transients were analyzed using the software IonWizard (IonOptix).

Statistical Analysis
The data are reported as mean ± SEM, unless otherwise stated. Analysis was carried out with Prism 9 software (Graphpad, San Diego, CA, USA). For comparisons of two groups, unpaired Student's t test was used in the case of parametric distribution of the data. Three or more groups including more than one differentiation experiment were compared using nested one-way ANOVA. The results were corrected for multiple comparisons by Sidak's correction. Fisher's exact test was used to statistically compare proportions. p values are two-sided and considered statistically significant if p < 0.05.

Conclusions
In conclusion, we showed that the neuronal sodium channel Na V 1.8, which contributes to the I NaL in the heart, is down-regulated in atrial SCN10A-KO iPSC-CMs and, importantly, contributes to I NaL formation in human atrial CMs. Na V 1.8 KO or the inhibition of Na V 1.8 modulates proarrhythmogenic triggers such as I NaL and diastolic SR-Ca 2+ leak in human atrial CMs. Therefore, Na V 1.8 might represent a novel treatment target for antiarrhythmic strategies.