NaV1.8 as Proarrhythmic Target in a Ventricular Cardiac Stem Cell Model

The sodium channel NaV1.8, encoded by the SCN10A gene, has recently emerged as a potential regulator of cardiac electrophysiology. We have previously shown that NaV1.8 contributes to arrhythmogenesis by inducing a persistent Na+ current (late Na+ current, INaL) in human atrial and ventricular cardiomyocytes (CM). We now aim to further investigate the contribution of NaV1.8 to human ventricular arrhythmogenesis at the CM-specific level using pharmacological inhibition as well as a genetic knockout (KO) of SCN10A in induced pluripotent stem cell CM (iPSC-CM). In functional voltage-clamp experiments, we demonstrate that INaL was significantly reduced in ventricular SCN10A-KO iPSC-CM and in control CM after a specific pharmacological inhibition of NaV1.8. In contrast, we did not find any effects on ventricular APD90. The frequency of spontaneous sarcoplasmic reticulum Ca2+ sparks and waves were reduced in SCN10A-KO iPSC-CM and control cells following the pharmacological inhibition of NaV1.8. We further analyzed potential triggers of arrhythmias and found reduced delayed afterdepolarizations (DAD) in SCN10A-KO iPSC-CM and after the specific inhibition of NaV1.8 in control cells. In conclusion, we show that NaV1.8-induced INaL primarily impacts arrhythmogenesis at a subcellular level, with minimal effects on systolic cellular Ca2+ release. The inhibition or knockout of NaV1.8 diminishes proarrhythmic triggers in ventricular CM. In conjunction with our previously published results, this work confirms NaV1.8 as a proarrhythmic target that may be useful in an anti-arrhythmic therapeutic strategy.


Introduction
Cardiac electrophysiology is a complex and precisely regulated process crucial for the normal functioning of the heart.Sodium channels are essential for the rapid depolarization phase of the cardiac action potential (AP) and contribute to initiating and propagating electrical signals [1,2].However, under certain pathological conditions, such as heart failure (HF), ischemia, and atrial fibrillation (AF), voltage-gated (Na V ) sodium channels exhibit increased reactivation or sustained opening, resulting in the relevant augmentation of a persistent inward current known as the late sodium current (I NaL ) [3][4][5][6][7][8][9][10][11].One particular sodium channel isoform, Na V 1.8, encoded by the SCN10A gene, has gained attention due to its potential involvement in cardiac electrophysiology.Extensive research has shown that Na V 1.8 contributes to nociceptive signaling and pain perception in peripheral sensory neurons [12][13][14][15].However, genome-wide association studies reported that the SCN10A gene is associated with cardiac arrhythmias [16][17][18].Although several studies have investigated the expression of SCN10A in ventricular myocardium [19][20][21][22][23][24], there is an ongoing debate regarding the mechanism responsible for the effects associated with Na V 1.8 and the specific cellular localization of these effects, whether it is in cardiac ganglia or cardiomyocytes (CM) [20,23].The present study addresses this knowledge gap using genetic ablation or knockout (KO) of SCN10A in human induced pluripotent stem cell-CM (iPSC-CM) as a tool to supplement the use of pharmacological agents that inhibit the channel.This approach can reveal the inherent impact of Na V 1.8 on I NaL in human CM without any confounding or unspecific effects of pharmacological inhibitors.This SCN10A iPSC-CM KO model was used in our previous study and demonstrated that Na V 1.8 is expressed in HF CM and contributes to I NaL formation in ventricular iPSC-CM [22].Since it is still unclear how Na V 1.8-dependentI NaL contributes to cellular arrhythmias, the objective of this study was to investigate potential triggers of arrhythmias in human ventricular CM.Therefore, we conducted additional cellular electrophysiological measurements, including the spontaneous release of Ca 2+ from the sarcoplasmic reticulum (SR), action potential duration (APD), delayed afterdepolarizations (DADs), and Ca 2+ transients to further characterize the arrhythmogenic potential of Na V 1.8 in the heart.By utilizing CRISPR/Cas9-based KO technology, we provide compelling evidence for the existence of a Na V 1.8-mediatedI NaL in human CM, contributing valuable information to the existing body of knowledge [22].

Effects of Na V 1.8 on I NaL in Human Ventricular iPSC-Cardiomyocytes
Homozygous SCN10A/Na V 1.8 KO iPSC were differentiated into 2-month-old ventricular CM as described elsewhere [25].In our previous study, we demonstrated that the knockout of Na V 1.8 in ventricular iPSC-CM after mild isoproterenol stimulation (Iso, 50 nmol/L) resulted in a substantial reduction (~70%) in the amplitude of I NaL compared to control iPSC-CM [22].The application of a Na V 1.8-specific blocker (PF-01247324) did not produce any additional effect on I NaL in Na V 1.8 KO iPSC-CM compared to untreated Na V 1.8 knockout cells [22].In the present study, we aimed to validate and expand upon these initial findings by conducting a larger-scale investigation involving a higher number of single ventricular iPSC-CM generated from a higher number of cardiac differentiations.To increase the relatively small amplitude of I NaL in healthy human iPSC-CM, we used 50 nmol/L Iso, which is consistent with our previous experiments.To assess potential non-specific effects associated with either genetic KO or pharmacological intervention, we utilized the specific Na V 1.8 blocker PF-01247324 at a concentration of 1 µmol/L [22].In functional voltage-clamp experiments, we showed that I NaL was significantly reduced by pharmacological inhibition and the genetic KO of Na V 1.8, validating the impact of both approaches on I NaL (Figure 1).The increased I NaL induced by Iso in control ventricular iPSC-CM (−108.4± 9.5 A*ms*F −1 ) was significantly reduced in KO iPSC-CM by 70.8% (−32.5 ± 4.4 A*ms*F −1 , p < 0.0001, Figure 1b) and by the specific Na V 1.8 inhibitor, PF-01247324 (1 µmol/L), by 70.0% (−31.6 ± 6.7 A*ms*F −1 ; p < 0.0001; Figure 1b).No further effects on I NaL were observed in KO iPSC-CM after the application of PF-01247324 (−36.3 ± 3.0 A*ms*F −1 ; p < 0.981; Figure 1b).These results demonstrated in a broader set of experiments based on statistical analyses of multiple cardiac differentiations and not on single iPSC-CM, confirm our previous results that Na V 1.8 contributes to I NaL formation in ventricular iPSC-CM, demonstrated in a broader set of experiments based on statistical analyses of multiple cardiac differentiations and not single iPSC-CM.

Effects of Na V 1.8 on APD and Arrhythmic Triggers in Ventricular Cells
To explore the potential effects of Na V 1.8 on the action potential in human ventricular iPSC-CM, we conducted current-clamp experiments on whole cells.The APD of KO iPSC-CM was not significantly different from that of control cells, both with and without additional application of the specific Na V 1.8 blocker PF-01247324 (Figure 2a,b, control at 0.5 Hz, APD 90 , 395.4 ± 42.0 ms vs. control + PF-01247324 420.9 ± 46.5 ms, an increase of 6%; KO control 340.4 ± 34.6 ms, a decrease of 13% vs. KO + PF-01247324 376.4 ± 34.8 ms, a reduction of 4.8%).Furthermore, the APD in KO cells at 20% repolarization (APD 20 ), at 50% repolarization (APD 50 ), and at 70% repolarization (APD 70 ) were also similar to that of control cells (Supplemental Figure S1, Supplemental Table S1).Potential non-specific effects associated with KO or pharmacological inhibition of Na V 1.8 were analyzed by comparing resting membrane potential (RMP), action potential amplitude (APA), and upstroke velocity (Vmax) between all groups.No significant effects of SCN10A-KO or pharmacological inhibition of Na V 1.8 were observed on these parameters.

Effects of NaV1.8 on APD and Arrhythmic Triggers in Ventricular Cells
To explore the potential effects of NaV1.8 on the action potential in human ventricular iPSC-CM, we conducted current-clamp experiments on whole cells.The APD of KO iPSC-CM was not significantly different from that of control cells, both with and without additional application of the specific NaV1.8blocker PF-01247324 (Figure 2a,b, control at 0.5 Hz, APD90, 395.4 ± 42.0 ms vs. control + PF-01247324 420.9 ± 46.5 ms, an increase of 6%; KO control 340.4 ± 34.6 ms, a decrease of 13% vs. KO + PF-01247324 376.4 ± 34.8 ms, a reduction of 4.8%).Furthermore, the APD in KO cells at 20% repolarization (APD20), at 50% repolarization (APD50), and at 70% repolarization (APD70) were also similar to that of control cells (Supplemental Figure S1, Supplemental Table S1).Potential non-specific effects associated with KO or pharmacological inhibition of NaV1.8 were analyzed by comparing resting membrane potential (RMP), action potential amplitude (APA), and upstroke velocity (Vmax) between all groups.No significant effects of SCN10A-KO or pharmacological inhibition of NaV1.8 were observed on these parameters.

Role of Na V 1.8 in the Generation of SR Ca 2+ Leak in the Ventricle
It is known that I NaL can induce arrhythmogenic diastolic SR Ca 2+ release [26].Furthermore, we demonstrated in our previous studies that the arrhythmogenic potential of Na V 1.8 in the ventricle is exerted through the enhancement of I NaL [22,23] and, consequently, the induction of spontaneous SR Ca 2+ release on a subcellular level.To further investigate these effects on a standardized larger scale at the CM-specific level and ultimately by KO of Na V 1.8 in human CM, we conducted confocal microscopy experiments in ventricular human control and Na V 1.8 KO iPSC-CM.
Diastolic confocal line scans using Fluo 4-AM revealed that KO of SCN10A in ventricular human iPSC-CM resulted in a decrease in the frequency of spontaneous arrhythmogenic Ca 2+ sparks compared to the corresponding control cells (control: 4.18 ± 0.25 vs. KO: 2.72 ± 0.19 sparks/100 µm/s, p = 0.02).When Na V 1.8 was pharmacologically inhibited using PF-01247324, a significant reduction in diastolic Ca 2+ sparks was observed in control cells (control + PF-01247324: 2.50 ± 0.22, p = 0.03) but not in KO cells (KO + PF-01247324: 2.36 ± 0.25, p = 0.99) (Figure 3a,b).Thus, the reduction in both I NaL and diastolic SR Ca 2+ release in Na V 1.8 KO or after Na V 1.8 inhibition confirms a significant role of this sodium channel in cellular arrhythmogenesis in the human ventricle.
However, the amount of systolic Ca 2+ release and the dynamics of Ca 2+ sequestration, as investigated in epifluorescence experiments using Fura 2-AM, were left unaffected by KO or pharmacological inhibition of Na V 1.8 (Supplemental Figure S2).

Role of NaV1.8 in the Generation of SR Ca 2+ Leak in the Ventricle
It is known that INaL can induce arrhythmogenic diastolic SR Ca 2+ release [26].Furthermore, we demonstrated in our previous studies that the arrhythmogenic potential of NaV1.8 in the ventricle is exerted through the enhancement of INaL [22,23] and, consequently, the induction of spontaneous SR Ca 2+ release on a subcellular level.To further investigate these effects on a standardized larger scale at the CM-specific level and ultimately by KO of NaV1.8 in human CM, we conducted confocal microscopy experiments in ventricular human control and NaV1.8 KO iPSC-CM.We further investigated the effects of pharmacological inhibition and genetic ablation of Na V 1.8 on the incidence of diastolic Ca 2+ waves, which constitute primary proarrhythmic triggers.The percentage of cells exhibiting diastolic Ca 2+ waves was significantly reduced in control ventricular iPSC-CM by inhibition of Na V 1.8 with PF-01247324 (percent of all cells exhibiting Ca 2+ waves: control 17% vs. control + PF-01247324 7% of all cells exhibiting Ca 2+ waves, p < 0.05).Genetic ablation of Na V 1.8 showed a slight tendency to reduce Ca 2+ wave frequency, but the effect was not statistically significant (control: 17% vs. KO: 9%; p = 0.14; Figure 3c,d).

Na V 1.8 Inhibition or KO Reduces Proarrhythmic Triggers in Ventricular Cardiomyocytes
To gain further insights into the contribution of I NaL to cellular arrhythmias, we investigated potential triggers of arrhythmias.Because afterdepolarizations occur infrequently under basal conditions, we augmented cellular SR Ca 2+ stores with isoproterenol (Iso) to increase cellular arrhythmias [27].Under these conditions, we observed a pro-nounced number of DADs in control iPSC-CM.This effect was significantly reduced by inhibiting Na V 1.8 with PF-01247324 and by genetically depleting Na V 1.8 (DADs/min., Figure 4a,b, at 0.5 Hz control iPSC-CM 12.9 ± 1.4 DADs/min.;control iPSC-CM + PF-01247324: 2.8 ± 0.7 DADs/min.;KO iPSC-CM control 5.4 ± 1.2 DADs/min.;KO iPSC-CM + PF-01247324: 4.1 ± 1.2 DADs/min.; Figure 4a,b).The application of PF-01247324 had no significant effect on Na V 1.8 KO lines, which showed an already reduced incidence of arrhythmic events.Thus, genetic KO or inhibition of Na V 1.8 markedly prevented cellular arrhythmias in human ventricular CM.We did not observe any early afterdepolarizations in control or Na V 1.8 KO iPSC-CM.

NaV1.8 Inhibition or KO Reduces Proarrhythmic Triggers in Ventricular Cardiomyocytes
To gain further insights into the contribution of INaL to cellular arrhythmias, we investigated potential triggers of arrhythmias.Because afterdepolarizations occur infrequently under basal conditions, we augmented cellular SR Ca 2+ stores with isoproterenol (Iso) to increase cellular arrhythmias [27].Under these conditions, we observed a pronounced number of DADs in control iPSC-CM.This effect was significantly reduced by inhibiting NaV1.8 with PF-01247324 and by genetically depleting NaV1.8 (DADs/min., Figure 4a,b, at 0.5 Hz control iPSC-CM 12.9 ± 1.4 DADs/min.;control iPSC-CM + PF-01247324: 2.8 ± 0.7 DADs/min.;KO iPSC-CM control 5.4 ± 1.2 DADs/min.;KO iPSC-CM + PF-01247324: 4.1 ± 1.2 DADs/min.; Figure 4a,b).The application of PF-01247324 had no significant effect on NaV1.8 KO lines, which showed an already reduced incidence of arrhythmic events.Thus, genetic KO or inhibition of NaV1.8 markedly prevented cellular arrhythmias in human ventricular CM.We did not observe any early afterdepolarizations in control or NaV1.8KO iPSC-CM.

Discussion
In this study, we employed CRISPR/Cas9 KO technology in human ventricular CM to demonstrate that the genetic KO of Na V 1.8 resulted in a substantial reduction in I NaL .No significant effects on APD were observed.The heightened frequency of spontaneous arrhythmogenic SR Ca 2+ sparks, and Ca 2+ waves elicited by Iso was significantly diminished through pharmacological intervention or genetic KO of Na V 1.8.Additionally, DADs and spontaneous action potentials, indirectly elicited by an increased SR Ca 2+ leak, were effectively mitigated through either pharmacological intervention or genetic KO of Na V 1.8.

Role of Na V 1.8 on I NaL Generation
In the current study, we validated the involvement of Na V 1.8 in Iso-augmented I NaL in human control ventricular iPSC-CM by observing a reduction in I NaL through the specific Na V 1.8 blocker PF-01247324 or genetic ablation.Our data support the findings of other studies including a report by Yang et al. (2012) [28].These authors inhibited SCN10A channels with a specific Na V 1.8 blocker in adult SCN10A-/mice and rabbit ventricular CM.Their results showed a reduction in I NaL and anti-arrhythmic effects on ventricular arrhythmias after pharmacologic inhibition of Na V 1.8 [28].In our earlier work, Dybkova et al. (2018) identified Na V 1.8 in human failing ventricular myocardium and demonstrated its significant contribution to I NaL formation [23].We also exhibited successful modulation of I NaL by Na V 1.8 inhibitors [23].Additionally, in another study, Na V 1.8 expression was significantly upregulated in human left ventricular hypertrophy (LVH), and increased Na V 1.8 expression was associated with enhanced I NaL , potentially contributing to the arrhythmogenic substrate and impaired cardiac function observed in LVH [20].Of note, in addition to its demonstrated presence in atrial and ventricular human CM (adult and iPSC-CM), Na V 1.8 associated I NaL has also been characterized in various other species (murine, canine and porcine models); thus, some species-and model-dependent differences should be considered [9,10,26,27, Most of the previous work regarding Na V 1.8-related characteristics of I NaL was obtained on the basis of pharmacological interventions.Drugs may have additional, unspecific effects, leaving the mechanism by which Na V 1.8 generates I NaL unclear.The present study addresses this gap in knowledge and confirms our previous results in human SCN10A-KO iPSC-CM by demonstrating that Na V 1.8 plays a significant role in generating I NaL by taking an alternative approach that is not purely pharmacological.Furthermore, our results demonstrate that Na V 1.8-inducedI NaL is not limited to ganglia, as assumed in the past, but is also present in ventricular CM, as shown by the results of our previous project [22].
Since I NaL is typically very small under healthy or basal conditions, we employed isoproterenol to enhance I NaL in our cellular model in order to facilitate a comparison between control and KO iPSC-healthy-CM.It is worth noting that without beta-adrenergic stimulation or structural disease, Casini et al. (2019) did not detect any Na V 1.8-dependentI NaL in non-diseased rabbit ventricular CM by using specific blockers [57].This underscores the dependence of Na V 1.8-associatedI NaL enhancement on stimulation by pharmacological (beta-adrenergic activation) or pathological circumstances (e.g., chronic CAMKII δc overexpression [22]), which likely explains the absence of Na V 1.8 effects in their study.The fact that I NaL is only enhanced under pathological conditions has to be taken into account in the interpretation of experiments and establishes I NaL as a disease-specific target.

Significance of Na V 1.8 in I NaL and Arrhythmogenesis
Previous studies have established the significant role of I NaL in determining the duration of APs in ventricular CM [27,45,[58][59][60][61][62].Considering that Na V 1.8 expression is upregulated in human HF, we investigated the impact of Na V 1.8-inducedI NaL on various AP parameters.Consistent with our previous findings, our current study demonstrates that Na V 1.8 has only minimal effects on ventricular AP parameters in ventricular control CM or in SCN10A-KO iPSC-CM.At first glance, the results seem to contradict previous findings regarding ventricular Na V 1.8 channels, their effect on APD, and their contribution to arrhythmias in the context of HF and hypertrophy.In the failing human heart and in a mouse model with a genetic SCN10A KO, Dybkova et al. (2018) demonstrated that Na V 1.8 expression was higher and that this was associated with a minimally prolonged APD, contributing to I NaL -induced arrhythmias [23].The contribution of Na V 1.8 to Na +and Ca 2+ -dependent cellular arrhythmias revealed that Na V 1.8 channels can promote abnormal electrical activity and Ca 2+ overload in CM.These proarrhythmic effects were mediated by enhanced Na + and Ca 2+ influx through Na V 1.8 channels [21].Moreover, the interaction between CaMKII δc and Na V 1.8 in HF leads to CaMKII δc phosphorylation of Na V 1.8 channels, exacerbating the proarrhythmic effects in the failing hearts [22].In human LVH, Ahmad et al. (2019) demonstrated that enhanced Na V 1.8 function was associated with AP prolongation [20].Overall, these studies suggest that the increased expression and activity of Na V 1.8 channels in failing hearts and hypertrophied ventricles, in contrast to the healthy CM investigated in the present study, may contribute to APD prolongation and proarrhythmic effects.Thus, further investigation of Na V 1.8 and its inhibition and of the characteristics of I NaL in injured or diseased human iPSC-CM models will be of interest to help clarify mechanisms that play a role under pathological conditions.
We show that the inhibition of Na V 1.8 by PF-01247324 of I NaL or Vmax was absent in Na V 1.8 KO ventricular iPSC-CM.Given that Vmax (dv/dt) serves as an indicator of the rapid influx of Na + and the peak Na + current, these findings indicate that Na V 1.8 does not play a relevant role in the peak Na + current in ventricular iPSC CM.
The association between enhanced I NaL and an increased susceptibility to arrhythmias is intricate [3,9,45].This complexity stems from the increased leakage of Ca 2+ from the SR, which can instigate a transient inward current, subsequently giving rise to arrhythmogenic DADs.In the current study, we observed a decrease in the frequency of spontaneous SR Ca 2+ sparks and spontaneous Ca 2+ waves in human ventricular iPSC-CM with SCN10A KO as well as in control CM after pharmacological inhibition of Na V 1.8.Since Ca 2+ waves are known to act as potent proarrhythmic triggers, our findings provide evidence for the involvement of Na V 1.8-inducedI NaL in cellular arrhythmogenesis in isolated human ventricular iPSC-CM.This is in line with our previous investigations, which demonstrated that the I NaL , mediated by the influx of Na + , can induce the influx of Ca 2+ via the reverse mode of the sodium-calcium exchanger (NCX) in the human ventricle.This process can lead to an elevation in the cytosolic concentration of Ca 2+ and an increased occurrence of ryanodine receptor (RyR 2 ) Ca 2+ release events [21].Furthermore, inhibiting I NaL by using specific Na V 1.8 channel inhibitors has been shown to reduce the reverse mode of the NCX and, consequently, mitigate diastolic proarrhythmogenic SR Ca 2+ leakage [20,21,23].Interestingly, we did not observe any effects on intracellular Ca 2+ transients in SCN10A-KO CM or following Na V 1.8 inhibition in control CM.This implies there are no negative inotropic effects of Na V 1.8 inhibition, which is particularly noteworthy considering the situation in HF patients.
Since I NaL -dependent diastolic SR Ca 2+ release has the potential to induce an NCXmediated depolarizing current (I Ti ), resulting in cellular arrhythmias [63][64][65], we examined the impact of Na V 1.8 on afterdepolarizations and spontaneous action potentials.In our current investigation, a substantial number of DADs was noted in control iPSC-CM following Iso application.This effect was significantly mitigated by either inhibiting Na V 1.8 with PF-01247324 or through genetic depletion.
It is widely recognized that an augmented I NaL results in cellular Na + accumulation [66,67], subsequently leading to Ca 2+ overload through activation of the NCX reverse mode [9,21,26,68,69].Our earlier investigations have elucidated that this sequence of events may initiate the diastolic release of Ca 2+ from the SR and the subsequent occurrence of DADs.These effects are attributed to an intensified phosphorylation of RyR 2 , regulated by CaMKII δc [27]; this may explain our current findings and how Na V 1.8 influences arrhythmogenesis via I NaL .

Clinical Relevance
Ventricular arrhythmias, such as ventricular tachycardia and ventricular fibrillation, are life-threatening conditions associated with a high risk of sudden cardiac death [60,70].Studies have shown that increased Na V 1.8-dependentI NaL plays a role in the development of arrhythmia in various pathological conditions, including HF, LVH, and other structural heart diseases in ventricular CM [20,22,23].Unfortunately, in HF patients, current anti-arrhythmic medications, such as amiodarone, have shown severe adverse effects, potentially including potentially organ toxicity [71].Therefore, there is a crucial need for safer and more effective compounds to treat arrhythmias, especially in patients with HF.In the context of trials testing oral selective inhibitors in patients, the findings of the aforementioned article suggest a promising and easily implemented strategy for drug repurposing [71].Targeting Na V 1.8 and specifically inhibiting I NaL emerges as a potential therapeutic approach to prevent or treat ventricular arrhythmias.The current study shows that either genetic elimination of Na V 1.8 through SCN10A KO in iPSC-CM or pharmacological inhibition of Na V 1.8 can effectively reverse the proarrhythmic effects at the cellular level in ventricular cells.

Materials and Methods
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 Medical Center of Göttingen (Az-10/9/15).Informed consent was signed by all tissue donors.

Generating Homozygous Knockout iPSC
The generation of the homozygous SCN10A-KO iPSC line was carried out following the previously described protocols [22,25].Briefly, we used a previously described wildtype iPSC line [72] as a template and edited it with the CRISPR-Cas9 technique as described in Maurer et al. [25].The wildtype iPSC were electroporated with a mixture of two guideRNAs, a tracrRNA, and the Cas9 protein using the Human Stem Cell Nucleofector Kit (Amaxa VPH-5022) and the Amaxa Nucleofection II Device (Lonza, program B-016).An electroporation enhancer was added to enhance electroporation efficiency.After electroporation, 72 colonies were expanded, and genomic DNA was analyzed by Sanger sequencing.A promising iPSC mixed clone was chosen for further singularisation.After successful singularisation and additional Sanger sequencing of genomic DNA, two homozygously edited SCN10A knockout iPSC clones were obtained.These clones were regularly sequenced to check for maintenance of the knockout gene editing.

IPSC-Cardiomyocyte Differentiation
Two Na V 1.8 KO iPSC lines (K62.1 and K62.4) and the corresponding isogenic control iPSC line [72] were cultured and differentiated into ventricular iPSC-CM as described previously [22,72].In brief, iPSCs were cultured in a monolayer on Geltrex ® -coated dishes in chemically defined E8 medium (Life Technologies) until 80-90% confluency.Then, we initiated cardiac differentiation by manipulating Wnt signaling.Wnt signaling was activated by a medium change to cardio differentiation medium (RPMI 1640 with GlutaMAX medium (Gibco) supplemented with 0.02% L-ascorbic acid 2-phosphate (Sigma-Aldrich) and 0.05% albumin (Sigma-Aldrich)), supplemented with the GSK3 inhibitor CHIR99021 (4 µmol/L, Millipore).Subsequently, Wnt signaling was inhibited by changing the medium 48 h later to cardio differentiation medium supplemented with 5 µmol/L of the inhibitor of Wnt production-2 (IWP2, Millipore) for another 48 h.The medium was changed to cardio differentiation medium without adding small molecules for 48 h.From day 6 onward, the medium was changed to cardio culture medium (RPMI 1640 with GlutaMAX medium supplemented with 2% B27 with insulin (Gibco)), which was changed every 2 to 3 days.From day 14 to 21, the cells began beating.They were then passaged as 600,000 cells/well onto 6-well culture dishes and metabolically selected for CM for 4 to 5 days by depletion of glucose but supplementation of lactate (4 mmol/L) in a specified cardio selection medium (RPMI 1640 w/o glucose supplemented with 0.02% L-ascorbic acid 2-phosphate (Sigma-Aldrich) and 0.05% albumin (Sigma-Aldrich)).IPSC-CM were matured in cardio culture medium, with a medium change twice a week.The purity of the cells was determined by flow cytometry (>90% cTNT+ cells), immunofluorescence staining, and qPCR analysis for cardiac ventricular sub-type markers.Measurements were performed roughly 60 to 90 days after the initiation of the differentiation process.For functional experiments, iPSC-CM were plated on glass dishes with different cell numbers (World Precision Instruments).

Standard Operating Procedures
All electrophysiological measurements were conducted following a standardized protocol.A total of 35,000 ventricular iPSC-CM were plated on glass-bottomed Fluoro Dishes.Spontaneously beating ventricular iPSC-CM were stimulated at 0.5 Hz during experiments to achieve a steady state.Single-cell measurements were performed at room temperature with a stimulation frequency of 0.5 Hz/1 Hz/2 Hz.To selectively inhibit sodium currents induced by Na V 1.8, a specific Na V 1.8 blocker, PF-01247324 (1 µmol/L, Sigma-Aldrich, Taufkirchen, Germany), was utilized.Cellular electrophysiological measurements were carried out with mild beta-adrenergic stimulation using isoproterenol (Iso) at a concentration of 50 nmol/L (Sigma-Aldrich, Taufkirchen, Germany).iPSC-CM were incubated with either Iso (50 nmol/L, or Iso + PF-01247324 (1 µmol/L, Sigma-Aldrich, Taufkirchen, Germany) for 15 min before starting measurements.Regarding the specificity of Na V 1.8 blockers, there are already published data that have demonstrated the specificity of PF-01247324 and showed that it is an efficacious inhibitor of Na V 1.8 [21,22,73,74].

Patch-Clamp-Experiments
Patch-clamp experiments were performed as previously described [21,22,74,75].For action potential recordings, we employed the whole-cell patch-clamp technique in the current clamp configuration.The microelectrodes (3-5 MΩ) were filled with the following solution (in mmol/L): 92 K-aspartate, 48 KCl, 1 Mg-ATP, 10 HEPES, 0.02 EGTA, 0.1 GTP-Tris, and 4 Na 2 -ATP (pH 7.2, adjusted with KOH).The bath solution consisted of the following components (in mmol/L): 140 NaCl, 4 KCl, 1 MgCl 2 , 2 CaCl 2 , 10 glucose, and 10 HEPES (pH 7.4, adjusted with NaOH).Action potentials were continuously elicited by square current pulses with an amplitude of 1-2 nA and a duration of 1-5 ms at a frequency ranging from 0.5 to 2 Hz.The access resistance typically ranged from ~5 to 15 MΩ after patch rupture.Fast capacitance was compensated for in a cell-attached configuration, while membrane capacitance and series resistance were compensated after patch rupture.Signals were filtered using 2.9 and 10 kHz Bessel filters and recorded with an EPC10 from HEKA Elektronik Dr. Schulze GmbH, Lambrecht, Germany.Both the measuring electrode and the bath electrode (earth conductor of the bath solution) were made of chlorinated silver wire (HEKA Elektronik Dr. Schulze GmbH, Lambrecht, Germany).
To measure the I NaL ruptured patch, whole-cell patch-clamping was performed at room temperature.The resistance of the pipette was between 2 and 3 mega-Ohm (MΩ) when filled with pipette solution containing (in mmol/L): 95 CsCl, 40 Cs-glutamate, 10 NaCl, 0.92 MgCl 2 , 5 Mg-ATP, 0.3 Li-GTP, 5 HEPES, 0.03 niflumic acid (to block Ca 2+activated chloride current), 0.02 nifedipine (to block Ca 2+ current), 0.004 strophanthidine (to block Na + /K + ATPase current) 1 EGTA, and 0.36 CaCl 2 (free[Ca 2+ ]i, 100 nmol/L) (pH 7.2 with CsOH at room temperature).iPSC-CM were maintained in the bath solution containing (in mmol/L): 135 NaCl, 5 tetramethylammonium chloride, 4 CsCl, 2 MgCl 2 , 10 glucose, and 10 HEPES (pH 7.4 with CsOH at room temperature).To minimize contaminating Ca 2+ currents during I NaL measurements, Ca 2+ was omitted from the bath solution.I NaL was measured only in those iPSC-CM where a seal of more than 1 giga-Ohm was achieved, and the access resistance remained < 7 MΩ.When the whole-cell patch configuration was achieved, iPSC-CM were given a period of 3 min to be stabilized before conducting measurements.Thereafter, iPSC-CM were held at −120 mV before depolarization to −35 mV for a duration of 1000 ms with 10 pulses and a basic cycle length of 2 s.I NaL was measured as integral current amplitude between 100 and 500 ms and was normalized to the membrane capacitance.

Confocal Ca 2+ Imaging and Epifluorescence Microscopy
Ca 2+ imaging and epifluorescence microscopy were performed as previously described [74].
In detail, for confocal Ca 2+ imaging, ventricular iPSC-CM plated on glass-bottom FluoroDishes were incubated with the Ca 2+ indicator Fluo 4-AM (10 µmol/L, Invitrogen) for 15 min at room temperature for de-esterification of the dye.The solution was substituted with Tyrode's solution and the respective pharmacological agents and incubated for 15 min.Confocal line scans were obtained with a laser scanning confocal microscope (LSM 5 Pascal, Zeiss).Scans were conducted after continuous electrical field stimulation at 0.5 Hz during a pause in 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.
For epifluorescence microscopy, ventricular iPSC-CM were dissociated and plated as described above and loaded with the ratiometric Ca 2+ indicator Fura 2-AM (5 µmol/L, Invitrogen) for 15 min at RT. Subsequently, cells were washed with Tyrode's solution for de-esterification and incubated with pharmacological agents as described above.Measurements were performed using a fluorescence detection system (IonOptix) connected to an inverted microscope with oil immersion lens (40×).CM were subjected to electrical field stimulation at 0.5 Hz for the duration of the experiment to ensure steady intracellular Ca 2+ concentration.Recording of Ca 2+ transients for analysis was performed at 0.5 Hz at steady state.The stimulation was paused for 30 s to evaluate the spontaneous beating frequency of iPSC-CM (Supplemental Figure S3).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).Three or more groups, including more than one differentiation experiment, were compared using nested one-way ANOVA to account for potential variance between iPSC-CM differentiations.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.

Limitations
Using an iPSC-CM model, it is not possible to discriminate between specific cell types located at different parts of the ventricle (epi-, mid-, endocardial).All experiments were performed on a single-cell level, limiting a direct translation of our results and conclusions regarding arrhythmias into an in-vivo situation (e.g., no electrical cell-to-cell conduction).

Conclusions
In conclusion, our study demonstrated that Na V 1.8 is involved in the formation of I NaL in human ventricular iPSC-CM under pathological conditions.Genetic knockout or pharmacological inhibition of Na V 1.8 effectively modulated proarrhythmogenic triggers, including I NaL , diastolic SR Ca 2+ leak, Ca 2+ waves, and DADs in human ventricular CM.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/ijms25116144/s1.Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

15 Figure 1 .
Figure 1.NaV1.8 contributes to INaL formation in ventricular iPSC-CM.(a) Original traces of INaL in control iPSC-CM vs. SCN10A KO-iPSC-CM cells according to the inserted protocol.(b) Mean values per differentiation ± SEM of INaL (control n = 31 cells/5 differentiations; control + PF n = 14 cells/4 differentiations; SCN10A KO iPSC-CM control n = 18 cells/5 differentiations, SCN10A KO-iPSC-CM + PF n = 24 cells/4 differentiations).Single cells are presented as individual symbols and different iPSC-CM differentiations are color-coded.The small horizontal lines indicate the mean values per differentiation.Data were compared using nested one-way ANOVA with Sidak's test for multiple comparisons to calculate p values.

Figure 1 .
Figure 1.Na V 1.8 contributes to I NaL formation in ventricular iPSC-CM.(a) Original traces of I NaL in control iPSC-CM vs. SCN10A KO-iPSC-CM cells according to the inserted protocol.(b) Mean values per differentiation ± SEM of I NaL (control n = 31 cells/5 differentiations; control + PF n = 14 cells/4 differentiations; SCN10A KO iPSC-CM control n = 18 cells/5 differentiations, SCN10A KO-iPSC-CM +PF n = 24 cells/4 differentiations).Single cells are presented as individual symbols and different iPSC-CM differentiations are color-coded.The small horizontal lines indicate the mean values per differentiation.Data were compared using nested one-way ANOVA with Sidak's test for multiple comparisons to calculate p values.

Figure 2 .
Figure 2. Na V 1.8 inhibition or KO has no significant impact on action potential parameters of iPSC-CM.(a) Original traces of APD 90 in ventricular control iPSC-CM vs. SCN10A-KO iPSC-CM at 0.5 Hz.Mean data ± SEM of (b) APD 90 , (c) AP amplitude, (d) RMP and (e) upstroke velocity (Vmax) of control and SCN10A-KO iPSCM (control n = 18 cells/4 differentiations; control + PF-01247324 [PF] n = 12 cells/3 differentiations; SCN10A-KO control n = 12 cells/3 differentiations, SCN10A-KO + PF n = 16 cells/3 differentiations).Single cells are presented as individual symbols and different iPSC-CM differentiations are color-coded.The small horizontal lines indicate the mean values per differentiation.Data were compared using nested one-way ANOVA with Sidak's test for multiple comparisons to calculate p values.

Figure 3 .
Figure 3. Contribution of Na V 1.8 to spontaneous diastolic SR Ca 2+ release in ventricular iPSC-CM.(a) Representative surface plots showing spontaneous diastolic Ca 2+ sparks (green) in ventricular iPSC-CM.(b) Mean values of Ca 2+ spark frequency (CaSpF) normalized to scan width and duration.Numbers indicate the total cell count of control CM (control, n = 75 cells/4 differentiations), control cells treated with Na V 1.8 inhibitor PF-01247324 (control + PF-01247324 [PF], n = 85/4), and KO of Na V 1.8 after control treatment and treatment with PF-01247324 (n = 68/4 vs. 67/4).(c) Original representative line-scans of ventricular iPSC-CM illustrating a spontaneous proarrhythmogenic diastolic Ca 2+ wave (green).(d) Percentage of cells exhibiting diastolic Ca 2+ waves (control: 17% vs. control + PF-01247324: 7% of cells exhibiting Ca 2+ waves, p < 0.05; KO: 9% vs. KO + PF-01247324: 7%).Values are presented as mean ± SEM or absolute numbers.Single cells are presented as individual symbols and different iPSC-CM differentiations are color-coded.The small horizontal lines indicate the mean values per differentiation.Data were compared using nested one-way ANOVA with Sidak's test for multiple comparisons to calculate p values.Proportions were compared using Fisher's exact test.

Figure 4 .
Figure 4. NaV1.8 KO and inhibition reduce cellular arrhythmogenic triggers (a) Original traces of DADs and triggered activity in control iPSC-CM vs. SCN10A-KO iPSC-CM at 0.5 Hz.(b) Mean values ± SEM of DAD/min.(control n = 18 cells/4 differentiations; ventricular control + PF n = 11 cells/3 differentiations; SCN10A-KO control n = 10 cells/3 differentiations, SCN10A-KO + PF n = 16 cells/3 differentiations).Single cells are presented as individual symbols and different iPSC-CM differentiations are color-coded.The small horizontal lines indicate the mean values per differentiation.Data were compared using nested one-way ANOVA with Sidak's test for multiple comparisons to calculate p values.

Figure 4 .
Figure 4. Na V 1.8 KO and inhibition reduce cellular arrhythmogenic triggers (a) Original traces of DADs and triggered activity in control iPSC-CM vs. SCN10A-KO iPSC-CM at 0.5 Hz.(b) Mean values ± SEM of DAD/min.(control n = 18 cells/4 differentiations; ventricular control + PF n = 11 cells/3 differentiations; SCN10A-KO control n = 10 cells/3 differentiations, SCN10A-KO + PF n = 16 cells/3 differentiations).Single cells are presented as individual symbols and different iPSC-CM differentiations are color-coded.The small horizontal lines indicate the mean values per differentiation.Data were compared using nested one-way ANOVA with Sidak's test for multiple comparisons to calculate p values.