Introduction: In heart failure (HF) and atrial fibrillation, persistent Na⁺ current (I_NaL) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. We have recently shown that the Na⁺ channel Na_v1.8 contributes to arrhythmogenesis by inducing I_NaL (Bengel et al., Nat Comm 2021). In addition, GWAS studies indicate that mutations in the SCN10A gene (Na_v1.8) are associated with increased risk for arrhythmias, Brugada syndrome, and sudden death. However, it is still controversially discussed whether these Na_v1.8-related effects are mediated by cardiac ganglia or cardiomyocytes. Therefore, we aimed to study the electrophysiological contribution of Na_v1.8 in proof of principle experiments by establishing a human CRISPR-Cas9-generated SCN10A-knock-out (KO) atrial pluripotent stem cell CM (iPS-CM).

Methods and results: We used CRISPR/Cas9 technology to generate homozygous SCN10A-KO-iPS lines. Two SCN10A KO clones were shown to maintain full pluripotency, and spontaneous in vitro differentiation capacity. Both SCN10A KO lines were directly differentiated into 2-month-old functional atrial iPSC-CM and downregulation of Na_v1.8 was confirmed by Western blot.

Voltage-clamp experiments were performed to analyze the impact of Na_v1.8 KO on I_NaL and showed that I_NaL is significantly reduced in Na_v1.8 KO iPSC-CM compared to control. Moreover, the specific Na_v1.8 inhibitor (PF-01247324, 1 µmol/l)  demonstrated a significant reduction of the I_NaL in atrial control iPSC-CM; (n=20-31 cells/4-5 differentiations). Importantly, we observed no effects of PF-01247324 on I_NaL in SCN10A KO iPSC-CM generally (n=14-26 cells/3-4 differentiations) thereby also either indicating the specificity of the drug. To assess the potential influence of SCN10A KO and pharmacological Na_v1.8 inhibition on action potential parameters (APD) in atrial iPSC-CM we performed whole-cell current-clamp experiments. Action potential duration, resting membrane potential, action potential amplitude and upstroke velocity were not altered in SCN10A KO iPSC-CM compared to control nor after application of the specific Na_v1.8 blocker (n=18-22 cells/4-5 differentiations). Furthermore, Ca²⁺ measurements (Fluo 4-AM) were performed to analyze the consequence of SCN10A KO on the proarrhythmogenic diastolic SR Ca²⁺ leak. We observed a significant decrease in SR Ca²⁺ spark frequency (CaSpF) in atrial SCN10A-KO-CM compared to WT iPSC-CM CM (n= 65-75 cells/3-5 differentiations. Application of Na_v1.8 specific blocker showed a significantly suppressed SR Ca²⁺ leak in control cardiomyocytes.

Conclusion: In conclusion, we were able to generate homozygous SCN10A-KO-iPSCs using CRISPR/Cas9 technology and confirmed downregulation of the neuronal Na⁺ channel Na_v1.8 in human atrial cardiomyocytes. Our data clearly demonstrate that Na_v1.8 is responsible for generation of the proarrhythmic I_NaL in human atrial cardiomyocytes. Moreover, inhibition of Na_V1.8 modulates well-accepted proarrhythmogenic triggers such as I_NaL and diastolic SR-Ca²⁺ leak. Therefore, targeting Na_v1.8 in atrial cardiomyocytes may be a novel antiarrhythmic treatment option, which merits further investigation.