DOI DOI https://doi.org/10.1007/s00392-021-01843-w
Introduction: In heart failure (HF), enhanced persistent Na+ current (late INa) exerts detrimental effects on cellular electrophysiology and can induce arrhythmias. We have shown that the Na+ channel Nav1.8 contributes to arrhythmogenesis by inducing the late INa. In addition, GWAS studies indicate that mutations in the SCN10A gene (Nav1.8) are associated with increased risk for arrhythmias, Brugada syndrome, and sudden death. However, it is still controversially discussed whether these Nav1.8-related effects are mediated by cardiac ganglia or cardiomyocytes. Therefore, we aimed to study the electrophysiological contribution of Nav1.8 in proof of principle experiments by using human CRISPR-Cas9-generated SCN10A-knock-out (KO) atrial und ventricular pluripotent stem cell CM (iPS-CM).
Methods and results: CRISPR/Cas9 technology was applied 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 and ventricular CM and downregulation of Nav1.8 was confirmed in both cardiac subtypes by Western blot.
To analyze the impact of Nav1.8 KO on late INa, whole-cell patch clamp was performed and showed a significant reduction of isoproterenol-(Iso)-induced late INa in atrial Nav1.8-KO-cardiomyocytes compared to control-cardiomyocytes (WT+Iso n=28 celles/4 differentiations vs KO+Iso 26/4, p<0.0001). In ventricular Nav1.8-KO-cardiomyocytes we also showed a significant reduction of Iso-induced late INa compared to control-cardiomyocytes (WT+Iso n=18/4 vs KO+Iso: n= 16/4, p<0.0001). Nav1.8 specific blocker PF-01247324 (1 nmol/L) revealed a significant abbreviation in atrial and ventricular (all 14-28/4, p<0.0001) Nav1.8-KO-cardiomyocytes compared to control-cardiomyocytes.
In atrial Nav1.8-KO-cardiomyocytes action potential duration, resting membrane potential, action potential amplitude and upstroke velocity were not altered compared to control. In this line in ventricular Nav1.8-KO-cardiomyocytes, no effect on any action potential parameters could be observed compared to control (n=19/4 control vs 16/3 KO).
Furthermore, Ca2+ measurements (Fluo4 AM) were performed to analyze the consequence of SCN10A KO on the proarrhythmogenic diastolic SR Ca2+ leak. We observed a significant decrease in Ca2+ spark frequency (CaSpF) in atrial and ventricular SCN10A-KO-cardiomyocytes (all 65-75/3-5 & p<0.0001). Application of Nav1.8 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 Nav1.8 in atrial and ventricular cardiomyocytes. Our data clearly demonstrate that Nav1.8 is responsible for generation of late INa in human cardiomyocytes. Moreover, inhibition of NaV1.8 modulates well-accepted proarrhythmogenic triggers such as late INa and diastolic SR-Ca2+ leak. Therefore, targeting Nav1.8 in cardiomyocytes may be a novel antiarrhythmic treatment option, which merits further investigation.https://dgk.org/kongress_programme/jt2021/aP1189.html