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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.

Rationale: Atrial fibrillation (AF) and heart failure often coexist, but their interaction is poorly understood. Clinical data indicate that the arrhythmic component of AF may contribute to left ventricular (LV) dysfunction. Objective: This study investigates the effects and molecular mechanisms of AF on the human LV. Methods and Results: Ventricular myocardium from patients with aortic stenosis and preserved LV function with sinus rhythm or rate-controlled AF was studied. LV myocardium from patients with sinus rhythm and patients with AF showed no differences in fibrosis. In functional studies, systolic Ca 2+ transient amplitude of LV cardiomyocytes was reduced in patients with AF, while diastolic Ca 2+ levels and Ca 2+ transient kinetics were not statistically different. These results were confirmed in LV cardiomyocytes from nonfailing donors with sinus rhythm or AF. Moreover, normofrequent AF was simulated in vitro using arrhythmic or rhythmic pacing (both at 60 bpm). After 24 hours of AF-simulation, human LV cardiomyocytes from nonfailing donors showed an impaired Ca 2+ transient amplitude. For a standardized investigation of AF-simulation, human iPSC-cardiomyocytes were tested. Seven days of AF-simulation caused reduced systolic Ca 2+ transient amplitude and sarcoplasmic reticulum Ca 2+ load likely because of an increased diastolic sarcoplasmic reticulum Ca 2+ leak. Moreover, cytosolic Na + concentration was elevated and action potential duration was prolonged after AF-simulation. We detected an increased late Na + current as a potential trigger for the detrimentally altered Ca 2+ /Na + -interplay. Mechanistically, reactive oxygen species were higher in the LV of patients with AF. CaMKII (Ca 2+ /calmodulin-dependent protein kinase IIδc) was found to be more oxidized at Met281/282 in the LV of patients with AF leading to an increased CaMKII activity and consequent increased RyR2 phosphorylation. CaMKII inhibition and ROS scavenging ameliorated impaired systolic Ca 2+ handling after AF-simulation. Conclusions: AF causes distinct functional and molecular remodeling of the human LV. This translational study provides the first mechanistic characterization and the potential negative impact of AF in the absence of tachycardia on the human ventricle.

Introduction: The effects of atrial fibrillation (AF) on left ventricular (LV) function are poorly understood. Clinical data indicate that the arrhythmic component of AF may contribute to LV dysfunction. This study investigated the effects of AF on the human LV. Methods and Results: LV myocardium from patients with preserved LV function (EF>50%) with sinus rhythm (SR, n=31) or rate-controlled AF (n=24) obtained during surgery for aortic stenosis was studied. Clinical data were mostly balanced without changes in LV function and remodeling. LV myocardium from SR and AF patients showed no changes in fibrosis. In functional studies, systolic Ca 2+ transient amplitude of human LV cardiomyocytes (CM) was reduced in AF patients, while diastolic Ca 2+ levels and Ca 2+ transient kinetics were unaltered. These results were confirmed in LV CM from non-failing donors with AF (n=4) vs. SR (n=8). In addition, normofrequent AF was simulated in vitro using arrhythmic or rhythmic electrical culture pacing (both at 60 bpm). After 24h of AF-simulation, human LV CM from non-failing donors (n=9) showed also an impaired Ca 2+ transient amplitude. For a standardized chronic AF-simulation, human iPSC-CM (n=22 differentiations/5 individuals) were tested. 7 days of AF-simulation caused reduced systolic Ca 2+ transient amplitude and sarcoplasmic reticulum Ca 2+ load, which could be explained by an increased diastolic Ca 2+ leak. Mechanistically, oxidative stress markers were higher in the LV of AF patients. Ca 2+ /calmodulin-dependent protein kinase IIδ (CaMKII) was found to be more oxidized at Met281/282 in the LV of AF patients leading to an increased CaMKII activity. Thus, RyR2 phosphorylation was elevated, which likely underlies the depression of LV Ca 2+ handling in AF. In translational contractility experiments of human ventricular trabecula from patients with heart failure (n=8), in vitro AF-simulation for 8h worsened systolic and diastolic contractile function. Conclusions: Here we could firstly show that AF impairs human LV function via increased oxidative stress and CaMKII activity in different patient populations and models. Thus, this translational study demonstrates the detrimental effects and mechanisms of AF in the absence of tachycardia on the human LV.