Background: Atrial fibrillation (AF) is one of the most important cardiac arrythmias. It is often associated with left ventricular systolic dysfunction (LVSD), which can be an underlying mechanism but also the result of AF. Although AF is the subject of intensive research, the cellular mechanisms leading to LVSD remain poorly understood. In particular, the effects of AF on ventricular cardiomyocytes warrant investigation. Recently, the CASTLE-AF trial could demonstrate that patients with AF and LVSD had reduced mortality, better QoL and reverse remodeling when treated with catheter ablation compared to medical therapy (N Engl J Med. 2018;378:417-427). In addition, further analysis of the patients included in CASTLE-AF showed an improved outcome when AF burden was reduced to <50% at 6 months after catheter ablation (JACC Clin Electrophysiol. accepted). Therefore, we aimed to explain these observations in term of AF burden in a back to bench approach using electrical cardiomyocyte stimulation with different AF burden.

Methods: To study the effects of different AF burden, experiments were performed using field stimulated, cultured induced pluripotent stem cell cardiomyocytes (iPSC-CM). iPSC-CM were divided into four groups and stimulated for 7 days with a frequency of 90 bpm.  Group 1 had an arrhythmia burden of 0%, group 2 of 30%, group 3 of 60% and group 4 of 90%. The beat-to-beat variability in groups with irregular stimulation was 40%. Epifluorescence microscopy for measurements of Ca²⁺ transients was performed using the ratiometric Ca²⁺ dye Fura-2 AM. Action potential measurements were conducted using ruptured-patch whole-cell current clamp technique. Experiments were conducted with stimulation frequencies of 30 bpm, 60 bpm and 120 bpm during measurements.

Results: Action potential duration (APD) prolongation is a known hallmark of heart failure. After 7 days, APD90 at 30 bpm increased significantly between Burden 0% and Burden 60/90% and between Burden 30% compared to Burden 60/90%. Furthermore, there was a significant APD90 increase at 60 bpm between Burden 30% and Burden 60% Apart from APD prolongation, there were also changes in cellular Ca2+ homeostasis. In line with the results from action potential measurements, a significant reduction of amplitudes of systolic Ca2+ transients was observed at 30 bpm between Burden 0/30% and Burden 60%. At 60 bpm, Ca2+ transients decreased significantly between Burden 0% and Burden 60% as well as Burden 30% and Burden 60%/90% (figure 1). Thus, we could indeed detect a threshold of AF burden of ~60% where cellular function starts to be impaired which clearly relates to the findings of the detailed analysis of CASTLE-AF.
 

Figure 1: APD90 was increased and Ca²⁺ transients were decreased at stimulation frequencies of 30 bpm and 60 bpm after 7 days (APD90: B0 n=19, B30 n=22, B60 n=25, B90 n=21, CaTransAmpl: B0 n=35, B30 n=32, B60 n=50, B90 n=34, data are presented with SEM, 2way ANOVA were performed, *p<0.05, ***p<0,001).

Conclusion: In this study, we could demonstrate that varied outcome in patients with different AF burden translates to changes at the cellular level. Significant prolongations of APD90 and reductions of Ca2+ transients could be detected at a threshold of 60% of AF-simulated cardiomyocyte stimulation as it was observed to be harmful in the CASTLE-AF trial. Therefore, these changes may help to understand the findings of the CASTLE-AF trial