Human atrium-sized in vitro modelling of atrial fibrillation: a new experimental scale for preclinical therapeutic testing

Abstract Background Atrial size strongly influences atrial fibrillation (AF) development, maintenance, and therapeutic outcome, including pharmacological cardioversion. Despite this, preclinical research relies heavily on models considerably smaller than the human atrium, such as cell-based and small-animal models. Alternatively, large-animal models—though closer in size—are costly, ethically challenging, and limited by species-specific differences. Consequently, there is a need for real-size models of human AF that enable therapeutic testing at a patient-relevant scale, encompassing intermediate and enlarged atrial sizes. Purpose To develop a full human atrium–sized model of AF, establishing a new experimental scale for preclinical therapeutic testing regarding improved pharmacological cardioversion. Methods Monolayers of conditionally immortalized human atrial myocytes (hiAM) were created in three sizes: standard in vitro size (9.5 cm²), young adult human atrium-sized (55 cm²) and enlarged human atrium-sized (152 cm²). Hereafter, these model sizes are referred to as small, intermediate and enlarged, respectively. To visualize electrical activity of these monolayers and characterize their electrophysiological features, we performed optical voltage mapping. For arrhythmia studies, re-entrant circuits were induced by tachypacing. Arrhythmia complexity was quantified by the number of concurring circuits, which cores were tracked to assess their trajectories. Subsequent pharmacological testing during arrhythmias included flecainide, and flecainide + ibutilide. Results Upon 1 Hz electrical pacing all monolayers showed homogeneous wave propagation and similar wavelength between sizes. Tachypacing-induced re-entrant circuits showed a clear size-dependent increase in arrhythmia complexity and circuit movement, while dominant frequency declined with increasing size (Figure 1). Subsequent therapeutic testing revealed size-dependent drug effects. Vehicle infusion (0.1% DMSO) had no effect on dominant frequency and arrhythmia complexity, and arrhythmias persisted in all models except for one termination in the small model. Yet, Flecainide (3 and 10 µM) reduced dominant frequency and arrhythmia complexity across all models. However, termination efficacy decreased with increasing size, with arrhythmia termination in the enlarged model occurring only at the higher dose. In contrast, combined treatment with 3 µM flecainide and 20 nM ibutilide enhanced re-entry termination in human-atrium sized models. Notably, this combination did terminate arrhythmias in the enlarged model—an effect not observed with 3 µM Flecainide alone—demonstrating synergistic antiarrhythmic effects. Conclusion Using human atrium-sized in vitro modelling of AF we demonstrate that size matters in the study of both arrhythmia dynamics and pharmacological responses, enabling identification of synergistic drugs as an improved pharmacological cardioversion at an enlarged atrial size.Figure 1For image description, please refer to the figure legend and surrounding text.