Although cell-based therapies show potential antiarrhythmic effects that could be mediated by their paracrine action, the mechanisms and the extent of these effects were not deeply explored. We investigated the antiarrhythmic mechanisms of extracellular vesicles secreted by cardiosphere-derived cell extracellular vesicles (CDC-EVs) on the electrophysiological properties and gene expression profile of HL1 cardiomyocytes. HL-1 cultures were primed with CDC-EVs or serum-free medium alone for 48 h, followed by optical mapping and gene expression analysis. In optical mapping recordings, CDC-EVs reduced the activation complexity of the cardiomyocytes by 40%, increased rotor meandering, and reduced rotor curvature, as well as induced an 80% increase in conduction velocity. HL-1 cells primed with CDC-EVs presented higher expression of SCN5A, CACNA1C, and GJA1, coding for proteins involved in INa, ICaL, and Cx43, respectively. Our results suggest that CDC-EVs reduce activation complexity by increasing conduction velocity and modifying rotor dynamics, which could be driven by an increase in expression of SCN5A and CACNA1C genes, respectively. Our results provide new insights into the antiarrhythmic mechanisms of cell therapies, which should be further validated using other models.

Background Stem cells and their secreted extracellular vesicles (EVs) have shown different cardioprotective effects. However, their impact on the electrophysiological properties of the heart tissue remains controversial. While the use of some progenitor cells seems to have antiarrhythmic potential, the use of cardiomyocyte-like cells may be proarrhythmic. The mechanisms behind, and whether these effects are linked to cell engraftment and not to their secreted products is not fully known. Purpose The aim of this study was to investigate the electrophysiological modifications induced by extracellular vesicles secreted by human cardiosphere-derived cells (CDC-EVs) in an in vitro model of atrial fibrillation in order to explore their potential antiarrhythmic effect. Methods CDCs were derived from cardiac biopsies of patients who underwent cardiac surgery for other reasons. Purified CDC-EVs resuspended in serum-free media (SFM) vs. SFM alone were added to HL-1 atrial myocyte monolayers presenting spontaneous fibrillatory activity. After 48 hours, the monolayers were fully confluent, and the electrophysiological properties were analysed through optical mapping in both the treated (n=9) and control plates (n=9). Optical mapping recordings of the monolayers were analysed with Matlab for the activation frequency, activation complexity, rotor dynamics (curvature and meandering) and conduction velocity. Results CDC-EVs reduced activation complexity of the fibrillating atrial monolayers by ∼40% (2.74±0.59 vs. 1.61±0.16 PS/cm2, p<0.01). This reduction in activation complexity was accompanied by larger rotor meandering (1.47±0.82 vs. 4.32±2.25 cm/s, p<0.01) and decreased curvature (1.79±0.40 vs. 0.87±0.24 rad/cm, p<0.01) in the treated group. Despite reduction in the activation complexity, activation frequency did not change significantly between both groups. This could be in part because CDC-EVs increased conduction velocity by 80% (1.32±0.57 vs. 2.65±0.87 cm/s, p<0.01). Low conduction velocity has been linked to higher reentry recurrence, and lower meandering and higher curvature to higher rotor stability and harder AF termination. Therefore, CDC-EVs seem to drive cardiomyocytes to a less arrhythmic profile reducing activation complexity and preventing remodelling by increasing conduction velocity and modifying rotor dynamics. Conclusions CDC-EVs significantly modify conduction velocity and rotor dynamics, therefore reducing fibrillation complexity and remodelling to drive atrial myocytes to a less arrhythmogenic profile. Testing CDC-EVs in more robust models of atrial fibrillation, the most common sustained arrhythmia in humans with significant morbidity and mortality, is of special interest. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III, Ministerio de Ciencia e Innovaciόn,CIBERCV, Spain Figure 1