miR-4324-containing extracellular vesicles released in pericardial drainage fluid facilitate postoperative atrial fibrillation

Aims Pericardial drainage fluid (PCF) offers a non-invasive and direct window to detect surgery-induced microenvironmental changes implicated in postoperative atrial fibrillation (POAF) onset. We hypothesized that cardiac surgery elicits release of extracellular vesicles (EVs) in PCF, serving as a conduit transmitting proarrhythmic and profibrotic miRNAs and contributing to atrial remodeling and POAF. Methods and Results We investigated the role of PCF-EVs derived miRNAs in POAF in prospective, multicenter discovery and validation cohorts and spontaneous POAF (sPOAF) mouse model. PCF-EVs from POAF patients elicited marked atrial collagen deposition and a higher incidence of sPOAF in mice compared to non-POAF controls. Strikingly, EV blockage in sPOAF mouse model provided complete sPOAF protection (50% vs 0). A distinct profibrotic miRNA signature was identified in PCF-EVs from POAF patients. Among these, miR-4324, a previously unreported murine miRNA, emerged as a potent inducer of AF, triggered sPOAF in 83% of treated mice, and demonstrating excellent predictive accuracy (AUC=0.921) in a clinical validation cohort. Visualization of miR-4324 trajectory using miRNAscope demonstrated that miR-4324-containing EVs, originating from the surgically-damaged left ventricle, rapidly transported to thin-walled atria via PCF, where miR-4324 activated TGF-β1/Smad3 signaling by targeting SKP1. This facilitated collagen deposition and a proarrhythmic atrial substrate. In contrast, miR-4324 inhibition blocked these effects. Conclusion These findings uncover a previously unrecognized high-speed PCF-EVs pathway that propagates proarrhythmic miR-4324 signals from surgical sites to reshape the atrial microenvironment into a proarrhythmic milieu via SKP1-TGF-β1/Smad3 axis. PCF, typically discarded, could be repurposed for POAF prophylaxis.