Myofibroblast-Derived Exosomes Contribute to Development of a Susceptible Substrate for Atrial Fibrillation.

Objective: Atrial fibrosis plays a critical role in atrial fibrillation (AF). A key event in the pathogenesis of fibrosis is the activation of fibroblasts (FBs) into myofibroblasts (MFBs). Paracrine factors released from MFBs lead to ion channel expression changes in cardiomyocytes (CMs). Downregulation of L-type calcium channel Ca(v)1.2 expression is a hallmark of AF-associated ionic remodeling. However, whether exosome (Exo)-mediated crosstalk between MFBs and CMs regulates Ca(v)1.2 expression remains unknown. Methods: Atrial FBs and CMs were isolated and cultured from neonatal rats by enzymatic digestion. The activation of FBs into MFBs was induced by angiotensin II. Co-culture assay and in vitro Exo treatment were used to determine the effect of MFB-derived Exos on Ca(v)1.2 expression. Confocal Ca2+ imaging was performed to examine the adrenergic stimulation-elicited Ca2+ influx signals. The levels of potential Ca(v)1.2-inhibitory microRNAs (miRNAs) were measured by qRT-PCR. Results: Untreated FBs expressed limited amounts of alpha smooth muscle actin (alpha-SMA), while angiotensin II induced a significant upregulation of alpha-SMA-expressing MFBs. Co-cultures of MFBs and CMs resulted in downregulation of Ca(v)1.2 expression in CMs, which was largely abolished by pretreatment of MFBs with exosomal inhibitor GW4869. More importantly, treatment with MFB-derived Exos caused repression of Ca(v)1.2 expression in CMs. Additionally, the adrenergic receptor agonist-elicited Ca2+ influx signals in CMs were remarkably attenuated by pretreatment with MFB-derived Exos, corresponding to the paralleled change in Ca(v)1.2 expression. Finally, miR-21-3p, a potential Ca(v)1.2-inhibitory miRNA, was enriched in MFB-derived Exos and upregulated in CMs in response to MFB-derived Exos. Conclusion: We uncover an Exo-mediated crosstalk between MFBs and CMs, contributing to increased vulnerability to AF by reducing the expression of Ca(v)1.2 in CMs.