Identification of Connexin 26 on Extracellular Vesicles from Human Cardiomyocytes and Plasma: Novel Insights into miRNA Loading and Oxidative Injury

Connexin 26 (Cx26), a gap junction protein, is poorly understood in the context of cardiac milieu, including extracellular vesicles (EVs). Here, we report for the first time the presence of Cx26 on EVs obtained from human induced pluripotent stem cell-derived cardiomyo-cytes and human plasma. Using an in vitro model of oxidative stress (OS) and apoptosis in dH9c2 cardiomyocytes, modeling some key molecular and cellular aspects of ischemic heart disease (IHD) and heart failure (HF), we observed a significant decrease in Cx26 lev-els in EVs released by injured cells, accompanied by changes in EV concentration, partic-ularly in exosomes. Our findings revealed that Cx26 modulates the selective loading of specific microRNAs, namely miR-1 and miR-30a, into EVs, suggesting a novel non-canonical, gap junction-independent role of Cx26 in EV-mediated cardiac signaling. Analysis of plasma EVs from healthy donors confirmed the presence of Cx26-positive EVs of cardiomyocyte origin, indicated by co-staining with cardiac troponin T. These results highlight circulating EV-associated Cx26 as a potential biomarker of cardiomyocyte damage in cardiomyopathies characterized by oxidative stress and apoptosis, such as IHD and HF.