Mesenchymal Stem Cells-derived Exosomal miR-148a-3p Alleviates Atrial Fibrosis and Vulnerability to Atrial Fibrillation Through Targeting ALK5/Smad2 Axis

Aims: Atrial fibrosis is the hallmark of atrial fibrillation (AF). Accumulating evidence have shown that mesenchymal stem cells (MSCs) can alleviate fibrosis in diverse organs. However, the role of MSCs-derived exosome (MSCs-Exo) in the pathogenesis of atrial fibrosis and vulnerability to AF was unknown.Methods: Exosomes were isolated from cultured MSCs. Transmission electron microscopy and western blot were used to examine the purity of MSCs-Exo. 8-week-old BALB/c mice were randomized into sham group, angiotensin-II (Ang-II) group, and Ang-II+MSCs-Exo group. A mini-pump was implanted for subcutaneous infusion of Ang-II for 4 weeks to induce atrial fibrosis. In the Ang-II+MSCs-Exo group, 300μg/150μl MSCs-Exo were injected into tail veins twice a week after establishing the animal model. Echocardiography was used to evaluate heart structure and function. Intracardiac electric stimuli was used to analyze AF inducibility. Masson staining was used to evaluate the degree of atrial fibrosis. Ang-II-induced proliferation, migration, phenotypic switching and the capacity of extracellular matrix synthesis were examined after MSCs-Exo preconditioning. We reanalyzed two public expression datasets of MSCs-Exo and selected a few most highly expressed microRNAs. After literature search and experimental validation, we narrowed down our candidates to miR-148a-3p. Then, we predicted the potential target genes of miR-148a-3p, and determined ALK5 and SMAD2 as the targets of miR-148a-3p. Western blot was used to quantify Ang-II-induced protein expressions of TGF-β1/ALK5/SMAD2 pathway in fibroblasts after exposure to miR-148a-3p-rich MSCs-Exo.Results: Compared with mice in Ang-II group, mice in Ang-II+MSCs-Exo group showed less atrial enlargement, atrial fibrosis, a lower AF inducibility and AF duration. Ang-II-induced proliferation, migration, phenotypic switching, and capacity of extracellular matrix synthesis were ameliorated in fibroblasts after co-incubation with MSCs-Exo. After exposure to miR-148a-3p inhibitor, MSCs derived Exosomes (MSCs-148a-3p inhibitor-Exo) can significantly reverse above phenomenon. As the targets of miR-148a-3p, the expressions of ALK5 and Smad2 were lower in Ang-II+MSCs-Exo group compared with the Ang-II group. Furthermore, MSCs-Exo treatment inhibited Ang-II-induced activation of TGF-β1/ALK5/Smad2 pathway, and MSCs-148a-3p inhibitor -Exo reverse above phenomenon.Conclusions: MSCs-Exo can alleviate Ang-II induced atrial fibrosis and AF vulnerability through transferring miRNA-148a-3p to inhibit the activation of TGF-β1/ALK5/Smad2 pathway, providing a new avenue to AF treatment.