FOXO3a Induces Myocardial Fibrosis by Upregulating Mitophagy

Abstract

Background: Atrial fibrillation is one of the most common cardiac arrhythmias. Myocardial fibrosis is closely associated with atrial remodeling, which leads to heightened risk of atrial fibrillation. This study aimed to explore whether forkhead box protein O3 (FOXO3a) impacts myocardial fibrosis incidence by regulating mitophagy.

Methods: Cell viability was assessed by cell counting kit-8 (CCK-8) assays. The expression of vimentin and cytochrome C was detected by immunofluorescence assays. Quantitative real-time polymerase chain reaction (PCR) was used to analyze the relative mRNA level of FOXO3a. Expression of FOXO3a, phosphorylated FOXO3a, Collagen I, Collagen III, alpha-smooth muscle actin (α-SMA), matrix metalloprotease 9 (MMP9), light chain 3 (LC3), phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1), Parkin, and sequestosome-1 (p62) proteins were determined by western blotting. 5-ethynyl-2'-deoxyuridine (EDU) incorporation was employed to measure cell proliferation. Changes in mitochondrial membrane potential were determined by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) staining. A wound healing assay was used to examine cell migration, and the levels of reactive oxygen species were determined by flow cytometry.

Results: The expression of FOXO3a was upregulated in cardiac fibroblasts treated with angiotensin II (AngII), while the expression of phosphorylated FOXO3a was downregulated under these conditions. FOXO3a knockdown significantly inhibited the proliferation, migration and collagen secretion of cardiac fibroblasts treated with AngII. The ratio of LC3 II/I as well as expression of PINK1 and Parkin was increased, and the expression of p62 was decreased, in cardiac fibroblasts treated with AngII. Moreover, these effects were limited by FOXO3a knockdown. Finally, the mitophagy inducer everolimus (RAD001) attenuated the suppressive effect of FOXO3a knockdown on cardiac fibroblast activation.

Conclusions: FOXO3a promotes the progress of myocardial fibrosis by triggering mitophagy in cardiac fibroblasts.

Keywords: FOXO3a; cardiac fibroblasts; mitophagy; myocardial fibrosis.