Fibroblast-derived exosomal miRNA-133 promotes cardiomyocyte-like differentiation

Abstract

Objective: To investigate the role of exosomal miRNA-133 secreted by cardiac fibroblasts (CFs) in promoting cardiomyocyte differentiation.

Methods: Neonatal rat CFs were cultured in vitro, and the cultured CFs were divided into three groups as follows: induction, miRNA-133 high expression, and miRNA-133 inhibition. miRNA-133 was transfected into CFs with lentivirus as a vector. CFs were transfected with the miRNA-133 inhibitor, and the markers of cardiomyocyte were detected through immunofluorescence staining, Western blotting, and real-time quantitative polymerase chain reaction (qRT-PCR) at 3, 8, and 14 days, respectively. The expression levels of cardiac troponin T (cTnT) and cardiac actin (α-actin) were determined, and qRT-PCR was used to detect the expression of miRNA-133 in the fibroblast exosomes.

Results: CFs subjected to immunofluorescence staining expressed vimentin and discoid domain receptor 2. The exosomes secreted by CFs were observed as small vesicles of 30-100 nm via transmission electron microscopy, and Western blotting was used to detect exosome-specific protein CD63 and CD9 expression. The expression levels of cTnT, α-actin, and exosomal miRNA-133 secreted into the supernatant of the miRNA-133 high-expression group increased gradually at different time points and reached the highest level at 14 days. The expression levels of cTnT, α-actin, and exosome miRNA-133 in the miRNA-133 inhibition group were the lowest.

Conclusion: The exosomal miRNA-133, which is derived from CFs, can promote the differentiation of fibroblasts into cardiomyocyte-like cells.

Keywords: Cardiac fibroblasts; Cardiomyocyte-like cells; Exosomes; cTnT; miRNA-133; α-actin.