miR-20a-5p inhibition attenuates post-myocardial infarction ventricular remodeling in association with mTORC1/S6 signaling

Abstract Maladaptive ventricular remodeling is the major pathological process linking myocardial infarction (MI) to heart failure, yet its upstream molecular drivers remain incompletely defined. Extracellular vesicle (EV)-enriched microRNAs (miRNAs) are emerging regulators of intercellular communication and cardiac pathology. This study investigated the association of miR-20a-5p with post-MI remodeling and mTORC1/S6-related signaling. MI was induced in male Sprague–Dawley rats by permanent ligation of the left anterior descending coronary artery. EV-enriched fractions were isolated from conditioned media of adult heart-derived adherent cultures established from Sham and post-MI hearts, and miR-20a-5p abundance was quantified. Neonatal cardiomyocytes and cardiac fibroblasts were used for labeled-particle exposure and functional assays. In vitro gain- and loss-of-function experiments used a miR-20a-5p mimic or inhibitor, with rapamycin used for pathway inhibition. The in vivo intervention study comprised five groups and did not include a miR-20a-5p mimic arm. Molecular responses were assessed by RT-qPCR, Western blotting, flow cytometry, and a dual-luciferase reporter assay; cardiac function and remodeling were evaluated by echocardiography and histology. miR-20a-5p abundance was higher in EV-enriched fractions from cultures established from post-MI hearts than in corresponding fractions from cultures established from Sham hearts, and cell-associated DiI fluorescence was observed after exposure of neonatal cardiomyocytes and cardiac fibroblasts to labeled preparations. In cardiac cells, miR-20a-5p overexpression increased apoptosis-related and fibroblast-activation markers and was accompanied by increased p-S6, whereas miR-20a-5p inhibition or rapamycin produced changes in the opposite direction. Separately, a dual-luciferase assay in HEK-293T cells showed that miR-20a-5p reduced reporter activity of the wild-type, but not the mutant, Ddit4 3′UTR construct. In vivo, the combined intervention group showed the largest observed changes among the tested groups, including reduced fibrosis and improved left ventricular functional parameters; a formal interaction analysis was not performed. The findings support associations among miR-20a-5p, interaction with the Ddit4 3′UTR, mTORC1/S6-related signaling, and adverse remodeling after MI. EV-specific miR-20a-5p manipulation, DDIT4 rescue, autophagic-flux assessment, and in vivo gain-of-function studies are required before a linear causal pathway or therapeutic strategy can be established.