Abstract:Aim To investigate the role of microRNA-101-3p (miR-101-3p) in a mouse model of heart failure after left anterior descending branch (LAD) coronary artery ligation-induced myocardial infarction (MI), and to analyze its association with Ras-related C3 botulinum toxin substrate 1/p21 activated kinase 1 (RAC1/PAK1) signaling, endothelial-to-mesenchymal transition (EndMT), and myocardial fibrosis. Methods Heart failure after MI was induced by LAD ligation in mice, which were then randomly assigned to the sham, MI model, miR-101-3p agomir (miR-101-3p-Ago) treatment, RAC1 inhibitor (NSC23766) treatment, combined treatment, and metoprolol positive-control groups. Four weeks after surgery, transthoracic echocardiography was performed to assess left ventricular ejection fraction (LVEF), fractional shortening (FS), left ventricular end-diastolic/systolic diameter (LVEDD/LVESD), and wall thickness. Hematoxylin-eosin, Masson and wheat germ agglutinin (WGA) staining were used to evaluate myocardial structural remodeling and fibrosis. Immunofluorescence co-staining for VE-cadherin/α-smooth muscle actin (α-SMA) and CD31/vimentin was performed to assess EndMT-related phenotypes. Western blot was used to detect the expression of RAC1, PAK1 and EndMT-related proteins. Transmission electron microscopy (TEM) was employed to observe myocardial ultrastructural changes in cardiomyocytes. Results Compared with the Sham group, cardiac function (LVEF, FS) in the model group was significantly decreased, and ventricular remodeling and myocardial fibrosis were aggravated (all P＜0.05). Meanwhile, the RAC1/PAK1 pathway was activated in myocardial tissue, accompanied by significantly enhanced EndMT-related phenotypes (all P＜0.01). Treatment with either miR-101-3p-Ago or the RAC1 inhibitor alone significantly improved cardiac function, suppressed RAC1/PAK1 activation, and attenuated EndMT-related abnormalities (P＜0.05). The combined intervention exerted the strongest protective effect, reducing the myocardial fibrotic area by more than 60% and significantly restoring functional indices such as LVEF (P＜0.01). Moreover, its anti-fibrotic and cardioprotective effects on key parameters were significantly superior to those of either monotherapy (P＜0.05), indicating a clear synergistic benefit. TEM findings further suggested that the combined intervention partially ameliorated myocardial ultrastructural injury. Conclusion miR-101-3p agomir improves myocardial fibrosis and cardiac function in mice after myocardial infarction by inhibiting RAC1/PAK1mediated EndMT, suggesting that it may serve as a potential molecular target for anti-fibrotic therapy after myocardial infarction.
