Enhancing heart regeneration and repair after myocardial infarction through inhibition of a specific MicroRNA by targeting the IFNy-JAK-STAT pathway

Following myocardial infarction (MI), endothelial cells and fibroblasts are essential for tissue repair and regeneration. Modulating their function may enhance healing and cardiac recovery. Given the limited effective therapies, microRNAs have emerged as promising therapeutic targets. This project investigates the role of microRNA-X (miR-X) in regulating endothelial and fibroblast function, focusing on angiogenesis, to evaluate its therapeutic potential for improving cardiac repair after MI in vivo. MiR-X expression was analyzed in vivo 24 and 72 hours after ultrasound-guided MI in C57BL/6J mice using qRT-PCR. Expression was further assessed in human endothelial cells (ECs) and human cardiac fibroblasts (hcF). After anti-miR-X transfection, cellular functions including migration, proliferation, angiogenesis, and cell death were examined. Immunofluorescence microscopy was performed. Potential downstream targets were identified by RNA sequencing and validated by qRT-PCR and western blot. Therapeutic effects were evaluated in C57BL/6 mice after MI and in a hind limb ischemia model. MiR-X was significantly upregulated 24 hours after MI in mice (p<0.05), with no change at 72 hours. Similarly, hypoxia induced a significant increase in miR-X expression in human ECs (p<0.05) and hcF at 24 hours, but not at 72 hours. Functional analyses showed no effect of miR-X on EC proliferation. However, its inhibition significantly enhanced EC migration after 6 hours (p<0.0001), reduced apoptosis (p<0.05), and improved the angiogenic capacity (p<0.05), without morphological alterations. HcF were activated in vitro by TGFβ-1 to myofibroblasts and by IL-1β to inflammatory fibroblasts. MiR-X downregulation reduced proliferation and migration in both phenotypes, while cell death was unaffected. RNA sequencing revealed significant downregulation of CMPK2 (p<0.0001) and XAF1 (p<0.05) in ECs after anti-miR transfection, confirmed by qRT-PCR. Western blot showed reduced CMPK2 and XAF1 protein levels (p<0.05). Upstream pathway analysis demonstrated significant downregulation of IFNGR1 and STAT1 proteins (p<0.05), without changes at the RNA level. Similar genes were regulated in hcF. In vivo, anti-miR-X treatment after MI significantly improved ejection fraction (EF) (p<0.05) in mice. Fractional shortening (FS) was also increased but did not reach statistical significance. After hind limb ischemia, treated mice showed significantly improved blood perfusion of the affected limb compared to controls (p<0.05). MiR-X represents a potential therapeutic target to enhance recovery after MI. Its inhibition modulates hcF function and improves EC function, promoting angiogenesis via the IFNγ–JAK–STAT pathway. In vivo data confirm pro-angiogenic effects and improved cardiac function after MI. Future studies will investigate direct microRNA binding, co-cultures of hcF and EC, and perform staining and RNA expression analyses of in vivo samples.