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Abstract: Following a myocardial infarction (MI), fibroblasts differentiate to myofibroblasts, which possess some of the characteristics of smooth muscle cells (SMCs) and contribute to wound healing. Previous studies suggested that the miR-143/-145 cluster plays a critical role in SMC differentiation. Therefore, we determined whether miR-145 promoted differentiation of cardiac fibroblasts to myofibroblasts. Following coronary occlusion in mice, myocardial miR-145 expression was downregulated at 3days but was restored at 7days. In vitro studies showed that hypoxia also downregulated miR-145 in cardiac fibroblasts. The number of α-smooth muscle actin (α-SMA) positive cells in fibroblast cultures was employed to determine their transdifferentiation to cardiac myofibroblasts and was increased by 73.5% after transient transfection with miR-145. Ultrastructural analysis of α-SMA stress fibers revealed that ~95% of the α-SMA+ cells treated with miR-145 organized their actin-filament bundles with a specific orientation compared to only 15% in the scrambled control group. This orientation of the SMA bundles and their integration with the filamentous actin fibers of the cytoskeleton permit infarct wound contraction. Structural and functional studies showed that miR-145 induced a myofibroblast phenotype, and miR-145 also potentiated the production of mature collagen by myofibroblasts. Repression of KLF5, a target of miR-145, was validated by a chimeric luciferase construct tagged with the full-length 3′-UTR of KLF5. A dramatic decrease in KLF5 and a corresponding increase in myocardin expression were observed after transfecting cultured fibroblasts with miR-145. Similar results were found in vivo: the transient decrease in miR-145 expression 3days post-MI was associated with an increase in KLF5 and a decrease in myocardin. In addition, in vivo delivery of a miR-145 antagomir 1day prior to and 2 and 6days after MI decreased myofibroblast formation and increased scar size. The antagomir also reversed the suppressed expression of KLF5 protein in the scar region at day 7 after MI. In summary, we describe a novel association between miR-145 and fibroblast differentiation toward myofibroblasts. These observations provide a new approach to promote endogenous scar healing and contracture by stimulating the transdifferentiation of cardiac fibroblasts to myofibroblasts. [Copyright &y& Elsevier]