Virtually every form of progressive heart failure (HF) is associated with increased fibrosis. However, currently, there is no approved therapy to specifically target myocardial fibrosis in the diseased heart. Until recently, HF studies were largely limited to cardiomyocytes (CMs), primarily due to unavailability of fibroblast (FB)-specific mouse models. The foundation of the current proposal is primarily based on our reports on the role of the GSK- 3 family of kinases in myocardial fibrosis. Recently, we used novel FB-specific mouse models to demonstrate the opposing function of the GSK-3 family of kinases in myocardial fibrosis. Specifically, GSK-3α mediates; however, GSK-3β inhibits myocardial fibrosis. Mechanistically, we identified that GSK-3α promotes fibrosis via the RAF-MEK-ERK pathway in a TGF-β1-SMAD-3 independent manner. In contrast, GSK-3β directly interacts with SMAD-3 to limit the profibrotic TGF-β1/SMAD-3 signaling. Herein, we are proposing translational studies with newly created conditional global GSK-3α KO mouse and recently developed GSK-3α-specific pharmacological inhibitor to determine if GSK-3α could be a viable therapeutic target for the diseased heart (Aim 1). Furthermore, we will delineate the mechanism of GSK-3β mediated myocardial fibrosis regulation by employing multiple conditional FB-specific KO models (Aim 2). We believe the proposed PeriostinMCM and TCF21MCM are currently the strongest tools available for FB-specific gene targeting in vivo. The proposed research is highly significant since it proposes novel strategies to prevent cardiac dysfunction and adverse remodeling in the diseased heart. The long-term goal of the proposed studies is to identify new therapeutic targets for the treatment of cardiac dysfunction, myocardial fibrosis, and subsequent HF.