While the cardiac kinome is comprised of several hundred kinases, the vast majority of the literature is focused on only a few kinases. Indeed, the function of numerous highly expressed cardiac kinases, many of which may be potential therapeutic targets, are unexplored. In order to identify novel cardiac kinase(s) potentially involved in HF development, we employed a combined transcriptome and bioinformatics approach (Expression2Kinases (X2K)). This integrated approach enabled us to discover the critical role of serine-threonine kinase Homeodomain-Interacting Protein Kinase 2 (HIPK2) in cardiomyocyte (CM) biology. Specifically, we employed three different mouse models and AAV9-mediated gene therapy approaches to define the CM-specific role of HIPK2 and underlying mechanisms. Since there was no literature connecting HIPK2 to cardiac biology, these studies were entirely novel. HIPK2 has been strongly implicated in multiple organ fibrosis. However, the role of HIPK2 in fibrotic remodeling of the ischemic heart is entirely unknown. Herein, we aim to define the role of HIPK2 in adult heart fibroblast (FB) biology and fibrotic remodeling in the ischemic heart. As a first step towards this goal, we have generated inducible conditional FB-specific mouse models (FB-HIPK2-KOs driven by periostinMCM and TCF21MCM). Our preliminary studies suggest that FB-specific deletion of HIPK2 leads to excessive fibrosis and accelerated cardiac dysfunction post-MI. At the cellular/molecular level, adenovirus-mediated expression of HIPK2 (Ad-HIPK2) reduced the pro-fibrotic TGF-β1-mediated SMAD-2/3 activation and myofibroblasts transformation. Therefore, we hypothesize that HIPK2 exerts a critical break on fibrotic remodeling by inhibiting the canonical TGF-β1-SMAD-3 signaling. The preliminary data strongly support our overall hypothesis that HIPK2 is a critical negative regulator of FB activation and adverse myocardial fibrotic remodeling in the diseased heart. Specific aims are designed to critically examine the role of HIPK2 in cardiac FB biology and fibrotic remodeling of the ischemic heart. The proposed PeriostinMCM and TCF21MCM models are widely accepted as the strongest tools available for FB-specific in vivo gene targeting. Furthermore, throughout the proposal, a wide range of in vitro assays have been incorporated to gain mechanistic insights into the in vivo findings. Thus, the proposed studies are of high significance, not only by contributing translationally-relevant information to manage ischemic cardiac injury but also by providing novel mechanistic insights into cardiac pathophysiology.