Cardiovascular disease (CVD) is a leading cause of morbidity and mortality and is especially prevalent among US veterans. Heart failure (HF) is one of the most common manifestations of CVD, and one of the main underlying pathologic mechanisms is cardiac fibrosis. Fibrosis is a reparative mechanism that follows any type of injury. In the heart, it leads to reduced tissue compliance, cardiomyocyte hypertrophy and apoptosis, chamber dilation, and eventually HF. HF patients undergoing implantation of a left ventricular assist device or heart transplantation present a unique opportunity as cardiac tissue becomes available at the time of the above operations. Our studies in HF patients have identified adipocyte enhancer binding protein 1 (AEBP1) as a major node associated with myofibroblast activation, abnormal extracellular matrix (ECM) homeostasis, and impaired wound healing following cardiac injury. Our goal is to investigate the mechanisms by which the aberrant expression of AEBP1 contributes to cardiac fibrosis and to exploit its inhibition as a potential antifibrotic therapy. Our recent studies indicated the correlation of elevated AEBP1 with increased cardiac fibrosis and myofibroblast activation. The overexpression of AEBP1 in primary fibroblasts led to the upregulation of myofibroblast markers and ECM. Chromatin immunoprecipitation and sequencing identified Runt-related transcription factor 1 (RUNX1) as a potential transcription factor regulating AEBP1 expression. We will investigate the mechanisms by which RUNX1 regulates AEBP1 expression during cardiac fibroblast activation and subsequent fibrosis development (Aim 1). We will perform manipulative expression (overexpression or shRNA knockdown) of RUNX1 in conjunction with quantitative determination of AEBP1 expression level. We will determine the cis regulatory element in the AEBP1 genomic region which RUNX1 occupies during cardiac myofibroblast activation and fibrosis development following cardiac injury. Our previous studies have shown that the overexpression of AEBP1 in human primary fibroblasts is associated with increased expression of RUNX1, mesenchyme homeobox (MEOX), and myocardin-related transcription factor A (MRTF-A). The knockdown of AEBP1 in fibroblasts stimulated with transforming growth factor beta (TGF-ß) led to a reduced expression of RUNX1, MEOX and MRTF-A. Our goal is to understand the mechanistic involvement of RUNX1, MEOX, and MRTF-A in AEBP1-mediated cardiac fibrosis (Aim 2). By overexpressing RUNX1, MEOX, or MRTFA in primary fibroblasts we will identify the gene and protein expression of myofibroblast markers and ECM in relation to AEBP1 expression and determine whether they are downstream of AEBP1, suggesting a possible feedback loop mechanism. We will test if the knockdown of RUNX1, MEOX, or MRTF-A in fibroblasts stimulated with TGF-ß will reduce myofibroblast activation and fibrosis. In a mouse model of myocardial infarction (MI) and phenylephrine/angiotensin 2 (PE/...