Understanding the mechanism of AEBP1-mediated fibrosis post-cardiac injury in heart failure

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/ANGT)-induced fibrosis, we will evaluate the protein expression of RUNX1 and MRTF-A, and if the knockdown of RUNX1 or MEOX, or the expression of MRTF-A will attenuate myofibroblast activation and cardiac fibrosis.

AEBP1 inhibition in mice undergoing MI or infusion of PE/ANGT led to a reduction in cardiac fibrosis and myofibroblast activation, in our recent studies. Our goal is to determine the therapeutic antifibrotic potential of inhibiting the AEBP1 pathway (Aim 3). Since AEBP1 is also implicated in wound healing, the

timing of intervention following organ injury will be critical in the development of such an antifibrotic therapy. We will determine the optimal timepoint of intervention by evaluating cardiac function, histological changes, myofibroblast markers, autophagy, and oxidative stress. Utilizing the novel long-term functional preservation of

human myocardium in vitro under continuous electromechanical stimulation (MyoDishTM), we can evaluate its therapeutic potential. This novel technology can help us develop a proof of concept for the treatment of cardiac fibrosis and avoid the limitations of animal models in recapitulating the chronicity and complexity of human HF.