PROJECT SUMMARY Heart failure with preserved ejection fraction (HFpEF) is the predominant form of heart failure and its incidence continues to rise. HFpEF disproportionately affects women after menopause and has a high mortality rate. Due to the lack of well-characterized mechanisms and targeted therapy, treatment of this underappreciated disease remains focused on symptoms and does not address the underlying structural changes of cardiac remodeling that are key to disease progression. I propose to identify the mechanisms underlying the primary structural cardiac changes that occur in HFpEF in a sex specific manner. Specifically, in this study, I propose that Thrombospondin-1 (TSP-1) is an attractive target in cardiac remodeling noted in HFpEF. Mechanistically, myocardial TGF-β activation is a common element in HFpEF. However, TGF-β inhibition is impractical due to the associated toxicity, necessitating the search for other options to influence this pathway. TSP-1 is a matricellular protein that has no structural role, but modulates cell-cell and cell-matrix interactions by interacting with growth factors and surface receptors. Moreover, it is induced in metabolic syndrome, activates TGF-β1, and inhibits metalloproteinase activity in the heart, which could modulate fibrotic pathways. My central hypothesis is that is that TSP-1 is a key matricellular protein and local mediator of TGF-β1-induced cardiac fibrosis whose expression and activation in cardiac fibroblasts is modified by sex hormones, resulting in augmented collagen deposition and crosslinking. I will investigate the effect of biological sex on TSP-1 activation, TGF-β signaling, and subsequent dysregulation of cell behavior, tissue mechanics, and ventricular function. I will also determine the therapeutic potential of targeting TSP-1 in a rodent model of HFpEF. In Aim 1, my goal is to determine the regulatory mechanisms of TSP-1 and downstream consequences of TSP-1 induction in cardiac fibroblasts. Using human cardiac fibroblasts cells, I will identify the mechanisms by which hypoxia and hyperglycemia induce TSP-1, which then activates TGF-β1 and fibrosis. In Aim 2, my goal is to determine if a gain of androgen signaling boosts TSP-1 expression. Using human cardiac fibroblasts, we will describe the relative gain of androgen signaling post-menopause TSP-1 levels in relation to TGF-β activation. In Aim 3, my goal is to establish TSP-1 as a targetable enzyme in HFpEF. Using an in vivo rat model of HFpEF we will test if TSP-1 inhibition prevents or reverses HFpEF ex vivo and in vivo. This study will establish 1) the therapeutic potential for TSP-1 inhibition in HFpEF, 2) that ratio of estrogen to androgen modifies cardiac remodeling as opposed to absolute hormone levels, and 3) introduce a new therapeutic approach and a new target to treat HFpEF by addressing matrix remodeling. In addition, completion of this project will allow me to align my clinical expertise as a cardiac anesthesiologist w...