PROJECT SUMMARY Heart failure (HF) remains a major public health concern as the number of American adults experiencing HF continues to rise. Engineered heart tissues (EHTs) have emerged as a significant advancement for disease modeling and drug screening, better replicating in vivo processes than many small-animal models. However, despite their potential in assessing the impact of drugs on CMs, these EHTs lack vasculature, a critical component of cardiac function. In vivo, cardiomyocytes (CMs) are surrounded by a dense network of vasculature which supplies oxygen and nutrients and regulates blood flow to the heart. Endothelial cells (ECs) lining these vessels emit protective signals, that govern vital cardiac processes including heart development, homeostasis, and tissue regeneration. As such, there is a critical need for a new EHT model which incorporates a perfusable vascular element to study interactions between vascular perfusion, the endothelium, and CMs, shedding light on their impacts on cardiac function and disease. One notable example of the interplay between vasculature and CMs that could be studied using this model is endothelial dysfunction. Linked to HF, endothelial dysfunction results from inflammation, triggered by stressors such as hypertension, hyperglycemia, hemodynamic changes, and viruses. While previous research has focused on impaired vasodilation as the primary link to HF, more recent work has begun to explore the role that disrupted autocrine and paracrine signaling may have on EC-CM communication, leading to cardiac stress, hypertrophy, death, and overall reduced cardiac function. However, no existing in vitro model allows for direct study of the impacts of endothelial dysfunction and its consequences on CMs. Our group recently developed a perfusable collagen-based EHT through injection molding techniques. With precise control of tube geometry, perfusable pressure and flow, and cellular and matrix composition, this new EHT model presents an opportunity to study the interaction of flow, endothelial function, and cardiac function, and the impact of endothelial dysfunction on this relationship. We hypothesize that the inclusion of perfusion within this EHT model will enhance EC survival and function and improve the physiologically relevant cardiac response to altered load and electrical stimulation. Additionally, we expect that induced endothelial dysfunction will lead to increased cardiac dysfunction. To test this hypothesis, we will assess the effect of flow on endothelial cell retention and, consequently, cardiac function within our perfusable EHT model, investigating the phenotypic, functional, and transcriptional changes that are induced by the addition of a vascular component. We will also use this perfusable EHT model to determine our ability to chemically and mechanically induce endothelial dysfunction and study the effect this dysfunction has on neighboring CMs. This model will deepen our understanding of vascular p...