Project Summary/Abstract Demand for new cardiac therapies is on the rise, as cardiac disease is the leading cause of morbidity and mortality in the US and around the globe. Engineering human myocardium as a therapeutic means to restore muscle mass has great promise to enhance heart function, yet the current lack of integration of engineered tissue into the native heart limits its efficacy. Our long-term goal is to re-engineer contractility into the heart by using human pluripotent stem cell (hPSC)-derived cardiomyocytes to create functionally competent engineered cardiac tissue for regenerating myocardium of injured hearts. The overall objective of this proposal is to develop parallel components of an engineered cardiac tissue therapy that address constituents requisite for translational implementation by using inventive hybrid biomaterial, biophysical, and biochemical approaches. Our central hypothesis is that designing an instructive, biomimetic microenvironment will improve implant integration and contractility, and reduce risks for arrhythmia. We will rigorously test this hypothesis with quantitative measures in vitro and in vivo, using hybrid biomaterials to customize the implant and instruct host- implant interactions. Aim 1 is to promote host vascularization of engineered myocardium implanted in vivo in a rat model of myocardial infarction through the localized release of angiogenic growth factors from alginate microspheres encapsulated within the engineered tissue in order to increase capillary density, vessel hierarchy, and global tissue perfusion. Aim 2 is to enhance the electromechanical integration of engineered hPSC-derived cardiac tissue in the injured host rat heart after epicardial implantation using connexin 43- overexpressing cardiac myocytes and fibroblasts to increase conduction between implant and host assessed by optical mapping and graft-autonomous calcium transients in order to reduce arrhythmic risk. Aim 3 is to examine the biophysical remodeling process of engineered cardiac tissue in vitro by altering matrix composition, including human cardiac fibroblasts to remodel the tissue, and preconditioning by mechanical creep in order to modulate cell-matrix morphogenesis and improve tissue contractility. The parallel and independent aims have synergy and focus on mechanistic approaches to develop engineered human myocardium as an angiogenic therapy, anti-arrhythmic therapy, and contractile therapy for the heart. We believe this project is significant for its potential benefits on the efficacy and translation of engineered cardiac tissue in vivo and is innovative in its use of biomaterials and quantitative approaches to develop sophisticated, state-of-the-art engineered human myocardium for heart regeneration.