Title: Myocardial remuscularization by cardiac patch delivery of epicardial FSTL1 and CCND2 overexpressing cardiomyocytes Project Summary Despite undergoing intensive treatment regimens, patients with severe acute myocardial infarction (AMI) often end up with end stage congestive heart failure (CHF). From the molecular and cellular perspective, heart failure occurs due to the loss of the contractile unit of the left ventricle: cardiomyocytes (CMs). Therefore, promotion of myocyte proliferation and understanding the regulators of myocyte cell cycle could have highly significant impact on the management of heart failure. In this proposal, we seek to develop 3D bioengineered cardiac muscle constructs that incorporate a functional vascular network and recapitulate some of the key microenvironmental cues of native heart tissue. Our recent studies have identified main biomechanical and molecular cues that can significantly enhance cell cycle re-entry of adult CMs. We demonstrated that epicardial application of a cardiac patch, laden with follistatin like-1 (FSTL1) protein, protected the mouse and pig heart against AMI, left ventricle dilatation, and heart failure. We recently reported that overexpression of a cell cycle gene, CCND2 (cyclin D2), induces proliferation of transplanted human induced pluripotent stem cell (hiPSC) derived-CMs. This proposal builds upon our recent technological achievements, enabling cast or bioprinting of major cardiac cells and hydrogels at high spatial resolution (20 µm) to fabricate 3D perfusable vascular constructs. Our central hypothesis is that 3D cardiac constructs, laden with FSTL1 and hiPSC-CCND2 CMs, can synergistically remuscularize ischemic myocardium. We test this hypothesis in three integrated Specific Aims (SAs). In SA1, we will utilize our cast/bioprinted 3D cardiac tissue models to identify the cellular and molecular mechanisms underlying myocyte pro-proliferative effect of FSTL1 treatment in vitro. In SA2, we will assess the identified signaling pathways, involved in FSTL1-CCND2 CM-patch effect, to promote remuscularization in mouse model of MI (both acute and chronic). In SA3a, we will assess the pre-clinical potential of bioengineered pre- vascularized muscle patch device in treating AMI in a pig model of ischemia-reperfusion. We will compare the efficacy of open chest delivery versus a novel minimally invasive, catheter-based, pericardial delivery of FSTL1 and CCND2 CM laden muscle patch to the epicardium. SA3b, we will evaluate the effectiveness of the engineered patch for preventing the LV dilatation without inducing arrhythmogenic complications. The panoramic optical mapping and transmural electrical EP mapping whole heart will assess the electromechanical integration between the muscle patch constructs and recipient myocardium. The findings from these EP studies will guide the design of new generations of cell lines and patch constructs with improved EP characteristics, thereby reducing the risk of ...