PROJECT SUMMARY Ischemic heart disease (IHD) is the leading cause of death worldwide, resulting in 9 million deaths yearly. Current treatments for IHD are medications or surgical procedures to improve blood flow, which cannot address the underlying permanent loss of cardiomyocytes (CMs) resulting from myocardial infarction (MI). After MI, the heart undergoes fibrotic repair rather than regeneration. This leads to maladaptive remodeling, further death of CMs, and eventual heart failure (HF) over time. In IHD patients with HF, heart transplant becomes the only therapy. However, new cases of HF far outpace the number of heart transplant procedures performed each year. Therefore, there is an urgent need for therapeutics that directly address the loss of CMs. In neonatal mammalian hearts post-MI and in adult hearts expressing active yes-associated protein 1 (YAP), a transcriptional co-regulator of cell proliferation, CMs can re-enter the cell cycle and divide to generate new CMs. I have uncovered that in both models, CM proliferation requires interplay between poised CMs, cardiac fibroblasts (CFs), and tissue-resident macrophages (MPs); poised CMs re-enter the cell cycle, while the Complement pathway, a component of innate immunity, is activated in CFs and MPs to facilitate CM cell cycle progression and de-differentiation. Specifically, C3 is activated in CFs and C3ar1 is activated in MPs, with C3 or C3ar1 knockout (KO) resulting in decreased CM proliferation. The central hypothesis of this proposal is activation of Complement alters the composition and metabolism of the cardiac microenvironment to promote CM proliferation. The first aim investigates the direct role of C3ar1 resident MPs in CM proliferation. C3ar1 MPs expand greatly during CM proliferation and express high levels of the insulin growth factor pathway gene Igf1 and genes involved in extracellular matrix (ECM) turnover such as Adam15. I will assay CM proliferation and metabolism in resident MP-specific Igf1 KO or Adam15 KO versus Control hearts, as well as ECM composition and stiffness. In the second aim, I will study how C3-CFs induce glycolytic metabolism within the cardiac microenvironment in response to MI. My preliminary data show that in neonatal C3 KO hearts, CFs failed to upregulate glycolysis after MI, resulting in impaired cardiac repair compared to Control hearts. I will use CF- specific C3 KO and Control hearts to determine if C3 is required for CF activation and whether the metabolic stress response in MPs is impaired after MI. Altogether, the proposed project aims to understand the interactions between different cell types of the renewal competent niche of in vivo models of CM proliferation. I believe that this work extends our CM-centric understanding of proliferation and may yield new insights and directions for cardiac renewal.