Project Summary Fibrotic remodeling after myocardial infarction involves temporal development of several fibroblast phenotypes, characterized by high proliferation rates, differentiation into myofibroblasts, and heightened extracellular matrix deposition. These processes require large increases in metabolic demand to achieve or maintain the activated fibroblast state. Nevertheless, how fibroblasts meet these phases of high metabolic demand and whether particular metabolic steps could be antifibrotic targets remains unclear. Previous studies indicate that profibrotic stimuli augment catabolic activity in fibroblasts and that lowering either glycolysis or oxidative phosphorylation diminishes myofibroblast differentiation. These metabolic pathways are important not only for ATP production, but also for their role in the synthesis of cellular building blocks and secreted proteins. In particular, several glycolytic intermediates serve as amphibolic metabolites capable of entering into pathways responsible for de novo nucleotide, phospholipid, and amino acid synthesis. These biosynthetic pathways could be particularly important in the fibrotic response because activated fibroblasts require higher anabolic output to provide biomolecular building blocks for cell division and ECM secretion. Our preliminary data suggest that the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase 2 (Pck2), is upregulated in activated fibroblasts. This enzyme is positioned to coordinate anabolic processes because it can deliver carbon from the Krebs cycle intermediate pool to the 3-carbon glycolytic pool, thereby increasing precursors for several biosynthetic pathways. We also find glutaminase to be upregulated in activated fibroblasts, which could augment glutamine- derived carbon for the Krebs cycle pool, which is then available to Pck2. Suggestive of its importance to fibroblast function, Pck2 deletion decreases fibroblast proliferation and appears to affect markers of myofibroblasts, indicating that it is a critical enzyme that may facilitate or uphold the activated fibroblast phenotype. We propose that Pck2 is a fundamental regulator of fibrosis that regulates fibroblast activation by meeting the biosynthetic demands required for proliferation or ECM deposition. Nevertheless, virtually nothing is known about how biosynthetic pathways change in fibroblasts in response to fibrogenic stimuli or how Pck2 influences fibroblast metabolism and cardiac fibrosis. To address these gaps in knowledge, we will: (1) elucidate how Pck2 influences cardiac fibroblast metabolism; and (2) determine the influence of Pck2 on fibroblast activation and MI-induced cardiac remodeling. This project will yield fundamental knowledge necessary for understanding how fibroblast metabolism modulates fibrotic remodeling after cardiac injury.