The ability of the heart to use multiple substrates provides the flexibility needed to balance catabolic demands with anabolic requirements; however, the failing heart shifts its energetic reliance toward glucose, and has diminished fuel flexibility. This switch in fuel use is associated with pathological remodeling, but it remains unclear how increased reliance on glucose catabolism affects cardiac health. We propose the general hypothesis that the inability of the failing heart to spare glucose-derived carbon for biosynthetic reactions causes pathological remodeling. We find that several collateral biosynthetic pathway metabolites are higher in the compensatory phase of hypertrophy, and that reductions in their abundance coincide with the early stages of heart failure. Nevertheless, how cardiac metabolic pathways are inter-regulated remains unclear, and how changes in metabolism elicit myocardial responses to stress remains unanswered. To span such gaps in knowledge, we will examine how collateral biosynthetic pathways change with cardiac remodeling in vivo by using deep network stable isotope tracing after pressure overload. We will also examine how physiologic stimuli for cardiac growth regulate cardiac biosynthetic pathway activity. We will correlate the changes in biosynthetic pathways with catabolic pathway activity. In Aim 2, we will determine how changes in the cardiac catabolism modulate collateral biosynthetic pathway activity in the heart. For this, we will force glucose, fat, or ketone oxidation using pharmacological and genetic approaches and measure glucose carbon fate in anabolic pathways using deep network stable isotope tracing. Under controlled metabolic conditions, we will construct an atlas demonstrating how glycolysis, mitochondrial activity, and substrate availability affect glucose carbon fate and anabolic pathway activity in cardiomyocytes. In Aim 3, we will augment biosynthetic pathway activity by genetically or allosterically regulating key metabolic steps in the heart or by introducing enzymes to activate metabolic pathways that are not typically operational in the mammalian heart. We will determine how these interventions regulate cardiac metabolism and affect myocardial structure and function during pressure overload-induced heart failure. We will delineate how these interventions affect the metabolism-guided decisions in cell signaling and gene expression that modulate cardiac hypertrophy and heart failure. Thus, this project will provide fresh perspectives about how metabolism regulates cardiac health and could identify innovative metabolic approaches to control cardiac remodeling. In particular, these studies will integrate our current understanding of cardiac catabolism with new knowledge of how cardiac anabolism is regulated in the heart. Such insights are conceptually novel and will contribute to understanding how metabolism regulates cardiac hypertrophy. Thus, these studies will identify: the metabolic pathway...