13C-Metabolic Flux Analysis for Understanding Cardiac Energy Homeostasis

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Strong evidence links the development of cardiac hypertrophy and heart failure (HF) to dramatic alterations in mitochondrial fuel metabolism and bioenergetics. Specifically, the capacity of the heart to oxidize its chief fuels, fatty acids (FA) and glucose, becomes constrained, causing compensatory shifts in metabolism to alternative substrates. Recent work in the Muoio lab using mouse models and a system biology approach has identified marked perturbations in short chain carbon metabolites (acetyl CoA, ketones and branched chain amino acids (BCAA)-derived intermediates) as a strong signature of HF. Likewise, emerging findings from the Newgard lab link cardiometabolic stress to aberrant BCAA metabolism and inactivation of anaplerotic pathways that refill intermediates of the tricarboxylic acid cycle. Whereas static assessment of metabolite concentrations can highlight pathways deserving of further attention, measurement of metabolic flux is necessary to precisely pinpoint sites of metabolic dysregulation. Current heart perfusion methods to measure fluxes have several limitations including: 1) restriction to a single 13C-tracer per perfusion, despite various substrates of interest; 2) inadequate assessment of anaplerosis, as pyruvate is considered while other potentially important sources are ignored (e.g. BCAA-derived propionyl CoA); and 3) limited analysis of only a small fraction of the 13C-enrichment data generated in each experiment. To overcome these limitations, this project aims to develop and validate a powerful 13C-based “multiplex” MFA method, wherein several 13C- substrates (namely glucose, lactate, FA, and BCAA) can be applied to a single heart perfusion to evaluate substrate oxidation and anaplerotic fluxes, with emphasis on fluxes around the pyruvate, acetyl CoA and propionyl CoA nodes. This “multiplex” technique will reduce mouse sample size required for perfusions, while simultaneously increasing quality and quantity of flux information obtained. Lastly, this project will apply the “multiplex” MFA method to the carnitine acetyltransferase (CrAT) knockout mouse, which is a well-characterized model of altered acetyl and propionyl CoA metabolism. BCAA will serve as tool compounds to probe both acetyl CoA and propionyl CoA metabolism in CrAT- deficient hearts. Overall, this study will advance long-term goals of defining potential roles of CrAT and BCAA metabolism as factors that contribute to adaptive/maladaptive remodeling of the failing heart.