Lactate is metabolic fuel utilized by the heart under healthy, fasted conditions. Research from our groups and others suggests lactate is also an essential fuel source during cardiac stress. The monocarboxylate transporter 1 (MCT1) has been localized to both the plasma membrane and mitochondrial membrane of cardiac cells, which allows the import into the cytosol and direct import and metabolism of lactate in the mitochondria under both healthy and stressed conditions. |How lactate transits the cell and in which compartment metabolic reactions occur to enable lactate oxidation is not completely understood. Lactate dehydrogenases (LDHA and LDHB) catalyze the interconversion of lactate and pyruvate in the cytosol and inside the mitochondria, respectively, while malate dehydrogenase 1 (MDH1) regenerates NAD+ for the glycolytic pathway that produces pyruvate. In order to obtain a more mechanistic understanding of lactate's metabolic role in the heart, we are conducting stable isotope tracing analysis within an MCT1 deletion cell line paired with knockouts of lactate dehydrogenases (LDHA and LDHB) and malate dehydrogenase (MDH1). We are using HCT116 cells as our initial cells to establish baseline methodology. We hypothesize that MCT1 enables the majority of lactate oxidation to pyruvate to occur directly in the mitochondria, thus directly supplying the cell with energy in the form of NADH and pyruvate. Our experiments are designed to test this by utilizing isotope labeled lactate to track energy production in our engineered knockout cell lines. We expect to observe a decrease in lactate's contribution to ATP production within the TCA cycle. Due to lactate's potential role as a preferential energetic substrate in respiration and oxidation in cardiomyocytes, expanding our understanding of lactate metabolism may offer new therapeutic possibilities to improve cardiac function in the failing heart.