Abstract Body: Introduction: Sodium glucose cotransporter-2 inhibitors (SGLT2i) have emerged as a foundational therapy for heart failure (HF). Despite profound clinical benefits, the mechanism remains unclear. Evidence from our lab indicates SGLT2i may act directly on the heart through a novel off-target, metabolic enhancing mechanism, directly targeting pantothenate kinase 1 (PANK1), the rate limiting enzyme initiating Coenzyme A (CoA) biosynthesis. Purpose: 1) To test if SGLT2i directly activate PANK1 and improves cardiac metabolism. Methods: Human donor and explanted hearts were arrested in-situ with ice-cold cardioplegia, procured, and stable isotope infusion studies were performed in ex vivo perfused human myocardial tissue wedges treated with DMSO or empagliflozin (EMPA) for 90-minutes succeeded by metabolomic analyses. Purified PANK1 and HepG2 cells expressing genetically encoded acetyl-CoA (AcCoA) biosensors were generated to assess the effects of SGLT2i. Additionally, cultured adult and neonatal rat ventricular myocytes (NRVMs) were treated with SGLT2i and hopantenate (HOPA, CoA biosynthesis inhibitor), to evaluate CoA biosynthesis and respiration. Results: Acute EMPA treatment of human cardiac wedges increased fuel uptake and oxidation, boosted TCA cycle activity, and increased ATP concentration (P=0.013). EMPA also increased incorporation of isotopically labeled pantothenate into AcCoA (P=0.018). Using NRVMs, EMPA increased labeled pantothenate into AcCoA and free CoA (both P<0.05) that was attenuated with HOPA. These changes were accompanied with increased ADP stimulated respiratory capacity (P=0.022) that was mimicked by a known PANK activator, PZ2891 (P=0.045), and mitigated by HOPA. Next, we tested different SGLT2i on PANK1 activity. EMPA, dapagliflozin (DAPA), and canagliflozin (CANA) all increased PANK1 activity (dose response, 20-40% increase). Additionally, we leveraged AcCoA biosensors expressed in HepG2s and observed EMPA and CANA treatment increased cytosolic AcCoA concentrations (P<0.001 and P=0.041 respectively). DAPA and CANA increased AcCoA levels in the mitochondria (both P<0.01) while EMPA treatment trended higher (P=0.064). Finally, PANK1 KD in NRVMs depressed ADP stimulated maximal respiratory function (~25%, P=0.031) Conclusion: This study implicates CoA biology in the pathogenesis of HF. The data provided suggest PANK1 as a direct target of SGLT2i and at least in part explain the direct benefits of SGLT2i in patients with HF.