Introduction: Β-hydroxybutyrate (BHB) is an oxidative fuel substrate, primarily produced to sustain cerebral metabolism when carbohydrate availability is low (1). BHB is usually only elevated with concomitant depletion of muscle glycogen, and the permissive role of glycogen content on BHB oxidation in the heart is therefore poorly understood. However, it is possible to elevate BHB using exogenous ketones (2), so we sought to determine whether low, control or high cardiac muscle glycogen content could influence ketone body oxidation in the isolated perfused rodent heart. Methods: Following euthanasia by sodium pentobarbitone injection in accordance with UK legislation, hearts from 150-200g male Wistar rats were perfused with modified Krebs-Henseleit buffer (KH) in a sealed apparatus using the Langendorff mode. Heart glycogen was manipulated to low (L), control (C) and high (H) levels using one of three ‘pre-experiment’ KH compositions: L = 20 mins + no substrate; C = 20 min + 10mM glucose; H = 60 mins + 10mM glucose, 1mM lactate, 4mM pyruvate + 50mU.min-1 insulin. All hearts were then switched to KH containing 4mM 14C-BHB and 4mM glucose for 40 mins. Gas and KH samples were collected every 10 mins to measure appearance of 14CO2 and [14C]-bicarbonate, which was used to calculate oxidation of 14C-BHB. Hearts were freeze-clamped at the end of the ‘pre-experiment’ (n=16) or experimental (n=19) periods and analyzed for glycogen by an enzymatic method; metabolomic analysis was completed using HPLC-GCMS. Repeated measures ANOVA with Tukey post hoc corrections were performed. Significance was taken at p<0.05. Values are mean ± SEM. Units for glycogen are: μM glycosyl-units/g wet weight. Results: Glycogen availability was significantly different between L, C and H groups following the ‘pre experiment’ period (L=5.4±1.6, C=16.3±3.4, H=43.5±5.1). Following perfusion with BHB, glycogen levels had risen in all three groups (L=33.9±3.6, C=41.4±6.4, H=64.4±10.7), indicating glycogen re-synthesis. Increased BHB oxidation correlated to increased glycogen availability (L=0.84±0.14 μmol/min/heart, C=0.93±0.15 μmol/min/heart, H=1.41±0.25 μmol/min/heart) (R2=0.35). In the H condition, glycolytic intermediates were higher than in L and C, apart from phosphoenolpyruvate, which was significantly lower. TCA cycle intermediates were higher with H glycogen vs. L and C. Conclusions: Our results demonstrate a permissive role of cardiac glycogen in the combustion of increasing quantities of BHB. These findings suggest an obligate requirement for glucose in the oxidation of BHB, possibly because BHB is not inherently anaplerotic, with ensuing cataplerosis limiting TCA capacity (3). Further work on the interactions between carbohydrate and BHB metabolism in working muscle is underway.