Under normal physiological conditions, fatty acids are the preferred metabolic substrate for the myocardium. However during ischaemia, fatty acid oxidation ceases and anaerobic glycolysis becomes the main source of ATP production (1). Upon reperfusion, due to the build up of fatty acids during ischaemia, inhibition of glucose oxidation can occur, this can be detrimental (2). Improved re-coupling of glycolysis and glucose oxidation via stimulation of pyruvate dehydrogenase can relieve this detrimental effect (2, 3 and 4). This study investigates the effects of elevated extracellular glucose in the absence or presence of the medium chain fatty acid octanoate, and whether pyruvate has any effect on the actions of octanoate. Ventricular cardiomyocytes were enzymatically isolated from adult male Wistar rats which had been culled humanely in accordance with Home Office guidelines. Contractile function of the isolated cardiomyocytes was assessed using a simulated ischaemia/reperfusion protocol, where cardiomyocytes were stimulated to contract at 1Hz by electrical field stimulation and continuously perfused with Tyrode solution at 32±2°C. Ischaemia was simulated by perfusing a metabolic inhibition (MI) solution of Substrate-Free Tyrode solution containing cyanide (2mM) and iodoacetic acid (1mM) for 7 minutes, followed by 10 minutes of ‘reperfusion’ with Tyrode solution. These data show that perfusing isolated cardiomyocytes with 0.5mM pyruvate and an elevated extracellular glucose concentration (20mM) significantly increased the percentage of cardiomyocytes regaining contractile recovery from 27.6 ± 3.6% (5mM glucose control) to 51.5 ± 2.2%*** (Results presented as mean ± S.E.M; N= 6 experiments, ≥169 cardiomyocytes for each experiment, ***P<0.001 Unpaired T-test). The replacement of pyruvate (0.5mM) with octanoate (0.5mM), under conditions of elevated extracellular glucose, significantly decreased the percentage of cardiomyocytes able to regain contractile function from 51.5 ± 2.2% to 12.9 ± 2.1% **** (Results presented as mean ± S.E.M; N= 6 experiments, ≥156 cardiomyocytes for each experiment, ****P<0.0001 Unpaired T-test). The addition of 5mM pyruvate to octanoate, under conditions of elevated extracellular glucose, increased the percentage of cardiomyocytes regaining contractile recovery from 12.9 ± 2.1% to 25.5 ± 6.1% (Results presented as mean ± S.E.M; N= 6 experiments, ≥156 cardiomyocytes for each experiment, P=0.08 Unpaired T-test). These data show that the cardioprotection imparted by perfusion with an elevated extracellular glucose concentration, is eliminated by octanoate and that the effect of octanoate could be attenuated by pyruvate. We hypothesis these effects are due to the coupling status of glycolysis and glucose oxidation upon reperfusion.