In patients hospitalized with acute myocardial infarction (MI), acute hyperglycaemia is common and is associated with adverse outcome(1,2). In this setting, mortality risk increases in proportion to blood glucose concentration at admission irrespective of a prior diagnosis of diabetes mellitus. Hyperglycaemia may adversely influence cardioprotective stimuli, such as ischaemic preconditioning (IPC), thought to be mediated by PKCε activation, and also causes perturbation of cardiac action potential (APD) repolarisation. While attenuation of IPC by hyperglycaemia has been demonstrated in vivo, the mechanisms remain unclear. It has been shown that elevated extracellular glucose can cause activation of PKC and so we used rat isolated cardiomyocytes to investigate the role of PKC in the deleterious effects of hyperglycaemia on cardiomyocyte function. Cardiomyocytes were enzymatically isolated from adult male Wistar rats culled in accordance with home office regulations. IPC cardiomyocytes were isolated from hearts after an IPC protocol of 3 cycles of 5 minutes of stopped perfusion. Cardioprotection was assessed using a contractile function protocol(3). Using 1 Hz electric field stimulation (EFS) the effects of glucose on cardiomyocyte contraction was investigated when exchanging the solution from 5 to 20 mM glucose. In control experiments the number of contractile cardiomyocytes was reduced by 40±4% in 20 mM glucose which occurs concurrently with an increase in the number of asynchronous contractions (additional to the EFS). Inhibition of PKCα (21±1%***) and βI (16±3%***), but not βII (35±5%), γ (54±1%), δ (45±4%) or ε (100%) using isoform-specific Tat-linked PKC inhibitor peptides attenuated this decrease in contractile cardiomyocytes (***P<0.001, n=6 experiments >100 cells for each experiment). The mean increase in APD in 20 mM glucose was also attenuated by inhibition of PKCα and β (139±33ms to 12±2ms respectively, P<0.001, n=8 for each). Finally, these inhibitors were used to reverse the attenuation of IPC in 20 mM glucose. Contractile recovery in IPC cells was 72±3% in 5 mM and 30±4% in 20 mM glucose abolishing the beneficial effects of IPC. Inhibition of PKCα and β in 20 mM glucose prior to the contractile function protocol reversed the attenuation of contractile recovery (58±5%) which was not significantly different to IPC cardiomyocytes in 5 mM glucose (P>0.05, n=6 experiment, >100 cells each). In summary, our findings provide a direct mechanistic link between hyperglycaemia at the time of MI and subsequent adverse prognosis via PKCα and βI-dependent mechanisms. Our data suggest specific PKCs to be potential therapeutic targets in the treatment of MI, and strengthen the case for the active management of elevated glucose in MI in man.