ATP-sensitive potassium channels (KATP) play a central role in protecting cardiac myocytes from metabolic stress. KATP channels are hetero-octomers consisting of four inward rectifier family Kir6 subunits (Kir6.1 or Kir6.2) and four sulphonyurea receptor subunits (SUR1, SUR2A or SUR2B). Current pharmacological tools do not allow distinction between the roles of each KATP channel subunit. We have therefore developed a molecular approach to specifically alter protein levels of individual KATP channel subunits and so define their role in cardioprotection. We designed shRNA constructs to selectively down-regulate the pore forming subunit KATP channel, Kir6.2. These constructs were engineered into an adenoviral vector system for efficient delivery into isolated adult rat cardiac myocytes. The down-regulation of KATP currents after 24h was confirmed by comparing the currents evoked by a KATP channel opener (p1075, 10μM) of myocytes infected with control virus (9.4±1.3 pA/pF, n=11) versus those infected with adenovirus encoding shRNA Kir6.2 (5.4±0.9 pA/pF, n=14). To assess the effect of Kir6.2 down-regulation on cardioprotection, myocytes were placed in a bath, superfused with Tyrode solution and synchronized to contract by field stimulation at 1Hz. Myocytes were challenged with a metabolic insult of 4 min perfusion with metabolic substrate free Tyrode solution containing cyanide (2mM) and iodoacetic acid (1mM) followed by reperfusion for 10 min. The specific down-regulation of Kir6.2 by shRNA resulted in a decrease in contractile recovery (control, 82±2%, n=147; shRNA Kir6.2, 39±1%, n=126). Calcium homeostasis during metabolic stress is a key factor in myocyte survival. Using the calcium indicator Indo1 we measured intracellular calcium levels in response to metabolic stress and reperfusion. Myocytes expressing shRNA constructs down-regulating Kir6.2 showed a higher calcium level after metabolic stress and reperfusion (Indo1 ratio; control, 1.0±0.01 n=43; shRNA Kir6.2, 1.2±0.02, n=43). The effect of Kir6.2 knockdown on ischaemic preconditioning was assessed with an ischaemic pelleting method in which a pellet of myocytes was layered with mineral oil to prevent gaseous exchange to mimic ischaemia, followed by a return to normal medium to mimic reperfusion injury. We found that ischaemic preconditioning was abolished in myocytes infected with Kir6.2 shRNA compared to those infected with control virus (control cell survival 36±1%, n=71 compared to 17±1%, n=102 survival of Kir6.2 knockdown cells). These results are consistent with a direct role for KATP channels containing the subunit Kir6.2 in the cardioprotective response to metabolic stress.