Diazoxide (DZX) exerts a powerful pharmacological preconditioning effect, protecting cardiac muscle from the effects of ischaemia and reperfusion, and has been proposed to act through a mitochondrial ATP-sensitive K⁺ (K_ATP) channel (Garlid et al. 1997). However, Kir6.2 knockout mice, which lack sarcolemmal K_ATP channel activity, do not show protection by DZX (Suzuki et al. 2003). Here, we have investigated DZX protection and its effects on sarcolemmal K_ATP channel activity in rat ventricular myocytes.

Myocytes were isolated enzymatically from humanely killed adult rats (Rodrigo et al. 2002). Contractions were measured with a video system, action potentials and single channel activity with patch clamp, and [Ca²⁺]_i with fura-2. Metabolic inhibition (MI) was induced by superfusion with substrate-free Tyrode solution containing 2 mM NaCN and 1 mM iodoacetate for 7 min, and all experiments were done at 34 °C.

Cells were stimulated at 1 Hz, treated with DZX (100 µM) for 5 min 2 min before MI, and contractile function was assessed 10 min after reperfusion. DZX pretreatment increased the proportion of cells that recovered contractile function from 16.8 ± 2.4 % (mean ± S.E.M., n = 21 expts) to 65.0 ± 2.2 % (n = 20, P < 0.001, Student’s unpaired t test), and also increased the proportion of cells in which diastolic [Ca²⁺]_i recovered to below 250 nM. DZX also accelerated action potential and contractile failure during MI. In both control and DZX-pretreated cells, loss of contraction accompanied failure of the action potential. Contraction failed after 192 ± 6 s (n = 57) in control cells and 138 ± 4 s (n = 23) in DZX-pretreated cells (P < 0.001). In cell-attached patches on unstimulated myocytes, MI led to sarcolemmal K_ATP channel activity, but this occurred earlier in DZX-pretreated cells than in control cells (145 ± 24 s, n = 8 versus 225 ± 15 s, n = 10 respectively, P < 0.01). However, local application of DZX by inclusion in the patch pipette did not accelerate channel opening. The blocker of sarcolemmal K_ATP channels HMR 1883 (10 µM) delayed action potential failure and also reduced the protective effect of DZX pre-treatment.

Our results show that pre-treatment with DZX increases recovery of function and Ca²⁺ homeostasis after MI and reperfusion in isolated myocytes. Early activation of sarcolemmal K_ATP channels leads to failure of the action potential and contraction and it is likely that this contributes to protection by DZX.

This work was supported by the British Heart Foundation.