Pharmacological preconditioning by diazoxide downregulates cardiac L-type Ca2+ channels

BACKGROUND AND PURPOSE Pharmacological preconditioning (PPC) with mitochondrial ATP-sensitive K+ (mitoK(ATP)) channel openers such as diazoxide, leads to cardioprotection against ischaemia. However, effects on Ca2+ homeostasis during PPC, particularly changes in Ca2+ channel activity, are poorly understood. We investigated the effects of PPC on cardiac L-type Ca2+ channels. EXPERIMENTAL APPROACH PPC was induced in isolated hearts and enzymatically dissociated cardiomyocytes from adult rats by preincubation with diazoxide. We measured reactive oxygen species (ROS) production and Ca2+ signals associated with action potentials using fluorescent probes, and L-type currents using a whole-cell patch-clamp technique. Levels of the alpha(1c) subunit of L-type channels in the cellular membrane were measured by Western blot. KEY RESULTS PPC was accompanied by a 50% reduction in alpha(1c) subunit levels, and by a reversible fall in L-type current amplitude and Ca2+ transients. These effects were prevented by the ROS scavenger N-acetyl-L-cysteine (NAC), or by the mitoK(ATP) channel blocker 5-hydroxydecanoate (5-HD). PPC signficantly reduced infarct size, an effect blocked by NAC and 5-HD. Nifedipine also conferred protection against infarction when applied during the reperfusion period. Downregulation of the alpha(1c) subunit and Ca2+ channel function were prevented in part by the protease inhibitor leupeptin. CONCLUSIONS AND IMPLICATIONS PPC downregulated the alpha(1c) subunit, possibly through ROS. Downregulation involved increased degradation of the Ca2+ channel, which in turn reduced Ca2+ influx, which may attenuate Ca2+ overload during reperfusion.