Pharmacological mitochondrial ATP-sensitive K⁺ channel (mitoK_ATP) opening protects against ischemic damage and mimmicks ischemic preconditioning. However, physiological agonists for this channel still have not been uncouvered.  In this study, we verified if reactive oxygen species (ROS) generated by mitochondria are able to activate mitoK_ATP. Using reverse electron transport to generate ROS by mitochondria and the swelling technique, we demonstrate here that ROS activate K⁺ entry into control and preconditioned mitochondria. Exogenous H₂O₂ also activated mitoK_ATP in a manner reversed by catalase.  This effect was not seen when using medium without K⁺.  The effect of diazoxide (a mitoK_ATP agonist) was impaired by thiol antioxidants (N-acetylcysteine, mercaptopropionylglycine and dithiothreitol), demonstrating that diazoxide activity requires oxidized thiols, and that these types of antioxidants are not useful to study the mechanism of mitoK_ATP opening during preconditioning. In order to avoid this problem, we used a cellular system (HL-1 cardiomyocytes) submitted to simulated preconditioning and diazoxide pretreatment in the presence of catalase and found that catalase reversed the beneficial effect of preconditioning, but not of diazoxide, indicating that ROS mediating preconditioning occur upstream of mitoK_ATP. Collectivelly, these results suggest that mitoK_ATP activity by diazoxide is impaired by thiol antioxidants. Furthermore, this channel acts as a ROS sensor that decreases mitochondrial ROS release and triggers the protective effects of ischemic preconditioning.

Supported by FAPESP and CNPq.