Bioenergetic and volume regulatory effects of mitoKATP channel modulators protect against hypoxia-reoxygenation-induced mitochondrial dysfunction

The mitochondrial ATP-sensitive K+ (mitoK(ATP)) channel plays a significant role in mitochondrial physiology and protects against ischemic reperfusion injury in mammals. Although fish frequently face oxygen fluctuations in their environment, the role of the mitoK(ATP) channel in regulating the responses to oxygen stress is rarely investigated in this class of animals. To elucidate whether and how the mitoK(ATP) channel protects against hypoxia-reoxygenation (H-R)-induced mitochondrial dysfunction in fish, we first determined the mitochondrial bioenergetic effects of two key modulators of the channel, diazoxide and 5-hydroxydecanoate (5-HD), using a wide range of doses. Subsequently, the effects of low and high doses of the modulators on mitochondrial bioenergetics and volume under normoxia and after H-R using buffers with and without magnesium and ATP (Mg-ATP) were tested. In the absence of Mg-ATP (mitoK(ATP) channel open), both low and high doses of diazoxide improved mitochondrial coupling, but only the high dose of 5-HD reversed the post-H-R coupling-enhancing effect of diazoxide. In the presence of Mg-ATP (mitoK(ATP) channel closed), diazoxide at the low dose improved coupling post-H-R, and this effect was abolished by 5-HD at the low dose. Interestingly, both low and high doses of diazoxide reversed H-R-induced swelling under mitoK(ATP) channel open conditions, but this effect was not sensitive to 5-HD. Under mitoK(ATP) channel closed conditions, diazoxide at the low dose protected the mitochondria from H-R-induced swelling and 5-HD at the low dose reversed this effect. In contrast, diazoxide at the high dose failed to reduce the swelling caused by H-R, and the addition of the high dose of 5-HD enhanced mitochondrial swelling. Overall, our study showed that in the presence of Mg-ATP, both opening of mitoK(ATP) channels and bioenergetic effects of diazoxide were protective against H-R in fish mitochondria, while in the absence of Mg-ATP only the bioenergetic effect of diazoxide was protective.