P1075 opens mitochondrial KATP channels and generates reactive oxygen species resulting in cardioprotection of rabbit hearts

We have recently proposed that opening of mitochondrial KATP channels (mitoKATP) acts as a trigger for preconditioning (PC) by causing mitochondria to produce reactive oxygen species (ROS). Controversy exists as to whether the putative sarcolemma-selective KATP channel opener P1075 also opens mitoKATP channels and may be cardioprotective. We purified mitoKATP channels from either rabbit heart, rat heart or rat brain and reconstituted the proteins into liposomes. mitoKATP channels from each of these tissues were opened by P1075 with EC50 values of 60–90 nM. We next tested whether P1075 causes rabbit cardiomyocytes to produce ROS in a KATP-dependent fashion. Mitochondrial ROS production was monitored by the appearance of fluorescence as reduced MitoTracker™ Red was oxidized. P1075 (100 μM) led to a 44 ± 9% increase in ROS generation (P < 0.001 vs. untreated cells), which was similar to the increase seen with 50 μM diazoxide, a selective mitoKATP channel opener (49 ± 9%, P < 0.001 vs. untreated cells). The effect of P1075 was equally potent at a concentration of 150 nM. The P1075-induced increase in ROS production was blocked by 50 μM glibenclamide (GLI), a non-selective KATP blocker, and by 5-hydroxydecanoate (1 mM), a highly selective mitoKATP blocker (–6 ± 14% and +4 ± 12%, respectively; P = n.s). In isolated rabbit hearts, P1075 (150 nM) markedly reduced infarct size compared to control animals (10.6 ± 8.1% of the area at risk vs. 31.5 ± 5.6%, P < 0.05). GLI (5 μM) as well as 5-hydroxydecanoate (200 μM) completely blocked P1075’s anti-infarct effect (31.7 ± 9.5% and 27.7 ± 4.6% infarction, respectively; P = n.s. vs. untreated hearts). These data provide strong evidence that P1075 does open mitoKATP channels and protects the ischemic rabbit heart in a mitoKATP-dependent manner.