Abstract 2509: Measurement of Mitochondrial Oxidant Stress and Superoxide Formation in the Intact Heart During Ischemia and Reperfusion
Mitochondria are a primary source of reactive oxygen species during reperfusion. We tested the hypothesis that mitochondrial oxidant stress and O2·− formation could be detected in the whole heart during ischemia-reperfusion using Mito-hydroethidine (Mito-HE). Mito-HE is selectively taken up by the mitochondria and oxidized to fluorescent mito-ethidium (Mito-Et) in the presence of a variety of mitochondrial oxidants. Superoxide (O2·−) oxidizes Mito-Et to oxyethidium (HO-Mito-Et), providing a specific marker for mitochondrial O2·− production.
Methods: Sprague-Dawley rat hearts were isolated and perfused with modified Krebs-Henseleit buffer using the Langendorff method. Left ventricular surface Mito-Et and HO-Mito-Et fluorescence and NADH autofluorescence were measured using a fiber-optic cable with appropriate filters and a photomultiplier tube. Hearts were loaded with Mito-HE (100 nM/min) for 20 minutes, rinsed and then received 1) normal perfusion (control, n=3), 2) diazoxide (100 μM/min, n=2), or 3) MnTBAP (10 μM/min, n=2) prior to 25 min ischemia followed by 15 min reperfusion.
Results: Confocal fluorescence microscopy in a separate group of hearts confirmed localization of Mito-Et and HO-Mito-Et in the mitochondria. Mito-Et fluorescence increased 16.6±2.6% during the 40 minute rinse period in all groups indicating continued localization of mito-Et into the mitochondria during rinsing. Mito-Et fluorescence increased 4.7% with the addition of diazoxide and was reduced 4.7% by addition of MnTBAP as compared to CTRL. Reperfusion caused an 8.1% increase in Mito-Et fluorescence in the CTRL group, a 2.6% increase in the diazoxide group and a 1.7% decrease in the MnTBAP group. A similar pattern of change was noted with HO-Mito-Et in all groups. NADH fluorescence was increased at the end of reperfusion in hearts receiving diazoxide (28%) and decreased in hearts receiving MnTBAP (−14%) as compared to CTRL.
Conclusions: As expected, diazoxide increased and MnTBAP decreased mitochondrial oxidant stress and O2·− production with loading, confirming the mitochondrial specificity of this probe in the beating heart. Both compounds reduced the mitochondrial oxidant stress and O2·− production at reperfusion, despite different mechanisms of action. This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).