Mitochondrial dysfunction in cardiac ischemia–reperfusion injury: ROS from complex I, without inhibition

Abstract

A key pathologic event in cardiac ischemia reperfusion (I-R) injury is mitochondrial energetic dysfunction, and several studies have attributed this to complex I (CxI) inhibition. In isolated perfused rat hearts, following I-R, we found that CxI-linked respiration was inhibited, but isolated CxI enzymatic activity was not. Using the mitochondrial thiol probe iodobutyl-triphenylphosphonium in conjunction with proteomic tools, thiol modifications were identified in several subunits of the matrix-facing 1alpha sub-complex of CxI. These thiol modifications were accompanied by enhanced ROS generation from CxI, but not complex III. Implications for the pathology of cardiac I-R injury are discussed.

Cardioprotection and mitochondrial S-nitrosation: Effects of S-nitroso-2-mercaptopropionyl glycine (SNO-MPG) in cardiac ischemia–reperfusion injury

“…were subfractionated (see below) and the homogenate, mitochondria and cytosolic fractions were analyzed by ozone chemiluminescence for SNO content. Mitochondria were traced through this fractionation protocol by monitoring the matrix marker enzyme citrate synthase [36]. Furthermore, S-nitrosation of complex I in these mitochondria was monitored by separating the respiratory complexes using blue-native electrophoresis, followed by ozone chemiluminescence analysis of the complex I bands, as previously described [27].…”

“…to obtain the nominal cytosolic fraction. Integrity pf mitochondria was checked by measurement of their respiratory properties with a Clark O 2 electrode [36], with mitochondria from cells exhibiting respiratory control ratios of 8.8 ± 0.5 respiring on glutamate plus malate, and 5.3 ± 0.3 respiring on succinate.…”

“…Pellets were also assayed for SNO content by the biotin switch analysis (see below, Figure 4B). Complex I activity of pellets was measured spectrophotometrically (340 nm) as the rotenone-sensitive rate of NADH oxidation in the presence of coenzyme-Q 1 (Eisai Pharmaceutical, Japan) as previously described [27,36].…”

“…ischemia without reperfusion, in the presence or absence of SNO-MPG. Mitochondrial content in subsequent experiments was normalized to citrate synthase activity, determined as previously described [36]. Mitochondrial Ca 2+ uptake was measured spectrophotometrically using Arsenazo III as previously described [39], with glutamate plus malate as respiratory substrates.…”

“…Mitochondrial Ca 2+ uptake was measured spectrophotometrically using Arsenazo III as previously described [39], with glutamate plus malate as respiratory substrates. Mitochondrial ROS generation was monitored spectrofluorimetrically using Amplex red (Molecular Probes, Eugene OR), as previously described [36].…”