Depolarized Mitochondrial Membrane Potential and Protection with Duroquinone in Isolated Perfused Lungs from Rats Exposed to Hyperoxia

Abstract

ObjectiveDissipation of mitochondrial membrane potential (Δψm) is a hallmark of mitochondrial dysfunction, which plays a key in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS). Our objective was to use a previously developed experimental‐computational approach to estimate tissue Δψm in isolated perfused lungs of rats exposed to hyperoxia as a rat model of human ARDS, and to evaluate the ability of the ubiquinone analog duroquinone (DQ) to reverse any hyperoxia‐induced depolarization of lung Δψm.MethodsRats were exposed to hyperoxia (>95% O2) or normoxia (room air) for 48 hrs, after which lungs were excised and connected to a ventilation‐perfusion system. The experimental protocol consisted of measuring the concentration of the fluorescent dye rhodamine 6G (R6G) during three single‐pass phases: loading, washing, and uncoupling, in which the lungs were perfused with and without R6G, and with the mitochondrial uncoupler FCCP, respectively. For normoxic lungs, the protocol was repeated with 1) rotenone (mitochondrial complex I inhibitor), 2) rotenone and either DQ or its vehicle (DMSO), and 3) rotenone, glutathione (GSH), and either DQ or DMSO added to the perfusate. Hyperoxic lungs were studied with and without DQ and GSH added to the perfusate. A physiologically‐based pharmacokinetic (PBPK) computational model was used to estimate lung Δψm from R6G data.ResultsFigure 1 shows that for hyperoxic lungs, R6G venous effluent concentrations tended to be higher during the loading phase, but significantly lower during the uncoupling phase compared to normoxic lungs, consistent with less uptake of R6G by hyperoxic lungs due to partial depolarization of Δψm. Figure 1 also shows that adding DQ to perfusate during the three phases almost completely reversed differences in R6G concentrations between normoxic and hyperoxic lungs. The PBPK computational model analysis of the R6G data show that exposure to hyperoxia depolarized lung tissue Δψm from ‐140 ± 9 (SE, n = 5) mV to ‐107 ± 9 (n = 5) mV, and complex I inhibition depolarized lung Δψm from ‐140 ± 9 (n = 5) to ‐57 ± 2 (n = 4) mV. Computational model analysis results also demonstrate the efficacy of DQ to fully reverse both rotenone‐induced and hyperoxia‐induced lung Δψm depolarization, consistent with the ability of the lungs to reduce DQ to its hydroquinone form (DQH2), which in turn bypasses mitochondrial complexes I and II to reduce mitochondrial complex III and restores Δψm.ConclusionsThis study demonstrates hyperoxia‐induced Δψm depolarization in isolated perfused lungs, and the utility of this approach for assessing the impact of potential therapies such as exogenous quinones that target mitochondria in isolated perfused lungs.