Dissipation of mitochondrial membrane potential (Δψm) is a hallmark of mitochondrial dysfunction. Our objective was to use a previously developed experimental-computational approach to estimate tissue Δψm in intact lungs of rats exposed to hyperoxia and to evaluate the ability of duroquinone (DQ) to reverse any hyperoxia-induced depolarization of lung Δψm. Rats were exposed to hyperoxia (>95% O2) or normoxia (room air) for 48 h, 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 6 G (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 (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. Computational modeling was used to estimate lung Δψm from R6G data. Rat exposure to hyperoxia resulted in partial depolarization (-33 mV) of lung Δψm and complex I inhibition depolarized lung Δψm by -83 mV. Results also demonstrate the efficacy of DQ to fully reverse both rotenone- and hyperoxia-induced lung Δψm depolarization. This study demonstrates hyperoxia-induced Δψm depolarization in intact lungs and the utility of this approach for assessing the impact of potential therapies such as exogenous quinones that target mitochondria in intact lungs.NEW & NOTEWORTHY This study is the first to measure hyperoxia-induced Δψm depolarization in isolated perfused lungs. Hyperoxia resulted in a partial depolarization of Δψm, which was fully reversed with duroquinone, demonstrating the utility of this approach for assessing the impact of potential therapies that target mitochondria such as exogenous quinones.
Keywords: acute lung injury; cationic rhodamine dyes; computational modeling; electrochemical transmembrane potential; fluorescence imaging.