(A-17) Quantitative Assessment of Mitochondrial Function in Lungs of Rats Susceptible to Hyperoxia-Induced Acute Respiratory Distress Syndrome (ARDS)

Introduction:: ARDS carries a mortality rate >30% due to lack of early detection tools and limited therapies. Exposure of adult rats to hyperoxia (100% O₂) is a well-established model of human ARDS, with the first clinical evidence of lung injury after >40 hours and death by 72 hours. Yet, adult rats exposed to 60% O₂ for 7 days (H-S) show no clinical signs of injury. However, when pre-exposed to H-S, rats become more susceptible to lung injury as evidenced by a decrease in their subsequent survival time in 100% O₂. The objective of this study is to use an experimental and computational approach to assess mitochondrial functions in lungs of H-S rats to elucidate their role in hyperoxia susceptibility.


Materials and Methods:: Adult Sprague-Dawley rats were exposed to 60% O₂ (H-S) or room air (normoxia) for 7 days. Lungs were isolated and the pulmonary vascular endothelial filtration coefficient (K_f) was determined as a measure of pulmonary vascular permeability, and lung tissue mitochondrial membrane potential (Dy_m) was determined using the cationic dye rhodamine 6G. Expression of the electron transport chain complexes was measured in lung tissue homogenate using western blotting. Finally, mitochondria were isolated from lung tissue and used to assess mitochondrial respiration and bioenergetics. For quantitative assessment of the data from isolated mitochondrial studies, we are expanding our current computational model of the bioenergetics of isolated lung mitochondria (PMID: 29889855) by incorporating key ions and their regulations along with regulations of various enzymes and transporters by substrates, products, and co-factors.


Results, Conclusions, and Discussions:: H-S rats gained body weight at the same rate as normoxic rats, with no difference in lung wet weight or wet/dry weight ratio. K_f was 178% larger in H-S lungs compared to normoxics. Lung tissue homogenate studies showed decreased complex I (-81%) and II (-23%) expression, with no change in complex III, IV, or V expression. Isolated mitochondria studies showed that state 3 O₂ consumption rate and respiratory control index were lower in isolated mitochondria from lungs of H-S rats as compared to normoxia rats in the presence of complex I or II substrates, consistent with decreased complex I and II expression and activity. Isolated mitochondria studies also showed that H-S resulted in an increase in the time needed for Dy_m repolarization following ADP-stimulated depolarization in the presence of complex I (+64%) or complex II (+23%) substrates, consistent with decreased complex I and II activity. In contrast, Dy_m in isolated perfused lungs was relatively unchanged in lungs of H-S rats. For the computational modeling aspects of this study, we have so far accounted for the effect of ions and regulations on the flux of the reactions catalyzed by various mitochondrial enzymes in our current model (PMID: 29889855).



Results suggest that excess of complex I, which is the case in health, and a larger contribution of complex II to Dy_m might be sufficient to overcome the effect of a decrease in complex I activity/expression and maintain Dy_m in lungs of H-S rats. However, after sensitization, complex I expression is low enough that additional stress (e.g., exposure to 100% O₂) which further decreases complex I expression and deteriorates lung tissue mitochondrial bioenergetics, may have significant effect on lung tissue cellular functions. The proposed computational model will allow us to assess the effect of measured changes in mitochondrial activity/expression of various complexes on mitochondrial bioenergetics. The results suggest a potential role for mitochondrial dysfunction and vascular permeability in the susceptibility of H-S rats to hyperoxia-induced ARDS, which is clinically relevant since ventilation with 60% O₂ is often required for prolonged periods of time, particularly with COVID-19.




Acknowledgements (Optional): : This study was funded by NHLBI grant 2R15HL129209-02, Department of Veterans Affairs Merit Review Award BX001681, and NSF grant DMS 2153387.









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