Abstract Rationale Alveolar-capillary crosstalk leads to barrier failure in pneumonia-induced Acute Lung Injury (ALI). We have previously shown that the loss of negative mitochondrial membrane potential (mitochondrial depolarization) in the capillary endothelium causes barrier failure in animal models of ALI. We identified mitochondrial Uncoupling Protein 2 (UCP2) as essential for barrier failure and a potential therapeutic target. Here, we sought to determine a link between the metabolic effects of mitochondrial depolarization and the cytoskeletal alterations that lead to barrier failure. Methods To measure ALI impacts on mitochondrial metabolism and the cytoskeleton, we instilled Pseudomonas aeruginosa strain K (2.5 x 105 colony forming units x 24h) or lipopolysaccharide (LPS, 5mg/kg x 48h) by the intranasal route in C57BL/6J mice. For metabolomics analysis, we flash froze freshly isolated endothelial cells, lysed cells in 80% methanol with 0.1% formic acid, and quantified tricarboxylic acid (TCA) cycle intermediates by liquid chromatography-mass spectrometry. We separated lung tissue or freshly isolated endothelial cells into filamentous (f)-actin and globular (g)-actin portions by solubilization with triton-x detergent. In cultured endothelial cells, we depolarized mitochondria with a 24-h exposure to LPS and inhibited UCP2 using RNA interference. We detected N-acetylated actin from cell lysates with immunoblot. Results Targeted metabolomics identified an increase in citrate in endothelium of mice with P. aeruginosa pneumonia, and a decrease in cis-aconitate that was blocked with Ucp2 knockdown (p 0.05). Lung tissue from mice that received intranasal LPS had a decrease in f-actin/g-actin ratio as compared to PBS-instilled controls. Endothelial cells from P. aeruginosa-treated VE-Cadherin Cre+ x UCP2f/f mice were protected against f-actin/g-actin decreases. LPS-treated endothelial cells in culture showed a significant decrease in uptake of mitotracker deep red (MTDR); the decrease was blocked with Ucp2 knockdown. Concurrently, N-terminal actin acetylation in cultured endothelia increased with LPS treatment and was blocked with Ucp2 knockdown. Conclusions Our results support a model in which UCP2-induced mitochondrial depolarization in the pulmonary endothelium increases citrate production, N-terminal acetylation of actin, and depolymerization of f-actin. Since acetyl-coenzyme A (acetyl-CoA) derived from citrate can acetylate actin, destabilizing f-actin, we suggest that UCP2-mediated mitochondrial depolarization increases citrate production and thereby cytosolic acetyl-CoA production. Acetyl CoA increases N-terminal acetylation of actin, decreasing f-actin and causing barrier failure. Our work lays the groundwork to understand the mechanistic connection between mitochondrial dysfunction and barrier failure in ALI. This abstract is funded by: HL148403 to RFH
Hough et al. (Fri,) studied this question.