Peroxynitrite induces depolarization and impairments of respiration in isolated murine brain mitochondria

Abstract

ObjectivePeroxynitrite, generated from nitric oxide and superoxide, plays a pathological role in both ischemic and neurodegenerative brain injury. Mitochondrial dysfunction is a key initiator of cerebrovascular and neurological pathologies. Thus, understanding the acute effects of peroxynitrite on brain mitochondrial physiology will help identify potential therapeutic targets. The objective of the study was to investigate the impact of peroxynitrite on respiratory function and membrane potential of isolated murine brain mitochondria.MethodsMitochondria were isolated from the brains of 12‐week‐old C57bl6 (n= 5–6) mice and treated with various concentrations of peroxynitrite (0.1μM, 1μM and 5μM) for one hour in assay buffer containing 10mM pyruvate and 2mM malate. Mitochondria (10μg/well) were plated into cell plates and oxygen consumption rate (OCR) was measured using Seahorse XFe24 Analyzer after serial injection of ADP (5mM), oligomycin (5μM), FCCP (5μM) and antimycin (10μM)/rotenone (2μM). Respiratory parameters were reported as OCR (pmoles/minute). Mitochondrial membrane potential was measured using fluorescence probe rhodamine 123 dye. Western Blots were then performed to detect the nitrotyrosine residues in the mitochondrial proteins.ResultsAcute peroxynitrite treatment decreased basal, or state II respiration, by 55.5 % (46.7±4.7 vs 102.5±2.5, n=6, p=0.0004) in brain mitochondria. Similarly, 5μM peroxynitrite treatment of isolated mitochondria diminished the uncoupler‐induced, or state III respiration, by 58% (94.5±12.9 vs 225.9±45.3, n=6, p=0.03). In addition, 5μM peroxynitrite reduced the mitochondrial membrane potential by 65.6% compared to untreated mitochondria (34.4 ±13.2 vs. 100, n=6, p‐value < 0.05). Notably, isolated mitochondria treated with peroxynitrite displayed increased nitrotyrosine content confirming the nitrotyrosination of the mitochondrial proteins.ConclusionsAcute treatment with peroxynitrite depolarizes isolated mitochondria leading to impairment of mitochondrial respiration which in turn impairs the ability of mitochondria to provide energy at the basal level and in response to increasing demand. Thus, peroxynitrite exhibits direct effects on the mitochondrial respiration which has deleterious consequences for neuronal survival in ischemic and neurodegenerative brain injury.Support or Funding InformationUndergraduate Summer Research Fellowship by American Physiological Society (Albuck); and National Institute of Health: National Institute of General Medical Sciences and National Institute of Neurological Disorders and Stroke (Katakam: R01NS094834).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.