Meszaros 2018 Mt Med Hinxton

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High-resolution mitochondrial oxygen kinetics as diagnostic tool in Complex IV impairments.

Link:

Meszaros AT, Haider M, Di Marcello M, Gnaiger E (2018)

Event: Mitochondrial Medicine 2018 Hinxton UK

Assessment of mitochondrial respiration at higher-than-physiological oxygen (O2) concentrations has become a standard in biomedical research. In contrast, the impact of mitochondrial O2 kinetics on (patho)physiology is largely neglected. To facilitate further studies, we developed the automatized software module O2kinetics. Using this advanced tool, we re-evaluated the O2 dependence of mitochondrial respiration in various respiratory states, experimental conditions, and in the presence of cytochrome c oxidase (CIV) inhibitors.

Mitochondria isolated from mouse brain, heart and liver were incubated at 37 °C in Oroboros O2k High-Resolution FluoRespirometers. Using substrate-uncoupler-inhibitor titration (SUIT) protocols with various fuel substrate combinations in OXPHOS-, LEAK- and ET-states, we investigated the effect of pathway and coupling control on mitochondrial p50 (O2 partial pressure at half-maximum O2 flux, JO2). Kinetic data was obtained during aerobic-anaerobic transitions with high time-resolution at data sampling intervals of 0.2 s. p50 values were calculated using the O2kinetics software for automatic correction and calibration steps. Depending on experimental temperature and respiratory state, p50 ranged from 0.006 to 0.07 kPa for NADH-linked LEAK respiration with glutamate&malate, N(GM)L, and NADH-&succinate-linked OXPHOS capacity including GM and pyruvate, NS(GMP)P, in agreement with and extending data published previously. In heart and liver mitochondria, the p50 was higher in OXPHOS- compared to LEAK-states, increasing proportionally with CIV turnover. Upon inhibition of CIV, p50 increased several-fold up to 0.15 kPa, NS(GM)P, which was not reflected in JO2 at kinetic O2 saturation. In diagnostic experiments, increasing p50 values correlated well with decreasing CIV activity, as measured upon addition of ascorbate and TMPD.

Mitochondrial p50 measurement is a quick, highly accurate and sensitive way for early detection of reduced CIV excess capacity, as affinity of mitochondria for O2 decreases already at a slight CIV inhibition. Determination of mitochondrial O2 dependence has implications in a wide range of biomedical research topics, particularly in studies with CIV mutations (e.g. Leigh syndrome), or with small inhibitory molecules (nitric oxide). O2kinetics provides a fast and simple method for the detection of CIV impairment and extends standard OXPHOS analysis to the intracellular O2 regime in vivo, reflecting low physiological O2 concentrations or tissue hypoxia.


Bioblast editor: Kandolf G, Meszaros AT O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck Oroboros


Labels: MiParea: Respiration, mt-Medicine  Pathology: Neurodegenerative 

Organism: Mouse  Tissue;cell: Heart, Nervous system, Liver  Preparation: Isolated mitochondria  Enzyme: Complex IV;cytochrome c oxidase  Regulation: Oxygen kinetics  Coupling state: LEAK, OXPHOS, ET  Pathway: N, CIV, NS  HRR: Oxygraph-2k 

Labels, 2018-04 

Affiliations

Meszaros AT(1), Haider M(2), Di Marcello M(1,3), Gnaiger E(1,3)
  1. Oroboros Instruments, Innsbruck, Austria
  2. Steinhauser & Haider Technology Consulting OG, Innsbruck, Austria
  3. Daniel Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria. - andras.meszaros@oroboros.at