Komlodi 2017 MiP2017

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Timea Komlodi
H2O2 production under hypoxia in brain and heart mitochondria: does O2 concentration matter?

Link: MiP2017

Komlodi T, Sobotka O, Gnaiger E (2017)

Event: MiP2017

COST Action MITOEAGLE
There is a large body of literature deciphering the role of mitochondria in reactive oxygen species-mediated signalling, which is mainly based on alterations in superoxide generation rate (further dismutated to H2O2) [1-3]. At sea level, the partial pressure of oxygen (O2) is around 20 kPa (concentration ≈200 µM) in the ambient air, but only < 1 kPa (≈10 µM) in the vicinity of mitochondria [4]. Quite importantly, ambient oxygen levels, also often applied in vitro, are hyperoxic for mitochondria. When reducing O2 levels towards the physiological intracellular range, contrasting results were reported either with increasing or decreasing H2O2 production rates. Such differences might arise from variability in species, tissue, detection method, respiration medium and O2 levels used. To shed light on some of the above cofounding factors, we set out to investigate the effect of various O2 concentrations on H2O2 production and O2 consumption in isolated mitochondria under well controlled conditions.

To this aim, isolated mouse brain and heart mitochondria respiring with the combination of NADH-linked substrates glutamate and malate and complex II-linked substrate, succinate in a closed-chamber respirometer combined with fluorimetry (Oroboros O2k High-Resolution FluoRespirometer). O2 concentration was varied from ≈170 µM to 15-20 µM with injection of N2 in the gas phase both in the LEAK and OXPHOS states. H2O2 production was determined by the highly specific Amplex UltraRed assay in MiR05 and KCl-based media. The rate of H2O2 generation was corrected for chemical background, changes of O2 level over time and for the sensitivity of the detection system.

Here we reveal that upon decrease of the O2 concentration from ≈170 µM to ≈20 µM, the rate of H2O2 formation is reduced as compared with normoxic H2O2 production in the LEAK but not in the OXPHOS state. After re-oxygenation, the rate of H2O2 generation again increases. At the same time, reducing the O2 concentration to ≈ 20 µM did not compromise O2 consumption, which is in line with the high affinity of mitochondrial respiration for O2 (p50 ≈ 0.03-0.04 kPa in OXPHOS).

Taken together, when reducing the O2 concentration to the physiological intracellular range in vitro, the rate of H2O2 production declines in a metabolic states-dependent manner. In contrast, respiration was not affected by the O2 concentration down to ≈ 20 µM neither in brain nor in heart. Based on this, we can assume that the H2O2 production is more sensitive to the changes of O2 concentration than the O2 consumption.


Bioblast editor: Kandolf G O2k-Network Lab: CZ Hradec Kralove Cervinkova Z, AT Innsbruck Gnaiger E, AT Innsbruck Oroboros


Labels: MiParea: Respiration 

Stress:Oxidative stress;RONS, Hypoxia  Organism: Mouse  Tissue;cell: Heart, Nervous system  Preparation: Isolated mitochondria 

Regulation: Oxygen kinetics  Coupling state: LEAK, OXPHOS  Pathway: N, NS  HRR: Oxygraph-2k 

Amplex UltraRed, MitoEAGLE 

Affiliations and support

Timea K(1), Sobotka O(3), Gnaiger E(1,2)
  1. Oroboros Instruments, Innsbruck, Austria
  2. D. Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria
  3. Charles Univ, Fac Medicine Hradec Kralove, Dept Physiology, Hradec Kralove, Czech Republic. - timea.komlodi@oroboros.at
Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE.

References

  1. Waypa GB and Schumacker PT (2005) Hypoxic pulmonary vasoconstriction: redox events in oxygen sensing. J Appl Physiol 98:404-14.
  2. Hoffman DL and Brookes PS (2009) Oxygen sensitivity of mitochondrial reactive oxygen species generation depends on metabolic conditions. J Biol Chem 284:16236-45.
  3. Hernansanz-Agustin P, Ramos E, Navarro E, Parada E, Sanchez-Lopez N, Pelaez-Aguado L, Cabrera-Garcia JD, Tello D, Buendia I, Marina A, Egea J, Lopez MG, Bogdanova A, Martinez-Ruiz A (2017) Mitochondrial complex I deactivation is related to superoxide production in acute hypoxia. Redox Biol 12:1040-51.
  4. Gnaiger E, Steinlechner-Maran R, Mende G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583-96.