Doerrier 2015 Abstract MiP2015

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Simultaneous measurement of mitochondrial respiration, hydrogen peroxide production, and NADH autofluorescence to assess mitochondrial function.


Doerrier C, Plangger I, Sumbalova Z, Fasching M, Tretter L, Gnaiger E (2015)

Event: MiP2015

An increasing number of studies point to mitochondria as key regulators of many physiological and pathological conditions, related to life style (including physical exercise and nutrition), neurodegenerative diseases, metabolic disorders, inflammatory diseases, cancer, heart failure, and aging. Moreover, mitochondria are an important source of reactive oxygen species (ROS), which are needed for cell signaling. However, an increase in ROS production generates an oxidative stress which is implicated in the pathogenesis of many diseases. In particular, the NADH redox state is related to ROS production. Succinate is a substrate of succinate dehydrogenase (CII). For analysis of mitochondrial function with CII-linked substrates, rotenone (inhibitor of CI) is added to prevent accumulation of oxaloacetate (Oa), which is a strong competitive inhibitor of CII [1]. Ischaemic accumulation of succinate has been related to mitochondrial ROS production during reperfusion by reverse electron transfer [2]. In the present study, we used succinate with and without rotenone as a model for pathophysiological mitochondrial ROS-production.

We investigated the effect of succinate (10 mM) alone, S, or succinate (10 mM) with rotenone (0.5 μM), S(Rot), on mitochondrial respiration, hydrogen peroxide (H2O2) production and NADH redox state in cardiac isolated mitochondria from C57BL/6 mice. Respiration media (37 °C) were optimized for the specific protocols. High-resolution respirometry (HRR) was applied with the O2k-Fluorometer (Oroboros , Innsbruck, Austria). H2O2 production was measured simultaneously using Amplex Ultrared [3]. NAD(P)H autofluorescence was monitored in a prototype NextGen-O2k, which combines HRR with O2k-Spectrofluorometry. Step changes of the fluorescence signal were calibrated with NADH and corrected for changes observed in chemical background tests. These methods allow analyzing simultaneously relevant bioenergetic parameters to assess mitochondrial function.

Oxygen consumption levels were similar for S and S(Rot) in the LEAK state (without adenylates). However, H2O2 production was substantially higher with S in LEAK state. Adding ADP (2 mM) to S(Rot) to induce OXPHOS capacity, mitochondrial respiration increased by 70%. In contrast ADP titration to S induced a decline in respiration by 30% with respect to the LEAK state, which is the so-called “succinate paradox” [4].

O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E, AT Innsbruck MitoCom, HU Budapest Tretter L, SK Bratislava Sumbalova Z

Labels: MiParea: Respiration, Instruments;methods, Exercise physiology;nutrition;life style 

Stress:Oxidative stress;RONS  Organism: Mouse  Tissue;cell: Heart  Preparation: Isolated mitochondria 

Regulation: Substrate  Coupling state: LEAK, OXPHOS  Pathway:HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Spectrophotometer  Event: D1, Oral  MiP2015 


1-Oroboros Instruments, Innsbruck, Austria; 2-Pharmacobiochem Lab, Fac Medicine, Comenius Univ, Bratislava, Slovakia; 3-Dept Medical Biochem, Semmelweis Univ, MTA-SE Laboratory Neurobiochem, Budapest, Hungary; 4-D.Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Med Univ Innsbruck, Austria. -

Abstract continued

H2O2 production, however, declined to similar low levels after addition of ADP in protocols S and S(Rot). Rot added after the “succinate paradox” state displayed a stimulatory effect on mitochondrial respiration, restoring OXPHOS capacity comparable to S(Rot). When Rot was added to isolated mitochondria in the absence of succinate and ADP, NADH levels increased, as expected when endogenous substrates support dehydrogenase activity at a low level of residual oxygen consumption. Addition of S to mitochondria increased NADH levels with and without Rot in the LEAK state. Addition of ADP to S(Rot) induced a significant increase of the NADH fluorescence signal, which was entirely explained by the chemical background effect of ADP titration, such that NADH levels remained identical in the LEAK and OXPHOS states. In contrast, NADH levels declined significantly upon ADP without Rot. Further titration of malate inhibited S(Rot)-OXPHOS capacity (with an unexpected concomitant decline of NADH), and inhibited further respiration with S alone without change in NADH.

Fluorometry and spectrofluorometry integrated into the NextGen-O2k provide bioenergetically relevant parameters to analyze mitochondrial function. Accumulation of Oa in the presence of S and absence of rotenone causes reverse electron transfer, which induces a pathological increase of ROS production. After addition of ADP to S(Rot), NADH did not change significantly, indicating that the malate dehydrogenase equilibrium was maintained at constant low Oa concentration, supporting high activity of CII. However, the decrease of NADH after addition of ADP to S indicated a shift to higher Oa levels, which then may explain the observed inhibition of CII-linked respiration. On the other hand, antioxidant systems are challenged in the S protocol and thus may contribute to the depletion of NAD(P)H. Taken together, the “succinate paradox” represents a relevant model for the study of physiological and pathological control of ROS production, redox and respiratory control.

References and acknowledgements

  1. Ackrell BA, Kearney EB, Mayr M (1974) Role of oxalacetate in the regulation of mammalian succinate dehydrogenase. J Biol Chem 249:2021-7.
  2. Chouchani ET, Pell VR, Gaude E, Aksentijević D, Sundier SY, Robb EL, Logan A, Nadtochiy SM, Ord EN, Smith AC, Eyassu F, Shirley R, Hu CH, Dare AJ, James AM, Rogatti S, Hartley RC, Eaton S, Costa AS, Brookes PS, Davidson SM, Duchen MR, Saeb-Parsy K, Shattock MJ, Robinson AJ, Work LM, Frezza C, Krieg T, Murphy MP (2014) Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS. Nature 515:431-5.
  3. Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38.
  4. Oroboros (2012) First O2k-Fluorometry and high-resolution respirometry workshop (IOC66) Innsbruck, Tirol, Austria; 2012 March 15 to 16. Mitochondr Physiol.

The project NextGen-O2k is supported by the "Technologieförderungsprogramm - Tiroler Innovationsförderung" of the Tyrolean Government.