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Abstract:	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 (H<sub>2</sub>O<sub>2</sub>) 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). H<sub>2</sub>O<sub>2</sub> 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, H<sub>2</sub>O<sub>2</sub> 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].
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MiP areas:	Gender Respiration Instruments;methods mt-Biogenesis;mt-density mt-Structure;fission;fusion mt-Membrane mtDNA;mt-genetics nDNA;cell genetics Genetic knockout;overexpression Comparative MiP;environmental MiP Developmental biology Exercise physiology;nutrition;life style mt-Medicine Patients Pharmacology;toxicology mt-Awareness
Pathological states:	COPD Aging;senescence Alzheimer's Autism Cancer Cardiovascular Diabetes Inherited Infectious Myopathy Neurodegenerative Obesity Parkinson's Sepsis Other
Stress conditions:	Hypoxia Cell death Cryopreservation Ischemia-reperfusion Permeability transition Oxidative stress;RONS Temperature Mitochondrial disease
Organism:	Pig Human Mouse Rat Guinea pig Bovines Horse Dog Rabbit Cat Other mammals Chicken Zebrafish Drosophila Artemia Caenorhabditis elegans Saccharomyces cerevisiae Birds Reptiles Amphibians Fishes Chelicerates Crustaceans Hexapods Myriapods Annelids Molluscs Nematodes Other invertebrates Fungi Plants Protists Eubacteria Archea Algae
Tissues;cells:	Fat Heart Skeletal muscle Nervous system Liver Kidney Lung;gill Islet cell;pancreas;thymus Endothelial;epithelial;mesothelial cell Blood cells Genital Other cell lines CHO HEK HeLa Neuroblastoma Fibroblast HUVEC Macrophage-derived Lymphocyte Platelet Stem cells
Preparations:	SMP Intact organism Intact organ Permeabilized cells Permeabilized tissue Homogenate Isolated mitochondria Chloroplasts Enzyme Oxidase;biochemical oxidation Intact cells
Enzymes:	Complex I Adenine nucleotide translocase Complex II;succinate dehydrogenase Complex III Complex IV;cytochrome c oxidase Complex V;ATP synthase Inner mt-membrane transporter Marker enzyme Supercomplex TCA cycle and matrix dehydrogenases Uncoupling protein
Regulation:	pH Aerobic glycolysis ADP ATP ATP production AMP Calcium Coupling efficiency;uncoupling Cyt c Flux control Inhibitor Ion;substrate transport mt-Membrane potential Oxygen kinetics PCr;Cr Phosphate Redox state Substrate Temperature Threshold;excess capacity Uncoupler Fatty acid Amino acid Q-junction effect
Coupling:	ET LEAK ROUTINE OXPHOS
Pathways:	F N S Gp DQ CIV NS Other combinations ROX
High-resolution respirometry:	pH Oxygraph-2k TIP2k O2k-Fluorometer NO TPP Ca O2k-Spectrophotometer O2k-Manual O2k-Protocol Theory NextGen-O2k
Event:	A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4 D1 D2 D3 D4 E1 E2 Oral Poster P-flash Review
Additional labels:	(separate values by colon)

Free text: Any additional information (e.g. affiliations of authors). == Affiliations == 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. - carolina.doerrier@oroboros.at == Abstract continued == H<sub>2</sub>O<sub>2</sub> 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 == #Ackrell BA, Kearney EB, Mayr M (1974) Role of oxalacetate in the regulation of mammalian succinate dehydrogenase. J Biol Chem 249:2021-7. #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. #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. #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.

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