Doerrier 2015 Abstract MiPschool Greenville 2015
|"Succinate paradox" study by high resolution respirometry combined with fluorometry and spectrofluorometry: NextGen-O2k.|
Event: MiPschool Greenville 2015
Substrate-uncoupler-inhibitor titration (SUIT) protocols are an important aid to improve our understanding of mitochondrial bioenergetics. Succinate is a substrate which supports electron flux through succinate dehydrogenase (CII). Adding rotenone (inhibitor of CI) prevents the accumulation of oxaloacetate (Oa) which is a strong competitive inhibitor of CII .
In the present study, high-resolution respirometry (HRR) was used to evaluate the effect of succinate (10 mM) alone, S, or succinate (10 mM) with rotenone (0.5 μM), S(Rot), on mitochondrial respiration in cardiac mitochondria isolated from wild-type C57BL/6 mice. Respiration and hydrogen peroxide (H2O2) production were measured simultaneously in the Oroboros O2k-Fluorometer using Amplex Ultrared . In addition, respiration and NADH redox state were measured with a prototype NextGen-O2k, which combines high-resolution respirometry with O2k-Spectrofluorometry.
Mitochondrial respiration was similar in the LEAK state for S and S(Rot). However, H2O2 production was much higher with S. ADP (2 mM) added to S(Rot) induced a 70% increase of respiration from the LEAK to the OXPHOS state. Surprisingly, however, ADP added to S inhibited oxygen consumption by 30% with respect to the LEAK state (SL), which is the so-called “succinate paradox” . This paradox was not observed for H2O2 production, which declined to similar low levels after addition of ADP in states S and S(Rot). When Rot was added to isolated mitochondria in the absence of succinate and ADP, NADH increased, which is expected when endogenous substrates support dehydrogenase activity at a low level of residual oxygen consumption. Stimulation of respiration by addition of S increased NADH with and without Rot. The response of NADH to addition of ADP was diametrically different for S (decrease of NADH) and S(Rot) (increase of NADH).
Spectrofluorometry integrated into the NextGen-O2k extends the scope of bioenergetic analyses. Understanding mitochondrial respiratory control can be improved by combining HRR with simultaneous measurement of additional parameters. Our results support the hypothesis  that accumulation of Oa in the presence of S and absence of rotenone causes reverse electron transfer (RET) which induces a pathological increase of reactive oxygen species (ROS) production. When NADH increases after addition of ADP to S(Rot), there is a shift of the malate dehydrogenase equilibrium reaction towards a lower Oa concentration, supporting high activity of SDH. Conversely, lower NADH after addition of ADP to S leads to a further increase of Oa, which then inhibits SDH and respiration. In addition, antioxidant systems could be responsible for depletion of NADH and a corresponding rise of NAD+. The succinate paradox, therefore, can be considered as a relevant model for the study of physiological and pathological control of ROS production and respiratory control.
Labels: MiParea: Respiration, Instruments;methods
Organism: Mouse Tissue;cell: Heart Preparation: Isolated mitochondria
Coupling state: LEAK, OXPHOS Pathway: S HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Spectrophotometer Event: Oral
1-Oroboros Instruments, Innsbruck, Austria; 2-Pharmacobiochem Lab, Fac Medicine, Comenius Univ, Bratislava, Slovakia; 3-Dep Medical Biochem, Semmelweis Univ, Budapest, Hungary; 4-D.Swarovski Research Lab, Dept.Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria. - [email protected]
References and acknowledgements
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The project NextGen-O2k is supported by the "Technologieförderungsprogramm - Tiroler Innovationsförderung" of the Tyrolean Government.