Cardoso 2022a EBEC
Event: EBEC2022 Marseille FR
Several electron transfer pathways into the Q-junction reduce coenzyme Q. Substrate combinations are used to study the bioenergetics of isolated mitochondria, such as (1) pyruvate & malate providing the CI substrate NADH (N-pathway); (2) succinate as the substrate of CII (S-pathway); and (3) fatty acids & malate to initiate fatty acid oxidation (F-pathway) . In addition, oxidative phosphorylation (OXPHOS) is impacted by coupling control, with electron transfer (ET) maximally stimulated in the presence of uncouplers (ET capacity) or by ADP (OXPHOS capacity), or inhibited in the absence of ADP or by oligomycin (LEAK respiration) . Theoretically, modulating pathway and coupling control upstream or downstream of the Q-junction, respectively, impacts the relation between the Q redox state and O2 flux in opposite directions. Therefore, our aim was to characterize the influence of pathway and coupling control on these two bioenergetic parameters.
We used the NextGen-O2k with Q-Module to measure O2 flux and the Q redox state simultaneously, using CoQ2 as a probe . Cryopreserved HEK 293T cells were incubated with digitonin (plasma membrane permeabilization) and combinations of substrates and inhibitors to measure ET capacity in different pathway-control-states. Coupling control was assessed in LEAK and OXPHOS states (absence and presence of 2.5 mM ADP) and ET capacity (CCCP uncoupler titration).
Pathway control exerted a push effect on the Q-junction, as Q became reduced proportional with respiration supported by F-, N-, S-, and combined NS-pathways. Coupling control, contrarily, had a pull effect on the Q-junction: with respiration increasing from LEAK to OXPHOS and ET capacity (S- and F-pathways), Q became increasingly oxidized. Combined measurements of respiration and Q-redox state provide new diagnostic tools based on a deeper understanding of mitochondrial bioenergetics.
- Luiza H.D. Cardoso1, Chris Donnelly1,2, Timea Komlódi1,3, Erich Gnaiger1
- Oroboros Instruments, Innsbruck, Austria
- Institute of Sport Sciences, University of Lausanne, Switzerland
- Current address: Department of Medical Biochemistry, Semmelweis University, Hungary
- Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. https://doi.org/10.26124/bec:2020-0002
- Gnaiger E et al ― MitoEAGLE Task Group (2020) Mitochondrial physiology. https://doi.org/10.26124/bec:2020-0001.v1
- Komlódi T, Cardoso LHD, Doerrier C, Moore AL, Rich PR, Gnaiger E (2021) Coupling and pathway control of coenzyme Q redox state and respiration in isolated mitochondria. https://doi.org/10.26124/bec:2021-0003
EBEC Poster session
- Abstract Booklet: https://ebec2022.org/ABSTRACT-BOOKLET/
List of abbreviations, terms and definitions - MitoPedia
Labels: MiParea: Respiration
Organism: Human Tissue;cell: HEK Preparation: Permeabilized cells
Coupling state: LEAK, OXPHOS, ET Pathway: F, N, S, NS HRR: Oxygraph-2k