Timon-Gomez 2024 MitoFit
| Timón-Gómez A, Doerrier C, Sumbalová Z, Garcia-Souza LF, Baglivo E, Cardoso LHD, Gnaiger E (2024) Analysis of mitochondrial respiratory pathway and coupling control by substrate-uncoupler-inhibitor titration reference protocols. MitoFit Preprints 2024.8. https://doi.org/10.26124/mitofit:2024-0008 |
» MitoFit Preprints 2024.8.
Timon-Gomez A, Doerrier Carolina, Sumbalova Zuzana, Garcia-Souza Luiz F, Baglivo Eleonora, Cardoso Luiza HD, Gnaiger Erich (2024) MitoFit Prep
Abstract: Capacities of electron transfer (ET) and oxidative phosphorylation (OXPHOS) are fundamental for mitochondrial function. Since the plasma membrane is impermeable to several compounds, such as ADP, used in OXPHOS analysis, respirometry with living cells provides limited information compared to mitochondrial preparations (isolated mitochondria, tissue homogenates, permeabilized tissues and cells).
We studied mouse mitochondria from brain as a glucose-dependent tissue, and from heart which relies highly on fatty acid oxidation. HEK 293T cells were analyzed as a widely used experimental model, and human peripheral blood mononuclear cells (PBMCs) and platelets from non-invasive liquid biopsies as mitochondrial biomarkers. 20 respiratory states were interrogated applying two substrate-uncoupler-inhibitor titration reference protocols in parallel.
In mouse heart and human PBMCs, OXPHOS capacities were identical to ET capacities in every pathway state, but this applied only to the NADH-linked N-pathway in platelets. Adding succinate S for NS or additional substrate combinations in platelets, ET capacity exceeded OXPHOS capacity indicating that the phosphorylation system was driven to saturation. N-linked respiration increased more than 2-fold from OXPHOS to ET in mouse brain mitochondria, but NS-linked excess ET capacity decreased. HEK 293T cells had variable ET capacities at constant OXPHOS capacity in all pathway states. In all models, incomplete additivity of the N- and S-pathway argued against tight channeling through supercomplexes.
OXPHOS analysis enables to attribute respiratory capacities to nutrient-specific pathways and distinguish metabolic adjustments from functional defects by formulating robust working hypotheses. Bioenergetic snapshots obtained by OXPHOS analysis gain perspective on the basis of a comparative mitochondrial database.
• Keywords: high-resolution respirometry HRR, substrate-uncoupler-inhibitor-titration SUIT protocol RP, OXPHOS, pathway control, coupling control, electron transfer system ETS
• Bioblast editor: Tindle-Solomon L
• O2k-Network Lab: AT Innsbruck Oroboros
ORCID: 
Timon-Gomez A, Doerrier Carolina, Sumbalova Zuzana, Garcia-Souza Luiz F, Baglivo Eleonora, Cardoso Luiza HD, Gnaiger Erich
Data availability
- Original files are available Open Access at Zenodo repository: https://zenodo.org/records/13774074
Labels: MiParea: Respiration, Instruments;methods
Organism: Human, Mouse
Tissue;cell: Heart, Nervous system, Liver, Blood cells, HEK
Preparation: Permeabilized cells, Homogenate, Isolated mitochondria, Intact cells
HRR: Oxygraph-2k, O2k-Protocol
