Abstract
Protocols for High-Resolution FluoRespirometry of intact cells, permeabilized cells, permeabilized muscle fibers, isolated mitochondria, and tissue homogenates offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques, small needle biopsies of muscle, and mitochondrial preparation methods. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation (OXPHOS) improve our understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial diseases. Respiratory states are defined in functional terms to account for the network of metabolic interactions in complex SUIT protocols with stepwise modulation of coupling control and electron transfer pathway states. A regulated degree of intrinsic uncoupling is a hallmark of oxidative phosphorylation, whereas pathological and toxicological dyscoupling is evaluated as a mitochondrial defect. The noncoupled state of maximum respiration is experimentally induced by titration of established uncouplers (CCCP, FCCP, DNP) to collapse the protonmotive force across the mitochondrial inner membrane and measure the electron transfer (ET) capacity (open-circuit operation of respiration). Intrinsic uncoupling and dyscoupling are evaluated as the flux control ratio between non-phosphorylating LEAK respiration (electron flow coupled to proton pumping to compensate for proton leaks) and ET capacity. If OXPHOS capacity (maximally ADP-stimulated O2 flux) is less than ET capacity, the phosphorylation pathway contributes to flux control. Physiological substrate combinations supporting the NADH and succinate pathway are required to reconstitute tricarboxylic acid cycle function. This supports maximum ET and OXPHOS capacities, due to the additive effect of multiple electron supply pathways converging at the Q-junction. ET pathways with electron entry separately through NADH (pyruvate and malate or glutamate and malate) or succinate (succinate and rotenone) restrict ET capacity and artificially enhance flux control upstream of the Q-cycle, providing diagnostic information on specific ET-pathway branches. O2 concentration is maintained above air saturation in protocols with permeabilized muscle fibers to avoid experimental O2 limitation of respiration. Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background O2 flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in High-Resolution FluoRespirometry.
Key words
- Substrate-uncoupler-inhibitor titration
- Human vastus lateralis
- Needle biopsy
- HEK
- HPMC
- HUVEC
- Fibroblasts
- PBMCs
- Platelets
- ROUTINE respiration
- Oxidative phosphorylation
- Q-junction
- Pyruvate
- Glutamate
- Malate
- Succinate
- Leak
- Coupling control
- Uncoupling
- O2k-FluoRespirometer
- O2 flux
- Residual O2 consumption
- Instrumental background
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- CCP :
-
Coupling control protocol
- ET :
-
Electron transfer
- E :
-
ET capacity
- FCR :
-
Flux control ratio
- FCF :
-
Flux control factor
- HRFR:
-
HRFR High-Resolution FluoRespirometry
- L :
-
LEAK respiration
- mt :
-
Mitochondrial
- O2k:
-
O2k Oxygraph-2k
- P :
-
OXPHOS capacity
- POS :
-
Polarographic oxygen sensor
- R :
-
ROUTINE respiration
- Rox:
-
Residual oxygen consumption
- SUIT:
-
Substrate-uncoupler-inhibitor titration
- W w :
-
Wet weight
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Acknowledgments
We thank Philipp Gradl and his team (WGT-Elektronik GmbH & Co KG) for O2k hardware and electronics development, Lukas Gradl for software development (DatLab 3 to 7), and Markus Haider for software development for p 50 analysis. Marielle Hansl and Stephanie Droescher performed some experiments with intact cells. This work is an extension of the original presentation by Pesta and Gnaiger [9], was supported by K-Regio project MitoFit, and is a contribution to COST Action CA15203 MitoEAGLE.
Competing Financial Interests
E.G. is the founder and CEO of Oroboros Instruments, Innsbruck, Austria.
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Doerrier, C., Garcia-Souza, L.F., Krumschnabel, G., Wohlfarter, Y., Mészáros, A.T., Gnaiger, E. (2018). High-Resolution FluoRespirometry and OXPHOS Protocols for Human Cells, Permeabilized Fibers from Small Biopsies of Muscle, and Isolated Mitochondria. In: Palmeira, C., Moreno, A. (eds) Mitochondrial Bioenergetics. Methods in Molecular Biology, vol 1782. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7831-1_3
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DOI: https://doi.org/10.1007/978-1-4939-7831-1_3
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