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High-Resolution FluoRespirometry and OXPHOS Protocols for Human Cells, Permeabilized Fibers from Small Biopsies of Muscle, and Isolated Mitochondria

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Mitochondrial Bioenergetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1782))

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.

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Abbreviations

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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Authors and Affiliations

  1. Oroboros Instruments, Innsbruck, Austria

    Carolina Doerrier, Gerhard Krumschnabel, Yvonne Wohlfarter, András T. Mészáros & Erich Gnaiger

  2. Daniel Swarovski Research Laboratory, Mitochondrial Physiology, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Innsbruck, Austria

    Luiz F. Garcia-Souza & Erich Gnaiger

Authors
  1. Carolina Doerrier
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  2. Luiz F. Garcia-Souza
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  3. Gerhard Krumschnabel
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  4. Yvonne Wohlfarter
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  5. András T. Mészáros
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  6. Erich Gnaiger
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Correspondence to Erich Gnaiger .

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Editors and Affiliations

  1. Department of Life Sciences, University of Coimbra, Coimbra, Portugal

    Carlos M. Palmeira

  2. Department of Life Sciences, University of Coimbra, Coimbra, Portugal

    António J. Moreno

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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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