Abstract
Protocols for high-resolution respirometry (HRR) of intact cells, permeabilized cells, and permeabilized muscle fibers offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques and small needle biopsies of muscle. Multiple substrate–uncoupler–inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation 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 and substrate control. 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 (FCCP, DNP) to collapse the proton gradient across the mitochondrial inner membrane and measure the capacity of the electron transfer system (ETS, open-circuit operation of respiration). Intrinsic uncoupling and dyscoupling are evaluated as the flux control ratio between nonphosphorylating LEAK respiration (electron flow coupled to proton pumping to compensate for proton leaks) and ETS capacity. If OXPHOS capacity (maximally ADP-stimulated oxygen flux) is less than ETS capacity, the phosphorylation system contributes to flux control. Physiological Complex I + II substrate combinations are required to reconstitute TCA cycle function. This supports maximum ETS and OXPHOS capacities, due to the additive effect of multiple electron supply pathways converging at the Q-junction. Substrate control with electron entry separately through Complex I (pyruvate + malate or glutamate + malate) or Complex II (succinate + rotenone) restricts ETS capacity and artificially enhances flux control upstream of the Q-cycle, providing diagnostic information on specific branches of the ETS. Oxygen levels are maintained above air saturation in protocols with permeabilized muscle fibers to avoid experimental oxygen 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 oxygen flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in HRR.
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Abbreviations
- CCP:
-
Coupling control protocol
- E :
-
Electron transfer system capacity
- FCR:
-
Flux control ratio
- HRR:
-
High-resolution respirometry
- L :
-
LEAK respiration
- mt:
-
Mitochondrial
- 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
References
Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583–596
Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277–297
Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Dykens JA, Will Y (eds) Mitochondrial dysfunction in drug-induced toxicity. Wiley, New York, pp 327–352
Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41:1837–1845
Gnaiger E (2003) Oxygen conformance of cellular respiration: a perspective of mitochondrial physiology. Adv Exp Med Biol 543:39–56
Gnaiger E, Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Steurer W, Margreiter R (2000) Mitochondria in the cold. In: Heldmaier G, Klingenspor M (eds) Life in the cold. Springer, New York, pp 431–442
Fasching M, Renner-Sattler K, Gnaiger E (2010) Mitochondrial respiration medium – MiR06. Mitochondr Physiol Netw 14(13): 1–4. http://www.oroboros.at
Veksler VI, Kuznetsov AV, Sharov VG, Kapelko VI, Saks VA (1987) Mitochondrial respiratory parameters in cardiac tissue: a novel method of assessment by using saponin-skinned fibers. Biochim Biophys Acta 892:191–196
Skladal D, Sperl W, Schranzhofer R, Krismer M, Gnaiger E, Margreiter R, Gellerich FN (1994) Preservation of mitochondrial functions in human skeletal muscle during storage in high energy preservation solution (HEPS). In: Gnaiger E, Gellerich FN, Wyss M (eds) What is controlling life? vol 3, Modern Trends in Biothermokinetics. Innsbruck University Press, Innsbruck, pp 268–271
Gnaiger E, Méndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc Natl Acad Sci USA 97:11080–11085
Steinlechner-Maran R, Eberl T, Kunc M, Margreiter R, Gnaiger E (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol 271:C2053–C2061
Renner K, Amberger A, Konwalinka G, Kofler R, Gnaiger E (2003) Changes of mitochondrial respiration, mitochondrial content and cell size after induction of apoptosis in leukemia cells. Biochim Biophys Acta 1642:115–123
Steinlechner-Maran R, Eberl T, Kunc M, Schröcksnadel H, Margreiter R, Gnaiger E (1997) Respiratory defect as an early event in preservation/reoxygenation injury in endothelial cells. Transplantation 63:136–142
Stadlmann S, Rieger G, Amberger A, Kuznetsov AV, Margreiter R, Gnaiger E (2002) H2O2-mediated oxidative stress versus cold ischemia-reperfusion: mitochondrial respiratory defects in cultured human endothelial cells. Transplantation 74:1800–1803
Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380:919–928
Hütter E, Unterluggauer H, Garedew A, Jansen-Dürr P, Gnaiger E (2006) High-resolution respirometry – a modern tool in aging research. Exp Gerontol 41:103–109
Aguirre E, Rodríguez-Juárez F, Bellelli A, Gnaiger E, Cadenas S (2010) Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells. Biochim Biophys Acta. doi:10.1016/j.bbabio.2010.01.033
Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. I. Kinetics of oxygen utilization. J Biol Chem 217:383–393
Stadlmann S, Renner K, Pollheimer J, Moser PL, Zeimet AG, Offner FA, Gnaiger E (2006) Preserved coupling of oxidative phosphorylation but decreased mitochondrial respiratory capacity in IL-1β treated human peritoneal mesothelial cells. Cell Biochem Biophys 44:179–186
Smolková K, Bellance N, Scandurra F, Génot E, Gnaiger E, Plecitá-Hlavatá L, Ježek P, Rossignol R (2010) Mitochondrial bioenergetic adaptations of breast cancer cells to aglycemia and hypoxia. J Bioenerg Biomembr. doi:10.1007/s10863-009-9267-x
Jones DP (1986) Intracellular diffusion gradients of O2 and ATP. Am J Physiol 250:C663–C675
Villani G, Attardi G (1997) In vivo control of respiration by cytochrome c oxidase in wild-type and mitochondrial DNA mutation-carrying human cells. Proc Natl Acad Sci USA 94:1166–1171
Gnaiger E, Rieger G, Kuznetsov A, Fuchs A, Stadlmann S, Lassnig B, Hengster P, Eberl T, Margreiter R (1997) Mitochondrial ischemia-reoxygenation injury and plasma membrane integrity in human endothelial cells. Transplant Proc 29:3524–3526
Gnaiger E, Kuznetsov AV, Rieger G, Amberger A, Fuchs A, Stadlmann S, Eberl T, Margreiter R (2000) Mitochondrial defects by intracellular calcium overload versus endothelial cold ischemia/reperfusion injury. Transpl Int 13:555–557
Vercesi AE, Bernardes CF, Hoffmann ME, Gadelha FR, Docampo R (1991) Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ. J Biol Chem 266:14431–14434
Gnaiger E, Kuznetsov AV, Lassnig B, Fuchs A, Reck M, Renner K, Stadlmann S, Rieger G, Margreiter R (1998) High-resolution respirometry. Optimum permeabilization of the cell membrane by digitonin. In: Larsson C, Påhlman I-L, Gustafsson L (eds) Biothermokinetics in the post genomic era. Chalmers Reproservice, Göteborg, pp 89–95
Saks VA, Veksler VI, Kuznetsov AV, Kay L, Sikk P, Tiivel T, Tranqui L, Olivares J, Winkler K, Wiedemann F, Kunz WS (1998) Permeabilised cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol Cell Biochem 184:81–100
Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am J Physiol Heart Circ Physiol 286:H1633–H1641
Kuznetsov AV, Strobl D, Ruttmann E, Königsrainer A, Margreiter R, Gnaiger E (2002) Evaluation of mitochondrial respiratory function in small biopsies of liver. Anal Biochem 305:186–194
Rasmussen UF, Rasmussen HN (2000) Human quadriceps muscle mitochondria: a functional characterization. Mol Cell Biochem 208:37–44
Palmer JW, Tandler B, Hoppel CL (1977) Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. J Biol Chem 252:8731–8739
Gnaiger E, Lassnig B, Kuznetsov AV, Margreiter R (1998) Mitochondrial respiration in the low oxygen environment of the cell: effect of ADP on oxygen kinetics. Biochim Biophys Acta 1365:249–254
Gnaiger E, Kuznetsov AV (2002) Mitochondrial respiration at low levels of oxygen and cytochrome c. Biochem Soc Trans 30:252–258
Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662:7–25
Dubowitz V, Sewry CA (2006) Muscle biopsy: a practical approach. Saunders Elsevier, Philadelphia
Dufour S, Rousse N, Canioni P, Diolez P (1996) Top-down control analysis of temperature effect on oxidative phosphorylation. Biochem J 314:743–751
Gnaiger E (ed) (2007) Mitochondrial pathways and respiratory control. OROBOROS MiPNet, Innsbruck. http://www.oroboros.at
Sun F, Huo X, Zhai Y, Wang A, Xu J, Su D, Bartlam M, Rao Z (2005) Crystal structure of mitochondrial respiratory membrane protein Complex II. Cell 121:1043–1057
Puchowicz MA, Varnes ME, Cohen BH, Friedman NR, Kerr DS, Hoppel CL (2004) Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria – case studies. Mitochondrion 4:377–385
Delhumeau G, Cruz-Mendoza AM, Lojero CG (1994) Protection of cytochrome c oxidase against cyanide inhibition by pyruvate and α-ketoglutarate: effect of aeration in vitro. Toxicol Appl Pharmacol 126:345–351
Boushel R, Gnaiger E, Schjerling P, Skovbro M, Kraunsøe R, Dela F (2007) Patients with type 2 diabetes have normal mitochondrial function in skeletal muscle. Diabetologia 50:790–796
Gnaiger E, Wright-Paradis C, Sondergaard H et al (2005) High-resolution respirometry in small biopsies of human muscle: correlations with body mass index and age. Mitochondr Physiol Netw 10(9):14–15. http://www.mitophysiology.org/index.php?gnaigere
Scheibye-Knudsen M, Quistorff B (2009) Regulation of mitochondrial respiration by inorganic phosphate; comparing permeabilized muscle fibers and isolated mitochondria prepared from type-1 and type-2 rat skeletal muscle. Eur J Appl Physiol 105:279–287
Aragonés J, Schneider M, Van Geyte K et al (2008) Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism. Nat Genet 40:170–180
Kuznetsov AV, Lassnig B, Margreiter R, Gnaiger E (1998) Diffusion limitation of oxygen versus ADP in permeabilized muscle fibers. In: Larsson C, Påhlman I-L, Gustafsson L (eds) Biothermokinetics in the post genomic era. Chalmers Reproservice, Göteborg, pp 273–276
Gnaiger E, Forstner H (eds) (1983) Polarographic oxygen sensors. aquatic and physiological applications. Springer, New York
Gnaiger E (2010) Oxygen calibration and solubility in experimental media. Mitochondr Physiol Netw 6(3):1–20. http://www.oroboros.at
Fasching M, Gnaiger E (2010) Instrumental background correction and accuracy of oxygen flux. Mitochondr Physiol Netw 14(6):1–12. http://www.oroboros.at
Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R, Kunz WS (2008) Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc 3:965–976
Gnaiger E, Steinlechner R, Keriel C, Leverve X, Rossi A, Saks V, Sibille B, Kay L, Novel V, Daneshrad Z, Gellerich FN, Eberl T, Skladal D, Sperl W, Margreiter R (1995) Oxygen sensitivity of respiration in endothelial cells, hepatocytes and permeabilized muscle fibers studied by high-resolution respirometry. J Mol Med 73:B39
Acknowledgments
This work was supported by OeNB Jubiläumsfond project 13476 and is a contribution to Mitofood COST Action FAO602. We thank Dr. Michael Schocke who was responsible for taking the human biopsies, and Drs. Robert Boushel, Flemming Dela, Steen Larson, Nis Stride, Dan Kane, and Darrel Neufer for advice in the technique of biopsy sampling.
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Pesta, D., Gnaiger, E. (2012). High-Resolution Respirometry: OXPHOS Protocols for Human Cells and Permeabilized Fibers from Small Biopsies of Human Muscle. In: Palmeira, C., Moreno, A. (eds) Mitochondrial Bioenergetics. Methods in Molecular Biology, vol 810. Humana Press. https://doi.org/10.1007/978-1-61779-382-0_3
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