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Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells

Abstract

The assessment of mitochondrial respiratory chain (RC) enzymatic activities is essential for investigating mitochondrial function in several situations, including mitochondrial disorders, diabetes, cancer, aging and neurodegeneration, as well as for many toxicological assays. Muscle is the most commonly analyzed tissue because of its high metabolic rates and accessibility, although other tissues and cultured cell lines can be used. We describe a step-by-step protocol for a simple and reliable assessment of the RC enzymatic function (complexes I–IV) for minute quantities of muscle, cultured cells and isolated mitochondria from a variety of species and tissues, by using a single-wavelength spectrophotometer. An efficient tissue disruption and the choice for each assay of specific buffers, substrates, adjuvants and detergents in a narrow concentration range allow maximal sensitivity, specificity and linearity of the kinetics. This protocol can be completed in 3 h.

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Figure 1: Potter tissue grinders for tissue and cell homogenization.
Figure 2: Quality control of 50 μM reduced cytochrome c solution.
Figure 3: Cytochrome c reduction.
Figure 4: Effect of freeze-thaw cycles on complex I activities in fibroblast-isolated mitochondria.
Figure 5: Representative traces of spectrophotometric assays for respiratory chain enzymes in muscle homogenates.
Figure 6: Troubleshooting: decreased linearity of the reaction with time because of excessive sample protein concentrations.

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References

  1. DiMauro, S. & Schon, E.A. Mitochondrial respiratory-chain diseases. N. Engl. J. Med. 348, 2656–2668 (2003).

    Article  CAS  Google Scholar 

  2. Balaban, R.S., Nemoto, S. & Finkel, T. Mitochondria, oxidants, and aging. Cell 120, 483–495 (2005).

    Article  CAS  Google Scholar 

  3. Szendroedi, J., Phielix, E. & Roden, M. The role of mitochondria in insulin resistance and type 2 diabetes mellitus. Nat. Rev. Endocrinol. 8, 92–103 (2011).

    Article  Google Scholar 

  4. Chandra, D. & Singh, K.K. Genetic insights into OXPHOS defect and its role in cancer. Biochim. Biophys. Acta 1807, 620–625 (2011).

    Article  CAS  Google Scholar 

  5. Eng, C., Kiuru, M., Fernandez, M.J. & Aaltonen, L.A. A role for mitochondrial enzymes in inherited neoplasia and beyond. Nat. Rev. Cancer 3, 193–202 (2003).

    Article  CAS  Google Scholar 

  6. Miro, O. et al. Mitochondrial DNA depletion and respiratory chain enzyme deficiencies are present in peripheral blood mononuclear cells of HIV-infected patients with HAART-related lipodystrophy. Antivir. Ther. 8, 333–338 (2003).

    CAS  PubMed  Google Scholar 

  7. Lebrecht, D., Setzer, B., Ketelsen, U.P., Haberstroh, J. & Walker, U.A. Time-dependent and tissue-specific accumulation of mtDNA and respiratory chain defects in chronic doxorubicin cardiomyopathy. Circulation 108, 2423–2429 (2003).

    Article  CAS  Google Scholar 

  8. Lin, M.T. & Beal, M.F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nature 443, 787–795 (2006).

    Article  CAS  Google Scholar 

  9. Winklhofer, K.F. & Haass, C. Mitochondrial dysfunction in Parkinson's disease. Biochim. Biophys. Acta 1802, 29–44 (2010).

    Article  CAS  Google Scholar 

  10. Hauptmann, S. et al. Mitochondrial dysfunction: an early event in Alzheimer pathology accumulates with age in AD transgenic mice. Neurobiol. Aging 30, 1574–1586 (2009).

    Article  CAS  Google Scholar 

  11. Reisch, A.S. & Elpeleg, O. Biochemical assays for mitochondrial activity: assays of TCA cycle enzymes and PDHc. Methods Cell Biol. 80, 199–222 (2007).

    Article  CAS  Google Scholar 

  12. Villani, G. & Attardi, G. Polarographic assays of respiratory chain complex activity. Methods Cell Biol. 80, 121–133 (2007).

    Article  CAS  Google Scholar 

  13. Kuznetsov, A.V. et al. Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat. Protoc. 3, 965–976 (2008).

    Article  CAS  Google Scholar 

  14. Vives-Bauza, C., Yang, L. & Manfredi, G. Assay of mitochondrial ATP synthesis in animal cells and tissues. Methods Cell Biol. 80, 155–171 (2007).

    Article  CAS  Google Scholar 

  15. Janssen, A.J. et al. Measurement of the energy-generating capacity of human muscle mitochondria: diagnostic procedure and application to human pathology. Clin. Chem. 52, 860–871 (2006).

    Article  CAS  Google Scholar 

  16. Solaini, G., Sgarbi, G., Lenaz, G. & Baracca, A. Evaluating mitochondrial membrane potential in cells. Biosci. Rep. 27, 11–21 (2007).

    Article  CAS  Google Scholar 

  17. Spinazzi, M. et al. Optimization of respiratory chain enzymatic assays in muscle for the diagnosis of mitochondrial disorders. Mitochondrion 11, 893–904 (2011).

    Article  CAS  Google Scholar 

  18. Medja, F. et al. Development and implementation of standardized respiratory chain spectrophotometric assays for clinical diagnosis. Mitochondrion 9, 331–339 (2009).

    Article  CAS  Google Scholar 

  19. Gellerich, F.N., Mayr, J.A., Reuter, S., Sperl, W. & Zierz, S. The problem of interlab variation in methods for mitochondrial disease diagnosis: enzymatic measurement of respiratory chain complexes. Mitochondrion 4, 427–439 (2004).

    Article  CAS  Google Scholar 

  20. Trounce, I.A., Kim, Y.L., Jun, A.S. & Wallace, D.C. Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines. Methods Enzymol. 264, 484–509 (1996).

    Article  CAS  Google Scholar 

  21. Salviati, L. et al. Copper supplementation restores cytochrome c oxidase activity in cultured cells from patients with SCO2 mutations. Biochem. J. 363, 321–327 (2002).

    Article  CAS  Google Scholar 

  22. Angelini, C. et al. Childhood encephalomyopathy with cytochrome c oxidase deficiency, ataxia, muscle wasting, and mental impairment. Neurology 36, 1048–1052 (1986).

    Article  CAS  Google Scholar 

  23. Zheng, X.X., Shoffner, J.M., Voljavec, A.S. & Wallace, D.C. Evaluation of procedures for assaying oxidative phosphorylation enzyme activities in mitochondrial myopathy muscle biopsies. Biochim. Biophys. Acta 1019, 1–10 (1990).

    Article  CAS  Google Scholar 

  24. Frezza, C., Cipolat, S. & Scorrano, L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat. Protoc. 2, 287–295 (2007).

    Article  CAS  Google Scholar 

  25. Palmer, J.W., Tandler, B. & Hoppel, C.L. Biochemical differences between subsarcolemmal and interfibrillar mitochondria from rat cardiac muscle: effects of procedural manipulations. Arch. Biochem. Biophys. 236, 691–702 (1985).

    Article  CAS  Google Scholar 

  26. Jonckheere, A.I., Smeitink, J.A. & Rodenburg, R.J. Mitochondrial ATP synthase: architecture, function and pathology. J. Inherit. Metab Dis. (2011).

  27. Barrientos, A., Fontanesi, F. & Diaz, F. Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using polarography and spectrophotometric enzyme assays. Curr. Protoc. Hum. Genet. 63, 19.3.1–1 (2009).

    Google Scholar 

  28. Grad, L.I., Sayles, L.C. & Lemire, B.D. Isolation and functional analysis of mitochondria from the nematode Caenorhabditis elegans. Methods Mol. Biol. 372, 51–66 (2007).

    Article  CAS  Google Scholar 

  29. Rowley, N. et al. Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding. Cell 77, 249–259 (1994).

    Article  CAS  Google Scholar 

  30. Janssen, A.J. et al. Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts. Clin. Chem. 53, 729–734 (2007).

    Article  CAS  Google Scholar 

  31. Moghaddas, S., Distler, A.M., Hoppel, C.L. & Lesnefsky, E.J. Quinol type compound in cytochrome c preparations leads to non-enzymatic reduction of cytochrome c during the measurement of complex III activity. Mitochondrion 8, 155–163 (2008).

    Article  CAS  Google Scholar 

  32. Fischer, J.C. et al. Investigation of mitochondrial metabolism in small human skeletal muscle biopsy specimens. Improvement of preparation procedure. Clin. Chim. Acta 145, 89–99 (1985).

    Article  CAS  Google Scholar 

  33. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    Article  CAS  Google Scholar 

  34. Chretien, D., Bourgeron, T., Rotig, A., Munnich, A. & Rustin, P. The measurement of the rotenone-sensitive NADH cytochrome c reductase activity in mitochondria isolated from minute amount of human skeletal muscle. Biochem. Biophys. Res. Commun. 173, 26–33 (1990).

    Article  CAS  Google Scholar 

  35. Kirby, D.M., Thorburn, D.R., Turnbull, D.M. & Taylor, R.W. Biochemical assays of respiratory chain complex activity. Methods Cell Biol. 80, 93–119 (2007).

    Article  CAS  Google Scholar 

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Acknowledgements

This work has been supported by a donation from Stevanato Group to M.S., in memory of its founder G. Stevanato; from Telethon Italy grant no. GGP09207; and from a grant from Fondazione Cariparo. This research is part of a project of the Telethon-funded Italian Collaborative Network on Mitochondrial Disorders (GUP09004). The funding source had no role in the conduction of the study. We are grateful to L. Santinello for her assistance as librarian.

AUTHOR CONTRIBUTIONS

M.S. and A.C. designed, and performed experiments, analyzed data and wrote the paper; V.P. performed experiments. L.S. and C.A. analyzed data and critically revised the paper.

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Correspondence to Marco Spinazzi.

Supplementary information

Supplementary Video 1

Muscle homogenization. (SWF 7352 kb)

Table 1

Respiratory chain enzyme activities in illustrative examples of control preparations in muscle and cultured fibroblasts. (PDF 51 kb)

Figure 1

Respiratory chain enzymatic activities in cultured fibroblasts from patients with different mitochondrial disorders. Patients with mutations in two different CIV assembly factors, SURF1 (white bar) and SCO2 (dark grey bar), have isolated CIV deficiency. The patient with mutations in COQ2, a gene required for CoQ10 biosynthesis, displays selective complex II+III deficiency (light grey bar). The patient with the C5545T mtDNA mutation in tRNAtrp, which impairs mitochondrial protein synthesis, has a generalized defect of complexes containing mtDNA encoded subunits (CI, II+III, III, IV; black bar). (PDF 7 kb)

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Spinazzi, M., Casarin, A., Pertegato, V. et al. Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells. Nat Protoc 7, 1235–1246 (2012). https://doi.org/10.1038/nprot.2012.058

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