Microscale Oxygraphy Reveals OXPHOS Impairment in MRC Mutant Cells

Overview

Journal Mitochondrion

Specialty Cell Biology

Date 2012 Feb 8

PMID 22310368

Citations 65

Authors

F Invernizzi

I DAmato

P B Jensen

S Ravaglia

M Zeviani

V Tiranti

Affiliations

Soon will be listed here.

Abstract

Given the complexity of the respiratory chain structure, assembly and regulation, the diagnostic workout for the identification of defects of oxidative phosphorylation (OXPHOS) is a major challenge. Spectrophotometric assays, that measure the activity of individual respiratory complexes in tissue and cell homogenates or isolated mitochondria, are highly specific, but their utilization is limited by the availability of sufficient biological material and intrinsic sensitivity. A further limitation is tissue specificity, which usually determines attenuation, or disappearance, in cultured fibroblasts, of defects detected in muscle or liver. We used numerous fibroblast cell lines derived from patients with OXPHOS deficiencies to set up experimental protocols required for the direct readout of cellular respiration using the Seahorse XF96 apparatus, which measures oxygen consumption rate (OCR) and extra-cellular acidification rate (ECAR) in 96 well plates. Results demonstrate that first level screening based on microscale oxygraphy is more sensitive, cheaper and rapid than spectrophotometry for the biochemical evaluation of cells from patients with suspected mitochondrial disorders.

Citing Articles

A novel MT-ATP6 variant associated with complicated ataxia in two unrelated Italian patients: case report and functional studies.

Sala D, Marchet S, Nanetti L, Legati A, Mariotti C, Lamantea E Orphanet J Rare Dis. 2024; 19(1):200.

PMID: 38755691 PMC: 11100036. DOI: 10.1186/s13023-024-03212-y.

Emerging variants, unique phenotypes, and transcriptomic signatures: an integrated study of COASY-associated diseases.

Cavestro C, Morra F, Legati A, DAmato M, Nasca A, Iuso A Ann Clin Transl Neurol. 2024; 11(6):1615-1629.

PMID: 38750253 PMC: 11187879. DOI: 10.1002/acn3.52079.

Genetic, metabolic and clinical delineation of an MRPS23-associated mitochondrial disorder.

Ittiwut C, Ittiwut R, Kuptanon C, Matsuhashi T, Shimura M, Sugiyama Y Sci Rep. 2023; 13(1):22005.

PMID: 38086984 PMC: 10716371. DOI: 10.1038/s41598-023-49161-7.

Evaluation of Mitochondrial Dysfunction and Idebenone Responsiveness in Fibroblasts from Leber's Hereditary Optic Neuropathy (LHON) Subjects.

Baglivo M, Nasca A, Lamantea E, Vinci S, Spagnolo M, Marchet S Int J Mol Sci. 2023; 24(16).

PMID: 37628761 PMC: 10454080. DOI: 10.3390/ijms241612580.

Mitochondrial Dysfunction in Peripheral Blood Mononuclear Cells as Novel Diagnostic Tools for Non-Alcoholic Fatty Liver Disease: Visualizing Relationships with Known and Potential Disease Biomarkers.

Garrafa E, Segala A, Vezzoli M, Bottani E, Zanini B, Vetturi A Diagnostics (Basel). 2023; 13(14).

PMID: 37510108 PMC: 10378438. DOI: 10.3390/diagnostics13142363.

References

Fernandez-Vizarra E, Bugiani M, Goffrini P, Carrara F, Farina L, Procopio E . Impaired complex III assembly associated with BCS1L gene mutations in isolated mitochondrial encephalopathy. Hum Mol Genet. 2007; 16(10):1241-52. DOI: 10.1093/hmg/ddm072. View

Bugiani M, Tiranti V, Farina L, Uziel G, Zeviani M . Novel mutations in COX15 in a long surviving Leigh syndrome patient with cytochrome c oxidase deficiency. J Med Genet. 2005; 42(5):e28. PMC: 1736058. DOI: 10.1136/jmg.2004.029926. View

Kirby D, Thorburn D, Turnbull D, Taylor R . Biochemical assays of respiratory chain complex activity. Methods Cell Biol. 2007; 80:93-119. DOI: 10.1016/S0091-679X(06)80004-X. View

Smeitink J, Zeviani M, Turnbull D, Jacobs H . Mitochondrial medicine: a metabolic perspective on the pathology of oxidative phosphorylation disorders. Cell Metab. 2006; 3(1):9-13. DOI: 10.1016/j.cmet.2005.12.001. View

Bugiani M, Invernizzi F, Alberio S, Briem E, Lamantea E, Carrara F . Clinical and molecular findings in children with complex I deficiency. Biochim Biophys Acta. 2004; 1659(2-3):136-47. DOI: 10.1016/j.bbabio.2004.09.006. View

Rustin P, Chretien D, Bourgeron T, Gerard B, Rotig A, Saudubray J . Biochemical and molecular investigations in respiratory chain deficiencies. Clin Chim Acta. 1994; 228(1):35-51. DOI: 10.1016/0009-8981(94)90055-8. View

Brand M, Nicholls D . Assessing mitochondrial dysfunction in cells. Biochem J. 2011; 435(2):297-312. PMC: 3076726. DOI: 10.1042/BJ20110162. View

Rogers G, Brand M, Petrosyan S, Ashok D, Elorza A, Ferrick D . High throughput microplate respiratory measurements using minimal quantities of isolated mitochondria. PLoS One. 2011; 6(7):e21746. PMC: 3143121. DOI: 10.1371/journal.pone.0021746. View

Acin-Perez R, Fernandez-Silva P, Peleato M, Perez-Martos A, Enriquez J . Respiratory active mitochondrial supercomplexes. Mol Cell. 2008; 32(4):529-39. DOI: 10.1016/j.molcel.2008.10.021. View

10.

Rodenburg R . Biochemical diagnosis of mitochondrial disorders. J Inherit Metab Dis. 2010; 34(2):283-92. PMC: 3063578. DOI: 10.1007/s10545-010-9081-y. View

11.

Janssen A, Trijbels F, Sengers R, Smeitink J, van den Heuvel L, Wintjes L . Spectrophotometric assay for complex I of the respiratory chain in tissue samples and cultured fibroblasts. Clin Chem. 2007; 53(4):729-34. DOI: 10.1373/clinchem.2006.078873. View

12.

DAurelio M, Vives-Bauza C, Davidson M, Manfredi G . Mitochondrial DNA background modifies the bioenergetics of NARP/MILS ATP6 mutant cells. Hum Mol Genet. 2009; 19(2):374-86. PMC: 2796897. DOI: 10.1093/hmg/ddp503. View

13.

Benit P, Goncalves S, Dassa E, Briere J, Martin G, Rustin P . Three spectrophotometric assays for the measurement of the five respiratory chain complexes in minuscule biological samples. Clin Chim Acta. 2006; 374(1-2):81-6. DOI: 10.1016/j.cca.2006.05.034. View

14.

Wittig I, Schagger H . Supramolecular organization of ATP synthase and respiratory chain in mitochondrial membranes. Biochim Biophys Acta. 2009; 1787(6):672-80. DOI: 10.1016/j.bbabio.2008.12.016. View

15.

Paumard P, Vaillier J, Coulary B, Schaeffer J, Soubannier V, Mueller D . The ATP synthase is involved in generating mitochondrial cristae morphology. EMBO J. 2002; 21(3):221-30. PMC: 125827. DOI: 10.1093/emboj/21.3.221. View

16.

Giorgio V, Petronilli V, Ghelli A, Carelli V, Rugolo M, Lenaz G . The effects of idebenone on mitochondrial bioenergetics. Biochim Biophys Acta. 2011; 1817(2):363-9. PMC: 3265671. DOI: 10.1016/j.bbabio.2011.10.012. View

17.

Mattiazzi M, Vijayvergiya C, Gajewski C, Devivo D, Lenaz G, Wiedmann M . The mtDNA T8993G (NARP) mutation results in an impairment of oxidative phosphorylation that can be improved by antioxidants. Hum Mol Genet. 2004; 13(8):869-79. DOI: 10.1093/hmg/ddh103. View

18.

Ghezzi D, Arzuffi P, Zordan M, Da Re C, Lamperti C, Benna C . Mutations in TTC19 cause mitochondrial complex III deficiency and neurological impairment in humans and flies. Nat Genet. 2011; 43(3):259-63. DOI: 10.1038/ng.761. View

19.

Valente L, Piga D, Lamantea E, Carrara F, Uziel G, Cudia P . Identification of novel mutations in five patients with mitochondrial encephalomyopathy. Biochim Biophys Acta. 2008; 1787(5):491-501. DOI: 10.1016/j.bbabio.2008.10.001. View

20.

Massa V, Fernandez-Vizarra E, Alshahwan S, Bakhsh E, Goffrini P, Ferrero I . Severe infantile encephalomyopathy caused by a mutation in COX6B1, a nucleus-encoded subunit of cytochrome c oxidase. Am J Hum Genet. 2008; 82(6):1281-9. PMC: 2427282. DOI: 10.1016/j.ajhg.2008.05.002. View