Doerrier 2020 PaduaMuscleDays

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Doerrier C, Di Marcello M, Gnaiger E (2020) Commitment to reproducibility in targeting mitochondrial respiratory control: basic and advanced applications of the O2k-FluoRespirometer. PaduaMuscleDays.

Link: Padova Muscle Days

Doerrier C, Di Marcello M, Gnaiger E (2020)

Event: 2020 PaduaMuscleDays Padova IT

Mitochondria play a crucial role in health and disease, imposing a growing demand for evaluation of mitochondrial function in inherited and preventable degenerative diseases. These are associated with lack of physical exercise and obesity, causing decline of mitochondrial fitness and early aging. High-resolution respirometry (HRR) is based on the state-of-the-art instrument (Oroboros O2k, Innsbruck, Austria) for mitochondrial and cell respiration[1]. The O2k combines real-time respirometry with simultaneous measurement of H2O2 production, mt-membrane potential, ADP-ATP exchange, pH, Ca2+, or nitric oxide. HRR can be applied with a wide spectrum of sample preparations (e.g. isolated mitochondria, tissue homogenate, permeabilized fibers, permeabilized cells, living cells). Optimized substrate-uncoupler-inhibitor titration (SUIT) protocols offer a powerful tool for comprehensive mitochondrial physiology analysis by HRR[2]. Peripheral blood mononuclear cells, platelets and permeabilized fibers are relevant models for diagnostic analysis of oxidative phosphorylation (OXPHOS) in clinical applications (e.g. aging, sarcopenia, obesity). Science and particularly mitochondrial respiratory research face a reproducibility crisis[3]. Consequently, Oroboros is strongly committed towards reproducibility by promoting:

  1. O2k quality control (i.e. calibrations, instrumental background, DatLab software);
  2. proficiency tests with reference samples for inter-laboratory harmonization[4,5]; and
  3. training by scientific experts.

Supported by the COST Action MitoEAGLE network, we generate reference values for an Open-Access mitochondrial respirometry database, which requires harmonization of nomenclature[6]. With a large O2k-Network, the Oroboros Ecosystem operates in the frame of collaborative Open-Innovation (NextGen-O2k with Q-redox sensor, NAD(P)H autofluorescence and PhotoBiology module) to promote quality and reproducibility in mitochondrial to promote quality and reproducibility in mitochondrial physiology.


Bioblast editor: Plangger M O2k-Network Lab: AT Innsbruck Oroboros, AT Innsbruck Gnaiger E, AT Innsbruck MitoFit


Labels: MiParea: Respiration, Instruments;methods 


Tissue;cell: Skeletal muscle, Blood cells  Preparation: Permeabilized cells, Permeabilized tissue, Homogenate, Isolated mitochondria, Intact cells 


Coupling state: OXPHOS 

HRR: Oxygraph-2k, O2k-Fluorometer 


Affiliations and support

Carolina Doerrier(1), Marco Di Marcello(1), Erich Gnaiger(1,2)
  1. Oroboros Instruments, Innsbruck
  2. D Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Med Univ Innsbruck; Austria. - erich.gnaiger@oroboros.at
Support: Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE. Supported by project NextGen-O2k which has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No. 859770.

References

  1. Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70.
  2. Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Mitochondr Physiol Network 25.05. Oroboros MiPNet Publications, Innsbruck.
  3. Baker M (2016) 1,500 scientists lift the lid on reproducibility. Survey sheds light on the ‘crisis’ rocking research. Nature 533:452–4.
  4. Doerrier C, Gama-Perez P, Distefano G, Pesta D, Soendergaard SD, Chroeis KM, Gonzalez-Franquesa A, Goodpaster BH, Coen P, Larsen S, Gnaiger E, Garcia-Roves PM (2019) Inter-laboratory harmonization of respiratory protocols in permeabilized human muscle fibers. MitoFit Preprint Arch. doi:10.26124/mitofit:ea19. MiPSchool .0009.
  5. Chabi B, Ost M, Gama-Perez P, Dahdah N, Lemieux H, Holody CD, Carpenter RG, Tepp K, Puurand M, Kaambre T, Dubouchaud H, Cortade F, Pesta D, Calabria E, Casado M, Fernandez-Ortiz M, Acuña-Castroviejo D, Villena JA, Grefte S, Keijer J, O'Brien K, Sowton A, Murray AJ, Campbell MD, Marcinek DJ, Nollet E, Wüst R, Dayanidhi S, Gnaiger E, Doerrier C, Garcia-Roves PM (2019) Generating reference values on mitochondrial respiration in permeabilized muscle fibers. MitoFit Preprint Arch. doi:10.26124/mitofit:ea19.MitoEAGLE.0001.
  6. Gnaiger E, Aasander Frostner E, Abdul Karim N, et al. (2019) Mitochondrial respiratory states and rates. MitoFit Preprint Arch. doi:10.26124/ mitofit:190001.v6.