Chabi 2019 MitoFit Preprint Arch EA

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Chabi 2019 MitoFit Preprint Arch EA

Chabi Béatrice, 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. https://doi.org/10.26124/mitofit:ea19.MitoEAGLE.0001

» MitoFit Preprint Arch EA19.1

Generating reference values on mitochondrial respiration in permeabilized muscle fibers

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 Marisol, Acuna-Castroviejo D, Villena JA, Grefte S, Keijer J, O'Brien K, Sowton A, Murray AJ, Campbell MD, Marcinek DJ, Nollet E, Wuest R, Dayanidhi S, Gnaiger Erich, Doerrier C, Garcia-Roves PM (2019) MitoFit Preprint Arch

Abstract: Version 1 (v1) 2019-10-10 doi:10.26124/mitofit:ea19.MitoEAGLE.0001

Permeabilized muscle fibers (pfi) are widely used to assess mitochondrial (mt) respiratory function in skeletal muscle of various models in different physiological and pathological conditions. Facing the numerous data available for mt-respiration from the literature, it remains challenging to determine what the right values are for a specific respiratory protocol. Moreover, mt-respiration values are highly dependent on pfi preparation, which required good technical skills.

In the frame of COST Action MITOEAGLE, one of the objectives of WG2 is the generation of reference values for mitochondrial respirometry in permeabilized skeletal muscle sample preparations. The idea is that new researchers in the field follow a reference protocol and check if their values are in an acceptable range. This approach could serve to test researchers’ technical skills and therefore determine if they are proficient enough to perform their own experiments with confidence. - Extended abstract

Affiliations

Chabi Béatrice (1), Ost M (2), Gama-Perez P (3), Dahdah N (3), Lemieux H (4), Holody CD (4), Carpenter RG (4), Tepp K (5), Puurand M (5), Kaambre T (5), Dubouchaud H (6), Cortade F (1), Pesta D (7,8), Calabria E (9), Casado M (1)0, Fernandez-Ortiz M (11), Acuña-Castroviejo D (11), Villena JA (12), Grefte S (13), Keijer J (13), O'Brien K (14), Sowton A (14), Murray AJ (14), Campbell MD (15), Marcinek DJ (15), Nollet E (16), Wüst R (16), Dayanidhi S (17), Gnaiger E (18,19), Doerrier C (18), Garcia-Roves PM (3)

1. DMEM, INRA, Univ Montpellier, France - [email protected]
2. German Institute of Human Nutrition Potsdam-Rehbruecke, Germany
3. Dept Physiological Sciences, Univ Barcelona and Bellvitge Biomedical Research Institute (IDIBELL) Spain
4. Faculty Saint-Jean, Univ Alberta, Canada
5. Lab of Chemical Biology, National Inst of Chemical Physics and Biophysics, Estonia
6. Lab Bioénergétique Fondamentale et Appliquée, Univ Grenoble Alpes, INSERM, U1055, France
7. Institute for Clinical Diabetology, German Diabetes Center, Leibniz Center for Diabetes Research at Heinrich-Heine Univ Düsseldorf, Germany
8. German Center for Diabetes Research, Munich, Neuherberg, Germany
9. Dept of Neurological and Movement Sciences, Univ of Verona, Italy
10. Dept of Molecular and Cellular Pathology and Therapy, Instituto de Biomedicina de Valencia, Spain
11. Biomedical Research Center, Univ of Granada, Spain
12. Metabolism and Obesity Lab, Vall d’Hebron Research Inst, Spain
13. Human and Animal Physiology, Wageningen Univ, The Netherlands;
14. Dept of Physiology, Development & Neuroscience, Univ of Cambridge, UK
15. Dept of Radiology, Univ of Washington, South Lake Union, USA
16. Dept of Human Movement Sciences, Faculty of Behavioural and Movement Sciences, Vrije Univ Amsterdam, The Netherlands
17. Rehabilitation Institute of Chicago, Feinberg School of Medicine, Northwestern Univ, USA
18. Oroboros Instruments, Austria
19. Dept Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ Innsbruck, Austria

Figures

Figure 1. Substrate-uncoupler-inhibitor titration protocol (SUIT-008 O2 pfi D014). Sequential titrations and respiratory states. 1PM: NADH-pathway (N-pathway) :in the presence of 5 mM pyruvate and 2 mM malate in the N-LEAK state. 2D: saturating ADP (N-OXPHOS state). 2c: 10 μM cytochrome c for evaluating the integrity :of the outer mitochondrial membrane. 3G: 10 mM glutamate as an additional NADH-linked substrate (N-OXPHOS state). 4S: 10 mM succinate (NS-OXPHOS capacity). :5U: uncoupler titrations to evaluate the electron transfer- (ET-) capacity (NS-ET capacity). 6Rot: inhibition of CI by rotenone (S-ET capacity). 7Ama: :inhibition of CIII by antimycin A (residual oxygen consumption, Rox). Oxygen concentration range in the experiment was maintained between 400-250 µM O2.

Figure 2. NS-OXPHOS capacity of permeabilized soleus muscle fibers. NS-OXPHOS capacity was measured in permeabilized soleus muscle fibers from male C57BL/6J mice by :16 research groups. Median with interquartile range show results from individual group with muscle fibers obtained from at least four soleus muscles.

References

1. Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep 7:2840, DOI:10.1038/s41598-017-02789-8. - https://www.bioblast.at/index.php/SUIT-8_O2_pfi_D14
2. Baker M (2016) 1,500 scientists lift the lid on reproducibility. Survey sheds light on the ‘crisis’ rocking research. Nature 533:452–4.

Support

This preprint is based upon work from COST Action CA15203 MitoEAGLE, supported by COST (European Cooperation in Science and Technology).

• Bioblast editor: Iglesias-Gonzalez J • O2k-Network Lab: FR Montpellier Wrutniak-Cabello C, ES Barcelona Garcia-Roves PM, CA Edmonton Lemieux H, EE Tallinn Kaambre T, EE Tallinn Saks VA, DE Duesseldorf Roden M, IT Verona Calabria E, ES Valencia Casado Pinna M, ES Granada Acuna-Castroviejo D, NL Nijmegen Koopman WJH, NL Wageningen Keijer J, US AK Fairbanks O'Brien K, UK Cambridge Murray AJ, US WA Seattle Marcinek DJ, NL Amsterdam Nollet E, NL Amsterdam Wuest RC, US IL Chicago Dayanidhi S, AT Innsbruck Gnaiger E, AT Innsbruck MitoFit, AT Innsbruck Oroboros.

Labels: MiParea: Respiration, Instruments;methods 

Organism: Human  Tissue;cell: Skeletal muscle  Preparation: Permeabilized tissue 

Coupling state: LEAK, OXPHOS, ET  Pathway: N, S, NS, ROX  HRR: Oxygraph-2k 

Preprints, MitoEAGLEPublication, soleus