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. MitoFit Preprint Arch EA19.1. https://doi.org/10.26124/mitofit:ea19.MitoEAGLE.0001 |
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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)
- DMEM, INRA, Univ Montpellier, France - beatrice.chabi@inra.fr
- German Institute of Human Nutrition Potsdam-Rehbruecke, Germany
- Dept Physiological Sciences, Univ Barcelona and Bellvitge Biomedical Research Institute (IDIBELL) Spain
- Faculty Saint-Jean, Univ Alberta, Canada
- Lab of Chemical Biology, National Inst of Chemical Physics and Biophysics, Estonia
- Lab Bioénergétique Fondamentale et Appliquée, Univ Grenoble Alpes, INSERM, U1055, France
- Institute for Clinical Diabetology, German Diabetes Center, Leibniz Center for Diabetes Research at Heinrich-Heine Univ Düsseldorf, Germany
- German Center for Diabetes Research, Munich, Neuherberg, Germany
- Dept of Neurological and Movement Sciences, Univ of Verona, Italy
- Dept of Molecular and Cellular Pathology and Therapy, Instituto de Biomedicina de Valencia, Spain
- Biomedical Research Center, Univ of Granada, Spain
- Metabolism and Obesity Lab, Vall d’Hebron Research Inst, Spain
- Human and Animal Physiology, Wageningen Univ, The Netherlands;
- Dept of Physiology, Development & Neuroscience, Univ of Cambridge, UK
- Dept of Radiology, Univ of Washington, South Lake Union, USA
- Dept of Human Movement Sciences, Faculty of Behavioural and Movement Sciences, Vrije Univ Amsterdam, The Netherlands
- Rehabilitation Institute of Chicago, Feinberg School of Medicine, Northwestern Univ, USA
- Oroboros Instruments, Austria
- 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
- 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
- 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 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