# Generating reference values on mitochondrial respiration in permeabilized muscle fibers

Béatrice Chabi, Mario Ost, Pau Gama-Perez, Nora Dahdah, Hélène Lemieux, Claudia Holody, Carpenter Rg, Kersti Tepp, Marju Puurand, Tuuli Kaambre, et al.

► To cite this version:

Béatrice Chabi, Mario Ost, Pau Gama-Perez, Nora Dahdah, Hélène Lemieux, et al. Generating reference values on mitochondrial respiration in permeabilized muscle fibers. MiP2019/MitoEAGLE - 14th Conference on Mitochondrial Physiology: Mitochondrial function: changes during life cycle and in noncommunicable diseases, Mitochondrial Physiology Society, Oct 2019, Belgrade, Serbia. 3p., 10.26124/mitofit:ea19.MitoEAGLE.0001. hal-02941367

HAL Id: hal-02941367

https://hal.inrae.fr/hal-02941367

Submitted on 17 Sep 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Generating reference values on mitochondrial respiration in
permeabilized muscle fibers

Chabi Béatrice¹✉, Ost M², Gama-Perez P³, Dahdah N³, Lemieux H⁴, Holody CD⁴, Carpenter RG⁴,
Tepp K⁵, Puurand M⁵, Kaambre T⁵, Dubouchaud H⁶, Cortade F¹, Pesta D⁷,⁸, Calabria E ⁹, Casado
M¹⁰, Fernandez-Ortiz M¹¹, Acuña-Castroviejo D¹¹, Villena JA¹², Grefte S¹³, Keijer J¹³, O'Brien
K¹⁴, Sowton A¹⁴, Murray AJ¹⁴, Campbell MD¹⁵, Marcinek DJ¹⁵, Nollet E¹⁶, Wüst R¹⁶, Dayanidhi
S¹⁷, Gnaiger E¹⁸,¹⁹, Doerrier C¹⁸, Garcia-Roves PM³✉

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

<figure><img src="image_2.png" /></figure>

beatrice.chabi@inra.fr pgarciaroves@ub.edu

https://wiki.oroboros.at/index.php/Crispim_2019_MitoFit_Preliminary_Arch

<figure><img src="image_3.png" /></figure>

MitoFit
Preprint Arch

Free pdf

© 2019 Chabi et al. This is an Open Access extended abstract (not peer-reviewed) distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are credited. © remains with the authors, who have granted MitoFit an Open Access preprint license in perpetuity.

Editor MitoFit Preprint Archives: Gnaiger E

**Introduction**

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.

## Materials and Methods

Sixteen international research groups participated to this study and received males (N=4) and females (N=4) C57BL/6J mice aged 14-16 weeks from the same provider. They performed permeabilized fibers from soleus muscle according to a common defined protocol.

Mt-respiration was measured in respiration media MiR05-Kit following the substrate-uncoupler-inhibitor titration (SUIT) protocol SUIT-008_O2_pfi_D014 represented in Figure1 [1]. Chemicals were provided by each group.

## Results

Preliminary analyses of oxygen fluxes showed variability between the groups with median values for NS-OXPHOS capacity ranging from 116 to 335 pmol·s⁻¹·mg tissue⁻¹ (Figure 2). Sources of this variability are currently under investigation through the

analysis of respiratory traces to define exclusion criteria as well as a questionnaire gathering technical information and operator skills.

This unique international study has the ambition to significantly contribute to the generation of a mt-database that will help research groups in the investigation of mitochondrial physiology and pathology. Moreover, such database will help in the harmonization of mt-respiratory protocols and the resolution of the reproducibility crisis in mt-research [2].

<figure><img src="image_4.png" /></figure>

**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><img src="image_5.png" /><figcaption>**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.</figcaption></figure>

## 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.