Chabi 2017 MITOEAGLE Obergurgl

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Optimization of permeabilized fibers preparation for mitochondrial respiration measurements using Design of Experiments methodology.

Link: MitoEAGLE

Cortade F, Gaillard V, Chabi B (2017)

Event: MitoEAGLE Obergurgl 2017

COST Action MitoEAGLE

Mitochondrial respiration is commonly studied using isolated mitochondria in skeletal muscle. Several data also showed that measuring respiration in permeabilized fibers (pf) that preserve mitochondrial morphology and intracellular interactions is of particular interest to get more information the regulation of this organelle activity in different knockout and transgenic mice models [1,2].

To optimize the pf preparation from mouse Tibialis anterior in our lab, we used the Design of Experiments (DoE) methodology that evaluates the impact of several experimental conditions or factors, on the pf respiration parameters (Pyruvate Malate Succinate respiration (PMS leak) and respiratory control ratio (RCRPMS). This DoE uses the Hadamard matrix of order 8, corresponding to eight experiments with 4 repetitions to evaluate the experiment variability. We measured the effect of 6 factors such as fiber types (red vs white), manual teasing (gentle vs rough), sapogenin content of saponin (8 - 25% vs 20 - 35%), saponin concentration for permeabilization (25 vs 50 µg/ml), incubation time with saponin (10 vs 30 mn) and muscle resting period in buffer before the teasing step (0h vs 6h).

Our results showed the PMS leak respiration rate was the highest for pf that were prepared from red fibers with a rough teasing and sapogenin content of 20-35%. Muscle resting period did not significantly affect the respiration, with no deleterious effect of the 6h resting period. The two remaining factors did not impact PMS leak respiration rate (saponin concentration and permeabilization time). In addition, RCRPMS variability was the lowest using the optimized conditions determined for PMS leak respiration.

Using a DoE analysis, we were able to optimize pf assay conditions with a reduced number of experiments and animals, and rapidly obtain valuable data in accordance with ethical recommendations. The optimization of pf preparation by DoE will be pursued with two goals (i) studying the possible interactions existing between the 3 factors related to saponin (sapogenin content, saponin concentration and incubation time), (ii) calculating the optimal sample size (n) needed to observe statistically significant differences between two animal groups.


Bioblast editor: Kandolf G


Labels: MiParea: Respiration 


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


Coupling state: LEAK  Pathway: NS 

Event: A1, Poster 


Affiliations

(1)Cortade F, (2)Gaillard V, (1)Chabi B
  1. INRA, UMR DMEM, Univ Montpellier
  2. ITG, Inst Temps Géré, Paris; France.- fabienne.cortade@inra.fr, beatrice.chabi@inra.fr

References

  1. Picard M, Ritchie D, Wright KJ, Romestaing C, Thomas MM, Rowan SL, Taivassalo T, Hepple RT (2010) Mitochondrial functional impairment with aging is exaggerated in isolated mitochondria compared to permeabilized myofibers. Aging Cell 9:1032-46.
  2. Britto F, Cortade F, Belloum Y, Blaquiere M, Gallot Y, Docquier A, Pagano A, Jublanc E, Bendridi N, Ramonatxo C, Chabi B, Francaux M, Casas F, Freyssenet D, Rieusset J, Giorgetti Peraldi S, Carnac G, Ollendorf V ,Favier F. REDD1 reduces muscle metabolism to foster adaptation under energetic stress. (submitted).