Garcia-Roves 2017 MiP2017
During our last WG2 meeting, MITOEAGLE Obergurgl July 2017, we agreed to start generating mitochondrial respirometry data sets following the same experimental procedure in different laboratories. The results obtained could serve as “reference values” for permeabilized skeletal muscle sample preparations. The idea is that new researchers in the field could perform the same experimental procedure and check if their values are in the range, for the different parameters assessed. It should serve to test researchers' technical skills and therefore determine if he/she is ready to perform his/her own experiments with confidence.
We suggested to run the pilot experiments initially in a few groups; to check that the experimental procedure works similarly in different labs and that the results obtained show, as we expect, low variability between labs. If we are satisfied with our initial approach and results, then we will open the invitation to all labs interested and ask them to actively participate. The final aim of this task is to collect all the data reported and write a first manuscript during 2018.
To obtain respirometry reference values in permeabilized skeletal muscle fibers all research groups involved use C57BL/6J mice, both male (N=4) and female (N=4), age 14-16 weeks, and dissect soleus muscle for our analysis. During the presentation, we will be giving detailed information of the experimental procedure followed to get permeabilized skeletal muscle fibers. The substrates, uncouplers, inhibitors titration (SUIT) protocol that we are assessing is represented in Figure 1. Finally, we will be presenting and discussing our preliminary data. This presentation should set the stage to initiate a discussion and define our next actions in this WG2 specific task.
• Bioblast editor: Kandolf G • O2k-Network Lab: ES Barcelona Garcia-Roves PM, ES Barcelona IDIBAPS Hospital Clinic, FR Montpellier Wrutniak-Cabello C, AT Innsbruck Oroboros, NL Nijmegen Koopman WJ, US LA Baton Rouge Irving BA
Labels: MiParea: Respiration
Organism: Mouse Tissue;cell: Skeletal muscle Preparation: Permeabilized tissue
Coupling state: OXPHOS, ET Pathway: N, S, NS, ROX HRR: Oxygraph-2k
Figure 1: SUIT protocol. Saturated concentrations of pyruvate (P); malate (M); ADP with MgCl2 (D*), saturated ADP must be tested in each experiment; cytochrome c (c); glutamate (G); succinate (S); ADP (D*): add 2.5 mM to check if ADP is saturated after the addition of succinate; FCCP/CCCP (U*): 0.5 µM steps to reach the maximum respiration (ETS), report the uncoupler used (FCCP or CCCP); rotenone (R); Antimycin A (Ama).
- Garcia-Roves PM(1), Chabi B(2), Doerrier C(3), Dubouchaud H(4), Grefte S(5), Irving B(6), Ost M(7), Pesta D(4)
- Dept Physiological Sciences, Univ Barcelona, Spain
- INRA, UMR866 Dynamique Musculaire Métabolisme, Univ Montpellier, France
- Oroboros Instruments, Austria
- Lab Bioénergétique Fondamentale Appliquée, Univ Grenoble Alpes, France
- Human Animal Physiol, Wageningen Univ, The Netherlands
- School Kinesiology, Louisiana State Univ, LA, USA
- German Inst Human Nutrition Potsdam-Rehbruecke, Germany
- German Center for Diabetes Research, Germany. - [email protected]