After euthanasia of 5- to 6-month-old mice, muscle tension and position were normalized by pinning the entire skinned hindlimb on a paraffin-coated dish, with the metatarsus making an angle of 90° with the tibia. The limb was then fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2), at room temperature for 4 hours. Muscles were cut in small pieces and kept overnight in the same fixation buffer at 4°C. Samples were then processed at the Microscopy and Imaging Facility for Microbes, Animals and Foods MIMA2 platform, INRAE. They were contrasted with 0.5% oolong tea extract in cacodylate buffer, postfixed with a solution of 1% osmium tetroxide and 1.5% potassium cyanoferrate, and gradually dehydrated in ethanol (30 to 100%) and substituted gradually in a mix of propylene oxyde-epon and embedded in Epon (Delta microscopy). Thin sections (70 nm) were collected onto 200-mesh copper grids, and counterstained with lead citrate. Grids were examined with a Hitachi HT7700 electron microscope operating at 80 kV (Elexience). Images were acquired with a charge-coupled device camera (AMT).

For cristae quantification, 20 images at ×12,000 magnification were analyzed per animal, and all visible cristae were assessed using the function plot profile on ImageJ (v1.47, NIH). Mean cristae width corresponded to the maximal diameter of the tubular part and was calculated for each crista of each analyzed mitochondrion. For each animal, a minimum of 100 cristae was measured for the superficialis gastrocnemius muscle and 800 cristae for the soleus muscle. All images were analyzed in a blinded manner.

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