Mice (4 months old) were euthanized, and mitochondria were isolated from tibialis anterior muscles as described above and immediately frozen in liquid nitrogen and protected from oxidation by a layer of argon gas and stored at −80°C until analysis. Quantitation of fatty acids and glycerophospholipids contents was performed as previously described for myoblasts (20, 54). Phospholipid species representation was performed as described hereafter. Briefly, total phospholipid amount was determined with Corona CAD detection by calculating the concentration of each phospholipid class (in microgram/ml) compared to calibration ranges of commercial standards. Chromatographic system from Thermo Fisher Scientific included a Dionex U-3000 Rapid Separation Liquid Chromatography (RSLC) system with two quaternary pumps, an autosampler, and a column oven. The RSLC system was coupled online to a charged aerosol detector, Corona-CAD Ultra for the quantitative part of the study and: a LTQ-Orbitrap Velos Pro for the lipidomic study (all from Thermo Fisher Scientific). Liquid chromatography, mass spectrometry, phospholipid species identification, and phospholipid species representation were performed as previously described (55). Statistical analysis was performed using multiple pairwise tests according to the Holm-Bonferroni method.

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