The Effect of FFAs on the Mitochondrial Respiration

To test the effect of FFAs on the mitochondrial respiration, patient and control myoblasts were plated (as described in Section 2.2.1) 16 h prior to Mito stress test measurements, Bovine serum albumin (BSA)-conjugated palmitic acid or oleic acid (ratio FFA: BSA = 6:1) was added to the media. The final concentration of oleate or palmitate was 300 µM. Comparable amounts of BSA were added in the untreated group.

#### 2.2.2. Fatty Acid Oxidation

To assess the ability of myoblasts to oxidize exogenous fatty acids, the oxygen-consumption rate (OCR) was analyzed using a XF96 Cell Analyzer according to the manufacturer's protocol (Seahorse Bioscience). For this purpose, the myoblast of patients and controls were plated (as described in Section 2.1) 16-h prior to measurements, growth medium was replaced by substrate-limited medium (Dulbecco's modified Eagle's medium (DMEM) with 0.5 mM glucose, 1.0 mM glutamine, 0.5 mM carnitine, and 1% FBS) and incubated at 37 ◦C or 40 ◦C. Moreover, 45 min before the beginning of OCR measurement, the medium was changed into pre-warmed FAO Assay Medium (111 mM NaCl, 4.7 mM KCl, 2.0 mM MgSO4, 1.2 mM Na2HPO4, 2.5 mM glucose, 0.5 mM carnitine, and 5 mM HEPES). Just before measurements, palmitic acid or oleic acid were added to a final concentration of 167 µM total FA (bound in a 6:1 molar ratio to BSA) as substrate. The data were normalized to cell numbers.

All Seahorse experiments were repeated at least twice. All data shown are the means ± SD of at least 3 different wells per group.

All chemicals used for these assays were obtained from Sigma Aldrich (St Louis, MO, USA).
