Sodium palmitate (Sigma) was conjugated to fatty acid–free bovine serum albumin (BSA) (Calbiochem) at 1 mM and pH adjusted to 7.4 (41). Seahorse medium was composed of unbuffered DMEM base (Sigma) containing 5 mM glucose, 50 μM palmitate, 200 μM carnitine, and 100 μM pyruvate, pH adjusted to 7.4 at 37°C (41). Adult cardiomyocytes were spun down at 100g for 30 s and exposed to Seahorse medium. Cells were then counted and plated at 1500 cells per well on laminin-coated Seahorse 96-well plates. Reperfusion was timed to 1 hour before protocol initiation. Measurements were conducted on the XF96 Seahorse Analyzer. OCR measurements in cardiomyocytes were assured to be stable for the duration of the experiment. Drug-optimized values for maximal responses were as follows: 1.3 μM oligomycin, 1 μM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 1 μM rotenone, 1 μM antimycin A, 25 mM 2-DG, and 50 μM etomoxir. At the end of the Seahorse assay, the plate was subjected to a protein assay (Thermo Fisher Scientific) for normalization parameters. Oligomycin was used to inhibit ATP synthase, FCCP was used to deplete the proton gradient, and rotenone/antimycin A was used to inhibit complexes 1 and 3, respectively, thus stopping all mitochondrial-mediated respiration. Glucose oxidation was inhibited by using 2-DG, and fatty acid oxidation was inhibited by using etomoxir. Baseline OCR was determined as the difference between baseline OCR minus nonmitochondrial OCR. ATP-linked OCR was determined as the difference between oligomycin-inhibited respiration (rate 4) and baseline OCR (rate 3). Glucose-mediated respiration was determined as the difference between 2-DG–OCR (rate 4) minus baseline OCR (rate 3). Palmitate-mediated respiration was determined as etomoxir-inhibited respiration (rate 6) minus etomoxir-free respiration (rate 5). For glucose and palmitate OCR graphs, all groups were normalized to WT recordings, which was set to 100%.

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