2.5. Agilent extracellular seahorse analysis of glycolysis, glucose oxidation and fatty acid oxidation

Isolated cardiomyocytes were seeded on matrix gel–coated cell culture microplates (Agilent Seahorse XF24) at the cell intensity of 8000 cells/well. Generally, before performing the experiment, culture medium was changed into 500 μL assay medium (Agilent Seahorse XF Base Medium), and then, cells were incubated in 37°C non‐CO₂ incubator for 1 hour.

Glycolysis Load 100mM glucose (56 μL), 10 μM oligomycin (62 μL) and 1 M 2‐deoxy‐glucose (2‐DG, 69 μL) into the corresponding injection port of sensor cartridge and do sensor cartridge calibration. Then, start the system to conduct basal extracellular acidification rate (ECAR) measurement [3 × (1.5 min mix, 2 min wait, 1.5 min measure)], which was followed by injection of glucose, oligomycin and 2‐DG successively, and each compound injection was followed by the ECAR measurement [3 × (1.5 min mix, 2 min wait, 1.5 min measure)].

Glucose and pyruvate oxidation Load 100 mM glucose (56 μL) or 10 mM pyruvate (56 μL) to injection port of sensor cartridge. After starting the measurement, the protocol consisted of basal oxygen consumption rate (OCR) measurement [3 × (1.5 min mix, 2 min wait, 1.5 min measure)] and fuel substrate–induced OCR measurement [3 × (1.5 min mix, 2 min wait, 1.5 min measure)] following glucose or pyruvate injection.

Palmitate acid oxidation Palmitate acid should be conjugated with BSA as previous report. 20 10mM palmitate‐BSA (56 μL) or BSA solution (56 μL) was loaded into ports of sensor cartridge and injected into the microplate following the basal OCR measurement [3 × (1.5 min mix, 2 min wait, 1.5 min measure)]. In addition, the palmitate acid–induced OCR measurement is 9 × (1.5 min mix, 2 min wait, 1.5 min measure).