Dmem medium

Mutant or WT cells, derived as described in supplemental methods, were directly seeded on the XF 24-plate at a density of 6 × 10⁴ cells per well in DMEM-F12 (1:1) medium supplemented with 10% fetal bovine serum (FBS) and 2 mM Glutamine. Media was replaced every 3–4 days. After 2 weeks in vitro cells were equilibrated with bicarbonate-free DMEM medium (without pyruvate, lactate, glucose, glutamine; Seahorse bioscience) supplemented with 2.5 mM glucose. The OCR and ECAR was measured using a Seahorse XFe24 Extracellular Flux Analyzer (Seahorse Bioscience)34 (link). After a baseline measurement, mitochondrial function and glycolytic flux was determined as previously described66 (link). Oxygen consumption and glycolytic rates in astrocytes were determined through sequential addition of 6 μM oligomycin, 0.5 mM 2,4-dinitrophenol, and 1 μM antimycin per 1 μM rotenone. After the experiment, protein concentration was determined for each well and OCR and ECAR values were normalized by mg. protein.

The Seahorse Bioscience XF24 Extracellular Flux Analyzer Kit (Seahorse Bioscience) was used to measure glucose dependency in vitro. The Mito Fuel Flex test was completed after subjecting cardiomyocytes to glucose oxidation pathway inhibition by UK5099 (4 μM) followed by long‐chain fatty acid oxidation inhibition by Etomoxir (2 μM). This order of inhibitors allows for calculation of glucose oxidation dependency. The protocol was modified to test dependency in Pdk4 inhibited cardiomyocytes. Prior to loading isolated cardiomyocytes to the Seahorse cell‐plate, cardiomyocytes were incubated with 10 μM Pdk4 inhibitor (MedChem Express, Cat# HY‐135954A) in DMEM medium (Seahorse Bioscience) for 5 min at 37°C. The cell‐plate was also loaded with cardiomyocytes suspended in 10 μM Pdk4 inhibitor in DMEM medium.

The Seahorse XF24 was used to measure the oxygen consumption rate (OCR) of isolated cardiomyocytes. Isolated cardiomyocytes and sectioned fresh brain tissues were differentiated in customized Seahorse 24-well. We applied DMEM Medium (Seahorse Bioscience), supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM D-glucose. OCR was measured using the Seahorse Bioscience XF24 Extracellular Flux Analyzer (Seahorse Bioscience). Measurements were taken as the cells were incubated sequentially under four conditions: 1) basal levels were measured with no additives; 2) oligomycin (1.5 μM) was added to reversibly inhibit ATP synthase and OXPHOS, showing glycolysis alone; 3) FCCP (1 μM), a mitochondrial uncoupler, was added to induce maximal respiration; and 4) Antimycin A (10 μM), a Complex III inhibitor, was added to obtain non-mitochondrial oxygen consumption background. The Seahorse software was used to plot the results. OCR was normalized to cell number per well.