G8270

To determine the optimal concentration of treatment with HG and treatment time, PCs were cultured in 6-well plates for 24 h, 48 h, and 72 h in RPMI-1640 medium containing 5.5 mM glucose supplemented with 0, 10, 20, 30, 40, or 50 mM of D-glucose (#G8270, Sigma, Oakville, ON, Canada). Subsequently, PCs were collected for detecting the level of the cytoskeleton-related protein, synaptopodin. Mannitol was used to achieve equivalent osmotic pressure, and L-glucose (#G5500, Sigma, Oakville, ON, Canada, a nonmetabolizable isomer of glucose) served as a control to determine the uniqueness of D-glucose.

The mice were fasted for 6 h with free access to water. Oral gavage was preformed using a specialized gavage needle (Sigma-Aldrich) affixed to a 1-mL syringe. Briefly, animals were gently restrained, and the gavage was carefully passed through the mouth, down to the depth of the last rib (~stomach). After obtaining a basal blood sample of ~30 μL from the tail vein in a EDTA-tubes, mice were given an oral dose of L-lysine (1 g/Kg of body weight, Sigma-Aldrich, L5501) or glucose (1 g/Kg of body weight, Sigma-Aldrich, G8270) in a volume of ~250 μL, and a second and third blood sample was obtained at 15 and 30 min. EDTA was used as an anticoagulant.

Using a Langendorff system, adult cardiomyocytes were isolated from male mice at 10 weeks of age^{39 (link)}. In brief, after anesthesia, the heart was quickly excised from the mice and cannulated via the aorta. Then, the heart was perfused at constant flow for 1 min at 37 °C with a buffer containing 120 mM NaCl (31319, Nakalai Tesque), 5.4 mM KCl (163-03545, FUJIFILM Wako Pure Chemical Corporation), 1.6 mM MgCl₂ (135-00165, FUJIFILM Wako Pure Chemical Corporation), 1.2 mM NaH₂PO₄ (197-02865, FUJIFILM Wako Pure Chemical Corporation), 5.6 mM glucose (G8270, Sigma-Aldrich), 20 mM NaHCO₃ (191-01305, FUJIFILM Wako Pure Chemical Corporation), and 5 mM taurine (T0625, Sigma-Aldrich), followed by collagenase buffer containing 1.2 mg/ml collagenase type 2 (CLS-2, Worthington Biochemical Corporation) and 0.020 mg/ml protease type XIV (P-5147, Sigma-Aldrich). After filtration into a sterilized tube, the supernatant containing the dispersed myocytes was gently centrifuged at 20 × g for 3 min. The cell pellet was resuspended in the buffer containing 200 μM Ca²⁺. The cardiomyocytes were pelleted by gravity for 10 min. After aspiration of the supernatant, the suspension of rod-shaped cardiomyocytes was then used for western blot analysis or reverse transcription and real-time quantitative polymerase chain reaction (qRT-PCR).

The day before the seahorse assay, 7.5 × 10⁴ cells were seeded into seahorse XF^e24 microplates (102340, Agilent), and the XF^e24 cartridge (102340, Agilent) was calibrated in the seahorse prep station (Agilent) overnight. Before the assay, the medium was replaced by 0.5 ml XF base Medium (102353, Agilent) supplemented with 2 mM glutamine (25030081, Gibco). Cells were incubated at 37 ˚C for 1 h in the seahorse prep station. 56 μl glucose (100 mM) (G8270, Sigma), 62 µl oligomycin (10 μM) (75351, Sigma) and 69 µl 2DG (1 M) (D6134, Sigma) were added into the cartridge wells. ECAR and OCR levels were determined using seahorse bioscience XF^e24 extracellular flux analyzer (Agilent) and each cycle of measurement involved mixing (3 min), waiting (2 min), and measuring (3 min) cycles.