Figure 3.

Protocols for oxygen glucose deprivation/reperfusion (OGD/R) stimulation in neonatal rat cardiomyocytes (NRCMs). NRCMs were starved for 24 h in serum-free high-glucose-containing Dulbecco’s modified Eagle’s medium (DMEM) before the experiment. ((Upper): Cont) The cells were cultured in high-glucose-containing DMEM under normoxic conditions (95% air and 5% CO₂ at 37 °C) for 15 min. In order to follow the condition of OGD/R stimulation, medium change was performed at 10 min. PBS, a vehicle, was treated throughout the experiment. ((Middle): OGD/R) The cells were cultured in glucose-free DMEM under hypoxic conditions (1% O₂, 94% N₂ and 5% CO₂ at 37 °C) for 10 min (OGD). Then, the cells were cultured with high-glucose-containing DMEM in normoxic conditions for 5 min (R). PBS was treated throughout the experiment. (Lower) The cells were stimulated with OGD/R. Canstatin (+canstatin: 250 ng/mL), gp91 ds-tat (+gp91 ds-tat: 2.5 µM) or tempol (+tempol: 1 mM) was treated throughout the experiment.