In Vitro H/R Experiments.

To simulate in vivo myocardial I/R injury in vitro, H9c2 rat cardiomyoblasts were subjected to H/R. Hypoxia [partial pressure of oxygen (PO₂) <10 mmHg] was achieved by placing cardiomyocyte cells into an hypoxic incubator (atmosphere of 5% CO₂ and 95% N₂) in serum-free/glucose-free Hepes-buffered medium (in millimoles per liter: NaH₂PO₄ 0.9, NaHCO₃ 6.0, CaCl₂ 1.0, MgSO₄ 1.2, sodium lactate 40, Hepes 20, NaCl 98.5, KCl 10, pH 6.8) at 37 °C. Reoxygenation was achieved by transferring the cells to normoxic H9c2 culture medium [DMEM (high glucose) supplemented with 10% (vol/vol) FCS, 100 IU/mL of penicillin and 100 µg/mL of streptomycin] and incubation in 95% air, 5% CO₂ to simulate reperfusion. Cells were subjected to 9 h hypoxia to simulate ischemia and 6 h reoxygenation to simulate reperfusion. Cells incubated in normoxic H9c2 culture medium were prepared in parallel for each condition and served as controls.