Enos sirna

Lipofectamine reagent was used for eNOS siRNA (purchased from Santa Cruz Biotechnology) and mock control oligonucleotides transfecting according to the manufacturer's instructions. siRNAs transfection concentration was 100 nM. Transfected cells were then incubated in new culture media for an additional treatment as indicated.

Embryonic rat cardiomyocyte-derived cell line H9C2 was maintained in DMEM (Dulbecco’s modified Eagle’s medium) containing 10 % FBS in a humidified atmosphere (5 % CO₂) at 37 °C. Commercial Cav-3 siRNA or eNOS siRNA (Santa Cruz Biotechnology) was used for inhibition of Cav-3 expression according to the manufacturer’s protocol. After transfection with control, Cav-3 siRNA or eNOS siRNA, cells were treated with N-acetylcysteine (NAC, 1 mmol/L) in the condition of LG or HG for 36 h, then the cells was exposed to 4 h of hypoxia (1 % O₂, 5 % CO₂, 94 % N₂) followed by 4 h of reoxygenation (H/R). LDH (lactate dehydrogenase) release was measured to evaluate the extent of cellular injury using a cytotoxicity assay kit (Jiancheng Bioengineering Institute, China) according to the manufacturer’s instructions.