Mechanism of asymmetric dimethylarginine-induced oxidative stress and autophagy in rat cardiomyocytes

Objective To investigate asymmetric dimethylarginine (ADMA)-induced changes in reactive oxygen species and beclin-1 expression and the mechanism of ADMA-mediated beclin-1 expression in rat cardiomyocytes.Methods Rat cardiomyocyes H9c2(2-1) were cultured in high glucose Dulbecco's modified Eagle medium (DMEM) and supplemented with 10%fetal bovine serum at 37℃ in a humidified 5%CO₂ incubator.Changes in reactive oxygen species (ROS) in H9c2(2-1) cells treated with different doses (3,5,10,15,20,and 30 μmol/L) of ADMA were examined with flow cytometry.Protein levels of beclin-1,PI3kinase-p110α and phosphorylation levels of serine/threonine kinase (AKT) ser473 were analyzed with Western blot and corresponding cell survival rates were detected.Results Compared with the control group,the levels of ROS in ADMA treated cells increased markedly (9.86±0.86,15.91±0.97,19.15±3.13,28.76±1.69,29.57±1.99,and 35.09±2.22;all P<0.05);the protein levels of beclin-1 in ADMA treated cells increased significantly(90.3±6.5,76.2±8.7,76.2±6.4,71.4±5.6,67.8±5.0,63.5±4.2,and 61.2±6.9,all P<0.05);levels of PI3kinase-p110α and phosphorlation levels of AKT ser473 decreased markedly (both P<0.05).ADMA significantly reduced cell viability (P<0.05),and the cell viability was correlated with ADMA levels in a dose-dependent manner.Conclusion ADMA enhances ROS generation in rat cardiac H9c2(2-1) cells.Upregulation of beclin-1 could be induced by ADMA through downregulation of PI3K-p110α and phosphorylation of AKT ser473.