Downregulation of Manganese Superoxide Dismutase by Angiotensin II in Cardiac Fibroblasts of Rats: Association With Oxidative Stress in Myocardium

BACKGROUND: The aim of this study was to determine whether angiotensin II (ANG II) affects the protein and mRNA expressions of the mitochondrial antioxidant manganese superoxide dismutase (Mn-SOD) in cardiac fibroblasts of rats through inducing the phosphorylation of the proteins Akt and FOXO3a, thereby contributing to the oxidative stress in the myocardium. METHODS: Cardiac fibroblasts (passage 2) from normal male adult rats were cultured to confluency and incubated in serum-free Dulbecco's modified Eagle's medium for 24 h. The cells were then preincubated with/without the tested inhibitors for 1 h and then further incubated with/without ANG II (1 µmol/l) for 24 h. RESULTS: ANG II increased the production of superoxide ions in the cardiac fibroblasts, and decreased the activity levels of both Mn-SOD and CuZn-SOD, but not the activity levels of catalase and glutathione peroxidase. ANG II also decreased the mRNA and protein expressions of Mn-SOD, but not those of CuZn-SOD, catalase, and glutathione peroxidase. The likely mechanism through which ANG II produces the effect of reducing Mn-SOD activity is by reducing the extent of binding of FOXO3a to the Mn-SOD promoter. In control fibroblasts, inhibition of FOXO3a transcription with small-interfering RNA (siRNA) led to a reduction in the binding of FOXO3a to the Mn-SOD promoter, and a concomitant reduction in Mn-SOD gene expression. Our data suggest that when Akt is phosphorylated by ANG II, P-Akt is translocated from the cytoplasm to the nucleus; subsequently, nuclear phosphorylation of FOXO3a by P-Akt leads to relocalization of FOXO3a from the nucleus to the cytosol, resulting in a decrease in its transcriptional activity, and consequently in Mn-SOD expression. The likelihood of such a mechanism of action is further strengthened by the fact that inhibition of phosphoinositide 3-kinase with wortmannin or LY 294002, and Akt inhibition, were shown to lead to a decrease in P-AKT and to a consequent increase in Mn-SOD mRNA expression. CONCLUSIONS: Our data indicate that ANG II inactivates FOXO3a by activating Akt, leading to a reduction in the expression of the antioxidant Mn-SOD, and thereby potentially contributing to oxidative stress in the myocardium.