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

Abstract

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. 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. 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. 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.