Figure 1

Iron-overload alters myocardial SIRT1/FOXO1 signaling which is restored by RSV.

(A,B) Western blot analysis and quantification of two major transcriptional factors, Nrf2 and FOXO1, showing no change in Nrf2 levels but increased nuclear levels of FOXO1 in early iron-overloaded hearts (A) with immunofluorescence staining in myocardial tissue with early iron-overload confirming increased nuclear FOXO1 levels as illustrated by the white arrows (B). (C,D) Immunoprecipitated cardiac acetylated FOXO1 increased in response to iron-overload which was markedly suppressed by resveratrol (RSV) with corresponding inverse changes in SIRT1 levels (C) while immunofluorescence staining for FOXO1 (green) and SIRT1 (red) in cultured and stretched murine LV cardiofibroblasts showing that in response iron exposure nuclear FOXO1 increased with reduced SIRT1 levels, while RSV (100 μM) prevents the loss of SIRT1 without affecting the increased total FOXO1 levels (D). Resveratrol therapy increased the phosphorylation of AMPK (threonine-172) in iron-overloaded myocardium (E). SIRT1 activator, SRT1720 (1 μM), prevents iron-induced oxidative stress in cardiomyocytes based on dihydroethidium (DHE) staining for superoxide levels, 4-hydroxynonenal (4-HNE) and nitrotyrosine immunofluorescence (F). R.R. = relative ratio; A.U. = arbitrary unit. n = 3 repeats from n = 2 hearts. *p < 0.05 compared with all other groups; ^#p < 0.05 compared with the placebo group.