Fig 2: Effect of prolonged moderate exercise on Nrf2/ARE-Antioxidants in the aging heart.Representative immunoblots of cytosolic extracts from the hearts of young and old mice under basal conditions and following 6-weeks of moderate exercise training (MET). Protein blots were probed with respective antibodies as indicated. Individual lanes represent separate animals (n = 4–6/group). A. Analysis of nuclear Nrf2 in young and old mice subjected to MET. In sedentary mice, Nrf2 protein levels were decreased significantly in old when compared to young (*p<0.05). Following MET, nuclear Nrf2 levels were significantly increased in old mice to levels equivalent to those of young mice (#, $p<0.05 in MET vs. respective basal). (B) Densitometry analysis of respective protein signals were performed using Image-J and expressed relative to mean values of the sedentary-young group. Under basal conditions, a significant decrease in the protein levels of GSR, G6PD, NQO1, catalase, GPX1 and GCS were observed in the heart tissues of old when compared to young mice. Following 6-weeks of moderate exercise, most of the antioxidants were significantly (*p<0.05) upregulated or stabilized in the aging heart.

Fig 3: Increased Nrf2 regulated antioxidant genes in HL-1 cardiomyocytes by 24 h of TNF-a treatment. Nrf2 regulated antioxidant Gene expression Profiles (Gclc, Gclm, G6pd, Gpx1, Cat, Nqo1, Sod1 and Sod2) in TNF-a treated HL-1 cell samples for 24 h were analyzed by quantitative PCR and gene levels were normalized with Gapdh (n=3). Mann Whitney test was used to establish statistical significance, where *p<0.05 vs control.

Fig 4: Lower concentration of TNF-a treatment for 2 h increases Nrf2 regulated antioxidant proteins in HL-1 cardiomyocytes. (A) Immunoblot analyses of Nrf2 regulated antioxidant proteins (GCLC, GCLM, G6PD, GPX1, CAT, NQO1, SOD1 and SOD2) in 2 h of TNFa treated HL-1 cells (n=3). (B) Relative intensity of the protein signal was calculated using Image-J software and normalized to GAPDH. Statistical significance was calculated by Mann Whitney test, where *p<0.05 vs control.

Fig 5: Immunoblot analyses of protein expression for Nrf2 and antioxidant enzymes.A. Analysis of nuclear Nrf2 in young and old mice subjected to EES. In sedentary mice, Nrf2 protein levels were decreased significantly in young versus old mice. EES exacerbated the decrease of nuclear Nrf2 in old mice. Blots/values represent n = 4–6 from each group. *p<0.05 between young vs. old and #p<0.05-between basal vs. EES. (B) Representative immunoblots of cytosolic extracts from the hearts of young and old mice under basal conditions and following EES. Protein blots were probed with anti-HO1, NQO1, GCLM, GCLC, Catalase, SOD1, SOD2, GSR, G6PD, GPX1 and GAPDH. Individual lanes indicate a single animal. Densitometry analysis of respective protein signals was performed using Image-J and expressed as relative intensity units calculated as mean values of young and old, *p<0.05. Individual lanes indicate each animal (n = 6). #p<0.05-between basal and EES.