Effects of Nrf2 deficiency on mitochondrial oxidative stress in aged skeletal muscle

Abstract

Oxidative stress and mitochondrial dysfunction are associated with the aging process. However, the role of nuclear factor erythroid 2 -related factor 2 (Nrf2) in skeletal muscle during aging remains to be clarified. In the current study, we assessed whether the lack of Nrf2, which is known as a master regulator of redox homeostasis, promotes age-related mitochondrial dysfunction and muscle atrophy in skeletal muscle. Here, we demonstrated that mitochondrial 4-hydroxynonenal and protein carbonyls, markers of oxidative stress, were robustly elevated in aged Nrf2 knockout (KO) mice because of the decreased expression of Nrf2-target antioxidant genes. Mitochondrial respiration declined with aging; however, there was no difference between Nrf2 KO and age-matched WT mice. Similarly, cytochrome c oxidase activity was lower in aged WT and Nrf2 KO mice compared with young WT mice. The expression of Mfn1 and Mfn2 mRNA was lower in aged Nrf2 KO muscle. Mitochondrial reactive oxygen species production per oxygen consumed was elevated in aged Nrf2 KO mice. There was no effect of Nrf2 KO on muscle mass normalized to body weight. These results suggest that Nrf2 deficiency exacerbates age-related mitochondrial oxidative stress but does not affect the decline of respiratory function in skeletal muscle.

Keywords: Aging; mitochondria; oxidative stress; skeletal muscle.

Figure 1

Body weight, skeletal muscle mass, and sarcopenic index in aged Nrf2 KO mice. (A) Body weight. (B) Gastrocnemius and tibialis anterior muscle mass. (C) Sarcopenic index (muscle mass per body weight). Data are presented as mean ± SEM. n = 6–7 in each group. *P < 0.05 **P < 0.01, significant difference versus WT‐YOUNG. ^# P < 0.05 ^## P < 0.01, significant difference versus WT‐AGED. WT, wild type; KO, knockout.

Figure 2

Nrf2 target antioxidant genes in aged Nrf2 KO mice. Data are presented as mean ± SEM n = 5–7 in each group. *P < 0.05 **P < 0.01, significant difference versus WT‐YOUNG. ^# P < 0.05 ^## P < 0.01, difference versus WT‐AGED. WT, wild type; KO, knockout.

Figure 3

Mitochondrial oxidative stress in aged Nrf2 KO mice. A) Mitochondrial 4‐HNE content. B) Mitochondrial protein carbonyls. Data are presented as mean ± SEM n = 5–7 in each group. *P < 0.05 **P < 0.01, significant difference versus WT‐YOUNG. ^# P < 0.05, difference versus WT‐AGED. WT, wild type; KO, knockout.

Figure 4

Enzyme activity in aged Nrf2 KO mice. Data are presented as mean ± SEM n = 5–7 in each group. *P < 0.05 **P < 0.01, significant difference versus WT‐YOUNG. WT, wild type; KO, knockout.

Figure 5

Mitochondrial function in aged Nrf2 KO mice. A) Oxygen consumption rate during mitochondrial respiration. B) Mitochondrial ROS production per oxygen consumed. Data are presented as mean ± SEM n = 5–7 in each group. *P < 0.05 **P < 0.01, significant difference versus WT‐YOUNG. ^# P < 0.05, difference versus WT‐AGED. WT, wild type; KO, knockout.

Figure 6

Mitochondrial dynamics genes in aged Nrf2 KO mice. Data are presented as mean ± SEM n = 5–7 in each group. *P < 0.05, significant difference versus WT‐YOUNG. ^## P < 0.01, difference versus WT‐AGED. WT, wild type; KO, knockout.

Figure 7

Mitochondrial protein levels in whole muscle lysate from aged Nrf2 KO mice. A) Mitochondrial OXPHOS proteins. B) Mitochondrial fusion and fission regulatory proteins. Data are presented as mean ± SEM n = 5–7 in each group. *P < 0.05, significant difference versus WT‐YOUNG. ^# P < 0.05, difference versus WT‐AGED. WT, wild type; KO, knockout.