Nrf2 deficiency promotes the increasing trend of autophagy during aging in skeletal muscle: a potential mechanism for the development of sarcopenia

Abstract

This study aims to explore the impact of nuclear factor erythroid 2-related factor 2 (Nrf2) deficiency on skeletal muscle autophagy and the development of sarcopenia. LC3b, P62, Bnip3, Lamp-1, and AMPK protein levels were measured in muscle from young, middle-aged, old Nrf2-/- (knockout, KO) mice and age-matched wild-type (WT) C57/BL6 mice. Autophagy flux was measured in young WT, young KO, old WT, old KO mice, using colchicine as autophagy inhibitor. There was a trend of higher accumulation of LC3b-II, P62, Bnip3, Lamp-1 induced by colchicine in old WT mice compared with young WT mice. Colchicine induced a significantly higher accumulation of LC3b-II, P62, Bnip3, Lamp-1 in KO mice compared with WT mice, both in the young and old groups. AMPK and reactive oxygen species (ROS) were unregulated following Nrf2 KO and increasing age, which was consistent with the increasing trend of autophagy flux following Nrf2 KO and increasing age. Nrf2 KO and increasing age caused decreased cross-sectional area of extensor digitorum longus and soleus muscles. We concluded that Nrf2 deficiency and increasing age may activate AMPK and ROS signals to cause excessive autophagy activation in skeletal muscle, which can be a potential mechanism for the development of sarcopenia.

Keywords: aging; autophagy; autophagy flux measurements; sarcopenia; skeletal muscle.

Figure 1

Expression of LC3b-II, LC3b-I, P62, Bnip3, and Lamp-1 proteins in young WT, middle-aged WT, and old WT mice. (A) Western blot images. (B–F) Statistical graphs. Data represent mean ± SE, (n=4-5). *P <0.05 effect of age by one-way ANOVA. #P <0.05 compared with young WT mice. &P <0.05 compared with middle-aged WT mice.

Figure 2

Expression of LC3b-II, LC3b-I, P62, Bnip3, and Lamp-1 proteins in young KO, middle-aged KO, and old KO mice. (A) Western blot images. (B–F) Statistical graphs. Data represent mean ± SE, (n=4-5). *P <0.05 effect of age by one-way ANOVA. #P <0.05 compared with young KO mice. &P <0.05 compared with middle-aged KO mice.

Figure 3

Expression of LC3b-II, LC3b-I, P62, Bnip3, and Lamp-1 proteins in young WT and young KO mice. (A) Western blot images. (B–F) Statistical graphs. Data represent mean ± SE, (n=5), *statistically significant.

Figure 4

Expression of LC3b-II, LC3b-I, P62, Bnip3, and Lamp-1 proteins in middle-aged WT and middle-aged KO mice. (A) Western blot images. (B–F) Statistical graphs. Data represent mean ± SE, (n=5), *statistically significant.

Figure 5

Expression of LC3b-II, LC3b-I, P62, Bnip3, and Lamp-1 proteins in old WT and old KO mice. (A) Western blot images. (B–F) Statistical graphs. Data represent mean ± SE, (n=5), *statistically significant.

Figure 6

Autophagy flux in skeletal muscle of young WT and old WT mice. (A) Western blot images. (B–E) Statistical graphs. Autophagy flux was calculated by the fold of changes in the expression of LC3b-II, P62, Bnip3, and Lamp-1 induced by colchicine. Data represent mean ± SE, n=3. *statistically significant.

Figure 7

Autophagy flux in skeletal muscle of young WT and young KO mice. (A) Western blot images. (B–E) Statistical graphs. Data represent mean ± SE, n=3-4. *statistically significant.

Figure 8

Autophagy flux in skeletal muscle of old WT and old KO mice. (A) Western blot images. (B–E) Statistical graphs. Data represent mean ± SE, n=3-4. *statistically significant.

Figure 9

AMPK phosphorylation level in skeletal muscle of different groups. (A, C, E, G, I) Western blot images. (B, D, F, H, J) Statistical graphs. Data represent mean ± SE, n=4-5. *P <0.05 main effect. #P <0.05 compared with young mice. &P <0.05 compared with middle-aged mice.

Figure 10

ROS level in skeletal muscle of different groups. (A) Mito-Tracker green is a marker of mitochondria, dihydroethidium (DHE) is a probe of reactive oxygen species (ROS). Images from green and red fluorescence were merged (yellow) to locate the mitochondrial source of ROS generation. The yellow color indicates ROS within the mitochondria. (B) Total ROS. (C) Mitochondrial ROS. Data represent mean ± SE, n=3. #P <0.05 main effect of age by two-way ANOVA. &P <0.05 main effect of genotype by two-way ANOVA.*P <0.05 Nrf2 KO vs WT mice of the same age.

Figure 11

Nrf2 deficiency exacerbated skeletal muscle loss during aging. (A, C) Representative images of the cross-sectional area (CSA) of extensor digitorum longus (EDL) and soleus (SOL) muscles by HE staining. (B, D) CSA of the EDL and SOL muscles in the young and old mice of WT and KO genotypes. Data represent mean ± SE, n=3. #P <0.05 main effect of age by two-way ANOVA. &P <0.05 main effect of genotype by two-way ANOVA.*P <0.05 Nrf2 KO vs WT mice of the same age.