Increased iron content and RNA oxidative damage in skeletal muscle with aging and disuse atrophy

Abstract

Muscle atrophy with aging or disuse is associated with deregulated iron homeostasis and increased oxidative stress likely inflicting damage to nucleic acids. Therefore, we investigated RNA and DNA oxidation, and iron homeostasis in gastrocnemius muscles. Disuse atrophy was induced in 6- and 32-month old male Fischer 344/Brown Norway rats by 14 days of hind limb suspension (HS). We show that RNA, but not DNA, oxidative damage increased 85% with age and 36% with HS in aged muscle. Additionally, non-heme iron levels increased 233% with aging and 83% with HS at old age, while staining for free iron was strongest in the smallest fibers. Simultaneously, the mRNA abundance of transferrin receptor-1 decreased by 80% with age and 48% with HS for young animals, while that of the hepcidin regulator hemojuvelin decreased 37% with age, but increased about 44% with disuse, indicating a dysregulation of iron homeostasis favoring increased intracellular free iron in atrophied muscles. RNA and DNA concentrations increased with age and were negatively correlated with muscle mass, whereas protein concentrations decreased with aging, indicating a preferential loss of protein compared to nucleic acids. Furthermore, xanthine oxidase activity increased with age, but not with HS, while mRNA abundance of the Y box-binding protein-1, which has been suggested to bind oxidized RNA, did not change with age or HS. These results suggest that RNA oxidation, possibly mediated by increased non-heme iron, might contribute to muscle atrophy due to disuse particularly in aged muscle.

Figure 1. Total RNA oxidation increases with atrophy in rat gastrocnemius muscle

RNA (A) and DNA (B) oxidation in gastrocnemius muscles of 6 and 32 month (mo) old control (unfilled bars) and hind limb suspended (HS; hatched bars) rats is shown. For RNA (A), a main effect for age and HS was observed. The * indicates a significant difference for HS within the age group.

Figure 2. Changes in non-heme iron, TfR1, and HJV mRNA levels with atrophy

Non-heme iron concentration (A) and relative TfR1 (B) and HJV (C) mRNA levels of gastrocnemius muscles from 6 and 32 month (mo) old control (open bars) and HS (hatched bars) rats are shown. For all three parameters, a main effect for age and HS was observed. The * indicates a significant difference for HS within the age group.

Figure 3. Muscle mass is correlated with RNA oxidation and iron levels

Correlation between muscle weight to body weight ratio (MW/BW) and RNA oxidation in gastrocnemius muscle (A): Pearson’s coefficient: r = − 0.84 (p < 0.0001). Correlation between muscle weight to body weight ratio (MW/BW) and non-heme iron (Fe) in gastrocnemius muscles (B); Pearson’s correlation: r = − 0.78 (p < 0.0001). Correlation between non-heme iron (Fe) and RNA oxidation in gastrocnemius muscles (C); Pearson’s coefficient: r = 0.85 (p < 0.0001).

Figure 4. Free iron content in muscle fibers

Representative histology pictures showing free iron content and muscle atrophy of the four groups. Gastrocnemius muscle cross sections of young (6 mo: A and B) and old (32 mo: C and D) control (A and C) and hind limb suspended (HS: B and D) rats were stained for free iron. Bar in D for all pictures 25µm.

Figure 5. Increased XOD activity with age

The XOD activity normalized to total protein concentration in gastrocnemius muscles of 6 and 32 month (mo) old control (open bars) and hind limb suspended (HS; hatched bars) rats is shown. A main effect for age was observed.