Long-term perturbation of muscle iron homeostasis following hindlimb suspension in old rats is associated with high levels of oxidative stress and impaired recovery from atrophy

Abstract

In the present study, we investigated the effects of 7 and 14 days of re-loading following 14-day muscle unweighting (hindlimb suspension, HS) on iron transport, non-heme iron levels and oxidative damage in the gastrocnemius muscle of young (6 months) and old (32 months) male Fischer 344×Brown Norway rats. Our results demonstrated that old rats had lower muscle mass, higher levels of total non-heme iron and oxidative damage in skeletal muscle in comparison with young rats. Non-heme iron concentrations and total non-heme iron amounts were 3.4- and 2.3-fold higher in aged rats as compared with their young counterparts, respectively. Seven and 14 days of re-loading was associated with higher muscle weights in young animals as compared with age-matched HS rats, but there was no difference in muscle weights among aged HS, 7 and 14 days of re-loading rats, indicating that aged rats may have a lower adaptability to muscle disuse and a lower capacity to recover from muscle atrophy. Protein levels of cellular iron transporters, such as divalent metal transport-1 (DMT1), transferrin receptor-1 (TfR1), Zip14, and ferroportin (FPN), and their mRNA abundance were determined. TfR1 protein and mRNA levels were significantly lower in aged muscle. Seven and 14 days of re-loading were associated with higher TfR1 mRNA and protein levels in young animals in comparison with their age-matched HS counterparts, but there was no difference between cohorts in aged animals, suggesting adaptive responses in the old to cope with iron deregulation. The extremely low expression of FPN in skeletal muscle might lead to inefficient iron export in the presence of iron overload and play a critical role in age-related iron accumulation in skeletal muscle. Moreover, oxidative stress was much greater in the muscles of the older animals measured as 4-hydroxy-2-nonhenal (HNE)-modified proteins and 8-oxo-7,8-dihydroguanosine levels. These markers remained fairly constant with either HS or re-loading in young rats. In old rats, HNE-modified proteins and 8-oxo-7,8-dihydroguanosine levels were markedly higher in HS and were lower after 7 days of recovery. However, no difference was observed following 14 days of recovery between control and re-loading animals. In conclusion, advanced age is associated with disruption of muscle iron metabolism which is further perturbed by disuse and persists over a longer time period.

Fig. 1

Changes in non-heme iron levels (μg/g wet tissue) and total non-heme iron amounts (μg/muscle) in the gastrocnemius muscle of young and old rats with HS and re-loading treatments. (A) HS was associated with higher non-heme iron concentration in the gastrocnemius muscle of old (p<0.05), but not young rats. (B) Total amounts of non-heme iron were significantly higher in the gastrocnemius muscle of old rats (age effect: p<0.0001). However, neither HS nor re-loading following HS affected non-heme iron amount in the gastrocnemius muscle for both young and old rats, indicating that disuse-induced muscle loss in aged animals contributes to non-heme iron accumulation. Values are means±SEM (n=7–8). ^a,b,cDifferent letters are significantly different from each other (p<0.05). Graph bar legends: Control, non-suspended control; HS, 14 days of HS; HS+7, 14 days of HS followed by 7 days of ambulatory re-loading; HS+14, 14 days of HS followed by 14 days of ambulatory re-loading.

Fig. 2

Protein and gene expression of hemojuvelin (HJV) in the gastrocnemius muscle of young and old rats in control, HS, HS+7 and HS+14 groups. (A) The protein expression levels of HJV were unchanged with age and treatments. (B) HJV mRNA levels significantly elevated by HS in gastrocnemius muscle of both young and old rats, which subsequently mitigated by re-loading. Values are means±SEM (n=7–8). ^a,b,cDifferent letters are significantly different from each other (p<0.05). Graph bar legends: Control, non-suspended control; HS, 14 days of HS; HS+7, 14 days of HS followed by 7 days of ambulatory re-loading; HS+14, 14 days of HS followed by 14 days of ambulatory re-loading.

Fig. 3

Protein and gene expression levels of key iron transport proteins in the gastrocnemius muscle of young and old rats in control, HS, HS+7 and HS+14 groups. There was a significant age-related decrease in TfR1 protein (A) and gene expression (B) levels in gastrocnemius muscle (p<0.0001). Post hoc analysis showed that re-loading following HS was associated with higher TfR1 protein levels in the gastrocnemius muscle of young rats, whereas TfR1 protein and mRNA levels did not differ between groups in old rats. Significant age, treatment and interaction effects have been observed in Zip14 protein levels (C), though mRNA levels (D) were fairly constant. (E) HS was associated with higher DMT1 protein levels in the gastrocnemius muscle of aged, but not young rats. Moreover, the mRNA levels of DMT1 nonIRE (F) and IRE (G) in the gastrocnemius muscle of young and old rats were determined. With respect to the only known iron export protein in mammals, the mRNA levels of FPN (H) didn’t change with age or HS in gastrocnemius muscles. However, 7 and 14 days of re-loading after HS in old rats was associated with lower FPN mRNA levels in comparison with their age-matched HS animals. Values are means±SEM (n=7–8). ^a,b,cDifferent letters are significantly different from each other (p<0.05). Graph bar legends: Control, non-suspended control; HS, 14 days of HS; HS+7, 14 days of HS followed by 7 days of ambulatory re-loading; HS+14, 14 days of HS followed by 14 days of ambulatory re-loading.

Fig. 4

Oxidative damage in the gastrocnemius muscle of rats as determined by dot blot analysis and HPLC-ECD. (A) HS was associated with higher HNE-modified protein levels (p<0.01) in the gastrocnemius muscle of old, but not young rats. Seven days of re-loading following HS was associated with lower levels of HNE-modified proteins in old HS rats (p<0.01). (B) There was a significant increase in nitrotyrosine levels with age (p<0.0001). (C–D) Oxidative damage to RNA and DNA in gastrocnemius muscle was assessed as their oxidation products 8-oxo-7,8-dihydroguanosine (8-oxoGuo) and 8-oxo-7,8-dihydro-2′-oxyguanosine (8-oxodGuo), respectively. (C) Greater RNA oxidation level in gastrocnemius muscle was associated with age (age, p<0.001). (D) The DNA oxidation levels did not change significantly over the course of aging and treatments. Values are means±SEM (n=7–8). ^a,b,cDifferent letters are significantly different from each other (p<0.05). Graph bar legends: Control, non-suspended control; HS, 14 days of HS; HS+7, 14 days of HS followed by 7 days of ambulatory re-loading; HS+14, 14 days of HS followed by 14 days of ambulatory re-loading.

Fig. 5

Changes of iron homeostasis with aging and muscle unweighting in the skeletal muscle of Fischer344×Brown Norway rats. Muscle iron import via TfR1 is lower in old rats, which may represent a compensatory mechanism aimed at countering excessive iron accumulation. However, age-related iron accumulation may be due to the extremely low expression of FPN in the skeletal muscle, which might result in an ineffective iron export with age. DMT1- and Zip14-mediated iron uptake during HS may be at least partly responsible for non-heme iron overload in the atrophying muscle of aged animals.