Regrowth after skeletal muscle atrophy is impaired in aged rats, despite similar responses in signaling pathways

Abstract

Skeletal muscle regrowth after atrophy is impaired in the aged and in this study we hypothesized that this can be explained by a blunted response of signaling pathways and cellular processes during reloading after hind limb suspension in muscles from old rats. Male Brown Norway Fisher 344 rats at 6 (young) and 32 (old) months of age were subjected to normal ambulatory conditions (amb), hind limb suspension for 14 days (HS), and HS followed by reloading through normal ambulation for 14 days (RE); soleus muscles were used for analysis of intracellular signaling pathways and cellular processes. Soleus muscle regrowth was blunted in old compared to young rats which coincided with a recovery of serum IGF-1 and IGFBP-3 levels in young but not old. However, the response to reloading for p-Akt, p-p70s6k and p-GSK3β protein abundance was similar between muscles from young and old rats, even though main effects for age indicate an increase in activation of this protein synthesis pathway in the aged. Similarly, MAFbx mRNA levels in soleus muscle from old rats recovered to the same extent as in the young, while Murf-1 was unchanged. mRNA abundance of autophagy markers Atg5 and Atg7 showed an identical response in muscle from old compared to young rats, but beclin did not. Autophagic flux was not changed at either age at the measured time point. Apoptosis was elevated in soleus muscle from old rats particularly with HS, but recovered in HSRE and these changes were not associated with differences in caspase-3, -8 or -9 activity in any group. Protein abundance of apoptosis repressor with caspase-recruitment domain (ARC), cytosolic EndoG, as well as cytosolic and nuclear apoptosis inducing factor (AIF) were lower in muscle from old rats, and there was no age-related difference in the response to atrophy or regrowth. Soleus muscles from old rats had a higher number of ED2 positive macrophages in all groups and these decreased with HS, but recovered in HSRE in the old, while no changes were observed in the young. Pro-inflammatory cytokines in serum did not show a differential response with age to different loading conditions. Results indicate that at the measured time point the impaired skeletal muscle regrowth after atrophy in aged animals is not associated with a general lack of responsiveness to changes in loading conditions.

Keywords: Apoptosis; Autophagy; Hind limb suspension; Inflammation; Protein degradation; Protein synthesis.

Figure 1. Restoration of muscle size after atrophy is impaired in soleus muscle of aged rats

Soleus muscle weight (A), muscle weight-to-body weight ratio (B), and mean muscle fiber cross sectional area (C) of ambulatory (amb, black bars), hind limb suspended (HS, white bars), and reloaded (HSRE, grey bars) rats at 6 and 32 months are depicted (n=8–10). Values are means ± SE; * indicates significant difference from ambulatory, # indicates significant difference from HS, and ‘a’ indicates a difference from young within the same group; p<0.05.

Figure 2. Serum IGF-1 and IGFBP-3 concentrations do not recover after disuse in the aged rat

Serum insulin (A), IGF-1 (B) and IGFBP-3 (C) of ambulatory (black bars), hind limb suspended (HS, white bars), and reloaded (HSRE, grey bars) rats at 6 and 32 months are depicted (n=8–10). Values are means ± SE; * indicates significant difference from ambulatory and # indicates significant difference from HS; p<0.05.

Figure 3. Akt response to reloading is similar in young adult and old rats

Representative bands for Western analysis of phospho(Ser473)-Akt, total Akt, and actin (used as loading control) are shown for soleus muscle of ambulatory (amb), hind limb suspended (HS) and reloaded (HSRE) rats at 6 and 32 months (A). Quantification of Western analysis is depicted for phospho- Akt (B), total Akt (B) and the ratio of phospho to total Akt (C) for ambulatory (black bars), hind limb suspended (HS, white bars), and reloaded (HSRE, grey bars) rats at 6 and 32 months (n=8–10). Values are means ± SE; * indicates a main effect for HSRE difference from HS and ambulatory, and # indicates a main effect for HSRE from HS; p<0.05.

Figure 4. p70s6k is not changed in response to disuse and re-ambulation

Representative bands for Western analysis of phospho(Thr389)-p70s6k, total p70s6k and actin (used as loading control) are shown for soleus muscle of ambulatory (amb), hind limb suspended (HS) and reloaded (HSRE) rats at 6 and 32 months (A). Quantification of Western analysis is depicted for phospho-p70s6k (B), total p706sk (B) and the ratio of phospho to total p70s6k (C) for ambulatory (black bars), hind limb suspended (HS, white bars), and reloaded (HSRE, grey bars) rats at 6 and 32 months (n=8–10). Values are means ± SE; ^ indicates a main effect for age; p<0.05.

Figure 5. Recovery of GSK3β is similar in young and old rats

Representative bands for Western analysis of phospho(Ser9)-GSK3β, total GSK3β, and actin (used as loading control) are shown for soleus muscle of ambulatory (amb), hind limb suspended (HS) and reloaded (HSRE) rats at 6 and 32 months (A). Quantification of Western analysis is depicted for phospho- GSK3β (B), total GSK3β (B) and the ratio of phospho to total GSK3β (C) for ambulatory (black bars), hind limb suspended (HS, white bars), and reloaded (HSRE, grey bars) rats at 6 and 32 months (n=8–10). Values are means ± SE; * indicates a main effect for age and # indicates a main effect for HS; p<0.05.

Figure 6. Protein degradation markers MAFbx and Murf-1 are similarly affected by reloading at different ages

Soleus muscle mRNA abundance for Murf-1 (A) and Mafbx (B) for ambulatory (black bars), hind limb suspended (white bars) and reloaded (grey bars) rats at 6 and 32 months is shown (n=8–10). Values are means ± SE; * indicates a main effect for HS different from ambulatory and # indicates a main effect for HSRE different from HS; p<0.05.

Figure 7. mRNA abundance of markers for autophagy show similar responses to different loading conditions in young adult and old rats

Soleus muscle mRNA abundance for ATG5 (A), ATG7 (B) and beclin (C) for ambulatory (black bars), hind limb suspended (white bars) and reloaded (grey bars) rats at 6 and 32 months is shown (n=8–10). Values are means ± SE; * indicates a main effect for HS different from other groups and # indicates difference from HS in young; p<0.05.

Figure 8. Marker of autophagic flux is lower with age, but not different with loading

Representative bands for Western analysis of LC3b-I (top band), LC3b-II (bottom band) and actin (used as loading control) are shown for soleus muscle of ambulatory (amb), hind limb suspended (HS) and reloaded (HSRE) rats at 6 and 32 months of age (A). Quantification of Western analysis for LC3b-II (B) and the ratio of LC3b-II to LC3b-I (C) is depicted for ambulatory (amb, black bars), hind limb suspended (HS, white bars), and reloaded (HSRE, grey bars) rats at 6 and 32 months of age (n=8–10). Values are means ± SE; p=0.052 indicates a trend for main effect for age.

Figure 9. Apoptosis is increased in soleus muscle of aged rats with disuse

Apoptotic index as a measure of DNA fragmentation of soleus muscle from ambulatory (amb, black bars), hind limb suspended (HS, white bars) and reloaded (HSRE, grey bars) rats at 6 and 32 months is shown (n=8–10). Values are means ± SE; * indicates a main effect for age and # indicates difference from ambulatory and HSRE in old; p<0.05.

Figure 10. ARC protein abundance is decreased with atrophy in response to age and hind limb suspension

Representative bands for Western analysis of ARC are shown for soleus muscle of ambulatory (amb), hind limb suspended (HS) and reloaded (HSRE) rats at 6 and 32 months of age (A). Ponceau S was used as a loading control (not shown). Quantification of Western analysis is depicted (B) for ambulatory (black bars), hind limb suspended (HS, white bars), and reloaded (HSRE, grey bars) rats at 6 and 32 months of age (n=8–10). Values are means ± SE; * indicates a main effect for age, and # indicates a main effect for HS; p<0.05.

Figure 11. Cytosolic EndoG is decreased with age and reloading

Representative bands for Western analysis of cytosolic EndoG are shown for soleus muscle of ambulatory (amb), hind limb suspended (HS) and reloaded (HSRE) rats at 6 and 32 months of age (A). Ponceau S was used as a loading control (not shown). Quantification of Western analysis is depicted (B) for ambulatory (black bars), hind limb suspended (HS, white bars), and reloaded (HSRE, grey bars) rats at 6 and 32 months of age (n=8–10). Values are means ± SE; * indicates a main effect for age, and # indicates a main effect for HSRE, different from other groups; p<0.05.

Figure 12. Cytosolic and nuclear AIF protein abundance is decreased with age

Representative bands for Western analysis of cytosolic (A) and nuclear (C) AIF are shown for soleus muscle of ambulatory (amb), hind limb suspended (HS) and reloaded (HSRE) rats at 6 and 32 months of age. Ponceau S was used as a loading control (not shown). Quantification of Western analysis for cytosolic (B) and nuclear (D) AIF is depicted for ambulatory (black bars), hind limb suspended (HS, white bars), and reloaded (HSRE, grey bars) rats at 6 and 32 months of age (n=8–10). Values are means ± SE; * indicates a main effect for age, and # indicates a main effect for HS different from ambulatory; p<0.05.

Figure 13. Macrophage (ED2) abundance in dysregulated in soleus muscle of aged rats

Representative cross sections immunoreacted for ED2 (CD163) of soleus muscle from 6 (A,C,E) or 32 (B,D,F) month old ambulatory (A,B), hind limb suspended (C,D), or reloaded (E,F) rats. CD163 is stained in red and nuclei are stained in blue (DAPI); arrows indicate regular staining and arrow heads point to diffuse CD163 positive staining. Bar in F represents 25μm for all pictures. Quantification of ED2 positive staining (G) in soleus muscle from ambulatory (amb, black bars), hind limb suspended (HS, white bars) and reloaded (HSRE, grey bars) rats at 6 and 32 months is shown (n=8–10). Values are means ± SE; * indicates a significant difference from young and # indicates a significant difference from ambulatory and HSRE in old; p<0.05.