Balanced Diet-Fed Fat-1 Transgenic Mice Exhibit Lower Hindlimb Suspension-Induced Soleus Muscle Atrophy

Abstract

The consequences of two-week hindlimb suspension (HS) on skeletal muscle atrophy were investigated in balanced diet-fed Fat-1 transgenic and C57BL/6 wild-type mice. Body composition and gastrocnemius fatty acid composition were measured. Skeletal muscle force, cross-sectional area (CSA), and signaling pathways associated with protein synthesis (protein kinase B, Akt; ribosomal protein S6, S6, eukaryotic translation initiation factor 4E-binding protein 1, 4EBP1; glycogen synthase kinase3-beta, GSK3-beta; and extracellular-signal-regulated kinases 1/2, ERK 1/2) and protein degradation (atrophy gene-1/muscle atrophy F-box, atrogin-1/MAFbx and muscle RING finger 1, MuRF1) were evaluated in the soleus muscle. HS decreased soleus muscle wet and dry weights (by 43% and 26%, respectively), muscle isotonic and tetanic force (by 29% and 18%, respectively), CSA of the soleus muscle (by 36%), and soleus muscle fibers (by 45%). Fat-1 transgenic mice had a decrease in the ω-6/ω-3 polyunsaturated fatty acids (PUFAs) ratio as compared with C57BL/6 wild-type mice (56%, p < 0.001). Fat-1 mice had lower soleus muscle dry mass loss (by 10%) and preserved absolute isotonic force (by 17%) and CSA of the soleus muscle (by 28%) after HS as compared with C57BL/6 wild-type mice. p-GSK3B/GSK3B ratio was increased (by 70%) and MuRF-1 content decreased (by 50%) in the soleus muscle of Fat-1 mice after HS. Balanced diet-fed Fat-1 mice are able to preserve in part the soleus muscle mass, absolute isotonic force and CSA of the soleus muscle in a disuse condition.

Keywords: Fat-1 mice; hindlimb suspension; muscle disuse atrophy; protein synthesis/degradation signaling; ω-3 PUFAs.

Figure 1

Muscle strength, contractile properties and muscle fatigue of the four groups studied (C57BL/6, C57BL/6 + HS, Fat-1, Fat-1 + HS) in the soleus muscle. Muscle strength: (A) Absolute isotonic force. (B) Absolute tetanic force. Contractile properties: (C) TTP; (D) HRT; (E) LRT. Muscle fatigue: (F) and (G) Resistance to fatigue (force vs contraction). Values are presented as mean ± SEM, n = 5–8 animals. The results were compared using two-way ANOVA and Bonferroni post-hoc test. In (A), p < 0.01 indicates significant difference using the Bonferroni post-hoc test. In (B), * p < 0.01 for C57BL/6 and Fat-1 groups vs. C57BL/6 + HS and Fat-1 + HS groups (main effect of HS), using two-way ANOVA only (no statistical differences using the Bonferroni post-hoc test). HS: hindlimb suspension; TTP: time to peak; HRT: half relaxation time; LRT: late relaxation time.

Figure 2

Cross-sectional areas (CSA) of the soleus muscle and soleus muscle fibers of the four groups studied (C57BL/6, C57BL/6 + HS, Fat-1, Fat-1 + HS). (A) Representative histological hematoxylin and eosin staining images of cross-sectional areas of the whole soleus muscle. a. C57BL/6; b. C57BL/6 + HS; c. Fat-1; d. Fat-1 + HS. Reference bar represents 200 µm. (B) Cross-sectional area of the soleus muscle. Values are presented as mean ± SEM, n = 7–8 animals. (C) Representative histological hematoxylin and eosin staining images of cross-sectional areas of soleus muscle fibers. a. C57BL/6; b. C57BL/6 + HS; c. Fat-1; d. Fat-1 + HS. Reference bar represents 50 µm. (D) Cross-sectional areas of soleus muscle fibers. Values are presented as mean ± SEM, n = 6–8. The results were compared using two-way ANOVA and Bonferroni post-hoc test. In (B) and (D), p values indicate significant differences using the Bonferroni post-hoc test. HS: hindlimb suspension; CSA: Cross-sectional area. 3.7. Activities of Cathepsin L and 26S Proteasome in the Gastrocnemius Muscle, and Contents of Atrogin-1/MAFbx and MuRF1 in the Soleus Muscle.

Figure 3

(A) Cathepsin L activity in the gastrocnemius muscle of the four groups studied (C57BL/6, C57BL/6 + HS, Fat-1, Fat-1 + HS). (B) 26S proteasome activity in the gastrocnemius muscle. (C) atrogin-1/MAFbx and (D) MuRF-1 content in soleus muscle. Values are presented as mean ± SEM on the basis of total protein loading as indicated by the Ponceau S measurement (C,D) and expressed relative to the C57BL/6 control group, n = 5–8 animals. The results were compared using two-way ANOVA and Bonferroni post-hoc test. In (A) and (D), p < 0.05 indicates significant differences using the Bonferroni post-hoc test. HS: hindlimb suspension.