Alterations in muscle weights following 6 days of denervation are illustrated in Figure 1 for muscles used for whole muscle protein extraction. Six days after denervation hemidiaphragm muscles were hypertrophic with a wet weight of 43.3 ± 0.7 mg (n = 8, p < 0.001 versus innervated and sham operated, one-way ANOVA with Tukey’s multiple comparisons test, Figure 1) compared to innervated controls with a wet weight of 28.2 ± 0.8 mg (n = 8) and sham operated control muscles with a wet weight of 29.7 ± 1.0 mg (n = 8). After six days of denervation anterior tibial muscles were atrophic with a wet weight of 51.6 ± 1.8 mg (n = 8), compared to innervated controls with a wet weight of 65.9 ± 1.8 mg (n = 8, p < 0.001, Student’s paired t-test, Figure 1). After six days of denervation pooled gastrocnemius and soleus muscles were atrophic with a wet weight of 149.1 ± 4.4 mg (n = 8), compared to innervated controls with a wet weight of 198.3 ± 5.7 mg (n = 8, p < 0.001, Student’s paired t-test, Figure 1).

Weights of muscles used for preparing separated nuclear and cytosolic fractions were as follows. 6-days denervated hemidiaphragm muscles were hypertrophic with a wet weight of 40.3 ± 1.5 mg (n = 8) compared to innervated controls with a wet weight of 29.6 ± 0.6 mg (n = 8, p < 0.001, Student’s t-test). 6-days denervated anterior tibial muscles were atrophic with a wet weight of 44.7 ± 1.8 mg (n = 8), compared to innervated controls with a wet weight of 60.8 ± 1.9 mg (n = 8, p < 0.001, Student’s paired t-test).

FoxO1 protein expression increased 0.8-3.7-fold in all 6-days denervated muscles studied, atrophic as well as hypertrophic (Figure 2).

The mean expression level of total FoxO1 protein in anterior tibial muscles was 383.8 ± 131.0 arbitrary units (n = 8) in denervated muscles compared to 100.0 ± 31.1 (n = 8) in innervated muscles (p < 0.05, Student’s paired t-test, Figure 2A). The mean protein expression level in pooled gastrocnemius and soleus muscles was 179.5 ± 10.7 arbitrary units (n = 8) in denervated muscles compared to 100.0 ± 11.9 (n = 8) in innervated muscles (p < 0.01, Student’s paired t-test, Figure 2B). The mean protein expression level in hemidiaphragm muscles was 471.0 ± 173.1 arbitrary units in denervated muscles (n = 8, p < 0.05 versus innervated and sham operated, Kruskal-Wallis test with Dunn’s multiple comparisons test, Figure 2C) compared to 100.0 ± 59.5 (n = 8) in innervated muscles and 70.7 ± 33.6 (n = 8) in sham operated control muscles (Figure 2C).

Levels of phosphorylated FoxO1 were unchanged in 6-days denervated anterior tibial muscle (atrophic) but increased about 0.8-1-fold in 6-days denervated hemidiaphragm (hypertrophic) and in pooled 6-days denervated gastrocnemius and soleus muscles (atrophic, Figure 3).

The mean expression level of phosphorylated FoxO1 in anterior tibial muscles was 99.3 ± 21.9 arbitrary units (n = 8) in denervated muscles compared to 100.0 ± 28.6 (n = 8) in innervated muscles (Figure 3A). The mean expression level in pooled gastrocnemius and soleus muscles was 196.1 ± 24.6 arbitrary units (n = 8) in denervated muscles compared to 100.0 ± 13.1 (n = 8) in innervated muscles (p < 0.01, Student’s paired t-test, Figure 3B). The mean expression level in hemidiaphragm muscles was 178.5 ± 27.9 arbitrary units in denervated muscles (n = 8, p < 0.05 versus innervated and p < 0.01 versus sham operated, one-way ANOVA with Tukey’s multiple comparisons test, Figure 3C) compared to 100.0 ± 12.6 (n = 8) in innervated muscles and 74.0 ± 12.7 (n = 8) in sham operated control muscles (Figure 3C).

The level of acetylated FoxO1 increased about 0.8-fold in 6-days denervated anterior tibial muscles (atrophic) but no statistically significant changes were seen in 6-days denervated pooled gastrocnemius and soleus muscles (atrophic) nor in 6-days denervated hemidiaphragm muscles (hypertrophic, Figure 4).

The mean expression level of acetylated FoxO1 in anterior tibial muscles was 183.5 ± 36.2 arbitrary units (n = 8) in denervated muscles compared to 100.0 ± 16.7 (n = 8) in innervated muscles (p < 0.01, Wilcoxon matched-pairs signed rank test, Figure 4A). The mean expression level of acetylated FoxO1 in pooled gastrocnemius and soleus muscles was 114.6 ± 10.6 arbitrary units (n = 8) in denervated muscles compared to 100.0 ± 11.6 (n = 8) in innervated muscles (Figure 4B). The mean expression level of acetylated FoxO1 in hemidiaphragm muscles was 99.8 ± 10.3 arbitrary units (n = 8) in denervated muscles compared to 100.0 ± 19.8 (n = 8) in innervated muscles and 109.5 ± 15.8 (n = 8) in sham operated control muscles (Figure 4C).

MuRF1 protein expression increased 0.3-0.9-fold in all 6-days denervated muscles studied, atrophic as well as hypertrophic (Figure 5).

The mean expression level of MuRF1 protein in anterior tibial muscles was 132.6 ± 21.2 arbitrary units (n = 8) in denervated muscles compared to 100.0 ± 17.2 (n = 8) in innervated muscles (p < 0.01, Wilcoxon matched-pairs signed rank test, Figure 5A). The mean protein expression level in pooled gastrocnemius and soleus muscles was 189.0 ± 15.6 arbitrary units (n = 7) in denervated muscles compared to 100.0 ± 13.0 (n = 7) in innervated muscles (p < 0.05, Wilcoxon matched-pairs signed rank test, Figure 5B). The mean protein expression level in hemidiaphragm muscles was 170.9 ± 10.4 arbitrary units in denervated muscles (n = 8, p < 0.05 versus innervated muscles, one-way ANOVA with Tukey’s multiple comparisons test, Figure 5C) compared to 100.0 ± 13.3 (n = 8) in innervated muscles and 111.3 ± 24.3 in sham operated control muscles (Figure 5C).

In innervated as well as in 6-days denervated atrophic anterior tibial muscle total and phosphorylated FoxO1 protein were mainly present in cytosolic fractions. Expression increased about 1-fold and 0.2-fold, respectively, in cytoplasmic fractions of 6-days denervated muscles (Figure 6).

The mean expression level of total FoxO1 protein in the cytosolic fraction of anterior tibial muscles was 136.7 ± 14.7 arbitrary units (n = 8) in denervated muscles compared to 66.9 ± 4.4 (n = 8) in innervated muscles (p < 0.01, Student’s paired t-test). In the nuclear fraction the expression level of total FoxO1 protein in denervated muscle was 23.7 ± 4.2 arbitrary units (n = 8) compared to 33.1 ± 5.0 (n = 8) in innervated muscles (Figure 6A).

The mean expression level of phosphorylated FoxO1 in the cytosolic fraction of anterior tibial muscles was 97.9 ± 5.6 arbitrary units (n = 8) in denervated muscles compared to 84.8 ± 5.1 (n = 8) in innervated muscles (p < 0.01, Student’s paired t-test). In the nuclear fraction the expression level of phosphorylated FoxO1 in denervated muscle was 16.7 ± 3.4 arbitrary units (n = 8) compared to 15.2 ± 4.9 (n = 8) in innervated muscles (Figure 6B).

In innervated as well as in 6-days denervated hypertrophic hemidiaphragm muscle total and phosphorylated FoxO1 protein were mainly present in cytosolic fractions. Expression of total FoxO1 protein increased about 1.7-fold and 1.4-fold, respectively, in cytoplasmic and nuclear fractions of 6-days denervated muscles. Expression of phosphorylated FoxO1 increased about 1.3-fold and 2.5-fold, respectively, in cytoplasmic and nuclear fractions of 6-days denervated muscles (Figure 7).

The mean expression level of total FoxO1 protein in the cytosolic fraction of hemidiaphragm muscles was 204.8 ± 32.2 arbitrary units (n = 8) in denervated muscles compared to 77.2 ± 16.6 (n = 8) in innervated muscles (p < 0.01, Student’s t-test). In the nuclear fraction the expression level of total FoxO1 protein in denervated muscle was 54.8 ± 10.1 arbitrary units (n = 8) compared to 22.8 ± 5.5 in innervated muscles (p < 0.05, Student’s t-test, Figure 7A).

The mean expression level of phosphorylated FoxO1 in the cytosolic fraction of hemidiaphragm muscles was 213.3 ± 41.8 arbitrary units (n = 8) in denervated muscles compared to 92.8 ± 25.3 (n = 8) in innervated muscles (p < 0.05, Mann–Whitney test). In the nuclear fraction the expression level of phosphorylated FoxO1 in denervated muscle was 25.2 ± 4.8 arbitrary units (n = 8) compared to 7.2 ± 2.0 in innervated muscles (p < 0.01, Student’s t-test, Figure 7B).

In innervated as well as in 6-days denervated atrophic anterior tibial muscle and hypertrophic hemidiaphragm muscle MuRF1 protein was mainly present in cytosolic fractions. Expression increased about 0.5-2.4-fold in cytoplasmic as well as nuclear fractions of 6-days denervated muscles (Figure 8).

The mean expression level of MuRF1 protein in the cytosolic fraction of anterior tibial muscles was 90.2 ± 12.2 arbitrary units (n = 8) in denervated muscles compared to 60.4 ± 10.2 (n = 8) in innervated muscles (p < 0.05, Student’s paired t-test). In the nuclear fraction the expression level of MuRF1 protein in denervated muscle was 62.0 ± 10.2 arbitrary units (n = 8) compared to 39.6 ± 6.1 (n = 8) in innervated muscles (p < 0.05, Student’s paired t-test, Figure 8A).

The mean expression level of MuRF1 protein in the cytosolic fraction of hemidiaphragm muscles was 134.5 ± 21.6 arbitrary units (n = 8) in denervated muscles compared to 80.5 ± 9.3 (n = 8) in innervated muscles (p < 0.05, Student’s t-test). In the nuclear fraction the expression level of MuRF1 protein in denervated muscle was 67.1 ± 10.4 arbitrary units (n = 8) compared to 19.5 ± 2.1 in innervated muscles (p < 0.001, Student’s t-test, Figure 8B).

Adult male NMRI mice (Scanbur, Sollentuna, Sweden) were used in this study. The mice were kept in cages with environment enrichment and with free access to a standard laboratory diet and tap water. The animals were anaesthetized by inhalation of isoflurane before surgery and received a subcutaneous injection of buprenorphine (50 μg/kg) for post-operative analgesia. Denervation of either the left hemidiaphragm or the left hind-limb was performed by sectioning and removing a few mm of the phrenic nerve or the sciatic nerve as described previously [50]. Six days after denervation the mice were killed by cervical dislocation. Hind-limb muscles (anterior tibial and gastrocnemius together with soleus) were rapidly dissected, weighed, frozen on dry ice and stored at −80°C. Innervated control hind-limb muscles were collected from the contralateral (right) leg of animals that were denervated by sectioning the left sciatic nerve. Innervated left control hemidiaphragms were collected from separate animals that had received no surgery. To control for this, eight animals used for hemidiaphragm studies went through sham surgery. These animals were anaesthetized by inhalation of isoflurane, had a subcutaneous injection of buprenorphine (50 μg/kg) and a unilateral thoracotomy without touching the phrenic nerve. For dissection of the hemidiaphragm muscle the diaphragm, attached to the rib cage, was quickly removed and placed in cold phosphate buffered saline (PBS) with calcium (2 mM). The left hemidiaphragm was then dissected under a dissecting microscope, blotted dry on filter paper, weighed, frozen on dry ice and stored at −80°C. The experimental manipulations have been approved by the Ethical Committee for Animal Experiments, Linköping, Sweden (permit number: 67–10).

Hemidiaphragm, anterior tibial and pooled gastrocnemius and soleus muscles were used for protein extractions. The muscles were homogenized using an Ultra-Turrax homogenizer (Janke and Kunkel, Staufen, Germany) in 1 ml (hemidiaphragm and anterior tibial muscles) or 2 ml (pooled gastrocnemius and soleus muscles) buffer containing 100 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% sodium deoxycholate and 1% Halt™ Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, Rockford, IL). The supernatant was recovered and the pellet was resuspended in 0.5 ml (anterior tibial and hemidiaphragm muscles) or 1.0 ml (pooled gastrocnemius and soleus muscles) of homogenization buffer and recentrifuged. The supernatants were combined and the protein concentration was determined using the Bradford assay [51].

Mouse hemidiaphragm and anterior tibial muscles were used for cytosolic and nuclear protein extraction. The method used was slightly modified from [52] and has been described separately [53]. In brief, muscles that had been stored at −80°C were homogenized using an Ultra-Turrax homogenizer (Janke and Kunkel, Staufen, Germany) in 1 ml low salt lysis buffer (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT); pH 7.9 with 1% Halt™ Protease and Phosphatase Inhibitor Cocktail from Thermo Scientific, Rockford, IL). The homogenized tissue was then vortexed (15 s), put on ice (10 min), vortexed again (15 s) and centrifuged (16.000 g for 15 s). The supernatant cytosolic extract was frozen at −80°C for subsequent analyses. The nuclear pellet was resuspended on ice in a high salt nuclear extraction buffer (20 mM HEPES, 420 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 25% glycerol; pH 7.9 with 1% Halt™ Protease and Phosphatase Inhibitor Cocktail from Thermo Scientific, Rockford, IL). Four μl of nuclear extraction buffer was used per mg muscle wet weight. Preparations were incubated on ice for 30 min and vortexed (10 s) every 5 min before centrifugation (16.000 g for 6 min). The supernatant nuclear extract was frozen at −80°C for subsequent analyses. Protein determinations for each fraction were obtained using the Bradford assay [51].

Western blots were prepared essentially as described in [50]. Fifteen to thirty μg protein were reduced, denatured and electrophoretically separated on a 12% polyacrylamide gel with a 5.2% polyacrylamide stacking gel on top. Gels were electroblotted onto PVDF Plus transfer membranes (Amersham Hybond-P, GE Healthcare, Buckinghamshire, England) and the membranes were blocked and then incubated with antibodies. Primary antibodies for detecting total-FoxO1 (rabbit monoclonal) (C29H4) [2880] and pFoxO1 S256 (rabbit polyclonal) [9461] were obtained from Cell Signaling Technology (Beverly, CA), MuRF1 (goat polyclonal) [AF5366] was obtained from R&D systems (Abingdon, England) and Ac-FoxO1 (rabbit polyclonal) (FKHR D19) [49437] from Santa Cruz Biotechnology (Santa Cruz, CA). All primary antibodies were used at a dilution of 1/800 – 1/1500. Antibodies were visualized with horseradish peroxidise conjugated secondary immunoglobulin diluted 1/1000 goat anti-rabbit IgG [P0448] and 1/1000-1/10000 rabbit anti-goat IgG [P0449] (Dako, Glostrup, Denmark). The bound immune complexes were detected using the ECL Plus Western blotting detection system and Hyperfilm ECL (Amersham International and Amersham Pharmacia Biotech, Buckinghamshire, England).

The expression levels of total, phosphorylated and acetylated proteins were studied semi-quantitatively using data from Western blots. Equal amounts of total, cytosolic or nuclear proteins from innervated or denervated muscles were loaded on the gels. Measured levels of total, phosphorylated or acetylated proteins are expressed without normalization to any specific protein. No loading controls were used and any difference in protein quantifications, pipettings steps, protein transfers etc. are included in the variations of the data sets.

Image analysis was performed using the gel plotting macro of the program ImageJ (Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij/, 1997–2007). Results were obtained in uncalibrated optical density units

In order to be able to compare data for whole muscle homogenates run on different gels, one innervated muscle sample (a reference sample) was included in all gels containing samples to be compared to each other. All other samples were measured relative to this reference, the signal of which was set to 100.0 in all gels. In order to more easily compare denervated and innervated muscles all data were finally normalized in such a way that the average signal from innervated muscles became 100.0.

For quantification of protein expression in separated cytosolic and nuclear fractions one of the cytosolic fractions from an innervated muscle was used as a reference sample and was included in all gels. All other samples were measured relative to this reference, the signal of which was set to 100.0. From the amount of protein loaded on gels in relation to the total amount of protein extracted in the nuclear and cytosolic fractions a total cytosolic and a total nuclear signal was calculated for whole muscles. In the final analysis total cytosolic and total nuclear signals were again normalized in such way that the sum of the nuclear and cytosolic signals became 100.0 in innervated muscle.

Data are presented as mean values ± standard error of the mean (SEM). For statistical comparisons of unfractionated hemidiaphragm muscles (innervated, sham operated and denervated) one way-ANOVA was used, followed by Tukey’s multiple comparisons test, for normally distributed data (according to D’Agostino-Pearson omnibus K2 normality test). Statistical significance for data not being normally distributed was determined using the Kruskal-Wallis test with Dunn’s multiple comparisons test. For other comparisons Student’s t-test (paired observations for hind-limb muscles, unpaired observations for hemidiaphragm muscles) was used for normally distributed data. Statistical significance for data not being normally distributed was determined using the Wilcoxon matched-pairs signed rank test (hind-limb muscles) or the Mann–Whitney test (hemidiaphragm muscles).