Abstract
BackgroundForkhead box O (FoxO) transcription factors and E3 ubiquitin ligases such as Muscle RING finger 1 (MuRF1) are believed to participate in the regulation of skeletal muscle mass. The function of FoxO transcription factors is regulated by post-translational modifications such as phosphorylation and acetylation. In the present study FoxO1 protein expression, phosphorylation and acetylation as well as MuRF1 protein expression, were examined in atrophic and hypertrophic denervated skeletal muscle.MethodsProtein expression, phosphorylation and acetylation were studied semi-quantitatively using Western blots. Muscles studied were 6-days denervated mouse hind-limb muscles (anterior tibial as well as pooled gastrocnemius and soleus muscles, all atrophic), 6-days denervated mouse hemidiaphragm muscles (hypertrophic) and innervated control muscles. Total muscle homogenates were used as well as separated nuclear and cytosolic fractions of innervated and 6-days denervated anterior tibial and hemidiaphragm muscles.ResultsExpression of FoxO1 and MuRF1 proteins increased 0.3-3.7-fold in all 6-days denervated muscles studied, atrophic as well as hypertrophic. Phosphorylation of FoxO1 at S256 increased about 0.8-1-fold after denervation in pooled gastrocnemius and soleus muscles and in hemidiaphragm but not in unfractionated anterior tibial muscle. A small (0.2-fold) but statistically significant increase in FoxO1 phosphorylation was, however, observed in cytosolic fractions of denervated anterior tibial muscle. A statistically significant increase in FoxO1 acetylation (0.8-fold) was observed only in denervated anterior tibial muscle. Increases in total FoxO1 and in phosphorylated FoxO1 were only seen in cytosolic fractions of denervated atrophic anterior tibial muscle whereas in denervated hypertrophic hemidiaphragm both total FoxO1 and phosphorylated FoxO1 increased in cytosolic as well as in nuclear fractions. MuRF1 protein expression increased in cytosolic as well as in nuclear fractions of both denervated atrophic anterior tibial muscle and denervated hypertrophic hemidiaphragm muscle.ConclusionsIncreased expression of FoxO1 and MuRF1 in denervated muscles (atrophic as well as hypertrophic) suggests that these proteins participate in the tissue remodelling occurring after denervation. The effect of denervation on the level of phosphorylated and acetylated FoxO1 differed in the muscles studied and may be related to differences in fiber type composition of the muscles.
Highlights
Forkhead box O (FoxO) transcription factors and E3 ubiquitin ligases such as Muscle RING finger 1 (MuRF1) are believed to participate in the regulation of skeletal muscle mass
All results reported are based on data from 7 sets of 8 animals generating 16 denervated anterior tibial muscles with 16 contralateral innervated controls (8 innervated and 8 denervated muscles used for whole muscle protein extraction and 8 innervated and 8 denervated muscles used for preparing separate cytosolic and nuclear fractions), 8 denervated pooled gastrocnemius and soleus muscles with 8 contralateral innervated controls, 16 denervated hemidiaphragm muscles, 16 innervated control hemidiaphragms from separate animals (8 innervated and 8 denervated muscles used for whole muscle protein extraction and 8 innervated and 8 denervated muscles used for preparing separate cytosolic and nuclear fractions) and 8 hemidiaphragm muscles from sham operated animals
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)
Summary
Forkhead box O (FoxO) transcription factors and E3 ubiquitin ligases such as Muscle RING finger 1 (MuRF1) are believed to participate in the regulation of skeletal muscle mass. Muscle inactivity leads to a decrease in mass (atrophy) whereas increased activity leads to an increase in mass (hypertrophy) Such changes in muscle mass are believed to occur as a result of alterations in a delicate balance between pathways regulating muscle protein synthesis and degradation [2]. FoxO transcription factors include the four members FoxO1 (FKHR), FoxO3 (FKHRL1), FoxO4 (AFX) and FoxO6 [5,6,7] These are reported to have important roles in e.g. stress resistance and metabolism by regulating the expression of target genes. In growing cells FoxO proteins are to a high extent located in the cytoplasm [9] since nuclear export is a response to growth signals and nuclear import is a response to stress signals such as oxidative stress [9,10]
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