Changes in REDD1, REDD2, and atrogene mRNA expression are prevented in skeletal muscle fixed in a stretched position during hindlimb immobilization

Abstract

Immobilized skeletal muscle fixed in a shortened position displays disuse atrophy, whereas when fixed in a stretched position it does not (Goldspink, D. F. (1977) J Physiol 264, 267-282). Although significant advances have been made in our understanding of mechanisms involved in development of atrophy in muscle fixed in a shortened position, little is known about why mass is maintained when muscle is immobilized in a stretched position. In the present study, we hypothesized that skeletal muscle immobilized in a stretched position would be protected from gene expression changes known to be associated with disuse atrophy. To test the hypothesis, male Sprague-Dawley rats were anesthetized using isoflurane and subjected to unilateral hindlimb immobilization for 3 days with the soleus fixed in either a shortened or stretched position. All comparisons were made to the contralateral nonimmobilized muscle. Soleus immobilized in a shortened position exhibited disuse atrophy, attenuated rates of protein synthesis, attenuated mTORC1 signaling, and induced expression of genes for REDD1, REDD2, MAFbx, and MuRF1. In contrast, immobilization of the soleus in a stretched position prevented these changes as it exhibited no difference in muscle mass, rates of protein synthesis, mTORC1 signaling, or expression of genes encoding REDD1, MAFbx or MuRF1, with REDD2 expression being reduced compared to control. In conclusion, fixed muscle length plays a major role in immobilization-induced skeletal muscle atrophy whereby placing muscle in a shortened position leads to induction of gene expression for REDD1, REDD2, and atrogenes.

Keywords: Casting; Ddit4; Ddit4l; disuse atrophy; mTORC1.

Figure 1.

The mass and rates of protein synthesis in the soleus are reduced only when the muscle is immobilized in a shortened position. Rates of protein synthesis were measured by the amount of puromycin incorporated into protein as assessed by immunoblot analysis. Rats had one hindlimb immobilized for 3 days in a position to place the soleus muscle in either a stretched or shortened position (Imm: Immobilized; N leg = Nonimmobilized limb). Bars represent (A) the mean mass of the soleus, and (B) the mean rates of protein synthesis in the soleus from an immobilized hindlimb expressed as a percentage of the values from the soleus from the contralateral nonimmobilized hindlimb. Data are mean percentages ± SEM, n =10 rats/group. *P <0.05 versus soleus from the nonimmobilized limb.

Figure 2.

Phosphorylation of p70S6K1 at Thr389 in the soleus is reduced only when the muscle is immobilized in a shortened position. Phosphorylation of p70S6K1 at Thr389 was assessed by protein immunoblot analysis. Rats had one hindlimb immobilized for 3 days in a position to place the soleus in either a stretched or shortened position (Imm: Immobilized; N leg = Nonimmobilized limb). Bars represent the mean p70S6K1 phospho/total protein ratio in the soleus from an immobilized hindlimb expressed as a percentage of that in the soleus from the contralateral nonimmobilized hindlimb. No changes in total p70S6K1 expression were observed in any group across 3 days of hindlimb immobilization. Data are mean percentages ± SEM, n =10 rats/group. Imm *P <0.05 versus soleus from the nonimmobilized limb.

Figure 3.

REDD1 and REDD2 mRNA expression in the soleus is induced only when the muscle is immobilized in a shortened position. REDD1 and REDD2 mRNA expression were assessed by Taqman gene expression assay. Rats had one hindlimb immobilized for 3 days in a position to place the soleus in either a stretched or shortened position. Bars represent the mean (A) REDD1 mRNA‐, or (B) REDD2 mRNA‐to‐Tbp mRNA ratio in the soleus from an immobilized hindlimb expressed as a percentage of the same ratio in the soleus from the contralateral nonimmobilized hindlimb. No changes in Tbp gene expression were observed in any group across 3 days of hindlimb immobilization. Data are mean percentages ± SEM, n =10 rats/group. *P <0.05 versus soleus from the non‐immobilized limb.

Figure 4.

Atrogene expression is induced in the soleus only when the muscle is immobilized in a shortened position. Atrogene mRNA expression was assessed by Taqman gene expression assay. Rats had one hindlimb immobilized for 3 days in a position to place the soleus muscle in either a stretched or shortened position. Bars represent the mean (A) MAFbx mRNA‐, or (B) MuRF1 mRNA‐to‐Tbp mRNA ratio in the soleus from an immobilized hindlimb expressed as a percentage of the same ratio in the soleus from the contralateral nonimmobilized hindlimb. No changes in Tbp gene expression were observed in any group across 3 days of hindlimb immobilization. Data are mean percentages ± SEM, n =10 rats/group. *P <0.05 versus soleus from the nonimmobilized limb.