Myogenin regulates denervation-dependent muscle atrophy in mouse soleus muscle

Abstract

Muscle inactivity due to injury or disease results in muscle atrophy. The molecular mechanisms contributing to muscle atrophy are poorly understood. However, it is clear that expression of atrophy-related genes, like Atrogin-1 and MuRF-1, are intimately tied to loss of muscle mass. When these atrophy-related genes are knocked out, inactive muscles retain mass. Muscle denervation stimulates muscle atrophy and Myogenin (Myog) is a muscle-specific transcription factor that is highly induced following muscle denervation. To investigate if Myog contributes to muscle atrophy, we have taken advantage of conditional Myog null mice. We show that in the denervated soleus muscle Myog expression contributes to reduced muscle force, mass, and cross-sectional area. We found that Myog mediates these effects, at least in part, by regulating expression of the Atrogin-1 and MuRF-1 genes. Indeed Myog over-expression in innervated muscle stimulates Atrogin-1 gene expression and Myog over-expression stimulates Atrogin-1 promoter activity. Thus, Myog and the signaling cascades regulating its induction following muscle denervation may represent novel targets for therapies aimed at reducing denervation-induced muscle atrophy.

Figure 1. Conditional ablation of Myog gene expression

Myog^flox/flox;CAGG-CreER^™ mice were injected with vehicle (Con) or tamoxifen (Tam) for 5 days. Left lower hindlimb muscles were denervated by sciatic nerve transection and 3–7 days later innervated and denervated muscles were dissected. Genomic DNA was isolated for PCR amplification of exon 1 from the Myog gene (top panel). RNA was isolated from satellite cells (middle panel) or from muscle (bottom panel). Shown are ethidium bromide stained gels used to resolve the amplified gene products. Note that tamoxifen treatment prevented amplification of genomic DNA containing exon 1 of the Myog gene (top panel) and also prevented denervation-dependent Myog mRNA induction (Den + Tam) (middle and bottom panels), while animals that did not receive tamoxifen showed a large increase in Myog mRNA expression following muscle denervation (Compare Inn and Den lanes in the middle and bottom panels). Experiments were done in duplicate.

Figure 2. Myog deletion inhibits denervation-dependent reductions in muscle mass and force

The left lower hindlimb muscles of Wt or Myog null (Myog^−/−) mice were denervated for 14 days. Innervated and denervated EDL and soleus muscles were then dissected and muscle mass (A) and force (B) assayed. Reported is the denervated mass and force relative to the contralateral innervated muscle. Note that Myog deletion attenuates denervation-dependent loss in muscle mass and force, especially for the soleus muscle. Error bars are standard error of the mean (n=4).

Figure 3. Myog deletion attenuates denervation-dependent reductions in muscle cross sectional area

The left lower hindlimb muscles of Wt or Myog null (Myog^−/−) mice were denervated for 14 days. Innervated and denervated EDL and soleus muscles were then dissected and muscle cross sectional areas measured. (A) Cross sectional areas relative to the innervated control are reported. Experiments were done in triplicate and 3 random areas from each muscle were sampled for fiber cross sectional areas. Error bars are standard error of the mean. (B) Typical cross section through an innervated and denervated soleus muscle from Wt and Myog null (Myog^−/−) mice. Note reduced denervation-dependent soleus muscle atrophy in Myog^−/− mice. (C) Adult hind limb muscles were denervated and 5 days later BrdU was injected (once daily) into animals for the next 8 days. Soleus muscles were collected and stained for laminin (green) to identify the extracellular matrix, BrdU (red) to identify dividing cells and DAPI (blue) to visualize nuclei.

Figure 4. Denervation-dependent regulation of Myog, Atrogin-1 and MuRF-1 gene expression

Lower hindlimb muscles of adult mice were denervated for the indicated times. Real-time PCR was used to quantify Myog, Atrogin-1, MuRF-1 and Actin mRNA levels. RNA levels were normalized to Actin RNA levels and then normalized to innervated RNA levels. Western blots were used to assay denervation-dependent Myog protein induction. Myosin protein levels were used to reveal differences in the amount of protein loaded onto the gel. Error bars are standard error of the mean (n=3).

Figure 5. Direct electrical stimulation of denervated soleus muscle suppresses denervation-dependent induction of Myog, Atrogin-1 and MuRF-1 mRNAs

Lower hind limbs of adult rats were bilaterally denervated and stimulating electrodes were implanted into one hind limb. Denervated soleus muscles were electrically stimulated for 0–48 hrs. RNA was isolated from innervated, 3-day denervated and 3-day denervated/stimulated soleus muscle and assayed by RT-PCR (representative ethidium bromide stained agarose gel is shown) and Real-time PCR (graph). Note that direct electrical stimulation of soleus muscle rapidly suppressed denervation-dependent Myog, Atrogin-1 and MuRF-1 gene expression. Experiments were repeated twice with almost identical results.

Figure 6. Regulation of atrophy gene expression in Wt and Myog^−/− mice

The left lower hind limb muscles of Wt or Myog null (Myog^−/−) mice were denervated for 3 days. Innervated and denervated soleus muscles were then dissected and RNA extracted for RT-PCR. Ethidium bromide stained gel shows the relative transcript levels. Real-time PCR was used to quantify these levels which are shown in the graph. Note that Myog deletion blocks denervation-dependent induction of Atrogin-1, MuRF-1 and Myog mRNA expression while FoxO3 mRNA expression is slightly increased in following muscle denervation. Error bars are standard error of the mean (n=3).

Figure 7. Myog regulates Atrogin-1 gene expression and Atrogin-1 promoter activity

(A) pCS2:Myog and pCS2:EGFP expression vectors were electroporated into innervated soleus muscle of Wt animals. Twelve days later soleus muscles were harvested and GFP-expressing fibers dissected for RNA analysis. Real-time PCR was used to quantify Myog and Atrogin-1 mRNA levels. Note Myog overexpression induced Atrogin-1 expression. Error bars are standard error of the mean (n=3). (B) Atrogin-1 promoter activity is regulated by Myog overexpression. HEK 293 cells were co-transfected with an Atrogin-1:luciferase reporter vector harboring various lengths of the Atrogin-1 promoter upstream of the firefly luciferase sequence and a normalization vector ubC:Renilla luciferase that harbors the human Ubiquitin promoter upstream of the Renilla luciferase sequence, with and without pCS2:Myog which allows for overexpression of Myog from the simian CMV promoter. (C) The soleus muscle of adult mice was electroporated with Atrogin-1:luciferase reporter plasmid[Sandri et al., 2004], pCS2:EGFP to identify electroporated fibers and ubC:Renilla luciferase for normalization. 12 days post-electroporation the left hindlimb muscle was denervated for 3 days and then GFP⁺ fibers were dissected from innervated and denervated electroporated muscles for luciferase assays. Black ovals in the promoter cartoon indicate E-boxes (CANNTG) that conform to the consensus Myog binding E-box, while grey ovals represent E-boxes that do not conform to this consensus sequence. Note the stimulation of Atrogin-1 promoter activity by Myog overexpression and muscle denervation and that as one deletes promoter sequences harboring E-boxes, Myog-dependent and denervation-dependent Atrogin-1 promoter activation is reduced. Experiments were performed in triplicate. Error bars are standard error of the mean (n=3).