Molecular profiles of Quadriceps muscle in myostatin-null mice reveal PI3K and apoptotic pathways as myostatin targets

Abstract

Background: Myostatin (MSTN), a member of the TGF-beta superfamily, has been identified as a negative regulator of skeletal muscle mass. Inactivating mutations in the MSTN gene are responsible for the development of a hypermuscular phenotype. In this study, we performed transcriptomic and proteomic analyses to detect altered expression/abundance of genes and proteins. These differentially expressed genes and proteins may represent new molecular targets of MSTN and could be involved in the regulation of skeletal muscle mass.

Results: Transcriptomic analysis of the Quadriceps muscles of 5-week-old MSTN-null mice (n = 4) and their controls (n = 4) was carried out using microarray (human and murine oligonucleotide sequences) of 6,473 genes expressed in muscle. Proteomic profiles were analysed using two-dimensional gel electrophoresis coupled with mass spectrometry. Comparison of the transcriptomic profiles revealed 192 up- and 245 down- regulated genes. Genes involved in the PI3K pathway, insulin/IGF pathway, carbohydrate metabolism and apoptosis regulation were up-regulated. Genes belonging to canonical Wnt, calcium signalling pathways and cytokine-receptor cytokine interaction were down-regulated. Comparison of the protein profiles revealed 20 up- and 18 down-regulated proteins spots. Knockout of the MSTN gene was associated with up-regulation of proteins involved in glycolytic shift of the muscles and down-regulation of proteins involved in oxidative energy metabolism. In addition, an increased abundance of survival/anti-apoptotic factors were observed.

Conclusion: All together, these results showed a differential expression of genes and proteins related to the muscle energy metabolism and cell survival/anti-apoptotic pathway (e.g. DJ-1, PINK1, 14-3-3epsilon protein, TCTP/GSK-3beta). They revealed the PI3K and apoptotic pathways as MSTN targets and are in favour of a role of MSTN as a modulator of cell survival in vivo.

Figure 1

Abundance of candidate proteins to be myostatin targets as revealed by western-blotting in the muscles of control and MSTN-null mice. A-D: control mice; E-H: MSTN-null mice. pAkt: phosphorylated akt; GSK-3β: Glycogen synthase kinase 3β; GSK-3β (ser9): Glycogen synthase kinase 3β (phosphorylated at serine 9); DJ-1: cancer- and Parkinson's disease-associated protein (park7); PINK1: PTEN-induced putative kinase 1; PTEN: tumour-suppressor phosphatase with tensin homology. TCTP: Translationally controlled tumor protein; 14-3-3E: 14-3-3ε protein. Actin was used as a loading control protein.

Figure 2

In MSTN-null mice, regulators of the PI3K pathway and cell survival/apoptosis likely contribute to muscle hypertrophy. PI3K signalling pathway: PI3K, Phosphatidyl Inositol 3 Kinase; Akt, protein kinase B; GSK-3β, Glycogen synthase kinase 3β; eIF2B, eukaryotic protein synthesis initiation factor 2B; PTEN, tumour-suppressor phosphatase with tensin homology (the most important negative regulator of the cell survival signalling initiated by PI3K); DJ-1, a cancer- and Parkinson's disease-associated protein (a novel regulator of the PI3K-PTEN pathway). Survival/Anti-apoptotic factors: PINK1, PTEN-induced putative kinase; Dad1, defender against apoptotic death; TCTP, Translationally controlled tumour protein; 14-3-3E, 14-3-3 protein epsilon; Birc5, survivin.