Identifying the signalling pathway of a novel Myostatin Splice Variant (MSV)

Myostatin (Mstn), a member of the transforming growth factor-β super family, is a potent negative regulator of skeletal muscle mass. Studies delineating the function of Mstn have identified multiple signal transduction pathways that convey the Mstn signal. Mstn has been shown to influence canonical TGF-β, mitogen activated protein kinase (MAPK) and the PI3K/AKT signal transduction cascades. The discovery in our laboratory of a novel splice variant of Mstn (MSV) that opposes Mstn and stimulates the proliferation of myoblasts provided the impetus for the investigations in this thesis. The splicing of MSV was restricted to the Cetartiodactyl clade of mammals, and MSV may represent an intragenic regulator of Mstn. Thus, the studies undertaken in this thesis were to delineate the signalling pathways used by Mstn and MSV in order to understand how their opposing roles in myoblasts regulate myogenesis. Initially, microarray analysis was used to investigate the transcriptional responses of ovine myoblasts following exposure to recombinant Mstn (eukaryotic) and MSV (prokaryotic) protein. Mstn treatment induced changes in number of transcripts, with changes consistent with previous investigations, for example increased interlukin-6 (IL6) and decreased MyoD. In addition, a novel transcriptional target of Mstn, the β1 subunit of the Na⁺-K⁺-ATPase was discovered. Treatment of ovine myoblasts with recombinant MSV induced a plethora of transcriptional responses. IPA analysis suggested a number of these were due to LPS (endotoxin) contamination, which could be attributed to the production of this protein in E. coli. This was confirmed using the Limulus amebocyte lysate assay. Phase separation using Triton-X 114 proved an effective method for the removal of LPS from the MSV preparation. Western blot analysis was performed following the treatment of ovine myoblasts with Mstn and purified MSV. Consistent with previous myoblast studies Mstn stimulated canonical TGF-β (Smad) signalling and the p38 and ERK components of the MAPK signalling cascade. In contrast to previous studies, Mstn also stimulated AKT signalling, with specific phosphorylation of serine 473 (AKTS⁴⁷³). In addition, Mstn altered the abundance of multiple myogenic transcription factors (MyoD, Myf5, MRF4, Pax7 and Mef2) and the abundance and/or the phosphorylation of targets that have a metabolic role in skeletal muscle (rps6, 4EBP1 and p70S6K). Treatment with MSV increased the abundance of Smad 3, Myf5, 4EBP1 and stimulated AKTS⁴⁷³and 4EBP1 phosphorylation. These data provided the foundation for confirmation of these pathways targeted by MSV in C₂C₁₂ myoblasts that stably expressed MSV. C₂C₁₂ cells expressing MSV had an increased proliferative capacity and showed increased mitochondrial activity (EZ4U assay) as compared to controls. These cells showed an increased abundance of the MRFs MyoD, MRF4 and Myogenin and an increased abundance or phosphorylation of signalling targets involved in canonical TGF-β, MAPK and PI3K/AKT signalling cascades. In addition, cells expressing MSV had an increased abundance and phosphorylation of acetyl co-enzyme A carboxylase (ACC) and 4EBP1, which have established roles in regulating metabolism and the synthesis of protein. The significant overlap of processes influenced by the Na⁺ , K⁺ ATPase complex and Mstn prompted an investigation on how Mstn regulates the β1 subunit of the Na⁺ , K⁺ ATPase. This transcriptional response was found to be dependent on the Smad pathway. In addition, these studies also show that Na⁺ K⁺ ATPase activity plays a role in proliferation and differentiation of ovine myoblasts and suggest that Mstn inhibits ion flow controlled through the function of this enzyme complex. In conclusion, these studies show that Mstn and MSV share a number of common signalling targets. In contrast to previous studies of Mstn, the stable expression MSV increases the activation of AKT signalling and increases the abundance of key myogenic transcription factors. In addition, MSV increases the abundance and phosphorylation of ACC and 4EBP1, molecules involved regulating the synthesis of protein and fatty acids. In addition, the β1 subunit of the Na⁺ K⁺ ATPase, was identified as a novel transcriptional target of Mstn, with this regulation controlled through a Smad dependant mechanism. These data confirm the postulate that Mstn and MSV have divergent signalling functions and suggest a role for MSV in the control of oxidative metabolism