Using microarray and IPA analyses, the changes in gene expression associated with Mstn and MSV treatment in ovine myoblasts was investigated. Both proteins induced significant gene expression changes after a 6 h treatment, and the integrity of the microarray data was confirmed by the validation of the metabolic targets using qPCR. With the use of IPA, the top biological functions associated with Mstn treatment appeared consistent with Mstn function. These included the regulation of genes involved in skeletal muscle disorders, cellular growth and proliferation, cellular development, the cell cycle and skeletal and muscular system development and function. The response to MSV on the other hand did not display any top biological functions associated with skeletal muscle. Rather, MSV induced changes in gene expression that had a greater connectivity with inflammatory responses, with three functions similar to those identified for Mstn (cellular movement, cellular growth and proliferation and the inflammatory response). Identification of the inflammatory response as a top biological function was a concern with 45 genes up-regulated by MSV vs. 9 for Mstn. Therefore, the purity of the MSV preparation was questioned. This was due primarily to the fact that it had been produced in E. coli, and no prior steps were taken to ensure it was LPS free. This was not a concern with the commercial Mstn preparation, which is produced in a eukaryotic system and has certified low concentrations of endotoxin (<0.1 EU/µ g protein). Upon investigating the top 10 genes up-regulated by MSV treatment, 8 have been previously shown to be regulated by LPS as opposed to 4 in the Mstn top 10 (Ingenuity® Systems, www.ingenuity.com). Interestingly, IPA upstream regulator analysis predicted LPS as an upstream regulator for both datasets, with MSV regulating 34 out of 45 and Mstn 10 out 12 identified LPS target genes from their respective data sets, in a manner consistent with LPS as an upstream activator. However, there were key differences in this analysis indicating that the regulation of Mstn targets occurred via different mechanisms. Importantly, the MSV data set also identified Toll like receptor 4 (TLR4), the LPS receptor, as an upstream regulator. In addition, TGFβ1, a close relative of Mstn, was identified as an upstream regulator in the Mstn but not the MSV data set. This is of particular importance, as activation of this pathway can regulate 7 of the 10 LPS target genes (referred to above) identified in the Mstn analysis, with the remaining three not influenced by MSV treatment in this analysis (data not
shown), illustrating that the changes induced following exposure to Mstn are consistent with TGF-β treatment.
Following my discovery that recombinant MSV preparations were likely to be contaminated with endotoxin, the extent of this problem had to be investigated. This required firstly determining if endotoxin could regulate the proliferation of myoblasts and, secondly, the removal of endotoxin from the MSV preparation. LPS significantly increased the proliferation of ovine myoblasts at concentrations consistent with those observed in the MSV preparation using the LAL assay. This had significant consequences on my interpretation of the microarray data. With no clear way to discriminate between an LPS and/or an MSV dependent target, the Mstn data was the only reliable portion of this analysis. As the differential regulation of the β1 subunit of the Na+-K+-ATPase by both Mstn and MSV containing LPS (Figure 3.1) had been confirmed, this target was selected for further investigation from the Mstn perspective (Chapter 5). This and other Mstn targets would be investigated following treatment with MSV, once an endotoxin free preparation was available. To determine if the purified MSV preparation influenced the transcription of Mstn targets identified from the microarray analysis, primers for qPCR were designed to amplify top molecules regulated by Mstn. Purified MSV did not alter the transcription of any of the top molecules influenced by Mstn that were investigated in this study.
Following the determination of the endotoxin concentration present in the MSV preparation, its removal became a strong focus. The use of Polymyxin resin and Triton X-114 phase separation were used as established methods for the removal endotoxin from bacterially produced recombinant proteins. Polymyxin reduced the concentration of endotoxin present in the MSV preparation by ~75%, but not enough to warrant its use as a purification method. Purification with Triton X-114 reduced endotoxin content to undetectable levels. However, this method introduced a caveat for ongoing studies using this preparation. This is due to residual Triton X-114 remaining in the preparation, which could potentially alter protein folding, have bioactivity of its own or interfere with the MSV contained in this preparation. This was addressed by including dialysis buffer and LPS solutions that had also been subjected to Triton X-114 as controls where appropriate. Given that concentration of endotoxin in the Triton X-114 purified
MSV was zero, this method was chosen to purify MSV for further investigations. It is noteworthy here that purification with Polymyxin was performed with a single incubation with MSV, while Triton X-114 involved 4 phase separations (see 2.2.16). Thus, it is possible that a refined Polymyxin protocol could yield similar results to that of Triton X-114. However, concerns regarding the use of Triton X-114 were lessened from other studies reporting that proteins treated with Triton X-114 maintain bioactivity in binding as well as physiological studies (Liu, Tobias et al. 1997; Magalhaes, Lopes et al. 2007). Furthermore, residual Triton X- 114 is reported to be less than 0.018% (Aida and Pabst 1990).
The proliferative response was the first bioassay used to determine if the LPS free MSV had retained its bioactivity. Interestingly, proliferation assays showed that the MSV preparation could still stimulate C2C12, but not ovine
myoblast proliferation as previously shown (unpublished data). This was done in parallel with a purified LPS preparation to ensure the Triton X-114 procedure yielded endotoxin free and biologically inactive medium. The species specific response to the MSV, suggests that there may be differing mechanisms that convey the function of MSV in myoblasts. Alternatively, primary ovine myoblasts may be more sensitive to residual Triton X-114 as compared to the immortalised C2C12 cells. Importantly, this resulted in a purified MSV preparation that could be
used for signal transduction and metabolic studies in ovine myoblasts.