Define the underlying mechanism of action of fertilised egg yolk as a nutritional supplement on muscle using an in vitro model and -omics technologies.
1. Determine the effects of fertilised egg yolk on the epigenome of myoblasts using small RNAseq and bioinformatics.
2. Determine the effects of fertilised egg yolk on myoblasts using an unbiased proteomics approach and bioinformatics, including data integration and meta-analysis with results from aim 1.
Muscle disorders, including age-related sarcopenia and disease-related cachexia are characterised by compromised skeletal muscle mass and function. This is due to the failure of satellite cells or myoblasts to fuse with damaged myofibres, or a decrease in their number and function. Muscle regeneration may be achieved by implementing new therapeutic strategies targeted at the cellular level. There are several characteristics of skeletal muscle regeneration that can contribute to regeneration of muscle tissue including myogenic stem cell proliferation/differentiation, regulation of myogenic regulatory molecules (including myogenin and MyoD), and alteration of muscle-specific genes.
Nutritional interventions, such as fertilised egg yolk, are a promising approach to promote muscle growth and development. Previous studies have demonstrated its effectiveness at combating muscle loss in vitro using C2C12 myoblasts. Treatment with both fertilised and unfertilised egg yolk increased cell proliferation and differentiation in a dose dependent manner characterised by increased cell metabolism, nuclei number, myotube number/fusion index and expression of myogenin. Additionally, there was a difference in cell morphology and fusion between fertilised and unfertilised egg yolk. Using TMT labelling the proteome of unfertilised and fertilised egg yolk found a higher abundance in fertilised egg yolk of proteins related to angiogenesis, indicating fertilised egg yolk may be useful to enrich pro-angiogenic proteins. Interestingly the stimulation of angiogenesis may increase the pool of myogenic stem cells that are available to drive muscle regeneration. These findings lead to the development of a nutritional supplement Fortetropin®, a high pressure pasteurised, freeze-dried, fertilised chicken egg yolk powder. Its use has been studied in human and veterinary clinical trials and it has been demonstrated to build muscle, reduce muscle loss and protein breakdown markers. Thus, it has the potential to increase muscle mass in ageing or following injury. A reduction of serum myostatin levels following Fortetropin® treatment significantly improved owner assessed mobility scores.
We hypothesise that fertilised egg yolk will alter the myoblast epigenome and proteome with downstream effects on biological pathways. Understanding the mechanisms by which fertilised egg yolk exerts its effects will increase our knowledge of muscle regeneration with clinical implications for its use in humans and companion animals.
Methodologies: C2C12 myoblasts will be cultured in 6-well plates. At 80% confluence, cells will be treated with PBS (control), 1mg/ml unfertilised egg yolk or 1mg/ml fertilised egg yolk (n=4 per group) for 2 days. Cell viability will be confirmed and myogenin/MYOD expression assessed using qRT-PCR. Myoblasts will then be differentiated to myotubes to examine the effect of unfertilised vs fertilised egg yolk on cell fusion and viability.
Myoblast/myotube protein will be subject to mass spectrometry-based proteomics using LC-MS/MS and label-free protein quantification using Progenesis QI (undertaken by Peffers so costs will be 25% of a service cost)12 and small RNAseq on the Illumina HiSeq4000 (n=4 each group). These techniques will define differentially expressed proteins and small non-coding RNA cargo of treated myoblasts which will then be subject to bioinformatics analysis15
Understanding the mechanisms by which fertilised egg yolk exerts its effects will increase our knowledge of muscle regeneration and have clinical implications for its use in humans and companion animals.
The research proposed is of high importance as we anticipate that the outcomes of this study will lead to a greater understanding of the role that supplements play on the processes underlying muscle regeneration, and hence to the logical development of interventions to correct/enhance these processes. Specifically, understanding the mechanisms by which fertilised egg yolk exerts its effects will increase our knowledge of muscle regeneration and have clinical implications for its use in humans and companion animals particularly during ageing. Developing this collaboration will increase the number of funding mechanisms available and expanding our professional networks.
Please enquire/apply to Professor Mandy Peffers.