Summary
The major objectives of this project were to develop a proteomics capability to provide new avenues for muscle development research in livestock species by applying new technology to investigate enhanced ovine muscle development and high growth phenotypes. Our purpose was to discover specific proteins and the critical cellular pathways that are involved in muscle development and growth that are genetically regulated. These findings are reported within the context of further understanding the cellular mechanisms driving muscle development.
The proteins and genes reported here can be used to provide new tools for identification of superior muscling and growth genotypes, or they can be used as targets for treatments that may enhance muscle development and/or growth. The Semitendinosus muscle samples used in this experiment were taken from lambs bred for enhanced genetic potential for muscling (M), growth rate (G) or neutral genetic potential for muscling and growth rate (C)'. Hegarty and co - workers' demonstrated in this lamb group, that breeding for enhanced genetic capacity for muscling resulted in increased hind quarter weight and crude protein content at a high plane of nutrition compared to both control and growth lambs. To elucidate changes in the underlying cellular mechanisms that support enhanced muscle development and growth rate, we enriched for populations of proteins of interest from within the complex tissue biopsies. Highly abundant contractile filament and extracellular matrix proteins were removed prior to protein expression analysis to enable enrichment of lower abundance proteins. Further subcellular fractionation reduced the complexity of the cellular protein solution and yielded a nuclear enriched (NUC), total cell membrane (TCM) and clarified cytosolic fraction (CYT).
The images presented here demonstrate that clear fractionation of cellular proteins into the NUC, CYT and TCM fraction with little duplication of proteins between fractions. Proteins in the NUC, TCM and CYT fractions of the M, G and C genotypes were separated using twodimensional electrophoresis and detected following staining with SYPRO Ruby fluorescent protein stain. Differential protein expression was determined using statistical analysis of relative concentration of matched proteins. Differentially expressed proteins were identified following peptide mass fingerprinting of proteins using Matrix Assisted Laser Desorption Ionisation (MALDI) - Time of Flight (TOF) mass spectrometry. Protein identities were determined using MS-Fit search software and ammalian protein and genome sequences. Proteome maps of the NUC, CYT and TCM fractions are presented, with differentially expressed proteins noted and proteins identified by peptide mass fingerprinting listed.
In total, 393 proteins were reproducibly detected in the NUC fraction. Of these proteins, 13 were differentially expressed between the M, G and C genotypes. Analysis using MALDI-TOF peptide mass fingerprinting identified 9 proteins. In the CYT fraction, 613 protein spots were reproducibly detected. Of these proteins, 24 were differentially expressed between the M, G and C genotypes. Analysis using MALDI-TOF peptide mass fingerprinting identified 11 proteins. In total, 639 proteins were reproducibly detected in the TCM fraction. Of these proteins, 24 were differentially expressed between the M, G and C genotypes. Analysis using MALDI-TOF peptide mass fingerprinting identified 14 proteins. Differences in hind-limb muscle development between M lambs and C and G lambs were reflected in changes in protein expression related to shifts in protein and energy metabolism pathways. Changes in net protein accretion can occur through reduction in protein turnover, or increase in protein synthesis. Increased protein accretion in M lambs is suggested to be via increased protein synthetic capacity and muscle cell hypertrophy accompanied by elevated IGF-1 sensitivity and transition towards glycolytic fibre type. Changes in expression of ribosomal and translational regulatory proteins suggests that increased capacity for ribosomal biogenesis and elevated protein translational activity was the mechanism driving increased protein synthesis in the M genotype.
Evidence was found for reduced recruitment of myonuclei in M lambs relative to C lambs, suggesting that elevated protein synthetic capacity was driven primarily through hypertrophy and not hyperplasia. This finding was supported by down regulation of some protein degradation pathway components, indicating that hypertrophy was supported by a reduction in energetic expenditure associated with protein turnover. Hegarty and co-workers' demonstrated in these lambs, that breeding on the basis of high EBVs for growth produced no increase in lean muscle mass or crude protein in the hind-limb of lambs fed at a high plane of nutrition. Despite no apparent difference in hind-limb muscle growth in G lambs compared to C, G lambs exhibited marked differences in regulation and function of energy metabolism pathways.
Evidence presented here demonstrates a shift towards elevated glucose metabolism in G lambs. This is supported by changes in regulatory proteins that influence insulin sensitivity in concert with elevated expression of proteins involved in glucose metabolism pathways. The shift in energy balance in G lambs towards increased efficiency of glucose utilisation may be regulated by small GTPase proteins including those of the mitogen activated protein-kinase pathway and their interaction with the phosphatidylinositol second messenger system. Despite no apparent difference in muscle mass at the high plane of nutrition between G and C lambs, energetic efficiency associated with muscle maintenance processes and growth may be increased in G lambs.
At low planes of nutrition these inherent shifts in metabolism may provide a mechanism for greater efficiency of energy utilisation to support growth. The research presented here details shifts in protein and energy metabolic and regulatory pathways associated with genetic capacity for muscling and growth in lambs. This information is of immediate use to the meat and livestock industry through furthering our understanding of the molecular mechanisms that underlie genetic selection for muscling and growth rate. The pathways detailed within this report present opportunities to specifically target the mechanisms that enhance muscle accretion and growth in lambs. Developing a treatment means by which to alter the function of these pathways may provide a method for further improving muscle development or growth rate in animals that have been bred on the basis of enhanced genetic capacity for muscling and/or growth.
Furthermore, development of mechanisms and treatments to specifically target these processes over coming years may yield greater gains in muscle accretion and growth rate than breeding on the basis for genetic capacity alone. These findings provide candidate genes from which to scan the current industry flock for sequence variation. Defining the chromosomal location of this list of genes in light of current information on chromosomal location of quantitative trait loci (QTL) for muscling and growth rate may provide opportunities to seek single nucleotide polymorphism's within single genes that associate with these traits.
Hence, this research has presented and opportunity to leverage further intellectual property from existing QTL projects. Validation of differential protein expression of the proteins identified here through the use of antibodies specific for the individual proteins would provide more confidence in the findings relating to individual proteins. However, given the consistency of trends defined within each of the muscling and growth genotypes, a degree of confidence in the importance of the biological pathways presented here in influencing the traits of interest can be gained.
This project formed part of research within Project 2.2.1 of the Sheep CRC, Understanding muscle and fat biology to better meet consumer requirements. The work detailed in this report was funded through Meat and Livestock Australia, the Victorian Department of Primary Industries and the Sheep Co-operative Research Centre. As such, knowledge and information developed through this experimentation will be shared between DPI, MLA and the Sheep CRC.
