Manipulation of animal growth by suppressing the functions of growth differentiation factors 8 (myostatin) and 11 with their propeptides

Muscle growth and bone growth are important for meat production as well as human health, since muscle and bone are two major body components in agricultural animals and abnormal development of the musculoskeletal system, which is formed by muscles and bones, will cause serious health problems to humans. Growth differentiation factor 8 (GDF8, also called myostatin) is a remarkable inhibitor for muscle growth, and growth differentiation factor 11 (GDF11) is a significant regulator that controls normal formation of skeletons. Myostatin and GDF11 are two highly homologous molecules that are antagonized by their own propeptide, myostatin propeptide and GDF11 propeptide, through a similar mechanism. Myostatin propeptide and GDF11 propeptide are generated from the N-terminal peptide present in the myostatin precursor and GDF11 precursor, respectively, following proteolytic processing. Mouse myostatin propeptide has been shown to promote muscle growth by blocking myostatin function. A mutated mouse myostatin propeptide carrying a mutation at the cleavage site of the BMP-1/TLD-like metalloproteinases was recently demonstrated to be more effective in promoting muscle growth than the wild-type mouse myostatin propeptide. GDF11 propeptide also was recently shown to antagonize GDF11 function in vitro. This project was designed to study the effect of a BMP-1/TLD-like metalloproteinases-resistant pig myostatin propeptide (a mutated pig myostatin propeptide) on muscle growth by depressing myostatin function, and the effects of mouse GDF 11 propeptide on bone growth by antagonizing GDF11 function. Recombinant wild-type and mutated porcine myostatin propeptides were produced in insect cells. Their purities and identities were confirmed by SDS-PAGE and Western Blot with anti-his.tag and anti-myostatin propeptide antibodies. Using the A204 cells reporter assay system, the abilities to depress myostatin activity in vitro were compared between wild-type and mutated pig myostatin propeptide. In 6h co-incubation analysis, mutated and wild-type porcine myostatin propeptides showed no difference in blocking myostatin activity, while in 24 h or 48 h co-incubation analyses, the former had a stronger effect on blocking myostatin activity than the later, suggesting the mutated pig myostatin propeptide has increased resistance to the degradation by BMP-1/TLD-like metalloproteinases. The mutated porcine myostatin propeptide was injected into neonatal mice to test its in vivo effects on muscle growth. Mice treated with mutated propeptide were 11--15% heavier than the controls from the age of 25 to 57 days. The weights of major skeletal muscles significantly increased by 13.5--24.8% in mice injected with mutated pig myostatin propeptide. Muscle histology analysis showed that enhanced muscle mass in mice administered with mutated pig myostatin propeptide was primarily caused by increase of muscle fiber size. These results indicate that the pig myostatin propeptide also is inhibited by the BMP-1/TLD-like metalloproteinases, the mutated pig myostatin propeptide may have application for improving muscle growth in pigs. To investigate the in vivo effects of GDF11 propeptide on bone growth by suppressing GDF11 function, transgenic mice over-expressing GDF11 propeptide specifically in bone tissue were generated by intracytoplasmic sperm injection (ICSI) in combination with piggyBac transposon-mediated gene transfer. Skeletal analyses showed that transgenic mice exhibited transformation of the seventh cervical vertebra (C7) into a thoracic vertebra, since extra ribs were formed on the C7 of transgenic mice. The effect of GDF11 propeptide transgene on forming the C7 ribs depended on transgene expression level, as the frequency of forming the C7 ribs was associated with the transgene mRNA abundance in transgenic mice. Reverse transcription PCR revealed that the transgene started to express at 12.5 dpc or between 10.5-12.5 dpc in transgenic mice. Altered expression patterns of Hoxa-4 and Hoxa-5 genes were found in 13 dpc transgenic mice by in situ hybridization, suggesting the transgene signals through these two Hox genes to regulate vertebral patterning. Earlier ossification was observed in 16.5 dpc transgenic mice, in comparison with wild-type littermates. X-rays analysis and bone histology analysis showed that bone mineral content, bone mineral density and relative trabecular bone volume were significantly increased by 11.12%, 9.68% and 60.9%, respectively, in adult transgenic male mice, and 14.96%, 8.69% and 57.7%, respectively, in adult female transgenic mice, compared to their sex-matched wild-type littermates. These results revel that GDF11 propeptide plays an important role in vertebral formation and postnatal bone growth. The overall results of this study enhanced our understanding on regulation of muscle growth and bone growth by myostatin, GDF11 and their propeptides. The knowledge obtained from this work supports further studies that aim to manipulate agricultural animal growth or improve human health.