Effects Of Maternal Dietary Protein On Protein Synthesis And Myostatn Transcriptional Regulation In Skeletal Muscle Of Meishan Piglets And Its Mechanisms

Myostatin (MSTN) is a negative regulator of skeletal muscle growth factor. Since MSTN was discovered, the researches about it were focused on the effects of gene knockout or over-expression on muscle and effects on cell proliferation and differentiation in vitro. Recent studies have shown that, MSTN can reduce muscle hypertrophy by inhibition of skeletal muscle protein synthesis, thereby affect muscle growth. However, the signaling pathways of MSTN regulating protein synthesis are not clear. Skeletal muscle is susceptible to nutritional programming, demonstrating high plasticity of muscle fiber phenotype in response to maternal undernutrition or protein restriction during pregnancy and/or lactation. Our previous study found that maternal dietary protein levels can change MyHCIIb gene expression in skeletal muscle of offspring Meishan pigs, and the effects are stage-specific, namely short-term and long-term effects.However, it is not clear whether maternal dietary protein affect protein synthesis and the expression of MSTN in LD muscle, or the effects are stage-specific. Furthermore, the mechanisms underlying such stage-dependent regulation of gene expression in response to maternal nutrition have not been elucidated. Therefore, the present study was aimed to investigate the effects of maternal dietary protein during gestation and lactation on offspring skeletal muscle MSTN activity and transcriptional regulation in Meishan pigs and its mechanism.1Effects of maternal dietary protein level on the levels of insulin and free amino acid in the plasma and cross-sectional area of muscle fiber of offspring at weaning and finishing stagesMeishan sows were fed traditionally on low-protein diets, whereas the standard commercial diets contain at least2folds higher crude protein. In this study,14primiparous purebred Meishan sows were fed on either low-protein (6%and7%) or standard-protein (12%and14%) diets, during gestation and lactation, respectively. Newborn pigs were counted and individually weighed at parturition. Litter size was adjusted to7to8pigs per litter at24h post farrowing in the same group. Newborn piglets were allowed free access to their mothers and weaned at35days (d) of age. After weaning, piglets were raised following the standard feeding regimen with the starter, grower and finisher diets recommended for the breed. Male pigs were killed at weaning (35days of age) and finishing (8months of age) stages, and the myofiber characteristics were observed.The longissimus dorsi (LD) was taken for measuring the cross-sectional areas using HE staining, determination muscle protein concentration using bicinchonininc acid (BCA). The levels of insulin and free amino acids in plasma were measured by radioimmunoassay and amino acid auto-analyzer, respectively. The results showed that:Body weight, LD muscle weights and muscle protein concentrations were significantly lower(p<0.05) in LP piglets at weaning stage with decreased cross-sectional area muscle fiber (CSA)(p<0.05). The levels of insulin and free amino acid were decreased significantly in LP piglets at weaning stage (p<0.01).At finishing stage, none of body weight, muscle weight or muscle protein concentration was affected by maternal dietary protein, so did insulin or free amino acid in plasma. However, cross-sectional area of muscle fiber was showed a tendency of increase (p=0.057) in LP pigs. These results suggest that maternal dietary protein level may regulate the growth of skeletal muscle by affecting skeletal muscle protein synthesis in pigs at weaning stage.2Effects of maternal dietary protein levels on the downstream signal of insulin and amino acid and MSTN downstream signal pathway in LD muscle of offspring at weaning and finishing stagesAs mentioned above, maternal dietary protein levels changed the levels of insulin and free amino acids in plasma, and did it affect their downstream signal or not? We investigated insulin receptor (Insulin receptor, IR) and Akt protein expression of insulin downstream signaling and protein aynthesis key factors of amino acid downstream signaling using Western blotting. We detected the expression of genes (MSTN,MyoD, MEF2and Myogenin) related to muscle development and amino acid downstream signal genes using Real-time PCR.We found that maternal dietary protein levels did not affect downstream of insulin signaling, but maternal low dietary protein decreased protein aynthesis key factors p70S6K and eIF4E activity of amino acid downstream signal significantly (p<0.05),and reduced eIF2a expression of amino acid downstream signal in LD muscle of offspring at weaning stage. We also found that the expression of ATF4, LAT1and CHOP of amino acid downstream signal were decreased significantly in LP piglets at weaning stage (p<0.05),but the expression of muscle development related genes were not affected except MSTN.MSTN is susceptive to amino acid, and it played an important role in skeletal muscle protein syntheis. Maternal low dietary protein decreased MSTN mRNA levels significantly in LP piglets at weaning stage, but the MSTN mRNA level was increased in LP pigs significantly at finishing stage (P<0.05). Thus, we examined the expression of MSTN receptor (ACVR2B and ALK5) and the changes of its downstream signal smad2,3, p38-MAPK, ERK, JNK and their phosphorylation forms. The results showed that ACVR2B mRNA levels was significantly increased (p<0.05) and ALK5mRNA levels showed a trend of increase in LP piglets at weaning stage (p=0.073), the phosphorylation levels of smad2and p38-MAPK were significantly increased in LP piglets at weaning stage. Nevertheless, there were no significant differences between the two groups at finishing stages.Our results suggested that maternal dietary protein levels may affect muscle protein synthesis by amino acid and MSTN signaling pathways at weaning stage.3The molecular mechanism of maternal dietary protein level affecting transcriptional regulation of MSTN geneFrom the above, we found that the effects of maternal dietary protein on MSTN expression were distinct at weaning and finishing stages. To investigate the mechanism of maternal programming, we detected the expression of MSTN transcription factors (FOXO1, MyoD, CREB and C/EBPβ) in nuclear lysates of LD muscle by western blot analysis. Significantly lower (p<0.05) C/EBPβ (LAP*) protein content was detected in LD muscle of LP piglets. It suggested that C/EBPP may be involved in transcriptional regulation of MSTN. But it was no difference between the two groups at finishing stage. In order to clarify whether diminished C/EBPβ protein content in nuclear lysates takes part in the transcriptional regulation of MSTN,we performed chromatin immunoprecipitation (ChIP) analysis with anti-C/EBPβ antibody followed by qPCR assay using2pairs of specific primers to amplify MSTN promoter regions containing3putative binding sites for C/EBPP, respectively. The ChIP-qPCR analysis revealed significantly (p<0.05) decreased C/EBPβ binding to all the three binding sites on MSTN promoter in LP piglets at weaning stage. The results indicate that maternal low-protein diet decreases both nuclear content of C/EBPβ protein and C/EBPβ binding to the promoter of MSTN gene, which may be responsible for the down-regulation of MSTN mRNA expression in offspring pigs at weaning. However, the binding of C/EBPβ to putative binding sites2/3at MSTN promoter region was significantly up-regulated (p<0.05) in LP pigs, which was in agreement with the up-regulated MSTN mRNA expression. To determine whether histone modifications, including histone acetylation and methylation, on the promoter of MSTN gene are involved in the transcriptional regulation of MSTN gene, we performed ChIP assay with specific antibodies against4respective histone modification marks, namely acetyl-H3(H3ac), H3K4me3, H3K27me3and H3K9me1.At finishing stage, LP pigs showed increased level of H3ac (p<0.05) and H3K27me3(p<0.01), but decreased H3K9mel (p<0.05) on the promoter of MSTN gene in LD muscle. H3K4me3did not differ between the two groups. None of the histone modification marks showed differences between SP and LP piglets at weaning. Those results imply the involvement of histone modifications in the programming effect of maternal dietary protein on transcriptional regulation of MSTN gene at finishing stage. We also checked the expression of miRNAs predicted to target MSTN. The expression of ssc-miR-136and ssc-miR-500was reduced significantly (p<0.05) in the skeletal muscle of LP pigs at finishing stage, with a trend of decrease for ssc-miR-27a (p=0.057). None was differently expressed between the two groups at weaning stage. These results indicate that maternal dietary protein affects expression of miRNAs predicted to target MSTN at finishing stage. In conclusion, our results indicate that maternal dietary protein affects MSTN expression through distinct transcriptional regulation mechanisms at different stages. The immediate effect at weaning is mediated by C/EBPβ binding without epigenetic modifications, whereas the long-term effect at finishing stage involves both C/EBPβ binding and epigenetic regulations including histone modification and microRNA expression.