Signaling Pathways Related to Protein Synthesis and Amino Acid Concentration in Pig Skeletal Muscles Depend on the Dietary Protein Level,Genotype and Developmental Stages

Muscle growth is regulated by the homeostatic balance of the biosynthesis and degradation of muscle proteins. To elucidate the molecular interactions among diet, pig genotype, and physiological stage, we examined the effect of dietary protein concentration, pig genotype, and physiological stages on amino acid (AA) pools, protein deposition, and related signaling pathways in different types of skeletal muscles. The study used 48 Landrace pigs and 48 pure-bred Bama mini-pigs assigned to each of 2 dietary treatments: lower/GB (Chinese conventional diet)- or higher/NRC (National Research Council)-protein diet. Diets were fed from 5 weeks of age to respective market weights of each genotype. Samples of biceps femoris muscle (BFM, type I) and longissimus dorsi muscle (LDM, type II) were collected at nursery, growing, and finishing phases according to the physiological stage of each genotype, to determine the AA concentrations, mRNA levels for growth-related genes in muscles, and protein abundances of mechanistic target of rapamycin (mTOR) signaling pathway. Our data showed that the concentrations of most AAs in LDM and BFM of pigs increased (P<0.05) gradually with increasing age. Bama mini-pigs had generally higher (P<0.05) muscle concentrations of flavor-related AA, including Met, Phe, Tyr, Pro, and Ser, compared with Landrace pigs. The mRNA levels for myogenic determining factor, myogenin, myocyte-specific enhancer binding factor 2 A, and myostatin of Bama mini-pigs were higher (P<0.05) than those of Landrace pigs, while total and phosphorylated protein levels for protein kinase B, mTOR, and p70 ribosomal protein S6 kinases (p70S6K), and ratios of p-mTOR/mTOR, p-AKT/AKT, and p-p70S6K/p70S6K were lower (P<0.05). There was a significant pig genotype-dependent effect of dietary protein on the levels for mTOR and p70S6K. When compared with the higher protein-NRC diet, the lower protein-GB diet increased (P<0.05) the levels for mTOR and p70S6K in Bama mini-pigs, but repressed (P<0.05) the level for p70S6K in Landrace pigs. The higher protein-NRC diet increased ratio of p-mTOR/mTOR in Landrace pigs. These findings indicated that the dynamic consequences of AA profile and protein deposition in muscle tissues are the concerted effort of distinctive genotype, nutrient status, age, and muscle type. Our results provide valuable information for animal feeding strategy.     

不同猪品种肌肉组织FoxO1与MyoD基因mRNA的表达及其相关性分析

分别取6月龄八眉猪、长白猪和长×八杂交猪背最长肌,提取总RNA,设计并合成猪FoxO1和MyoD 引物,以β肌动蛋白作为内参,优化反应条件和体系,利用RT-PCR方法检测八眉、长白和长×八杂交猪肌肉组织中FoxO1和MyoD基因mRNA 的表达.结果表明,在不同经济类型猪品种肌肉组织中,FoxO1和MyoD基因mRNA的表达丰度均存在显著差异,FoxO1在八眉猪肌肉组织中的表达普遍高于长白猪（P<0.01）,在杂交组合长×八肌肉组织中的表达也高于长白猪（P<0.01）;而MyoD恰好相反,即在长白猪肌肉组织中的表达普遍高于八眉猪（P<0.01）,在杂交组合长×八肌肉组织中的表达也高于八眉猪（P<0.01）．结果提示,FoxO1和MyoD基因mRNA的表达在肌肉发育中存在负相关（r = 0.728 , P < 0.05）,FoxO1在肌肉组织中的上调作用,可能是造成的MyoD基因mRNA表达降低的原因之一,进而负调控肌肉的发育和骨骼肌的量．     

The expression of myogenic regulatory factors and muscle growth factors in the masticatory muscles of dystrophin-deficient (MDX) mice

The activities of myogenic regulatory factors (MRF) and muscle growth factors increase in muscle that is undergoing regeneration, and may correspond to some specific changes. Little is known about the role of MRFs in masticatory muscles in mdx mice (the model of Duchenne muscular dystrophy) and particularly about their mRNA expression during the process of muscle regeneration. Using Taqman RT-PCR, we examined the mRNA expression of the MRFs myogenin and MyoD1 (myogenic differentiation 1), and of the muscle growth factors myostatin, IGF1 (insulin-like growth factor) and MGF (mechanogrowth factor) in the masseter, temporal and tongue masticatory muscles of mdx mice (n = 6 to 10 per group). The myogenin mRNA expression in the mdx masseter and temporal muscle was found to have increased (P < 0.05), whereas the myostatin mRNA expressions in the mdx masseter (P < 0.005) and tongue (P < 0.05) were found to have diminished compared to those for the controls. The IGF and MGF mRNA amounts in the mdx mice remained unchanged. Inside the mdx animal group, gender-related differences in the mRNA expressions were also found. A higher mRNA expression of myogenin and MyoD1 in the mdx massterer and temporal muscles was found in females in comparison to males, and the level of myostatin was higher in the masseter and tongue muscle (P < 0.001 for all comparisons). Similar gender-related differences were also found within the control groups. This study reveals the intermuscular differences in the mRNA expression pattern of myogenin and myostatin in mdx mice. The existence of these differences implies that dystrophinopathy affects the skeletal muscles differentially. The finding of gender-related differences in the mRNA expression of the examined factors may indicate the importance of hormonal influences on muscle regeneration.     

Molecular expression of myostatin and MyoD is greater in double-muscled than normal-muscled cattle fetuses

Excessive muscling in double-muscled cattle arises from mutations in the myostatin gene, but the role of myostatin in normal muscle development is unclear. The aim of this study was to measure the temporal relationship of myostatin and myogenic regulatory factors during muscle development in normal (NM)- and double-muscled (DM) cattle to determine the timing and possible targets of myostatin action in vivo. Myostatin mRNA peaked at the onset of secondary fiber formation (P < 0.001) and was greater in DM (P < 0.001) than in NM. MyoD expression was also elevated throughout primary and secondary fiber formation (P < 0.001) and greater in DM (P < 0.05). Expression of myogenin peaked later than MyoD (P < 0.05); however, it did not differ between NM and DM. These data show that myostatin and MyoD increase coincidentally during formation of muscle fibers, indicating a coordinated role in the terminal differentiation and/or fusion of myoblasts. Myostatin mRNA is also consistently higher in DM than NM, suggesting that a feedback loop of regulation is also disrupted in the myostatin-deficient condition.     

Myogenic gene expression at rest and after a bout of resistance exercise in young (18-30 yr) and old (80-89 yr) women.

The purpose of this study was to investigate mRNA expression of several key skeletal muscle myogenic controllers; myogenic differentiation factor (MyoD), muscle regulatory factor 4 (MRF4), myogenic factor 5 (Myf5), myogenin, myostatin, and myocyte enhancer factor 2 (MEF2) at rest and 4 h after a single bout of resistance exercise (RE) in young and old women. Eight young women (YW; 23 +/- 2 yr, 67 +/- 5 kg) and six old women (OW; 85 +/- 1 yr, 67 +/- 4 kg) performed 3 sets of 10 repetitions of bilateral knee extensions at 70% of one repetition maximum. Muscle biopsies were taken from the vastus lateralis before and 4 h after RE. Using real-time RT PCR, mRNA from the muscle samples was amplified and normalized to GAPDH. At rest, OW expressed higher (P < 0.05) levels of MyoD, MRF4, Myf5, myogenin, and myostatin compared with YW. In response to RE, there was a main time effect (P < 0.05) for the YW and OW combined in the upregulation of MyoD (2.0-fold) and MRF4 (1.4-fold) and in the downregulation of myostatin (2.2-fold). There was a trend (P = 0.08) for time x age interaction in MRF4. These data show that old women express higher myogenic mRNA levels at rest. The higher resting myogenic mRNA levels in old women may reflect an attempt to preserve muscle mass and function. When challenged with RE, old women appear to respond in a similar manner as young women.     

Myostatin inhibits myoblast differentiation by down-regulating MyoD expression

Myostatin, a negative regulator of myogenesis, is shown to function by controlling the proliferation of myoblasts. In this study we show that myostatin is an inhibitor of myoblast differentiation and that this inhibition is mediated through Smad 3. In vitro, increasing concentrations of recombinant mature myostatin reversibly blocked the myogenic differentiation of myoblasts, cultured in low serum media. Western and Northern blot analysis indicated that addition of myostatin to the low serum culture media repressed the levels of MyoD, Myf5, myogenin, and p21 leading to the inhibition of myogenic differentiation. The transient transfection of C(2)C(12) myoblasts with MyoD expressing constructs did not rescue myostatin-inhibited myogenic differentiation. Myostatin signaling specifically induced Smad 3 phosphorylation and increased Smad 3.MyoD association, suggesting that Smad 3 may mediate the myostatin signal by interfering with MyoD activity and expression. Consistent with this, the expression of dominant-negative Smad3 rescued the activity of a MyoD promoter-reporter in C(2)C(12) myoblasts treated with myostatin. Taken together, these results suggest that myostatin inhibits MyoD activity and expression via Smad 3 resulting in the failure of the myoblasts to differentiate into myotubes. Thus we propose that myostatin plays a critical role in myogenic differentiation and that the muscular hyperplasia and hypertrophy seen in animals that lack functional myostatin is because of deregulated proliferation and differentiation of myoblasts.     

Satellite cells on isolated myofibers from normal and denervated adult rat muscle.

Satellite cells (SCs) in normal adult muscle are quiescent. They can enter the mitotic program when stimulated with growth factors such as basic FGF. Short-term denervation stimulates SC to enter the mitotic cycle in vivo, whereas long-term denervation depletes the SC pool. The molecular basis for the neural influence on SCs has not been established. We studied the phenotype and the proliferative capacity of SCs from muscle that had been denervated before being cultured in vitro. The expression of PCNA, myogenin, and muscle (M)-cadherin in SCs of normal and denervated muscle fibers was examined at the single-cell level by immunolabeling in a culture system of isolated rat muscle fibers with attached SCs. Immediately after plating (Day 0), neither PCNA nor myogenin was present on normal muscle fibers, but we detected an average of 0.5 M-cadherin(+) SCs per muscle fiber. The number of these M-cadherin(+) cells (which are negative for PCNA and myogenin) increased over the time course examined. A larger fraction of cells negative for M-cadherin underwent mitosis and expressed PCNA, followed by myogenin. The kinetics of SCs from muscle fibers denervated for 4 days before culturing were similar to those of normal controls. Denervation from 1 to 32 weeks before plating, however, suppressed PCNA and myogenin expression almost completely. The fraction of M-cadherin(+) (PCNA(-)/myogenin(-)) SCs was decreased after 1 week of denervation, increased above normal after denervation for 4 or 8 weeks, and decreased again after denervation for 16 or 32 weeks. We suggest that the M-cadherin(+) cells are nondividing SCs because they co-express neither PCNA or myogenin, whereas the cells positive for PCNA or myogenin (and negative for M-cadherin) have entered the mitotic cycle. SCs from denervated muscle were different from normal controls when denervated for 1 week or longer. The effect of denervation on the phenotypic modulation of SCs includes resistance to recruitment into the mitotic cycle under the conditions studied here and a robust extension of the nonproliferative compartment. These characteristics of SCs deprived of neural influence may account for the failure of denervated muscle to fully regenerate. (J Histochem Cytochem 47:1375-1383, 1999)     

MyoD cannot compensate for the absence of myogenin during skeletal muscle differentiation in murine embryonic stem cells

myogenin (-/-) mice display severe skeletal muscle defects despite expressing normal levels of MyoD. The failure of MyoD to compensate for myogenin could be explained by distinctions in protein function or by differences in patterns of gene expression. To distinguish between these two possibilities, we compared the abilities of constitutively expressed myogenin and MyoD to support muscle differentiation in embryoid bodies made from myogenin (-/-) ES cells. Differentiated embryoid bodies from wild-type embryonic stem (ES) cells made extensive skeletal muscle, but embryoid bodies from myogenin (-/-) ES cells had greatly attenuated muscle-forming capacity. The inability of myogenin (-/-) ES cells to generate muscle was independent of endogenous MyoD expression. Skeletal muscle was restored in myogenin (-/-) ES cells by constitutive expression of myogenin. In contrast, constitutive expression of MyoD resulted in only marginal enhancement of skeletal muscle, although myocyte numbers greatly increased. The results indicated that constitutive expression of MyoD led to enhanced myogenic commitment of myogenin (-/-) cells but also indicated that committed cells were impaired in their ability to form muscle sheets without myogenin. Thus, despite their relatedness, myogenin's role in muscle formation is distinct from that of MyoD, and the distinction cannot be explained merely by differences in their expression properties.     

Myostatin and MyoD family expression in skeletal muscle of IGF-1 knockout mice

Insulin-like growth factor-1 (IGF-1) is a positive regulator in proliferation and differentiation of skeletal muscle cells, while myostatin (MSTN) is a member of transforming growth factor beta superfamily that acts as a negative regulator of skeletal muscle mass. The present study was performed to detail whether a correlation exists between MSTN and IGF-1 in skeletal muscle of IGF-1 knockout mice (IGF-1(-/-)) and their wild type (WT; i.e., IGF-1(+/+)) littermates. The body weight of IGF-1(-/-) animals was 32% that of WT littermates. The fiber cross-sectional areas (CSA) and number of fibers in M. rectus femoris of IGF-1(-/-) animals were 49 and 59% those of WT animals, respectively. Thus, muscle hypoplasia of IGF-1(-/-) undoubtedly was confirmed. Myostatin mRNA levels and protein levels were similar between M. gastrocnemius of IGF-1(-/-) and WT animals. Myostatin immunoreactivity was similarly localized in muscle fibers of both IGF-1(-/-) and WT M. rectus femoris. The mRNA levels of MyoD family (Myf5, MyoD, MRF4, myogenin) were differentially expressed in IGF-1(-/-)M. gastrocnemius, in which the mRNA expression of MRF4 and myogenin was significantly lower, whereas there were no changes in the mRNA expression of Myf5 and MyoD. These findings first describe that myostatin expression is not influenced by intrinsic failure of IGF-1, although MRF4 and myogenin are downregulated.     

Formononetin ameliorates muscle atrophy by regulating myostatin-mediated PI3K/Akt/FoxO3a pathway and satellite cell function in chronic kidney disease

Muscle atrophy is a common complication in chronic kidney disease (CKD). Inflammation and myostatin play important roles in CKD muscle atrophy. Formononetin (FMN), which is a major bioactive isoflavone compound in Astragalus membranaceus, exerts anti-inflammatory effects and the promotion of myogenic differentiation. Our study is based on myostatin to explore the effects and mechanisms of FMN in relation to CKD muscle atrophy. In this study, CKD rats and tumour necrosis factor α (TNF-α)-induced C2C12 myotubes were used for in vivo and in vitro models of muscle atrophy. The results showed that FMN significantly improved the renal function, nutritional status and inflammatory markers in CKD rats. Values for bodyweight, weight of tibialis anterior and gastrocnemius muscles, and cross-sectional area (CSA) of skeletal muscles were significantly larger in the FMN treatment rats. Furthermore, FMN significantly suppressed the expressions of MuRF-1, MAFbx and myostatin in the muscles of CKD rats and the TNF-α-induced C2C12 myotubes. Importantly, FMN significantly increased the phosphorylation of PI3K, Akt, and FoxO3a and the expressions of the myogenic proliferation and differentiation markers, myogenic differentiation factor D (MyoD) and myogenin in muscles of CKD rats and the C2C12 myotubes. Similar results were observed in TNF-α-induced C2C12 myotubes transfected with myostatin-small interfering RNA (si-myostatin). Notably, myostatin overexpression plasmid (myostatin OE) abolished the effect of FMN on the phosphorylation of the PI3K/Akt/FoxO3a pathway and the expressions of MyoD and myogenin. Our findings suggest that FMN ameliorates muscle atrophy related to myostatin-mediated PI3K/Akt/FoxO3a pathway and satellite cell function.     

Dietary Leucine Supplementation Improves the Mucin Production in the Jejunal Mucosa of the Weaned Pigs Challenged by Porcine Rotavirus

The present study was mainly conducted to determine whether dietary leucine supplementation could attenuate the decrease of the mucin production in the jejunal mucosa of weaned pigs infected by porcine rotavirus (PRV). A total of 24 crossbred barrows weaned at 21 d of age were assigned randomly to 1 of 2 diets supplemented with 1.00% L-leucine or 0.68% L-alanine (isonitrogenous control) for 17 d. On day 11, all pigs were orally infused PRV or the sterile essential medium. During the first 10 d of trial, dietary leucine supplementation could improve the feed efficiency (P = 0.09). The ADG and feed efficiency were impaired by PRV infusion (P<0.05). PRV infusion also increased mean cumulative score of diarrhea, serum rotavirus antibody concentration and crypt depth of the jejunal mucosa (P<0.05), and decreased villus height: crypt depth (P = 0.07), goblet cell numbers (P<0.05), mucin 1 and 2 concentrations (P<0.05) and phosphorylated mTOR level (P<0.05) of the jejunal mucosa in weaned pigs. Dietary leucine supplementation could attenuate the effects of PRV infusion on feed efficiency (P = 0.09) and mean cumulative score of diarrhea (P = 0.09), and improve the effects of PRV infusion on villus height: crypt depth (P = 0.06), goblet cell numbers (P<0.05), mucin 1 (P = 0.08) and 2 (P = 0.07) concentrations and phosphorylated mTOR level (P = 0.08) of the jejunal mucosa in weaned pigs. These results suggest that dietary 1% leucine supplementation alleviated the decrease of mucin production and goblet cell numbers in the jejunal mucosa of weaned pigs challenged by PRV possibly via activation of the mTOR signaling.     

The effect of hyperammonemia on myostatin and myogenic regulatory factor gene expression in broiler embryos

Myogenesis is facilitated by four myogenic regulatory factors and is significantly inhibited by myostatin. The objective of the current study was to examine embryonic gene regulation of myostatin/myogenic regulatory factors, and subsequent manipulations of protein synthesis, in broiler embryos under induced hyperammonemia. Broiler eggs were injected with ammonium acetate solution four times over 48 h beginning on either embryonic day (ED) 15 or 17. Serum ammonia concentration was significantly higher (P<0.05) in ammonium acetate injected embryos for both ED17 and ED19 collected samples when compared with sham-injected controls. Expression of mRNA, extracted from pectoralis major of experimental and control embryos, was measured using real-time quantitative PCR for myostatin, myogenic regulatory factors myogenic factor 5, myogenic determination factor 1, myogenin, myogenic regulatory factor 4 and paired box 7. A significantly lower (P<0.01) myostatin expression was accompanied by a higher serum ammonia concentration in both ED17 and ED19 collected samples. Myogenic factor 5 expression was higher (P<0.05) in ED17 collected samples administered ammonium acetate. In both ED17 and ED19 collected samples, myogenic regulatory factor 4 was lower (P⩽0.05) in ammonium acetate injected embryos. No significant difference was seen in myogenic determination factor 1, myogenin or paired box 7 expression between treatment groups for either age of sample collection. In addition, there was no significant difference in BrdU staining of histological samples taken from treated and control embryos. Myostatin protein levels were evaluated by Western blot analysis, and also showed lower myostatin expression (P<0.05). Overall, it appears possible to inhibit myostatin expression through hyperammonemia, which is expected to have a positive effect on embryonic myogenesis and postnatal muscle growth.     

The myostatin gene is a downstream target gene of basic helix-loop-helix transcription factor MyoD

Myostatin is a negative regulator of myogenesis, and inactivation of myostatin leads to heavy muscle growth. Here we have cloned and characterized the bovine myostatin gene promoter. Alignment of the upstream sequences shows that the myostatin promoter is highly conserved during evolution. Sequence analysis of 1.6 kb of the bovine myostatin gene upstream region revealed that it contains 10 E-box motifs (E1 to E10), arranged in three clusters, and a single MEF2 site. Deletion and mutation analysis of the myostatin gene promoter showed that out of three important E boxes (E3, E4, and E6) of the proximal cluster, E6 plays a significant role in the regulation of a reporter gene in C(2)C(12) cells. We also demonstrate by band shift and chromatin immunoprecipitation assay that the E6 E-box motif binds to MyoD in vitro and in vivo. Furthermore, cotransfection experiments indicate that among the myogenic regulatory factors, MyoD preferentially up-regulates myostatin promoter activity. Since MyoD expression varies during the myoblast cell cycle, we analyzed the myostatin promoter activity in synchronized myoblasts and quiescent "reserve" cells. Our results suggest that myostatin promoter activity is relatively higher during the G(1) phase of the cell cycle, when MyoD expression levels are maximal. However, in the reserve cells, which lack MyoD expression, a significant reduction in the myostatin promoter activity is observed. Taken together, these results suggest that the myostatin gene is a downstream target gene of MyoD. Since the myostatin gene is implicated in controlling G(1)-to-S progression of myoblasts, MyoD could be triggering myoblast withdrawal from the cell cycle by regulating myostatin gene expression.