The effect of nutritional status on myogenic gene expression of satellite cells derived from different muscle types.

Satellite cells (SC) are a multipotential stem cell population responsible for facilitating posthatch muscle fiber hypertrophy. The proliferation and differentiation of SC is sensitive to nutritional regimen, and the SC response to nutrition varies depending upon their muscle type of origin. The objective of the current study was to determine the effect of altering protein synthesis on the expression of several key genes regulating SC activity and the effect of muscle type. Satellite cells isolated from the fast glycolytic pectoralis major and the fast oxidative and glycolytic biceps femoris were studied. These genes included the myogenic regulatory factors myogenic determination factor 1 (MyoD) and myogenin, the cell-membrane associated proteoglycans syndecan-4 and glypican-1, the extracellular matrix proteoglycan decorin, and the transcription factor paired box 7. Protein synthesis potential varied by the concentration of the sulfur amino acids Met and Cys during SC proliferation and differentiation. The SC were cultured and treated with 1 of 6 Met/Cys concentrations: 60/192, 30/96 (control), 7.5/24, 3.0/9.6, 1.0/3.2, or 0/0 mg/L. A consistent pattern of gene expression emerged following Met/Cys manipulation as increasing reductions in mRNA expression for all genes were observed as Met/Cys concentration decreased, whereas increased Met/Cys concentration caused either no change or had a small negative effect on mRNA expression. Reduced paired box 7 expression would limit myogenic specification of SC, whereas decreased myogenic regulatory factor expression would affect subsequent myogenic development of the SC. Decreased levels of decorin affect SC response to growth factors like myostatin and transforming growth factor ß, and extracellular matrix organization. These data highlight the importance of nutrition on the expression of genes critical for satellite cell activation, proliferation and differentiation, and growth factor signal transduction.

The effect of nutritional status and muscle fiber type on myogenic satellite cell fate and apoptosis.

Satellite cells (SC) are multipotential stem cells that can be induced by nutrition to alter their cellular developmental fate, which may vary depending on their fiber type origin. The objective of the current study was to determine the effect of restricting protein synthesis on inducing adipogenic transdifferentiation and apoptosis of SC originating from fibers of the fast glycolytic pectoralis major (p. major) and fast oxidative and glycolytic biceps femoris (b. femoris) muscles of the chicken. The availability of the essential sulfur amino acids Met and Cys was restricted to regulate protein synthesis during SC proliferation and differentiation. The SC were cultured and treated with 1 of 6 Met/Cys concentrations: 60/192, 30/96 (control), 7.5/24, 3/9.6, 1/3.2, or 0/0 mg/L. Reductions in Met/Cys concentrations from the control level resulted in increased lipid staining and expression of the adipogenic marker genes peroxisome proliferator-activated receptor gamma and stearoyl-CoA desaturase during differentiation in the p. major SC. Although b. femoris SC had increased lipid staining at lower Met/Cys concentrations, there was no increase in expression of either adipogenic gene. For both muscle types, SC Met/Cys, concentration above the control increased the expression of peroxisome proliferator-activated receptor gamma and stearoyl-CoA desaturase during differentiation. As Met/Cys concentration was decreased during proliferation, a dose-dependent decline in all apoptotic cells occurred except for early apoptotic cells in the p. major, which had no treatment effect (P < 0.05). During differentiation, decreasing Met/Cys concentration caused an increase in early apoptotic cells in both fiber types and no effect on late apoptotic cells except for an increase in the p. major 7.5/24 mg/L of Met/Cys treatment. In general, the viability of the SC was unaffected by the Met/Cys concentration except during proliferation in the p. major 0/0 mg/L of Met/Cys treatment, which increased SC viability. These data demonstrate the effect of nutrition on SC transdifferentiation to an adipogenic lineage and apoptosis, and the effect of fiber type on this response in an in vitro context.

The effect of nutritional status on myogenic satellite cell proliferation and differentiation.

Early posthatch satellite cell (SC) mitotic activity is a critical component of muscle development and growth. Satellite cells are stem cells that can be induced by nutrition to follow other cellular developmental pathways. The objective of the current study was to determine the effect of restricting protein synthesis on the proliferation and differentiation of SC, using variable concentrations of Met and Cys to modulate protein synthesis. Broiler pectoralis major SC were cultured and treated with 1 of 6 different Met/Cys concentrations: 60/192, 30/96 (control), 7.5/24, 3/9.6, 1/3.2, or 0/0 mg/L. The effect of Met/Cys concentration on SC proliferation and differentiation was measured, and myonuclear accretion was measured by counting the number of nuclei per myotube during differentiation. The 30/96 mg/L Met/Cys treatment resulted in the highest rate of proliferation compared with all other treatments by 72 h of proliferation (P < 0.05). Differentiation was measured with Met/Cys treatments only during proliferation and the cultures receiving normal differentiation medium (R/N), normal proliferation medium and differentiation medium with variable Met/Cys (N/R), or both proliferation and differentiation receiving variable Met/Cys treatments (R/R). Differentiation responded in a dose-dependent manner to Met/Cys concentration under all 3 of these treatment regimens, with a degree of recovery in the R/N regimen cells following reinstatement of the control medium. Reductions in both proliferation and differentiation were more pronounced as Met/Cys concentrations were further reduced, whereas increased differentiation was observed under the increased Met/Cys concentration treatment when applied during differentiation in the N/R and R/R regimens. The number of nuclei per myotube was significantly decreased in the severely Met/Cys restricted treatments (P < 0.05). These data demonstrate the sensitivity of pectoralis major SC to nutritional availability and the importance of optimal nutrition during both proliferation and differentiation for maximizing SC activity, which will affect subsequent muscle mass accretion.

Adenovirus-mediated interference of FABP4 regulates mRNA expression of ADIPOQ, LEP and LEPR in bovine adipocytes.

Fatty acid binding protein 4 (FABP4) is an important adipocyte gene, with roles in fatty acid transport and fat deposition in animals as well as human metabolic syndrome. However, little is known about the functional regulation of FABP4 at the cellular level in bovine. We designed and selected an effective shRNA (small hairpin RNA) against bovine FABP4, constructed a corresponding adenovirus (AD-FABP4), and then detected its influence on mRNA expression of four differentiation-related genes (PPAR(y), CEBPA, CEBPB, and SREBF1) and three lipid metabolism-related genes (ADIPOQ, LEP and LEPR) of adipocytes. The FABP4 mRNA content, derived from bovine adipocytes, decreased by 41% (P < 0.01) after 24 h and 66% (P < 0.01) after 72 h of AD-FABP4 infection. However, lower mRNA content of FABP4 did not significantly alter levels of differentiation-related gene expression at 24 h following AD-FABP4 treatment of bovine-derived preadipocytes (P = 0.54, 0.78, 0.89, and 0.94, respectively). Meanwhile, knocking down (partially silencing) FABP4 significantly decreased ADIPOQ (P < 0.05) and LEP (P < 0.01) gene expression after 24 h of AD-FABP4 treatment, decreased ADIPOQ (P < 0.01) and LEP (P < 0.01) gene expression, but increased LEPR mRNA expression (P < 0.01) after a 72-h treatment of bovine preadipocytes. We conclude that FABP4 plays a role in fat deposition and metabolic syndrome by regulating lipid metabolism-related genes (such as ADIPOQ, LEP and LEPR), without affecting the ability of preadipocytes to differentiate into adipocytes.

Modulation of turkey myogenic satellite cell differentiation through the shedding of glypican-1.

Glypican-1 is a cell membrane heparan sulfate proteoglycan. It is composed of a core protein with covalently attached glycosaminoglycan, and N-linked glycosylated (N-glycosylated) chains, and is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) linkage. Glypican-1 plays a key role in the growth and development of muscle by regulating fibroblast growth factor 2 (FGF2). The GPI anchor of glypican-1 can be cleaved, resulting in glypican-1 being secreted or shed into the extracellular matrix environment. The objective of the current study was to investigate the role of glypican-1 shedding and the glycosaminoglycan and N-glycosylated chains in regulating the differentiation of turkey myogenic satellite cells. A glypican-1 construct without the GPI anchor was cloned into the mammalian expression vector pCMS-EGFP, and glypican-1 without the GPI anchor and glycosaminoglycan and N-glycosylated chains were also cloned. These constructs were co-transfected into turkey myogenic satellite cells with a small interference RNA targeting the GPI anchor of endogenous glypican-1. The soluble glypican-1 mutants were not detected in the satellite cells but in the cell medium, suggesting the secretion of the soluble glypican-1 mutants. Soluble glypican-1 increased satellite cell differentiation and enhanced myotube formation in the presence of exogenous FGF2. The increase in differentiation was supported by the elevated expression of myogenin. In conclusion, the shedding of glypican-1 from the satellite cell surface acts as a positive regulator of satellite cell differentiation and sequesters FGF2, permitting further differentiation.

Heparan sulfate proteoglycans, syndecan-4 and glypican-1, differentially regulate myogenic regulatory transcription factors and paired box 7 expression during turkey satellite cell myogenesis: implications for muscle growth.

The heparan sulfate proteoglycans have been shown to play essential roles in the proliferation and differentiation of myogenic satellite cells. Myogenic regulatory factors (MRF) and paired box 7 (Pax7) are essential transcription factors for satellite cell myogenesis. The objective of the current study was to investigate whether the expression of the MRF and Pax7 is, in part, regulated by the heparan sulfate proteoglycans, syndecan-4, and glypican-1, whose expression has been shown to differentially affect satellite cell proliferation and differentiation. To test this objective, small interfering RNA was used to knockdown the gene expression of glypican-1 and syndecan-4. The effect on the expression of MRF and Pax7 was measured at the mRNA level by real-time quantitative PCR. The knockdown of the glypican-1 gene decreased mRNA expression of MyoD, myogenin, MRF4, and Pax7 expression during proliferation and differentiation of turkey satellite cells; whereas knockdown of the syndecan-4 gene increased mRNA expression of MyoD and MRF4 expression during cell proliferation but not during differentiation and had no effect on myogenin and Pax7 expression. These data suggested that the precise expression of the MRF are dependent upon the appropriate expression of glypican-1 and syndecan-4 during satellite cell proliferation and differentiation, and Pax7 expression is influenced by glypican-1.

Changes in satellite cell proliferation and differentiation during turkey muscle development.

Posthatch muscle growth is determined by the activation, differentiation, and fusion of satellite cells. Satellite cells composing an individual muscle are heterogeneous, which will differentially affect muscle growth. The proliferation and differentiation of turkey primary pectoralis major muscle cells were investigated in vitro at 1 d of age and at 4, 8, 16, 26, 35, 45, and 54 wk of age. The turkey was selected for these studies because turkey skeletal muscle fibroblasts do not grow in primary muscle cell cultures. Results from the proliferation analysis showed a decrease in proliferation by 8 wk of age. Differentiation into myotubes was significantly decreased by 4 wk of age and myotube diameter was decreased. The changes in muscle weight relative to total BW were measured for the anterior latissimus dorsi, biceps brachii, pectoralis major, sartorius, biceps femoris, and gastrocnemius muscles to compare the relative growth of different muscles. The age at which the muscles reached their maximum relative weight was muscle-dependent, with the biceps brachii plateauing the earliest at 4 wk and the sartorius the latest at 45 wk of age. These data suggested that changes in myogenic cells begin to occur early in muscle development and the activity of the satellite cells during these initial stages of posthatch growth is critical in overall muscle mass accumulation.

Effect of glypican-1 covalently attached chains on turkey myogenic satellite cell proliferation, differentiation, and fibroblast growth factor 2 responsiveness.

Glypican-1 is a cell membrane heparan sulfate proteoglycan that is composed of a core protein and covalently attached glycosaminoglycan (GAG) chains and N-linked glycosylated (N-glycosylated) chains. The glypican-1 GAG chains are required for cell differentiation and responsiveness to fibroblast growth factor 2 (FGF2). The role of glypican-1 N-glycosylated chains in regulating cell activities has not been reported. The objective of the current study was to investigate the role of glypican-1 N-glycosylated chains and the interaction between N-glycosylated and GAG chains in turkey myogenic satellite cell proliferation, differentiation, and FGF2 responsiveness. The wild-type turkey glypican-1 and turkey glypican-1 with mutated GAG chain attachment sites were cloned into the pCMS-EGFP mammalian expression vector and were used as templates to generate glypican-1 N-glycosylated 1-chain and no-chain mutants with or without GAG chains by site-directed mutagenesis. The wild-type glypican-1 and all glypican-1 N-glycosylated 1-chain and no-chain mutants with or without GAG chains were transfected into turkey myogenic satellite cells. Cell proliferation, differentiation, and FGF2 responsiveness were measured. The overexpression of glypican-1 N-glycosylated 1-chain and no-chain mutants without GAG chains increased cell proliferation and differentiation compared with the wild-type glypican-1 but not the glypican-1 N-glycosylated mutants with GAG chains attached. Cells overexpressing glypican-1 N-glycosylated mutants with or without GAG chains increased cell responsiveness to FGF2 compared with wild-type glypican-1. These data suggest that glypican-1 N-glycosylated chains and GAG chains are critical in regulating turkey myogenic satellite cell proliferation, differentiation, and responsivness to FGF2.

Perspectives on the formation of an interdisciplinary research team.

As research funding becomes more competitive, it will be imperative for researchers to break the mentality of a single laboratory/single research focus and develop an interdisciplinary research team aimed at addressing real world challenges. Members of this team may be at the same institution, may be found regionally, or may be international. However, all must share the same passion for a topic that is bigger than any individual's research focus. Moreover, special consideration should be given to the professional development issues of junior faculty participating in interdisciplinary research teams. While participation may be "humbling" at times, the sheer volume of research progress that may be achieved through interdisciplinary collaboration, even in light of a short supply of grant dollars, is remarkable.

Transforming growth factor-beta1-induced satellite cell apoptosis in chickens is associated with beta1 integrin-mediated focal adhesion kinase activation.

Transforming growth factor-beta1 (TGF-beta1) induces apoptosis in many types of cells. The cell adhesion receptor, beta1 integrin subunit, prevents apoptosis and may be involved in TGF-beta1-induced muscle cell apoptosis. In the current study, chicken primary satellite cells, myogenic precursors, were used to investigate the apoptotic effect of TGF-beta1 on muscle cells. The data from the current study showed that the addition of exogenous TGF-beta1 reduced beta1 integrin expression and altered its localization. Treatment of the satellite cells with TGF-beta1 increased the number of apoptotic cells indicated by annexin-V using flow cytometry. The number of caspase-positive cells was increased in the TGF-beta1-treated immunostained cells, which supported that TGF-beta1 induced satellite cell apoptosis. It has been shown that beta1 integrin is involved in muscle cell survival. In response to the activation of beta1 integrin, focal adhesion kinase (FAK) phosphorylates tyrosine at residue 397 and activates cell survival signal transduction. The phosphorylation of FAK was significantly reduced from 30 min to 4 h after TGF-beta1 treatment during both satellite cell proliferation and differentiation. These data suggested that the apoptotic effect of TGF-beta1 on satellite cells is likely associated with a beta1 integrin-mediated FAK signaling pathway during satellite cell proliferation and differentiation.

Extrinsic regulation of domestic animal-derived myogenic satellite cells II.

The existence of myogenic satellite cells was reported some 47 years ago, and, since that time, satellite cell research has flourished. So much new information is generated (daily) on these cells that it can be difficult for individuals to keep abreast of important issues related to their activation and proliferation, the modulation of the activity of other cell types, the differentiation of the cells to facilitate normal skeletal muscle growth and development, or to the repair of damaged myofibers. The intent of this review is to summarize new information about the extrinsic regulation of myogenic satellite cells and to provide specific mechanisms involved in altering satellite cell physiology. Where possible, examples from agriculturally important animals are used for illustrative purposes.

Effect of Smad3-mediated transforming growth factor-beta1 signaling on satellite cell proliferation and differentiation in chickens.

Transforming growth factor-beta1 (TGF-beta1) is a potent inhibitor of muscle cell proliferation and differentiation. The TGF-beta1 signal is carried by Smad proteins into the cell nucleus, resulting in the regulation of the expression of key myogenic regulatory factors including MyoD and myogenin during myogenesis. However, to date, the molecular mechanism of the inhibition by Smad-mediated TGF-beta1 signaling on the function of the myogenic regulatory factors has not been well understood. The present study was designed to investigate the effect of TGF-beta1 on satellite cell proliferation and differentiation by a Smad3-dependent signaling pathway. A chicken line, low score normal (LSN) with reduced muscling and upregulated TGF-beta1 expression, was used and compared with a normal chicken line. In LSN satellite cell cultures, both MyoD and myogenin expression was significantly decreased compared with the normal cells. Furthermore, in response to exogenous TGF-beta1, the normal satellite cells had a significant decrease in both MyoD and myogenin expression, which suggests that TGF-beta1 inhibited MyoD and myogenin expression, resulting in decreased satellite cell proliferation and differentiation. The expression of Smad3 and Smad7, key proteins of the Smad family, was greater in the LSN cultures than that measured in the normal culture. The addition of TGF-beta1 reduced Smad3 expression, but did not affect the expression of Smad7. The reduction of Smad3 in response to TGF-beta1 suggests that a negative regulatory feedback is likely involved in LSN satellite cell proliferation and differentiation. The overexpression of Smad3 inhibited both MyoD and myogenin expression in normal and LSN satellite cells. In contrast, the underexpression of Smad3 increased the expression of MyoD and myogenin in the LSN cells. However, in the normal cells, only myogenin expression was increased by Smad3 overexpression, but not MyoD. These data together suggest that LSN satellite cells are more responsive to a Smad3-dependent TGF-beta1 signaling pathway than normal satellite cells, and a Smad3-independent pathway is also likely involved in the regulation of satellite cell proliferation and differentiation.

The effect of fibroblast growth factor 2 on the in vitro expression of syndecan-4 and glypican-1 in turkey satellite cells.

The membrane-associated heparan sulfate proteoglycan families, consisting of the syndecans and glypicans, are low-affinity receptors for fibroblast growth factor 2 (FGF2) that are essential in regulating the cellular response to FGF2. Fibroblast growth factor 2 is a potent stimulator of skeletal muscle cell proliferation and a strong inhibitor of differentiation. The regulation of the expression of the syndecans and glypicans will likely play a role in modulating the effects of FGF2 on cellular growth properties. In the present study, the effect of FGF2 on the expression of syndecan-4 and glypican-1 was measured by real-time PCR during turkey myogenic satellite cell proliferation and differentiation in vitro. Both syndecan-4 and glypican-1 transcription were influenced by the addition of exogenous FGF2. Syndecan-4 mRNA expression was reduced only during proliferation, whereas glypican-1 expression was reduced during both proliferation and differentiation. These results suggest that FGF2 growth factor signaling is, in part, regulated by an autoregulatory loop involving FGF2 regulation of syndecan-4 and glypican-1 expression and will affect the growth of skeletal muscle by modulating the proliferation and differentiation of satellite cells.

The effect of glypican-1 glycosaminoglycan chains on turkey myogenic satellite cell proliferation, differentiation, and fibroblast growth factor 2 responsiveness.

The glypicans are a family of cell-surface heparan sulfate proteoglycans consisting of a core protein covalently attached with glycosaminoglycans (GAG). Only glypican-1 is expressed in skeletal muscle and increases in expression during myoblast differentiation. Previous studies have suggested that glypican-1 influences fibroblast growth factor 2 (FGF2) signaling pathway by its heparan sulfate chains. Fibroblast growth factor 2 is a potent stimulator of muscle cell proliferation and an intense inhibitor of differentiation. To investigate the functional contribution of each GAG chain attachment site, a turkey glypican-1 full length cDNA (1,650 bp, Gen-Bank accession number AY551002) was cloned into the pCMS-EGFP vector and mutated at 2 or all 3 potential GAG attachment sites at Ser(483), Ser(485), and Ser(487) to obtain 1-chain and no-chain mutants, respectively. The unmutated glypican-1, 1-chain, and no-chain mutants, and the pCMS-EGFP vector without an insert were transfected into turkey myogenic satellite cells. The transfected cell cultures were assayed for cell proliferation, differentiation, and FGF2 responsiveness. The overexpression of glypican-1 increased FGF2 responsiveness during proliferation compared with the 1-chain, no-chain mutants, and the pCMS-EGFP vector without an insert, but there was no significant interaction between FGF2 and glypican-1. The overexpression of glypican-1 also increased differentiation but did not affect proliferation when compared with the 1-chain, no-chain mutants, and the pCMS-EGFP vector without an insert. To support the overexpression data, glypican-1 expression was reduced using a small interfering RNA against turkey glypican-1. Inhibition of glypican-1 expression decreased myogenic satellite cell proliferation, differentiation, and FGF2 responsiveness during proliferation. These data indicate that glypican-1 function requires the GAG chain attachment sites for myogenic satellite cell FGF2 responsiveness during proliferation and to affect the process of differentiation.

Effect of syndecan-1, syndecan-4, and glypican-1 on turkey muscle satellite cell proliferation, differentiation, and responsiveness to fibroblast growth factor 2.

The membrane-associated heparan sulfate proteoglycan families consisting of the syndecans and glypicans are low-affinity receptors for fibroblast growth factor 2 (FGF2). Fibroblast growth factor 2 is a potent stimulator of skeletal muscle cell proliferation and a strong inhibitor of differentiation. Because syndecan-1, syndecan-4, and glypican-1 potentially play unique, but pivotal, roles in muscle cell proliferation and differentiation, these proteoglycans were examined for their effect on muscle cell proliferation and differentiation and FGF2 responsiveness. In the present study, turkey Randombred Control 2 line myogenic satellite cells were transfected with expression vector constructions of syndecan-1, syndecan-4, or glypican-1 to assay their role during muscle development and the effect on FGF2 responsiveness. During proliferation, only syndecan-1 increased proliferation. Both syndecan-4 and glypican-1 decreased proliferation at 72 h but generally did not affect the proliferation process. There was no interaction between the transfected gene and cell proliferation response to FGF2. Glypican-1 increased differentiation early in the process (24 h), and at later times differentiation was decreased by glypican-1. Both syndecan-1 and syndecan-4 overexpression decreased differentiation. During differentiation, except for glypican-1 at 48 h of differentiation, there was no interaction between gene treatment and FGF2 responsiveness. This result indicates that FGF2 responsiveness was not affected by the overexpression of syndecan-1, syndecan-4, and glypican-1 during differentiation. These data demonstrate that syndecan-1, syndecan-4, or glypican-1 differentially affect the processes of turkey muscle cell proliferation and differentiation, and can regulate these developmental stages in an FGF2-independent manner.

Differential expression of membrane-associated heparan sulfate proteoglycans in the skeletal muscle of turkeys with different growth rates.

Heparan sulfate proteoglycans (HSPG) are key components of the cell membrane and extracellular matrix of skeletal muscle cells. Two major groups of membrane-associated HSPG found in skeletal muscle are syndecans (SYN) and glypicans (GPC), both of which can regulate growth factor activities and, thus, modulate cell proliferation and differentiation. In the current study, the mRNA expression of a group of membrane-associated HSPG (SYN 2 through 4 and GPC 1) was investigated in embryonic pectoralis major muscle [embryonic days (ED) 14, 16, 18, 20, 22, 24] and myogenic satellite cells isolated from males of a turkey genetic line selected for increased 16-wk BW (F line) and an unselected randombred control (RBC2 line) from which the F line was developed. The mRNA expression was measured by a real-time quantitative PCR approach. The SYN 2 and SYN 4 expression exhibited a similar pattern during embryonic p. major muscle development, which remained constant from ED 14 to ED 22 and declined sharply from ED 22 to ED 24 to a very low level. In contrast, the SYN 3 and GPC 1 expression showed a continuous decline from ED 14 to ED 24. The F line had higher SYN 2 (ED 14, 18, 20, 22), SYN 3 (ED 22), and SYN 4 (ED 22) expression than the RBC2 line. In myogenic satellite cells, initiating differentiation resulted in a decrease in SYN 2 expression and an increase in GPC 1 expression. Both SYN 3 and SYN 4 expression stayed almost constant through both the proliferation and differentiation stages. The proliferating satellite cells from the F line displayed higher SYN 4 expression than those from the RBC2 line. Collectively, the results from the current study suggest that membrane-associated HSPG are differentially expressed in both embryonic p. major muscle tissue and satellite cells isolated from F-line and RBC2-line male turkeys, implying their distinct roles in myogenesis and differing influence on muscle growth properties.

Effect of transforming growth factor-beta on decorin and beta1 integrin expression during muscle development in chickens.

Myoblast-extracellular matrix interactions play a pivotal role in skeletal muscle development. Transforming growth factor-beta (TGF-beta) is a key regulator of muscle cell proliferation and differentiation. The level of TGF-beta expressed will affect the concentration of the extracellular matrix proteoglycan decorin and the cell surface beta1 integrin subunit. The decorin proteoglycan is a regulator of cell growth as well as the organization of the extracellular matrix. The beta1 integrin plays a role in muscle cell attachment, migration, and the formation of multinucleated myotubes. In the current study, chicken myogenic satellite cells isolated from the pectoralis major muscle from the chicken genetic muscle weakness, low score normal (LSN), and normal pectoralis major muscle were used to investigate TGF-beta expression as it relates to decorin and beta1 integrin mRNA expression. The LSN muscle defect is characterized by altered myotube formation and sarcomere structure, and the satellite cells have reduced proliferation and differentiation. The mRNA expression was measured by real-time quantitative reverse transcription PCR. The LSN condition has elevated expression of TGF-beta2 and TGF-beta4 with increased expression of decorin and decreased beta1 integrin during myogenic satellite cell proliferation and differentiation. Normal satellite cell cultures were treated with the addition of exogenous TGF-beta during differentiation to determine if the altered expression of LSN decorin and beta1 integrin was associated with TGF-beta expression. The addition of exogenous TGF-beta decreased decorin expression during differentiation and reduced beta1 integrin expression at 24 and 48 h of differentiation. These results suggested that alteration of decorin expression in the LSN myogenic satellite cells may occur by a mechanism involving factors in addition to TGF-beta, but the addition of exogenous TGF-beta did affect both decorin and beta1 integrin expression. These data, therefore, suggested that TGF-beta might play a pivotal role in chicken skeletal muscle formation through modulation of the expression of both extracellular matrix molecules and cellular receptors important in the control of cell migration and growth regulation.

Effect of genetic selection on MyoD and myogenin expression in turkeys with different growth rates.

Skeletal muscle development requires the ordered expression of specific myogenic regulatory factors, which include MyoD, Myf5, myogenin, and MRF4. The MyoD and Mrf5 factors are required for the determination of myoblasts, whereas myogenin and MRF4 play a pivotal role in terminal differentiation. In the current study, males and females of a turkey genetic line selected only for increased 16-wk BW (F line) and an unselected randombred control (RBC2 line) from which the F line was developed were used to investigate the developmental expression of MyoD and myogenin mRNA in embryonic pectoralis major muscle and myogenic satellite cells. Pectoralis major muscle was isolated at embryonic d (ED) 14, 16, 18, 20, 22, and 24. The mRNA levels of MyoD and myogenin were measured using a real-time quantitative polymerase chain reaction method. Both MyoD and myogenin expression declined during embryonic development. The decrease in MyoD expression started at ED 16 for the F line and at ED 18 for the RBC2 line for both sexes. Myogenin expression in both lines began to decline at ED 14. The F line males had lower myogenin expression at ED 14, 16, and 18 than the RBC2 line males, which was similar for the F line females compared with the RBC2 line females except there was no significant difference at ED 18. The RBC2 line males had greater expression than the females for myogenin at ED 16 and 18 for the RBC2 line. Proliferating myogenic satellite cells in both lines and sexes expressed low levels of MyoD and myogenin. After the initiation of differentiation in both lines and sexes, there was a sharp surge in MyoD expression at 24 h followed by a decrease at 48 h and then an increase in expression through 72 or 96 h of differentiation. There were line and sex differences in myogenin expression during the differentiation process. These data are suggestive of growth- and sex-related differences in the expression of myogenic regulatory factors key to muscle cell proliferation and differentiation, which will affect muscle growth rate.

Effects of syndecan-1 and glypican on muscle cell proliferation and differentiation: implications for possible functions during myogenesis.

The heparan sulfate proteoglycans, syndecan-1 and glypican, are low-affinity receptors for fibroblast growth factor 2 (FGF2). Because FGF2 is a potent stimulator of skeletal muscle cell proliferation and a strong inhibitor of differentiation, it is likely that changes in syndecan-1 and glypican expression will affect myogenesis as both, in part, regulate FGF-dependent signaling. In the current study, expression vector constructs containing either syndecan-1 or glypican were transfected into turkey myogenic satellite cells resulting in the overexpression of these genes. The amount of expression of each of these genes was measured by semiquantitative reverse transcriptase polymerase chain reaction. The satellite cell cultures overexpressing syndecan-1 were unable to fuse to form multinucleated myotubes after differentiation was induced. The syndecan-1-transfected cells maintained a rounded morphology typical of cells during proliferation. In contrast, the satellite cells transfected with glypican formed larger myotubes. These results suggest that both syndecan-1 and glypican play pivotal, but different, roles in both muscle cell proliferation and differentiation.