The effect of hepatocyte growth factor on turkey satellite cell proliferation and differentiation.

The effect of hepatocyte growth factor (HGF) on turkey satellite cell proliferation and differentiation was examined in cell culture. Satellite cell clones were established from one muscle of an individual turkey. The results showed that HGF is a potent activator and mitogen of turkey satellite cells and embryonic myoblasts with maximal stimulation at 1 ng/mL. HGF is also an inhibitor of differentiation of turkey satellite cells. Heterogeneity in the responsiveness to HGF in the turkey satellite cell population was observed between clones selected for fast (Early) or slow (Late) rates of proliferation. However, two other Early clones exhibited responses similar to those of two other Late clones. When combined with insulin-like growth factor (IGF) and fibroblast growth factor (FGF), singularly or in combination, HGF did not exert any additive or synergistic effects on Early or Late clone proliferation. Whereas when combined with IGF, FGF, and platelet-derived growth factor (PDGF), HGF significantly stimulated proliferation of the Late clone but not the Early clone. Addition of anti-HGF antibody to culture media diminished proliferation and provided evidence of autocrine production of HGF by turkey satellite cell cultures. Heterogeneity also exists in the turkey satellite cell population with respect to autocrine production of HGF.

The effect of insulin-like growth factor analogs on turkey satellite cell and embryonic myoblast proliferation.

The effects of several human and chicken insulin-like growth factor (IGF) analogs on turkey satellite cell and embryonic myoblast proliferation were examined in serum-free medium. Similar rates of proliferation were observed when human or chicken IGF-I or IGF-II (13.1 nM) was administered to satellite cells. The biopotency of two analogs, which were modified to prevent interaction with IGF-binding proteins, was also examined. Human Des(1-6)IGF-II was equipotent to native human and chicken IGF-II. However, the chicken LR3 IGF-I analog was significantly less active toward satellite cells and embryonic myoblasts compared with chicken IGF-I. Human [Leu27] IGF-II, an analog designed to have reduced affinity to the IGF Type I receptor but unaltered binding to IGF-binding proteins, had a diminished effect on cell proliferation. Examination of IGF receptor binding characteristics revealed that chicken LR3 IGF-I had reduced ability to compete with [125I]hIGF-I for binding to satellite cells or embryonic myoblasts compared with chicken IGF-I. The observed biological responses to IGF suggest that IGF-binding proteins have little effect on Type I IGF receptor action in these cell types in serum-free medium. The results also suggest that alterations of the IGF molecule to prevent interaction with binding proteins may also alter receptor binding affinity.

Isolation and characterization of myogenic satellite cells from the muscular dystrophic hamster.

Myogenic satellite cells were isolated from control and dystrophic hamster diaphragms to examine cellular mechanisms involved in the physiology of muscular dystrophy. The Bio 14.6 dystrophic hamster, which possesses a defect in the delta-sarcoglycan gene, develops biochemical and physical symptoms of Duchenne-like and limb girdle muscular dystrophies. Because primary cultures of the control and dystrophic satellite cells became extensively contaminated with non-myogenic cells during proliferation, cell clones were developed to provide pure cultures for study. Cell culture conditions were optimized with the use of Ham's F-12K medium containing 10% fetal bovine serum +5% horse serum + 10 ng/mL basic fibroblast growth factor + 50 microg/mL porcine gelatin. Proliferation rates of the two clonal cultures were similar between the two lines. Satellite cell-derived myotubes from both primary cultures and clones differed between control and dystrophic animals. Dystrophic myotubes tended to be long and narrow, while the control-derived myotubes were broader. Measurement of muscle-specific creatine kinase during differentiation revealed that the dystrophic myotubes possessed higher creatine kinase levels than control myotubes (up to 146-fold at 168 h). The results demonstrate that satellite cells can be isolated from the hamster and may provide a useful tool to study muscular dystrophies associated with defects in the sarcoglycan complex and the involvement of sarcoglycans in normal skeletal muscle growth and development.

Variation in response to growth factor stimuli in satellite cell populations.

Variation in response to growth factor stimuli in myogenic satellite cell populations was investigated using a clonal-derived satellite cell culture system. Satellite cell clones were established from one muscle from one individual animal. One clone ("Early") which reached confluence on day 19 and one clone ("Late"), which reached confluence on day 29, were chosen for further examination. In previous studies, these two clones were found to differ in their growth rates in serum-containing medium. In the present study, the influence of growth factors on the proliferation of the two clones was compared in serum-free defined medium. Although basic fibroblast growth factor (bFGF), insulin-like growth factor-I (IGF-I), insulin, and platelet-derived growth factor-BB (PDGF-BB) stimulated proliferation of both clones, the Early clone was more responsive to all growth factors tested than the Late clone (P < or = 0.05). The Early clone was also more responsive to the proliferative and differentiative depressing effects of administered transforming growth factor-beta (P < or = 0.05). Examination of properties of the PDGF, FGF, and IGF-I receptors on these two clones revealed no differences in either dissociation constants or receptor numbers (P > or = 0.05). The results suggest that there is heterogeneity in satellite cell response to growth factors.

Comparison of growth factor receptors and metabolic characteristics of satellite cells derived from the biceps femoris and pectoralis major muscles of the turkey.

Myogenic satellite cells were isolated from the turkey pectoralis major (PM), a muscle composed largely of white fibers, and the biceps femoris (BF), a muscle composed largely of red fibers, and their properties were compared in culture. Satellite cells derived from the PM and BF muscles exhibited differences in metabolic parameters, growth factor receptor characteristics, and mitogenic responses. PM satellite cells exhibited greater responsiveness to platelet-derived growth factor (PDGF), and the PDGF receptor on these cells had a higher affinity toward ligand compared to BF cells (P < 0.05). Protein synthesis, protein degradation, and glucose uptake rates were higher in BF satellite cell cultures (P < 0.05), correlating with previously reported in vivo measurements using red and white muscle fibers.

Characterization of satellite cells derived from chickens with the low score normal (LSN) muscle weakness.

Myogenic satellite cell clones were established from the pectoralis major muscle of chickens with the low score normal (LSN) muscle weakness and controls. The percentage of cells which attached to substrata and began to proliferate was higher for the control line than the LSN line (55% vs 30%). Furthermore, of those clones which initiated growth, 63% of the control cells and only 32% of the LSN cells proliferated to confluence in 25 cm2 tissue culture flasks. Proliferation rates were significantly lower with LSN satellite cells than with controls (p < 0.05). LSN satellite cells were less responsive to the mitogenic effects of chicken serum (p < 0.05) and differentiation rates were lower compared with controls (p < 0.05). There was a greater (p < 0.05) number of insulin-like growth factor receptors on LSN satellite cells compared with controls. The IGF receptor binding affinities (Kds) between the two cell lines were similar (p > 0.05). The results suggest that a defect in satellite cell physiology may contribute to the skeletal muscle weakness seen in the LSN line.

In vitro characteristics of myogenic satellite cells derived from the pectoralis major and biceps femoris muscles of the chicken.

Satellite cells were isolated from the pectoralis major (PM) and biceps femoris (BF) muscles of 5-week-old broiler chickens to compare growth and differentiation characteristics in vitro. BF cells proliferated at greater rates in the growth medium and were more responsive to the mitogenic effects of chicken serum than PM cells at all levels tested (p < or = 0.05). When low serum-containing medium was administered, the levels of creatine kinase, a marker of differentiation, increased at greater rates in PM cultures than in BF cultures (p < or = 0.05). Administration of increasing levels of fibroblast growth factor in serum-free medium resulted in similar responsiveness of the two lines to this mitogen. No differences were detected in rates of protein synthesis or degradation in myotube cultures from the two muscle sources. The results suggest that satellite cells derived from PM and BF muscles of the chicken have different responsiveness to serum mitogens.

The role of platelet-derived growth factor in turkey skeletal muscle development.

The role of platelet-derived growth factor (PDGF) isoforms in the proliferation of turkey skeletal muscle cells was examined using turkey myogenic cells. To compare the effects of PDGF during different developmental stages, postnatal myogenic satellite cell and embryonic myoblast cultures were developed for in vitro comparisons. Satellite cell cultures from turkeys selected and unselected for skeletal muscle accretion rates were also established to compare the role of PDGF in turkeys with different genetic origins. The results demonstrated that the BB and AB isoforms of PDGF enhanced satellite cell and embryonic myoblast proliferation, while the AA isoform had no effect. Satellite cells and embryonic myoblasts were more responsive to the BB isoform than to the AB isoform. Competitive binding assays demonstrated that there were no differences between PDGF receptor affinities or receptor numbers on either embryonic myoblasts and satellite cells or satellite cells derived from selected and unselected turkeys. The results suggest that PDGF may be an important mitogenic factor in turkey skeletal muscle development.

The response to growth factors of cultured satellite cells derived from turkeys having different growth rates.

Satellite cells were isolated from the skeletal muscles of turkey varieties which grow at different rates to explore cellular mechanisms that may influence the rate of muscular growth. Satellite cells from the fast growing variety (Nicholas) were more responsive to insulin-like growth factors (IGFs) and insulin, and less responsive to fibroblast growth factor than were cells derived from the slow growing (Merriam's) turkey. IGF receptor affinities were similar between the Nicholas and Merriam's turkey satellite cells. The results suggest that differences in the responses of satellite cells to growth factors may influence rates of skeletal muscle accretion and whole body growth.

Comparison of protein metabolism and glucose uptake in turkey (Melegaris gallopavo) satellite cells and embryonic myoblasts in vitro.

Protein synthesis, protein degradation, and glucose uptake were compared in clonal-derived turkey myogenic satellite cells (clone D5-SC) and embryonic myoblasts (EM) and between satellite cell cultures from Nicholas (DN) and Merriam's (WM) turkeys. Protein synthesis rates were higher and degradation rates were lower in myotube cultures of D5-SC compared with EM (P < or = 0.05). Protein synthesis and degradation rates did not differ between cultures of DN and WM (P > or = 0.05). Glucose transport rates were significantly higher in EM than D5-SC cultures and did not differ between DN and WM cultures. Insulin-like growth factors and insulin stimulated protein synthesis, decreased protein degradation, and increased glucose uptake in all cell lines.

The influence of growth factors on turkey embryonic myoblasts and satellite cells in vitro.

The influence of growth factors on the proliferation and differentiation of clonal-derived embryonic myoblasts and satellite cells was examined in hormonally controlled serum-free media. Although insulin-like growth factor-I (IGF-I) and IGF-II each stimulated proliferation of both cell types, IGF-II was the more potent mitogen for embryonic myoblasts (P < or = 0.05). Exogenously added IGFs also stimulated differentiation of embryonic myoblasts (P < or = 0.05) but did not influence the differentiation of satellite cells (P > or = 0.05). Fibroblast growth factor (FGF) was required for proliferation of either cell type to occur. Platelet-derived growth factor-BB acted synergistically with IGF-I and FGF to stimulate proliferation. Unlike human muscle cells, neither avian satellite cells nor embryonic myoblasts responded to epidermal growth factor. The influence of IGFs and insulin on IGF-binding protein (IGFBP) release from myogenic cells was also examined. Both cell types secreted a major binding protein of 34 kDa and a second protein of 36 kDa. Satellite cells secreted a prominent IGFBP at 30.8 kDa, while embryonic myoblasts secreted higher molecular weight forms of 37 and 41 kDa. Highest IGFBP release was seen when cells from either type were administered IGF-II. Equimolar levels of IGF-I or insulin stimulated IGFBP release, however, at levels lower than that induced by IGF-II. Release of IGFBP was nearly undetectable in the presence of cycloheximide (50 micrograms/ml), suggesting that protein synthesis was necessary for IGFBP release.

Comparison of the proliferation and differentiation of myogenic satellite cells derived from Merriam's and commercial varieties of turkeys.

1. Myogenic satellite cells were isolated and cloned from the Pectoralis major muscles of 6-week-old Nicholas and Merriam's tom turkeys to compare in vitro properties of muscle development between turkeys with markedly different growth rates. 2. Although only small differences (P < or = 0.05) were noted between proliferation rates of the two cell populations in McCoy's 5A medium-15% chicken serum, satellite cells derived from the Nicholas variety were more responsive (P < or = 0.05) to mitogenic stimuli from serum at all levels tested. 3. When satellite cells were stimulated by low serum levels to differentiate into multinucleated myotubes, cells from the Merriam's turkey fused more rapidly (P < or = 0.05).

Tissue distribution of insulin-like growth factor receptors in the turkey.

1. The distribution and relative insulin-like growth factor (IGF) binding capacities of membranes derived from 14 tissues of the turkey were examined. 2. Affinity cross-linking analyses using [125I]IGF-I and [125I]IGF-II with membranes derived from the liver, pectoralis major muscle, gizzard, heart and brain indicated that both IGFs interact with only type-I IGF receptors on these tissues. 3. There was no evidence for the existence of a type-II IGF receptor in any tissue. 4. Although considerable variation was detected in the molecular weights of the IGF receptor alpha subunits between tissues (112.2-132.9 kDa), these differences did not appear to influence receptor-ligand affinities.