Effects of 17ß-estradiol on turkey myogenic satellite cell proliferation, differentiation, and expression of glypican-1, MyoD and myogenin.

The hypothesis of this study was that 17ß-estradiol (estradiol) stimulates turkey skeletal muscle growth by influencing myogenic satellite cell proliferation, differentiation, and the gene expression of selected proteins important in regulating growth and development. Increasing levels of estradiol were administered in basal medium containing additional nutrients. Female-derived pectoralis major (PM) satellite cell proliferation was stimulated by estradiol at a level of 10(-9)M following 4days of treatment. Male PM and biceps femoris (BF) satellite cell proliferation was increased at 10(-12)M estradiol. Turkey embryonic myoblast proliferation, however, decreased with 10(-9)M and 10(-5)M estradiol following 3days under these conditions. Estradiol had no effect on the differentiation of any of the 4 groups of cells. Likewise, glypican-1 expression was unaffected by estradiol treatment. MyoD expression decreased in male PM but not BF cells. MyoD expression in female PM cells and embryonic myoblasts were also unaffected by estradiol administration. Estradiol decreased myogenin expression in male satellite cells, but had no effect on female cells. There was a slight decrease in myogenin expression in embryonic myoblasts. The results demonstrate a direct effect of estradiol on avian satellite cell proliferation independent of glypican-1, and decreased expression of MyoD and myogenin in some myogenic cells, coinciding with increased cellular proliferation.

Nampt/visfatin/PBEF affects expression of myogenic regulatory factors and is regulated by interleukin-6 in chicken skeletal muscle cells.

Nicotinamide phosphoribosyltransferase (Nampt/visfatin/PBEF) has been identified as a rate-limiting NAD(+) biosynthetic enzyme and an adipokine found in the circulation. Human and chicken skeletal muscles are reported to have the highest level of Nampt expression among various tissues whose functional significance remains undetermined. Expression of Nampt is regulated by interleukin-6 (IL-6), an essential cytokine for postnatal muscle growth in mammals. The objective of the current study was to characterize expression of Nampt in chicken (Gallus gallus) myogenic cells and to determine the effect of Nampt on expression of IL-6, myogenic transcription factors, and glucose uptake. We also sought to determine the effect of IL-6 on Nampt expression in chicken myogenic cells. Nampt mRNA and protein were identified in both myoblasts and myocytes, although expression did not differ between the two cell types. Treatment with recombinant human Nampt was found to decrease myoD and mrf4 expression but to increase myf5 expression in myocytes, while glucose uptake was unaffected. In response to treatment with recombinant Nampt, IL-6 expression in myocytes was increased at 24h but decreased when treated for 48 or 72 h. Forced over-expression of chicken Nampt cDNA significantly decreased myf5 expression in myoblasts. Treatment of myogenic cells with lower levels (1 ng.mL(-1)) of recombinant IL-6 increased Nampt expression, whereas a higher IL-6 concentration (100 ng.mL(-1)) decreased Nampt mRNA abundance. Collectively, these results demonstrate that Nampt, regulated in part by IL-6, alters the expression of key myogenic transcription factors and thereby may influence postnatal myogenesis.

Effect of lipids on avian satellite cell proliferation, differentiation and heparan sulfate proteoglycan expression.

The objective of this study was to determine the effects of fatty acids on the proliferation, differentiation, and expression of syndecan-4 and glypican-1 in avian myogenic satellite cells (SC). SC derived from the pectoralis major (PM) and biceps femoris (BF) muscles of the turkey and chicken were individually administered 8 different fatty acids in defined medium during proliferation. A parallel set of turkey SC was induced to differentiate. Highest levels of proliferation of turkey PM and BF SC occurred in cultures containing oleate. Linoleate and oleate were equipotent in supporting proliferation of chicken SC. Microscopic examination revealed that inclusion of docosahexaenoate or eicosapentaenoate was toxic towards both PM and BF SC from both species. Linolenate and arachidonate diminished levels of differentiation. Expression of glypican-1 varied between treatments to a greater extent with turkey BF than with PM SC. Expression in chicken PM and BF SC demonstrated a similar pattern in response to treatments. Turkey PM syndecan-4 expression varied between treatments, whereas expression in turkey BF SC was similar between treatments. Expression in chicken SC varied little between treatments. The results demonstrate species and muscle-specific differences in the parameters examined. It is proposed that changes in lipid raft receptor interactions may contribute to these observed differences.

Phospho-MAPK as a marker of myogenic satellite cell responsiveness to growth factors.

To determine if differential response to growth factor stimuli between subpopulations of satellite cells was due to variation in the levels of activated intracellular signaling proteins, the levels of phospho-MAPK (phospho-ERK 1/2) were determined in clonal populations of turkey (Meleagris gallopavo) satellite cells. Relative levels of phospho-ERK 1/2 between clones were determined by Western blotting of extracts from satellite cells exposed to growth factor stimuli. Initial measurements using serum mitogenic stimuli showed differences in phospho-MAPK levels between the clonal subpopulations, but the responses did not correlate with proliferation rates of the individual clones (P>0.05). IGF-I alone did not increase phospho-MAPK levels compared to unstimulated controls (P>0.05), whereas FGF-2 did increase levels (P<0.05). A synergistic response was seen in satellite cells as well as embryonic myoblasts administered both IGF-I and FGF-2. When administered FGF-2 and IGF-I, 2 of the slow growing satellite cell clones exhibited lowest levels of phospho-MAPK (P<0.05). One of the slow growing clones had levels of phospho-MAPK similar to the three fast growing clones (P>0.05). The results suggest that variation in responsiveness to growth factor stimuli among satellite cell populations within muscles may be due to several different reasons. Some differences in cell responsiveness appear to be due to variation in phospho-MAPK generation.

The role of myostatin in chicken (Gallus domesticus) myogenic satellite cell proliferation and differentiation.

Myostatin (GDF-8) inhibits the activation, proliferation, and differentiation of myogenic satellite cells. The relative importance of this growth factor is demonstrated in myostatin-null mice and cattle possessing defective myostatin genes. These defects result in greatly enhanced musculature. In the present study, the effect of myostatin on chicken myogenic satellite cells derived from two different skeletal muscles was studied. The effect of anti-myostatin antibodies on cellular responses was also examined. Satellite cells isolated from the pectoralis major (PM) muscles were more responsive to the proliferation depressing effects of myostatin compared to cells from the biceps femoris (BF; P or=0.05). Myostatin administered to proliferating cells depressed the synthesis of decorin (P or= 0.05). Administration of anti-myostatin antibodies to proliferating cultures increased cell proliferation by 6-7% over 3 days. There was no effect on differentiation of either PM or BF cells. The present study demonstrates that there are differences in the responsiveness to myostatin of chicken satellite cells derived from different muscles. Evidence is also given to support the role of endogenous myostatin in autocrine regulation of muscle growth.

Effect of myostatin on turkey myogenic satellite cells and embryonic myoblasts.

Myostatin (GDF-8) inhibits the activation, proliferation, and differentiation of myogenic satellite cells. The relative importance of this growth factor is demonstrated in myostatin-null mice and cattle possessing defective myostatin genes. These defects result in greatly enhanced musculature. In the present study, we examined the effect of myostatin on turkey myogenic satellite cells and embryonic myoblasts. Compared with controls (P<0.05), proliferation of both turkey embryonic myoblasts and satellite cells was inhibited between 26 and 45% in serum-free medium containing 20 ng/mL myostatin. While individual turkey satellite cell clones differed in their responsiveness to myostatin, there were no significant differences in the responsiveness of fast and slow growing cells as groups (P>0.05). A slow growing clone that exhibited the greatest response to myostatin also exhibited the greatest depression of differentiation with this growth factor (P<0.05). All other turkey satellite cell clones exhibited similar responses to the differentiation depressing effects of myostatin (P>0.05). However, myostatin had no effect on differentiation of turkey embryonic myoblasts (P>0.05). When exposed to myostatin, 4 of 6 proliferating clones and all differentiating clones increased their expression of decorin, a growth inhibitor (P<0.05). The present study demonstrates that myostatin inhibits the proliferation and differentiation of satellite cells and suggests a role for decorin in myostatin action in muscle development.

Variation in fibroblast growth factor response and heparan sulfate proteoglycan production in satellite cell populations.

Variation in the responsiveness of myogenic satellite cell subpopulations to mitogenic stimuli was examined in cells isolated from the turkey pectoralis major muscle. Faster growing clonal cell populations were more responsive to fibroblast growth factor (FGF-2) and expressed greater levels of FGF-2 and FGF receptor-1 at the onset of proliferation than did slower growing cells. Faster growing clones also expressed higher levels of heparan sulfate proteoglycans (HSPG), especially during differentiation, than did slower growing clones. HSPG, which is important in FGF receptor signaling, increased during proliferation of all clones tested and decreased in all but one of the clones during differentiation. Slower growing clones increased their expression of FGF receptor-1 through proliferation and differentiation. However, expression of the receptor in faster growing clones decreased during differentiation. The FGF receptors-2 and -3 were not detected on turkey satellite cells or myotubes by reverse transcriptase-polymerase chain reaction methodology. These results demonstrate that there is heterogeneity in the properties and responsiveness of satellite cell populations derived from single muscles. Satellite cells that differ in proliferation rates differ in responsiveness to FGF-2, and in the expression of FGF-2, FGF receptor-1, and HSPG.