Effects of thermal stress and mechanistic target of rapamycin and wingless-type mouse mammary tumor virus integration site family pathways on the proliferation and differentiation of satellite cells derived from the breast muscle of different chicken lines.

Satellite cells (SCs) are muscle stem cells responsible for muscle hypertrophic growth and the regeneration of damaged muscle. Proliferation and differentiation of the pectoralis major (p. major) muscle SCs are responsive to thermal stress in turkeys, which are, in part, regulated by mechanistic target of rapamycin (mTOR) and Frizzled7 (Fzd7)-mediated wingless-type mouse mammary tumor virus integration site family/planar cell polarity (Wnt/PCP) pathways in a growth dependent-manner. It is not known if chicken p. major SCs respond to thermal stress in a manner similar to that of turkey p. major SCs. The objective of the current study was to investigate the effects of thermal stress and mTOR and Wnt/PCP pathways on the proliferation, differentiation, and expression of myogenic transcriptional regulatory factors in SCs isolated from the p. major muscle of a current modern commercial (MC) broiler line as compared to that of a Cornish Rock (BPM8) and Randombred (RBch) chicken line in the 1990s. The MC line SCs had lower proliferation and differentiation rates and decreased expression of myoblast determination factor 1 (MyoD) and myogenin (MyoG) compared to the BPM8 and RBch lines. Heat stress (43°C) increased proliferation and MyoD expression in all the cell lines, while cold stress (33°C) showed a suppressive effect compared to the control temperature (38°C). Satellite cell differentiation was altered with heat and cold stress in a cell line-specific manner. In general, the differentiation of the MC SCs was less responsive to both heat and cold stress compared to the BPM8 and RBch lines. Knockdown of the expression of either mTOR or Fzd7 decreased the proliferation, differentiation, and the expression of MyoD and MyoG in all the cell lines. The MC line during proliferation was more dependent on the expression of mTOR and Fzd7 than during differentiation. Thus, modern commercial meat-type chickens have decreased myogenic activity and temperature sensitivity of SCs in an mTOR- and Fzd7-dependent manner. The decrease in muscle regeneration will make modern commercial broilers more susceptible to the negative effects of myopathies with muscle fiber necrosis requiring satellite cell-mediated repair.

Differential effects of temperature and mTOR and Wnt-planar cell polarity pathways on syndecan-4 and CD44 expression in growth-selected turkey satellite cell populations.

Satellite cells (SCs) comprise a heterogeneous population of muscle stem cells. Thermal stress during the first week after hatch alters proliferation, myogenesis, and adipogenesis of SCs of turkey pectoralis major (p. major) muscle via mechanistic target of rapamycin (mTOR) and wingless-type mouse mammary tumor virus integration site family/planar cell polarity (Wnt/PCP) pathways. Pivotal genes in mTOR and Wnt/PCP pathways are mTOR and frizzled-7 (Fzd7), respectively. The objective of this study was to determine the differential effects of thermal stress on SDC4 and CD44 expression in turkey p. major muscle SCs and how the expression of SDC4 and CD44 is modulated by the mTOR and Wnt/PCP pathways. Satellite cells were isolated from the p. major muscle of 1-week-old faster-growing modern-commercial (NC) turkeys and slower-growing historic Randombred Control Line 2 (RBC2) turkeys, and were challenged with hot (43°C) and cold (33°C) thermal stress for 72 h of proliferation followed by 48 h of differentiation. The NC line SCs were found to contain a lower proportion of SDC4 positive and CD44 negative (SDC4+CD44-) cells and a greater proportion of SDC4 negative and CD44 positive (SDC4-CD44+) cells compared to the RBC2 line at the control temperature (38°C) at both 72 h of proliferation and 48 h of differentiation. In general, at 72 h of proliferation, the proportion of SDC4+CD44- cells decreased with heat stress (43°C) and increased with cold stress (33°C) relative to the control temperature (38°C) in both lines, whereas the proportion of SDC4-CD44+ cells increased with heat stress and decreased with cold stress. In general, the expression of SDC4 and CD44 in the NC SCs showed greater response to both hot and cold thermal stress compared to the RBC2 cells. Knockdown of mTOR or Fzd7 expression increased the proportion of SDC4+CD44- cells while the proportion of SDC4-CD44+ cells decreased during differentiation with line differences being specific to treatment temperatures. Thus, differential composition of p. major muscle SCs in growth-selected commercial turkey may be resulted, in part, from the alteration in SDC4 and CD44 expression. Results indicate differential temperature sensitivity and mTOR and Wnt/PCP pathway responses of growth-selected SC populations and this may have long-lasting effect on muscle development and growth.

Temperature and Growth Selection Effects on Proliferation, Differentiation, and Adipogenic Potential of Turkey Myogenic Satellite Cells Through Frizzled-7-Mediated Wnt Planar Cell Polarity Pathway.

Satellite cells (SCs) are a heterogeneous population of multipotential stem cells. During the first week after hatch, satellite cell function and fate are sensitive to temperature. Wingless-type mouse mammary tumor virus integration site family/planar cell polarity (Wnt/PCP) signaling pathway is significantly affected by thermal stress in turkey pectoralis major (p. major) muscle SCs. This pathway regulates the activity of SCs through a frizzled-7 (Fzd7) cell surface receptor and two intracellular effectors, rho-associated protein kinase (ROCK) and c-Jun. The objective of the present study was to determine the effects of thermal stress, growth selection, and the Fzd7-mediated Wnt/PCP pathway on proliferation, myogenic differentiation, lipid accumulation, and expression of myogenic and adipogenic regulatory genes. These effects were evaluated in SCs isolated from the p. major muscle of 1-week faster-growing modern commercial (NC) line of turkeys as compared to SCs of a slower-growing historic Randombred Control Line 2 (RBC2) turkey line. Heat stress (43°C) increased phosphorylation of both ROCK and c-Jun with greater increases observed in the RBC2 line. Cold stress (33°C) had an inhibitory effect on both ROCK and c-Jun phosphorylation with the NC line showing greater reductions. Knockdown of the expression of Fzd7 decreased proliferation, differentiation, and expression of myogenic regulatory genes: myoblast determination factor-1 and myogenin in both lines. Both lipid accumulation and expression of adipogenic regulatory genes: peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein-ß, and neuropeptide-Y were suppressed with the Fzd7 knockdown. The RBC2 line was more dependent on the Fzd7-mediated Wnt/PCP pathway for proliferation, differentiation, and lipid accumulation compared to the NC line. Thus, thermal stress may affect poultry breast muscle growth potential and protein to fat ratio by altering function and fate of SCs through the Fzd7-mediated Wnt/PCP pathway in a growth-dependent manner.

Effect of vitamin E and alpha lipoic acid on intestinal development associated with wooden breast myopathy in broilers.

Intestinal development is closely associated with inflammatory wooden breast (WB) myopathy. Vitamin E (VE) and alpha lipoic acid (ALA) with antioxidant and anti-inflammatory effects were used independently and in combination to evaluate their effects on intestinal developmental changes in ileal morphology and expression of genes related with gut nutrient transport, structure, and inflammation in broilers during the first 3 wk posthatch. A total of 160 newly hatched Ross 708 broiler chicks were randomly assigned into a control and 3 dietary treatments with 10 replicates of 4 birds each. Supplementation of VE (160 mg/kg) and ALA (500 mg/kg) independently and in combination were fed during the first 3 wk. At 1, 2, and 3 wk of age, one chick from each pen was harvested. Plasma VE concentration and ileal morphology were determined. Gene expression was measured by real-time quantitative PCR. Broilers in VE and combination of ALA and VE group had higher plasma VE concentration than the control and ALA group at 1, 2, and 3 wk of age (P < 0.01). All dietary treatments increased ileal villus height at 1 wk of age (P < 0.01) and decreased intraepithelial lymphocytes at 3 wk of age compared to the control (P ≤ 0.05). Combination of VE and ALA increased collagen type IV alpha 1 chain expression (P ≤ 0.05) and improved basement membrane structure indicating increased gut basement membrane integrity at 2 and 3 wk of age compared to the control. Expression of lipopolysaccharide-induced tumor necrosis factor-alpha factor associated with inflammation was decreased in all dietary treatments at 3 wk of age compared to the control (P < 0.01). Ileal morphology and gene expression were closely correlated with breast muscle morphology and gene expression. These results suggest that VE and ALA especially when they were combined in the diet had positive effects on mitigating intestinal inflammation and improving nutrient transport beginning at 1 wk of age, which is likely critical in reducing the severity of WB.

Effect of growth selection of broilers on breast muscle satellite cell function: Response of satellite cells to NOV, COMP, MYBP-C1, and CSRP3.

The wooden breast (WB) myopathy is characterized by the palpation of a hard pectoralis major muscle that results in the necrosis and fibrosis of muscle fibers in fast-growing heavy weight meat-type broiler chickens. Necrosis of existing muscle fibers requires the repair and replacement of these myofibers. Satellite cells are responsible for the repair and regeneration of myofibers. To address how WB affects satellite cell function, top differentially expressed genes in unaffected and WB-affected pectoralis major muscle determined by RNA-Sequencing were studied by knocking down their expression by small interfering RNA in proliferating and differentiating commercial Ross 708 and Randombred (RBch) satellite cells. RBch satellite cells are from commercial 1995 broilers before WB appeared in broilers. Genes studied were: Nephroblastoma Overexpressed (NOV); Myosin Binding Protein-C (MYBP-C1); Cysteine-Rich Protein 3 (CSRP3); and Cartilage Oligomeric Matrix Protein (COMP). Ross 708 satellite cells had greatly reduced proliferation and differentiation compared to RBch satellite cells. MYBP-C1, CSRP3, and COMP reduced late proliferation and NOV did not affect proliferation in both lines. The timing of the knockdown differentially affected differentiation. If the expression was reduced at the beginning of proliferation, the effect on differentiation was greater than if the knockdown was at the beginning of differentiation. These data suggest, appropriate gene expression levels during proliferation greatly impact multinucleated myotube formation during differentiation. The effect of slow myofiber genes MYBP-C1 and CSRP3 on proliferation and differentiation suggests the presence of aerobic Type I satellite cells in the pectoralis major muscle which contains anaerobic Type IIb cells.

Blood Gas Disturbances and Disproportionate Body Weight Distribution in Broilers With Wooden Breast.

Wooden breast syndrome is a widespread and economically important myopathy and vasculopathy of fast growing, commercial broiler chickens, primarily affecting birds with high feed efficiency and large breast muscle yield. To investigate potential systemic physiological differences between birds affected and unaffected by wooden breast, a total of 103 market-age Cobb 500 broilers were sampled for 13 blood parameters and the relative weights of the pectoralis major muscle, pectoralis minor muscle, external oblique muscle, wing, heart, lungs, liver, and spleen. Blood analysis was performed on samples taken from the brachial vein of live birds and revealed significant differences in venous blood gases between affected and unaffected chickens. Chickens with wooden breast exhibited significantly higher potassium (K+) and lower partial pressure of oxygen (pO2), oxygen saturation (sO2), and pH. Additionally, affected males had significantly higher partial pressure of carbon dioxide (pCO2) and total carbon dioxide (TCO2) than unaffected males. Wooden breast affected broilers also possessed a significantly heavier pectoralis major muscle and whole feathered wing compared to unaffected broilers. Blood gas disturbances characterized by high pCO2 and low pH are indicative of insufficient respiratory gas exchange, suggesting that wooden breast affected broilers have an elevated metabolic rate that may also be inadequately compensated due to cardiovascular deficiencies such as poor venous return or respiratory insufficiency. Lung tissues from 12 birds with extreme sO2 values were subsequently examined to assess whether lung pathology contributed to the observed blood gas disturbance. Comparison of lung morphology between affected and unaffected birds revealed no apparent differences that could contribute to decreased parabronchial gas exchange. However, an interesting finding was the detection of pulmonary phlebitis in one of the wooden breast-affected samples consistent with vascular changes observed in pectoralis major muscle exhibiting the wooden breast phenotype. Our results suggest that the effects of wooden breast are not limited to the pectoralis major muscle and further indicate the importance of research into metabolic changes associated with the myopathy.

The effect of syndecan-4 and glypican-1 expression on age-related changes in myogenic satellite cell proliferation, differentiation, and fibroblast growth factor 2 responsiveness.

Satellite cells are multipotential stem cells responsible for muscle growth and regeneration. Satellite cell proliferation, differentiation, and responsiveness to fibroblast growth factor 2 (FGF2) is, in part, regulated by the heparan sulfate proteoglycans syndecan-4 and glypican-1. Syndecan-4 and glypican-1 expression declines with satellite cell age and may be associated with decreased satellite cell activity. The objective of the current study was to determine if overexpression of syndecan-4 and glypican-1 would increase proliferation, differentiation and FGF2 responsiveness in satellite cells isolated from pectoralis major muscle from 16-wk-old turkeys. Overexpression of syndecan-4 and glypican-1 did not have a significant effect on proliferation and differentiation in 1d, 7 wk, and 16 wk satellite cells, and did not affect FGF2 responsiveness during proliferation. Expression of syndecan-4 and glypican-1 increased differentiation at 48 h in 1d, 7 wk, and 16 wk cells treated with FGF2. Expression of myogenic regulatory factors MyoD, myogenin, and MRF4 was affected by the overexpression of syndecan-4 and glypican-1. However, changes in myogenic regulatory factor expression did not have a significant effect on proliferation or differentiation. These data demonstrate that syndecan-4 and glypican-1 are likely not directly associated with the age related decrease in satellite cell activity.

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.