Expression of miRNAs in turkey muscle satellite cells and differential response to thermal challenge.

Thermal stress alters the transcriptome and subsequent tissue physiology of poultry; thus, it can negatively impact poultry production through reduced meat quality, egg production, and health and wellbeing. The modulation of gene expression is critical to embryonic development and cell proliferation, and growing evidence suggests the role of non-coding RNAs (RNA:RNA interaction) in response to thermal stress in animals. MicroRNAs (miRNAs) comprise a class of small regulatory RNAs that modulate gene expression through posttranscriptional interactions and regulate mRNAs, potentially altering numerous cellular processes. This study was designed to identify and characterize the differential expression of miRNAs in satellite cells (SCs) from the turkey pectoralis major muscle and predict important miRNA:mRNA interactions in these developing SCs under a thermal challenge. Small RNA sequencing was performed on RNA libraries prepared from SCs cultured from 1-week-old male Nicholas commercial turkeys (NCTs) and non-selected Randombred Control Line 2 turkeys during proliferation and differentiation at the control temperature (38°C) or under a thermal challenge (33°C or 43°C). A total of 353 miRNAs (161 known and 192 novel) were detected across the sequenced libraries. Expression analysis found fewer differentially expressed miRNAs in the SCs of NCT birds, suggesting that the miRNA response to heat stress has been altered in birds selected for their modern commercial growth traits. Differentially expressed miRNAs, including those with described roles in muscle development, were detected both among temperature treatments and between genetic lines. A prominent differential expression of miR-206 was found in proliferating turkey SCs with a significant response to thermal challenges in both lines. In differentiating SCs, isoforms of miR-1 had significant differential responses, with the expression of miR-206 being mainly affected only by cold treatment. Target gene predictions and Gene Ontology analysis suggest that the differential expression of miRNAs during thermal stress could significantly affect cellular proliferation and differentiation.

Broiler breast muscle myopathies: association with satellite cells.

Heavy weight fast-growing meat-type broiler chickens have largely been selected for growth rate, muscle mass yield especially for the breast muscle, and feed conversion. Substantial improvements have been made, but in recent years breast meat quality issues resulting in product downgrades or condemnation have occurred especially from necrotic and fibrotic myopathies like Wooden Breast. In general, the morphological structure of the broiler breast muscle has changed in the modern commercial broiler with muscle fiber diameters increased, circulatory supply decreased, and connective spacing between individual fibers and fiber bundles decreased. Satellite cells are the primary cell type responsible for all posthatch muscle growth, and the repair and regeneration of muscle fibers. Recent evidence is suggestive of changes in the broiler satellite cell populations which will limit the ability of the satellite cells to regenerate damaged muscle fibers back to their original. These changes in the cellular biology of broiler satellite cells are likely associated with the necrosis and fibrosis observed in myopathies like Wooden Breast.

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.

Transcriptome Response of Differentiating Muscle Satellite Cells to Thermal Challenge in Commercial Turkey.

Early muscle development involves the proliferation and differentiation of stem cells (satellite cells, SCs) in the mesoderm to form multinucleated myotubes that mature into muscle fibers and fiber bundles. Proliferation of SCs increases the number of cells available for muscle formation while simultaneously maintaining a population of cells for future response. Differentiation dramatically changes properties of the SCs and environmental stressors can have long lasting effects on muscle growth and physiology. This study was designed to characterize transcriptional changes induced in turkey SCs undergoing differentiation under thermal challenge. Satellite cells from the pectoralis major (p. major) muscle of 1-wk old commercial fast-growing birds (Nicholas turkey, NCT) and from a slower-growing research line (Randombred Control Line 2, RBC2) were proliferated for 72 h at 38 °C and then differentiated for 48 h at 33 °C (cold), 43 °C (hot) or 38 °C (control). Gene expression among thermal treatments and between turkey lines was examined by RNAseq to detect significant differentially expressed genes (DEGs). Cold treatment resulted in significant gene expression changes in the SCs from both turkey lines, with the primary effect being down regulation of the DEGs with overrepresentation of genes involved in regulation of skeletal muscle tissue regeneration and sarcomere organization. Heat stress increased expression of genes reported to regulate myoblast differentiation and survival and to promote cell adhesion particularly in the NCT line. Results suggest that growth selection in turkeys has altered the developmental potential of SCs in commercial birds to increase hypertrophic potential of the p. major muscle and sarcomere assembly. The biology of SCs may account for the distinctly different outcomes in response to thermal challenge on breast muscle growth, development, and structure of the turkey.

Transcriptome response of proliferating muscle satellite cells to thermal challenge in commercial turkey.

Thermal stress poses a threat to agricultural systems through increased risk to animal growth, health, and production. Exposure of poultry, especially hatchlings, to extreme temperatures can seriously affect muscle development and thus compromise subsequent meat quality. This study was designed to characterize transcriptional changes induced in turkey muscle satellite cells (SCs) cultured from commercial birds under thermal challenge to determine the applicability of previous results obtained for select research lines. Satellite cells isolated from the pectoralis major muscle of 1-week old commercial fast-growing birds (Nicholas turkey, NCT) and from a slower-growing research line (RBC2) were proliferated in culture at 38°C or 43°C for 72 h. RNAseq analysis found statistically significant differences in gene expression among treatments and between turkey lines with a greater number of genes altered in the NCT SCs suggesting early myogenesis. Pathway analysis identified cell signaling and regulation of Ca2+ as important responses. Expression of the intercellular signaling Wnt genes, particularly Wnt5a and 7a was significantly altered by temperature with differential response between lines. The peripheral calcium channel RYR3 gene was among the genes most highly upregulated by heat stress. Increased expression of RYR3 would likely result in higher resting cytosolic calcium levels and increased overall gene transcription. Although responses in the calcium signaling pathway were similar among the RBC2 and NCT lines, the magnitude of expression changes was greater in the commercially selected birds. These results provide evidence into how SC activity, cellular fate, and ultimately muscle development are altered by heat stress and commercial selection.

Thermal stress and selection for growth affect myogenic satellite cell lipid accumulation and adipogenic gene expression through mechanistic target of rapamycin pathway.

Satellite cells (SCs) are multipotential stem cells having the plasticity to convert to an adipogenic lineage in response to thermal stress during the period of peak mitotic activity (the first week after hatch in poultry). The mechanistic target of rapamycin (mTOR) pathway, which regulates cellular function and fate of SCs, is greatly altered by thermal stress in turkey pectoralis major muscle SCs. The objective of the present study was to determine the effects of thermal stress, selection for growth, and the role of the mTOR pathway on SC intracellular lipid accumulation and expression of adipogenic regulatory genes. These effects were analyzed using SCs isolated from the pectoralis major muscle of 1-wk-old modern faster-growing commercial turkey line (NC) selected for increased growth and breast muscle yield as compared with SCs of a historic slower-growing Randombred Control Line 2 (RBC2) turkey. Heat stress (43 °C) of SCs during proliferation increased intracellular lipid accumulation (P < 0.001), whereas cold stress (33 °C) showed an inhibitory effect (P < 0.001) in both lines. Knockdown of mTOR reduced the intracellular lipid accumulation (P < 0.001) and suppressed the expression of several adipogenic regulatory genes: peroxisome proliferator-activated receptor-γ (PPARγ; P < 0.001), CCAAT/enhancer-binding protein-ß (C/EBPß; P < 0.001), and neuropeptide-Y (NPY; P < 0.001) during both proliferation and differentiation. The NC line SCs showed fewer reductions in lipid accumulation compared with the RBC2 line independent of temperature. Both intracellular lipid accumulation (P < 0.001) and PPARγ expression (P < 0.001) were greater at 72 h of proliferation than at 48 h of differentiation in both the RBC2 and NC lines independent of temperature. Thus, hot and cold thermal stress affected intracellular lipid accumulation in the pectoralis major muscle SCs, in part, through the mTOR pathway in wea growth-dependent manner. Altered intracellular lipid accumulation could eventually affect intramuscular fat deposition, resulting in a long-lasting effect on the structure and protein to fat ratio of the poultry pectoralis major muscle.

Turkey breast muscle growth and development are sensitive to temperatures immediately after hatch due to an immature thermoregulatory system. Meat yield or quality problems may arise from external thermal stress during this period. Modern commercial turkeys are selected for increased growth and breast muscle yield. However, with excessive enlargement of muscle fibers, there are increased incidences of muscle damage and fat deposition in the breast muscle. The breast meat can be downgraded due to the meat quality problems. Satellite cells (SCs) are the only source of cells responsible for post-hatch muscle growth in poultry, and they are sensitive to temperature. This study identifies the cellular mechanisms in regulating thermal stress-induced fat synthesis in turkey breast muscle SCs. The results of the current study provide insight into how thermal stress and selection for rapid growth affect the fat content in SCs. These results have potential application in the development of temperature manipulation strategies to control fat production by SCs, which will impact poultry breast meat quality.

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 expression of PPARG, DNM2L, RRAD, and LINGO1 on broiler chicken breast muscle satellite cell function.

Disorders affecting the breast muscle of modern commercial broiler chickens have increased in recent years. Wooden Breast (WB) myopathy is characterized by a palpably hard breast muscle with increased fat deposition. WB is a metabolic disorder with lipid accumulation considered to be a primary causal factor. The adult myoblasts, satellite cells, are a partially differentiated stem cell population and primarily function in muscle growth and regeneration. The satellite cells also express adipogenic genes. The objective of this study was to determine the expression of the adipogenic genes PPARG, DNM2L, RRAD, and LINGO1 in commercial Ross 708 (708) and Randombred (RBch) satellite cells. RBch satellite cells are from commercial 1995 broilers before WB and 708 broilers are a modern commercial line. In general, expression of these genes was different between the 708 and RBch satellite cells during proliferation and differentiation. Expression of PPARG and RRAD were both significantly increased during both proliferation and differentiation in the 708 cells (P ≤ 0.05). Knocking down the expression of these genes with small interfering RNAs did not greatly affect either proliferation or differentiation. Lipid accumulation was affected by the knockdown of these genes with significant line effects from 48 h of proliferation through 72 h of differentiation. In general, 708 satellite cells had higher lipid levels. Knockdown treatment effect was specific to each gene. The results demonstrate that lipid biosynthesis has been affected in breast muscle satellite cells which may contribute to the increased lipid deposition in modern day commercial broiler chickens.

Thermal stress affects proliferation and differentiation of turkey satellite cells through the mTOR/S6K pathway in a growth-dependent manner.

Satellite cells (SCs) are stem cells responsible for post-hatch muscle growth through hypertrophy and in birds are sensitive to thermal stress during the first week after hatch. The mechanistic target of rapamycin (mTOR) signaling pathway, which is highly responsive to thermal stress in differentiating turkey pectoralis major (p. major) muscle SCs, regulates protein synthesis and the activities of SCs through a downstream effector, S6 kinase (S6K). The objectives of this study were: 1) to determine the effect of heat (43°C) and cold (33°C) stress on activity of the mTOR/S6K pathway in SCs isolated from the p. major muscle of one-week-old faster-growing modern commercial (NC) turkeys compared to those from slower-growing Randombred Control Line 2 (RBC2) turkeys, and 2) to assess the effect of mTOR knockdown on the proliferation, differentiation, and expression of myogenic regulatory factors of the SCs. Heat stress increased phosphorylation of both mTOR and S6K in both turkey lines, with greater increases observed in the RBC2 line. With cold stress, greater reductions in mTOR and S6K phosphorylation were observed in the NC line. Early knockdown of mTOR decreased proliferation, differentiation, and expression of myoblast determination protein 1 and myogenin in both lines independent of temperature, with the RBC2 line showing greater reductions in proliferation and differentiation than the NC line at 38° and 43°C. Proliferating SCs are more dependent on mTOR/S6K-mediated regulation than differentiating SCs. Thus, thermal stress can affect breast muscle hypertrophic potential by changing satellite cell proliferation and differentiation, in part, through the mTOR/S6K pathway in a growth-dependent manner. These changes may result in irreversible effects on the development and growth of the turkey p. major muscle.

Histological Analysis and Gene Expression of Satellite Cell Markers in the Pectoralis Major Muscle in Broiler Lines Divergently Selected for Percent 4-Day Breast Yield.

Muscle development during embryonic and early post-hatch growth is primarily through hyperplastic growth and accumulation of nuclei through satellite cell contribution. Post-hatch, muscle development transitions from hyperplasia to hypertrophic growth of muscle fibers. Commercial selection for breast yield traditionally occurs at ages targeting hypertrophic rather than hyperplastic growth. This has resulted in the production of giant fibers and concomitant challenges with regard to muscle myopathies. The current study investigates the impact of selection during the period of hyperplastic growth. It is hypothesized that selection for percentage breast yield during hyperplasia will result in an increased number of muscle cells at hatch and potentially impact muscle fiber characteristics at processing. This study characterizes the breast muscle histology of three broiler lines at various ages in the growth period. The lines include a random bred control (RAN) as well as lines which have been selected from RAN for high (HBY4) and low (LBY4) percentage 4-day breast yield. Post-rigor pectoralis major samples from six males of each line and age were collected and stored in formalin. The sample ages included embryonic day 18 (E18), post-hatch day 4 (d4), and day 56 (d56). The samples were processed using a Leica tissue processor, embedded in paraffin wax, sectioned, and placed on slides. Slides were stained using hematoxylin and eosin. E18 and d4 post-hatch analysis showed advanced muscle fiber formation for HBY4 and immature muscle development for LBY4 as compared to RAN. Post-hatch d56 samples were analyzed for fiber number, fiber diameter, endomysium, and perimysium spacing. Line HBY4 had the largest muscle fiber diameter (54.2 ± 0.96 µm) when compared to LBY4 (45.4 ± 0.96 µm). There was no line difference in endomysium spacing while perimysium spacing was higher for HBY4 males. Selection for percentage 4-day breast yield has impacted the rate and extent of muscle fiber formation in both the LBY4 and HBY4 lines with no negative impact on fiber spacing. The shift in processing age to later ages has exposed issues associated with muscle fiber viability. Selection during the period of muscle hyperplasia may impact growth rate; however, the potential benefits of additional satellite cells are still unclear.

Data Mining Identifies Differentially Expressed Circular RNAs in Skeletal Muscle of Thermally Challenged Turkey Poults.

Precise regulation of gene expression is critical for normal muscle growth and development. Changes in gene expression patterns caused by external stressors such as temperature can have dramatic effects including altered cellular structure and function. Understanding the cellular mechanisms that underlie muscle growth and development and how these are altered by external stressors are crucial in maintaining and improving meat quality. This study investigated circular RNAs (circRNAs) as an emerging aspect of gene regulation. We used data mining to identify circRNAs and characterize their expression profiles within RNAseq data collected from thermally challenged turkey poults of the RBC2 and F-lines. From sequences of 28 paired-end libraries, 8924 unique circRNAs were predicted of which 1629 were common to all treatment groups. Expression analysis identified significant differentially expressed circRNAs (DECs) in comparisons between thermal treatments (41 DECs) and between genetic lines (117 DECs). No intersection was observed between the DECs and differentially expressed gene transcripts indicating that the DECs are not simply the result of expression changes in the parental genes. Comparative analyses based on the chicken microRNA (miRNA) database suggest potential interactions between turkey circRNAs and miRNAs. Additional studies are needed to reveal the functional significance of the predicted circRNAs and their role in muscle development in response to thermal challenge. The DECs identified in this study provide an important framework for future investigation.

Effect of Temperature and Selection for Growth on Intracellular Lipid Accumulation and Adipogenic Gene Expression in Turkey Pectoralis Major Muscle Satellite Cells.

As multipotential stem cells, satellite cells (SCs) have the potential to express adipogenic genes resulting in lipid synthesis with thermal stress. The present study determined the effect of temperature on intracellular lipid synthesis and adipogenic gene expression in SCs isolated from the pectoralis major (p. major) muscle of 7-day-old fast-growing modern commercial (NC) turkeys compared to SCs from unselected slower-growing turkeys [Randombred Control Line 2 (RBC2)]. Since proliferating and differentiating SCs have different responses to thermal stress, three incubation strategies were used: (1) SCs proliferated at the control temperature of 38°C and differentiated at 43° or 33°C; (2) SCs proliferated at 43° or 33°C and differentiated at 38°C; or (3) SCs both proliferated and differentiated at 43°, 38°, or 33°C. During proliferation, lipid accumulation increased at 43°C and decreased at 33°C with the NC line showing greater variation than the RBC2 line. During proliferation at 43°C, peroxisome proliferator-activated receptor-γ (PPARγ) and neuropeptide-Y (NPY) expression was reduced to a greater extent in the NC line than the RBC2 line. At 33°C, expression of PPARγ, NPY, and CCAAT/enhancer-binding protein-ß (C/EBPß) was upregulated, but only in the RBC2 line. During differentiation, both lines showed greater changes in lipid accumulation and in C/EBPß and NPY expression if the thermal challenge was initiated during proliferation. These data suggest that adipogenic gene expression is more responsive to thermal challenge in proliferating SCs than in differentiating SCs, and that growth-selection has increased temperature sensitivity of SCs, which may significantly affect breast muscle structure and composition.