Progress 10/01/10 to 09/30/15
Outputs
Target Audience:The target audience comprises scientists working in the field of muscle growth and differentiation, as well as the broader areas of meat science, and meat animal growth and development Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project provided employment for a lab assistant, Mr. Jon Weiss, who worked part time on the project before beginning medical school in fall 2015. In addition, a new M.S. graduate student, Galen George, has started on the project effective in August 2014. How have the results been disseminated to communities of interest?Results of satellite cell culture work were communicated at the Annual Meeting of the Poultry Science Association in Louisville, Kentucky on July 30, 2015. The abstract is published in the Poultry Science Journal. What do you plan to do during the next reporting period to accomplish the goals?e are exposing muscle satellite cells from two different turkey genetic lines (RBC2 and F) from Ohio State University to various temperatures ranging from 31C to 43C. We are studying the effect of calcium ion concentration in these cells as a function of temperature to determine whether altered temperature alters the cytoplasmic [Ca2+], which in turn, could alter trancriptional rated. In addition, we are analyizing results of the exposure of posthatch turkeys to different brooding temperatures on gene expression during the first 3 days, as well as muscle ultrastructure and meat quality characteristics at 16 weeks of age. Studies are ongoing in collaboration with Dr. Kent Reed at the University of Minnesota to analyze effects on gene expression of thermal challenge of cultured satellite cells from RBC2 and F line turkeys. In a parallel study, we are analyzing effects of thermal challenge on breast muscle gene expression from birds exposed to different brooding temperatures. In the next phase of the study, we will analyze the effect of embryonic thermal challenge on development of thermotolerance in posthatch RBC2 and F-line turkeys. Eggs will be exposed to brief thermal challenge late in development, and posthatch poults will be exposed to different brooding thermal challenge conditions. Muscle tissue from market weight birds will be analyzed for muscle ultrastructure and meat quality characteristics.
Impacts
What was accomplished under these goals?
The importance of muscle as a food is exemplified by the turkey meat processing industry. In the USA, there has been a continual increase in consumption of turkey meat from 4.9 pounds of boneless equivalent in 1960 to 14.4 pounds in 2002. Breast meat yield is the primary profit center for the commercial poultry industry. A predominant factor leading to this increase in consumer consumption is that poultry breast meat is regarded as the ideal lean meat for a healthy diet. This is coupled to its affordability and ease of preparation. A 1% increase in breast yield will amount to at least $75 million increase in revenues to the poultry industry. Commercial geneticists have placed a tremendous amount of selection pressure on breast yield and conformation, but not the mechanisms regulating the growth of muscle. The ability to regulate the growth and development of muscle depends on understanding the cellular regulatory mechanisms and cellular interactions that occur. Changes in muscle fiber size, extracellular areas available between the individual fibers and fiber bundles, and increased fat deposition will alter meat quality as have been illustrated by problems like pale, soft, and exudative turkey and pork. The results from this study will provide information on the role of specific genes whose function is currently poorly understood, on the growth and development of muscle. Results from these experiments will also enable breeders to develop strategies for genetic improvement of animals which will yield higher quality meat at low cost. Climate change forecasts indicate that temperature extremes are increasing in frequency and intensity. Temperature extremes (hot and cold) have been shown to increase intramuscular fat accumulation and change breast muscle structure in poultry. Recent studies suggest that poultry selected for growth and muscle mass accretion are more sensitive to temperature extremes. Satellite cells, the only myonuclei source after hatch, are responsible for mediating posthatch hypertrophic growth. The objective of this study was to determine how temperature affects satellite cells from the pectoralis major muscle (breast muscle) of tur- keys previously selected for increased 16-wk body weight (F line) in comparison to an unselected Randombred Control line (RBC2) from which the F line was selected. Breast muscle satellite cells were cultured within a 5° range from 38°C to quantify both cold and hot temperature effects on proliferation, differentiation, and expression of myogenic regulatory factors: myogenic determination factor 1 (MYOD1) and myogenin. Proliferation significantly increased (P < 0 .05) in both lines at elevated temperatures (41° and 43°C) and decreased (P < 0 .05) at reduced temperatures (33°C, 35°C, and 37°C) compared with 38°C . Similarly, differentiation, the formation of multinucleated myotubes, significantly increased (P < 0 .05) at elevated temperatures (39°C and 41°C) and decreased (P < 0 .05) at reduced temperatures (33°C and 35°C) . Satellite cells isolated from F-line turkeys were more sensitive to both hot and cold temperatures as proliferation and differentiation increased at a greater rate when compared with the RBC2 line. Expression of MYOD1, required for proliferation, and myogenin, required for differentiation, increased at elevated temperatures and decreased at reduced temperatures compared with 38°C for both lines. These results demonstrate that satellite cell function is sensitive to both cold and hot temperatures and breast muscle satellite cells from the growth selected F line are more
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Clark, D.L., Coy, C.S., Strasburg, G.M., Reed, K.M., and Velleman, S.G. 2015. Influence of temperature and growth selection on turkey breast muscle cell proliferation, differentiation, and myogenic regulatory factor expression. Poultry Science 94: (E-Suppl. 1) page 104
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Progress 10/01/13 to 09/30/14
Outputs
Target Audience: The target audience comprises scientists working in the field of muscle growth and differentiation, as well as the broader areas of meat science, and meat animal growth and development Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project has provided employment for a lab assistant, Mr. Jon Weiss, who is working part time on the project before beginning medical school in fall 2015. In addition, a new M.S. graduate student, Galen George, has started on the project effective in August 2014. How have the results been disseminated to communities of interest? The results of the study have been communicated to the Journal of Animal Science. In addition, the goals of our newly funded project on effects of temperature variation on skeletal muscle growth and differentialn were communicated through two radio interviews (WTVN-AM in Columbus, Ohio, and KCRW-FM in Santa Monica, California) and a newspaper article syndicated internationally by the Los Angeles Times. What do you plan to do during the next reporting period to accomplish the goals? We are exposing muscle satellite cells from two different turkey genetic lines (RBC2 and F) from Ohio State University to various temperatures ranging from 31C to 43C. We are studying the effect of calcium ion concentration in these cells as a function of temperature to determine whether altered temperature alters the cytoplasmic [Ca2+], which in turn, could alter trancriptional rated. Complementing these studies, we will initiate studies that evaluate the effect of cold stress and heat stress during the first three days post mortem. Muscle tissues will be collected immediately following the research treatments and analyzed for RNA levels correspeonding to different genes. In addition, we will be treating another group of birds post hatch to determine the long term effect of thermal challenge on muscle structure and function in market age turkeys.
Impacts
What was accomplished under these goals?
The importance of muscle as a food is exemplified by the turkey meat processing industry. In the USA, there has been a continual increase in consumption of turkey meat from 4.9 pounds of boneless equivalent in 1960 to 14.4 pounds in 2002. Breast meat yield is the primary profit center for the commercial poultry industry. A predominant factor leading to this increase in consumer consumption is that poultry breast meat is regarded as the ideal lean meat for a healthy diet. This is coupled to its affordability and ease of preparation. A 1% increase in breast yield will amount to at least $75 million increase in revenues to the poultry industry. Commercial geneticists have placed a tremendous amount of selection pressure on breast yield and conformation, but not the mechanisms regulating the growth of muscle. The ability to regulate the growth and development of muscle depends on understanding the cellular regulatory mechanisms and cellular interactions that occur. Changes in muscle fiber size, extracellular areas available between the individual fibers and fiber bundles, and increased fat deposition will alter meat quality as have been illustrated by problems like pale, soft, and exudative turkey and pork. The results from this study will provide information on the role of specific genes whose function is currently poorly understood, on the growth and development of muscle. Results from these experiments will also enable breeders to develop strategies for genetic improvement of animals which will yield higher quality meat at low cost. Previous studies from our laboratory suggested that differential expression of genes between normal and pale, soft, and exudative (PSE) turkey is associated with development of the PSE syndrome. However, a detailed understanding of molecular mechanisms responsible for the development of this meat defect remains unclear. The objective of this study was to extend and complement our previous work by using deep transcriptome RNA sequence analysis to compare the respective transcriptome profiles and identify molecular mechanisms responsible for the etiology of PSE turkey meat. Turkey breasts (n = 43) were previously classified as normal or PSE using marinade uptake as an indicator of quality (high = normal; low = PSE). Total RNA from breast muscle samples with the highest (n = 4) and lowest (n = 4) marinade uptake were isolated and sequenced using the Illumina GA(IIX) platform. The results indicated differential expression of 494 loci (false discovery rate < 0.05). Changes in gene expression were confirmed using quantitative real-time PCR. Pathway analysis of differentially expressed genes suggested abnormalities of calcium homeostasis and signaling pathways regulating actin cytoskeleton structure as well as carbohydrate metabolism and energy production in PSE samples. Dysregulation of postmortem glucose oxidation in PSE turkey was suggested by both dramatic downregulation of pyruvate dehydrogenase kinase, isozyme 4 (PDK4) mRNA, the most downregulated gene, and a decrease in the protein product (P = 0.0007) as determined by immunoblot analysis. These results support the hypothesis that differential expression of several genes and their protein products contribute to development of PSE turkey meat. As the molecular basis for these differences becomes clear, our intent is to develop means for breeders to select turkeys that do not overexpress or downregulate specific genes that are associated with the PSE condition.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Malila Y, Carr KM, Ernst CW, Velleman SG, Reed KM, Strasburg GM. 2014. Deep transcriptome sequencing reveals differences in global gene expression between normal and pale, soft, exudative turkey meat. J. Anim. Sci. 92:1250-1260
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: The target audience comprises scientists working in the field of muscle growth and differentiation, as well as the broader areas of meat science, meat animal growth and development. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Support from this project partially supported a doctoral student, Yuwares Malila. How have the results been disseminated to communities of interest? Results from this project have been communicated through publications in the peer-reviewed scientific literature. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The importance of muscle as a food is exemplified by the turkey meat processing industry. In the USA, there has been a continual increase in consumption of turkey meat from 4.9 pounds of boneless equivalent in 1960 to 14.4 pounds in 2002. Breast meat yield is the primary profit center for the commercial poultry industry. A predominant factor leading to this increase in consumer consumption is that poultry breast meat is regarded as the ideal lean meat for a healthy diet. This is coupled to its affordability and ease of preparation. A 1% increase in breast yield will amount to at least $75 million increase in revenues to the poultry industry. Commercial geneticists have placed a tremendous amount of selection pressure on breast yield and conformation, but not the mechanisms regulating the growth of muscle. The ability to regulate the growth and development of muscle depends on understanding the cellular regulatory mechanisms and cellular interactions that occur. Changes in muscle fiber size, extracellular areas available between the individual fibers and fiber bundles, and increased fat deposition will alter meat quality as have been illustrated by problems like pale, soft, and exudative turkey and pork. The results from this study will provide information on the role of specific genes whose function is currently poorly understood, on the growth and development of muscle. Results from these experiments will enable breeders to develop strategies for genetic improvement of animals which will yield higher quality meat at low cost. In previous studies, we identified death-associated protein (DAP) as a candidate gene of interest for its potential role in turkey skeletal muscle development. Knockdown of DAP expression in cultured turkey satellite cells profoundly affected proliferation and differentiation. To investigate the mechanisms affected by DAP knockdown in proliferating and differentiating turkey satellite cells, small interfering RNA was used to knock down expression of DAP. Differential gene expression was determined using a turkey skeletal muscle long oligonucleotide microarray. Microarray data were validated using quantitative real-time PCR. In proliferating cells, a total of 397 genes showed differential expression (false discovery rate; FDR<0.05). Pathway analysis supports DAP involvement in mammalian target of rapamycin signaling. AKT1 and several eukaryotic translational initiation factors showed altered gene expression, suggesting DAP involvement in the regulation of protein synthesis. In differentiating cells, 175 genes showed differential expression (FDR<0.05). Pathway analysis at this stage identified DAP involvement in regulation of calcium signaling. Down-regulation of proteins involved in sarcoplasmic reticulum calcium flux and a majority of myofibrillar proteins suggests DAP may affect regulation of calcium homeostasis and muscle mass accumulation. This study provides the first evidence of global gene expression changes upon the knockdown of the DAP gene. In subsequent experiments, chicken satellite cells were transfected with DAP1 cloned into the pCMS-enhanced green fluorescent protein vector or pcDNA3.1 vector, or a small interference RNA against the endogenous DAP1 gene. The cells were assayed for proliferation, differentiation, and apoptosis. The overexpression of DAP1 increased proliferation, differentiation, and myotube diameter, but it had no effect on satellite cell apoptosis. In contrast, knockdown of DAP1 in chicken satellite cells significantly decreased proliferation, differentiation, and number of nuclei per myotube, and it increased apoptosis of the cells. These results suggest that DAP1 is required for regulating myogenesis and apoptosis of satellite cells, which may affect muscle mass accretion and regeneration, and ameliorate muscle sarcopenia.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Shin, J., McFarland, D. C., Strasburg, G. M. and Velleman, S. G. 2013. Function of death-associated protein 1 in proliferation, differentiation, and apoptosis of chicken satellite cells. Muscle Nerve. 48:777-790.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Our work over the past year focused on the second specific aim of the project, namely, to characterize the cellular and molecular basis of myogenesis. Outputs completed during the past year were focused on aims one and three. For the first specific aim, we continued our analysis of the role of death-associated protein-1 (DAP1) in the regulation of muscle growth and differentiation. We used Ingenuity Pathway Analysis to identify canonical pathways that were affected by knockdown of DAP1 gene expression. Subsequent experiments were focused on correlation of DAP1 and AKT1 expression with that of mRNA levels. The results of these studies were presented at the 2012 annual meeting of the Biophysical Society in San Diego, CA. A manuscript summarizing our findings is in preparation for submission to a peer-reviewed journal. In addition, a paper was published in Poultry Science earlier this year summarizing our findings from the previous year (2010-11) on the role of several novel genes including DAP1 in muscle growth and development. PARTICIPANTS: Gale Strasburg, Professor, Michigan State University. Project Director. Oversaw all aspects of projects, designed experiments, analyzed results, author/co-authored manuscripts and presentations. Sandra Velleman, Professor, Ohio State University. Project co-director. Contributed expertise in growth biology; designed satellite cell culture and knockdown experiments. Cathy Ernst, Professor, Michigan State University. Project co-director. Contributed expertise in functional genomics. Played a key role in analysis of microarray and RNA sequencing analysis. Kent Reed, Professor, University of Minnesota. Project co-director. Contributed expertise in the turkey genome, analysis of microarray data. Robert Tempelman, Professor, Michigan State University. Project co-director. Statistician; experimental design and analysis of microarray results. Kelly Sporer, Postdoctoral Associate, Michigan State University. Trained graduate students in laboratory techniques including microarrays and PCR, conducted microarray analysis of DAP1, versican, and MGP gene knockdown experiments. Katherine Stanchak Horton, Masters Student, Michigan State University. Conducted microarray experiments, analyzed data using Ingenuity Pathway Analysis, wrote manuscript and presented poster at Biophysical Society meeting. Lidia Nierobisz, Doctoral Student, North Carolina State University. Conducted microarray experiments on gene expression in muscles of different fiber types, analyzed data, primary author of manuscript. Chris Ashwell, Professor, North Carolina State University. Provided facilities for Nierobisz experiments and expertise and on-site training for microarray study. Paul Mozdziak, Professor, North Carolina State University. Served as mentor for Ms. Nierobisz, assisted experimental design, analysis of data and writing of manuscript. TARGET AUDIENCES: Our primary audience is the research community interested in growth and development of skeletal muscle Manuscripts were submitted for publication in peer-reviewed journals. Poster presentations of our most recent findings were made at the annual meeting of the Biophysical Society. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
From our previous studies, published in BMC Genomics in 2011, we undertook a focused series of studies to analyze the roles of 3 specific genes in muscle development. The genes were versican, Matrix-gla protein (MGP) and death-associated protein 1 (DAP1). These genes are differentially expressed at critical stages of development related to hyperplasia, hypertrophy, and market age of the turkey. Using small interfering RNA to knockdown the expression of these genes during proliferation and differentiation, each of the genes was found to differentially affect proliferation and differentiation. Versican and MGP predominantly affected proliferation with line effects, but later stages of differentiation were affected by the knockdown of versican and MGP. The underexpression of DAP inhibited myotube formation, which is a necessary stage in the development of muscle fibers. Without myotube development, muscle fiber formation will be inhibited or abolished. This is the first report that these genes with no previously documented functions with regard to muscle development play a critical role in muscle cell proliferation and differentiation.
Publications
- Velleman, S.G., Sporer, K.R.B., Ernst, C.W., Reed, K.M.,Stanchak, K., and Strasburg, G.M. 2012. Versican, Matrix-Gla Protein, and Death-associated Protein Expression Affect Muscle Satellite Cell Proliferation and Differentiation. 2012 Biophysical Society Meeting Abstracts, Biophysical Journal 102(3):512a
- Velleman, S.G., Sporer, K.R., Ernst, C.W., Reed, K.M.,and Strasburg, G.M. 2012. Versican, matrix Gla protein, and death-associated protein expression affect muscle satellite cell proliferation and differentiation. Poultry Science 91(8): 1964-1973.
- Nierobisz. L.S., Sporer, K.R., Strasburg, G.M., Reed, K.M., Velleman, S.G., Ashwell, C.M., Felts, J.V., and Mozdziak, P.E. 2012. Differential expression of genes characterizing myofibre phenotype. Animal Genetics 43(3): 298-308.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The focus of our work on this project is to characterize the cellular and molecular basis of myogenesis. We used small interfering RNA techniques to characterize the role of death-associated protein 1 in regulation of satellite cell proliferation and differentiation. Transfected cells were analyzed for changes in morphology, proliferation rate, and differentiation rate. RNA was isolated from muscle cells, amplifed to cDNA, and analyzed for differential gene expression. Bioinformatics software was used to identify pathways that were affected as a result of these knockdown experiments. An abstract of this work has been submitted for presentation at the annual meeting of the Biophysical Society in February 2012. This conference is regularly attended by a large segment of the community of muscle biology researchers. PARTICIPANTS: Dr. Gale Strasburg, Project Director, Michigan State University, overall supervision of project, design and analysis of microarray and PCR experiments. Dr. Sandra Velleman, collaborator, Ohio State University, conducted and analyzed satellite cell culture siRNA transfection experiments. Dr. Catherine Ernst,collaborator, Michigan State University, assisted in design and analysis microarray experiments. Dr. Robert Tempelman, collaborator, Michigan State University, statistician and microarray analysis. Dr. Kelly Sporer, postdoctoral research associate, project oversight and assisted in training graduate students in microarray analysis. Katherine Stanchak, graduate student, Michigan state University, trained in and conducted microarray experiments and bioinformatics analysis of data. TARGET AUDIENCES: Primary target audience for this work is the community of muscle biologists. The long-term goal is to develop a more comprehensive understanding of the role of specific muscle genes in growth and development which may then be utilized by the meat animal industry to improve efficiency of muscle growth and meat quality. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Approximately 100 genes exhibited differential expression in proliferating and differentiating satellite cells in culture. Bioinformatics analysis indicated that affected pathways include calcium signaling, skeletal muscle growth and development, and apoptosis.
Publications
- Sporer, K.R., Tempelman, R.J., Ernst, C.W., Reed, K.M., Velleman, S.G., and Strasburg, G.M. 2011. Transcriptional profiling identifies differentially expressed genes in developing turkey skeletal muscle. BMC Genomics 12:143.
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