NC131: Molecular Mechanisms Regulating Skeletal Muscle Growth and Differentiation

(Multistate Research Project)

Status: Inactive/Terminating

NC131: Molecular Mechanisms Regulating Skeletal Muscle Growth and Differentiation

Duration: 10/01/2000 to 09/30/2005

Administrative Advisor(s):

NIFA Reps:

Statement of Issues and Justification

This multistate research project is designed to meet the need for new, fundamental information about the molecular and cellular processes that regulate growth and differentiation of skeletal muscle in meat animals. Skeletal muscle, as meat, is the primary product of animal agriculture. Rational approaches for improving the efficiency of lean meat production and enhancing the quality of the final product rely implicitly on fundamental knowledge of the mechanisms that regulate growth of skeletal muscle tissue in the animal. Development of management and nutrition strategies, nutritional approaches, and genetic approaches to improving lean tissue growth all rely on a fundamental understanding of the molecular regulatory processes involved in skeletal muscle differentiation and growth.

JUSTIFICATION: This proposal describes a cooperative, multistate, multidisciplinary, basic research project with the overall goal of increasing the efficiency of lean meat production in domestic animals. This proposal for renewal of the NC-131 project describes the collaborative effort of 13 units and 24 principal investigators to characterize specific aspects of the molecular and cellular mechanisms that regulate skeletal muscle growth. Major points that support the continuation of this important, fundamental research projects for the next five years are:

  • The project relates to an important problem. Meeting consumer needs for a high quality product while maintaining profitability of production, decreasing environmental impacts, and minimizing use of natural resources will require improvements in the efficiency of meat production in domestic animals. These efforts rely directly on fundamental knowledge of the biological mechanisms that regulate muscle growth.
  • The project relates directly to identified national and regional agricultural research priorities:
    • Goal 1 of the CSREES National Program Goals is "To achieve an agricultural production system that is highly competitive in the global economy." Improving the efficiency of meat production in domestic animals, based on fundamental knowledge of the mechanisms of muscle growth, is directly related to this goal.
    • The crosscutting research areas established by the NCRA Committee in the Agricultural Production, Processing and Distribution area include "Develop improved animal, plant, and microbial production, production and marketing systems that are competitive, profitable and environmentally sound over the long term." This project provides the basic research necessary to achieve this goal.
    • The project supports current efforts to map and characterize genes important in animal production, by providing fundamental information about the regulation and expression of genes during muscle differentiation and growth.
  • The NC-131 Committee continues to be highly productive. Attachment III.B. lists 215 refereed papers, 57 book chapters/conference proceedings and 156 abstracts that have resulted from the first four years of the current five-year NC-131 project, and additional publications are expected by the September 30, 2000, termination date. Many of these papers are in high quality basic science journals, attesting to the quality of the work and justifying the basic approach of this project. As described in the Critical Review, substantial progress has been made under each of the specific objectives of the current project.
  • The NC-131 project is both a multistate and a multidisciplinary project, involving the effort of investigators at 11 different State Agricultural Experiment Stations and 2 USDA-ARS laboratories. The 24 Principal Investigators listed in Attachment I represent a variety of basic science disciplines that complement each other and provide the expertise necessary to complete the objectives.
  • The project-continues to involve a strong cooperative effort between the various units. Cooperative efforts during the current project have involved activities such as exchange of reagents, including well-characterized monoclonal and polyclonal antibodies and cDNA probes, sharing of knowledge and techniques, joint use of equipment and techniques available at particular stations, and joint publication of research results. Numerous collaborative projects are described in the procedures for the proposed revision. The committee feels strongly that the collaborations in this project would have been substantially more difficult to establish and maintain outside the framework of a funded regional project.
  • The members of the NC-131 committee have been highly successful in obtaining outside support to fund the research. Funding from the USDA NRICGP Program, NIH, NSF, and industry sources has been essential to carry out the work and to maintain the high level of productivity of the group, and this record of outside funding is expected to continue.

In summary, this project describes a fundamental research approach to an important agricultural problem. The investigators at the cooperating stations have demonstrated a high level of productivity, and are, thus, capable of making substantial progress towards the objectives outlined in this revised project proposal.

Related, Current and Previous Work

There is no duplication between this project and any other funded multistate (regional) project. Searching the CRIS Multi-State Projects database using the keywords "muscle and (growth or differentiation or development)" retrieved only a single project - NC-131 Searching this database with the single keyword "growth" yielded only 8 projects: NC-131, 6 projects dealing with plants, and a poultry project of limited scope (SR-DC-97-7), about which no information was available. Thus, both the current and the proposed NC-131 projects are unique among currently funded multistate projects. The only related Regional project is NE-148 "Regulation of Nutrient Use in Food Producing Animals," and there is little real overlap, as NE-148 deals primarily with nutrient use and hormonal regulation, does not focus on skeletal muscle and does not focus on understanding the molecular and cellular mechanisms regulating muscle growth. Funded projects dealing with identification of growth-related genes as part of gene mapping efforts do exist, but these do not focus on regulation. Thus, there is no duplication between this project and other multistate/regional projects. Muscle development is a major area of medical research in relation to human muscle diseases. However, the emphasis of this project is on studies related to muscle growth in domestic animals, rather than human disease, and every attempt has been made to identify areas in which the NC-131 committee can make important contributions to basic knowledge of muscle growth without duplicating efforts of the medical research community. Medical research organizations (NIH, MDA, AHA, etc.) are, however important sources of funding to support the research proposed by NC-131 project leaders.

The remainder of this section is a brief review of the current state of research in the area of molecular regulation of muscle development. This is not a comprehensive review, but rather a brief summary intended to describe our current knowledge and to point out those areas in which research by the NC-131 committee can have a major impact. Also, because the NC-131 committee has been working productively in this area under the current project, additional relevant information and references can be found in the Critical Review (Attachment III).

The major steps in embryonic differentiation of skeletal muscle in animals are known (see [76, 81,97] for review). Precursors cells arising from embryonic mesoderm undergo a sequence of patterning and commitment steps to produce populations of myoblasts in the somites, which then give rise to the various adult muscles [13, 20]. Terminal myoblast differentiation involves withdrawal from- the cell cycle, fusion to form myotubes, and expression of muscle-specific proteins [17, 56]. Myotubes then mature into muscle fibers. The number of fibers in a muscle is determined prenatally, while postnatal growth involves fiber hypertrophy with additional nuclei contributed by satellite cells [10]. The overall process is regulated in a complex manner by extracellular signals that activate intracellular signaling pathways leading to expression of specific genes at each step [2, 13]. Postnatal growth also involves synthesis, assembly and turnover of myofibrillar proteins. Thus, the three objectives of this proposal correspond to three major processes involved in muscle differentiation and growth, namely: control by extracellular factors; expression of specific genes; and assembly and turnover of myofibrillar proteins.

Myogenesis is regulated by signaling pathways that are initiated by binding of growth factors (GFs) or hormones to receptors [12, 24, 75, 92]. The GFs known to affect myogenesis include the insulin-like growth factors (IGFs) [7, 23, 30, 42, 51, 82], fibroblast growth factor (FGF-2 and FGF-6) [25, 31, 40, 50, 70, 94], platelet-derived growth factor (PDGF) [47, 93, 98], transforming growth factor-? (TGF-P) [14, 41, 51], myostatin [53, 62, 79] and hepatocyte growth factor (HGF) [1, 33, 84]. One example demonstrating the importance of GFs in regulation of muscle growth in meat animals is the discovery that a deletion in the myostatin gene is responsible for double muscling in cattle [37, 63]. The in vivo activity of GFs is affected by several variables. For example, the bioactivity of the IGFs in muscle is dramatically affected by nutritional status [88], the level of IGF in muscle tissue [7, 35, 49], presence of IGF receptors [71, 72] and interaction with a family of IGF binding proteins (IGFBPs) [9, 21, 22]. Many cell types, including myogenic cells and fibroblasts, can synthesize specific GFs, and muscle tissue contains mRNAs for IGFs [35, 49], IGFBPs [48, 61, 74, 96], FGF-6 [38], myostatin [62] and TGF-P [14]. Thus, GFs can act locally to regulate proliferation and differentiation of myogenic cells. Additional research is needed to characterize the function and expression of specific GFs in muscle and to define their endocrine, autocrine, and/or paracrine actions in regulating muscle growth in meat-producing animals.

In addition to GFs, the extracellular matrix (ECM) also has a profound influence on the differentiation and proliferation of myogenic cells [15, 28, 64]. The extracellular matrix may serve as a storage site for growth-regulating peptides, including FGF [19, 26, 60, 91], TGF-P [32, 67, 87], and the IGFBPs [52]. The nonaggregating proteoglycan decorin binds to TGF-P and suppresses its activity [95], and furthermore, TGF-P increases the expression of the decorin core protein and alters the size of the attached glycosaminoglycan side chains [8]. Thus, ECM can regulate GF function in skeletal muscle, but additional studies are needed to determine the extent to which the ECM itself regulates muscle growth and differentiation.

Each step in myogenesis involves specific changes in gene expression. Known early genes involved in patterning include those of the Hox and Pax gene families' [13]. Terminal myogenic differentiation is characterized by expression of four transcription factors that are members of the myogenic determination factor (MDF) family [4, 59, 65, 97], myogenin, MyoD, Myf-5 and MRF4. Each of these basic helix-loop-helix (bHLH) family members can activate myogenesis in nonmuscle cells, but analysis of embryonic expression and knockout studies show that each has a unique function in myogenesis [3, 90]. Other factors, such as pRb and p21, are involved in the inhibition of cell division that occurs simultaneously with terminal myogenesis [68, 89]. Additional transcription factors, particularly the MEF2 family [11, 66] cooperate with the MDFs to activate muscle-specific gene transcription. Action of MDFs also can be inhibited by factors such as the Id proteins [46] and the newly discovered inhibitory factor MyoR [57], to prevent myogenesis when or where it is not appropriate. Myogenesis is followed by fiber hypertrophy and maturation to yield adult muscle fiber types [77]. Fiber-type specialization is important to this project, as there is a significant correlation between fiber-type proportions and muscle growth and meat quality [16, 29], but little is known about how this process is regulated.

Our current knowledge is based primarily on studies in mice or chickens. Additional studies are needed to examine gene expression during myogenesis in livestock animals. Furthermore, much less is known about gene expression during growth, and thus, analysis of muscle-specific gene expression in hypertrophy models will provide new information that can be used to manipulate growth in meat animals. Studies are also needed to examine muscle-specific gene regulation during maturation, to provide information about control of fiber-type determination.

Gene expression during differentiation and growth in response to extracellular signals is mediated by cell signaling pathways. Sequential expression not only of transcription factors, but of signaling molecules such as receptors, protein kinases, and phosphatases is characteristic of the entire myogenic pathway [13]. For example, signaling molecules of the Sonic hedgehog, Wnt, and BMP families are key factors in early myogenesis, and are needed for MDF expression [13]. Although characterization of signal transduction pathways for GF responses is an active area of current study, additional information is needed to understand how GFs regulate myogenesis. In addition, the calpains, Ca2+-activated proteases that have been studied as part of NC-131 for several years, also appears to be involved in signaling during myogenesis. Several experiments have shown that the calpain system is involved in myoblast fusion [5, 6, 85], and calpains can degrade transcription factors such as c-fos and c-jun [18, 44]. However, the role of calpains in signaling during myogenesis is not known. ADP-ribosylation also appears to be involved in myogenesis [54], but the specific role of this protein modification is not understood.

Muscle growth involves synthesis and turnover of muscle proteins. Myofibrils constitute most of the muscle cell protein, and are responsible for major eating qualities of meat. Assembly of newly synthesized proteins into myofibrils, their integration into the cytoskeletal apparatus of the muscle cell, and their continual turnover are, thus, important aspects of muscle growth. Current models [69, 73] of myofibrillogenesis suggest that the initial steps involve coalescence of the proteins vinculin, a-actinin, and a-actin at the cell membrane, in structures similar to the costameres of adult muscle [58, 86]. This complex is attached to stress-fiber-like structures or premyofibrils consisting of actin filament bundles and non-muscle myosin [73]. Premyofibrils transform into nascent myofibrils and then into fully formed myofibrils, which become aligned and integrated into the overall muscle cytoskeleton [73]. The giant protein titin appears to be a major participant, possibly as a scaffold for assembly [36, 39, 73, 86]. However, many details, such as the nature of the many protein interactions involved, remain to be characterized.

Turnover requires either exchange of myofibrillar proteins with soluble pools or disassembly of myofibrils, or possibly both, depending on the metabolic state and health of the muscle. Both disassembly and further degradation of myofibrils involves participation of specific proteases. Three major proteolytic systems have been identified in muscle, namely: (1) cathepsins, located in lysosomes and/or the sarcoplasmic reticulum [78]; (2) the calpain system [80]; and (3) the ATP and ubiquitin dependent proteosome [43]. The cathepsin system appears to have only a minor role in normal myofibrillar protein turnover [27, 83]. On the other hand, the calpain system is thought to have a major role in turnover of myofibrillar protein [34, 45, 55]. This system consists of two proteases, u- and m-calpains, which differ in their Ca2+ requirements, and their biological inhibitor, calpastatin. Studies on the calpain system have been a major part of studies done as part ofNC-131, as noted above and thus, the proposed studies in this project will focus primarily on the calpains. Additional study is needed to determine how muscle cells segregate the proteins of the calpain system, and control their respective activities.

While this brief review cannot cover all of the hundreds of papers related to muscle growth and differentiation, several major areas where additional experimental work is needed have been noted. These areas are the focus of the experimental work to be done under this proposal.


  1. Characterize extracellular signaling mechanisms that regulate skeletal muscle growth and differentiation.
  2. Determine molecular mechanisms that control gene expression in skeletal muscle.
  3. Characterize mechanisms of cytoskeletal protein assembly and degradation in skeletal muscle.


The objectives for this revised project are similar to those for the currently active NC-131 project, and they remain broadly defined to allow for changes in the direction of the work that will likely occur based on results of current studies both within and outside of the project. The procedures have been substantially updated to reflect the current state of knowledge, and specific approaches are based on the expertise available within the participating units. Because of page limitations, detailed methods are not described. Documentation regarding methods and the expertise needed to complete the studies can be found in the Critical Review (Attachment III).

Procedures for Objective 1 - Characterize extracellular signaling mechanisms that regulate skeletal muscle growth and differentiation. Work will be focused in two areas: (a) Characterize the function and expression of growth factors, receptors and binding proteins involved in signaling pathways that influence skeletal muscle development; and (b) Determine the role of the extracellular matrix in signaling pathways in developing muscle. Units responsible for this objective are the Arizona, Indiana, Michigan, Minnesota, Ohio, South Dakota, Utah, and Washington Stations and the USDA/ARS Fargo Biosciences Research Center.

a. Characterize the function and expression of growth factors, receptors and binding proteins involved in signaling pathways that influence skeletal muscle development. This area represents a major portion of the studies proposed in this project. Several experimental models, most of which were developed as part of NC-131, will be used to examine the mechanisms of autocrine and paracrine control of myogenesis. Comparison of results obtained using each of these models will provide important insights into general mechanisms of myogenic regulation.

Studies at the Arizona Station will utilize the rat satellite cell culture system developed by this group to examine the mechanism of action of HGF and IGF specifically on satellite cell activation. Analysis of the role of HGF will include: (1) Characterization of HGF synthesis, secretion, storage and processing, using immunochemical analysis of HGF, RT-PCR to quantitate mRNA; and protease inhibitors to analyze processing; (2) Use of specific HGF neutralizing antibodies in the presence or absence of exogenous HGF to examine potential autocrine effects; (3) Analysis of the ability of IGF, FGF and TGF-(3 to modulate the HGF effect; and (4) Determination of signaling pathways that mediate HGF action by use of specific kinase inhibitors. Studies on the role of IGF in satellite cell activation will involve: (1) Characterization of the expression patterns of IGF-I and II and the Type-1 IGF receptor during activation; (2) determination of when satellite cells first become responsive to IGFs; and (3) examination of the effects of blocking IGF receptors with specific antibodies on activation, to determine if specific events in activation require IGF. In addition, these investigators will examine the role of GFs in satellite cell activation and muscle regeneration in vivo, by use of a muscle injury model.

Investigators at the Michigan station will characterize developmental changes in the proportion of proliferating and differentiating cells in porcine and bovine satellite cell cultures by use of immunofluorescence detection of markers for these two classes of myogenic cells. This method will also be used to examine satellite cell responses to exogenous growth promotants. In collaboration with investigators at the Minnesota Station, changes in satellite cell activity will be correlated with changes in muscle GF expression, allowing determination of when satellite cell activity declines developmentally and providing information about mechanisms regulating this decline. This information will be used to devise strategies to stimulate satellite cell activity at appropriate times during development to optimize skeletal muscle growth.

Studies at the Minnesota Station will utilize a porcine myogenic cell culture model to analyze the role of IGF-binding protein-3 (IGFBP-3) in regulation of myogenesis. Results from the current project have shown that differentiation of the porcine myogenic cells is accompanied by a transient decrease in production of IGFBP-3. Four series of experiments will be used to determine the function of this differentiation-associated reduction in IGFBP-3 levels. First, recombinant porcine IGFBP-3 will be used to analyze the effects of IGFBP-3 on the cultures, to analyze binding of IGFBP-3 to specific cell receptors, and to produce antibodies to IGFBP-3 that can be used to alter IGFBP-3 function. Second, antisense oligonucleotides will be used to inhibit IGFBP-3 synthesis in myogenic cell cultures, to determine if inhibiting expression alters differentiation. Third, to complement the antisense inhibition studies, IGFBP-3 will be over- expressed in myogenic cells, using the ecdysone-regulated promotor system. These studies will utilize L6 myoblasts, because this cell line does not normally synthesize IGFBP-3. Finally, receptors for IGFBP-3 cells will be characterized. In combination, these approaches will allow determination of the role of IGFBP-3 in myogenic differentiation.

In collaboration with the South Dakota Station, investigators at the Minnesota Station will also examine the role of satellite cells and IGF-I in anabolic-steroid-induced muscle growth in cattle. Preliminary studies suggest that an increase in number of activated satellite cells and muscle IGF-I levels may be involved in the enhanced muscle growth of steers implanted with a combined trenbolone acetate and estradiol implant. Thus, the time course of the steroid-induced increases in activated satellite cells, serum IGF-I, liver IGF-I mRNA, and levels of muscle IGF-I, HGF, FGF-2 and FGF-6 mRNA will be analyzed to determine the mechanism for the increase in activated satellite cells. Effects of the steroids and IGFs, FGF-2, HGF on isolated satellite cells from implanted and control steers also will be determined, to provide direct information on the effects of these compounds on satellite cells. These studies will provide significant new information about mechanisms of anabolic steroid action and satellite cell activation.

Investigators at the Washington Station will examine the role of interactions among the different cell types found in muscle tissue in the regulation of muscle development and growth. To address the hypothesis that the immune system is a major contributor to the regeneration of muscle tissue after exercise or injury, satellite cells will be co-cultured with leukocytes activated with various mitogens that stimulate different populations of cytokines, and the growth and differentiation of the satellite cells analyzed. In addition, leukocyte-conditioned media will be characterized by SDS-PAGE, chromatography, and antibody inhibition studies to identify the presence of known cytokines. Expression of cytokines and GFs will be analyzed by immuno- blotting, PCR, ELISA, or bioassay. The sheep preadipocyte/myogenic cell co-culture system developed at this Station will be used to define factors released by muscle cells that regulate fat cell development and to address the hypothesis that fat cells from muscle produce leptin that operates on other cells of muscle to regulate growth responses. To address the hypothesis that specific satellite cell populations are involved in specific periods of muscle growth, investigators at the Washington Station will continue to isolate and characterize satellite cells from all types of meat animals and to treat them with GFs and cytokines to determine if a developmental switch occurs in the ability of the cells to respond to specific anabolic agents. Finally, studies will be done to investigate the mechanisms of muscle atrophy in older animals, by examining the changes in responses to growth factors that occur with aging.

Investigators at the Utah Station will analyze satellite cells from lambs with the callipyge mutation, which results in extreme muscling, particularly in the hindquarters. Determination of the mechanisms that cause this muscle hypertrophy will provide new information about muscle growth regulation. Studies will focus on the role of satellite cells, because of their importance in muscle growth and because muscle hypertrophy in callipyge lambs is not seen until after birth. Experiments will include: (1) Determination of the effects of satellite cell genotype, muscle type, and serum type on properties of cultured satellite cells; (2) Isolation of representative clonal satellite cell lines and determination of effects of genotype and muscle type on the profile of satellite cell colonies and on properties of individual clonal lines; (3) Analysis of the composition of serum from callipyge and normal lambs and investigation of the effects of each serum on myoblast and fibroblast cell cultures; (4) Analysis of the effects of genotype on muscle histochemistry and on properties of satellite cells isolated from newborn lambs. In addition to providing new information about the role of satellite cells in callipyge hypertrophy, these studies will compliment the analysis of muscle gene expression in callipyge lambs to be done under objective 2.a of this proposal. Satellite cell analysis will be done in collaboration with the Washington, Michigan, and South Dakota Stations.

The South Dakota Station and the USDA/ARS-Fargo Research Center will collaborate to examine the role of p-adrenergic agonists in skeletal muscle development. These studies will utilize clonal avian satellite cell lines isolated at the South Dakota Station. Effects of P-agonists on proliferation and differentiation of myogenic cells will be investigated using enzyme-linked immunoculture assay (ELICA). Expression of proteins associated with the cytoskeleton (e.g., desmin), proliferation (proliferating cell nuclear antigen) and differentiation (sarcomeric myosin) will be evaluated when cells are grown with and without P-agonists.

Investigators at the Indiana Station will use three approaches to examine the roles of GFs in regulation of muscle development in whole animals. First, direct DNA injection will be used to obtain local production of recombinant, epitope-tagged IGF-I in porcine skeletal muscle, to examine the paracrine/autocrine roles of IGF-I in muscle growth. Effects of IGF-I on muscle fiber types will be examined by analysis of myosin heavy chain isoform expression. Second, myoblast-mediated gene transfer will be used in a compensatory muscle hypertrophy model in pigs, allowing implantation of myoblasts into muscle undergoing hypertrophy. Use of this model should facilitate fusion of implanted myoblasts with myofibers, especially if muscles are subjected to irradiation prior to cell implantation. The third approach will involve the use of transgenic mice that express GFs in a tissue-specific manner. In combination, these approaches will be useful in delineating the role of GFs in muscle development and will complement the cell culture studies, to provide a general picture of myogenic regulation by GFs.

b. Determine the role of the extracellular matrix in signal transduction pathways in developing muscle. Because the extracellular matrix (ECM) is involved in the regulation of muscle development and growth, investigators at the Ohio and South Dakota Stations and the USDA- Fargo Research Center will examine selected aspects of the role of ECM.

The Ohio Station, in collaboration with the South Dakota Station, will continue to investigate how proteoglycan expression is related to growth of skeletal muscle in a turkey line selected for increased 16 week body weight in comparison to the unselected control line. Research will focus on heparan sulfate proteoglycan expression in satellite cells derived from both lines of turkeys. In vivo studies will be done to determine the developmental pattern of expression of the heparan sulfate proteoglycans from 14 days of embryonic development through 16 weeks posthatch, by RNA analysis, detection of protein by ELISA and immunoblotting, and immunocytochemical localization. Additional studies at the Ohio Station will be focused on examination of cell-matrix interactions during muscle differentiation, utilizing the avian genetic muscle weakness model. Low Score Normal. The Low Score Normal birds exhibit decreased P 1 integrin expression. Integrins are transmembrane proteins that act as receptors for ECM, and the PI integrin subunit, in particular, links ECM to the cell cytoskeleton. The effect of this weakness on myosin filament assembly and isoform expression will be determined using myosin isoform-specific antibodies obtained from the California Station, to provide new information about sarcomere assembly in a model with altered integrin expression.

Studies at the South Dakota Station will focus on elucidating the role of sarcoglycans in satellite cell and myotube function. For these studies, satellite cells will be isolated and cloned from the Bio 14.6 muscular dystrophic hamster, which possesses a defect in the delta- sarcoglycan gene, and from normal control hamsters. Because administration of thyroid hormone to the dystrophic hamster has been shown to improve the contractile properties of the diaphragm muscle, experiments will include examination of the role of thyroid hormone in satellite cell and myotube physiology using this cell system. Preliminary studies have also shown that the morphology of myotubes derived from normal and dystrophic satellite cells differs greatly. In collaboration with the USDA-Fargo Center, the possible role of soluble mediators, including GFs and thyroid hormone, on myotube morphology and biochemical function will be examined. Immunochemical procedures will be used to measure and identify ECM protein elaboration by satellite cell and myotube cultures. The influence of secreted soluble factors on dystrophic and control satellite cell and myotube physiology will be examined using a co-culture system, using cells grown on permeable filter inserts, to allow for collection and analyses of secreted factors that may affect the functioning of cells grown in the lower chamber. In combination, these studies will provide new information about the function of specific ECM molecules in myogenic regulation.

See attached for continuation of methods.

Measurement of Progress and Results


Outcomes or Projected Impacts

  • Completion of these proposed studies will provide major advances in our knowledge of the molecular mechanisms that regulate muscle growth - advances that can be directly applied to improve efficiency of lean meat production in domestic animals. Dissemination of the results of the research will be done via the timely publication of refereed articles in established journals. Because this a basic research project, publications will be the primary tangible result expected from the project. It is anticipated that utilization of these published results will lead to improved efficiency of lean meat production in domestic livestock and poultry. To enhance dissemination of the research resulting from this project. Dr. Brad Johnson, an extension ruminant nutritionist and beef feedlot specialist at the South Dakota Station, has been added to the NC-131 committee. Dr. Johnson has had extensive experience in the study of basic mechanisms of skeletal muscle growth, and he will serve as liaison for the outreach role of the committee.



Projected Participation

View Appendix E: Participation

Outreach Plan


The Technical Committee will consist of at least one representative from each participating unit, appointed as described in the Manual for Cooperative Regional Research, with one representative from each unit designated as the voting member. The North Central Regional Association of Directors will appoint one Director to serve as Administrative Advisor and a representative of the USDA/CSREES will also serve as an Advisor; both will be ex officio member of this committee. The Technical Committee will elect a Chair and a Secretary to serve for a period of one year, and these officers will continue to be voting members. In succeeding years, the Secretary will become Chair, and only a new Secretary will be elected. The Chair, the Secretary, the immediate Past Chair and the Administrative Advisor will serve as the Executive Committee, and they will have the authority to act on behalf of the Technical Committee during the periods between meetings. Because this is a proposal for renewal of the NC-131 project, the current Chair will serve as Past Chair for the first year of the revised project and the current Secretary will become Chair if the renewal is approved.

The Chair, with approval of the Administrative Advisor, will call yearly meetings of the Technical Committee and interim meetings of the Executive Committee if needed. A report of research results from each unit will be presented orally and in writing at the Technical Committee meetings. The written report should include a list of publications resulting from research related to the project for the year. Reports will be critically reviewed by the Technical Committee and recommendations will be made for future research and coordination of research between units to maximize attainment of the Objectives. The Secretary will prepare minutes and an Annual Report for distribution to Technical Committee members, Directors and Department Chairs of participating State Agricultural Experiment Stations and USDA Laboratories, and the Regional Research Office, CSREES. Because submission of a yearly progress report is essential to assess progress, ensure participation, and prepare the Annual Report, any unit that does not submit a written progress report for two consecutive years will be considered not to be participating and will be dropped from the project.

A station, agency, or institution not currently listed as a participant in the project can petition to join by submitting an addendum to the Procedures section of this proposal. This addendum should describe the proposed work and how it contributes to the project. The Administrative Advisor will circulate the addendum to the voting members of the Technical Committee for their consideration and approval. If approved by a majority of the voting members of the Technical Committee, the addendum will be added to the official project outline.

Officers for the final year of the current NC-131 project are:

Chair: Michael V. Dodson, Washington State University

Secretary: William R. Dayton, University of Minnesota

Past Chair: Sandra G. Velleman, The Ohio State University

Administrative Advisor: Eton Aberle, University of Wisconsin

CSREES Advisor: Larry R. Miller, USDA/CSREES

Literature Cited

  • Alien, R. E., S. M. Sheehan, R. G. Taylor, T. L. Kendall, and G. M. Rice. 1995. Hepatocyte growth factor activates quiescent skeletal muscle satellite cells in vitro. J Cell Physio! 165:307-12.
  • Arnold, H. H., and T. Braun. 2000. Genetics of muscle determination and develop. Curr. Top. Develop. Biol. 48:155-65.
  • Arnold, H. H., and T. Braun. 1996. Targeted inactivation of myogenic factor genes reveals their roles during myogenesis: a review. Int. Dev. Biol. 40:345-363.
  • Arnold, H. H., and B. Winter. 1998. Muscle differentiation: more complexity to the network of myogenic regulators. Curr. Opin. Genet. Dev. 8:539-44.
  • Balcerzak, D., P. Cottin, S. Poussard, A. Cucuron, J. J. Brustis, and A. Ducastaing. 1998. Calpastatin-modulation of m-calpain activity is required for myoblast fusion Eur J Cell Biol. 75:247-53.
  • Bamoy, S., T. Glaser, and N. S. Kosower. 1998. The calpain-calpastatin system and protein degradation in fusing myoblasts. Biochim. Biophys. Acta 1402:52-60.
  • Barton-Davis, E. R., D. I. Shoturma, A. Musaro, N. Rosenthal, and H. L. Sweeney. 1998. Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function. Proc. Natl. Acad. Sci. USA 95:15603-7.
  • Bassols, A., and J. Massague. 1988. Transforming growth factor beta regulates the expression and structure of extracellular matrix chondroitin/dermatan sulfate proteoglycans J. Biol. Chem. 263:3039-45.
  • Binoux, M. 1996. Insulin-like growth factor binding proteins (IGFBPs): physiological and clinical implications. J. Pediatr. Endocrinol. Metab. 9 (Suppi 3):285-8.
  • Bischoff, R. 1994. The satellite cell and muscle regeneration, p. 97-118. In A. G. Engel, and C. Franzini-Armstrong (ed.), Myology. McGraw-Hill, New York.
  • Black, B. L., and E. N. Olson. 1998. Transcriptional control of muscle develop. by myocyte enhancer factor-2 (MEF2) proteins. Annu. Rev. Cell Dev. Biol. 14:167-96.
  • Blakesley, V. A., A. Scrimgeour, D. Esposito, and D. Le Roith. 1996. Signaling via the insulin-like growth factor-I receptor: does it differ from insulin receptor signaling? Cytokine Growth Factor Rev. 1:153-9.
  • Borycki, A.-G., and C. P. Emerson. 2000. Multiple tissue interactions and signal transduction pathways control somite myogenesis. Curr. Top. Dev. Biol. 48:165-224.
  • Bosche, W. J., D. Z. Ewton, and J. R. Florini. 1995. Transforming growth factor-beta isoform expression in insulin-like growth factor stimulated myogenesis. J Cell Physiol 164:324-33.
  • Brandan, E., M. E. Fuentes, and W. Andrade. 1991. The proteoglycan decorin is synthesized and secreted by differentiated myotubes. Eur. J. Cell Biol. 55:209-16.
  • Brocks, L., B. Hulsegge, and G. Merkus. 1998. Histochemical characteristics in relation to meat quality properties in the Longissimus Limborus of fast and lean growing lines of Large White pigs. Meat Sci. 50:411-20.
  • Buckingham, M. E. 1994. Muscle: the regulation of myogenesis. Curr. Opin Genet Dev 4:745-51.
  • Carillo, S., M. Pariat, A. M. Steff, P. Roux, M. Etienne-Julan, T. Lorca, and M. Piechaczyk. 1994. Differential sensitivity of FOS and JUN family members to calpains Oncosene 9:1679-89.
  • Chintala, S. K., R. R. Miller, and C. A. McDevitt. 1994. Basic fibroblast growth factor binds to heparan sulfate in the extracellular matrix of rat growth plate chondrocytes. Arch Biochem. Biophys. 310:180-6.
  • Christ, B., and C. P. Ordahl. 1995. Early stages of chick somite development. Anat. Embryol. 191:381-96.
  • Clemmons, D. R. 1997. Insulin-like growth factor binding proteins and their role in controlling IGF actions. Cytokine Growth Factor Rev. 8:45-62.
  • Clemmons, D. R., W. Busby, J. B. Clarke, A. Parker, C. Duan, and T. J. Nam. 1998. Modifications of insulin-like growth factor binding proteins and their role in controlling IGF actions. Endocr. J. 45 (Suppl):Sl-8.
  • Coleman, M. E., F. DeMayo, K. C. Yin, H. M. Lee, R. Geske, C. Montgomery, and R. J. Schwartz. 1995. Myogenic vector expression of insulin-like growth factor I stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice. J. Biol. Chem. 270:12109-16.
  • Coolican, S. A., D. S. Samuel, D. Z. Ewton, F. J. McWade, and J. R. Florini. 1997. The mitogenic and myogenic actions of insulin-like growth factors utilize distinct signaling pathways. J. Biol. Chem. 272:6653-62.
  • Crisona, N. J., K. D. Alien, and R. C. Strohman. 1998. Muscle satellite cells from dystrophic (mdx) mice have elevated levels of heparan sulphate proteoglycan receptors for fibroblast growth factor. J. Muscle Res. Cell Motil. 19:43-51.
  • DiMario, J., N. Buffinger, S. Yamada, and R. C. Strohman. 1989. Fibroblast growth factor in the extracellular matrix of dystrophic (mdx) mouse muscle. Science 244:688-90.
  • Fernandez, C., and R. D. Sainz. 1997. Pathways of protein degradation in L6 myotubes. Proc. Soc. Exp. Biol. Med. 214:242-7.
  • Femandez, M. S., J. E. Dennis, R. F. Drushel, D. A. Carrino, K. Kimata, M. Yamagata, and A. I. Caplan. 1991. The dynamics of compartmentalization of embryonic muscle by extracellular matrix molecules. Dev. Biol. 147:46-61.
  • Fiedler, I., K. Ender, M. Wicke, S. Maak, G. V. Lengerken, and W. Meyer. 1999. Structural and functional characteristics of muscle fibres in pigs with different malignant hyperthermia susceptibility (MRS) and different meat quality. Meat Sci. 53:9-15.
  • Florini, J. R., D. Z. Ewton, and S. A. Coolican. 1996. Growth hormone and the insulin-like growth factor system in myogenesis. Endocr. Rev. 17:481-517.
  • Floss, T., H. H. Arnold, and T. Braun. 1997. A role for FGF-6 in skeletal muscle regeneration. Genes Dev. 11:2040-51.
  • Fukushima, D., R. Butzow, A. Hildebrand, and E. Ruoslahti. 1993. Localization of transforming growth factor beta binding site in betaglycan. Comparison with small extracellular matrix proteogly cans. J. Biol. Chem. 268:22710-5.
  • Gal-Levi, R., Y. Leshem, S. Aoki, T. Nakamura, and 0. Halevy. 1998. Hepatocyte growth factor plays a dual role in regulating skeletal muscle satellite cell proliferation and differentiation. Biochim. Biophys. Acta 1402:39-51.
  • Goll, D. E., V. F. Thompson, R. G. Taylor, and J. A. Christiansen. 1992. Role of the calpain system in muscle growth. Biochimie 74:225-37.
  • Grant, A. L., W. G. Helferich, S. A. Kramer, R. A. Merkel, and W. G. Bergen. 1991. Administration of growth hormone to pigs alters the relative amount of insulin-like growth factor-I mRNA in liver and skeletal muscle. J. Endocrinol. 130:331-8.
  • Gregorio, C. C., H. Granzier, H. Sorimachi, and S. Labeit. 1999. Muscle assembly: a titanic achievement? Curr. Opin. Cell Biol. 11:18-25.
  • Grobet, L., L. J. Martin, D. Poncelet, D. Pirottin, B. Brouwers, J. Riquet, A. Schoeberlein, S. Dunner, F. Menissier, J. Massabanda, R. Fries, R. Hanset, and M. Georges. 1997. A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat. Genet. 17:71-4.
  • Han, J. K., and G. R. Martin. 1993. Embryonic expression of Fgf-6 is restricted to the skeletal muscle lineage. Dev. Biol. 158:549-54.
  • Handel, S. E., S. M. Wang, M. L. Greaser, E. Schultz, J. C. Bulinski, and J. L. Lessard. 1989. Skeletal muscle myofibrillogenesis as revealed with a monoclonal antibody to titin in combination with detection of the alpha- and gamma- isoforms ofactin. Dev. Biol. 132:35-44.
  • Hannon, K., A. J. Kudia, M. J. McAvoy, K. L. Clase, and B. B. Olwin. 1996. Differentially expressed fibroblast growth factors regulate skeletal muscle development through autocrine and paracrine mechanisms. J. Cell Biol. 132:1151-9.
  • Hathaway, M. R., J. R. Hembree, M. S. Pampusch, and W. R. Dayton. 1991. Effect of transforming growth factor beta-1 on ovine satellite cell proliferation and fusion. J. Cell. Physiol. 146:435-41.
  • Hembree, J. R., M. R. Hathaway, and W. R. Dayton. 1991. Isolation and culture of fetal porcine myogenic cells and the effect of insulin, IGF-I, and sera on protein turnover in porcine myotube cultures. J. Anim. Sci. 69:3241-50.
  • Hershko, A., and A. Ciechanover. 1998. The ubiquitin system. Annu. Rev. Biochem. 67:425-79.
  • Hirai, S., H. Kawasaki, M. Yaniv, and K. Suzuki. 1991. Degradation of transcription factors, c-Jun and c-Fos, by calpain. FEBS Lett. 287:57-61.
  • Huang, J., and N. E. Forsberg. 1998. Role of calpain in skeletal-muscle protein degradation. Proc. Natl. Acad. Sci. USA 95:12100-5.
  • Jen, Y., H. Weintraub, and R. Benezra. 1992. Overexpression of Id protein inhibits the muscle differentiation program: in vivo association of Id with E2A proteins. Genes Dev. 6:1466-1479.
  • Jin, P., K. Farmer, N. R. Ringertz, and T. Sejersen. 1993. Proliferation and differentiation of human fetal myoblasts is regulated by PDGF-BB. Differentiation 54:47-54.
  • Johnson, B. J., N. Halstead, M. E. White, M. R. Hathaway, A. DiCostanzo, and W. R. Dayton. 1998. Activation state of muscle satellite cells isolated from steers implanted with a combined trenbolone acetate and estradiol implant. J. Anim. Sci. 76:2779-86.
  • Johnson, B. J., M. E. White, M. R. Hathaway, C. J. Christians, and W. R. Dayton. 1998. Effect of a combined trenbolone acetate and estradiol implant on steady- state IGF-I mRNA concentrations in the liver of wethers and the longissimus muscle of steers. J. Anim. Sci. 76:491-7.
  • Johnson, S. E., and R. E. Alien. 1995. Activation of skeletal muscle satellite cells and the role of fibroblast growth factor receptors. Exp. Cell Res. 219:449-53.
  • Johnson, S. E., and R. E. Alien. 1990. The effects of bFGF, IGF-I, and TGF-beta on RMo skeletal muscle cell proliferation and differentiation. Exp. Cell Res. 187:250-4.
  • Jones, J. I., A. Gockerman, W. H. Busby, Jr., C. Camacho-Hubner, and D. R. Clemmons. 1993. Extracellular matrix contains insulin-like growth factor binding protein-5: potentiation of the effects of IGF-I. J. Cell Biol. 121:679-87.
  • Kambadur, R., M. Sharma, T. P. Smith, and J. J. Bass. 1997. Mutations in myostatin (GDF8) in double-muscled Belgian Blue and Piedmontese cattle. Genome Res. 7:910-6.
  • Kharadia, S. V., T. W. Huiatt, H. Y. Huang, J. E. Peterson, and D. J. Graves. 1992. Effect of an arginine-specific ADP-ribosyltransferase inhibitor on differentiation of embryonic chick skeletal muscle cells in culture. Exp Cell Res. 201:33-42.
  • Koohmaraie, M., S. D. Shackelford, and T. L. Wheeler. 1996. Effects of a beta-adrenergic agonist (L-644,969) and male sex condition on muscle growth and meat quality of callipyge lambs. J. Anim. Sci. 74:70-9.
  • Lassar, A. B., S. X. Skapek, and B. Novitch. 1994. Regulatory mechanisms that coordinate skeletal muscle differentiation and cell cycle withdrawal. Curr. Opin. Cell Biol. 6:788-94.
  • Lu, J., R. Webb, J. A. Richardson, and E. N. Olson. 1999. MyoR: a muscle-restricted basic helix-loop-helix transcription factor that antagonizes the actions of MyoD. Proc. Natl. Acad. Sci. USA 96:552-7.
  • Lu, M. H., C. DiLullo, T. Schultheiss, S. Holtzer, J. M. Murray, J. Choi, D. A. Fischman, and H. Holtzer. 1992. The vinculin/sarcomeric-alpha-actinin/alpha-actin nexus in cultured cardiac myocytes. J. Cell Biol. 117:1007-22.
  • Ludolph, D. C., and S. F. Konieczny. 1995. Transcription factor families: muscling in on the myogenic program. FASEB J. 9:1595-1604.
  • Majors, A., and L. A. Ehrhart. 1993. Basic fibroblast growth factor in the extracellular matrix suppresses collagen synthesis and type III procollagen mRNA levels in arterial smooth muscle cell cultures. Arterioscler. Thromb, 13:680-6.
  • McCusker, R. H., and D. R. Clemmons. 1998. Role for cyclic adenosine monophosphate in modulating insulin-like growth factor binding protein secretion by muscle cells. J. Cell. Physiol. 174:293-300.
  • McPherron, A. C., A. M. Lawler, and S. J. Lee. 1997. Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature 387:83-90.
  • McPherron, A. C., and S. J. Lee. 1997. Double muscling in cattle due to mutations in the myostatin gene. Proc. Natl. Acad. Sci. USA 94:12457-61.
  • Mehul, B., M. A. Doyennette-Moyne, M. Aubery, P. Codogno, and H. G. Mannherz. 1992. Enzymatic activity and in vivo distribution of 5'-nucleotidase, an extracellular matrix binding glycoprotein, during the development of chicken striated muscle. Exp. Cell Res 203:62-71.
  • Molkentin, J. D., and E. Olson. 1996. Defining the regulatory networks for muscle development. Curr. Opin. Genet. Dev. 6:445-453.
  • Naya, F. J., C. Wu, J. A. Richardson, P. Overbeek, and E. N. Olson. 1999. Transcriptional activity of MEF2 during mouse embryogenesis monitored with a MEF2-dependent transgene. Development 126:2045-52.
  • Newton, L. K., W. K. Yung, L. C. Pettigrew, and P. A. Steck. 1990. Growth regulatory activities of endothelial extracellular matrix: mediation by transforming growth factor-beta. Exp. Cell Res. 190:127-32.
  • Novitch, B. G., D. B. Spicer, P. S. Kirn, W. L. Cheung, and A. B. Lassar. 1999. pRb is required for MEF2-dependent gene expression as well as cell- cycle arrest during skeletal muscle differentiation. Curr. Biol. 9:449-59.
  • Ojima, K., Z. X. Lin, Z. Q. Zhang, T. Hijikata, S. Holtzer, S. Labeit, H. L. Sweeney, and H. Holtzer. 1999. Initiation and maturation of I-Z-I bodies in the growth tips of transfected myotubes.J. Cell Sci. 112:4101-12.
  • Pizette, S., F. Coulier, D. Bimbaum, and 0. DeLapeyriere. 1996. FGF6 modulates the expression of fibroblast growth factor receptors and myogenic genes in muscle cells. Exp. Cell Res. 224:143-51.
  • Quinn, L. S., and J. S. Roh. 1993. Overexpression of the human type-1 insulin-like growth factor receptor in rat L6 myoblasts induces ligand-dependent cell proliferation and inhibition of differentiation.^. Cell Res. 208:504-8.
  • Quinn, L. S., B. Steinmetz, A. Maas, L. Ong, and M. Kaleko. 1994. Type-1 insulin-like growth factor receptor overexpression produces dual effects on myoblast proliferation and differentiation. J. Cell. Physiol. 159:387-98.
  • Rhee, D., J. M. Sanger, and J. W. Sanger. 1994. The premyofibril: evidence for its role in myofibrillogenesis. Cell. Motil. Cytoskel. 28:1-24.
  • Rotwein, P., P. L. James, and K. Kou. 1995. Rapid activation of insulin-like growth factor binding protein-5 gene transcription during myoblast differentiation. Mol. Endocrinol 9:913-23.
  • See attached for additional literature cited.


    Land Grant Participating States/Institutions

    AZ, CA, HI, IA, IL, MI, MN, NC, NE, OH, OR, SD, UT, VA, WA, WI

    Non Land Grant Participating States/Institutions

    Log Out ?

    Are you sure you want to log out?

    Press No if you want to continue work. Press Yes to logout current user.

    Report a Bug
    Report a Bug

    Describe your bug clearly, including the steps you used to create it.