Duration: 10/01/2005 to 09/30/2010

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

The overall goal of this cooperative, multi-state, multidisciplinary, basic research project is to increase the efficiency of lean meat production in domestic animals. Meat, derived from skeletal muscle, is one of the most economically important outputs of animal agriculture. Rapid, efficient deposition of lean muscle tissue is essential to economical production of high-quality meat which is critical to both the economic success of producers and the health of consumers. Development of successful strategies to increase efficiency of muscle production requires increased understanding of the biological processes regulating differentiation and growth of muscle in meat animals. Although we have made significant progress toward this goal under the auspices of the current NC-131 project, recent advances in molecular and cellular biology methods (microarrays, siRNA, gene transfer, real-time RT-PCR, etc) have provided many of the tools necessary to dramatically advance our understanding of these processes. Consequently the goal of this multi-state, multidisciplinary, basic research project is to utilize these new tools to elucidate molecular and cellular processes that regulate differentiation and growth of skeletal muscle; thereby, providing the basic knowledge necessary to increase the efficiency of lean meat production in meat-producing animals.

This proposal for renewal of the NC-131 project describes the collaborative effort of reseachers from 18 different Agricultural Experiment Stations 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 project for the next five years are:

1. The project relates to a major agricultural 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.
   
2. The project relates directly to identified national agricultural research priorities: Objective 1.2 of the CSREES National Program Goals is "To increase the global competitiveness of the U.S. agricultural production system." 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.
   
3. CSREES Objective 1.3 is "To recruit and educate a diverse set of individuals for careers as future scientists, professionals, and leaders who are well-trained in agricultural sciences." An important component of this project has been and will continue to be the training of graduate students and postdoctorates in basic fields that will relate to long-term improvements in agricultural productivity.
   
4. 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.
   
5. Broadening and enriching the knowledge base about genomics, including the utilization of molecular techniques (gene mapping, est sequencing, functional genomics) is another goal of the NCRA that research conducted in this project will impact. The project supports current efforts to map genes important in animal production by providing fundamental information about the regulation and expression of genes during muscle differentiation and growth. New cutting-edge methodology, such as microarrays to assay gene expression of thousands of genes simultaneously and mass spectroscopy for protein and peptide identification, open new avenues for determination of biological mechanisms.
   
6. The NC-131 Committee continues to be highly productive. A total of 184 refereed papers, 41 book chapters/conference proceedings and 140 abstracts have resulted from the first four years of the current five-year NC-131 project, and additional publications are expected by the September 30, 2005, 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. Substantial progress has been made under each of the specific objectives of the current project.
   
7. The NC-131 project is both a multi-state and a multidisciplinary project, involving the effort of investigators at 18 different State Agricultural Experiment Stations. The Principal Investigators represent a variety of basic science disciplines that complement each other and provide the expertise necessary to complete the objectives.
   
8. 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.
   
9. 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, health-related granting agencies, 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 multi-state (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 3 projects: NC-131 and 2 projects dealing with plants. Thus, both the current and the proposed NC-131 projects are unique among currently funded multi-state 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 multi-state/regional projects.

Muscle differentiation (myogenesis) and growth are regulated by a number of growth factors (GF). During the previous project period members of the NC-131 regional project have made significant contributions to our understanding of the roles of insulin-like growth factors (IGFs) (1-9), fibroblast growth factors (9-11), transforming growth factor-beta (TGF-beta) and myostatin (1; 2; 4; 12; 13), anabolic steroids (1-4; 7; 14), hepatocyte growth factor (HGF) (15-19), leptin (20-22) and Insulin-like growth factor binding proteins (IGFBP) (5; 13; 23-28) in regulating muscle proliferation and differentiation in meat animals. However, much remains to be done. The discovery that muscle growth in cattle is dramatically affected by a deletion mutation in the myostatin gene illustrates the significant impact that even a single growth factor can have on muscle growth (29; 30). Similarly, although anabolic steroid implants have been used commercially for over 40 years to increase rate and efficiency of muscle deposition in feedlot steers (31), only recently has research done cooperatively by NC-131 project members begun to elucidate their mechanism of action on skeletal muscle growth (1-4; 7; 14). This illustrates the fact that, although the general effects of many GFs on muscle differentiation and growth have been recognized for a number of years, their exact mechanism of action is not known, partially because the effects of specific GFs on muscle are impacted by complex interactions of several factors unique to muscle tissue. For example, the bioactivities of the IGFs in muscle are dramatically affected by nutritional status (32), the level of IGF in muscle tissue (1; 33; 34), presence of IGF receptors (35-37) and interaction with a family of IGF binding proteins (IGFBPs) (1; 26; 27; 38-40). Although growth factors and hormones may reach muscle via the circulatory system, many cell types found in muscle tissue, including myogenic cells and fibroblasts, can synthesize specific GFs including IGFs (1; 34), IGFBPs (13; 26; 41), FGF-6 (42), myostatin (43), TGF-beta (44), and HGF (15). Thus, GFs produced locally in muscle can act to regulate proliferation and differentiation of myogenic cells. For example, mice overexpressing IGF-I in muscle tissue show increased muscle deposition and increased satellite cell numbers as compared to control mice. Additionally, mice overexpressing specific IGFBPs show alterations in muscle mass and growth rate. Furthermore, mice in which myostatin expression in muscle has been knocked out have 30 - 40% greater muscle mass than litter mate controls and double muscled cattle in which myostatin function has been impaired by a naturally occurring mutation in the myostatin gene have significantly increased muscle mass. Additional research is needed to characterize the expression of specific GFs in muscle and to define their endocrine, autocrine, and/or paracrine actions in regulating muscle growth in meat-producing animals.

Growth factors affect muscle through complex intracellular signal transduction pathways that are initiated by binding of growth factors (GFs) or hormones to specific intracellular or cell surface receptors (45-48). The formation of the ligand receptor complex initiates intracellular signal transduction mechanisms resulting in activation of specific molecules and/or expression of specific genes that regulate muscle differentiation and growth. The phosphatidylinositol 3-kinase (PI 3-kinase) pathway has been reported to stimulate differentiation of myogenic cells (46; 47; 49; 50) while the mitogen-activated protein kinase (MAPK) pathway stimulates proliferation at the expense of differentiation (45; 51; 52). Members of the Rho GTPase family have been shown to either stimulate or suppress differentiation depending on identity of the family member (53). Additionally, members of the TGF-beta family, including TGF-beta and myostatin, suppress both proliferation and differentiation of cultured muscle cell lines and primary myogenic cells via the Smad pathway (54-56). Recently, there is increasing evidence that the giant muscle protein titin, which constitutes nearly 10% of the protein in myofibrils, may participate in signal transduction, using mechanical stretch signals to affect gene expression (57). Some titin binding proteins re-distribute from the myofibril to the nucleus to provide signals for hypertrophy. Most of the evidence for this latter phenomenon has been obtained in the heart, but similar mechanisms are likely to also function in skeletal muscle. There is also evidence that the P94 calpain is tightly bound to titin; this suggests that titin breakdown may be a key initial step in muscle protein turnover and myofibril remodeling that occurs during growth (58). Signal transduction pathways interact with each other (cross-talk) in complex ways that are not completely understood at present. Although the general intracellular pathways utilized by a number of growth factors have recently been identified, little is currently known about how these specific pathways function in muscle to control proliferation and differentiation of embryonic myogenic cells and satellite cells or hypertrophy of muscle fibers in postnatal muscle. Furthermore, although the previous NC-131 project yielded significant new information on Raf/MEK/MAPK (59-61) and Smads (62), the majority of what is known has been determined in laboratory animals such as mice and rats and may not be directly applicable to meat-producing animals. These reports along with a large number of other reports establish the importance of identifying and characterizing intracellular signal transduction pathways activated by specific growth factors in muscle and elucidating their role in muscle differentiation and growth in meat animals. Consequently, more emphasis will be placed on this research area in the renewal proposal.

Results obtained during the previous NC-131 project (63-69) as well as the work of other researchers have shown that the extracellular matrix (ECM) surrounding muscle cells may play an important role in muscle growth and differentiation (70; 71). The ECM functions by directly interacting with membrane receptors and by modulating growth factor activities. The activity of growth factors such as Fibroblast growth factor type (FGF)-2, hepatocyte growth factor (HGF), transforming growth factor-beta (TGF-beta) and IGFBP are regulated by binding to proteoglycans (72-74). Cell surface heparan sulfate proteoglycans may modulate terminal myogenesis by acting as low affinity receptors for growth factors such as FGF-2 (75-77) and HGF (78). The fact that expression and cellular localization of proteoglycans is highly regulated during muscle differentiation (79) also suggests that these molecules may play a role in the differentiation process.

Myogenic regulatory factors (MRFs) are basic helix-loop-helix (HLH) transcription factors that initiate myogenesis and regulate the transcription of muscle specific genes (reviewed by (80-82)). There are four members of this family; MyoD1 (83), myogenin (84; 85), myf-5 (86) and MRF4/herculin/myf-6 (87-89). MRFs form heterodimers with ubiquitously expressed E2A, E12 and E47 gene products (90; 91), and these heterodimers bind to the DNA consensus sequence CANNTG, also referred to as an E box. Forced expression of any one of this family of transcription factors (myoD, myf-5, myogenin or MRF-4) in nearly every cell type can force cells to reprogram their chromatin and establish gene expression profiles similar to that of normal skeletal muscle cells (92). In fact, some argue that mechanisms underlying control of these "powerful" transcription factors may be a nodal point for understanding and controlling muscle development. The target of these dimers, the E box motif, is found in the regulatory regions of numerous muscle-specific genes including the MyHC isoform genes (93-97). Skeletal muscle consists of a heterogeneous collection of muscle fibers differing in their ability to contract and metabolize energy (98). Classification of each fiber type in muscle is a rather nebulous process but relies on the functional capabilities of each fiber. Speed of contraction is related to the type of myosin heavy chain (MyHC) isoform contained within a muscle fiber (99), and therefore, changes in the expression of these genes collectively will ultimately impact the muscle fiber functionality. Muscle fibers change to accommodate a myriad of physiological stimuli, such as hormonal changes, innervation state, aging, nutrition, and other physiological parameters. From a practical stand point, the relative proportion of these fibers in a muscle is related to its inherent ability to grow and its susceptibility to disuse atrophy. Although the aforementioned stimuli are well-documented, the molecular mechanisms controlling changes in MyHC gene expression as well as that of other muscle specific genes are not understood.

Transcription factors affecting gene expression in muscle work in concert with several growth factors, as evidenced by the fact that bHLH-mediated up-regulation of muscle specific genes only occurs when growth factor presence and abundance in the environmental milieu is permissible. These growth factors include such well-known proteins as the insulin-like growth factors, the family of fibroblast growth factors, transforming growth factors and many others. In addition, expression of extracellular matrix proteins may play a significant role in regulating differentiation and proliferation of myogenic cells. As discussed earlier in this review, myostatin provides a good illustration of the biological significance and the potential impact of growth factors in the biomedical and agriculture communities. Mutations in the myostatin gene are responsible for the well-known double-muscled syndrome in cattle. Myostatin is a member of the transforming growth factor-beta superfamily and is a negative regulator of skeletal muscle growth (100). Mice and cattle deficient for myostatin by naturally occurring or knock-out mutations of the gene exhibit dramatic increases in skeletal muscle mass (29; 30; 101-103). The condition in several cattle breeds such as Belgian Blue and Piemontese is associated with mutations in the myostatin gene and results in excessive muscle fiber formation (hyperplasia). Skeletal muscles of these cattle contain almost double the number of fibers compared with other cattle breeds, whereas fiber size is nearly equal or slightly larger than normal animals (104). This tremendous increase in muscle fiber number is associated with increases in muscle mass of > 20% and decreases in fat (104). Although other growth factors likely work in concert to modulate muscle growth, the myostatin "story" serves to illustrate the tremendous gains in muscle growth that are possible by elucidating the mechanisms controlling the expression and action of a specific protein regulating muscle growth (105-107). The recent development of techniques such as microarray analysis and real-time RT-PCR make it feasible to more completely characterize the expression of known molecules such as myostatin and to identify other proteins that may regulate proliferation and differentiation of muscle cells. During the current NC-131 project, a porcine complementary DNA (cDNA) microarray was produced that contained expressed sequence tags (EST) derived from whole embryo and adult skeletal muscle, and differential display PCR products from fetal and postnatal muscle (108-110). This microarray will be used to identify and characterize specific genes responsible for controlling prenatal myogenesis and postnatal myonuclear accretion.

Accumulation of muscle protein mass depends on the balance between rate of muscle protein synthesis and rate of muscle protein degradation. It is now well-established that muscle protein turnover has an important and sometimes the predominant role in rate of skeletal muscle growth. Decreasing rate of muscle protein turnover also is directly related to increasing the efficiency with which ingested nutrients are converted to muscle tissue. There are several classes of proteins in skeletal muscle; the contractile or myofibrillar proteins constitute over 60% of total muscle protein and are responsible for the contractile properties and many of the functional properties of muscle. Hence, studies on muscle protein turnover need to focus on the myofibrillar proteins. The myofibrillar proteins, however, also present unique challenges to their turnover. After synthesis, the myofibrillar proteins are rapidly assembled into myofibrils, which are tubular-like structures that extend continuously from one end of the muscle cell to the other. The contractile properties of myofibrils require that this continuous structure be maintained during turnover; hence turnover of the myofibrillar proteins must be done without disrupting this structure. Moreover, the myofibril is a dynamic structure that contains a number of proteins, some that are directly responsible for contractions (actin and myosin), others that serve to regulate contractile activity (e.g., troponin, tropomyosin) and others that seem to be involved in maintaining the myofibrillar structure (e.g., alpha-actinin, titin, nebulin). In addition, a number of proteins such as the intermediate filament (IF) proteins, bind to the periphery of the myofibril and are important for linking the contractile proteins to the sarcolemma and maintaining lateral registration of the myofibrils within a muscle cell. The dynamic interactions among these different groups of proteins, and the mechanism used by cells to accomplish their turnover without disrupting myofibrillar function is poorly understood.

Myofibrillar protein turnover, as is turnover of all proteins, is mediated by proteases. There are four proteolytic systems in muscle cells that are present in sufficient quantities to catalyze intracellular protein turnover.

1. The lysosomal system. Proteases in this system, the cathepsins, are located inside lysosomal structures and have acidic pH optima. The primary role of lysosomal cathepsins is degradation of extracellular proteins that have been taken up by the cells via pinocytosis or receptor-mediated endocytosis and then transported to the lysosome where they are degraded at the acidic pH values (3-5) in lysosmes. Skeletal muscle has a low level of catheptic enzymes when compared with other tissue such as liver or spleen, and cells, including muscle cells, contain cystatin, a potent inhibitor of cysteine cathepsins in their cytoplasm. Myofibrils are too large to be engulfed by lysosomes (which would result in severing the myofibril and loss of function), so lysosomal cathepsins do not have a primary role in metabolic turnover of myofibrillar proteins, although they may be involved in necrotic degradation, especially during times of macrophage invasion (111-113).

2. The Calpain system. The calpain system includes 14 different members plus calpastatin, a protein inhibitor that is specific for inhibition of the two, ubiquitous, well-characterized calpains, µ-calpain and m-calpain. Skeletal muscle contains appreciable quantities of the calpains, and evidence from a variety of sources indicates that the two ubiquitous calpains have an important role in metabolic turnover of myofibrillar proteins.

3. The Proteasome. The proteasome has a major role in intracellular protein turnover in all cells, including muscle cells. The proteasome has a major role in metabolic turnover of the sarcoplasmic protein fraction in skeletal muscle cells, and likely also has a role in turnover of myofibrillar protein.

4. The Caspase system. Proteases in the caspase family are responsible for degradation of proteins during apoptosis. The caspases are cysteine protease, like µ-and m-calpain are, but they do not require Ca2+ for activity. Because the caspases are activated only during periods of apoptosis, they do not have a role in the normal metabolic turnover of muscle proteins, although they could conceivably become activated during periods of muscle wasting.

Consequently, of the four protease systems in skeletal muscle, only the proteasome and the calpain family are likely to have important roles in metabolic turnover of myofibrillar proteins. The mechanism of how this turnover is accomplished, however, still remains unclear. A plausible mechanism for turnover of the myofibrillar proteins was first proposed nearly 30 years ago (114). It was suggested that the calpains, which are located on the Z-disk and I-band areas of the myofibril (115), cleave those polypeptides, such as titin, nebulin, and desmin that are responsible for maintaining the myofibrillar structure. This cleavage would release the outer layer of filaments from the surface of the myofibril, leaving a myofibril with a diameter that was smaller by one layer or filaments (Figure 1). This model is consistent with observations that atrophying muscle in different muscular dystrophies, after denervation, or during fasting has smaller diameter myofibrils than unaffected muscle has (116).The model is also consistent with the observation that approximately 5-15% of total myofibrillar protein in striated muscle can be released by gentle agitation in an ATP-containing solution (117). The release of these filaments, easily released myofilaments (ERM), does not require hydrolysis of ATP. The amount of ERM increases significantly in the presence of Ca2+, and available evidence indicates that the ERM are an intermediate in the turnover of myofibrillar proteins.

The calpains do not degrade polypeptides to amino acids but do rapidly degrade those proteins that are responsible for maintaining the myofibrillar assembly. The proteasome, on the other hand, cannot degrade intact myofibrils or even intact myofilaments but can rapidly degrade individual polypeptides to small, 4-10-amino acid peptides. Hence, althought the mechanism described in the dissociation /proteasome degradation model in Figure 1 has been widely quoted as being the mechanism used to turnover myofibrillar proteins in striated muscle, this model has never been critically examined.

Several studies have reported that monomeric actin (118) or myosin molecules (119) can exchange with actin monomers or myosin molecules in myofibrils in the absence of proteolysis, and recently it has been shown that purified alpha-actinin in solution also exchanges with alpha-actinin in myofibrils (120). Therefore, myofibrils may use more than one mechanism to turnover the proteins that constitute the myofibrillar structure. Possibly, those proteins on the surface of the myofibrils, such as the IF protein, turnover via direct exchange, whereas calpain proteolysis is used to release filaments.

Regardless of the mechanism that releases filaments/proteins from the myofibril, it is clear that some other mechanism is needed to degrade the released filaments/proteins to amino acids.

In addition to studying the mechanism of myofibril turnover, the previous NC-131 project has examined the assembly and organization of the cytoskeleton in developing and adult muscle cells, with emphasis on the proteins associated with the intermediate filament (IF) system. Results obtained during this project demonstrated that IFs have an important role in maintaining the structural integrity of the muscle fiber (121-127). The IFs encircle myofibrils at their Z-lines, link adjacent myofibrils, and help attach the peripheral layer of myofibrils to the cell membrane at sites called costameres. However, the protein interactions involved in organizing the IF network and attaching the IFs to other muscle cell cytoskeletal components are not well characterized. Elucidation of these protein interactions may identify rate-limiting steps in myofibril assembly that influence the rate of muscle growth. Additionally, these studies will contribute to our understanding of molecular interactions affecting meat tenderness.

As described above, members of the NC-131 regional project have made significant contributions to our fundamental understanding of the roles of growth factors, cell signaling, protein turnover and myofibril assembly in regulating proliferation, differentiation and growth of muscle cells in meat-producing animals. The studies proposed for the project renewal build upon these findings and will further increase our knowledge of the basic processes regulating muscle growth and differentiation. This fundamental knowledge will provide the framework necessary to develop strategies to increase the efficiency of muscle growth in meat animals.

Objectives

1. Characterize the signal transduction pathways 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.
   

Methods

The objectives of the currently active NC-131 project were broadly defined in order to allow for changes in direction of work based on results of current studies both within and outside of the project. Consequently, the overall objectives of the revised project are similar to those for the current NC-131 project. However, approaches and methodologies have been changed to reflect advances in knowledge during the five year duration of the currently active project. Because of page limitations general approaches rather than detailed methods are described. Objective 1: Characterize the signal transduction pathways that regulate skeletal muscle growth and differentiation. Studies in this objective focus primarily on autocrine and paracrine mechanisms regulating muscle cell proliferation and differentiation in economically important animals. Nine stations are involved in studies contributing to this objective and several of the proposed studies involve collaboration between two or more of the participating stations. The Arizona Station will examine the role of hepatocyte growth factor (HGF) in activation of quiescent satellite cells in bovine muscle. Initial experiments will examine whether HGF can stimulate activation of quiescent satellite cells in bovine muscle and whether myostatin and TGF-beta1 antagonize this activity. Although evidence suggests that HGF may be the physiological activator of quiescent satellite cells, the hypothesis has not been tested in vivo. Consequently, a second set of experiments will assess whether HGF is responsible for activation of quiescent satellite cells in vivo. Transgenic mice that express a dominant-negative form of HGF in muscle fibers will be generated and satellite cell activation will be examined following muscle injury. It is anticipated that satellite cell activation will be diminished or inhibited following injury in muscle that has expressed the inhibitory dominant negative form of HGF. A third set of experiments will examine what gene expression pattern characterizes the quiescent state of satellite cells and changes in this pattern during activation. The Minnesota Station will examine the mechanism by which IGF-I independent actions of insulin-like growth factor binding proteins (IGFBP)-3 and -5 mediate the proliferation-suppressing actions of TGF-beta and myostatin in porcine embryonic myogenic cell (PEMC) and porcine satellite cell (PSC) cultures. Both IGFBP-3 and -5 affect proliferation and differentiation of cultured myogenic cells and these IGFBPs mediate the proliferation-suppressing actions of TGF-beta and myostatin on PEMC (8; 13; 23; 27; 28). Proposed studies will utilize recombinant porcine (rp) IGFBP-3, rpIGFBP-5, neutralizing antibodies specific for each IGFBP (26), and PEMC and PSC culture models developed at Minnesota to accomplish the following experiments: 1) elucidation of the role of IGFBP-3 and IGFBP-5 in the proliferation-suppressing activity of TGF-beta and myostatin; 2) examination of the effects of TGF-beta and myostatin on localization of IGFBP-3 and IGFBP-5; 3) examination of the effects of IGFBP-3 and IGFBP-5 on TGF binding to the TGF receptor, receptor phosphorylation, and phosphorylation of down-stream components of the TGF beta signaling pathway; 4) examination of the effect of IGFBP-3 and IGFBP-5 on other intracellular signaling pathways that may effect proliferation. The goal of the Kansas Station is to utilize bovine muscle satellite cell and C2C12 cell culture models to assess specific non-genomic effects of anabolic steroids, such as progestins, on muscle cell proliferation, differentiation, and metabolism. Addition of melengestrol acetate (MGA), a synthetic progestin, to cultured bovine muscle satellite cells resulted in a dose-dependent decrease in DNA synthesis. MGA and progesterone (P4) also increased myogenin and a membrane-bound receptor (PRMC-1) mRNA in C2C12 cells. Since cultured muscle cells and skeletal muscle tissue do not possess the classical progesterone receptor and progesterone-receptor antagonists do not ameliorate the effects, research will investigate the role of the membrane-bound receptor in signal transduction of progestins in skeletal muscle and culture muscle cells. Since any of the non-genomic effects of steroids result in rapid increases in second-messenger molecules such as cAMP, changes in cAMP concentration will be assessed in cultured muscle cells exposed to progestins. Finally, the association of changes in second messenger molecules and transcriptional regulation of factors important to muscle cell differentiation, such as myogenin, will be studied in this model. During the previous NC-131 project the Kansas and Minnesota Stations have collaborated on studies of the mechanism of action of anabolic steroids in muscle growth in feedlot steers (1; 2; 4). These stations will continue their collaboration in this research area, focusing on the mechanism by which anabolic steroids stimulate increased muscle production of IGF-I mRNA. Animal facilities available at the Kansas Station will be used to generate muscle samples that will be evaluated at both the Kansas and Minnesota Stations. Bovine muscle satellite cell and C2C12 cell culture models will be utilized to assess specific non-genomic and genomic effects of anabolic steroids, such as estradiol and trenbolone acetate, on muscle cell proliferation, differentiation, metabolism and production of IGF-I mRNA. The Montana Station will evaluate how the genetic potential of an animal in addition to growth compounds affect satellite cell culture proliferation and differentiation. These researchers will examine the effects of genetic growth potential, tenderness or myostatin gene mutation on proliferation and differentiation of isolated satellite cells. Studies at the Wisconsin Station will focus on the role of titin in signal transduction in muscle cells. First, Wisconsin researchers will examine whether the distributions of titin binding proteins are altered during muscle development. A number of proteins have been shown to bind to titin in cardiac muscle, and many of these same proteins may also interact with the skeletal muscle titin isoforms. Initial studies will be conducted with antibodies against the protein Ankrd2, a protein that binds titin in skeletal muscle and to several transcription factors in the nucleus (128); in addition the protein responds to muscle stretch (129). Researchers will use Western blotting and immunofluorescence to compare protein expression and localization as they relate to muscle development. Wisconsin researchers hypothesize that the relative content of this protein will be higher during periods of initial rapid muscle growth. Additionally, it is expected that a larger proportion of the protein will be found in the nucleus during the rapid early periods of growth. The Washington Station will explore the effects of nutrition on muscle satellite cell proliferation and differentiation. An oral dietary supplement (creatine monohydrate; CM) possesses some ability to influence satellite cell differentiative activity in vitro and other commercially available compounds will be screened in a satellite cell culture system. The Washington Station will also conduct experiments utilizing co-cultures of muscle tissue-derived cells and fat tissue-derived cells. Signaling (regulation) between the two cell forms will be studied, and putative regulatory molecules will be identified. The Washington Station is also examining how myostatin influences the growth and developmental of skeletal muscle from a commercially important fish, the rainbow trout. The immediate goals of this research are to determine the mechanisms of myostatin action at the cellular level. The specific objectives of these studies are (i) to determine the effects of myostatin and IGF-I on myoblast proliferation and differentiation, (ii) to outline the basic signal transduction pathways of both, highlighting the sites of cross-talk, and (iii) to describe the respective receptor binding kinetics. Research efforts at the North Carolina Station will be focused on understanding the effects of different nutritional paradigms on skeletal muscle growth in poultry. A traditional poultry management practice has been to delay providing feed to birds until 48 to 72 hours post-hatch. To assess the effects of this practice on muscle growth, research at the North Carolina station is focused on studying the dynamics of satellite cell number/activity and myonuclear apoptosis during different feeding regimens (starvation/dietary lysine levels) over the early post-hatch period in relation to meat yield in poultry (130-133). Studies at the Idaho Station, in cooperation with the Washington Station, will focus upon leptin effects on partitioning of energy substrate utilization. This effect is to favor oxidation of fatty acids, sparing glucose. Thus, there is a mechanistic link between leptin and insulin in modulating substrate utilization. Researchers will target two mechanisms to identify key signaling molecules in these interactions. 1) Modulation of leptin actions in muscle by leptin binding proteins and other paracrine factors. 2) Activation of common intra-cellular pathways, and key messengers including insulin receptor substrates-1 and -2, phosphatidylinositol-3 kinase, and mitogen-activated phospho-kinase by leptin and insulin. The Idaho Station is will also conduct research to define the population characteristics and cellular and molecular regulation, of post-hatch fish cells. Researchers will 1) isolate representative populations of cells of fish muscle, that are involved in muscle growth, development or quality; 2) define specific cellular and molecular characteristics of the isolated cells and the tissues from which they are derived; and 3) develop viable cell lines for use in studies to define the cellular and molecular regulation of fish growth and development. The Indiana Station will examine molecular mechanisms controlling changes in muscle fiber type using changes in myosin heavy chain (MyHC) expression as a marker. Researchers will use various chemicals to delineate the role of the mitogen-activated protein kinsase (MAPK), protein kinase B and C and the Akt/mTorr pathways in MyHC gene regulation. These studies will be used to identify pathways that are instrumental in establishing muscle fiber type, as defined by MyHC expression. Once these pathways have been identified, electroporation procedures will be used to transfer reporter gene constructs and other controllable genes into rodent and pig skeletal muscle and validate the existence and functionality of these pathways in vivo. The Indiana Station will also examine signaling mechanisms involved in beta-adrenergic agonist (BAA) changes in MyHC expression in order to further understanding of the role of these mechanisms in BAA-augmented growth. Objective 2: Determine molecular mechanisms that control gene expression in skeletal muscle. Analysis of muscle-specific gene regulation is an active area of research in the agricultural research community and in the larger medical research community. Characterization of the entire sequence of gene regulatory steps that control myogenesis, and characterization of the all of the signaling pathways that regulate muscle-specific genes is clearly beyond the scope of this project. Thus, efforts will be focused on systems that fall within the expertise of the investigators in the NC-131 team and on systems that are of particular relevance for understanding muscle growth in meat animals. Cooperating units responsible for the work under this objective are the Arizona, Indiana, Illinois, Hawaii, Michigan, Nebraska, Ohio, Utah, South Dakota and Washington Stations. Investigators at the Utah Station will employ real-time quantitative PCR (Q-PCR) to profile gene expression in the hypertrophy-responsive gluteus medius muscle compared to gene expression in the hypertrophy-nonresponsive supraspinatus muscle from callipyge lambs. The profile will include several key genes important to muscle growth and development, an imprinted gene linked to expression of the callipyge phenotype, and a typically employed housekeeping gene. These data will then be analyzed using established methods to determine if expression each gene is linked to muscle hypertrophy. As part of this effort, this team will also develop and validate new statistical methods for analyzing these types of bioinformatics experiments. The goal of the Michigan Station is to identify and characterize specific genes responsible for controlling prenatal myogenesis and postnatal myonuclear accretion. Researchers will examine biochemical indices of myoblast proliferation and differentiation in vitro and in vivo, and use cDNA microarray technology to determine patterns of expression for 3,300 genes during myogenesis in vitro and during muscle development and growth in pigs. Simultaneous identification of differences in gene expression will enable the complex mechanisms controlling muscle development and growth to be deciphered. This fundamental knowledge will enable development of strategies to increase the number of myofibers that form prenatally, and increase postnatal myonuclear accretion via manipulation of satellite cell activity. Work at the Nebraska Station will focus on characterization of muscle proteins associated with calcium regulation in muscle. Researchers will characterize the protein profile of SR-associated proteins in PSE and unaffected (unimproved) meat animals and develop protein microarrays for characterization the calcium regulatory proteome. The North Carolina Station is actively focused on studying the dynamics of satellite cell number/activity and myonuclear apoptosis during different feeding regimens over the early embryonic and post-hatch periods. These researchers will examine the proportion of muscle cells lying inside and outside the basal lamina (based upon anti-laminin staining) expressing Pax7, c-met, myoD, and myogenin in relation to mitotic activity (BrdU incorporation). Five stations, (Illinois, Indiana, Ohio, South Dakota, and Washington), are studying the mechanism(s) by which the muscle growth inhibitory cytokine, myostatin exerts an effect on muscle growth using three separate approaches. The Washington group is exploiting the fact that some fish species have two myostatin genes. These researchers plan to determine the developmental expression profile of the different myostatin paralogues using molecular techniques and will further test the biological action of these paralogues by generating a myostatin null fish line and by developing transgenic fish overexpressing different dominant-negative myostatin isoforms. Additional studies will employ cloning the myostatin gene from rainbow trout and identifying skeletal muscle-specific transcriptional regulatory elements within the promoters of all three trout paralogues using DNase footprinting and gel-shift assays with nuclear protein from different tissues. Using quite a different approach, the Hawaii Station possesses a transgenic line of mice that over-express the myostatin prodomain. As a result, these mice experience muscle hypertrophy postnatally. These researchers plan to characterize the global gene expression of muscle from these mice and compare it to wild type using microarray analysis which will provide some pictures of what genes or gene groups are regulated by myostatin. Using yet another approach, the Illinois and Indiana Stations will utilize the classic myostatin null mouse line (extreme muscling) and mate these mice to leptin null mice (extreme obesity) to study how muscle responds to various perturbations in whole body metabolism. Expression and ontogeny of myogenic genes, along with the differentiation and development of classic muscle cell lineages will be evaluated using this unique mating. Using yet another approach, the South Dakota, Ohio and Arizona Stations have joined efforts to test the hypothesis that satellite cell subpopulations that differ in their responsiveness to growth factors differ in their expression of myostatin and their responsiveness to this polypeptide. These investigators plan to examine the responsiveness of cultured turkey satellite cells and embryonic myoblasts to myostatin during proliferation and differentiation. Muscles differing in contractile and biochemical characteristics will be used to address the effects of muscle type on this process. Researchers at the Indiana Station plan to test whether expression of MyHC is related to muscle growth or to mediation of muscle hypertrophy in growing animals. These scientists intend to use various MyHC null mice lines and myostatin null mice to determine the role of muscle fiber type in muscle hypertrophy that occurs in response to double muscling. Moreover, they intend to understand the mechanisms underlying up-regulation of various reporter gene constructs under control of MyHC-specific promoters. These studies will validate the relationship between muscle hypertrophy and muscle fiber type and unmask mechanisms that modulate the plasticity of muscle. The Illinois Station will investigate the role of apoptosis in changes in protein/myonuclei ratios along with other cellular responses during hypertrophy and atrophy. Using a chicken muscle model of hypertrophy and atrophy induced by stretch and release of stretch, respectively, researchers have observed a rapid reduction in muscle mass and myonuclei number. The observed changes in caspase activity were indicative of apoptosis. This model will be used to identify factors involved in the mechanism for selecting nuclei for apoptosis. In collaboration with the South Dakota Station, the Ohio Station proposes to continue to explore the involvement of the extracellular matrix in the stimulation of muscle development. Specifically, these scientists plan to determine how syndecan-1 and glypican expressions affect MyoD and myogenin expression during turkey satellite cell proliferation and differentiation. Syndecan-1 and glypican genes will be transfected and overexpressed in F-line turkey satellite cells and total RNA will be reverse transcribed and amplified by PCR for MyoD and myogenin expression. Objective 3: Characterize mechanisms of cytoskeletal protein assembly and degradation in skeletal muscle. Work will focus on determining the proteolytic mechanisms used to turnover myofibrillar proteins in skeletal muscle of domestic animals and the role of titin and intermediate filaments in the assembly and disassembly of myofibrillar proteins. Five stations, Arizona, Indiana, Iowa, Oregon, and Wisconsin, will use a coordinated, complementary approach to test different aspects of the hypothesis in Figure 1 The Arizona Station will utilize 3 approaches to determine whether the calpains are involved in generating easily releasable myofilaments (ERM) from myofibrils, both in vitro and in vivo, and then whether the proteasome can degrade the myofilaments released. 1) Cultured myogenic cells incubated with selected inhibitors of the proteasome or the calpains (all studies will use µ- and m-calpain), will be used to determine the effects of this incubation on the amount of ERM, the level of ubiquitinated myofibrillar proteins, the release of tyrosine (estimate of general protein degradation), and the release of 3-methylhistidine (estimate of myofibrillar protein degradation). 2) Purified myofibrils will be incubated with purified calpain to learn whether calpain can release from these myofibrils myofilaments that can be degraded by the proteasome. 3) Muscle strips obtained from animals that have been treated to increase the rate of myofibrillar protein turnover (unweighting or glucocorticoid administration) will be incubated with proteasome or calpain inhibitors as in Study 1. The Indiana Station will determine whether interaction of intermediate filament (IF) proteins on the myofibrillar with other myofibrillar proteins is altered by posttranslational events such as phosphorylation, whether these events affect the interaction/release of the proteins from the myofibril; whether interaction of alpha-actinin with actin in developing muscle alters interactions of other actin-binding proteins with actin; and whether degradation of specific cytoskeletal proteins changes the rate of exchange of myofibrillar proteins as is predicted by the model in Figure 1. The Iowa Station will continue to examine the assembly and organization of the cytoskeleton in developing and adult muscle cells, with emphasis on the proteins associated with the intermediate filament (IF) system. Studies will focus on two areas: (1) Characterization of protein interactions that occur at cytoskeletal attachment sites, and (2) examination of the role of covalent modifications, particularly ADP-ribosylation, on the properties of the IFs. Protein interaction studies will utilize both purified proteins and expressed protein domains, and interactions will be characterized by using several current methods for analysis of protein interactions. Studies on ribosylation will focus on analysis of the role this modification in regulating IF assembly in muscle cells and on characterization of the function of the ADP-ribosyltransferase enzymes. The Oregon Station will use two transgenic mice lines, a MURF-1-/-. and a MAFbx-/- line, to determine the effects of knocking out two E-3 ubiquitin ligases on proteasomal degradation of myofibrillar proteins and on the expression of calpains 1 and 2 (the large subunits of µ- and m-calpain, respectively), of cathepsins B and L, and of selected proteasomal subunits. The Oregon Station has the two lines of transgenic mice and is ready to proceed with the experiments as soon as this project is activated. The studies will test the hypothesis that expression level of the two muscle specific E-3 ligases, which have been shown to have important roles in muscle protein turnover, affects expression of other proteolytic systems involved in muscle protein turnover. The Wisconsin Station will use circular dichroism spectra and surface plasmon resonance to characterize interactions between repeating PPAK sequences and regions in the titin molecule rich in glutamic acid and to determine the role of these interactions in passive tension and whether these interactions affect assembly and/or disassembly of actin from myofibrils. The five stations will collaborate in these studies. Muscle samples from the transgenic and control mice will be assayed for calpain activity by the Arizona Station; the Indiana station will use fluorescence microscopy to learn whether myofibrillar structure and exchange of myofibrillar proteins are altered in the transgenic mice, and will exchange calpain-treated myofibrils with the Arizona Station. The Arizona Station will provide µ-and m-calpain purified from bovine muscle to the Indiana, Oregon and Wisconsin Stations. Titin peptides from the Wisconsin Station will be used by the Indiana Station to determine whether segments of the titin molecule affect exchange of myofibrillar proteins and where in the myofibrillar structure these segments bind (FRET). The Arizona and Indiana Stations will collaborate in characterizing the initial calpain-cleavages that release proteins to obtain some insights into which proteins/domains are important in maintaining myofibrillar structure.

Measurement of Progress and Results

Outputs

• Research supported by this project will result in training of new researchers equipped to use molecular and cell biology methods to study factors regulating proliferation and differentiation of muscle cells in meat-producing animals.
• Elucidation of the autocrine, paracrine and endocrine mechanisms by which specific growth factors such as IGF-I, TGF beta, FGF, HGF, IGFBP and leptin regulate proliferation and differentiation of skeletal muscle cells in meat animals.
• Increased knowledge of the role of specific genes in regulating proliferation and differentiation of muscle cells in meat animals.
• Increased understanding of the role of the extracellular matrix in regulating proliferation and differentiaton of muscle cells in meat animals.
• Increased understanding of the role of specific proteases in protein degradation in skeletal muscle.

Outcomes or Projected Impacts

• Publications will be the primary tangible result expected from the project because it focuses on basic research. It is anticipated that utilization of these published results will lead to improved efficiency of lean meat production in domestic livestock and poultry.
• The Committee will sponsor a Symposium on Molecular Mechanisms Regulating Skeletal Muscle Growth and Differentiation. This symposium will provide a mechanism to convey new research information and will increase the visibility of the project.
• Increased understanding of the role of growth factors and cell signaling pathways in regulating proliferation and differentiation of muscle cells may lead to molecular- and cellular biology-based strategies for more efficient production of lean meat which will benefit both producers and consumers. For example, it may be possible to alter fiber type distribution in muscle to increase muscle mass and/or meat quality. Additionally, it may be possible to increase or prolong the proliferative activity of muscle satellite cells so that they provide more DNA to support postnatal muscle fiber growth.
• Elucidation of mechanisms responsible for muscle protein degradation may provide information necessary to utilize molecular- or cell biology-based methods to reduce protein degradation in growing muscle. This will allow more rapid muscle growth without increasing energy requirements.
• Gene expression studies may lead to identification of genes that may be used to produce transgenic animals that more efficiently produce muscle. Additionally, because this project combines the skills of muscle biologists and molecular biologists, collaboration between committee members should result in elucidation of the function in muscle growth and differentiation of proteins produced by identified genes.

Milestones

(2009): Sponsor a sympossium on Molecular Mechanisms Regulating Skeletal Muscle Growth and Differentiation at ASAS/ADSA National Meeting. This will allow us to convey research findings to interested individuals in these organizations.

Projected Participation

View Appendix E: Participation

Outreach Plan

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. Additionally, in 2009 the committee will orangainze a sympossium on Molecular Mechanisms Regulating Skeletal Muscle Growth and Differentiation at the ASAS/ADSA National Meeting that will convey recent research findings to the members of these organizaitons.

Organization/Governance

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 Methods 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.

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