2.Procedures for Objective 2 - Determine molecular mechanisms that control gene expression in 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. Studies under this objective fall into two specific areas: (a) Analysis of the of growth-related gene
expression in muscle and (b) Characterization of specific intracellular pathways that transmit receptor signals to the nucleus in myogenic cells. Cooperating units
responsible for the work under this objective are the Alabama, Arizona, California, Iowa, Michigan, Nebraska, South Dakota, Utah, Washington, and
Wisconsin Stations and the US Meat Animal Research Center (US-MARC).
a. Analysis of the of growth-related gene expression in muscle. Studies
here will involve identification of muscle-specific genes, characterization of genes expressed in different muscle growth models, and analysis of specific gene
regulatory mechanisms.

One particular useful experimental system for the analysis of muscle gene expression during growth is the callipyge lamb model described earlier. Analysis of the
callipyge lamb model will be done at the US-MARC and the Utah and Michigan Stations. Studies at the US-MARC will involve direct analysis of genes differentiallyexpressed in callipyge lambs. Total cellular RNA will be extracted from two affected muscles, two unaffected muscles and the heart and liver from 8-week-old callipyge and non-callipyge lambs using established protocols. Callipyge-inducible genes will be identified using mRNA differential display, gene expression fingerprinting, and representational, difference analysis of cDNA. Once callipyge-inducible genes have been identified, tissue and genotype/phenotype specificity of these genes will be determined by northern blot analysis on RNA from various tissues and organs of control and callipyge lambs, and the genes will be cloned and sequenced to identify encoded proteins. As an alternative approach, the Utah Station, in collaboration with the Michigan Station, will characterize differential expression of genes from satellite cells isolated from normal and callipyge lambs, utilizing differential display RT-PCR and microarray analysis methods, both of which are currently in use at the Michigan Station. In combination with the analysis of the satellite cells from callipyge lambs by the Utah Station, as described earlier, these studies should provide substantial characterization of the genetic and regulatory mechanisms involved in callipyge.

Investigators at the Michigan Station also plan to characterize gene expression patterns in developing skeletal muscle using differential display reverse transcription
polymerase chain reaction (DD-RTPCR) and DNA microarrays. Gene expression patterns will be evaluated in fetal and postnatal pig skeletal muscle tissue and in
myogenic cell culture models from Michigan and other cooperating stations. Expected outcomes include the identification of novel genes and elucidation of how gene
expression patterns are regulated during skeletal muscle growth.

Studies at the Iowa Station will be directed towards defining the molecular mechanism by which work-overload induces skeletal muscle hypertrophy, using a rat
gastrocnemius-ablation model. Currently, very little is known about how physical activity is transformed into a chemical signal that activates or inactivates intracellular
signaling pathways to ultimately regulate gene transcription. In initial studies, integrin-linked kinase (ILK) was identified as a possible regulator of skeletal muscle
hypertrophy. When activated, ILK is capable of increasing skeletal alpha- actin promoter activity. Thus, studies will be directed towards attempting to induce skeletal muscle hypertrophy by activating ILK in cell cultures and in transgenic mice. Subsequently, the global mRNA expression pattern in response to work-overload will be evaluated by microarray analysis and suppressive subtraction hybridization, and genes that are differentially expressed between control and overloaded muscles will be sequenced and identified.

The Washington Station will isolate and characterize cytokine genes expressed in muscle tissue. To test the hypothesis that satellite cells express cytokines and these cytokines act as autocrine GFs, the genes for equine and canine cytokines will be isolated and characterized. Genes for canine and equine muscle-specific proteins will also be isolated for sequence comparison. The expression of muscle genes by satellite cells will be determined by ELICA under different culture conditions to determine the effect of those conditions on differentiation. The results of this work will identify GFs originating from the immune system.

During the current project, the Alabama Station has developed a whole embryo culture system that has been used to demonstrate that maternal treatment with porcine somatotropin enhances embryonic development. investigators at this station will use this system to identify genes expressed during embryonic development. Porcine, bovine, and when needed, chick and mouse embryos at perisomite stages of development will be used for these whole embryo culture experiments, and initial studies will be done to optimize culture conditions. Appropriately blocked experiments will then be conducted to allow assessment of second messenger inhibitors and GFs in the cultures. Following culture, embryos will be evaluated for growth and morphology and myogenic/developmental gene activity will be determined by quantitative RT- PCR and whole embryo in situ hybridization. Experiments will be done to determine effects of hormones, GFs, nutrients, second messenger pathway inhibitors, and blockers of myogenic gene activity on embryo growth. Results will provide a better understanding of factors and signaling mechanisms that regulate gene expression in meat animals during embryonic growth.

Studies at the Nebraska Station will focus on potential translational regulation during myogenesis as a. mechanism to regulate protein expression during muscle growth. To determine changes in mRNA translation between cultures of actively dividing C2C12 myoblasts and fused, myotube cultures, ribosomes will be isolated and fractionated on a sucrose gradient. The mRNA will be released, separated and analyzed using a differential display method. Candidate genes expressed exclusively in myoblasts or myotubes will be identified and sequenced to determine their identity, and genes not previously identified will be studied further to determine their action. A second study will examine if IGF-I, IGF-II, PDGF or FGF alter which mRNA is translated. Polyribosomes will be isolated from myotubes and myoblasts that have been treated with GFs to identify differentially expressed genes. A third study will determine the location where specific mRNA's are translated within the cell by isolation of cytoskeletal bound, membrane bound and free polyribosomes followed by identification of mRNA from each group using the differential display methodology.

In combination, these studies will provide for identification of muscle-expressed genes from several different important model systems. Cooperation and collaboration between the stations involved will be essential for the timely completion of the work. In particular, cooperation will be essential for construction of microarrays and identification of expressed genes.

Finally, studies at the California and Wisconsin Stations will take a somewhat different approach to that described up to this point, namely, the analysis of expression of genes for known myofibrillar proteins. Work at the California Station will involve analysis of the sarcomeric myosin genes and their developmental regulation. Specific studies will include: (1) Analysis of the organization and structure of the fast myosin heavy chain (MyHC) gene cluster; (2) Physical mapping and sequence analysis of fast and slow MyHC genes using PCR- based strategies; (3) Cloning and characterization of 5' flanking regions of MyHC genes for promoter and regulatory analyses using reporter constructs; (4) Identification of the regulatory sequences and binding proteins that control neural regulation of the Cemb3 fast MyHC gene in nerve-muscle co-cultures; (5) Transcriptional analyses of gene expression of newly identified fast and slow MyHC genes using quantitative PCR methodologies; and (6) Determination of the molecular mechanism responsible for the continued expression of neonatal MyHC at the ends of muscle fibers in adult muscle and the gradient of down regulation of neonatal MyHC isoform that extends from the region of the neuromuscular junction toward the ends of the fiber.

Studies to be done at the Wisconsin Station will focus on analysis of expression of the giant myofibrillar protein titin, which is thought to have important roles in
myofibrillogenesis and in muscle elasticity. Investigators at this station plan to sequence the 5' region of the porcine titin gene. PCR probes will be prepared based on the most 5' coding region of human titin cDNA and used to isolate clones from a porcine genomic library. A suitable porcine genomic library is available at the Indiana Station. Transient transfection assays in skeletal muscle cell lines will be conducted with DNA constructs containing titin DNA fragments upstream from the start site fused to the chloramphenicol acetyltransferase (CAT) reporter for analysis of promoter activity. Such studies should lead to new insights on the control of titin gene expression. b. Elucidate intracellular pathways that transmit receptor signals to the nucleus in myogenic cells. Studies will be focused specifically on areas with which members of the NC-131 committee have significant expertise. The three specific areas to be examined are: (1) The role of the calpain protease system in signal transduction and myogenic regulation, at the Arizona Station and US-MARC; (2) Differences in signal transduction in different clonal lines of avian satellite cells, at the South Dakota Station, and (3) Mono-ADP-ribosylation in muscle, at the Iowa Station.

Several lines of evidence, described in the critical review (Appendix III.A.) suggest a roles for the calpains, particularly m-calpain, in myoblast proliferation and
differentiation, and in signal transduction pathways, particularly the integrin-mediated mitogenic pathway. To examine further the role of calpains in myogenesis,
investigators at the Arizona Station and the US- MARC will utilize overexpression of the calpains and the inhibitor calpastatin in myogenic cells. Stable transfections
will be done with the C2C12 myoblast cell line, using a regulated expression system (Stratagene Lac-switch). Cells will be analyzed for expression of active calpastatin or calpain, myogenic differention, and cleavage of specific proteins. Additional studies at the Arizona Station will involve analysis of the regulation of calpain in response to ligand-binding to integrins, focusing on: (1) determination of which residues are phosphorylated in u- and m- calpain, (2) whether phosphorylation alters calpain activity in vitro, and (3) whether ligand binding to the integrins elicits phosphorylation/dephosphorylation at specific sites on calpain. Results of the studies at these two units will provide needed new information about the role of calpains in myogenesis.

Investigators at the South Dakota Station will test the hypothesis that myogenic satellite cells derived from a single skeletal muscle but that differ in their mitogenic
responses to GF stimuli, differ with respect to intracellular signaling activities. Preliminary work, using clonal cultures of avian satellite cells, has demonstrated that such cells do not differ in either the numbers or affinities of GF receptors. The proposed work will determine whether there are differences in the tyrosine kinase activities of IGF, FGF and PDGF receptors on the cell surfaces, levels and activities of intracellular kinase signaling proteins, and levels of several key phosphatase enzymes.

The Iowa Station will examine the role of arginine-specific mono-ADP-ribosylation as a signaling mechanism in myogenesis. This protein modification, catalyzed by
ADP-ribosyl- transferases (ARTs) is a ubiquitous cellular signaling mechanism, and previous results support a role for this signaling mechanism in myogenic
differentiation. Studies to further characterize ADP-ribosylation in myogenesis are: (1) Analysis of the expression of the three known muscle ART enzymes during
myogenesis in cultures of C2C12 and embryonic chick myogenic cells via quantitative RT-PCR; (2) Analysis of expression of the ART enzymes during embryo genesis in the mouse, via in situ hybridization; (3) Determination of which of the three ART enzymes is necessary for myogenesis of C2C12 myoblasts, via inhibiting expression of each of the three enzymes selectively with antisense oligonucleotides coupled with overexpression of each of the ART enzymes in the same culture system, and (4) Identification of substrates for the ART enzymes via labeling studies and use of specific antibodies for the modified proteins.

3. Procedures for Objective 3 - Characterize mechanisms of cytoskeletal protein assembly and protein degradation in skeletal muscle. Growth of skeletal muscle involves not only the accumulation of protein but the assembly of newly synthesized protein into myofibrils, along with the continued turnover of the cellular protein. Thus, characterization of mechanisms regulating skeletal muscle growth includes studies on the mechanisms of protein assembly and degradation in muscle. Work will focus on: (a) Characterization of the functional domains of muscle proteins involved in cytoskeletal assembly, and (b) Analysis of muscle protein degradation. Participating units are the California, Iowa, Nebraska and Wisconsin Stations and the US-Meat Animal Research Center.

a. Characterization of the functional domains of muscle proteins involved in cytoskeletal assembly. This area represents a major area of study in both the current and previous NC-131 projects, and one in which significant progress has been reported. Nonetheless, the mechanisms by which myofibrillar proteins are assembled into well-organized myofibrils, and by which these myofibrils are -integrated into the cellular cytoskeleton, remain incompletely characterized. Work in this area will concentrate on assembly of specific myofibrillar proteins and on the role of specific cytoskeletal attachment sites in the overall process.

The California Station will examine the mechanism of assembly of the myofibrillar protein myosin. Specific studies include: (1) Analysis of the regions that control
myosin filament formation using recombinant site-directed mutant LMM proteins. Regions that are responsible for parallel and anti-parallel myosin associations will be analyzed in paracrystalline aggregates; (2) Construction of model "synthetic myosins" using full length myosin rods fused to globular proteins (to mimic myosin S 1
domain) will be used to study the mechanism of myosin bipolar filament formation; (3) The amino acids that determine alpha-helical coiled-coil specificity at the N-end of the myosin rod will be determined by mutating specific amino acids at the core of the myosin rod and determining the specificity of dimerization of the coiled-coil using his-tagged proteins and metal-chelation chromatography; and (4) The mechanism and sites at which myosin binding proteins, such as C-protein and titin, interact with the myosin rod will be determined using expressed tagged fragments of both proteins and specific binding assays. Collaboration with the Wisconsin Station will provide the needed expressed fragments of titin.

The Wisconsin Station will continue to examine the role of titin in myofibril assembly. Proposed studies will involved examination of the binding of expressed A-band titin fragments f with expressed myosin rod segments and the interaction of expressed titin PEVK fragments with actin. The technique of surface plasmon resonance using the BIACORE instrument will be employed to determine protein-protein interactions. This instrument can measure both association and dissociation kinetics as well as provide information on binding stoichiometry. The research in this area will be conducted jointly with the Iowa and California Stations since the investigators at these two stations each have unique sets of cloned titin and myosin cDNA fragments. These three stations also have a number of characterized titin and myosin monoclonal antibodies, which will be made available to other stations in the project.

Studies at the Iowa Station will focus on understanding mechanisms by which the myofibril is integrated into the overall muscle cell cytoskeleton. Current evidence
shows that intermediate filaments wrap around the Z-lines of individual myofibrils, where they attach and align adjacent myofibrils and connect the peripheral layer of
myofibrils to the cell membrane skeleton at costameres. Costameres are also important sites for cell signaling involving the integrins, which are concentrated at these
attachment sites. Continued studies in this area will involve analysis of specific interactions among protein components of intermediate filaments, Z-line proteins, and
proteins associated with costameres, along with specific domains obtained by bacterial expression. Interactions will be analyzed by several methods including solid-phase binding studies, cosedimentation analysis and the yeast two-hybrid method.

b. Analysis of muscle protein degradation. Only a limited number of studies, to be done by the Nebraska Station and the US-MARC unit, are included in this revised project proposal. Nonetheless, these studies will help provide a more complete picture of the overall mechanisms that control muscle growth and an important complement to the studies described in other areas.

Work at the Nebraska Station will be directed towards understanding an essential step in the overall process of protein turnover in muscle, the process of myofibril
disassembly. Studies will be done to investigate the role of gelsolin and calpain in myofibril disassembly in mouse muscle cell cultures, by use of 2D electrophoresis to
determine gelsolin expression levels during differentiation. In addition, work at this Station will involve development of a proteomic analysis system and an associated
database for mouse muscle cell differentiation. Proteomic analysis can be applied to display only those polypeptides up or down regulated between control and treatment samples, and thus, this system will provide an important collaborative resource for analysis of protein synthesis and degradation in skeletal muscle.

Studies at the US-MARC will involve analysis of the effects of overexpression of bovine calpastatin cDNA on muscle growth and metabolism in transgenic mice, to
determine the consequences of overexpression of bovine skeletal muscle calpastatin on muscle growth. This experiment will be done in collaboration with Drs. R. J.
Schwartz and M. L. Fiorotto of the Baylor College of Medicine. Parameters to be measured include body weight, skeletal muscle calpastatin activity, fiber-typing,
protein, RNA and DNA contents of several muscles, and fractional synthesis and degradation rates. Analysis of these parameters will provide a more complete picture of the function of calpastatin in protein turnover and growth.

In summary, this proposal describes an integrated, collaborative approach to basic research in the area of skeletal muscle development and growth. The proposed studies are, for the most part, based on the considerable progress made during the currently active project, and the studies are focused in areas in which the participants have the expertise to complete the work within the time period for the project. For most of the project leaders, support for the work is supplemented by peer-reviewed, outside grants (USDA, NIH, NSF, etc.). Nonetheless, the resources of a funded. Regional Research Project are needed to form a basis of support for the collaborations necessary to carry out the proposed research. While numerous specific collaborations have been specified above, additional collaborative efforts will form an integral part of this project. These additional collaborations include sharing of specific reagents, such as monoclonal antibodies, cDNA probes, and expression constructs, sharing of expertise in specific areas, and joint use of equipment, for example the Quixcell cell manipulator at the South Dakota Station, which has been used by several other units for preparation of clonal cell lines.

This research project will involve the use of vertebrate animals, and the committee recognizes the importance of complying with the Animal Welfare Act of 1966 and 9 CFR Subchapter A (Laboratory Animals), as amended. The project will also involve recombinant DNA research, and the committee acknowledges the importance of complying with the NIH "Guidelines for Research Involving Recombinant DNA Molecules" and other applicable federal and state guidelines and regulations. Approved assurance forms for the use of vertebrate animals and recombinant DNA research will be completed by the individual participating units upon approval of the regional project. This project does not involve the use of human subjects.