NC131: Molecular Mechanisms Regulating Skeletal Muscle Growth and Differentiation

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

Annual/Termination Reports:

[01/03/2003] [01/08/2004] [12/16/2004] [03/30/2006]

Date of Annual Report: 01/03/2003

Report Information

Annual Meeting Dates: 10/04/2002 - 10/05/2002
Period the Report Covers: 10/01/2001 - 09/01/2002

Participants

Arizona - Darrel Goll; California - Evertt Bandman; Hawaii - Yong Soo Kim; Idaho - Rod Hill; Indiana - David Gerrard, Alan Grant; Iowa - Ted Huiatt, James Reecy; Michigan - Matt Doumit, Catherine Ernst; Minnesota - Bill Dayton, Michael White; Kansas - Brad Johnson; Nebraska - Mike Zeece; Ohio - Sandra Velleman; Oregon - Neil Forsberg; Washington - Michael Dodson; Wisconsin - Marion Greaser; Administrative Advisor - Elton Aberle, Wisconsin; CSREES Representative - Larry Miller

Brief Summary of Minutes

The annual meeting, chaired by Dave Gerrard, was held at Purdue University, West Lafayette, IN. Alan Grant, Animal Sciences Department Head, provided a welcome to Purdue University. Larry Miller described the reorganization of CSREES, the forthcoming meeting of the International Congress of Meat Science and Technology at Baltimore, MD in 2005, and a history of meat science research he has published. Dr. Debora Hamernik will replace Larry as CSREES liaison to NC-131. The committee thanked Larry for his years of participation and service to NC-131. Elton Aberle discussed the current federal budget deliberations for FY03. He indicated that Hawaii, Idaho, and Oregon were new participants in NC-131. John Killefer has resigned his position at West Virginia to accept a position at the University of Illinois and Illinois will be joining the project. He reminded the committee that mid-term reviews of NC-131 by the Administrative Advisor and the NCA-6 committee would be submitted before February, 2003. The committee should plan to discuss revision of the project at its meeting in October, 2003 and plan to have a revision completed by December 1, 2004, if it wishes to continue beyond September 30, 2005. Katherine Byrne has left Washington State University and will no longer participate in this project. Matt Doumit, who served as secretary for 2001-02, was elected chair and Marion Greaser was elected secretary for 2002-03.

The full annual report and minutes are available on the NC-131 web page,

http://ars.sdstate.edu/nc131/main.htm

Minutes Attachment:

Attachment

Accomplishments

Progress reports were presented by all institutions in attendance; in addition, written reports were distributed from the Pennsylvania, South Dakota and Utah stations, whose representatives were not able to attend the meeting. Project participants continue to aggressively acquire information about the fundamental biological process that regulate muscle growth and development of meat-producing animals. Substantial progress was made to better understand the extracellular signals, intracellular signal transduction pathways, and nuclear mechanisms that govern myoblast and satellite cell activity. These include signals that stimulate satellite cells to enter the cell cycle and the roles of insulin-like growth factors (IGFs), IGF binding proteins, and fibroblast growth factors in satellite cell and myoblast proliferation. Anabolic steroid effects on muscle growth appear to be mediated through the IGF pathway. Significant progress was also made to document differences in extracellular matrix proteins in skeletal muscles of rapid and slow growing genetic strains. Investigations of the intracellular roles of the calpains in living cells were continued. Inhibition of calpain activity prevents formation of integrin clusters and the ability of cells in culture to spread, form focal adhesions, actin filament networks, and stress fibers. Several stations continue to investigate gene expression and control in myogenic cells. Down regulation of important muscle genes was demonstrated in myoblasts expressing elevated levels of Raf and MAPK; the mechanism of down regulation is being investigated. The gene JAK2 is markedly elevated during work induced hypertrophy. When this gene is blocked with a specific inhibitor, however, myoblast differentiation and myotube maturation were prevented, demonstrating the importance of this gene in differentiation of skeletal muscle. Investigation continued of the biochemistry and molecular biology of the myosin heavy chain multi-gene family. It was demonstrated that a muscle fiber contains a series of nuclear domains, each responding to distinct localized signaling mechanisms that may result in differential gene expression within a single fiber. For example, the tapered ends of fibers contain neonatal myosin heavy chain isoform in addition to the adult isoform found throughout the length of the fiber. It appears that during development, neonatal myosin is initially repressed near the nerve motor endplate and that trophic factors emanating from the vicinity of the motor endplate represent a potential localized signaling pathway that differentially modulates myosin heavy chain gene expression in nuclear domains along the length of the muscle fiber. There are significant increases in the size of nuclear domains within a muscle fiber as it transforms from neonatal to adult type. Project participants continue to investigate how calpain is regulated in living cells. Significant progress was made in purification of m- and 5-calpains and calpastatins on immunoaffinity columns and their subsequent quantification in various muscles and tissues. Cell lines have been developed in which the activities of m- and 5-calpains may be regulated and their functions in muscle wasting identified. These cell lines are being used to delineate the roles of calpains in muscle protein degradation. The roles of two novel intermediate filament proteins, synemin and paranemin, in developing skeletal are being investigated. Paranemin is expressed first and localized in intermediate filaments and then co-localized with synemin and desmin throughout myofibrillogenesis in myotubes. Thus, paranemin and synemin may play a role in integrating intermediate filaments into the cytoskeleton during myofibrillogenesis. Finally, atomic force microscopy is being used to characterize single molecule force-extension curves of titin molecules. Titin contains unique repeated amino acid sequences that are responsible for physiological levels of passive force in moderately to highly stretched muscle fibers. These techniques are demonstrating differences among titin molecules from different species and types of muscle that account for differing levels of passive force. Committee members continue to be highly collaborative through the sharing of techniques, culture models, antibodies, molecular probes, and expertise. They use and frequently improve upon current methods in cell and molecular biology, genomics, and proteomics to address fundamental questions. They are highly productive as indicated by the large number of publications, including a significant number of joint publications among institutions. A full description of project accomplishments may be found on the NC-131 web page, <a href="http://ars.sdstate.edu/nc131/main.htm">http://ars.sdstate.edu/nc131/main.htm</a>.

Publications

Committee members published 42 full-length papers in refereed journals during the period of this report; 17 other full-length papers were accepted for publication. In addition, they published 40 abstracts of papers presented at professional meetings and 15 non-referred publications as proceedings and book chapters. The full listing of publications can be found in the NC-131 annual report for 2001-02, which is available on the NC-131 web page, <a href="http://ars.sdstate.edu/nc131/main.htm">http://ars.sdstate.edu/nc131/main.htm </a>.

Impact Statements

Information gained in the project continues to provide the foundation for the development of novel strategies to improve muscularity, the rate and efficiency of muscle growth, and meat quality. For example, understanding how proteolytic activity of calpains is regulated may provide the opportunity to increase the rate of muscle growth with little or no increase in ingested energy, just as our current understanding of calpain activation has provided novel methods to improve meat tenderization.
Understanding regulation of myoblast activation, proliferation, and differentiation by growth factor signaling offers potential to manipulate prenatal muscle fiber formation as well as postnatal satellite cell-mediated accumulation of myofiber nuclei.
Elucidating the mechanisms that control myofiber gene expression and subsequent phenotype may provide opportunities to improve live animal efficiency.
Likewise, manipulation of muscle protein isoform distribution may alter postmortem metabolism and subsequent meat quality.

Date of Annual Report: 01/08/2004

Report Information

Annual Meeting Dates: 10/24/2003 - 10/25/2003
Period the Report Covers: 10/01/2002 - 09/01/2003

Participants

Goll, Darrel(darrel.goll@arizona.edu)-Univ.of Arizona; Kim, Y.S. (ykim@hawaii.edu)-Univ. of Hawaii; Gerrard, David (dgerrard@purdue.edu)-Purdue Univ.; Reecy, Jim (jreecy@iastate.edu)-Iowa State Univ.; Johnson, Brad (bjohnson@ksu.edu)-Kansas State Univ.; Doumit, Matthew (doumitm@msu.edu)-Michigan State Univ.; Ernst, Catherine (ernstc@msu@edu)-Michigan State Univ.; Dayton, William (wdayton@umn.edu)-Univ. of Minnesota; Hathaway, Marcia (hathawaym@umn.edu)-Univ. of Minnesota; White, Michael (mwhite@umn.edu)-Univ. of Minnesota; Jones, Steven (sjones@unl.edu)-Univ. of Nebraska-Lincoln; Mozdziak, Paul (pemozdzi@unity.ncsu.edu)-North Carolina State Univ.; Velleman, Sandra (velleman.1@osu.edu)-Ohio State Univ.; Forsberg, Neil (neil.forsberg@orst.edu)- Oregon State Univ.; Johnson, Sally (sej4@psu.edu)-Pennsylvania State Univ.; Rodgers, B. Dan (danrodgers@wsu.edu)-Washington State Univ.; Greaser, Marion (mgreaser@facstaff.wisc.edu)-Univ. of Wisconsin-Madison; Aberle, Elton (eaberle@cals.wisc.edu)-Univ. of Wisconsin-Madison, Admin.Advisor; Hamernik, Debora (dhamernik@csrees.usda.gov)-USDA-CSREES

Brief Summary of Minutes

The annual meeting was held at Michigan State University, October 24-25, 2003 and chaired by Matt Doumit.

Deb Hamernik, CSREES Representative, provided update information on USDA personnel changes and competitive grant programs. A major change in competitive grant programs is an increase in size to grants to a range of $300,000 to $500,000 each. Unfortunately, this will have the effect of reducing the number of grants funded. Elton Aberle, Administrative Advisor, reminded the committee that the project is funded through September 30, 2005. A revised project proposal must be submitted and approved by the NCRA if the project is to continue beyone 2005. Aberle reviewed the process for a project renewal/revision and the timelines that must be met in 2004-05. Use of the NIMSS system in the writing of a revised/new project was reviewed. The revised project must be submitted by December 1, 2004.

The committee agreed to seek renewal of the NC131 project for the period 2005-2010. The same three objectives will be used for the next project period. These objectives and the stations that plan to address them are as follows:

(1) Characterize the signal transduction pathways that regulate skeletal muscle growth and differentiation

Arizona, Hawaii, Idaho, Indiana, Kansas, Michigan, Minnesota, North Carolina, Ohio, South Dakota, Pennsylvania, Utah, and Washington

(2) Determine the nuclear mechanisms that control gene expression in skeletal muscle

Arizona, Illinois, Iowa, Minnesota, Pennsylvania, Nebraska, South Dakota, Utah, Washington, Wisconsin

(3) Characterize muscle proteins and their functional domains involved in myofibrillar assembly and disassembly

Arizona, Iowa, Nebraska, Oregon, and Wisconsin

Each station is to prepare a one-page summary of the 5-year plans for their station. Literature citations and methods descriptions should also be included. These summaries should be submitted to the following committee members for assembly:

Objective 1 Minnesota Station (Dayton, White, Hathaway)

Objective 2 Purdue Station (Gerrard)

Objective 3 Arizona and Oregon Stations (Goll, Forsberg)

Marion Greaser has agreed to prepare a draft of the Statement of Issues and Justification. Bill Dayton has been appointed to serve as the overall coordinator of the project re-write.

Marion Greaser will move from secretary to chair for next year and the committee meeting will be held in Wisconsin in mid-October. Dr. Neil Forsberg (Oregon State) was unanimously elected secretary for next year.

The business meeting was adjourned. The committee continued with individual station reports.

Accomplishments

The goal of this project is to increase the efficiency of lean meat production in domestic animals through the use of basic research into the biological mechanisms that regulate muscle growth and differentiation. The results summarized in this project report demonstrate that the project participants continue to aggressively acquire information about the fundamental biological processes that regulate muscle development and growth of meat-producing animals. The committee continues to be highly productive as indicated by the number of publications appearing in the last year. Collaboration through sharing of techniques, culture models, antibodies, molecular probes, and expertise continues to be an important aspect of the project. Committee members use and frequently improve current methods in cell and molecular biology, genomics and proteomics to address fundamental problems relevant to meat animal production. Members have made substantial contributions toward understanding the extracellular signals, intracellular signal transduction pathways, and nuclear mechanisms that govern both myoblast/satellite cell activity and muscle protein accumulation. The activation of satellite cells to proliferate when muscle is stretched appears to involve both hepatocyte growth factor and nitric oxide synthase. Over-expression of fibroblast growth factor 1 receptor in muscle after in vivo transfection protects muscle against disuse atrophy. Janus kinase 2 and MEKK1 pathways affect muscle cell growth and muscle gene expression. Each of these pathways offers possibilities for pharmacological intervention or targets for genetic selection to increase muscle accretion. A number of experiments also point to the importance of IGFBP-3 in muscle cell proliferation and differentiation. Both estrogenic and androgenic steroids have been demonstrated to show anabolic effects on cultured satellite cells. Elevation of IGF-1 and IGFBP may be the mechanism by which sub-therapeutic doses of anti-microbials improve muscle growth. Zinc has been shown to be an important in the binding of insulin-like growth factor II (IGF-II) to its receptor. Procedures have been developed to generate transgenic chickens. This is the first report where such transgenes actually express protein from the inserted gene. These chickens, with inserted beta-galactosidase, appear to be able to digest lactose, and thus have the potential to utilize high lactose containing feedstuffs. A normalized porcine skeletal muscle cDNA library was developed and clones from this library have been used to construct a cDNA microarray. Initial results demonstrate that this technology will provide a rapid and powerful tool for identifying differentially expressed genes that are important for skeletal muscle development and growth. In addition, a cDNA macroarray developed at Iowa State University has been used to identify putative differentially expressed genes in proliferating and differentiating porcine skeletal muscle satellite cells. These genes can now be evaluated to further elucidate their roles in myogenesis. Microarray and reporter gene studies have identified several proteins that are up-regulated by ractopamine, including calmodulin, a major calcium regulatory protein of the cell. The committee has also made significant progress in understanding muscle protein characteristics and the regulation of myofibrillar assembly and disassembly. A number of studies have provided new insights on the role of calpains in muscle function. Proteolytic cleavage experiments have demonstrated that the shape of m- and m-calpains changes from a compact, fairly protease resistant to an open, easily cleaved arrangement in the presence of calcium. A quantitative immunofluorescence method has been developed to track postmortem changes in calpain 3. A new electrophoretic method has also been developed using agarose and it should prove valuable for characterizing high molecular weight muscle proteins and their fragments. Information gained by the project continues to provide the foundation for the development of novel strategies to improve muscularity, the rate and efficiency of muscle growth, and meat quality. For example, understanding how proteolytic activity of calpains is regulated may provide the opportunity to increase the rate of muscle growth with little or no increase in ingested energy, just as current understanding of calpain activation has providing novel methods to improve meat tenderization. Understanding regulation of myoblast activation, proliferation and differentiation by growth factor signaling offers potential to manipulate prenatal muscle fiber formation as well as postnatal satellite cell-mediated accumulation of myofiber nuclei. Elucidating the mechanisms that control myofiber gene expression and subsequent phenotype may provide opportunities to improve live animal efficiency. Likewise, manipulation of muscle protein isoform distribution may alter postmortem metabolism and subsequent meat quality. Developments based on fundamental research done by the committee are essential to the viability of animal agriculture. For the next year, studies will continue as described in the approved proposal. No changes in the approach or objectives are anticipated at this time.

Publications

Refereed Journal Articles Published Full-Length Articles Arizona Goll, D.E., V.F. Thompson, H. Li, W. Wei, and J. Cong. 2003. The calpain system. Physiol. Rev. 83: 731-801. Thompson, V.F., K.R. Lawson, K.R., J. Barlow, J., and D.E. Goll. 2003. Digestion of m- and m-calpain by trypsin and chymotrypsin. Biochim. Biophys. Acta 1648: 140-153. Idaho Hendricksen, R.E., C. Gazzola, M.M. Reich, R.F. Roberton, D.J. Reid, and R.A.Hill. 2003. Using molasses as an alternative to controlled release devices for administering n-alkane markers to cattle. Animal Sci.76: 471-480. Illinois Kocamis, H., and J. Killefer. 2003. Expression profiles of IGF-1, IGF-II, bFGF and TGFB2 growth factors during chicken embryonic development. Turk. J. Vet. Anim. Sci. 367-372. McCusker, R. H., R. L. Mateski, J. Novakofski. 2003. Zinc alters the kinetics of IGF-II binding to cell surface receptors and binding proteins. Endocrine 21(3): 279-288. McCusker, R. H. and J. Novakofski. 2003. Zinc partitions Insulin-Like Growth Factors (IGFs) from soluble IGF Binding Protein (IGFBP)-5 to the cell surface receptors of BC3H-1 muscle cells. J. Cell. Physiol. 197(3): 388-399. Indiana Hammelman, J.E., B.C. Bowker, A.L. Grant, J.C.Forrest, A.P. Schinckel, and D.E. Gerrard. 2003. Early postmortem electrical stimulation simulates PSE pork development Meat Sci. 63: 69-77. Hatem, L., J. Tan, and D.E. Gerrard. 2003. Determination of animal skeletal maturity by image processing. Meat Sci. 65: 999﷓1004. Iowa Carlsson, L., C. Fischer, G. Sjoberg, R. M. Robson, T. Segersen and L.﷓E. Thomell. 2002. Cytoskeletal derangements in hereditary myopathy with desmin L345P mutation. Acta Neuropathol. 104: 493﷓504. Kansas Johnson, B. J., M.E. White, M.R. Hathaway, and W.R. Dayton. 2003. Effect of differentiation on levels of insulin-like growth factor binding protein mRNAs in cultured porcine embryonic myogenic cells. Dom. Anim. Endocrinol. 24: 81-93. White, M. E., B.J. Johnson, M.R. Hathaway, and W.R. Dayton. 2003. Growth factor mRNA levels in muscle and liver of steroid-implanted and nonimplanted steers. J. Anim. Sci. 81: 965-972. Pampusch, M. S., B. J. Johnson, M. E. White, M. R. Hathaway, J. D. Dunn, A. T. Waylan, and W. R. Dayton. 2003. Time course of changes in growth factor mRNA levels in muscle of steroid-implanted and non-implanted steers. J. Anim. Sci. 81: 2733-2740. Michigan Scheffler J.M., D.D. Buskirk, S.R. Rust, J.D. Cowley and M.E. Doumit. 2003. Effect of repeated administration of combination trenbolone acetate and estradiol implants on growth, carcass traits, and beef quality of long﷓fed Holstein steers. J. Anim. Sci. 81: 2395﷓2400. Yao, J., P.M. Coussens, P. Saama, S. Suchyta, and C.W. Ernst. 2002. Generation of expressed sequence tags from a normalized porcine skeletal muscle cDNA library. Anim. Biotech. 13: 211﷓222. Farber, C.R., N.E. Raney, D.L. Kuhlers, K. Nadarajah and C.W. Ernst. 2003. Identification of genetic markers between two pig populations using representational difference analysis. Anim. Biotech. 14: 87﷓102. Zhao, S., D. Nettleton, W. Liu, C.W. Fitzsimmons, C.W. Ernst and C.K. Tuggle. 2003. cDNA macroarray analyses of differential gene expression in porcine fetal and postnatal skeletal muscle. J. Anim. Sci. 81: 2179﷓2188. Minnesota Pampusch, M. S., E. Kamanga-Sollo, M. E. White, M. R. Hathaway, and W. R. Dayton. 2003. Effect of recombinant porcine IGF-binding protein-3 on proliferation of embryonic porcine myogenic cell cultures in the presence and absence of IGF-I. J. Endocrinol. 176: 227-235. Kamanga-Sollo, E., M. S. Pampusch, M. E. White, and W. R. Dayton. 2003. Role of insulin-like growth factor binding protein (IGFBP)-3 in TGF-beta- and GDF-8 (myostatin)-induced suppression of proliferation in porcine embryonic myogenic cell cultures. J. Cell Physiol. 197: 225-231. Johnson, B. J., M. E. White, M. R. Hathaway, and W. R. Dayton. 2003. Effect of differentiation on levels of insulin-like growth factor binding protein mRNAs in cultured porcine embryonic myogenic cells. Domest. Anim Endocrinol. 24: 81-93. Pampusch, M. S., B. J. Johnson, M. E. White, M. R. Hathaway, J. D. Dunn, A. T. Waylan, and W. R. Dayton. 2003. Time course of changes in growth factor mRNA levels in muscle of steroid-implanted and non-implanted steers. J. Anim. Sci. 81: 965-972. White, M. E., B. J. Johnson, M. R. Hathaway, and W. R. Dayton. 2003. Growth factor messenger RNA levels in muscle and liver of steroid-implanted and nonimplanted steers. J. Anim Sci. 81: 965-972. Hathaway, M. R., W. R. Dayton, M. E. White, and M. S. Pampusch. 2003. Effects of antimicrobials and weaning on porcine serum insulin-like growth factor binding protein levels. J. Anim Sci. 81: 1456-1463. North Carolina Mozdziak P.E., Pophal S., Borwornpinyo S., J.N. Petitte. 2003. Transgenic chickens expressing beta﷓galactosidase hydrolyze lactose in the intestine J. Nutr. 133 (10): 3076﷓3079. Pophal, S., J.J. Evans, and P.E. Mozdziak. 2003. Myonuclear apoptosis occurs during early posthatch starvation Comp. Biochem. Physiol. B Biochem. Mol. Biol. 135 (4): 677﷓681. Giamario, C., J.N. Petitte, and P.E. Mozdziak. 2003. Hatchability of chicken embryos following somite manipulation Biotechniques 34 (6): 1128﷓1130. Greaser M.L., M. Berri, C.M. Warren, and P.E. Mozdziak. 2002. Species variations in cDNA sequence and exon splicing patterns in the extensible I﷓band region of cardiac titin: relation to passive tension J. Muscle Res. Cell Motil. 23 (5﷓6): 473﷓482. Mozdziak P.E, J.J. Dibner, and D.W. McCoy. 2003. Glyceraldehyde﷓3﷓phosphate dehydrogenase expression varies with age and nutrition status. Nutrition 19 (5): 438﷓440. Mozdziak, P.E., C. Giamario, and J.N. Petitte, 2003. Myonuclear accretion-a brief review. An. Sci. Papers and Reports 21 (Suppl. 1) 121-131. Mozdziak, P.E., S. Borwornpinyo, D.W. McCoy, and J.N. Petitte. 2003. Development of transgenic chickens expressing bacterial beta﷓galactosidase. Dev. Dynam. 226 (3): 439﷓445. Mozdziak, P.E., T.J. Walsh, and D.W. McCoy. 2002. The effect of early posthatch nutrition on satellite cell mitotic activity. Poult. Sci. 81 (11): 1703﷓1708. Mozdziak, P.E., J.J. Dibner, and D.W. McCoy. 2002.The effect of early posthatch starvation on calpain mRNA levels. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 133 (2): 221﷓226. Mozdziak, P.E., J.J. Evans, and D.W. McCoy. 2002. Early posthatch starvation induces myonuclear apoptosis in chickens J. Nutr. 132 (5): 901﷓903. Ohio McFarland, D.C., X. Liu, S.G. Velleman, Z.Caiyun, C.S. Coy, and J.E. Pesall. 2003. Variation in fibroblast growth factor response and heparan sulfate proteoglycan production in satellite cell populations. Comp. Biochem. Physiol. C Toxicol. Pharmacol. 134:341﷓351. Wick, M., S. G. Velleman, C. S. Coy, D. C. McFarland, and C. I. Pretzman. 2003. Myosin heavy chain isoform expression is altered during in vitro myogenesis in low score normal chickens. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 136(2): 401-408. Velleman, S.G. J.W. Anderson, C.S.Coy, and K.E. Nestor. 2003. Effect of selection for growth rate on muscle damage during turkey breast muscle development. Poult. Sci. 82: 1069﷓1074. Liu, X., D.C. McFarland, K.E. Nestor, and S.G. Velleman, S.G. 2003. Expression of fibroblast growth factor 2 and its receptor during skeletal muscle development from turkeys with different growth rates. Dom. Anim. Endocrinol. 25: 215﷓229. Velleman, S.G. and K.E. Nestor, K.E. 2003. Effect of selection for growth rate on myosin heavy chain temporal and spatial localization during turkey breast muscle development. Poult. Sci. 82: 1373﷓1377. Velleman, S.G., C.S.Coy, J.W. Anderson, R.A. Patterson, and K.E. Nestor. 2003. Effect of selection for growth rate and inheritance on post hatch muscle development in turkeys. Poult. Sci. 82: 1365﷓1372. Oregon Xiao, Y.Y., M.C. Wang, J. Purintrapiban and N.E. Forsberg. 2003. Roles of u-calpain in cultuired L8 muscle cells: application of a skeletal muscle-specific gene expression system. Comp. Biochem. Physiol. C Toxicol Pharmacol. 134: 439-450. Xiao, Y.Y., M.A. Beilstein, M.C.Wang J. Purintrapiban, and N.E. Forsberg. 2003. Development of a ponasterone A-inducible expression system for application in cultured skeletal muscle cells. Int. J. Biochem. Cell. Biol. 35: 79-85. Purintrapiban, J., M.C. Wang, and N.E. Forsberg. 2003. Degradation of sarcomeric and cytoskeletal proteins in cultured skeletal muscle cells. Comp. Biochem. Physiol. B Biochem Mol Biol. 136(3): 393-401. Azuma, M., Y. Tamada, S. Kanaami, E. Nakajima, Y. Nakamura, C. Fukiage, N.E. Forsberg, M.K. Duncan, and T.R. Shearer. 2003. Differential influence of proteolysis by calpain 2 and Lp82 on in vitro precipitation of mouse lens crystallins. Biochem. Biophys. Res. Commun. 307(3): 558-63. South Dakota Wick, M., S. G. Velleman, C. S. Coy, D. C. McFarland, and C. I. Pretzman. 2003. Myosin heavy chain isoform expression is altered during in vitro myogenesis in low score normal chickens. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 136(2): 401-408. Liu, X., K. E. Nestor, D. C. McFarland, and S. G. Velleman. 2002. Developmental expression of skeletal muscle heparan sulfate proteoglycans in turkey lines with different growth rates. Poult. Sci. 81: 1621-1628. McFarland, D. C., X. Liu, S. G. Velleman, C. Zeng, C. S. Coy, and J. E. Pesall. 2003. Variation in fibroblast growth factor and heparan sulfate proteoglycan production in satellite cell populations. Comp. Biochem. Physiol. C 134: 341-351. Liu, X., D. C. McFarland, K. E. Nestor, and S. G. Velleman. 2003. Expression of fibroblast growth factor 2 and its receptor during skeletal muscle development in turkeys with different growth rates. Dom. Anim. Endocrin. 25(2): 215-219. Wisconsin Harris, S.P., W.T. Heller, M.L. Greaser, R.L. Moss, and J. Trewhella. 2003. Solution structure of heavy meromyosin by small-angle scattering. J. Biol. Chem. 278: 6034-6040. Huang, C., Q. Zhou, P. Liang, M. Hollander, F. Sheikh, X. Li, M. Greaser, G. D. Shelton, S. Evans, and J.Chen. 2003. Short or long isoforms of Cypher can rescue lethality resulting from loss of Cypher function. J. Biol. Chem. 278: 7360-7365. Warren, C.M., P.R. Krzesinski, and M.L. Greaser. 2003. Vertical agarose gel electrophoresis and electroblotting of high molecular weight proteins. Electrophoresis 24: 1695-1702. Warren, C.M. and M.L. Greaser. 2003. Method for cardiac myosin heavy chain separation by SDS gel electrophoresis. Anal. Biochem. 320: 149-151. Washington Vierck, J., D. Icenoggle, L. Bucci and M.V. Dodson. 2003. The effects of ergogenic compounds on satellite cells. Med. Sci. Sports Exer. 35: 769-776. Kuber, P.S., S.K. Duckett, J.R. Busboom, G.D. Snowder, M.V. Dodson, J.L. Vierck and J.F. Bailey. 2003. Measuring the effects of phenotype and mechanical restraint on proteolytic degradation and rigor shortening in callipyge lamb longissimus muscle during extended aging. Meat Sci. 63(3): 325-331. Published Abstracts Arizona Cong. J., V.F. Thompson, and D.E. Goll. 2003. Phosphorylation of dephosphorylated m-calpain by protein kinase C. FASEB J. 17: A1006. Cong, J., V.F. Thompson, and D.E. Goll. 2003. Phosphorylation of the calpains. 3rd General Meeting of the International Proteolysis Society, Nagoya, Japan. (in press). Saido, M., H. Li, V.F. Thompson, N. Kunisaki, and D.E. Goll, D.E. 2003. Properties of the calpain system in rainbow trout. 3rd General Meeting of the International Proteolysis Society, Nagoya, Japan. (in press). Hawaii H.Y.Jin, Y.S. Kim and M.A. Dunn. 2003. Refolding and purification of unprocessed porcine myostatin expressed in E. coli. J. Anim. Sci. 81 (Suppl. 1): 305. Idaho Cochrane, J., A.L. Strat, M.V. Dodson, A. Gertler, and R.A. Hill. 2003. The impact of serine phosphorylation on the insulin pathway. Proc. Am. Chem. Soc. (in press). Hunt, M.L., T.A. Kokta, T. OShea, R.A. Hill, and J.R. McFarlane. 2003. Leptin clearance rates during the early follicular phase are higher than during the luteal phase of the estrius cycle in ewes. Proc. Endocrin. Soc. Aust. 46: 77. Hill, R.A., M.V. Dodson, a. Gertler, N.J. Hughes, and D. Henderson. 2003. Insulin signaling in bovine myogenic cells. J. Animal Sci. 81(Suppl. 1): 96. Indiana Eash, J.K., A. Olsen, A.L. Grant, D.E. Gerrard, and K.M. Hannon. 2003. Fibroblast growth factor receptor I mediates disuse atrophy in gastrocnemius and soleus muscles of mice. J. Anim. Sci. 81: (Suppl. 2): 57. Guo, Q., A.L. Grant, B.T. Richert, A.P. Schinckel and D. E. Gerrard. 2003. Ractopamine may improve meat quality by altering postmortem metabolism. J. Anim. Sci. 81: (Suppl. 2) 53. Iowa Lee, H.﷓S., R. M. Bellin, D. L. Walker, M. H. Stromer, D. R. Critchley, and R. M. Robson. 2002. Characteristics of an actin﷓binding site (ABS) in the talin head domain and comparison to an ABS near the C﷓terminal of the molecule. Mol. Biol. Cell 13: 56a. Lex, S.A., T. W. Huiatt, M. H. Stromer and R. M. Robson. 2003. Localization of intermediate filament proteins in developing avian skeletal muscle cells. J Anim. Sci. 81 (Suppl. 2): 28. Kansas Sissom, E. K., J. P. Kayser, A. T. Waylan, J. D. Dunn, and B. J. Johnson. 2003. Effect of melengestrol acetate (MGA) on bovine muscle satellite cell proliferation and differentiation. J. Anim. Sci. 81 (Suppl. 1): 305. Dunn, J. D., A. T. Waylan, J. P. Kayser, E. K. Sissom, and B. J. Johnson. 2003. Effect of flax supplementation and a combined trenbolone acetate and estradiol implant on muscle satellite cell activity in beef cattle. J. Anim. Sci. 81 (Suppl. 1): 305. Dunn, J. D., J. P. Kayser, A. T. Waylan, E. K. Sissom, J. S. Drouillard, and B. J. Johnson. 2003. Effect of flax supplementation and a combined trenbolone acetate and estradiol implant on circulating IGF-1 and muscle IGF-1 mRNA levels in finishing cattle. J. Anim. Sci. 81 (Suppl. 2): 20. Waylan, A. T., J. P. Kayser, J. D. Dunn, E. K. Sissom, and B. J. Johnson. 2003. Effect of flax supplementation and growth promotants on steady-state lipoprotein lipase and glycogenin mRNA concentrations in finishing cattle. J. Anim. Sci. 81 (Suppl. 2): 30. Kayser, J. P., J. D. Dunn, A. T. Waylan, S. S. Dritz, J. C. Nietfeld, J. E. Minton, and B. J. Johnson. 2003. Effects of acute enteric disease challenge on the insulin-like growth factor system in nursery pigs. J. Anim. Sci. 81 (Suppl. 2): 30. Burkey T. E., S. S. Dritz, J. C. Nietfeld, B. J. Johnson, and J. E. Minton. 2003. Effect of dietary mannanoligosaccharide (MOS) and sodium chlorate (CHL) on bacterial shedding in weaned pigs challenged with Salmonella enterica serotype typhimurium (ST). J. Anim. Sci. 81(Suppl. 2): 38. Dunn, J. D., J. P. Kayser, A. T. Waylan, E. K. Sissom, J. S. Drouillard, and B. J. Johnson. 2003. Effect of flax supplementation and a combined trenbolone acetate and estradiol implant on circulating IGF-1 and muscle IGF-1 mRNA levels and satellite cell activity in beef cattle. Proc. Plains Nutrition Spring Conference. Pub. AREC 03-13. Texas A & M Research and Extension Center, Amarillo. P. 106. Waylan, A. T., J. P. Kayser, J. D. Dunn, E. K. Sissom, and B. J. Johnson. 2003. Effect of flax supplementation and growth promotants on steady-state lipoprotein lipase and glycogenin mRNA concentrations in finishing cattle. Proc. Plains Nutrition Spring Conference. Pub. AREC 03-13. Texas A & M Research and Extension Center, Amarillo. P. 119. Epp, M. P., D. A. Blasi, B. J. Johnson, and J. P. Kayser. 2003. Steroid hormone profiles and brain monoamine oxidase type A (MAO-A) activity of buller steers. Proc. Plains Nutrition Spring Conference. Pub. AREC 03-13. Texas A & M Research and Extension Center, Amarillo. P. 107. Michigan Zhao, S., D. Nettleton, W. Liu, C. Fitzsimmons, C.W. Ernst, N.E. Raney and C.K. Tuggle. 2003. cDNA macroarray analyses of differential gene expression in porcine fetal and postnatal muscle. Plant and Animal Genome XI Conference. San Diego, CA. http://www.intl﷓pag.org/l1/abstracts/P5m_P623_XI.html. Nebraska Novak, C., S. Scheideler, and S. Jones. 2003. Effect of dietary level and TSAA:Lysine ratio on body composition, egg protein and magnum tissue of the laying hen. Poultry Sci. 80: (Suppl. 1) 390. North Carolina Evans, J.J., S. Pophal, and P.E. Mozdziak. 2003. Myonuclear apoptosis during early post﷓hatch starvation. Poult. Sci. (Suppl. 1)82: 45. Mozdziak, P.E. 2003. An undergraduate laboratory course on animal cell culture techniques Poult. Sci. (Suppl. 1) 82: 255. Mozdziak, P.E., J.J. Dibner, and D.W. McCoy. 2003. Glyceraldehyde﷓3﷓dehydrogenase expression in skeletal muscle is altered by nutritional status. Poult Sci. (Suppl. 1) 82: 324 Moore, D.T., P.R. Ferket, and P.E. Mozdziak, P.E. 2003. BrdU administration to avian embryos. Poult. Sci. (Suppl. 1) 82: 351 Giamario, C., J.N. Petitte, and P.E. Mozdziak. 2003. Hatchability of chicken embryos following intrasomite injection. Poult Sci. (Suppl. 1) 82: 354. Mozdziak, P.E., S. Pophal, S. Borwornpinyo, S. Pardue, and J.N. Petitte. 2003. Transgenic chickens expressing beta﷓galactosidase. Transgen. Res (in press). Mozdziak, P.E., S. Borwornpinyo, D.W. McCoy, and J.N. Petitte. 2002. Development of transgenic poultry expressing nuclear﷓located lacZ. Poult. Sci. 81 (Supplement 1): 376. Evans, J.J., D.W. McCoy, and P.E. Mozdziak. 2002. Early post﷓hatch starvation induces myonuclear apoptosis. Poult. Sci. 81 (Supplement 1): 100. Wisconsin Watanabe, K., P. Nair, D. Labeit, M. Kellermayer, M. Greaser, S. Labeit, and H. Granzier. 2003. Molecular mechanics of cardiac titins PEVK and N2B spring elements. Biophys. J. 84: 244a. Warren, C.M. and M.L. Greaser. 2003. Vertical agarose gel electrophoresis and electroblotting of high molecular weight proteins. Biophys. J. 84: 564a. Washington He, M.L., P.S. Mir, E. Okine, and M.V. Dodson. 2003. Effect of culture media containing conjugated linoleic acid (CLA)cis (c)9,trans(t)11 and CLAt10,c12 on proliferation of 3T3-L1 preadipocytes. Can. J. Anim. Sci. 83: #55. He, M.L., P.S. Mir, E. Okine, and M.V. Dodson. 2003. Effect of culture media containing conjugated linoleic acid (CLA)cis (c)9,trans(t)11 and CLAt10,c12 on lipid accumulation in 3T3-L1 preadipocytes. Can. J. Anim. Sci 83: #56. Hill, R.A., M.V. Dodson, A. Gertler, N.J. Hughes and D. Henderson. 2003. Insulin signaling in bovine myogenic cells. J. Anim. Sci. 81: 96. Cochrane, J., L. Strat, M.V. Dodson, A. Gertler and R.A. Hill. 2003. The impact of serine phosphorylation on the insulin pathway. Proc. Am. Chem. Soc. 143: 93. Fernyhough, M.E., L.R. Bucci, J. Feliciano, J.L. Vierck and M.V. Dodson. 2003. Ergogenic supplements and their effects on muscle satellite cells. J. Am. Coll. Nutr. 22(5): 482. Accepted Full-Length Articles Arizona Li, H., V.F. Thompson, V.F., and D.E. Goll. Effects of autolysis on properties of :- and m-calpain. Biochim. Biophys. Acta (in press). Illinois McCusker, R. H. and J. Novakofski. 2003. Zinc partitions IGFs From soluble IGFBP-5, but not soluble IGFBP-4, to myoblast IGF Type 1 receptors. J. Endo. (in press). Gahr, S.A., H. Kocamis, J.J. Richter and J. Killefer. 2003. The effects of in ovo rhIGF-I administration on expression of the growth hormone secretagogue receptor (GHSR) during chicken embryonic development. Growth Dev. Aging (in press). Indiana Bowker, B.C., D.R. Swartz, A.L. Grant, and D.E. Gerrard. Method of isolation, rate of postmortem metabolism, and myosin heavy chain isoform composition influence ATPase activity of isolated porcine myofibrils. Meat Sci. (in press). Alzghoul, M.B., D.E. Gerrard, B.A. Watkins and K.M. Hannon. Ectopic expression of IGF﷓I and Shh by skeletal muscle inhibits disuse﷓mediated skeletal muscle atrophy and bone osteopenia in vivo. FASEB J. (in press). Bowker, B.C., D.R. Swartz, A.L. Grant, and D.E. Gerrard. Myosin heavy chain isoforms influence myofibrillar ATPase activity under simulated postmortem pH, calcium, and temperature conditions. Meat Sci. (in press). Iowa Potts, J.K., S.E. Echternkamp, T.P.L. Smith, and J.M. Reecy.. Characterization of Gene Expression In Double﷓Muscled and Normal﷓Muscled Bovine Embryos. Anim. Genet. (in press). Kansas Dunn, J. D., B. J. Johnson, J. P. Kayser, A. T. Waylan, E. K. Sissom, and J. S. Drouillard. Effects of flax supplementation and a combined trenbolone acetate and estradiol implant on circulating insulin-like growth factor-I (IGF-I) and muscle IGF-I messenger RNA levels in beef cattle. J. Anim. Sci. (in press). Burkey T. E., S. S. Dritz, J. C. Nietfeld, B. J. Johnson, and J. E. Minton. Effect of dietary mannanoligosaccharide and sodium chlorate on growth performance, acute phase response and bacterial shedding of weaned pigs challenged with Salmonella enterica Serotype typhimurium. J. Anim. Sci. (in press). Anderson, P. T. and B. J. Johnson. Effects of metabolic modifiers in food animal production. Encyclopedia of Meat Sciences (in press). Michigan Wesolowski, S.R., N.E. Raney and C.W. Ernst. Developmental changes in the fetal pig transcriptome. Physiol.Genom. (in press). Farber, C.R., N.E. Raney, V.D. Rilington, P.J. Venta and C.W. Ernst. Comparative mapping of genes flanking the human chromosome 12 evolutionary breakpoint in the pig. Cytogenet. Genome Res. (in press). Martinez, M.M., G.M. Hill, J.E. Link, N.E. Raney, R.J. Tempelman and C.W. Ernst. Impact of pharmacological zinc and phytase on liver metallothionein concentration and mRNA abundance in the young pig. J. Nutr. (in press). Minnesota Kamanga-Sollo, E, M. S. Pampusch, G. Xi, M. E. White, M. R. Hathaway, and W. R. Dayton. IGF-I mRNA Levels in Bovine Satellite Cell Cultures: Effects of Fusion and Anabolic Steroid Treatment. J. Cell Physiol. (In press). Ohio Velleman, S.G., Anderson, J.W., and Nestor, K.E. Possible maternal inheritance of breast muscle morphology in turkeys at sixteen weeks of age. Poultry Sci. (in press). Velleman, S.G., and McFarland, D.C. Beta I integrin mediation of myogenic differentiation: Implications for satellite cell differentiation. Poultry Sci. (in press). Pennsylvania Wang, X., S.R. Thomson, J.D. Starkey, J.L. Page, A.D. Ealy and S.E. Johnson. Transforming growth factor beta-1 (TGF - beta 1) is up-regulated by activated Raf in skeletal myoblasts but does not contribute to the differentiation-defective phenotype. J. Biol. Chem. (in press). South Dakota Wick, M., S.G. Velleman, C.S. Coy, D.C. McFarland, and C.I. Pretzman. Myosin heavy chain isoform expression is altered during in vitro myogenesis in low score normal chickens. Comp. Biochem. Physiol. (in press). Velleman, S.G., Anderson, J.W., and Nestor, K.E. Possible maternal inheritance of breast muscle morphology in turkeys at sixteen weeks of age. Poult. Sci. (in press). Velleman, S.G., and McFarland, D.C. Beta I integrin mediation of myogenic differentiation: Implications for satellite cell differentiation. Poult. Sci. (in press). Washington Kuber, P.S., J.R. Busboon, E. Huff-Lonergan, S.K. Duckett, P.S. Mir, Z. Mir, R.G. McCormick, M.V. Dodson, C.T. Gaskins, J.D. Cronrath, D.J. Marks, and J.J. Reeves. Effects of biological type and dietary fat treatments on factors associated with tenderness I. Measurements on beef longissimus muscle. J. Anim. Sci. (in press). Cochrane, J., L. Strat, M.V. Dodson, A. Gertler and R.A. Hill. 2003. The impact of serine phosphorylation on the insulin pathway. Proc. Am. Chem. Soc.(in press). Dodson, M.V. Publishing in BAM is an act of benevolence, vanity, stupidity or just good science: Reflection of a BAM supporter. Basic and Appl. Myol. 13(6): (in press). Holman, B. and M.V. Dodson. Creatine monohydrate attenuates satellite cell proliferation. McNair J. 11(in press). Fernyhough, M.E., L.R. Bucci, J. Feliciano, J.L. Vierck and M.V. Dodson. Ergogenic supplements and their effects on muscle satellite cells. J. Am. Coll. Nutr. (in press). Non-refereed Publications Proceedings, Book Chapters, etc. Arizona Allen, R.E. and D.E. Goll. 2003. Cellular and developmental biology of skeletal muscle as related to muscle growth. In: Scanes, C.G., (ed.) Biology of Growth of Domestic Animals, Iowa State University Press, Ames, IA. pp 148-169. Idaho Hill, R.A., F.R. Dunshea, and M.V. Dodson. 2003. Growth and livestock. In: Scanes, C.G. (ed.) Biology of Growth of Domestic Animals. Iowa State University Press, Ames, IA. pp 342-364. Hill, R.A., J.M. Pell, and D.J. Flint. 2003. Immunomodulation of growth. In: Scanes, C.G. (ed) Biology of Growth of Domestic Animals. Iowa State University Press, Ames, IA. pp 316-341. Ohio Velleman, S.G. 2003. Extracellular matrix and growth. In: Scanes, C.G. (ed.) Biology of Growth of Domestic Animals, Iowa State University Press, Ames, IA. pp 134-147. South Dakota McFarland, D.C. 2003. Nutrition and growth. In: Scanes, C.G. (ed.) Biology of Growth of Domestic Animals, Iowa State University Press, Ames, IA. pp 249-262. Washington Calza, R.E. and M.V. Dodson. 2003. Approaches to assess animal growth potential. In: Scanes, C.G. (ed.) Biology of Growth of Domestic Animals, Iowa State University Press, Ames, IA. pp 27-58. Hill, R., F. Dunshea and M. Dodson. 2003. Growth and livestock. In: Scanes C.G. (ed). Biology of Growth of Domestic Animals, Iowa State University Press, Ames, IA. pp 342-364. Dissertation/Thesis Hawaii Lee, Y.K. 2003. Production and characterization of anti-myostatin antibody. M.S. Thesis, University of Hawaii, Honolulu, Hawaii, 101p. Indiana Bowker, B.C. 2003. Effects of myosin heavy chain isoform on postmortem myofibril functionality and meat quality. PhD Thesis, Purdue University, West Lafayette, Indiana, 193p. Nebraska Edeal, J. B. 2003. Identifying differentially expressed genes in C2C 12 myogenic cells using differential display PCR. M. S. Thesis, University of Nebraska, Lincoln, Nebraska, 101p.

Impact Statements

Several pathways have been identified and futher characterized whereby satellite cells are activated to proliferate. Each of these pathways offers possibilities for pharmacological intervention or targets for genetic selection to increase muscle protein accretion and muscle growth.
Procedures have been developed to generate transgenic chickens. This is the first report where such transgenes actually express protein from an inserted gene. Chickens with the inserted beta-galactosidase gene appear to be able to digest lactose, and thus have the potential to utilize high lactose-containing feedstuffs.
cDNA libraries were constructed and used to develop cDNA microarrays. This technology can be used to identify differentially expressed genes in skeletal muscle development and growth or in proliferating and differentiating satellite cells. These genes can now be evaluated further for their roles in myogenesis and may be targets for genetic selection to increase muscle growth.
A number of studies have provided new insights on the role of calpains in muscle function. Proteolytic cleavage experiments have demonstrated that the shape of m- and m-calpains changes from a compact, fairly protease resistant to an open, easily cleaved arrangement in the presence of calcium. A quantitative immunofluorescence method has been developed to track postmortem changes in calpain 3.
A new electrophoretic method has been developed using agarose and it should prove valuable for characterizing high molecular weight muscle proteins and their fragments.

Date of Annual Report: 12/16/2004

Report Information

Annual Meeting Dates: 10/29/2004 - 10/30/2004
Period the Report Covers: 10/01/2003 - 09/01/2004

Participants

Goll, Darrel (darrel.goll@arizona.edu)-Arizona; Bandman, Evertt (edbandman@ucdavis.edu); Kim, Yong Soo (ykim@hawaii.edu)-Hawaii; Hill, Rodney (rodhill@uidaho.edu)-Idaho; Killefer, John (jkillef@uiuc.edu)-Illinois; Gerrard, David (dgerrard@purdue.edu)-Indiana; Doumit, Matt (doumitm@msu.edu)-Michigan; Dayton, Bill (wdayton@umn.edu)-Minnesota; Hathaway, Marcia (hathaway@umn.edu)- Minnesota; White, Michael (mwhite@umn.edu)-Minnesota; Zeece, Michael (mzeece@unl.edu)-Nebraska ; Mozdziak, Paul (pemozdzi@unity.ncsu.edu)-North Carolina; Velleman, Sandra (velleman.1@osu.edu)-Ohio; Forsberg, Neil (neil.forsberg@orst.edu)-Oregon; McFarland, Douglas (Douglas_McFarland@sdstate.edu) -South Dakota; Carpenter, Charles (chuckc@cc.usu.edu)-Utah; Dodson, Michael (dodson@wsu.edu)-Washington; Greaser, Marion (mgreaser@facstaff.wisc.edu) -Wisconsin; Aberle, Elton (eaberle@cals.wisc.edu) Wisconsin

Brief Summary of Minutes

The annual meeting of the NC-131 technical committee was held in Madison, Wisconsin on October 29-30, 2004. The meeting was called to order by committee chair, Marion Greaser.

Discussions focused on progress necessary to re-submit the NC-131 project for funding. Status of each of the subsections was discussed and methods for submitting the individual segments into the NIMSS program were discussed. The individual objectives of the revised NC-131 program would include:

1. Characterize signal transduction pathways that regulate skeletal muscle growth and differentiation.

2. Determine the nuclear mechanisms that control gene expression in skeletal muscle.

3. Characterize muscle proteins and their functional domains involved in myofibrillar protein assembly and disassembly.

Each of the stations has, by now, submitted draft versions of accomplishments and objectives for each of the objectives. Chairs for each section were to collate this information and to provide information to Dr. Greaser within 2 weeks.

In the later business meeting, it was decided that next years meeting would be in Corvallis, Oregon and that Neil Forsberg would serve as Chair. Dr. Kim agreed to serve as Secretary of NC-131 for 2005 and to host the NC-131 meeting in Hawaii in 2006. Costs for travel to Hawaii were not considered to be burdensome as many discount flights are available.

Annual reports were due on 12/1/04 and should be sent to Dr. Aberle.

Dr. Doug McFarland requested electronic versions of reports to be sent to him for posting on the NC-131 website.

Dr. Aberle then presented a detailed report on changes in nomenclature proposed for NC reports and updated the group on progress of congressional appropriation bills. He reported that the NRI budget in Senate and House versions stood at $180 million, a slight increase from past years. He also apprised the group on discussions regarding development of a National Institute of Food and Agriculture which would grow to an annual budget of $1 billion over 5 years, if approved. Dr. Aberle queried the group on progress of the NC-131 renewal and determined that only Objective 3 remained to be submitted. Dr. Darrel Goll agreed to spearhead this task.

The business meeting was adjourned.

A complete version of the minutes can be found at the project's website, http://ars.sdstate.edu/nc131/main.htm.

Accomplishments

The mechanisms by which hepatocyte growth factor (HGF) regulates myogenic cell proliferation and differentiation were further defined. HGFstimulates satellite cell proliferation and is present in the extracellular compartment of skeletal muscle, as are serine proteases that can cleave pro-HGF. Nitric oxide synthase is stimulated by calcium and calmodulin to produce nitric oxide, which stimulates release of active HGF. Several experiments indicate that insulin-like growth factor binding protein-5 (IGFBP-5) affects muscle cell proliferation. IGFBP-5 suppresses proliferation in porcine embryonic myogenic cell cultures through both independent effects and in concert with myostatin and transforming growth factor-b1. In ovo administration of anti-myostatin antibody did not affect growth of broiler chicks but tended to increase the proportion of breast muscle. There seems to be cross-talk between the insulin and leptin signaling pathways in bovine myogenic cells and mouse Sol8 myogenic cells. Progestins have an anti-proliferative effect on myogenic cells which seems to be mediated through a mechanism other than transcriptional regulation by the progesterone receptor. Skeletal muscle fiber hypertrophy induced by b-agonists is fiber type-dependent and coupled to fiber type transitions to the fast-twitch phenotype. Activation of ERK and Akt/Gsk3 signaling pathways is greater in fast twitch muscles than slow twitch muscles during b-agonist induced hypertrophy. Adenosine monophosphate-activated protein kinase and the ERK-MAPK signaling pathways act synergistically to determine fiber type specification. Procedures have been developed to detect differentially expressed genes in developing pig skeletal muscle. High density oligonucleotide arrays showed 62 genes were differentially expressed in muscles between day 60 of gestation and 7 weeks of age. Measurements of differential gene expression are underway in the hypertrophy-responsive gluteus medius muscle compared to the hypertrophy-nonresponsive supraspinatus muscle of callipyge lambs. A c-DNA clone of chicken heterogeneous nuclear ribonucleoprotein was prepared and will be employed to study the role of these proteins in avian species. New insights continue to emerge into the role of the calpains and calpastatin in muscle function. While only one calpastatin gene has been identified in all species studied, as many as 8 or 9 different calpastatin polypeptides can be produced by the single gene by the use of different promoters and alternative splicing events. Six different calpastatin isoforms were expressed in E. coli. All isoforms are tight binding inhibitors of calpains, but differ in their ability to inhibit calpains. The presence of different isoforms in a single tissue may serve to modulate inhibition of calpain activity. Calpain from fish species seems to have different functional and activation mechanisms than mammalian calpain. Thin filament lengths differ significantly between myofibrils from beef rabbit, and chicken muscles. These differences may explain differences in muscle texture between species. High resolution agarose electrophoresis systems have allowed analysis of developmental changes in titin isoform expression. Alternative splicing patterns of titin change predictably during development from the neonate to the adult. The time course of titin isoform switching is similar to that occurring in myosin and troponin I during development. A more detailed annual report can be found at the project's website, <a href="http://ars.sdstate.edu/nc131/main.htm">http://ars.sdstate.edu/nc131/main.htm</a>.

Publications

Referreed Papers: Arizona: Li, H., V. F. Thompson, and D. E. Goll. 2004. Effects of autolysis on properties of the calpains. Biochim. Biophys. Acta 1691: 91-103. (Listed as in press in last years report). Mendias, C. L., R. Tatsumi, and R. E. Allen. 2004. The role of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in skeletal muscle satellite cell proliferation, differentiation, and fusion. Muscle and Nerve 30:497-500. Tatsumi, R., K. Mitsuhashi, K. Ashida, A. Haruno, A. Hattori, Y. Ikeuchi, and R. E. and Allen, R. E. 2004. Comparative analysis of mechanical stretch-induced activation activity of back and leg muscle satellite cells in vitro. Animal Science Journal (Japan) 75:345-351. Tatsumi, R., and R. E. Allen. 2004. Active hepatocyte growth factor is present in skeletal muscle extracellular matrix. Muscle and Nerve 22:654-658. Wendt, A., V. F. Thompson, and D. E. Goll. 2004. Interaction of calpastatin with calpain: a review. Biol. Chem. 385: 465-472. Zalewska, T., V. F. Thompson, and D. E. Goll. 2004. Effect of phosphatidylinositol and inside-out erythrocyte vesicles on autolysis of µ- and m-calpain from bovine skeletal muscle. Biochim. Biophys. Acta 1693: 125-133. Hawaii: H.J. Jin, M.A. Dunn, D. Borthakur and Y.S. Kim. 2004. Refolding and purification of unprocessed porcine myostatin expressed in Escherichia coli. Protein Expression and Purification 35:1-10. Idaho: Kinkel, A.D., Fernyhough, M.E., Helterline, D.L., Vierck, J.L., Oberg, K.S., Vance, T.J., Hausman, G.J., Hill, R.A. and Dodson, M.V. (2004) Oil red-O stains non-adipogenic cells: A precautionary note. Cytotechnology. (accepted). Hill, R.A., Strat, A.L., Hughes, N.J., Kokta, T.A., Dodson, M.V., and Gertler, A. (2004). Early Insulin Signaling Cascade In A Model of Oxidative Skeletal Muscle: Mouse Sol8 Cell Line. Biochimica et Biophysica Acta 1693: 205-211. Fernyhough, M.E., Helterline, D.L., Vierck, J.L., Hill, R.A. and Dodson, M.V. (2004). Coulter Counter use in the enumeration of muscle and fat stem cells. Methods in Cell Science 25(3-4):221-225. Kokta, T.A., Dodson, M.V., Gertler, A. and Hill, R.A. (2004). Intercellular Signaling Between Adipose Tissue and Muscle Tissue. Domestic Animal Endocrinology. (in press). Hendricksen, RE, Gazzola, C, Reich, MM, Roberton, RF, Reid, DJ, and Hill, RA (2003) Using molasses as an alternative to controlled release devices for administering n-alkane markers to cattle Animal Science 76:471-480. Illinois: Gahr, S.A., Kocamis, J.J., J. Killifer. The effects of in ovo rhIGF-1 administration oin expression of the GH secretagogue receptor (GHSR) during chicken embryonic development. Growth, Development and Aging. 68:3-10, 2004. McCusker, R. and J. Novakofski. Zinc partitions IGFs from soluble IGF-binding proteins (IGFBP)-5, but not soluble IGFBP-4, tpo myoblast IGF type 1 receptors. J. Endocrinol. 180:227-246, 2004. Salem, M., J.Killifer. Cloning of the calpain regulatory subunit cDNA from fish revelas a divergent domain V. Anim. Biotech. 15:1-13, 2004. Indiana: Bowker, B.C., Botrel, C., Swartz, D.R., Grant, A.L., Gerrard, D.E. (2004) Influence of myosin heavy chain isoform expression and postmortem metabolism on the ATPase activity of muscle fibers. Meat Science. 68:587-594. Bowker, B.C., Grant, A.L., Swartz, D.R., Gerrard, D.E. (2004) Myosin heavy chain isoforms influence myofibrillar ATPase activity under simulated postmortem pH, calcium, and temperature conditions. Meat Science. 67:139-147. Bowker, B.C., Swartz, D.R., Grant, A.L., Gerrard, D.E. (2004) Method of isolation, rate of postmortem metabolism, and myosin heavy chain isoform composition influence ATPase activity of isolated porcine myofibrils. Meat Science. 66:742-753. Alzghoul M.B., Gerrard D., Watkins B.A., Hannon K. (2004) Ectopic expression of IGF-I and Shh by skeletal muscle inhibits disuse-mediated skeletal muscle atrophy and bone osteopenia in vivo. FASEB Journal. 18(1):221-3. Kansas: Dunn, J. D., B. J. Johnson, J. P. Kayser, A. T. Waylan, E. K. Sissom, and J. S. Drouillard. 2003. Effects of flax supplementation and a combined trenbolone acetate and estradiol implant on circulating insulin-like growth factor-I (IGF-I) and muscle IGF-I messenger RNA levels in beef cattle. J. Anim. Sci. 81:3028-3034. Burkey T. E., S. S. Dritz, J. C. Nietfeld, B. J. Johnson, and J. E. Minton. 2004. Effect of dietary mannanoligosaccharide and sodium chlorate on growth performance, acute phase response and bacterial shedding of weaned pigs challenged with Salmonella enterica Serotype typhimurium. J. Anim. Sci. 82:397-404. Waylan, A. T., J. D. Dunn, B. J. Johnson, J. P. Kayser, and E. K. Sissom. 2004. Effect of flax supplementation and growth promotants on lipoprotein lipase and glycogenin messenger RNA concentrations in satellite cells and finishing cattle. J. Anim. Sci. 82:1868-1875. Johnson, B. J., S. S. Dritz, K. A. Skjolaas-Wilson, T. E. Burkey, and J. E. Minton. 2004. Interactive reponses in gut immunity, and systemic and local changes in the IGF system in nursery pigs in response to Salmonella enterica serovar Typhimurium. J. Anim. Sci. (accepted, 10/19/04). Anderson, P. T. and B. J. Johnson. 2004. Growth of meat animals/Metabolic modifiers. In: Encyclopedia of Meat Sciences. pp 538-546. Michigan: Ernst, C.W., N.E. Raney, V.D. Rilington, G.A. Rohrer, J.A. Brouillette and P.J. Venta. 2004. Mapping of the FES and FURIN genes to porcine chromosome 7. Anim. Genet. 35:142-143. Farber, C.R., N.E. Raney, V.D. Rilington, P.J. Venta and C.W. Ernst. 2003. Comparative mapping of genes flanking the human chromosome 12 evolutionary breakpoint in the pig. Cytogenet. Genome Res. 102:139-144. Pagan M., M.E. Davis, D.A. Stick, R.C.M. Simmen, N.E. Raney, R.J. Tempelman and C.W. Ernst. 2003. Evaluation of serum insulin-like growth factor binding proteins (IGFBP) in Angus cattle divergently selected for serum IGF-I concentration. Domest. Anim. Endocrinol. 25:345-358. Wesolowski, S.R., N.E. Raney and C.W. Ernst. 2004. Developmental changes in the fetal pig transcriptome. Physiol. Genomics. 16:268-274. Allison, C.P., R.C. Johnson and M.E. Doumit. 2004. The effects of halothane sensitivity on carcass composition and meat quality in HAL-1843-free pigs. J. Anim. Sci. (In Press). Minnesota: Kamanga-Sollo, E., M. S. Pampusch, G. Xi, M. E. White, M. R. Hathaway, and W. R. Dayton. 2004. IGF-I mRNA levels in bovine satellite cell cultures: Effects of fusion and anabolic steroid treatment. J. Cell Physiol 201:181-189. Xi, G., E. Kamanga-Sollo, M. S. Pampusch, M. E. White, M. R. Hathaway, and W. R. Dayton. 2004. Effect of recombinant porcine IGFBP-3 on IGF-I and Long-R3-IGF-I-stimulated proliferation and differentiation of L6 myogenic cells. Journal of Cellular Physiology 200, 387-394. North Carolina: Mozdziak, P. E., J. N. Petitte, and S. D. Carson, 2004 An introductory undergraduate course covering animal cell culture techniques. Biochemistry and Molecular Biology Education. 32: 319Ó322 Mozdziak PE and J. N. Petitte, 2004. Status of transgenic chicken models for developmental biology. Dev Dyn 229: 414Ó421. Mozdziak P.E., Giamario C., Dibner J.J., and D.W. McCoy, 2004. A chicken mRNA similar to heterogeneous nuclear ribonucleoprotein H1.Comp Biochem Physiol B 137: 89Ó94. Moore D.T., P. R. Ferket PR, and P. E.Mozdziak, 2004. In ovo intraperitoneal administration of bromodeoxyuridine to avian fetuses. Biotechniques 36: 50. Ohio: Ding, S.T., Li, Y.C., Nestor, K.E., Velleman, S.G., and Mersmann, H.J. 2003. Expression of turkey transcription factors and acyl CoA oxidase in different tissues and genetic populations. Poultry Sci. 82:17-24. Velleman, S.G., Coy, C.S., and Bacon, W.L. 2003. Temporal and spatial localization of the proteoglycan decorin transcripts during the progression of cholesterol induced atherosclerosis in Japanese quail. Connect. Tissue Res. 44:69-80. McFarland, D.C., Liu, X., Velleman, S.G., Caiyun, Z., Coy, C.S., and Pesall, J.E. 2003. Variation in fibroblast growth factor response and heparan sulfate proteoglycan production in satellite cell populations. Comp. Biochem. Physiol. Part C 134:341-351. Velleman, S.G. Anderson, J.W., Coy, C.S., and Nestor, K.E. 2003. Effect of selection for growth rate on muscle damage during turkey breast muscle development. Poultry Sci. 82:1069-1074. Liu, X., McFarland, D.C., Nestor, K.E., and Velleman, S.G. 2003. Expression of fibroblast growth factor 2 and its receptor during skeletal muscle development from turkeys with different growth rates. Dom. Anim. Endocrinol. 25:215-229. Velleman, S.G., and Nestor, K.E. 2003. Effect of selection for growth rate on myosin heavy chain temporal and spatial localization during turkey breast muscle development. Poultry Sci. 82:1373-1377. Velleman, S.G., Coy, C.S., Anderson, J.W., Patterson, R.A., and Nestor, K.E. 2003. Effect of selection for growth rate and inheritance on post hatch muscle development in turkeys. Poultry Sci. 82:1365-1372. Wick, M., Velleman, S.G., Coy, C.S., McFarland, D.C., and Pretzman, C.I. 2003. Myosin heavy chain isoform expression is altered during in vitro myogenesis in low score normal chickens. Comp. Biochem. Physiol. Part A 136:401-408. Velleman, S.G., Anderson, J.W., and Nestor, K.E. 2003. Possible maternal inheritance of breast muscle morphology in turkeys at sixteen weeks of age. Poultry Sci. 82:1479-1484. Velleman, S.G., and McFarland, D.C. 2004. Beta 1 integrin mediation of myogenic differentiation: Implications for satellite cell differentiation. Poultry Sci. 83:245-253. Liu, X., McFarland, D.C., Nestor, K.E., and Velleman, S.G. 2004. Developmentally regulated expression of syndecan-1 and glypican in pectoralis major muscle in turkeys with different growth rates. Dev. Growth Diff. 46:37-51. Velleman, S.G., Liu, X., Coy, C.S., and McFarland, D.C. 2004. Effects of syndecan-1 and glypican on muscle cell proliferation and differentiation: Implications for possible functions during myogenesis. Poultry Sci. 83: 1020-1027. Liu, X., Nestor, K.E., and Velleman, S.G. 2004. The influence for increased body weight and sex on pectoralis major muscle weight during the embryonic and post-hatch periods. Poultry Sci. 83: 83: 1089-1092. Nestor, K.E., Anderson, J.W., Patterson, R.A., and Velleman, S.G. 2004. Genetic variation in body weight and egg production in an experimental line selected long term for increased egg production, a commercial dam line, and reciprocal crosses between lines. Poultry Sci. 83: 83:1055-1059. Velleman, S.G. and Nestor, K.E. 2004. Inheritance of breast muscle morphology in turkeys at sixteen weeks of age. Poultry Sci. 83:1060-1066. Oregon: Xiao, Y.Y., M.C. Wang, J. Purintrapiban and N.E. Forsberg. (2003) Roles of u-calpain in cultured L8 muscle cells: application of a skeletal muscle-specific gene expression system. Comp. Biochem. Physiol. 134:439-450. Xiao, Y.Y., M.A. Beilstein, and N.E. Forsberg. (2003) Development of a ponasterone A-inducible expression system for application in cultured skeletal muscle cells. Int. J. Biochem. Cell. Biol. 35:79-85. Forsberg, N.E., Jan-Shiang Taur, Ying-yi Xiao and H. Chesbrough. (2003) Internationalization of the Animal Science curriculum: a survey of its current status, barriers to its implementation and its value. J. Anim. Sci. 81:1088-1094. Purintrapiban, J., M.C. Wang, N.E. Forsberg. (2003) Degradation of sarcomeric and cytoskeletal proteins in cultured skeletal muscle cells. Comp. Biochem. Physiol. Vol 136/3 pp 393-401. South Dakota: Liu, Xiaosong, Douglas C. McFarland, Karl E. Nestor, and Sandra G. Velleman. 2004. Developmental Regulated Expression of Syndecan-1 and Glypican in Pectoralis major Muscle with Different Growth Rates. Dev. Growth Differ. 46:37-51. Velleman, Sandra G. and Douglas C. McFarland. 2004. Beta1 Integrin Mediation of Myogenic Differentiation: Implications for Satellite Cell Differentiation. Poultry Science 83:245-252. Velleman, Sandra G., Xiaosong Liu, Cynthia S. Coy, and Douglas C. McFarland. 2004. Effects of Syndecan-1 and Glypican on Muscle Cell Proliferation and Differentiation: Implications for Possible Functions During Myogenesis. Poultry Science 83:1020-1027. Washington: Kokta, T, M.V. Dodson, A. Gertler and R.A. Hill. 2004. Intercellular signaling between adipose tissue and muscle tissue. Domestic Animal Endocrinology 27(4):301-331. Fernyhough, M.E., D. Helterline, J.L. Vierck, R.A. Hill and M.V. Dodson. 2004. Coulter counter use in enumeration of muscle and fat stem cells. Methods in Cell Science 25(3-4):221-225. Hill, R.A., A.L. Strat, N.J. Hughes, T.J. Kokta, M.V. Dodson and A. Gertler. 2004. Early insulin signaling cascade in a model of oxidative skeletal muscle: Mouse Sol8 cell line. Biochemica et Biophysica ACTA (Molecular Cell Research) 1693(3):205-211. Fernyhough, M.E., L.R. Bucci, J. Feliciano, J.L. Vierck, D. Helterline, and M.V. Dodson. 2004. Myogenic satellite cell proliferative and differentiative responses to components of common oral ergogenic supplements. Research in Sports Medicine 12(3):161-190. Dodson, M.V., M. Fernyhough, J. Antonio and L. Bucci. 2004. New ideas: Ordinary is extraordinary. Sports Nutrition Review Journal 1(1):67-68. Fernyhough, M.E., L.R. Bucci, J. Feliciano, J.L. Vierck, D. Icenoggle, and M.V. Dodson. 2004. Commonly consumed oral herbal supplements do not influence satellite cell activity. Research in Sports Medicine 12(2):71-93. Dodson, M.V. 2004. Publishing in BAM is an act of benevolence, vanity, stupidity or just good science: Reflection of a BAM supporter. Basic and Applied Myology 14(2):95-97. Kuber, P.S., J.R. Busboon, E. Huff-Lonergan, S.K. Duckett, P.S. Mir, Z. Mir, R.G. McCormick, M.V. Dodson, C.T. Gaskins, J.D. Cronrath, D.J. Marks, and J.J. Reeves. 2004. Effects of biological type and dietary fat treatments on factors associated with tenderness: I. Measurements on beef longissimus muscle. Journal of Animal Science 82(3):770-778. Kinkel, A.D., M.E. Fernyhough, D.L. Helterline, J.L. Vierck, K.S. Oberg, T.J. Vance, G.J. Hausman, R.A. Hill and M.V. Dodson. (accepted, 28 Sept). Oil red-O stains non-adipogenic cells: A precautionary note. Cytotechnology (accepted) Wisconsin: Ringkob, T.P., D.R. Swartz, and M.L. Greaser. 2004. Light microscopy/image analysis of thin filament lengths utilizing dual probes on beef, chicken and rabbit myofibrils. J. Anim. Sci. 82:1445-1453. Warren, C.M., P. R. Krzesinski, K. S. Campbell, R. L. Moss, and M. L. Greaser. 2004. Titin isoform changes in rat myocardium during development. Mech. Dev. 121:1301-1312. Dissertations Michigan: Allison, C.P. 2004. Use of halothane gas to identify pigs with novel polymorphisms in the skeletal muscle calcium release channel. PhD Dissertation. Michigan State University. Nebraska: Schulz, J. 2004. Development of protein microarrays for measurement of sarcoplasmic reticulum-associated proteins: A tool for studying calcium regulation. MS Thesis 12/04 Indiana: Dilger, Anna Carol, M. S. Purdue University, December (2004). Muscle hypertrophy and obesity. Purdue Univerisity, West Lafayette, Indiana Oregon: Mei-chuan Wang. Purification and characterization of recombinant calpain-5. PhD, 2004. Gene sequences recorded: Ohio: Liu, C. and Velleman, S.G. 2004. Meleagris gallopavo glypican-1 precursor mRNA, complete cds. GenBank Accession number AY551002. Liu, C. and Velleman, S.G. 2004. Meleagris gallopavo myogenin mRNA, complete cds. GenBank Accession number AY560111. Liu, C. and Velleman, S.G. 2004. Melagris gallopavo MyoD mRNA, complete cds. GenBank Accession number AY641567. Abstracts Arizona: Goll, D.E., J. Cong, and V.F. Thompson. 2003. Phosphorylation of the calpains. 3rd General Meeting of the International Proteolysis Society, Nagoya, Japan. p. 109. Li, H., V. F. Thompson, and D. E. Goll. 2004. Inhibitory properties of different calpastatin constructs. 4th International Conference on Cysteine Proteinases and Their Inhibitors. Portoro~, Solvania. p.55. Saido, M., H. Li, V. F. Thompson, N. Kunisaki, and D. E. Goll. 2003. Properties of the calpain system in rainbow trout. 3rd General Meeting of the International Proteolysis Society, Nagoya, Japan. P. 312. Idaho: Szasz, J.I., Hunt, C.W., Baker, S.D., Klein, T., Kuber, P.S., Glaze, B., Falk, D., Richard, R., Miller, J., Battaglia, R.A., and Hill, R.A. (2004) Correlations among ultrasonic carcass estimates, growth performance measures, and residual feed intake in Angus steers. Journal of Animal Science 82 (supplement 1) 409. Kokta, T.A., Strat, A.L., Dodson, M.V., Gertler, A. and Hill, R.A. (2004). Preliminary Characterization of Differentiating Bovine Adipofibroblasts. The Endocrine Society's 86th Annual Meeting, A163. Strat, A.L., Dodson, M.V., Gertler, A., and Hill, R.A. (2004) Leptin-Insulin Interactions: Effects on Insulin Receptor Substrate-1 Modulation in Bovine Myogenic Cells. The Endocrine Society's 86th Annual Meeting, A473. Strat, A.L., Rodgers, B.D., Dodson, M.V., Gertler, A., and Hill, R.A. (2004). Leptin Induced or Suppressed Genes in Myogenic Cells using High-Density Oligonucleotide Arrays. 64th Annual Meeting of the American Diabetes Association Diabetes 53: 267. Strat, A.L., Holder, M.C., Wallace, A., Dodson, M.V., Gertler, A. and Hill, R.A. (2003). Models of Insulin Resistance: Towards Defining Pathological Changes in the Insulin Signaling Cascade. 4th Annual Rachmiel Levine Diabetes and Obesity Symposium. p.112. City of Hope California. Cochrane, J., Strat, A.L., Dodson, M.V., Gertler, A. and Hill, R.A. (2003). The Impact of Serine Phosphorylation on the Insulin Pathway. Proceedings of the American Chemical Society 143:93. Hunt, M.L., Kokta, T.A., OShea, T., Hill, R.A. and McFarlane, J.R. (2003) Leptin clearance rates during the early follicular phase are higher than during the luteal phase of the estrus cycle in ewes. Proceedings of the Endocrine Society of Australia. 46: 77. Hill, R.A., Dodson, M.V., Gertler, A., Hughes, N.J. and Henderson, D. (2003). Insulin signaling in bovine myogenic cells. Journal of Animal Science 81(supplement 1): 96. Indiana: Rehfeldt, C., Ott, G., Gerrard, D.E., Varga, L., Schlote, W., Williams, J.L., Bünger, L. (2004) Effects of the Compact mutant myostatin allele MstnCmpt-dl1Abc introgressed into a high growth mouse line on skeletal muscle cellularity. J. Anim. Sci. 82, Suppl. 1, 378. Kansas: Johnson, B. J., S. S. Dritz, K. A. Skjolaas-Wilson, T. E. Burkey, and J. E. Minton. 2004. Interactive responses in gut immunity, and systemic and local changes in the IGF system in nursery pigs in response to Salmonella enterica serovar Typhimurium. J. Anim. Sci. 82 (Suppl. 1):444. Sissom, E. K., and B. J. Johnson. 2004. Potential non-genomic effects of progestins on C2C12 muscle cell proliferation and differentiation. 16th International Symp. of the J. Steroid Biochem. and Mol. Bio. 137-P. Waylan, A. T., B. J. Johnson, D. P. Gnad, and J. C. Woodworth. 2004. Effect of L-carnitine on porcine embryonic myoblast proliferation and the IGF-system. FASEB J. 18:(4) A547, Abstract #373.1. Rachakatla, R. S., M. L. Weiss, D. L. Davis, D. L. Troyer, and B. J. Johnson. 2004. Umbilical cord matrix stem cells express muscle specific markers and can be induced to differentiate into the myogenic pathway in vitro. FASEB J. 18(4) A6, Abstract #LB26. Sissom, E. K., and B. J. Johnson. 2004. Effect of progestins on bovine satellite cell insulin-like growth factor-I and myogenin messenger RNA (mRNA) abundance. J. Anim. Sci. 82 (Suppl. 2):18. Waylan, A. T., B. J. Johnson, D. P. Gnad, and J. C. Woodworth. 2004. Feeding L-carnitine to gestating sows alters the insulin-like growth factor system in cultured porcine embryonic myoblasts isolated from fetal skeletal muscle. J. Anim. Sci. 82 (Suppl. 2):20. Kayser, J. P., A. T. Waylan, and B. J. Johnson. 2004. The relationship between porcine uterine and placental IGF-I, IGF-II, IGFBP-3, and IGFBP-5 messenger RNA (mRNA) levels at mid-gestation. J. Anim. Sci. 82 (Suppl. 2):59. Epp, M. P., D. A. Blasi, B. J. Johnson, J. P. Kayser, and D. M. Grieger. 2004. Steroid hormone profiles and brain monoamine oxidase type A (MAO-A) activity of buller steers. J. Anim. Sci. 82 (Suppl. 2):1. Michigan: Allison, C.P., R.C. Johnson and M.E. Doumit. 2004. The effects of halothane sensitivity on carcass composition and meat quality in HAL-1843-normal pigs. J. Anim. Sci. 82(Suppl. 2):58. Allison, C.P., A.L. Marr, N.L. Berry, D.B. Anderson, D.J. Ivers, L.F. Richardson, K. Keffaber, R.C. Johnson, M.E. Doumit. 2004. Impact of halothane sensitivity on mobility status and blood metabolites of HAL-1843-free pigs following an aggressive handling model. J. Anim. Sci. 82(Suppl. 2). Bach, L.H., N.E. Raney, M.E. Doumit, E.E. Helman, S. Zhao, C.K. Tuggle and C.W. Ernst. Differential gene expression in proliferating and differentiating porcine skeletal muscle satellite cells. J. Anim. Sci. 82(Suppl. 2):107. Berry, N.L., E.E. Helman, C.P. Allison, and M.E. Doumit. 2004. Relationship between glycolytic oscillations and pork color and water-holding capacity. J. Anim. Sci. 82(Suppl. 2). Kim, K.-S., S. Costello, G.J.M. Rosa, A.M. Mullen, N.E. Raney and C.W. Ernst. Evaluation of microsatellite markers on bovine chromosomes 1 and 5 for potential allelic associations with meat characteristics and growth traits in beef cattle. J. Anim. Sci. 82(Suppl. 1):414. Rilington, V.D., N.E. Raney, P.M. Coussens, X. Ren, S.S. Sipkovsky and C.W. Ernst. Identification and mapping of differentially expressed genes in fetal and postnatal pig skeletal muscle. J. Anim. Sci. 82(Suppl. 2):43. Minnesota: Kamanga-Sollo, E., M. S. Pampusch, G. Xi, M. E. White, M. R. Hathaway, and W. R. Dayton. 2004. IGF-I mRNA levels in bovine satellite cell cultures: Effects of fusion and anabolic steroid treatment. J. Anim. Sci. 82(Suppl. 2):201 (Abstract). Pampusch, M, G. Xi, E. Kamanga-Sollo, M. White, M. Hathaway, and W. Dayton. 2004. Production of recombinant porcine IGF binding protein-5 (IGFBP-5) and its effect on proliferation of porcine embryonic myoblast cultures (PEMC) and L6 cells and on differentiation of L6 cells in the presence and absence of IGF-I . J. Anim. Sci. 82(Suppl. 2):201 (Abstract). Xi, G., E. I. Kamanga-Sollo, M. S. Pampusch, M. E. White, M. R. Hathaway, and W. R. Dayton. 2004. Effect of recombinant porcine IGFBP-3 on IGF-I and Long-R3-IGF-I-stimulated proliferation and differentiation of L6 myogenic cells. J. Anim. Sci. 82(Suppl. 2):201 (Abstract). Nebraska Creamer, B.A., J.M. Scheffler, and S.J. Jones 2004 Effects of insulin and glucose on translation rates in primary porcine satellite cells. J. Anim. Sci 82 (suppl 1) abs T 157. Schulz, J.S., S.J. Jones, and M. Zeece. 2004 Development of a microarray-based ELISA for the assessment of SR-associated calcium regulatory protein in skeletal muscle. J. Anim. Sci. 82 (suppl 1) abs T158 North Carolina: Pophal, S., P.E. Mozdziak, and S. Viera, 2004. Satellite cell mitotic activity of broilers receiving lysinedeficient diets. Poultry Sci. (Suppl. 1) 83: 2767. Pophal, S. P. E. Mozdziak, S. Borwornpinyo, and J. N. Petitte, 2004. Transgenic Chickens Expressing BetaÓGalactosidase Hydrolyze Lactose In The Intestine. Poultry Sci. (Suppl.1) 83: 2810. Ohio: Balog, J.M., Pavlidis, H.O., Wolfenden, R.E., Stamps, L., Nestor, K.E., Velleman, S.G., and Huff, W.E. 2003. Correlated responses to divergent selection for ascites incidence in broilers: Evaluation of meat quality characteristics. Poultry Sci. 82 (suppl 1):367. Coy, C.S., and Velleman, S.G. 2004. Expression of the anti-apoptotic gene Bcl-2 during skeletal muscle development in normal and Low Score Normal chickens. Poultry Sci. 83 (suppl 1). Washington: Kokta, T., A.L. Strat, J. Vierck, M.V. Dodson, A. Gertler, G. Hausman and R.A. Hill. 2004. Preliminary characterization of differentiating bovine adipofibroblasts. The Endocrine Society's 86th Annual Meeting, A163. Strat, A.L., M.V. Dodson, A. Gertler and R.A. Hill. 2004. Leptin-insulin interactions: Effects on insulin receptor substrate-1 modulation in bovine myogenic cells. The Endocrine Society's 86th Annual Meeting, A473. Strat, A.L., B.D. Rodgers, M.V. Dodson, A. Gertler and R.A. Hill. 2004. Leptin induced or suppressed genes in myogenic cells using high density oligonucleotide arrays. Diabetes 64th Annual Meeting. Diabetes 53 (suppl-2):A267 Fernyhough, M., J. Feliciano, L. Bucci, J.L. Vierck and M.V. Dodson. 2004. Cellular effects of the direct administration of oral ergogenic compounds on cultured myogenic satellite cells. Sports Nutrition Review Journal 1(1):S-5,6 Fernyhough M, L. Bucci, J. Feliciano, J.L. Vierck and M.V. Dodson. 2004. Nutrients found in some bodybuilding supplements do not influence postnatal muscle stem cell activity. FASEB J 18:616.14 Wisconsin: SantAnna Pereira, J., B.A. Huang, D. Pavlov, M. Greaser, A.C.-L. Wang, E. Homsher, and R.L. Moss. 2004. Post translation modification confers different kinetic modulation to the ²-myosin in heart and skeletal muscle. Biophys. J. 86:29a (Abst.). Warren, C.M., P.R. Krzesinski, K. S. Campbell, R. L. Moss, and M. L. Greaser. 2004. Titin isoform changes in rat myocardium during development. Biophys. J. 86:210a (Abst.). Greaser, M.L. C. M. Warren, and P. R. Krzesinski.2004. A rat mutation that alters cardiac titin isoform transitions during development. Biophys. J. 86:210a (Abst.).

Impact Statements

Substantial contributions toward understanding the extracellular signals, intracellular signal transduction pathways, and nuclear mechanisms that govern myogenic cell activity.
Identification of differentially expressed genes during muscle development.
Progress to understand muscle protein characteristics (particularly titin) and the regulation of myofibrillar assembly and disassembly.
Identification of possibilities for pharmacological intervention or targets for genetic selection to increase muscle growth.
Provide a foundation for the development of novel strategies to improve muscularity, increase the rate and efficiency of muscle growth, or improve meat quality.

Date of Annual Report: 03/30/2006

Report Information

Annual Meeting Dates: 10/21/2005 - 10/22/2005
Period the Report Covers: 10/01/2000 - 09/01/2005

Participants

Participants Attachment:

Attachment

Brief Summary of Minutes

Minutes Attachment:

Attachment

Accomplishments

Objective 1: Characterize the signal transduction pathways that regulate skeletal muscle growth and differentiation. Arizona Station. The Station demonstrated that hepatocyte growth factor (HGF) may be involved in linking mechanical perturbation of muscle to satellite cell activation. Involvement of NO in HGF release and satellite cell activation during stretch was also demonstrated, indicating that stretch triggers an intracellular cascade of events including NO synthesis, resulting in HGF release and satellite cell activation. Subsequent results indicated that calcium and calmodulin stimulate the activity of NOS, leading to the production of NO and HGF release from isolated satellite cells. The Station elucidated the functions of calpains in living cells. Inhibition of calpain activity prevented formation of integrin clusters and the ability of bovine aortic endothelial cells to spread, form focal adhesions, actin filament networks, and stress fibers, demonstrating that calpain activity is required for formation of integrin clusters. Results also indicated that ¼-calpain cleaves RhoA to produce an inactive form of RhoA that inhibits stress fiber assembly. The Station in collaboration with Dr. Kathryn Wagner at Johns Hopkins used myostatin-null mice to investigate the long-term effects of myostatin blockade on muscle growth and function. Senescent myostatin null mice continued to have normal muscle with increased mass and strength relative to controls. Muscles of senescent myostatin-null mice regenerated robustly from both chronic and acute injury. Early markers of regeneration were enhanced in the absence of myostatin, suggesting a mechanism for the attenuation of dystrophic features found in mdx mice lacking myostatin. Hawaii Station. The Station produced rabbit polyclonal anti-myostatin antibodies, using E-coli expressed chicken unprocessed and mature forms of myostatin. The two types of anti-myostatin antibodies did not show a binding affinity to the expected active form of myostatin in Western blot of skeletal muscle, while they showed a strong affinity to a commercial mysotatin, suggesting that the abundance of mature myostatin in skeletal muscle is too low to be detected by the current Western blot analysis. The Station also produced a monoclonal anti-myostatin antibody using recombinant mature myostatin to examine the effects of in-ovo administration of the antibody on post-hatch broiler growth and skeletal muscle mass. Results from in-ovo administration of the antibody suggest that immunoneutralization of myostatin during embryonic development is a potential means to improve growth potential of broilers. The Station examined the effect of maternal immunization against myostatin in broiler hens on post-hatch broiler growth and skeletal muscle mass. Results suggest that maternal immunization against myostatin is a potential means to improve skeletal muscle growth of broilers. Idaho Station. The Station established a muscle and muscle-derived fat cell co-culture system in collaboration with the Washington Station. This innovative adipocyte/myogenic cell co-culture system demonstrated its usefulness in examining the interaction of the adipocyte as a source of leptin and leptin binding proteins with muscle and in evaluating the developmental expression of fat cell differentiation markers from both fish and beef-derived adipofibroblasts. The Station has been studying leptin-insulin interactions with the goal of improving production efficiency in ruminants. Initial effort was to characterized primary bovine myogenic cells (BMC, provided by Washington Station) and mouse Sol8 myogenic cell line to determine signaling interactions between the insulin and leptin axes. Illinois Station. The Station has investigated the changes in protein/myonuclei ratios during hypertrophy and atrophy in a stretch-induced model of muscle hypertrophy of both young and old chickens and subsequent atrophy after release of stretch. Results demonstrated that individual fibers or clusters of fibers had localized concentrations of apoptotic nuclei, suggesting that a mechanism may exist for selecting nuclei for apoptosis. The Station also found Zn2+ acts in vivo to prevent secreted IGF-II from binding to IGFBP-3 and IGFBP-5, thus maintaining the IGF-II in an active state for receptor association. The Station also examined effects of in ovo administration of rhIGF-1 on the expression of GHSR during chicken embryonic development. Results suggest that GHSR may be active in regulating GH secretion during early embryonic development, and up-regulation of the GHSR gene following IGF-1 administration may have an important role in the determination of postnatal muscle growth. Indiana Station. The Station examined the ability of sonic hedge hog (Shh) and IGF-I to stimulate muscle hypertrophy and to inhibit disuse atrophy when over-expressed in skeletal muscle. Ectopic expression of IGF I and/or Shh significantly stimulated muscle fiber hypertrophy and increased muscle size, and attenuated the loss of muscle fiber area, muscle mass and muscle mass density. The Station generated an expression construct producing an epitope-tagged HGF protein to investigate the mechanism by which HGF modulates muscle growth via changes in satellite cell activity. The Station investigated the role of myofiber type in ²-agonist-induced skeletal muscle hypertrophy using C57BL/6, myosin heavy chain (MyHC) IIX-/- and IIB-/- mice. Results suggest that an orderly, step-wise progression through various MyHC is necessary for complete transition to the fast phenotype. The Station demonstrated that the increased lean growth in beta-agonist treated animals may be partially due to changes in fiber type composition. In addition, results indicated that a differential participation of ERK and/or Akt/Gsk signaling pathways in ²-adrenergic receptor signaling may account for the distinct responsiveness of slow and fast myofibers to ²-agonists. The Station is also investigating energy metabolism and myofiber type specification. Results suggest that activation of AMPK can lead to changes in muscle MyHC. The Station examined partitioning of nutrients between muscle and fat by crossing myostatin null mice with mice possessing the leptin db mutation to produce mice homozygous for both mutations. Results suggest that muscle hypertrophy may interact with adipose tissue in the development of obesity. The Station investigated the energy metabolism in Halothane (HAL) and Rendement Napole (RN) genotypes, which are characterized by accelerated postmortem energy metabolism and poor pork quality. Results demonstrated that elevated muscle glycogen content did not further aggravate rapid early postmortem metabolism, indicating that glycogen phosphorylase and other upstream glycolytic enzymes are sufficient to sustain rapid metabolism in HAL mutants. Kansas Station. The Station demonstrated that an acute enteric disease can lower circulating IGF-1, but more chronic conditions may be necessary to affect local IGF-1 levels in skeletal muscle, thus suggesting that alterations in IGF-1 levels during a diseased-state may contribute to the reduced skeletal muscle protein accretion during these periods of stress. The Station found that exposure of cultured cells to PUFA increased membrane fluidity compared to cells subjected to saturated and monounsaturated fatty acid and that prior incubation of muscle cells with an omega-3 fatty acid may alter the sensitivity of these cells to various growth factors. The Station has also studied the interaction between an anabolic-steroid growth promotant and a-linolenic acid (LA) in cattle. Results indicate that TBA + E2 implantation increases circulating IGF-1 levels and local IGF-1 mRNA levels, but this effect may be attenuated by flax (aLA) supplementation. Muscle cell culture experiments suggested that ±LA was not responsible for the IGF-I mRNA down-regulation, but the mammalian lignins, EL and ED abundant in flaxseed may act as the component responsible for IGF-I mRNA down-regulation in flax-fed steers. The Station determined that carnitine may alter mRNA levels of IGF-II and myogenin, thus affecting muscle cell proliferation and differentiation. The Station recently conducted several experiments to understand the potential interactions between the use of both anabolic steroids and ²-adrenergic agonists in beef cattle. Michigan Station. The Station used fluorescent immunostaining to determine satellite cell activity in cattle. Results indicate that the proportions of proliferating and differentiating satellite cells remain relatively constant in growing cattle from 200 to 500 kg body weight, and TBA/E2 implants had little effect on satellite cell activity 14 d after implantation. Minnesota Station. The Station produced recombinant IGFBP-3 and IGFBP-5 in a baculovirus expression system, and the purified rpIGFBP-3 and rpIGFBP-5 were used in antibody production. These antibodies were utilized to determine the role of IGFBP-3 and IGFBP-5 in regulating proliferation and differentiation of porcine embryonic myogenic cells (PEMC). The Station established that IGFBP-3 and IGFBP-5 play a necessary role in the proliferation-suppressing action of TGF-beta 1 and myostatin in PEMC cultures. The Station is using RNA interference technology to silence the IGFBP-3 and IGFBP-5 production in PEMC cells in a recent effort to develop a new valuable research tool to assess the role of the IGFBPs in mediating the anti-proliferative effects of myostatin and TGF-beta 1. The Station demonstrated that a combined E2/TBA steroid implant significantly increased muscle IGF-I mRNA levels in muscles of implanted steers. In vivo and in vitro results suggested a mechanism of steroid-enhanced muscle growth that involves both increased local production of IGF-I (independent of GH) and an increased number of actively proliferating muscle satellite cells. Nebraska Station. The Station has used a proteomics approach to gain a more global picture of biological phenomena related to muscle growth and development. SDS-PAGE examination of fractions obtained by differential sedimentation of pale, soft and exudative (PSE) and normal muscle samples suggested that significant differences between normal and PSE muscle may be found in the membrane fraction. The Station also investigated factors influencing protein synthesis in myotube cultures. Results show regulation by insulin and leucine of RNA synthesis as well as the formation of polyribosomes in both PSC and PDM in vitro. North Carolina Station. The Station examined the effect of early posthatch starvation on myonuclear apoptosis in chickens. Apoptosis appeared to be a mechanism contributing to the smaller myofiber size observed when feed is not provided early posthatch. The Station examined mechanisms by which early posthatch nutrition affects skeletal muscle growth in a series of experiment. Results suggest that it is possible to nutritionally stimulate the satellite cell population in the early post-hatch chick and that early post-hatch period is an important target for nutritional manipulations aimed at improving skeletal muscle growth and meat yield. The calpain system appeared not be affected by the presence of oral nutrition, but there is an age-related down regulation of p94 calpain mRNA expression. The Station observed that GAPDH was developmentally up-regulated with advancing age and changed with nutrition status in the early posthatch chick, suggesting that GAPDH is not a proper internal standard for muscle studies using quantitative northern analysis. The Station achieved the development of a protein (beta-galactosidase) expressing transgenic chicken for the first time using a replication defective retroviral vectors. The transgenic chicken could utilize lactose as an energy source, demonstrating that transgenic technology can be used to modify nutrient utilization in domestic poultry. Viral insertion carrying the lacZ gene was found on chromosome 11 within the predicted gene for neurotactin/fractalkine (CX3CL1), and it appears that neurotactin mRNA expression is missing from the brain of homozygous individuals. Ohio Station. Results from a series of experiments using a turkey line selected for increased growth (F-line) and its unselected randombred control line (RBC2-line) demonstrated changes of proteoglycan expression in skeletal muscles during embryonic development. Furthermore, the Station observed that feed depriviation at hatching in turkey and chicken resulted in changes in proteoglycan exporession. A collaborative effort by the South Dakota Station and the Ohio Station has shown that faster growing clonal cell populations were more responsive to fibroblast growth factor (FGF-2) and expressed greater levels of FGF-2 and FGF receptor at the onset of proliferation than did slower growing cells isolated from the same muscle. Faster growing clones also expressed higher levels of heparan sulfate proteoglycans (HSPG), especially during differentiation, than did slower growing clones. These results demonstrate that satellite cells that differ in proliferation rates differ in responsiveness to FGF-2, and in the expression of FGF-2, FGF receptor-1 and HSPG. The Station cloned a full length turkey glypican cDNA into the expression vector pCMS-EGFP to understand the mechanism of how the heparan sulfate family of proteoglycans is involved in the regulation of muscle growth properties. The Station also constructed glypican mutants for each of the glycosaminoglycan chains. Results showed that the overexpression of glypican-1 increases proliferation and FGF2 responsiveness of satellite cells. As differentiation proceeded, the transfected cells began to exhibit a reduction in differentiation. Oregon Station. The Station has focused on the roles that two E3 ubiquitin ligases play in coordination of muscle growth using two lines of mice which lack muscle-specific E3 ligases (MuRF-1 and MAFbx). Two strains of mice (MuRF-1 -/- and MAFbx -/-) were subjected to a 48 hour fasting challenge and effects of the induced atrophy on expression of mRNAs encoding specific proteolytic enzymes is underway. South Dakota Station. The Station examined the properties of satellite cell subpopulations within single muscles that differ in their responses to growth factors by focusing on determining if the variation in growth factor responses is due to differences in the activity of the receptors. In a collaborative project with the Ohio Station, the South Dakota Station developed serum-free medium that supports the growth of avian satellite cells. Since the amount of growth factor added in the medium is known, the serum-free medium is very useful in examining the role of the extracellular matrix in growth factor activity on myogenic satellite cells. Results also suggest a role for decorin in myostatin action in muscle development. The Station is using a microarray analysis to determine the gene expression of myogenic satellite cells having different proliferation rates. Utah Station. The Station has initiated a project employing 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 profiled genes included several that are key to muscle growth and development, an imprinted gene linked to expression of the callipyge phenotype, and a typically employed housekeeping gene. Primers have been developed for candidate genes to achieve optimal performance in real time Q-PCR. Washington Station. The Station isolated satellite cells from aged and young dogs of two different breeds to investigate the mechanisms of muscular atrophy, specifically focusing on components of the immune system. A subtractive cDNA library was made to identify genes unique to differentiated muscle. The Station examined the effects of oral supplements and dietary components on muscle cell physiology by examining their effects on muscle cell proliferation and differentiation in culture system. Results showed that a few of the tested compounds were capable of inducing proliferation and/or differentiation, but nearly all of the herbal extracts were toxic to satellite cells, and results suggest that regulatory agents other than growth factors and hormones may influence myogenesis. The Station has begun to examine how control of somatic tissue growth and development is coordinated through the collaborative efforts of hormones, growth factors and cytokines. The current goals are to determine the mechanisms of myostatin action at the cellular level using an alternative model organism, the rainbow trout Oncorhynkus mykiss. West Virginia Station. The Station observed that the ontogeny of myostatin gene expression coincided with the major mitogenic and myogenic events during chick embryonic development. Follistatin gene expression during pectoralis muscle development displayed a reverse expression pattern compared to that of activin-B, supporting the concept of inhibitory functions of follistatin on activins. The Station has also demonstrated that in ovo administration of recombinant human IGF increases the live, breast, and leg weights of 6-wk-old broilers. Objective 2: Determine the nuclear mechanisms that control gene expression in skeletal muscle. California Station. The Station has continued to focus on the biochemistry and the molecular biology of the chicken myosin heavy chain multigene family. Results suggest that during development neonatal myosin is initially suppressed near the motor endplate and that trophic factors emanating from the vicinity of the motor endplate represent a potential localized signaling pathway that may differentially modulate myosin heavy chain gene expression in nuclear domains along the length of the muscle fiber. The Station also tested the hypothesis that myonuclear domains expressing neonatal myosin within end regions of maturing fibers will be smaller than domains elsewhere in the fibers. Results demonstrate that myonuclear domains are smaller at the terminal tips of maturing muscle fibers than the other regions. Iowa Station. The Station defined the global changes in muscle gene expression in response to work overload in the rat. The Station also compared the gene expression between double-muscled (mh) and wild-type (wt) bovine embryos using subtractive hybridization analysis. Results showed that of the first 58 expressed sequence tags (EST), 72% of the EST were homologous to known bovine EST, whereas 18% of the EST were novel. In addition, 24% of these 58 EST had no homology to any known gene in any other species. The Station demonstrated that ractopamine, which when fed to pigs increases skeletal alpha-actin mRNA abundance in muscle, does not directly regulate the porcine skeletal alpha-actin promoter in vitro using porcine satellite cells. Results also indicate that the accumulation of skeletal alpha-actin mRNA in response to ractopamine treatment may be due to posttranscriptional regulation. In another experiment, the Station used the suppression subtractive hybridization (SSH) procedure to examine pre translational gene expression after ractopamine administration in porcine skeletal muscle. Nine genes and one expressed sequence tag were identified as being differentially expressed in longissimus dorsi muscle after ractopamine stimulation. Northern blot analysis performed on the original RNA samples confirmed that calmodulin 1 mRNA abundance increases approximately 2 fold after 3 d of ractopamine treatment. This is the first study in which the expression of a calcium modulated protein has been implicated in the phenotypic adaptations of skeletal muscle to ractopamine in the pig. The Station examined the role of JAK2 in muscle growth. Microarray analysis of gene expression in skeletal muscle after three days of work overload demonstrated that JAK2 expression increased 8.7-fold. Results from in vitro cell culture system support that JAK2 plays a critical role in the differentiation of skeletal muscle. In addition, results indicate that JAK2 is required for both normal and stretch induced proliferation in C2C 12 myoblasts. Michigan Station. The Station performed cDNA macroarray experiments to identify genes that are differentially expressed in pig skeletal muscle at several fetal and postnatal ages, as well as in proliferating and differentiating pig skeletal muscle satellite cells. Sequence has been obtained and evaluated for 782 clones. A publicly accessible database has been developed for the storage of clone data (http://www.cafg.msu.edu). In a subsequent experiment, nine genes were identified to be differentially expressed between fetal and neonatal skeletal muscles. Results demonstrate that microarray analysis can reveal putative differentially expressed genes in developing skeletal muscle. In another microarray results indicated that genes for two extracellular matrix proteins, fibronectin I and collagen type I alpha 2 were differentially expressed between fetal and postnatal muscle. The Station used halothane gas to identify pigs with novel polymorphisms in the skeletal muscle calcium release channel (RYR1). Results indicate that the SNPs in RYR1 may be useful markers for functional differences in other proteins that affect stress susceptibility, ambulatory status, and meat quality. The Station has continued to work on developmental changes in myogenic cell activity in an effort to modify cell activity and effectively improve muscle growth. Initial efforts were to identify appropriate markers for porcine satellite cells. Results demonstrate that use of NCAM staining to distinguish porcine myogenic from non-myogenic cells is apparently more conservative than c-met labeling. North Carolina Station. The Station examined the influence of the leucine metabolite, ß hydroxy ß methylbutyrate (HMB), and feed deprivation on muscle development in the early posthatch poultry. Results demonstrate that dietary supplementation of HMB may have an anabolic effect on early post hatch muscle. The Station clones a cDNA for a chicken. The deduced amino acid sequence was approximately 86% identical to corresponding regions of human, mouse, or zebrafish proteins similar to heterogeneous nuclear ribonucleoprotein HI. The expression data confirmed the size of the predicted mRNA sequence. The newly identified sequence may be employed in future studies aimed at understanding the role of heterogeneous nuclear ribonucleoproteins in avian species. Pennsylvania Station. The Station demonstrated that activated Raf inhibits myogenesis through downstream induction of MAPK. Moreover, extreme levels of Raf activity lead to the creation of a TGF beta 1 autocrine loop that contributes to inhibition of myogenesis. In another study, the Station demonstrated that Raf CAAX negatively impacts transcriptional regulators in addition to MEF2. To gain insight into the mechanism of inhibition of skeletal muscle growth, the Station examined gene expression profiles of mouse myocytes and differentiation-defective myocytes using microarray analysis. Results revealed the down-regulation of muscle genes in myoblasts expressing elevated levels of activated Raf and MAPK. Other results indicate that TGF-b1 is not entirely responsible for the Raf-imposed block to myogenesis. The Station also demonstrated the presence of a MEK-independent signaling module for Raf, confirming the presence of both MEK-dependent and independent signaling events for Raf. Objective 3: Characterize muscle proteins and their functional domains involved in myofibrillar assembly and disassembly Arizona Station. The Station has examined the effect of phosphorylation of the calpains to determine whether this event had any effect on proteolytic activity. Results from a series of experiments indicate that phosphorylation has an important role in the regulation of calpain activity. The Station also developed immunoaffinity columns for purifying both ¼- and m-calpain and calpastatin. The Station has examined the structures of m- and m-calpains in different calcium states using partial proteolytic cleavage. The Station has expressed in E. coli six different calpastatin isoforms and assayed their ability to inhibit the calpains. Results suggest that the different isoforms have some function other than a differential ability to inhibit the calpains, although the presence of different calpastatin isoforms in a single tissue could serve to modulate inhibition of the calpain in that tissue. The Arizona Station attempted to make ERMs from both rat soleus and extensor digitorum longus and from bovine diaphragm muscle using slight modifications of the published procedures. The ERM from either species have no desmin nor ±-actinin, as expected, but do have tropomyosin and troponin. Incubation of purified myofibrils with purified calpain results in ~ 2-fold increase of yield of ERM; but results suggest that calpain at the levels that have used by the researchers at this station caused removal of Z-disks and release of sarcomeres rather than ERM. Illinois Station. The Station has examined the calpain proteolytic system in rainbow trout skeletal muscle. Results suggest that the calpain system may be important in postmortem proteolysis and reduction in muscle integrity in post harvest rainbow trout. Additionally, the calpain 4 (catalytic subunit) cDNA from rainbow trout has been cloned and sequenced. This represents the first reported fish and lower vertebrate full-length cDNA of Capn4. Starvation of fingerlings led to increased expression of both calpain 1 and 2 indicating that these isozymes may be involved in protein mobilization for energy during starvation. Furthermore, two forms of the calpain inhibitor calpastatin were identified (CAST-long and CAST-short) derived from two different genes. CAST-L and CAST-S expression were correlated with growth and fillet quality being lowered in rainbow trout strains with slower growth and softer fillets. Another project at the Station focuses on antemortem and postmortem muscle metabolism using cull cows administered with beta-adrenergic agonists. Results indicate that feeding concentrate to cull cows improved growth rate and ractopamine supplementation had no added benefit to the concentrate feeding. Additional ongoing research at the Illinois station involves the interaction of muscle and adipose tissue in growth. Myostatin null-leptin db/db mice exhibit both increased muscle growth and obesity. The increase in muscle growth, however, is delayed by adipose tissue gain, as if adipose tissue growth is restricting the growth of muscle. Further work is being undertaken to understand the interplay between these two tissues. Indiana Station. The Station determined the influence of pH on the basal and Ca2+ activated myofibrillar ATPase activity in myofibrils from porcine red (RST) and white semitendinosus (WST) muscles. Results suggest that myofibrils with predominantly fast myosin heavy chain have a higher actin-activated myosin ATPase activity than myofibrils with primarily slow myosin heavy chain isoforms at Ca2+ concentrations and pH values characteristic of postmortem muscle. The Station determined the effects of postmortem pH and temperature declines on the actomyosin ATPase activity of muscle fibers expressing different MyHC isoforms. Maximum actomyosin ATPase activity was determined on individual fibers classified by MyHC expression. Results suggest that fibers expressing fast MyHC isoforms have a higher ATPase activity early postmortem than slow muscle fibers but are more prone to inactivation by a rapid pH decline. The Station established that exchange of TnI between a free pool and that in Tn on the myofibrillar thin filament is very slow using fluorescent labeling method. The station continues to use the method to determine protein dynamics within the contractosome. Iowa Station. The Station has been investigating the interaction of synemin with proteins in the Z-line and the costamere. Results indicate that synemin is directly involved in maintaining the structural integrity of muscle cells. Paranemin and synemin may play a role in integrating the IFs into the cytoskeleton during myofibrillogenesis. Michigan Station. The Station has undertaken studies to quantify the rate and extent of postmortem tenderization in porcine muscle and examine indices of proteolysis that correspond to mechanical measures of tenderness. Results suggest that tenderization of most pork loin chops is complete by d 7 postmortem, and the rate of tenderization corresponds to the rate of desmin degradation and m-calpain inactivation. Nebraska Station. A goal of the Nebraska Station has been to conduct microarray-based analysis for profiling of SR-associated proteins. Microarrays were developed to quantitatively assess the level of a selected set of proteins associated with calcium regulation in muscle. Results found that a number of significant differences exist in the level of SR proteins between Hal + and Hal- animals. A similar quantitative microarray analysis was conducted on SR proteins from turkey muscle. Results showed significant differences between improved and unimproved lines. Oregon Station. The Station has developed cell lines in which the activities of ¼- and m-calpains may be regulated and their functions in muscle wasting identified. Results with the cell lines demonstrated that calpains account for roughly 60% of total protein degradation and that m-calpain accounts for about half of that. Additionally, calpain substrates in muscle include nebulin, fodrin and desmin. The Station also used an in vivo model for muscle wasting (hind-limb suspended rat). Activities of u- and m-calpains and mRNA concentrations encoding these proteases are increased in hind-limb suspended rats and reloaded rats. Another project at the Station has focused on calpain 5. The protein autolyzes in the presence of calcium, but the calcium concentration dependence is extremely high (>20 mM). The activity of the enzyme is insensitive to E64 or calpastatin. Studies are also being conducted on the role of MuRF-1 and MAFbx on protein turnover. Both these proteins have been found to be up-regulated in immobilized, denervated, and hind limb suspension muscle. Both are E3 ligases that function in preparing proteins for proteasome degradation. MuRF-1 binds to titin near the M-line of the myofibril, and is also near a site of calpain binding. Wisconsin Station. The Station investigated the importance of various muscle fiber types in controlling pork quality. Results indicated very low correlations between fiber type and several measures of meat quality (ultimate pH, Minolta color, etc.). The Station expressed recombinant fragments representing the sub-domains of the extensible region of cardiac N2B titin to understand the binding of titin to the thin filaments. Results indicate that a dynamic interaction between the PEVK domain and F-actin makes a significant contribution to the passive tension and that S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, may provide a mechanism to free the thin filament from titin and reduce titin-based tension prior to active contraction. In another study, the Station used atomic force microscopy (AFM) to characterize the single molecule force-extension curves of the PEVK and N2B-unique sequence (N2B-Us), spring elements of titin, which together are responsible for physiological levels of passive force in moderately to highly stretched myocardium. Using the molecular characteristics and those determined earlier for the tandem Ig segment (assuming folded Ig domains), the Station modeled cardiac titins extensible region in the sarcomere and calculated the extension of the various spring elements and the forces generated by titin, both as a function of sarcomere length. The Station examined species differences in the extensible I-band region of cardiac titin. Results demonstrated that in addition to the higher proportion of the shorter N2B isoform found in rat compared with dog cardiac muscle observed previously, shorter N2B unique and N2BA PEVK segments may also contribute to the greater passive tension in cardiac muscle from rats. Results on titin phosphorylation study suggest that titin phosphorylation may modulate cardiac function in vivo. The Station also investigated the thermal properties of titin isolated from porcine and bovine longissimus muscles using differential scanning calorimetry. Results suggest that titin may be partially responsible for meat toughening when muscle tissue is heated above 60oC. Combination of a high-resolution SDS-agarose electrophoresis system and cDNA analysis was used to examine developmental changes in titin isoforms and alternative splicing patterns. The mechanism for controlling titin alternative splicing remains yet to be determined. The Station recently identified a rat with a mutation that affects the developmental program of titin isoform switching. Experiments are in progress to determine the expression patterns of titin. The Station developed an immunofluorescence microscopy method to follow changes in myofibrillar-bound calpain 3. Results indicate that the method is useful in examining the changes and location of calpains in various postmortem conditions. The Station, in a collaborative work, characterized four additional splice isoforms of Cypher genes, a striated muscle specific PDZ-LIM domain protein, in addition to the previously known two forms. The Station completed light microscopic analysis of filament lengths. The method has shown to be useful to study sarcomere structure and to explain species and muscle differences in meat texture. The Station examined the skeletal muscle phenotype of MLP knockout (MLPKO) mice in terms of the response to ECs. Results suggest that MLP play a subtle role in the maintenance of normal muscle characteristics and in the early events of the recovery process of skeletal muscle to injury, serving both structural and gene regulatory roles.

Publications

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Impact Statements

Information gained by the project continues to provide the foundation for the development of novel strategies to improve muscularity, the rate and efficiency of muscle growth, and meat quality.
During the project period (2000-2005), the NC-131 group made considerable progress in addressing each of these objectives as reflected by the various forms of research outputs: " 217 Refereed Publications " 163 Published Abstracts " 39 Non-refereed Publications, Reports and Book Chapters " 22 Theses, MS/Ph.D Programs Completed " 21 Gene Sequences Deposited
The project participants have expanded our information about the fundamental biological processes that regulate muscle development and growth of meat-producing animals. The importance of satellite cell proliferation and the involvement of IGF-1 for this process in postnatal skeletal muscle growth has been established. Differential responsiveness of muscle fiber types to hypertrophic stimuli such as beta-adrernergic agonists has been firmly established. At the same time, pathways leading to the expression of differential myofiber type genes have been elucidated.
The projects participants contributed toward understanding the extracellular signals, intracellular signal transduction pathways, and nuclear mechanisms that govern both myoblast/satellite cell activity and muscle protein accumulation.
The project participants have also made significant progress in understanding muscle protein characteristics, such as titin and costamere-associated protein, and the regulation of myofibrillar assembly and disassembly.