Annual Report

Regional Research Project NC-131

2001

Project Title: Molecular Mechanisms Regulating Skeletal Muscle Growth and Differentiation

Project Period: 10-1-00 to 9-30-2001

COOPERATING AGENCIES AND PRINCIPAL LEADERS

*Denotes voting member of the Technical Committee

PROGRESS OF THE WORK AND PRINCIPAL ACCOMPLISHMENTS

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

            The Arizona Station began using a device that mechanical stretches growing muscle cells in culture to simulate satellite cell activation.  Cultured quiescent satellite cells, subjected to mechanical stretch, were stimulated to enter the cell cycle earlier than control cultures.  Only a brief period of stretch, as short as 2 h, was necessary to stimulate activation.  Conditioned medium from stretched cells could activate unstretched satellite cells.  Presence of hepatocyte growth factor (HGF) on c-met-positive myogenic cells was detected by immunofluorescence at 12 h in culture, and immunoblots demonstrated that HGF was released by stretched satellite cells into medium. Also, stretch activation could be abolished by the addition of anti-HGF antibodies to stretch cultures, and activity in conditioned medium from stretched cultures, and activity in conditioned medium from stretched cells could be neutralized by anti-HGF antibodies. In addition, stretch appeared to cause release of preexisting HGF from the extracellular matrix. These experiments suggest that HGF may be involved in linking mechanical perturbation of muscle to satellite cell activation. 

            Using the same approach, the Iowa Station investigated the role of cyclin dependent kinase inhibitors, such as p21^WAF1 in cell cycle progression.  C2C12 myoblasts were plated at 15,000 cells/cm², transiently transfected with a p21-Luciferase promoter construct, and induced to differentiate with low serum media for 15 h.  Differentiating myoblasts were mechanically stretched for up to 48 h.  p21^WAF1 promoter activity increased 6.5-fold during myoblast terminal differentiation, and increased 2.1-fold in stretched vs. non-stretched myotubes at 48 h.  In contrast, c-jun promoter activity was not responsive to cyclic stretch.  Furthermore, p21 promoter activity tended to decrease in cyclically stretched myoblasts.  Creatine kinase activity increased in stretched cells vs. non-stretched cells after 36 (2.4-fold) and 48 (2.8-fold) h of stretch.  These results suggest that in skeletal myotubes the p21^WAF1 promoter is responsive to stretch, independent of terminal differentiation.

Studies to determine whether calpains have a role in cytokinesis during cell proliferation also have continued at the Arizona Station.  Studies using bovine aortic endothelial (BAE) cells showed that calpain activity is required for formation of integrin clusters. These clusters form unstream of Rac activition and are involved in activation of Rac and the subsequent formation of Rac-induced focal complexes. These Rac-induced focal complexes do not require calpain for their formation. Rac activation is followed by RhoA activation and formation of focal adhesions. Focal adhesion formation also does not require calpain activity. Calpains cleave b₃ integrins in the integrin clusters, but it is not clear whether calpain cleavage of b₃ integrin is required for formation of the integrin clusters or has some other function. These results indicate that integrin clusters require calpain activity for their formation and that these clusters provide a means to recruit Rac to sites of receptor-occupied integrin.

The Kansas Station has investigated the role of polyunsaturated fatty acids on proliferation and differentiation of muscle cells. Exposure of cultured cells to polyunstaturated fatty acids (PUFA) increased membrane fluidity compared to cells subjected to saturated and monounsaturated fatty acid.  Increased membrane fluidity potentiated insulin and insulin like growth factor-I (IGF-1) binding to their receptors.  Incubation of L6 cells with a-linolenic acid (1uM) for 12 h prior to the addition of a growth medium rich in IGF-1 increased the rate of ³H-thymidine incorporation approximately 27% as compared to control cultures.  These data suggest that prior incubation of muscle cells with an omega-3 fatty acid may alter the sensitivity of these cells to various growth factors present in a proliferation-inducing medium. 

The Kansas Station is studying the effect of disease on muscle growth using Salmonella typhimurium-challenged pigs.  Challenged pigs showed a precipitous decrease in circulating IGF-1 and IGF binding protein (IGFBP) -3 concentrations.  These data suggest that alterations in IGF/IGFBP levels during a diseased-state may contribute to the reduced skeletal muscle protein accretion during these periods of stress. 

The Ohio Station has continued studying the role of proteoglycans, specifically syndecan-1 and glypican, in modulating growth of skeletal muscle, using selected turkey lines.  Studies have shown that the embryonic pectoralis major muscles of highly selected birds have higher levels of heparan sulfate than unselected control birds.  This difference continues throughout posthatch development. Syndecan expression is high early in skeletal muscle differentiation and is subsequently downregulated whereas glypican expression is opposite that observed for syndecan. 

Communication between the cell and the extracellular environment is important in the formation of tissues and cell migration.  In collaborative studies between the Ohio Station and the South Dakota Station, integrin temporal and spatial localization was measured in Low Score Normal (LSN) satellite cells.  LSN satellite cells exhibit decreased proliferation and differentiation, and did not form multinucleated myotubes in culture.  Instead the LSN satellite cells formed rounded cellular structures.  LSN satellite cells expressed reduced levels of the beta 1 integrin subunit during differentiation.  Temporal and spatial localization studies have shown the beta 1 integrin subunit to be localized on the plasma membrane of normal satellite cells at points of cellular contact whereas in the LSN satellite cells, the beta 1 integrin is internalized within the cell.  Decreased contact of a cell with the substratum has often been associated with apoptosis.  During differentiation, the rounded LSN cells have been shown to be undergoing apoptosis, which is not occurring in the normal satellite cell cultures.

The Washington Station is studying mechanisms of muscular atrophy, specifically focusing on components of the immune system.  Satellite cells were isolated from aged and young dogs of two different breeds.  A subtractive cDNA library is being made to identify genes unique to differentiated muscle. 

            The Minnesota Station has continued studies on the role of IGFBP in regulating proliferation and differentiation of porcine myogenic cells.  To that end, these scientists have shown that primary porcine satellite cells produce IGFBP-3 and - 5 while porcine muscle-fibroblasts produce IGFBP-2 and - 4 but not IGFBP-3 or -5.  Because of this, their investigations have focused on the regulation of IGFBP-3 and - 5 production by IGF-1, transforming growth factor beta (TGF-b), and basic fibroblast growth factor (bFGF) in primary porcine satellite cell cultures. Both IGF-1 and bFGF exhibited dose-dependent increases in [³H]Thymidine incorporation with increasing concentration from 1-50 ng/ml, whereas TGF-b causes a dose-dependent decrease in [³H]Thymidine in corporation from 0.01 - 0.5 ng/ml.  When 20 ng/ml of IGF-1 was added to the media, IGFBP-3 was increased approximately 65% and IGFBP-5 was increased approximately 2 fold.  The addition of 0.5 ng/ml TGF-b caused over a two fold increase in IGFBP-3 and approximately an 80% increase in IGFBP-5, while50 ng/ml of bFGF caused approximately a 40% and 70% increase in IGFBP-3 and –5 respectively.  IGFBP-3 and -5 were not detectable in the CM from porcine muscle-derived fibroblasts whether IGF-1, TGF-b1 or bFGF were present or not.  These data suggest that the affects of IGF-1, TGF-b and bFGF on porcine satellite cell growth and differentiation may in part be through the autocrine/paracrine production of IGFBP-3 and –5 by porcine satellite cells.

Researchers from the Minnesota Station have continued their efforts in trying to understand the role of IGFBP-3 by producing tagged, recombinant porcine (rp) IGFBP-3.  The rpIGFBP-3 protein is a dimmer binding between 40 and 43 kd and ¹²⁵I-IGF-I Western ligand blots show that it binds IGF-I.  Both IGF-I- and Des (1,3) IGF-I-stimulated proliferation of cultured porcine embryonic myogenic cells are suppressed in a concentration-dependent manner by 5 to 100 ng of rpIGFBP-3/ml media. Anti-rpIGFBP-3 inhibited the actions of IGFBP-3 as evidenced by the fact that addition of 20 mg of anti-rpIGFBP-3 IgG to culture media completely abolishes the proliferation suppressing action of 50 ng of rpIGFBP-3.  Furthermore, addition of anti-IGFBP-3 IgG to TGF b-treated cells abolishes approximately 50% of the TGF b-induced suppression of proliferation. Thus, it appears that increased levels of IGFBP-3 play a significant role in the ability of TGF b to suppress proliferation of cultured myogenic cells. This finding is of particular interest in muscle in light of the importance of myostatin in developing muscle.

The West Virginia Station has continued studying the developmental expression pattern of myostatin, follistatin, and activin-B gene expression in the whole embryo and pectoralis muscle of the developing chicken embryo using semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR).  The ontogeny of myostatin gene expression coincides 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, which supports the concept of inhibitory functions of follistatin on activins.

The West Virginia Station has also demonstrated that in ovo administration of recombinant human IGF increases the live, breast, and leg weights of 6-wk-old broilers.

In a new initiative, the Nebraska Station has elected to use a proteomics approach to gain a more global picture of biological phenomena related to muscle growth and development.  Fractions obtained by differential sedimentation of pale, soft and exudative (PSE) and normal muscle samples were examined by SDS-PAGE.  Samples from PSE and normal muscle were sedimented at 2,000g,  20,000g  and 100,000g.  Western blots of these fractions were performed using a monoclonal antibody to the ryanodine receptor protein, which is intimately associated with intracellular calcium regulation.  The strongest antibody staining was found in the 20,000g and 100,000g pellets for both PSE and normal muscle samples. However, substantially less antibody staining was found in the PSE sample fractions than in the corresponding normal sample. These results suggest that significant differences between normal and PSE muscle may be found in the membrane fraction.  Further, these researchers subjected sarcoplasmic reticulum preparations from normal and PSE muscle to 2-D electrophoresis.  Comparison of 2-D sarcoplasmic reticulum separations from normal and PSE muscle by image analysis showed a number of differentially expressed proteins. A group of 20 spots corresponding to a portion of these proteins of interest were excised from the gels and subjected to mass spectrometry.

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

The Pennsylvania Station has examined the effects of constitutive Raf expression on chick embryonic day 10 (ED10) myoblasts in vitro.  To further clarify the means by which Raf CAAX, a membrane localized protein, inhibits muscle formation, myogenic regulatory factor (MRF) mRNA levels in chick myoblasts expressing the activated allele were examined.  Myogenic cells overexpressing Raf BXB transcribed the full complement of MRFs.  However, RT-PCR and Western analysis revealed that Raf CAAX severely repressed the expression of myogenin.   Unexpectedly, myogenin transcription was restored by treatment of the cells with PD98059, a chemical inhibitor of MEK, indicating that a region within the myogenin gene was responsive to MEK activity.  To identify the region of MEK responsiveness, a panel of 5’ deletion mutants of the proximal 829 bp myogenin promoter was constructed by PCR and cloned upstream of the luciferase gene.  Each of the promoter-reporters was active in myogenic cells and inactive in fibroblasts.  In addition, each of the promoter-reporters was inhibited by overexpression of Raf CAAX.  Thus, the smallest promoter-reporter (-228mgn-Luc) containing an E-box and MEF2 site, includes the region affected by Raf CAAX.  However, cotransfection of CMV-Raf CAAX and -228mgn-Luc with either pBABE-MEKA221 or pCDNA-RKIP, dominant inhibitors of MEK, did not restore myogenin promoter activity.  These results indicate that the MEK responsive region lies outside of the 829 bp myogenin promoter.  To determine the role of MEF2 in the Raf imposed block to avian myogenesis, ED10 myoblasts were transfected with MEF2-Luc or MHC-Luc and Raf CAAX.  After 48 hours the cells were analyzed for reporter activity.  MEF2-Luc, a reporter plasmid comprised of the minimal MHC promoter fused to two MEF2 binding sites, directs high level luciferase expression that is inhibited by Raf CAAX.  In a similar manner, the minimal MHC reporter that lacks MEF2 binding sites (MHC-Luc) is inhibited by Raf CAAX.  These results argue that Raf CAAX negatively impacts transcriptional regulators in addition to MEF2.  Alternatively, we cannot rule out the possibility that the reduction in MHC-Luc activity by activated Raf is not a consequence of disrupted MRF:MEF2 interactions.

In other work, the Pennsylvania Station has screened a library to identify genes differentially regulated in response to activated Raf and identified a partial cDNA encoding for a basic leucine zipper protein with homology to Tax responsive element binding protein 107 (TaxREB107).  The full-length cDNA was cloned by RACE and found to be the chicken homologue of TaxREB107 (GenBank Accession number AY032864).  Northern analysis revealed that cTaxREB107 is abundantly expressed in all muscle types (cardiac, skeletal, smooth) and to a much lesser extent in brain and liver.   Functional studies revealed that overexpression of cTaxREB107 in chick myoblasts caused a modest 70% increase in muscle-reporter gene activity.  However, co-transfection of CMV-cMyoD, CMV-cTaxREB107 and 4Rtk-Luc, a multimerized muscle E-box reporter, did not significantly alter luciferase expression levels above controls.  Thus, cTaxREB107 likely enhances muscle gene expression through a mechanism independent of direct binding to the MRFs.  Immunolocalization experiments revealed that cTaxREB107 is located primarily in the nucleolus structures in mononucleated myoblasts with faint staining evident in the cytoplasm.  As the cells differentiate into the mature myocyte, the degree of immunostaining intensifies within the cytoplasmic compartment.  However, the nucleus remains immunopositive for cTaxREB107 expression.  Our results are supportive of a dual role for cTaxREB107 as a ribosomal and DNA binding protein.  

In yet other studies, the Pennsylvania Station has addressed the role of the E-protein heterodimer partnering with MRF function.  A Drosophilae Engrailed repressor domain was substituted for the E47 transcriptional activation domain to create EnDE47.   Stable mouse myoblast lines were developed that express EnD47 and the lines were analyzed for subsequent expression of muscle proteins.  C2C12EnDE47 myoblasts failed to fuse into large myocytes and directed low-level expression of myosin heavy chain and troponin T.  However, the cells maintained their ability to synthesize desmin and a-actinin suggesting that the chimeric repressor affects subsets of muscle genes.  To identify the heterodimer partners of E47, EnDE47 was immunoprecipitated from C2C12EnDE47 myocytes and the precipitate was analyzed by Western blotting for MyoD and myogenin expression.   Chimeric protein interacted readily with myogenin.  However, no MyoD:EnDE47 complexes were detected.  In vitro translated MyoD:EnDE47 formed and bound DNA as well as control, MyoD:E47 indicating that the repressor domain did not disturb the capacity of EnDE47 to form heterodimers.  Blotting with anti-MyoD demonstrated a strong MyoD immunoreactive band that was not complexed to EnDE47.  This work argues that MyoD heterodimers do not exist in C2C12 muscle cells and that MyoD homodimers are responsible for mediating MyoD-directed transcription. 

The Iowa Station is also attempting to define the global changes in muscle gene expression in response to 3-d of work overload in the rat.  Using robust statisitical analyses, researchers found 125 genes with adjusted P-values less than 0.05.  Theoretically, 5% of these genes are false positives.  This approach has increased the robustness of detecting differentially expressed genes.

            One gene identified by the Iowa Station, integrin-linked kinase (ILK) was shown to increase expression during work overload hypertrophy.  Using a transgenic mouse that overexpresses ILK in heart myocardium showed increased a-actin gene expression.  However, no myocardium hypertrophy was evident, suggesting that up-regulation of ILK in hypertrophying myocardium is not a causal response.

The Iowa Station has begun work to identify of genes downstream of myostatin using double-muscled (mh) and wild-type (wt) bovine embryos using subtractive hybridization analysis.  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 Michigan Station has employed a combination of differential display reverse transcription PCR and cDNA microarray experiments to identify genes that are differentially expressed in pig skeletal muscle at several fetal and postnatal ages.  Clones from this library were sequenced and used for the development of cDNA microarrays.  Microarrays, containing 171 sequenced clones, were subjected to total RNA from skeletal muscle of pigs at 60 d of gestation and 7 weeks of age, labeled with both Cy3 and Cy5, respectively and used to screen the microarray.  Fifty-five clones were overexpressed by at least 2-fold in 60 d fetal skeletal muscle as compared to 7 wk postnatal skeletal muscle.  Forty-one clones were overexpressed by at least 2.5-fold.  No clones were overexpressed in 7 wk postnatal muscle.  Our results demonstrate that cDNA microarray technology provides a powerful and rapid means for identifying differentially expressed genes.

Objective 3: Characterize muscle proteins and their functional domains involved in myofibrillar assembly and disassembly

The Arizona Station has re-examined the effect of phosphorylation of the calpains to determine whether this event had any effect on proteolytic activity. Studies revealed that ³²P was incorporated into a band that migrated to the same location as the 80kDa subunit of m-calpain in 2-D gels of human platelets. Subsequent studies using antibodies specific for phospo-Ser, phospho-Thr, and phospho-Try residues showed that both m- and m-calpain, as purified from tissues (bovine skeletal muscle, bovine cardiac muscle, human platelets, human placenta), contained phospho-Ser, phospho-Thr, and phospho-Tyr residues. Using Western analysis and proteolytic degradation of the calpains, these researchers showed that both m- and m-calpain had at least 6 phosphorylated residues, two each of phospho-Ser, phospho-Thr, and phospho-Tyr, and that these phosphorylated residues were concentrated in two areas of the calpain molecule; one having phospho-Ser, phospho-Thr and phospho-Tyr residues in the domain I/IIa part of the 80-kDa subunit and one, also having phospho-Ser, phospho-Thr, and phospho-Tyr residues in domain IIb/N-terminal part of domain III. These researchers documented residues on the calpains that are phosphorylated.  Hence, m-calpain has nine phosphorylated and m-calpain has eight phosphorylated residues, as they are purified from tissues. Not all calpain molecules in a preparation are phosphorylated to the same degree.  Phosphate analysis of 8 different calpain preparations showed that they contained 2.2 to 4.0 phosphate residues/molecule. The phosphates could not be removed by incubation with a number of phosphatases including alkaline phosphate, acid phophatase, calcineurin, etc. in the absence of Ca²⁺(EDTA), but could be removed completely in the presence of 2 mM Ca²⁺. Removal of all phosphorylated residues resulted in a proteolytically inactive calpain. Removal of all phospho-Ser and phospho-Thr residues with calcineurin also resulted in a proteolytically inactive enzyme. Hence, it seems likely that phosphorylation has an important role in regulation of calpain activity.  

The Wisconsin Station has begun to address the importance in various muscle fiber types in controlling pork quality.  Comparisons of eight different strains of pigs indicated that variations exist in the proportions of type I and IIA fibers.  Correlations between fiber type and several measures of meat quality (ultimate pH, Minolta color, etc.) were, however, generally low.  The Indiana Station has approached the same question using isolated myofibrils from different muscles at different times postmortem. Results of their studies suggest that the method of myofibril isolation may alter the composition of (fast vs. slow) myofibrils isolated.  In addition, the relative contribution of myofibrils in a mixed preparation may alter their susceptibility to pH inactivation. 

The Wisconsin Station has continued efforts to understand the binding of titin to the thin filaments.  These researchers have expressed recombinant fragments representing the sub-domains of the extensible region of cardiac N2B titin (tandem-Ig segments, the N2B splice element, and the PEVK domain), and assayed them for binding to F-actin.  The PEVK fragment bound F-actin, while no binding was detected for other fragments.  Significance of PEVK-actin interaction was investigated using in vitro motility and single-myocyte mechanics.  As F-actin slid relative to titin in the motility assay, a dynamic interaction between the PEVK domain and F-actin retarded filament sliding.  Myocyte results suggest that a similar interaction makes a significant contribution to the passive tension.  The effect of calcium on PEVK-actin interaction was also studied.  Although calcium alone had no effect, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibited PEVK-actin interaction in a calcium-dependent manner.  Gel overlay analysis revealed that S100A1 bound the PEVK region in vitro in a calcium-dependent manner, and S100A1 binding was observed at several sites along titin's extensible region in situ, including the PEVK domain.  In vitro motility results indicate that S100A1-PEVK interaction reduces the force that arises as F-actin slides relative to the PEVK domain, and suggest that S100A1 may provide a mechanism to free the thin filament from titin and reduce titin-based tension prior to active contraction.

The Wisconsin Station is investigating the role of myosin binding protein C (MyBP-C) in contraction, using a knockout mouse that lacks MyBP-C in heart.  Adult (3-4 mos) cMyBP-C^-/-, but not cMyBP-C^+/- mice displayed significant cardiac hypertrophy (two-fold increase of LV to body mass ratio) compared to wild type littermates. Ca²⁺ sensitivity of tension, measured in single skinned myocytes, was reduced in cMyBP-C^-/- but not cMyBP-C^+/- mice. Microscopic observation of myofibrils from homozygous knockout mice revealed minimal structural alteration.  These results establish that cMyBP-C, although important for normal cardiac physiology, is not essential for thick filament assembly. 

The Iowa Station is investigating the interaction of synemin with proteins in the Z-line and the costamere.  Blot overlay experiments were conducted to show an interaction between synemin and vinculin, a protein located in costameres of muscle cells.  Yeast two-hybrid assays were used to further characterize the interactions of synemin with desmin, vimentin, a-actinin and vinculin in an intracellular context, and to define the specific protein domains involved in these interactions.  Results demonstrated that: (1) the rod domain of synemin interacts strongly with both desmin and vimentin, but the synemin tail does not interact with either desmin or vimentin; (2) the interaction of synemin with a-actinin is specific for the tail domain of synemin, specifically the C-terminal one-fourth of the tail (tail domain IIb), and involves both the N-terminal head domain and the central rod domains of a-actinin; and (3) both the rod domain and the C-terminal one-fourth of the tail domain of synemin interact with vinculin, specifically only the tail domain of vinculin.  These results support a model for synemin function in which the synemin rod domain is responsible for the interactions involved in forming heteropolymeric intermediate filaments (IF) with desmin or vimentin, whereas the synemin tail extends from the IFs to bind to a-actinin or vinculin.  Synemin could thus bind IFs to myofibrils at the Z-line via the interaction with a-actinin, and to costameres at the sarcolemma via the interactions with vinculin and/or a-actinin.  These linkages would enable the IF network to link together all of the myofibrils at their Z-lines, and to tie the peripheral layer of myofibrils to the costameres.  Thus, synemin appears to be directly involved in maintaining the structural integrity of muscle cells.

The Michigan 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.  Using loin chops from two breeds of pigs, these researchers have shown that tenderness differs with breed or location within the loin.  Tenderness increased with storage time. Immunoblot analysis of tender and less tender samples at d 1 revealed that desmin degradation paralleled changes in shear force.  Intact desmin was typically undetectable in tender samples by d 7 and in less tender samples by d 14.  a-actinin declined during postmortem storage, although a-actinin was detectable at d 14 postmortem in tender pork longissimus.  Calpain activity using casein zymography indicated that m-calpain activity remained unchanged during postmortem storage.  Activity of m-calpain declined more rapidly in tender vs less tender pork.  These data 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.

Usefulness of Findings

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.  Many of the results summarized in the aforementioned report provide the basis for how growth occurs in economically important, meat-producing animals.  Genes and gene products investigated by this committee have been or likely will be targeted my many for exploiting animal growth efficiency.  Improving the biological efficiency of meat animals has been made possible by contributions of past and present members of this committee.  Much effort by this committee has been exerted to determine the mechanism by which satellite cells can be activated and exploited to improve muscle growth.  More recently, using more powerful techniques, these researchers have uncovered many new genes that will likely result in dramatic changes in our understanding of how muscle grows and functions.  Not only has this committee produced substantial gains in the understanding of animal growth, but also has expanded greatly the mechanism controlling product quality.  For example, studies regarding the role of calpains in controlling postmortem tenderization may ultimately lead to technologies to impact meat tenderness.  Likewise, identification of a desirable muscle fiber type composition could maximize meat production as well as quality.  These developments are essential to viability of animal agriculture.  

Work Planned for Next Year

Studies will continue as described in the recently approved renewal proposal.  No changes in the approach or objectives are anticipated at this time.

Publications for the Year

Refereed Journal Articles

            Published Full-Length Articles

Arizona

Bialkowska, K., S. Kulkarni, X. Du, D.E. Goll, T.C. Saido, and J.E.B. Fox. 2000. Evidence that β3 integrin-induced Rac activation involves the calpain-dependent formation of integrin clusters that are distinct from focal complexes and focal adhesions that form as Rac and RhoA become active. J. Cell Biol. 151: 685-695.

Cottin, P., V.F. Thompson, S.K. Sathe, A. Szpacenko, and D.E. Goll. 2001. Autolysis of μ- and m-calpain from bovine skeletal muscle. Biol. Chem. 382: 767-776.

Delgado, E.F., G.H.Geesink, J.A. Marchello, D.E. Goll, and M. Koohmaraie. 2001. The calpain system in three muscles of normal and callipyge sheep. J. Anim. Sci. 79:398-412.

Delgado, E.F., G.H. Geesimk, J.A. Marchello, D.E. Goll, and M. Koohmaraie. 2001. Properties of myofibril-bound  calpain activity in longissimus muscle of callipyge and normal sheep. J. Anim. Sci. 79: 2097-2107.

Tatsumi, R., Sheehan, S.M., Iwasaki, H., Hattori, A., and Allen, R.E. 2001. Mechanical stretch induces activation of skeletal muscle satellite cells in vitro. Exp. Cell Res. 267, 107-114.

Indiana

Depreux, F.F.S., A.L. Grant and D.E. Gerrard.  2002.  Influence of halothane genotype and body-weight on myosin heavy chain composition in pig muscle as related to meat quality Livestock Prod. Sci.  73:265-273.

Iowa

Bellin, R. M., T. W. Huiatt, D. R. Critchley, and R. M. Robson. 2001. Synemin may function to directly link muscle cell intermediate filaments to both myofibrillar Z-lines and costameres. J. Biol. Chem. 276:32330-32337.

Schweitzer, S. C., M. W. Klymkowsky, R. M. Belin, R. M. Robson, Y. Capetanaki, and R. M. Evans. 2001. Paranemin and the organization of desmin filament networks. J. Cell Sci. 114:1079-1089.

Minnesota

Yi, Z., M. R. Hathaway, W. R. Dayton, and M. E. White 2001. Effects of growth factors on insulin-like growth factor binding protein (IGFBP) secretion by primary porcine satellite cell cultures. J Anim. Sci. 79:2820-2826.

Nebraska

Gu, X., G. Whipple-Van Patter, M. O’Dwyer and M.G. Zeece. 2001. Capillary electrophoretic analysis of µ- and m-calpain using fluorescently labeled substrates. Electrophor. 22: 2336-2342.

Ohio

Velleman, S.G. 2000.  The role of the extracellular matrix in skeletal development.  Poultry Sci.  79:985-989.

Velleman, S.G., Liu, X., Nestor, K.E., and McFarland, D.C. 2000.  Heterogeneity in growth and differentiation characteristics in male and female satellite cells isolated from turkey lines with different growth rates.  Comp.  Biochem.  Physiol.  Part A 125:503-509.

Velleman, S.G., Coy, C.S., Gannon, L., Wick, M., and McFarland, D.C. 2000.   Beta 1 integrin expression during normal and low score normal avian myogenesis.  Poultry Sci.  79:1179-1182.

Pedersen, M.E., Kulseth, M-A., Kolset, S.O., Velleman, S., and Eggen, K.H. 2001.  Decorin and fibromodulin expression in two bovine muscles (M.  Semitendinosus and M. Psoas Major) differing in texture.  J. Muscle Foods 12: 1-17.

Velleman, S.G. and Nestor, K.E. 2001.  Mode of inheritance of the low score normal condition in chickens.  Poultry Sci.  80:1273-1277.

Pennsylvania

Becker, J.R., C.M. Dorman, T.M. McClafferty and S.E. Johnson.  2001.  Characterization of a dominant inhibitory E47 protein that suppresses C2C12 myogenesis.  Exp. Cell Res. 267:135-143.

South Dakota

Velleman, S. G., X. Liu, K. E. Nestor, and D. C. McFarland. 2000.  Heterogeneity in Growth and Differentiation Characteristics in Male and Female Satellite Cells Isolated from Turkey Lines with Different Growth Rates. Comparative Biochemistry and Physiology Part A. 125:503-509.

McFarland, D. C., Y. N. Singh, A. D. Johnson, J. E. Pesall, K. K. Gilkerson, and L. S. Vander Wal. 2000. Isolation and Characterization of Myogenic Satellite Cells from the Muscular Dystrophic Hamster.  Tissue and Cell 32(3): 257-265.

Pesall, J. E., D. C. McFarland, J. P. McMurtry, J. A. Clapper, G. L. Francis, and K. K. Gilkerson. 2001. The Effect of Insulin-like Growth Factor Analogs on Turkey Satellite Cell and Embryonic Myoblast Proliferation.  Poultry Science 80:944-948.

Kocamis, H., D. C. McFarland, and J. Killefer. 2001. Temporal Expression of Growth Factor Genes during Myogenesis of Satellite Cells Derived from the Biceps Femoris and Pectoralis Major Muscles of the Chicken. Journal of Cellular Physiology 186(1): 146-153.

Washington

McGuire, T.C., S.T. Leib, S.M. Loning, W. Zhang, K.M. Byrne and R.H. Mealey. Equine infectious anaemia virus proteins most frequently recognized by cytotoxic T lymphocytes from infected horses. Journal of General Virology  81:2735-2739

Byrne, K.M., K. Bynum, L. Robinette and L. Brownlee.  2000. Calcium oxalate stones in feline littermates. Journal of Feline Medicine and Surgery 2:111-114

Byrne, K.M., G.A. Reinhart and M.G. Hayek.  2000. Standardized flow cytometry gating in veterinary medicine. Methods in Cell Science  22:191-198

Ebling, T.L., L.K. Fox and K.M. Byrne.  2000.  Isolation of bovine mammary lymphocytes for fluorescent activated flow cytometry.  Methods in Cell Science 22:239-245

Mir, P.S., J.L. Vierck, Z. Mir and M.V. Dodson. 2000. Quantification of lipid in cultured 3T3-L1 adipocytes. Animal Science 71(6):521-526

Dodson, M.V., A. Zimmerman and K. Byrne.  2000.  Establishment of an international research team for the study of myogenic satellite cells derived from fish. Cell Biology International 24(11):849-850

Dodson, M.V. 2000. Are we making progress in defining the role and regulation of myogenic satellite cells? Basic and Applied Myology 10(4):201-202

Wisconsin

Greaser, M.L. 2001. Identification of new repeating motifs in titin.  Proteins 43:145-149.

Sant'Ana Pereira, J., D. Pavlov, M. Nili, M. Greaser, E. Homsher and R. L. Moss. 2001. Kinetic differences in cardiac myosins with identical loop 1 sequences.  J. Biol. Chem. 276:4409-4415.

Yamasaki, R., M. Berri, Y. Wu, K. Trombitas, M. McNabb, Kellermayer, M.S.Z., Witt, C., Labeit, D., Labeit, S., Greaser, M., and Granzier, H. 2001. Titin-actin interaction in mouse myocardium: passive tension modulation ad its regulation by calcium/S100A1.  Biophys. J. 81:2297-2313.

Published Abstracts

Arizona

Li, H., V.F. Thompson, and D.E. Goll. 2000. Efficacy of different calpastatin constructs. Mol. Biol. Cell 11:385a.

Cong,J. V.F. Thompson, and D.E. Goll. 2000. Phosphorylation of the calpains. Mol. Biol. Cell 11:386a.

Iowa

Bellin, R. M., T. W. Huiatt, and R. M. Robson. 2000. Mapping the protein interaction domains of the intermediate filament protein synemin. Mol. Biol. Cell 11:532a.

Stromer, M., and M. S. Mayes. 2000. The domain structure in three different smooth muscles. Mol. Biol. Cell 11:69a.

Tong, W., D. H. Burke, D. J. Graves, R. M. Robson, and T. W. Huiatt. 2000. ADP-ribosylation of muscle cell desmin detected with nucleic acid aptamers. Mol. Biol. Cell 11:533a.

Bellin R, T. Huiatt, and R. Robson. 2001. Use of the yeast two-hybrid system to elucidate the multiple protein interaction domains within the muscle cell intermediate filament protein synemin. J. Anim. Sci.79 (Suppl. 2):54.

Potts, J. K., T. P. L. Smith, and J. M. Reecy. 2001. Identification of genes downstream of myostatin in the developing bovine embryo. J. Anim. Sci. 79 (Suppl. 2):51.

Tong W, D. Burke, R. Robson, and T. Huiatt. 2001. The intermediate filament protein desmin is ADP-ribosylated in skeletal muscle cells.  J Anim. Sci. 79 (Suppl. 2):54.

Webster, M., and J. M. Reecy. 2001. Cyclic Stretch influences p21WAF1 promoter activity in myoblasts and myotubes. J. Anim. Sci. 79 (Suppl. 1):28.

Webster, M., and J. M. Reecy. 2001. Cyclic Stretch increases p21WAF1 promoter activity independent of terminal differentiation in C2C12 myotubes. FASEB Summer Research Conference: Muscle Satellite and Stem Cells, July 2001.

Michigan

Allison, C.P., R.J. Tempelman and M.E. Doumit. 2001. Analysis of postmortem tenderization in porcine longissimus dorsi muscle.  J. Anim. Sci. 79 (suppl. 1):20.

Ritter, M.J., C.P. Allison, S.R. Debar, J.M. Scheffler, R.J. Tempelman and M.E. Doumit. 2001.  Evaluation of growth rate, carcass composition and meat quality of Berkshire- and Yorkshire-sired progeny.  Proc. Reciprocal Meat Conf. 54:367.

Scheffler, J.M., D.D. Buskirk, S.R. Rust, J.D. Cowley and M.E. Doumit. 200l. Repeated administration of implants to Holstein steers increases daily gain, longissimus muscle area and the percentage of USDA Select carcasses.  J. Anim. Sci. 79 (suppl. 1):275.

Ohio

Velleman, S.G., Coy, C.S., and McFarland, D.C. 2000.  Beta 1 integrin expression and localization during normal and low score normal avian myogenesis.  Mol.  Biol. Cell 11:54a.

Yilmaz, A., Wick, M., and Velleman, S.G. 2001.  Altered myosin heavy chain isoform transitions in satellite cells and pectoralis major muscle from LSN chickens.  J. Anim. Sci. (suppl 1) 79:598.

Hanagan, M.J., Roe, C.M., Velleman, S.G., and Reiser, P.J. 2001.  Sinusoidal stress analysis of skeletal muscle containing an altered extracellular matrix.  OSU Undergraduate Research Symposium.

Velleman, S.G. 2001.  Role of the extracellular matrix in muscle growth and development.  J. Anim.  Sci (suppl. 1)79:598.

South Dakota

Velleman, S. G., C. S. Coy, and D. C. McFarland. 2000. Beta 1 Integrin Expression and Localization during Normal and Low Score Normal Avian Myogenesis. Mol. Biol. Cell 11:54a.

Washington

Kuber, P., J. Busboom, E. Lonergan, S. Duckett, P. Mir, Z. Mir, R. McCormick, M. Dodson, C. Gaskins, J. Cronrath and D. Marks. 2001. Does troponin-t degradation, collagen percentage or collagen crosslinking explain differences in tenderness between Wagyu and Limousin cattle? Proc. Am. Soc. Anim. Sci. (Western Section) 52:17-20.

Byrne, K.M.  Flow cytometry applications.  Methods in Cell Science 22:165

Wisconsin

Harris, S.P, Gallagher, S., Greaser, M.L., Moss, R.L., and Trewella, J. 2001. Small angle X-ray scattering by heavy meromyosin in solution.  Biophys. J. 80:577a.

Accepted Full-Length Articles

Indiana

Eggert, J.M., F.F.S. Depreux, A.P. Schinckel, A.L. Grant and D.E. Gerrard.  2001.  Myosin heavy chain isoforms account for variation in pork quality. Meat Sci.(in Press)

Short, R.E., M.D. MacNeil, M.D. Grosz, D.E. Gerrard and E.E. Grings.  Pleiotropic effects in hereford, limousin, and piedmontese F2 crossbred calves of genes controlling muscularity including the piedmontese myostatin allele. J. Anim. Sci. (in Press)

Iowa

Lee, H.-S., R. M. Bellin, and R. M. Robson. 2001. Talin. In: Creighton, T. E. (ed.) Encyclopedia of Molecular Medicine, John Wiley & Sons, Inc., New York. (in press).

Minnesota

Schoonmaker, J. P., S. C. Loerch, W. R. Dayton, and M. R. Hathaway.  Effect of an accelerated finishing program on performance, carcass characteristics, and circulating IGF-1 concentration of early-weaned bulls and steers.  J. Anim. Sci. (in press).

Pennsylvania

Thomson, S.R. and S.E. Johnson.  2001.  Isolation and characterization of chicken TaxREB107, a putative DNA binding protein abundantly expressed muscle.  Gene (in press).

Washington

Vierck, J.L., D. Dal Porto and M.V. Dodson. 2001. Induction of preadipocyte differentiation by a defined treatment medium without DMI. Basic and Applied Myology (in press)

Dodson, M.V. 2001. Balancing research, teaching and service in an academic environment. NACTA Journal (in press).

Non-refereed Publications

                                Proceedings, Book Chapters, etc.

Arizona

Goll, D.E., V.F.Thompson, H. Li, and J. Cong. 2001. The role of the calpain system in neuromuscular disease. In: Banik, N., and Lajtha, A,, eds.), The Role of Proteinases in the Pathophysiology of Neurodegenerative Disease, Kluwer Academic/Plenum Publishers, New York, NY. pp. 63-75.

Michigan

Doumit, M.E., C.P. Allison and M.J. Ritter. 2001. Control of glycolysis in relation to pork quality traits.  Invited paper in sympsium: Beyond pH:  Metabolic factors affecting pork quality.  Proceedings of the 2^nd Annual Pork Quality Symposium.   

Yao, J., P.M. Coussens, J.L. Burton and C.W. Ernst. 2001. Development of high quality cDNA libraries and landmarks for cDNA microarrays to study complex disease and production traits in cattle and swine. Plant and Animal Genome IX meetings. San Diego, CA. http://www.intl-pag.org/pag/9/abstracts/P01_46.html.

Ohio

Hanagan, M.J., Roe, C.M., Velleman, S.G., and Reiser, P.J. 2001.  Sinusoidal stress analysis of skeletal muscle containing an altered extracellular matrix.  OSU Undergraduate Research Symposium.

South Dakota

Guan, X., B. N. Hoffman, D. C. McFarland, K. K. Gilkerson, C. Dwivedi, A. K. Erickson, S. Bebensee, and J. Pellegrini. 2001. Glutathione Conjugation and its Contribution to the Anticancer Activity of Sulofenur. Annual Meeting of the American Chemical Society – Proceedings.

Wisconsin

Greaser, M.L., H. Okochi, and A.A. Sosnicki. 2001. Role of fiber types in meat quality. Proc. 47^th Inter. Cong. Meat Sci Technol. pp 34-37.

Pospiech, E.  M. Szalata, R. L.J.M. van Laack, A. A. Sosnicki,  and M. L. Greaser. 2001. Tenderness and protein changes of pork in relation to pig genotype and postmortem glycolysis phenotype.  Proc. 47^th Inter. Cong. Meat Sci Technol. pp 258-259.

Van Laack, Riëtte L.J.M., R. G. Kauffman, and M.L. Greaser.  2001.  Determinants of ultimate pH of meat.  Proc. 47^th Inter. Cong. Meat Sci Technol. pp 22-26.

Greaser, M.L. 2001. Post-mortem muscle chemistry. In: Y.H. Hui, W.-K. Nip, R.W. Rodgers, & O.A. Young (ed.) Meat Science and Applications, Marcel Dekker, Inc., New York pp 21-37.

APPROVED:

                                                                                                                                      12-26-01

Dr. David Gerrard, Secretary, 2001-2002                                                                        Date

                                                                                 01/11/02

Dr. Elton Aberle, Administrative Advisor                                                                       Date