Annual Report

Regional Research Project NC-131

Project Title: Molecular Mechanisms Regulating Skeletal Muscle Growth and Differentiation

Project Period: 10-1-03 to 9-30-04

State                            Institution                                                              Project Leaders

Arizona                        University of Arizona                                                Goll, D.E.*

                                                                                                                    Allen, R.E.

California                    University of California, Davis                                     Bandman, E.*

Hawaii                        University of Hawaii                                                    Kim, Y.S.*

Idaho                           University of Idaho                                                    Hill, R.A.*

Indiana                         Purdue University                                                     Gerrard, D.E.*

                                                                                                                    Grant, A.L.

Iowa                            Iowa State University                                                Huiatt, T.W.*

                                                                                                                    Robson, R.M.

                                                                                                                    Reecy, J.M.

Kansas                         Kansas State University                                            Johnson, B.J.*

Michigan                     Michigan State University                                           Doumit, M.E.*

                                                                                                                    Ernst, C.W.

Minnesota                   University of Minnesota                                             Dayton, W.R.*

                                                                                                                    Hathaway, M.R.

                                                                                                                    White, M.E.

North Carolina            North Carolina State University                                  Mozdziak, P.

Ohio                            Ohio State University                                                Velleman, S.G.*

Oregon                        Oregon State University                                            Forsberg, N.E.

Pennsylvania               Pennsylvania State University                                     Johnson, S.E.*

South Dakota              South Dakotas State University                                 McFarland, D.C.*

Utah                            Utah State University                                                Carpenter, C.E.*

Washington                 Washington State University                                      Dodson, M.V.*

                                                                                                                    Rodgers, D.

Wisconsin                    University of Wisconsin                                             Greaser, M.L.*

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

Work at the Arizona Station continues to focus on the activation of satellite cells by HGF and the release of HGF from muscle cells in response to mechanical perturbation. One of the key questions is whether muscle stretch stimulates release of active HGF or proteolytic activation of pro-HGF and its subsequent release. In a published set of experiments, the presence of active and pro-hepatocyte growth factor (HGF) in the extracellular compartment of un-injured skeletal muscle was examined.  Extensor digitorum longus (EDL) muscles were excised from 9-month-old male rats and release of HGF was examined by Western blot following various extraction treatments. HGF was released into phosphate buffered saline (PBS) extraction solution containing 1 M NaCl or heparinases I and III, plus a cocktail of serine protease inhibitors.  Active HGF was the predominant form released.  Exogenous pro-HGF was cleaved when incubated with un-injured or crush-injured skeletal muscle in the absence of serine protease inhibitor cocktail but was not cleaved when inhibitors were included in incubation solution.  When muscle was incubated with Triton X-100 or crush-injured, both pro- and active HGF were released into the extract.  Finally, inclusion of the nitric oxide (NO) donor, sodium nitroprusside (SNP), resulted in release of HGF, and it was predominantly found in the active form.  These experiments indicate that active HGF is present in the extracellular compartment of uninjured skeletal muscle and that endogenous serine proteases are present in muscle tissue that can cleave pro-HGF.  Finally, the study demonstrates that NO stimulates release of active HGF from un-stretched and un-injured skeletal muscle, as previously reported with cultured muscle cells and stretched muscle in vivo.

In a set of preliminary set of experiments employing calcium ionophores, calmodulin inhibitors and nitric oxide synthase inhibitors, evidence was that calcium and calmodulin stimulate the activity of NOS which results in production of NO and HGF release from isolated satellite cells.  The mechanism through which NO stimulates HGF release from the extracellular matrix remains unknown.

At the Hawaii Station, myostatin, a negative regulator of muscle growth is under investigation. Myostatin is a member of the transforming growth factor-β (TGF-β) superfamily. In this study, effects of in-ovo administration of anti-myostatin antibody on post-hatch broiler growth and skeletal muscle mass were examined. To produce monoclonal antibodies against myostatin, semi-purified recombinant C-terminal myostatin fragment (18 kD) containing the mature form of myostatin was sent to the UH Manoa Monoclonal Antibody Facility. Among 120 hybridoma clones generated, 6 clones showed an affinity to the recombinant myostatin in immunoblotting.  A hybridoma clone that showed the strongest affinity to the C-terminal myostatin was used to produce ascites fluid, and the ascites fluid was subjected to the protein A affinity column to purify monoclonal anti-myostatin antibody (mAb72).

The mAb72 demonstrated an affinity to the monomer and dimmer from of recombinant mature myostatin (R&D Systems, Minneapolis, MN) in the Western blot analysis, with higher affinity to the monomer form than to the dimmer form of myostatin. When the cross reactivity of mAb72 with other members of the TGF-b superfamily was tested using the Western blot analysis, the mAb72 cross-reacted with rhBMP2 but not with rhTGF-b3 and pTGF-b1. The affinity of mAb72 to rhBMP2 was lower than the affinity to myostatin. Immunoblotting of skeletal muscle homogenates with mAb72 detected four proteins with approximate molecular wt at 50, 37, 31 and 17 kDa, but not detected a band presumed to be the mature myostatin (12.5 kDa). Results from a competitive ELISA supported the binding of mAb72 to mature myostatin.

To examine the effects of in-ovo administration of anti-myostatin antibody (mAb72) on post-hatch broiler growth, 90 fertilized broiler eggs were divided into three groups: control (no injection), mouse IgG injection and mAb72 injection.  20 µl of mouse IgG (2.5 µg protein) or mAb72 solutions (3 µg protein) were injected into the albumen through the blunt side of the eggs at 3 days after incubation.  The hole drilled for injection was sealed with parafilm, followed by melting of the parafilm with torch.  Eggs were set in incubator/hatcher until hatching.  After hatching, chicks were grown for 24 days, sacrificed, and the wt of breast muscle was measured.  The in-ovo-administration of mAb72 appeared not to affect the growth of broiler.  However, mAb72 injection tended to increase the ratio of breast muscle wt to body wt in male broilers.

In the past year, the Idaho Station has studied leptin-insulin interactions with the goal of improving production efficiency in ruminants.  This project, funded by the NRI commenced in December 2002.  Idaho is the lead laboratory with subcontracts to Washington and to Israel – Dr Gertler, University of Jerusalem.  Studies have been further supported by additional funds from the Idaho BRIN program (NIH umbrella) for synergistic studies using a mouse model.

The Idaho Station is characterizing 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. Studies have been conducted at both the protein level, investigating signaling mechanisms and at the level of gene expression, to determine whether cross-regulation of leptin and insulin signaling pathways is transcriptionally regulated in the two models.

In the two cell models, recruitment of PI 3-kinase to IRS-1 in response to insulin appears to follow a similar time-course.  Interestingly, relative abundance of PI 3-kinase to IRS-1 appears to be much less in the insulin-resistant bovine model, and suggests that the efficacy of bovine IRS-1 recruitment of PI 3-kinase is less than in the insulin-sensitive Sol 8 model.  Downstream activation potential of the IRS-1-PI 3-kinase complex, indicated via available pY, suggests that the bovine cells have additional capacity, which remains unbound by downstream messengers, whereas in Sol 8, there is little change in unbound pY in response to insulin (Strat et al., 2003).  Despite apparently normal insulin sensitivity in Sol8, recruitment of PI 3-kinase directly to the insulin receptor increased three-fold in response to insulin, whereas recruitment of PI 3-kinase to IRS-1 increased by only two-fold (Hill et al., 2004).

There appears to be functional antagonism between leptin and insulin in modulating energy substrate utilization.  A mechanism involved in this interaction appears to be cross-talk of signaling pathways.  Treatment of BMC with insulin, leptin or a sequential treatment: leptin then insulin and investigation of the targets: IRS-1 total protein, IRS-1 phosphorylated at serine 307 (pS307-IRS-1) and p70S6 kinase recruitment on IRS-1 revealed that insulin or leptin treatment alone induced only minor changes.  The increase in total IRS-1 in response to the sequential treatment, was paralleled by an increase in pS307-IRS-1 phosphorylation which is an accepted mechanism directing IRS-1to the proteosome degradation pathway.  Furthermore, the synchronous increase in p70S6 kinase recruited to IRS-1 suggests that this may be the serine-threonine kinase which is the major contributor to modulation of IRS-1 degradation following phosphorylation at S307.  Because neither hormone treatment alone resulted in induction of these phenomena observed in the sequential treatment, we speculate that p70S6 kinase may be a linking molecule in the cross-talk of the leptin and insulin signaling pathways (Strat et al., 2004a). 

Leptin effects on gene expression in BMC were investigated using cross-species hybridization to Human Genome U133A Array (HG-U133A).  The analysis showed the following pattern of detection: from 22283 probe sets 14.9% were detected as present, 83.3% as absent and 1.7% marginal. The change in the functional status of these genes was: 46 (0.2%) were induced de novo and included structural, functional and metabolic muscle tissue genes; expression of 90 (0.4%) genes was decreased and included cell cycle progression, extracellular matrix, adhesion molecules and functional genes; 215 (1%) increased and included mainly structural, functional and metabolic genes; 129 (0.6%) decreased including functional genes. The profiling pattern revealed no change of genes for the molecules involved in the leptin signaling pathway but only in pathway modulators (decreased serine-threonine kinase and tyrosine phosphatase), induction of the metabolic genes involved in known leptin effects and major induction of myogenic differentiation genes (Strat et al., 2004b).

The Indiana Station is currently investigating the role of myofiber type in β-agonist-induced skeletal muscle hypertrophy. It has been well documented that β-agonists can induce skeletal muscle hypertrophy and fiber type transition in rats, mice, pigs, cattle and sheep. However, the relationship between hypertrophy and fiber type and/or fiber type transition remains largely unknown. Toward this end, we fed C57BL/6, myosin heavy chain (MyHC) IIX^-/- and IIB^-/- mice 20 ppm clenbuterol for 2 wks. Soleus (slow), TA (fast) and EDL (fast) muscles were weighed and serial cryosections were cut and immunostained with MyHC-specific antibodies. Clenbuterol increased soleus, TA and EDL weights by 30%, 48% and 50%, respectively. Furthermore, the clenbuterol-induced increase in cross sectional area (CSA) was fiber type-dependent (type I, IIA, IIX and IIB increased by 6%, 28%, 66% and 32%, respectively). Lack of the type IIX and IIB genes had no effect on muscle weight of soleus, but blunted the clenbuterol effect in TA and EDL muscles. Further study of myofiber isoforms suggested that a compensatory increase in CSA induced by the gene knockout is limited. Specifically, knocking out the MyHC IIX gene blocks the normal β-agonist-stimulated transition to the IIB phenotype. Taken together, these findings confirm that β-agonist induced skeletal muscle hypertrophy is fiber type-dependent and coupled to fiber type transition to the fast-twitch phenotype, and suggest that an orderly, step-wise progression through various MyHC is ‘somehow’ necessary for complete transition to the fast phenotype.

In addition, the Indiana Station is examining signaling pathways in β-agonist-induced skeletal muscle hypertrophy. To understand more fully the mechanisms the underlying the β-agonist-induced skeletal muscle hypertrophy in slow versus fast muscles, the station has begun to explore the pathways involved in IGF-1-induced skeletal muscle hypertrophy. In vitro and in vivo reporter gene assay indicates that ERK signaling is differentially involved in slow versus fast myofiber-specific gene expression. Red and white semitendinosus muscles from pigs fed ractopamine for 3 wks were sampled for immunoblot analysis. ERK and Akt/Gsk3 signaling pathways were activated in both slow and fast muscles. Further analysis revealed that the activation of these signaling pathways is greater in fast muscles. The differential involvement of ERK signaling in slow and fast muscles in response to β-agonist can be explained, at least in part, by a greater presence of phophorylated ERK1/2 in muscle cells, and a greater responsiveness of fiber-type specific genes to ERK ablation in fast than in slow myofibers. These data suggest that a differential participation of ERK and/or Akt/Gsk signaling pathways in β-adrenergic receptor signaling may account for the distinct responsiveness of slow and fast myofibers to ractopamine.

The Indiana Station is also investigating energy metabolism and myofiber type specification.

AMP-activated protein kinase (AMPK) acts as an energy sensor in living cells. In part, AMPK controls glucose transport and mitochondrial energy metabolism and is tightly regulated by cellular concentrations of ATP and AMP. The relationship between AMPK signaling and muscle fiber type phenotype, however remains unknown.  In an effort to understand the role of AMPK in myofiber specification, C2C12 myotube cultures were treated with 5-aminoimidazole-4-carbozamide-β-D-ribofuransoside (AICAR), an AMP analog. After 4 days of AICAR treatment, type IIB MyHC protein increased 2-fold, while type I and total MyHC remained relatively unchanged. Moreover, the role of MAPK in muscle fiber type specification using porcine satellite cell cultures was examined.  Addition of the MEK inhibitor, U0126 increases type I MyHC 2-fold and decreases fast MyHC content.  Further analysis revealed that the increase of type IIB MyHC induced by AICAR can be blocked by the addition of a MEK inhibitor suggesting ERK signaling is downstream of AMPK activation and fiber type determination.  These data suggest that activation of AMPK can lead to changes in muscle MyHC composition and form the hypothesis that AMPK and the ERK-MAPK signaling pathways act synergistically to determine myofiber type specification.

Finally, the Indiana Station has examined partitioning of nutrients between muscle and fat. The objective of these studies was to employ a model of increased muscle and adipose tissue growth to further elucidate the interplay between these two diverse tissues. In the first study, myostatin null mice were interseminated with mice possessing the leptin db mutation to produce mice homozygous for both mutations.  Myostatin null-leptin db/db mice (DKO) were heavier than leptin db/db (LKO) and myostatin null (MKO) mice. While body fat content was decreased 10% in DKO compared with LKO mice, it was increased 400% compared with MKO mice. Body protein percentage was not different between DKO and LKO, but was decreased compared with wild type (WT) and MKO mice. The second study subjected MKO mice to high fat diets to determine if MKO mice were susceptible to high fat diet induced obesity. MKO and WT mice were assigned to high fat (HF) diets in which 60% of calories were derived from fat or low fat (LF) diets in which 10% of calories were derived from fat. After 4 weeks of ad libitum intake, WT mice consuming the HF diet were 11% heavier than WT mice on the LF diet. Additionally, HF feeding increased body fat content 57%. In contrast, while KO mice consuming the HF diet were also 11%  heavier than their LF counterparts, body fat content was increased only 28%. Comparing MKO on the HF diet with WT on the HF diet, MKO mice were heavier and had increased body protein content compared with WT, however body fat content of WT mice was increased nearly 200% compared with MKO. These data suggest that MKO mice may be more resistant to high fat diet induced obesity as their response to HF feeding was less severe than WT.  Together, these data suggest that muscle hypertrophy may interact with adipose tissue in the development of obesity.

Research at the Kansas Station has shown that progestins can elicit an anti-proliferative effect on cells through a non-genomic mechanism.  These non-genomic mechanisms are classified as an action by which the progestin does not work through the typical pathway involving transcriptional regulation by the progesterone receptor.  Previous results at the Kansas Station  have shown the addition of melengestrol acetate (MGA), a synthetic progestin, to cultured bovine muscle satellite cells resulted in a dose-dependent decrease in DNA synthesis as measured by [³H]-thymidine incorporation rate.  Effects of melengestrol acetate (MGA) and progesterone (P4) on insulin-like growth factor-I (IGF-I), myogenin, progesterone receptor (PR), and progesterone receptor membrane component 1 (PRMC) mRNA abundance in C₂C₁₂ muscle cells, as well as their effects on DNA synthesis were studied.  Cells in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum were plated on tissue culture plates.  Either MGA or P4 (0, 10 pM, 1 nM, 10 nM, 100 nM or 1 µM) in phenol-red-free DMEM was added 24 h after plating.  At 72 h, total RNA was isolated and reverse transcribed for complimentary DNA (cDNA) synthesis.  Real-time quantitative PCR was performed on the cDNA to estimate mRNA concentration. Melengestrol acetate or P4 addition had a dose effect on myogenin and PRMC mRNA abundance, with a significant increase (P<0.05) of myogenin mRNA in 100 nM MGA cultures.  There was no significant effect on IGF-I mRNA levels.  PR mRNA could not be detected, suggesting that it may not be present in C₂C₁₂ muscle cells.  In a separate set of experiments, C₂C₁₂ cells were plated in phenol-red-free DMEM containing 0.5 mg IGFBP-3 free swine serum/mL.  Melengestrol acetate or P4 (0, 10 pM, 1 nM or 100 nM) was added 24 h after plating, and DNA synthesis was estimated at 72 h by [³H]-thymidine incorporation (TI) rate.  Melengestrol acetate and P4 addition resulted in a dose-dependent decrease in TI.   RU486 (10 nM), in combination with either MGA or P4 (1 nM) also resulted in anti-proliferative effects.   The addition of RU486 to MGA or P4 treated cultures did not attenuate the reduction in TI caused by progestin addition.  RU486 alone had an anti-proliferative effect similar to that of the progestins.  These studies suggest that progestins reduce cell proliferation and concomitantly stimulate markers of muscle cell differentiation.  Furthermore, the inability to detect PR mRNA, coupled with the antiproliferative effect of RU486 in C₂C₁₂ cells suggests that these effects are mediated through a non-genomic mechanism.

At the Minnesota Station, several studies were completed in 2004. Insulin-like growth factor (IGF)-binding protein-5 (IGFBP-5) is produced by cultured porcine embryonic myogenic cell (PEMC) cultures and is secreted into the medium. IGFBP-5 may play some role in myogenesis and/or in changes in myogenic cell proliferation that accompany differentiation. IGFBP-5 reportedly may either suppress or stimulate proliferation or differentiation of cultured cells depending on cell type and culture conditions. Additionally, IGFBP-5 has been shown to possess both IGF-dependent and IGF-independent actions in some cell types. The goal of the first study was to produce recombinant porcine IGFBP-5 and assess its IGF-I-dependent and IGF-I-independent actions on proliferation of PEMCs. To accomplish this, porcine IGFBP-5 was expressed using the baculovirus system, purified and characterized the expressed recombinant porcine IGFBP-5 (rpIGFBP-5) and produced an anti-porcine IGFBP-5 antibody that neutralizes the biological activity of porcine IGFBP-5. rpIGFBP-5, purified to 98% homogeneity using nickel affinity chromatography and IGF-I affinity chromatography suppressed IGF-I-stimulated proliferation of PEMCs in a concentration-dependent manner (P < 0.05). rpIGFBP-5 also suppressed Long-R3-IGF-I-stimulated proliferation of PEMCs (P < 0.05), even in the presence of significant molar excess of Long-R3-IGF-I compared with rpIGFBP-5.  These results indicate that rpIGFBP-5 possesses IGF-independent activity in porcine myogenic cell cultures. The anti-rpGIFBP-5 IgY produced against rpIGFBP-3 specifically recognized native porcine IGFBP-5 in PEM culture media that also contained porcine IGFBP-2, -3, and -4.  This antibody is capable of neutralizing the effects of rpIGFBP-5 on PEMCs as well as detecting IGFBP-5 in Western blots.  The production of rpIGFBP-5 and an neutralizing antibody to porcine IGFBP-5 provide powerful tool to investigate the role of IGFBP-5 in porcine myogenic cell proliferation and differentiation. These data demonstrate that rpIGFBP-5 has the potential to affect proliferation of PEMCs during critical periods of muscle development that may impact the capacity for ultimate postnatal muscle mass development.

In an additional study at the Minnesota Station, treatment of cultured porcine embryonic myogenic cells (PEMC) with either TGF- β or myostatin results in significantly increased levels of IGFBP-5 mRNA; however, there was no significant difference between the IGFBP-5 mRNA level of PEMC cultures treated with TGF- β as compared to cultures treated with myostatin. In addition to increasing IGFBP-3 and IGFBP-5 expression, both TGF-β₁ and myostatin suppress IGF-I-stimulated proliferation in PEMC cultures. Since IGFBP-5 suppresses proliferation of PEMC, we were interested in determining whether IGFBP-5 plays a role in the ability of TGF-β₁ and myostatin to suppress proliferation in these cultures. Antibody neutralization of IGFBP-5 resulted in a 40 - 50%  and 30% decrease in the proliferation-suppressing activity of TGF-β₁ and myostatin, respectively. Thus, increased IGFBP-5 levels appear to play a significant role in both TGF-β₁- and myostatin-induced suppression of IGF-I-stimulated proliferation in PEMC cultures. Antibody neutralization of IGFBP-3 in cultures treated with either TGF- β₁ or myostatin resulted in decreases in proliferation-suppressing activity that were significantly greater than those observed with antibody neutralization of IGFBP-5. Furthermore, antibody neutralization of both IGFBP-3 and IGFBP-5 resulted in return of proliferation rates to control (untreated) levels in TGF- β₁ treated PEMC cultures and to 88% of control levels in myostatin treated cultures.

The final on-going study at the Minnesota Station investigates roles of IGFBP-5. In addition to IGF-I-dependent effects on cells, IGFBP-5 has also been shown to have IGF-I-independent effects in some cell types. Long-R3-IGF-I is an IGF-I analog that has very low affinity for the IGFBPs but retains its ability to bind to the type I IGF receptor and thereby stimulate proliferation. Consequently, suppression of Long-R3-IGF-I -stimulated proliferation by IGFBP-5 is believed to result from IGF-I independent association of IGFBP-5 with cell surface receptors rather than binding and inactivation of IGF-I. Thus, if anti-rpIGFBP-5 reduces the effects of myostatin or TGF-β₁ on Long R3-IGF-I-stimulated proliferation in PEMC cultures, it is reasonable to infer that IGF-I independent actions of IGFBP-5 play a role in myostatin and TGF-β₁–induced suppression of proliferation in these cultures.  Consequently, the effect of neutralizing IGFBP-5 activity on both TGF-β₁ and myostatin-induced suppression of Long-R3-IGF-I -stimulated proliferation was examined. Both TGF β₁ and myostatin suppress Long-R3-stimulated proliferation of in PEMC cultures. Additionally, neutralization of IGFPB-5 activity with anti-rpIGFBP-5 results in inhibition of both TGF-β₁- and myostatin-induced suppression of proliferation in these cultures.  These results strongly infer that IGF-I-independent actions of IGFBP-5 play a role in the ability of both TGF-β₁ and myostatin to suppress proliferation of PEMCs. Antibody neutralization of IGFBP-3 in cultures treated with either TGF- β₁ caused a decrease in proliferation-suppressing activity that was similar to that observed for antibody neutralization of IGFBP-5. In contrast, antibody neutralization of IGFBP-3 in myostatin treated PEMC cultures resulted in decreases in proliferation-suppressing activity that were significantly greater than those observed with antibody neutralization of IGFBP-5. Furthermore, antibody neutralization of both IGFBP-3 and IGFBP-5 resulted in return of proliferation rates to control (untreated) levels in TGF- β₁ treated PEMC cultures and to 93% of control levels in myostatin treated cultures.

At the Nebraska Station, Dr  Jones has been investigating factors influencing protein synthesis in myotube cultures. This study was conducted to determine whether insulin, glucose, and leucine cause an increase in RNA and recruitment of ribosomes for protein synthesis.  It was hypothesized that all three work independently to increase polyribosome formation in porcine satellite cells (PSC) and in porcine satellite cell derived myotubes (PDM), in vitro.  In both PSC and PDM, the addition of insulin or leucine at or above physiological levels or baseline levels, caused RNA and the percentage of polyribosome to increase compared to controls (p<.05).  A diminution of insulin or leucine below physiological or baseline levels, caused a decline (p<.05) in total RNA and polyribosome formation. When glucose was excluded from the medium, RNA and polysomes were reduced (p<.05).  However, there was no difference in either total RNA or percent polysome formation (p>.05) between glucose added media.  These data indicate that 1) insulin and leucine increase total RNA production and percent polysome formation, and 2) the absence of glucose decreases total RNA production and polysome formation.  When insulin and leucine were used in combined treatment levels, RNA concentration increased as leucine increased above baseline levels.  In PSC cells exposed to baseline leucine levels, as increase in RNA was observed when insulin was at least at physiological levels, and RNA levels tended to plateau at higher levels.  Polyribosome formation within PSC cells was not affected by an interaction of the insulin and leucine treatments.  Regardless of leucine levels, polyribosome initiation increased with increasing levels of insulin for PSC and PDM.  The results for all experiments showed insulin increases RNA synthesis as well as the formation of polyribosomes in both PSC and PDM in vitro.  When leucine levels drop below baseline, the production of RNA and polyribosomes significantly decreased.  While insulin increased RNA and polyribosomes, it does not appear to work synergistically with leucine to increase RNA production in either cell type.

Also at the Nebraska Station, Dr Zeece has been investigating the mechanism of calcium regulation in skeletal muscle. His lab is also developing proteomic tools to characterize the constituent calcium regulatory proteins. Calcium is intimately tied to many cellular activities. Calcium level is linked to protein degradation through activation of the calpain system. Calpain activity in turn is involved in a variety of cellular functions including:  myofibrillar protein turnover, pathogenic states of protein degradation e.g., muscular dystrophy and cell division and myofibrillogenesis. Abnormalities in calcium regulation are also of relevance to malignant hyperthermia in humans and its corollary in meat animals that results in poor quality meat. Dr Zeece’s laboratory has developed a protein microarray approach for simultaneous assessment of seven SR-associated proteins. The hypothesis tested was whether significant differences exist in the amounts of key calcium regulatory proteins between normal and PSE prone pigs. 

At the Ohio Station, goals have been characterize the role of the extracellular matrix in avian skeletal muscle development. During skeletal muscle growth and differentiation, proteoglycans are expressed in a dynamic fashion with large chondroitin sulfate proteoglycans being expressed embryonically followed by a late embryonic shift to smaller chondroitin, dermatan, and heparan sulfate proteoglycans. It is currently unknown how this shift in proteoglycan expression is related to skeletal muscle growth properties. To study how proteoglycan expression is affected by growth selection in turkeys, a turkey line selected for increased growth (F-line) and its unselected random bred control line (RBC2) are being used in studies.

Research at the Oregon Station is focusing on the roles that two E3 ubiquitin ligases play in coordination of muscle growth. Specifically, two lines of mice which lack muscle-specific E3 ligases (MuRF-1 and MAFbx) have been obtained from a collaborative arrangement with Regeneron Pharmaceuticals. Because MuRF-1 and MAFbx are up-regulated in all muscle wasting conditions, the hypothesis under investigation is that this up-regulation is a pre-requisite for changes in activities of the proteases which mediate muscle protein wasting. 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. Specifically, effects of fasting on expression of the calpains system and the proteasomal system in control (+/+), MuRF-1 and MAFbx mice is underway.

During the past year, research in collaboration with the South Dakota Station has focused on the heparan sulfate proteoglycans syndecan-1 and glypican. Both syndecan-1 and glypican are cell surface heparan sulfate proteoglycans that function as low affinity receptors for fibroblast growth factor 2 (FGF2). Syndecan-1 is expressed earlier in embryonic development than glypican whereas glypican is expressed later in embryonic development and during posthatch pectoralis major muscle development. Furthermore, syndecan-1 expression is higher in the F line male than the RBC2 line male.  It has been hypothesized that syndecan functions by presenting FGF2 to its receptor stimulating proliferation whereas glypican sequesters FGF2 allowing differentiation to proceed.

This year (Ohio Station) a full length turkey glypcian cDNA of 1653 bp coding for 550 amino acids which has been cloned into the expression vector pCMS-EGFP (Clontech, Palo Alto, CA) in both the sense and antisense orientation was cloned. Studies are in progress on how the overexpression and reduced expression of glypican affects satellite cell proliferation and differentiation, and responsiveness to FGF2.  Using Clonfectin, transfecting satellite cells derived from the F and RBC2 lines have been transfected. The data obtained to date is preliminary and will be reported next year. Glypican has three potential sites for the attachment of glycosaminoglycan residues and three potential N-glycosylation sites. The biological function of these sites is unknown.  Fibroblast growth factor 2 binds to the heparan sulfate glycosaminoglycan chains but it is unknown if it binds to one or all of the sites.  To ascertain the function of these regions of turkey glypican, we are in the process of constructing glypican mutants using site-directed mutagenesis. After the mutants are obtained, the glypican mutants will be verified for type of glycosaminoglycan by heparitinase and chondroitinase ABC digestion. The digested and undigested samples will be run on western blots and the glypcian will be detected using a turkey peptide antibody that is being made by In Vitrogen. The N-glycan mutants will be confirmed by PNGase F digestion and ConA lectin binding. The PNGase F digestion will be confirmed by Western blotting. After the site-directed mutant constructs are obtained, they will be tested by transfecting turkey satellite cells and measuring cell proliferation, differentiation, and FGF2 responsiveness. 

Additionally there are limited data concerning how the expression of heparan sulfate proteoglycans may be associated with the muscle specific transcriptional regulatory factors. In brief, the transfected cells with the various glypican constructs will be measured for MyoD and myogenin expression by real-time PCR at 72 h of proliferation and 0 h, 24 h, 48 h, and 72 h of differentiation.  Preliminary data on MyoD and myogenin expression in F and RBC2 satellite cells and embryonic muscle tissue from 14 through 24 days of development shows differences in the expression of both of these transcriptional regulatory factors. 

A second effort at the Ohio Station is examining the effect of feed deprivation on the expression of heparan sulfate, syndecan-1 and glypican in broiler pectoralis major muscle. In collaboration with the North Carolina State Station research is progress to determine how feed deprivation affects the expression of heparan sulfate proteoglycans, syndecan-1 and glypican in broiler pectoralis major muscle.  In commercial poultry operations, it is common for newly hatched poults to be without feed for an average of 48 h during the transportation period to farms.  During this immediate posthatch period, the bird uses nutrients from the yolk.  Both feed deprived turkey and chick poults have reduced growth and muscle growth is also decreased.  Research from the North Carolina station has shown that feed deprivation reduces satellite cell activity.  How the expression of proteoglycans that regulate growth factor responsiveness are affected is not known.  In the present study, the expression of the heparan sulfate proteoglycans, syndecan-1, and glypican were examined due to their role in the regulation of FGF2.  At hatch chick poults were separated into control and feed deprived groups.  Feed was withheld from the feed deprived birds for 3 d and the poults were returned to feed and sacrificed on day 7. Heparan sulfate proteoglycan expression was reduced at all stages analyzed in the feed deprived birds.  Syndecan-1 expression was unaffected by the feed deprivation. Glypican expression was decreased in the feed deprived birds on day 3 and after returning the birds to feed was higher than the control birds at day 7.

Research at the South Dakota Station continues to examine the properties of satellite cell subpopulations within single muscles that differ in their responses to growth factors. In previous work it was shown that rapidly growing satellite cell clones are more responsive to the mitogenic effects of IGF, FGF and PDGF.  They are also more responsive to the proliferation and differentiation depressing effects of TGF-β.  However, no differences were detected in either receptor numbers or affinities.  Currently the station is focused on determining whether the variation in growth factor responses is due to differences in the activity of the receptors.  The South Dakota Station has been developing a Western Blotting technique to measure the phosphorylation of the receptor docking protein Shc as a measure of receptor tyrosine kinase activity. As there are no commercial antibodies produced against the turkey Shc protein, they have had to screen many antibodies against Shc from other species.  They have recently found one that seems to work very well and are now screening numerous antibodies for their ability to recognize the tyrosine phosphate residues on turkey Shc.    

Scientists at the South Dakota Station have recently begun to examine whether these satellite cell subpopulations differ in their expression of myostatin and their responsiveness to this polypeptide.  In preliminary studies, they demonstrated that myostatin depresses the proliferation of all turkey satellite cell subpopulations examined.  However, there doesn’t appear to be any uniform differences between the Early and Late clones in terms of relative responsiveness.  Myostatin also depresses proliferation of turkey embryonic myoblasts. Chicken pectoralis major muscle-derived satellite cells are more responsive to myostatin than satellite cells from the biceps femoris.

Another project at South Dakota continues to be an active collaboration with Dr. Sandra Velleman at Ohio Station who is examining the role of the extracellular matrix in growth factor activity on myogenic satellite cells. Significant changes have been made in our serum-free medium formulation for avian satellite cells. The basal medium now utilizes Advanced DMEM which provides higher levels of many amino acids and vitamins as well as several other components compared to McCoy’s 5A medium.  In addition to insulin, this medium also includes the following growth factors: PDGF-BB, HGF, IGF-I, and bFGF.  This medium also is more suitable for the chicken satellite cell than the previous formulation.  Using this new medium formulation, chicken biceps femoris derived satellite cells proliferated approximately 6 fold in 3 days, while pectoralis major satellite cells proliferated approximately 10- fold in this same period of time. 

The Utah 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 include 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.  These data will then be analyzed using established methods to determine if expression each gene is linked to muscle hypertrophy.  As part of this effort, the Station will also work to develop and validate new statistical methods for analyzing these types of bioinformatics experiments.  Most specifically, the Station will explore the use of analysis methods that establish normalization procedures based on interrelationships amongst the many genes, thereby precluding the pitfalls associated with the currently employed methods that rely on normalization to one or two housekeeping genes. 

Test gene selection.  Although the molecular basis of muscle growth and development undoubtedly involves the complex interplay of numerous genes, many key genes have been identified. Amongst these, ovine mRNA sequences are known for genes involved with commitment to the myogenic lineage [myogenin, myf5], intracellular signal transduction [calmodulin 2], hypertrophy and protein turnover [calpains I and II, calpastatin, myostatin], muscle function [lactate dehydrogenase  A (LDH-A), myosin heavy chains (MHC) 2x, s, and 2a], autocrine/paracrine hormones [insulin like growth factors (IGF) I and II, basic fibroblast growth factor (bFGF), insulin like growth factor I receptor (IGF I-R)], and a commonly used housekeeping gene [glyceraldehyde 3 phosphate dehydrogenase (G3PDH)].  Additionally, Chris Bidwell at the Indiana Station will provide primers for an imprinted and muscle-expressed gene associated with muscle hypertrophy in callipyge lambs, DLK-1 (delta, drosphilia homolog-like 1).  DLK-1 is a transmembrane protein that belongs to the EGF-like homeotic protein family thought to play an important role in differentiation.

Research at the Washington Station is examining properties of fish satellite cells. Some fish species experience skeletal muscle hyperplasia throughout their lifetimes. In an attempt to define the regulation of fish satellite cells the Dodson laboratory was one of the first to isolate myogenic satellite cells from fish (steelhead trout) nearly 17 years ago. While their preliminary research helped to optimize culture conditions for trout-derived satellite cells, the satellite cell fraction did not proliferate to any appreciable extent in vitro, and our studies were limited to primary culture work. Without cell expansion, long-term culture work was, and has been, nearly impossible with fish cells. Although numerous laboratories have subsequently initiated research in this area, including the Mulvaney laboratory (Auburn) and the Fauconneau laboratory (France), no laboratory that is capable of sufficiently propagating fish-derived satellite cells in long term cell culture to allow for comprehensive regulatory\signaling studies to be performed. This research is indirectly applicable to the aquaculture industry of the Pacific Northwest. The overall scope of this project in 2004 was to screen a wide variety of relevant growth factors and/or hormones in (fast growth--rainbow trout and slow growth--yellow perch) fish-derived satellite cell cultures to assess potency in prompting cell proliferation. As large numbers of satellite cells are required, this is an important first step in acquiring proper cell numbers in order to conduce more mechanistic (signaling\regulatory) studies. A variety of different modifications were applied to the traditional satellite cell isolation procedure that we employed previously. Using collagenase, pronase, only slight centrifugation steps, and two different filtration steps the Washington Station isolated rather large numbers of satellite cells from both fish types. Some of these isolates have been frozen in liquid nitrogen; however, viability of frozen cells has not yet been studied.

In a second project the Washington Station is examining propagation of isolated cells. They previously determined that trout satellite cells could be maintained at 22 °C in non-bicarbonate buffered L-15 medium, supplemented with serum. Fetal calf serum and\or horse serum have widely been used as supplements (in basal media) to effect the proliferation of isolated satellite cells. This same formulation, however, did not support the proliferation of rainbow trout (or yellow perch) satellite cells--nor did any lot of horse serum that was added to the L-15 medium. They are now in the process of adding specific growth regulators [IGF-I, GH, FGF, HGH, IL-15, and others] to determine if these factors can induce fish satellite cells to proliferate in vitro. Future studies will be designed to determine initial mechanism(s) of growth factor induction of proliferation.

In a third project at the Washington Station, scientists are examining the interaction of dietary components and satellite cells. Amino acids, carbohydrates, lipids and other metabolic "building blocks" are required for satellite cell growth and development in vivo and in vitro. Chemically defined media, whereby all of the individual components are known, have been formulated for optimizing satellite cell activity in vitro. However, not just one defined medium exists. The requirement for some metabolic "building blocks" change--depending on the exact satellite cell physiology desired. Considering in vitro observations, scientists are examining whether individual dietary components might interact with satellite cells to alter growth factor-induced satellite cell activity in vivo. Evidence from some of their 2003 studies suggested that five (5) of twenty-eight (28) screened compounds; contained in simple serum-containing medium, possess some ability to alter satellite cell activity in vitro. This research has application to both the animal industry and the nutraceutical industry. As a first step towards identifying specific dietary components that might alter satellite cell physiology (+\- growth regulators), they have developed a simple in vitro assay in which oral dietary agents are exposed to satellite cells. The intent is to determine if a compound has regulatory potential in vitro, when added to IGF-I, then to evaluate application of the dietary agent in vivo.

A fourth project at the Washington Station is studying adipocyte de-differentiation. As early as 1974, reports have periodically appeared in the scientific literature regarding the apparent ability of mature adipocytes to lose lipid, then dedifferentiate to form proliferative-competent preadipocytes. Scientific terms such as asymmetric cell division, ceiling culture and adipofibroblast have been coined because of this research. Recently, reports have surfaced to suggest that mature bone-derived adipocytes possess the capability to dedifferentiate and form preadipocyte-type cells, as well as numerous other cell types in vitro. During attempts to define culture conditions that would allow for the co-culture of muscle and fat cells, Washington scientists were curious as to whether cells of the adipocyte lineage possess the ability to lose lipid and become proliferative preadipocytes, the progeny of which might (subsequently) differentiate and fill with lipid. This research has application to both the animal industry (altering carcass composition), as well as with human health (obesity and diabetes). The overall scope of this research in 2004 was to develop specific culture conditions that would result in pure cultures of mature adipocytes from fish and beef cattle. Additional goals were to develop points in the dedifferentiation of the mature cells, whereby markers of fat cell activity could be assessed. Mature adipocyte cultures were established from both beef animals (subcutaneous, visceral and intermuscular fat depots) and from trout (visceral fat depot only), and parallel cultures of traditional stromal vascular cells were also established from trout. Presently, the group is incapable of prompting mature adipocytes from trout to dedifferentiate, but beef-derived mature adipocytes (obtained from floating "ceiling cultures") spontaneously dedifferentiate and readily form adipofibroblasts. Progeny cultures of beef-derived adipofibroblasts were evaluated for ability to redifferentiate into lipid-filled fat cells. Adipofibroblasts are capable of both proliferating and differentiating to form lipid-filled adipocytes in vitro. The group is presently attempting to decipher the regulation of the dedifferentiation process, as well as the conversion of progeny adipofibroblasts to form adipocytes. Methods used for present studies include cell\gradient centrifugation, cell cloning, immunocytochemistry, using markers developed to detect specific developmental periods of fat-type cells [proliferation and early differentiation (AD-3, Pref-1), and mid differentiation (PPAR-g, LPL, GDPH, lipid) and late differentiation (Glut-4, HSL, perilipin, leptin)].  In addition, the group is using Western blot to validate cellular results, as well as initiating studies to transfect cells with YFP\GFP-Pref-1 or Glut-4 to examine switching events in adipocyte differentiation into adipocytes.

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

Scientists at the Michigan Station are focused on detection of differentially expressed genes in developing pig skeletal muscle using a long oligonucleotide microarray. This experiment examined differential expression of genes in hind limb skeletal muscle tissue of pigs at 60 d of gestation and 7 wk of age.  Oligonucleotide microarrays used for this study consisted of 13,297 70mer oligos (Pig Array-Ready Oligo Set v.1.0, Qiagen, Inc., Valencia, CA) and were printed at the University of Minnesota Advanced Genetic Analysis Center. Total skeletal muscle RNA from three pigs at 60 d of gestation and three pigs at 7 wk of age were reverse transcribed and labeled with both Cy3 and Cy5.  For microarray screening, each 60 d sample was randomly paired with two 7wk samples for a total of six slides. The fluorescence intensity data was LOESS normalized and analyzed using a mixed model. Sixty-two genes were revealed to be significantly differentially expressed (fold change >= 1.5, p-value <= 0.01) with 36 genes found to be more highly expressed at 7 wk of age and 26 more highly expressed at 60 d of gestation. Differential expression of titin-cap was validated by relative real-time RT-PCR analysis confirming higher expression in the 7 wk samples vs. the 60 d gestation samples. Thus, high density oligonucleotide arrays provide a powerful tool for examining gene expression patterns in developing pig skeletal muscle.

Investigators at the Michigan Station are also using halothane gas to identify pigs with novel polymorphisms in the skeletal muscle calcium release channel. The objective of this study was to determine if novel polymorphisms exist in the skeletal muscle calcium release channel gene (RYR1) that affect stress susceptibility, ambulatory status, and meat quality of commercial swine.  Greater than 25% of the pigs (n = 46/184) in two HAL-1843-free-sire lines exhibited a severe abnormal response to halothane (HS-H), whereas less than 2% of the pigs (n = 2/181) from two other lines were characterized as HS-H.  Pigs classified as HS-H were more prone to becoming non-ambulatory during rigorous handling compared with pigs exhibiting no abnormal response (HS-L; P < 0.02).  Following an 8 h rest after handling, pigs were transported to harvesting facilities.  Lower ultimate pH values and higher drip losses were observed in longissimus muscles of HS-Intermediate and HS-H pigs compared to HS-L pigs (P < 0.05).  No differences in ryanodine binding were observed between halothane categories. Twenty-two single nucleotide polymorphisms (SNPs) were identified in the RYR1. Although these SNPs are not predicted to alter the amino acid sequence of the calcium release channel, some SNPs appear to be associated with HS, mobility status, ryanodine binding, and meat quality. Collectively, these data demonstrate that some HAL-1843-normal pigs are sensitive to halothane anesthesia and are more susceptible to becoming NA than HS-L or HS-I pigs. The SNPs in RYR1 may be useful markers for functional differences in other proteins that affect stress susceptibility, ambulatory status, and meat quality.

Scientists at the North Carolina Station report that post-hatch myofiber growth is dependent upon the addition of new nuclei from the mitotically active satellite cell population. The objective of this study was to examine the relationship between different levels of dietary lysine and satellite cell mitotic activity during the early post-hatch period. Broiler chicks were split into five groups of 10 birds each immediately post-hatch. One group was not provided any feed or water for the first three days post-hatch, whereas the other groups were provided a standard starter diet with different levels of lysine (0.82%, 0.99%, 1.16%, 1.33%) for the first three days post-hatch. All birds were injected with 5-Bromo-2’-deoxyuridine (BrdU)  2 hours before they were killed on the third day post-hatch. Mitotically active satellite cells were identified in the Pectoralis thoracicus and quantified using BrdU immunohistochemistry in combination with computer-based image analysis. Satellite cell mitotic activity was significantly (P < 0.05) lower in the starved compared to any of the fed groups. However, satellite cell mitotic activity was highest (P < 0.05) in the birds that were provided a lysine deficient diet (0.82%). The current study suggests that it is possible to nutritionally stimulate the satellite cell population in the early post-hatch chick, and that it is an important endeavour to re-examine the nutritional requirements of the early post-hatch chick to optimize meat yield.

Early post‑hatch satellite cell mitotic activity is an important aspect of muscle development. An understanding about the interplay between nutrition and satellite cell mitotic activity will lead to more efficient meat production. The objective of this second study at the North Carolina Station was to test the influence of the leucine metabolite, ß‑hydroxy ß‑methylbutyrate (HMB), and feed deprivation on muscle development in the early post‑hatch poult. Male Nicholas poults were placed into one of four treatments:  Immediately fed a starter diet with 0.1% HMB (IF‑HMB); Immediately fed a starter diet containing 0.1% Solka Floc for a control (IF‑control); Withheld feed and water for 48 hours immediately post‑hatch and then fed the HMB diet (WF‑HMB); Withheld feed and water for 48 hours immediately post‑hatch and then fed the control starter diet (WF‑control). 5‑bromo‑2'‑deoxyuridine (BrdU) was injected intra‑abdominally into all poults to label mitotically active satellite cells. The pectoralis thoracicus was harvested two hours following the BrdU injection. Immunohistochemistry for BrdU, Pax7, and laminin along with computer‑based image analysis was used to study muscle development.  IF‑HMB poults had higher body weights (P < 0.01) at 48 hours and one week of age, and had higher satellite cell mitotic activity at 48 hours of age (P <  0.01) compared to the IF‑control and WF poults. However, the WF poults had the lowest amount of satellite cell mitotic activity (P < 0.01) at 48 hours post‑hatch than the other two groups. Therefore, dietary supplementation of HMB may have an anabolic effect on early post‑hatch muscle.

Before the sequencing of the chicken genome; the North Carolina Station had interest in calpastatin mRNA expression in chicken skeletal muscle. However, there was limited DNA sequence information available for chicken calpastatin. It was determined that the previously reported sequence for chicken calpastatin in Genbank was actually heterogeneous nuclear ribonucleoprotein, which is a  predominantly nuclear RNA‑binding protein that function in a variety of cellular activities. The objective of these experiments was to clone a cDNA for a chicken protein similar to other previously reported heterogeneous ribonucleoproteins for other species. The 5' and 3' ends of the chicken mRNA were cloned using Rapid Amplification of cDNA Ends (RACE). Subsequently, the expression of the mRNA sequence was confirmed via Northern analysis. 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.

Animal cell culture is a core laboratory technique in many molecular biology, developmental biology, and biotechnology laboratories. Cell culture is a relatively old technique that has been sparingly taught at the undergraduate level. The traditional methodology for acquiring cell culture training has been through trial and error, instruction when undertaking the first graduate student position, or instruction when hired for a specific industrial cell culture position. However, cell culture is an important candidate course for any biotechnology-related training program because it is a technique that must be performed by investigators before they perform many molecular procedures, and vertebrate cell culture is becoming increasingly important for biomanufacturing of therapeutic proteins. Therefore, a cell culture techniques course is an important offering for undergraduate students who aspire to graduate training, and also undergraduate students who will seek employment with biotechnology companies immediately after graduation. Recently, a cell culture techniques course was developed and delivered to students at the North Carolina Station as a component of an undergraduate Biotechnology minor curricula. Currently, the instructors at North Carolina State University are seeking to provide students with the necessary technical and critical reasoning skills to successfully perform animal cell culture.

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

At the Arizona Station the biology of calpastatin has been actively pursued for the past several years. Calpastatin, the only known protein inhibitor specific for the calpains, was first identified in 1976, but it was not until 1982 that an isoform of calpastatin was purified from human erythrocytes. Calpastatin evidently is unique to vertebrates because the genomic sequences of Drosophila and C. elegans do not contain a calpastatin gene. Although there is a single calpastatin gene in the species that have been studied thus far, up to 8-9 different calpastatin polypeptides can be produced from this single gene by use of different promoters and alternative splicing events. Many tissues contain more than one calpastatin polypeptide, and the physiological significance of these different calpastatins remains unclear. The “prototypal” calpastatin had four repetitive inhibitory domains, domains I, II, III, and IV, and a N-terminal domain named domain L.  The different calpastatin isoforms contain different versions of domain L, some formed by alternative splicing and some formed by use of an upstream promoter that transcribes a 68-amino-acid domain XL.  Other isoforms have different numbers of the repetitive domains: the human erythrocyte calpastatin contains only domains II-IV, the human placenta calpastatin contains only domains III-IV, and a human testis calpastatin contains only domain IV.

Because several calpastatin isoforms are often present in the same tissue, it has been impossible to study their inhibitory properties individually. Therefore, the Arizona Station has expressed in E. coli six different calpastatin isoforms and assayed their ability to inhibit the calpains: 1) a “full-length” calpastatin, XL-IV,  containing domain XL, domain L, and all four repetitive inhibitory domains; 2) L-IV, the prototypal calpastatin containing the L domain and all four inhibitory domains ; 3) I-IV, containing the four repetitive inhibitory domains but not domain L; 4) II-IV, containing domains II to IV, the “human” erythrocyte calpastatin”; 5) III-IV, containing domains III and IV; and 6) domain IV. The 6 calpastatin isoforms were assayed for their ability to inhibit the autolyzed and unautolyzed forms of µ- and m-calpain. The IC₅₀’s for the XL-IV, L-IV, I-IV, II-IV, and IV were similar, ranging from 5.3-14 nM, whereas the IC₅₀ for IV was significantly higher at 24.1 nM (all assays used 45.4 nM calpain). Activity of autolyzed m-calpain was inhibited by significantly lower concentrations of calpastatin than activity of the other calpains: the IC₅₀ for autolyzed m-calpain was 6.58 nM, whereas the IC₅₀‘s for the unautolyzed calpains were 13.9 (m-calpain) and 13.5 (µ-calpain) nM. The XL-IV, L-IV, and I-IV all contain four inhibitory domains, and these constructs inhibited 1.90, 3.07, and 3.73 molecules of calpain, respectively (averages for the four calpains). Also averaged for the four calpains, the II-IV, III-IV, and IV constructs inhibited 2.90, 1.36, and 0.96 molecules of calpain, respectively.  The IC₅₀’s indicate that all calpastatin isoforms tested are tight-binding inhibitors of all four calpains, and that the six isoforms assayed in this study do not differ by more than 3-fold in their ability to inhibit the calpains. It seems 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.

Goals of research at the Illinois Station involves further characterization of the calpain proteolytic system of the rainbow trout. Components of the trout system were isolated by chromatographic separation and their in vitro activities examined. Activities of calpains in post-harvest muscle were also determined. Incubation of trout fillets with CaCL2 was shown to reduce shear force suggesting that the calpain system may be important to post-mortem proteolysis and reduction of muscle integrity in post-harvest trout. The Illinois Station also examined catalytic subunits of rainbow trout calpains (Capn1 and Capn2) and expression during muscle wasting induced by starvation. Cloning and sequencing of the two isozymes revealed 65%identity with mouse calpains. Starvation of trout for 35 days upregulated Capn1 and Capn2, calpastatin short form and long form. Calpain enzyme activity was also increased suggesting involvement of the calpain system as an energy source during fasting. Cloning of calpain domain-V revealed divergence from mammalian calpains. This suggests different functional and activation mechanisms for fish calpains.

Scientists at the Illinois Station also examined effects of in ovo rhIGF-1 administration on e-day 3 on expression of GHSR during chicken embryonic development. Expression of GHSR increased slightly through e-day 5 and remained relatively constant throughout embryogenesis in the control chick. Expression patterns of GHSR, after in ovo administration of rhIGF-1 on e-day 3 demonstrated similar profiles to controls with the exception of a significant up-regulation of mRNA on day 8. These data 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 post-natal muscle growth.

The Illinois Station is also investigating roles of zinc in modulating IGF-1 mediated signaling and is currently studying effects of ractopamine on global gene expression profiles in skeletal muscle taken from pigs.

A goal of the Nebraska Station has been to conduct microarray-based ELISA for quantitation of SR-associated proteins. Microarrays were developed to quantitatively assess the level of a selected of set of proteins associated with calcium regulation in muscle. Specifically, monoclonal antibodies to:  RyR1, DHPR, calsequestrin, triadin, junctin, and  SERCA1 and 2 were used to probe the level of these target proteins in SR preparations. Microarrays were printed with 5 ng of SR protein preparations from 23 pigs of known genotype. Each array containing all 23 samples was incubated with antibodies to the above set of proteins. Fluorescently labeled detection antibody was used to identify the respective target protein in the printed complex.  The signal generated by laser-induced fluorescence was quantitated. The result for each target was quantitated and expressed as fluorescence intensity per unit (ng) protein. Porcine results are summarized in the table below.

Table 1. Normalized means for porcine microarray

^{a, b.c} Differing superscripts indicate significant difference within columns (P<0.05). Genotypes were halothane & RN negative (- -), halothane negative & RN carrier (- c), halothane & RN positive (+ +), halothane positive & RN carrier (+ c) and halothane & RN carrier (c c).  

Analyses of these data are not complete, but a number of significant differences have been identified thus far. For example, the levels of DHP, CSQ  and TRI were different between animals genotyped as negative for halothane response and RN and those that were positive for these defects.  More complete analysis will be reported in a manuscript currently in preparation. A similar quantitative microarray analysis was conducted on SR proteins from turkey muscle. These data showed significant differences between improved and unimproved lines (results not shown).

At the Wisconsin Station light microscopic analysis of filament lengths has been completed.

Image analysis procedures for immunofluorescence microscopy were developed to measure muscle thin filament lengths of beef, rabbit and chicken myofibrils. Strips of beef cutaneous trunci, rectus abdominis, psoas and masseter, chicken pectoralis and rabbit psoas muscles were excised 5 to 30 min post mortem. Fluorescein phalloidin and rhodamine myosin subfragment 1 (S1) were used to probe the myofibril structure. Digital images were recorded with a cooled charge-coupled device (CCD) controlled with IPLab Spectrum software on a Macintosh operating system.  The camera was attached to an inverted microscope utilizing both the phase contrast and fluorescence illumination modes.  Unfixed myofibrils incubated with fluorescein phalloidin showed fluorescence primarily at the Z-line and the tips of the thin filaments in the overlap region.  Images were processed utilizing IPLab and NIH Image software. A region of interest (ROI) was selected and scaled by a factor of 18.18 which enlarged the image from 11 pixels per micron to approximately 200 pixels per micron. An XY plot was exported to Spectrum 1.1 where the signal was processed with a second derivative routine so a cursor function could be used to measure length. Fixation prior to phalloidin incubation resulted in greatest intensity at the Z lines but a more uniform staining over the remainder of the thin filament zone. High resolution image capture and processing showed that thin filament lengths were significantly different (P < 0.01) between beef, rabbit, and chicken with lengths of 1.28 to 1.32 mm, 1.16 mm, and 1.05 mm, respectively. Measurements using the S1 signal confirmed the phalloidin results. Fluorescent probes may be useful to study sarcomere structure plus help explain species and muscle differences in meat texture.

Also at the Wisconsin Station, scientists are examining developmental changes in titin isoforms and alternative splicing patterns. The high-resolution agarose electrophoresis system we developed has allowed us to analyze changes in titin isoform expression. Developmental changes in the alternative splicing patterns of titin were observed in rat cardiac muscle. Titin from 16-day fetal hearts consisted of a single 3710 kDa band on SDS agarose gels, and it disappeared by 10 days after birth. The major adult N2B isoform (2990 kDa) first appeared in 18-day fetal hearts and its proportion in the ventricle increased to approximately 85% from 20 days of age and older. Changes in three other intermediate-sized N2BA isoform bands also occurred during this same time period. The cDNA sequences of fetal cardiac, adult ventricle, and adult soleus were different in the PEVK and alternatively spliced middle Ig domain. Extensive heterogeneity in splice patterns was found in the N2BA PEVK region. The extra length of the fetal titin isoforms appeared to be due to both a greater number of middle Ig domains expressed plus the inclusion of more PEVK exons. Passive tension measurements on myocyte-sized fragments indicated a significantly lower tension in neonate versus adult ventricles at sarcomere lengths greater than 2.1 um, consistent with the protein and cDNA sequence results. The time course of the titin isoform switching was similar to that occurring with myosin and troponin I during development.

Impact of Findings

Objectives of the research conducted via the NC-131 members include 1) characterization of the signal transduction pathways that regulate skeletal muscle growth and differentiation, 2) determination of the nuclear mechanisms that control gene expression in skeletal muscle and 3) characterization of muscle proteins and their functional domains involved in myofibrillar assembly and disassembly. In 2004, the NC-131 group made considerable progress in addressing each of these objectives as reflected by high publication rate and the dynamic exchange of information which occurred at this year’s meeting. In 2004, scientists associated with the NC-131 published 64 peer-reviewed articles, 43 abstracts, nine non-technical publications and book chapters and three gene sequences. Four dissertations were completed in 2004. A strength of the NC-131 group is the evidence of collaboration which exists among participating scientists. Several of the collaborative efforts among the group are reported above and in 2004, several additional collaborative opportunities arose. NC-131 committee 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 synthesis and degradation. The committee has also made significant progress in understanding muscle protein characteristics (particularly titin) and the regulation of myofibrillar assembly and disassembly. 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. The group has been active and successful in soliciting grant support from the USDA-NRICGP and from other sources and it is expected that the group will continue to function as the premier group developing and promoting more efficient muscle growth in domestic animal species.

Work Planned for Next Year

A major effort for 2004 and into 2005 will be the submission of a renewal application to support the NC-131. The group has actively developed this throughout the year and is expected to submit the final version in mid-December 2004. Studies will continue as described in the approved proposal. No changes in the approach or objectives are anticipated at this time.

Publications

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 year’s 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: 319‑322

Mozdziak PE and J. N. Petitte, 2004. Status of transgenic chicken models for developmental biology.  Dev Dyn 229: 414‑421.

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: 89‑94.

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:

 SEQ CHAPTER \h \r 1Ding, 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 isofor