Multistate Research Project NC1131

Project No. and Title: NC1131: Molecular Mechanisms Regulating Skeletal Muscle Growth and Differentiation Period Covered: 10-2008 to 09-2009 Date of Report: 15 Apr 2010 Annual Meeting Dates: 23-Oct-2009 to 24-Oct-2009 Participants: R. E. Allen – University of Arizona Robert Rhoads – University of Arizona Yong Soo Kim – University of Hawaii John Killefer – University of Illinois William Dayton – University of Minnesota Shihuan Kuang – Purdue University Douglas McFarland – South Dakota State University Keith Underwood – South Dakota State University Penny Riggs – Texas A&M University, Texas AgriLife Research David Gerrard – Virginia Polytechnic Institute and State University Marion Greaser – University of Wisconsin Min Du – University of Wyoming Alan Grant – Administrative Advisor Mark Mirando – NIFA Representative

Brief Summary of Minutes of Annual Meeting: Members not attending: Joy Winzerling – Arizona; Sally Johnson – Florida; Rod Hill – Idaho; Darl Swartz – Indiana; Joshua Selsby – Iowa; J.E. Minton – Kansas; Catherine Ernst – Michigan; Gale Strasburg – Michigan; Marcia Hathaway – Minnesota; Michael White – Minnesota; Steven Jones – Nebraska; Michael Zeece – Nebraska; Paul Mozdziak – North Carolina; Sandra Velleman – Ohio; Michael Dodson-Washington State;

The annual meeting of the NC-1131 technical committee meeting was held at the University of Wyoming, Laramie, WY, on 23-24 October 2009, and was hosted by Dr. Min Du, Department of Animal Science, University of Wyoming. On 23 October, the group was welcomed by Dr. Doug Hixon, Head of the Department of Animal Science. NIFA Representative, Dr. Mark Mirando, made brief remarks to the group regarding changes at NIFA, the USDA funding outlook, and status of funding RFA revisions. The business meeting was chaired by the Administrative Advisor, Dr. Alan Grant. Dr. John Killefer gave an update regarding the NC-1131 project renewal proposal. Dr. Killefer reported that the proposal had been posted and was assigned the temporary number NC-temp-1184. Project members were encouraged to complete Appendix E documents before December 1, 2009. Dr. Keith Underwood was confirmed as a new member of the group. Next year's annual meeting of the NC-1131 committee will be held at Texas A&M University, to be hosted by Dr. Penny Riggs. The group decided that the 2011 meeting would most likely be held in Virginia, followed by San Diego State in 2012. The remainder of the day was filled by oral station reports summarizing each station's contributions to the objectives of the NC-1131 project. The meeting adjourned, and the group toured the University of Wyoming campus on 24 Oct.

Accomplishments

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

The Arizona station’s area of interest within their skeletal muscle growth and development focus involves the identification of additional roles for satellite cells within the muscle regeneration process. For example, previous studies indicate that muscle regeneration involves the coordination of myogenesis and revascularization in order to restore proper muscle function. Myogenesis is driven by resident stem cells termed satellite cells (SC) whereas angiogenesis arises from endothelial cells and perivascular cells of pre-existing vascular segments and the collateral vasculature. Without proper revascularization of damaged muscle, myogenic cells do not survive and myogenesis ceases. Communication between myogenic and angiogenic cells seems plausible, especially given the number of growth factors produced by SC. To characterize these interactions, they developed an in vitro co-culture model composed of SC and microvascular fragments (MVF). In this system, isolated MVF suspended in collagen gel are cultured over a rat SC monolayer culture. In the presence of SC, MVF exhibit greater indices of angiogenesis than MVF cultured alone. A positive dose- dependent effect of SC conditioned medium (CM) on MVF growth was observed suggesting that SC secrete soluble-acting growth factor(s). Next, they specifically blocked VEGF action in SC CM and this was sufficient to abolish satellite cell-induced angiogenesis. Finally, hypoxia inducible factor-1alpha (HIF-1α), a transcriptional regulator of VEGF gene expression, was found to be expressed in cultured SC and in putative SC in sections of stretch-injured muscle. Hypoxic culture conditions increased SC HIF-1α activity which was positively associated with SC VEGF gene expression and protein levels. Another likely candidate protein is hepatocyte growth factor (HGF). To identify a role for HGF in satellite cell mediated angiogenesis, we blocked HGF action using various levels of anti-HGF antibody added to rat satellite cell conditioned medium and fed to MVF cultures. Following 5 days of culture, MVF were analyzed for indices of angiogenesis. Sprout number and length were decreased in a dose- dependent manner by increasing levels of HGF inhibition. Addition of recombinant HGF during HGF inhibition sufficiently rescued angiogenesis in the MVF cultures. This data indicates that satellite cell-derived VEGF and HGF play an important role in satellite cell mediated angiogenic processes.

The Hawaii station reported on their investigation of a recombinant activin receptor. Background: Myostatin (Mstn) binds to activin type IIB (ActRIIB) receptor to initiate its signaling process. Soluble form of extracellular domain of ActRIIB (ActRIIB-ECD) was shown to be an effective vehicle to suppress Mstn activity, as was demonstrated by an increase in muscle mass in mice injected with ActRIIB-ECD (Lee et al., 2005). To examine whether similar response would occur in meat-producing animals, they initiated a project to produce recombinant ActRIIB-ECD of chicken and pig. Methods: Coding sequences of chicken and pig ActRIIB-ECD were inserted into the pPICZA vector included in the EasySelect™ Pichia Expression Kit (Invitrogen), then transformed into the GS115 strain of P. pastoris. Transformed colonies were screened for expression of the recombinant proteins using SDS-PAGE and western blot analysis of the culture media after induction. Proteins were purified using an affinity chromatography with Ni-NTA agarose column. Mstn-suppressing capacity of the recombinant proteins was estimated using a pGL3-(CAGA)12 luciferase gene reporter assay in A204 rhabdomyosarcoma cell culture. Results: Clones expressing pig and chicken ActRIIB- ECD were identified, and recombinant proteins were purified. N-glycosylation of the ActRIIB-ECD was confirmed by the SDS-PAGE analysis of the reaction products of the proteins with peptide-N-glycosidase F (PNGase F). Two stage pattern of Mstn inhibition was observed by the glycosylated pig ActRIIB-ECD: a dose-dependent increase in Mstn inhibition (from 0% to 50%) from 0 to 0.15 ng/mL of the protein, and leveling off the inhibition up to 111 ng/mL of the protein, then another dose-dependent increase in Mstn inhibition (50% to 90%) beyond 111 ng/mL of the protein. The pattern of Mstn inhibition by the deglycosylated pig ActRIIB-ECD in the concentration range from 0 to 111 ng/mL was similar to that of the glycosylated pActRIIB-ECD, but beyond this concentration, a dose-dependent decrease in MSTN inhibition was observed by the deglycosylated pActRIIB-ECD.

The Illinois station investigated obesity resistance of myostatin null mice fed high fat diets. They have demonstrated that myostatin null mice fed diets containing 60% of calories from fat exhibit resistance to fat accumulation. In contrast to previous reports, however, myostatin null mice are not completely resistant to fat accumulation and in their study, the myostatin null mutation did not alter insulin resistance induced by high-fat feeding. Body fat content of wild type mice fed high fat diets increased 210% compared with low-fat fed wild type mice. In myostatin null mice fed high fat diets, body fat content increased 140% compared with low-fat fed myostatin null mice. Food and caloric intake, however, of high-fat fed wild type and myostatin null mice was similar suggesting energy wastage in myostatin null mice. They investigated the expression of several genes involved in adaptive thermogenesis in muscle (gastrocnemius and bicep femoris muscles) and white adipose tissue. Expression of PGC1α and β were reduced in myostatin null muscles compared with wild type and UCP3 expression was unchanged. In white adipose tissue, PGC1β, adiponectin and adipose triglyceride lipase expression was increased in high-fat fed myostatin null mice compared to all other treatments while UCP2 expression was increased by high-fat feeding in both genotypes. These results suggest but do not confirm increased thermogenesis in myostatin null mice fed high-fat diets. It does not appear, however, that under conventional feeding conditions that thermogenesis in white adipose tissue or muscle tissue differs between myostatin null and wild type mice. Similar to the results above, this study confirmed reduced expression of Lpl and slow myosin heavy chain isoform genes in myostatin null compared with wild type mice.

The Indiana station reported on the regulation of satellite cell function by notch signaling in the skeletal muscle. Postnatal muscle growth and hypertrophy requires addition of myonuclei into existing muscle fibers. This process is primarily mediated by muscle specific stem cells called satellite cells. In response to growth signals, quiescent satellite cells are activated, enter the cell cycle and proliferate as myoblasts. Proliferating myoblasts can differentiate into mononuclear muscle cells called myocytes, which then fuse with existing fibers to mediate muscle growth or hypertrophy. Alternatively, proliferating myoblasts can also withdraw from cell cycle, return to quiescence, and self-renew to replenish the satellite cell compartment. They are particularly interested in the signaling pathways that regulate the cell fate choice between self-renewal and differentiation in activated satellite cells. To this end, they have established a mouse model to investigate the regulation of satellite cell fate by an evolutionary conserved signaling transduction pathway – the ‘Notch’ signaling. In this transgenic mouse model, the Notch signaling can be visualized as it exhibits green fluorescence (GFP). The self-renewal and differentiation of satellite cells were dissected using in vitro single myofiber culture and in vivo muscle regeneration models, in which self-renewal and differentiating progenies can be readily identified with specific markers. They discovered an unexpected heterogeneity and dynamics in Notch activation in both quiescent and activated satellite cells. They have also established a strategy to purify satellite cells exhibiting Notch signaling and analyzed their gene expression profiles. Several genes were found to be uniquely expressed in the satellite cells that display Notch signaling. They are currently examining whether Notch signaling is correlated to stem cell fate and self-renewal in both quiescent and regenerating muscles. Furthermore, they have established mouse models that have constitutively activated or de-activated Notch signaling in the satellite cell lineage. The North Carolina station has studied apoptosis and macrophage infiltration that occur simultaneously and present a potential sign of muscle injury in skeletal muscle of nutritionally compromised, early post-hatch turkeys. Physical stress and malnutrition may cause elimination of myonuclei and produce inflammatory response in muscle. The objective of this study was to histochemically determine the association of apoptosis and/or macrophage infiltration with changes in Muscle satellite cell mitotic activity in pectoralis thoracicus muscle of early post-hatch turkey toms, Feed-deprived birds and birds provided with three different levels of crude protein and amino acids (0.88 NRC, 1.00 NRC, and 1.12 NRC) were used in this model. The number of apoptotic nuclei was significantly elevated (P<0.05) and presence of macrophage infiltration was readily detectable in feed-deprived and 0.88 NRC treatment groups 72 h and 96 h post- hatch Suggesting potential muscle injury and/or muscle remodeling. The number of apoptotic nuclei was the same (P>0.05), and there was no detectable macrophage infiltration present in birds placed on 1,00 NRC and 112 NRC diet 72 h, 96 h, and 120 It post-hatch. At 120 h post-hatch, feed-deprived and 0.88 NRC birds were characterized by no detectable levels of macrophage infiltration and a significant drop (P<0.05) in apoptotic nuclei. Understanding mechanisms that correlate early nutrition with skeletal muscle growth and development may present a useful tool in optimizing muscle health and improving meat quality and yield.

The Ohio station investigated the role of the extracellular matrix in avian skeletal muscle development. Their current research efforts are focused on understanding the mechanism of how the heparan sulfate family of proteoglycans may be involved in the regulation muscle growth properties. Fibroblast growth factor 2 (FGF2) is a potent stimulator of muscle cell proliferation and a strong inhibitor of muscle cell differentiation. Heparan sulfate proteoglycans function as a low affinity receptor for FGF2 thus permitting a high affinity interaction of FGF2 with its receptor. Our research is focused on the heparan sulfate proteoglycan syndecan and glypican families. The syndecans consist of syndecan 1-4 which are differentially expressed in skeletal muscle. The precise role each of these syndecans plays during skeletal muscle development is not well understood. The glypicans have six members, glypican-1 through 6, and only glypican-1 has been identified in skeletal muscle.

As previously reported, they cloned a full length turkey glypican cDNA of 1653 bp coding for 550 amino acids. This year they cloned the syndecan-4 cDNA which is 595 bp coding for 197 amino acids. Both of these cDNAs have been cloned into the expression vector pCMS-EGFP. Glypican-1 has three potential sites for the attachment of glycosaminoglycan (GAG) residues and three potential N-glycosylation sites. Syndecan-4 has three GAG attachment sites and two N-glycosylation sites. Last year they reported the development of site directed mutants of syndecan-4 and glypican-1 to determine the function of the GAG attachment sites. The GAG chains are thought to be important in growth factor signal transduction. By using a site directed mutagenesis approach the glycosaminoglycan attachment sites were mutated to create a series of site directed mutants for glypican-1 and syndecan-4. For glypican-1, the GAG attachment sites at Ser483, Ser485, and Ser487 were mutated to threonine to obtain 1-chain and no- chain mutants. A similar approach was done for syndecan-4 mutating serine sites 38, 65, and 67. The various constructs of syndecan-4 and glypican-1 were transfected into turkey satellite cells to assay the effect of the GAG chains on the proliferation, differentiation, and responsiveness to FGF2. The data obtained for glypican-1 suggests that glypican-1 function requires the GAG chain attachment sites for myogenic satellite cell FGF2 responsiveness during proliferation and to affect the process of differentiation. In contrast syndecan-4 may affect satellite cell behavior independent of the attached GAG chains and function in a manner independent of FGF2.

Also attached to the proteoglycan core protein are N-glycosylation chains. The function of the glycosylated chains has not been well studied to date. To begin to address the function of the N-glycosylation chains a site directed mutagenesis approach was used to create all possible N-glycan chain combinations and a no-chain construct. The N- glycans are attached to asparagine in the core protein at the sequence Asn-Xaa-Ser/Thr where Xaa is any amino acid but not proline. Glypican-1 has 3 N-glycan attachment sites at aspartate residues 76, 113, and 383 which been converted to alanine. Syndecan-4 has 2 N-glycan sites at Asn 124 and 136. The site-directed mutants have been confirmed by DNA sequencing. Preliminary data for both glypican-1 and syndecan-4 suggests that the N-glycan chains do not affect satellite cell proliferation, but differentiation is affected by the absence of the N-glycan chains attached to the core protein. The absence of the N-glycan chains increases differentiation for glypican-1 and in contrast differentiation is decreased by the absence of N-glycan chains for syndecan-4. The Ohio Station also investigated the role of transforming growth factor beta 1 signaling. Transforming growth factor-β1 (TGF-β1) induces apoptosis in many cell types. The cell adhesion receptor, β1 integrin subunit, prevents apoptosis and may be involved in TGF-β1-induced muscle cell apoptosis. Chicken primary satellite cells were used to investigate the apoptotic effect of TGF-β1 on muscle cells. The data showed that the addition of exogenous TGF-β1 reduced β1 integrin expression and altered its localization. Treatment of the satellite cells with TGF-β1 increased the number of apoptotic cells and increased the number of caspase positive cells. Expression of the anti-apoptotic gene Bcl-2 was significantly reduced by TGF-β1. These data suggest that the apoptotic effect of TGF-β1 on satellite cells was likely associated with a β1 integrin- mediated signaling pathway. In response to β1 activation, focal adhesion kinase (FAK) phosphorylates at tyrosine residue 397, and activates cell survival signal transduction. The phosphorylation of FAK was significantly reduced from 30 min to 4 h following TGF-β1 during both satellite cell proliferation and differentiation. These data suggest that TGF-β1-induced apoptosis is associated with β1 integrin-mediated FAK survival signaling pathway during satellite cell proliferation and differentiation.

The South Dakota investigated the effect of thyroid hormones on avian satellite cell proliferation, differentiation and mitochondrial metabolism. In vivo studies have demonstrated that thyroid hormones induce skeletal muscle differentiation and maturation and are involved in the shift from slow-tonic to fast twitch myofibers (Moore et al., 1984; Carpenter et al., 1987). Previous studies with bovine satellite cells demonstrated increased proliferation and differentiation with T3 administration (Cassar-Malex et al., 1999). Thyroid hormones also stimulate oxygen consumption and mitochondrial metabolic activity (Florini, 1987). This project was designed to determine what effects thyroid hormones have on pure populations of avian embryonic myoblasts and satellite cells produced by cloning. Using serum-free medium, addition of 0.25 – 32 ng/ml T-3 to turkey embryonic myoblasts derived from the pectoralis muscle bed resulted in decreased proliferation. Proliferation was decreased by 22% at 0.25 ng/ml and remained nearly level with higher concentrations. Similar results were seen with satellite cells from the pectoralis major muscle of a 6-week-old tom turkey. However when T-3 was administered to satellite cells from a 5-week-old broiler chicken pectoralis major muscle, proliferation decreased by 31% at 0.25 ng/ml and decreased by 53% at 32 ng/ml. Administration of increasing levels of T-4 resulted in a gradual decrease in proliferation of all cell types. This resulted in similar levels of inhibition with the turkey embryonic myoblast and turkey satellite cells at the highest hormone level. However, chicken satellite cells were less responsive to the proliferation depressing effect of T-4 compared to T-3 (33% vs. 53%). Administration of T-3 at the same levels (0.25 – 32 ng/ml) resulted in a gradual (17%) increase in the differentiation level of turkey embryonic myoblasts, while there were no changes in the differentiation of turkey or chicken satellite cells at any level of T-3. T-4 administration to turkey embryonic myoblasts or chicken satellite cells had no effect on differentiation at any level. However, turkey satellite cells demonstrated an increase of 21% in differentiation at the highest level of T-4 (32 ng/ml), while there was no effect at the lower levels (0.25 – 16 ng/ml).

The effect of thyroid hormones on mitochondrial activity was assessed by measuring formazan dye generation by dehydrogenase reactions. T-3 and T-4 had no effect on the mitochondrial activity of any of the 3 muscle cell types. However, mitochondrial activity decreased during differentiation on a DNA basis in all cells (range = 21%-43%). The effect of fatty acid composition of growth media on turkey satellite cell proliferation and differentiation was studied. Using serum-free defined medium, they previously demonstrated that turkey satellite cell membrane phospholipid composition could be changed significantly by providing different fatty acid species to the media formulation. To determine if membrane phospholipid composition and fluidity might alter the responsiveness of satellite cells to growth factor stimuli, fibroblast growth factor-2 (FGF-2) was administered at 20 ng/ml in serum-free medium containing one of 8 fatty acids at 5 uM (Linoleic, Stearic, Palmitic, Oleic, Linolenic, DHA, EPA, Arachidonic). With turkey pectoralis major (PM) derived satellite cells, greatest proliferation compared to zero fatty acid controls was seen in oleic acid (5.0 -fold greater) > linoleic acid (3.7-fold greater) > stearic acid (2.1-fold greater) = palmitic acid (1.9-fold greater). Other fatty acids either supported growth at the same rate as control cells or were inhibitory. Cells administered DHA or EPA showed very low proliferation rates and poor cell health (microscopic observation). With turkey biceps femoris (BF) derived satellite cells, greatest proliferation compared to zero fatty acid controls was seen in oleic acid (3.9-fold greater) > linoleic acid (2.9-fold greater) > stearic acid (1.5-fold greater) = palmitic acid (1.6-fold greater). Cells administered the other fatty acids performed similar to the PM cells receiving those treatments. To assess the effect of fatty acid type on satellite cell differentiation, cells were grown in the respective fatty acid containing serum-free media for 3 days and administered low serum containing medium (3% horse serum) daily for 2 days to initiate differentiation. DHA and EPA treatments were not applied to the differentiation treatments because of the poor performance and cell health noted above. Due to scattered myotube lifting and loss, results were somewhat variable between trials. Cells administered linoleic acid, stearic acid, palmitic acid, and oleic acid differentiated to a similar extent as the control zero fatty acid administered cells. Cells administered linolenic acid or arachidonic acid prior to fusion performed poorly compared to controls. BF satellite cells administered fatty acids and induced to differentiate performed similarly to PM cells, except that cells administered linoleic acid consistently differentiated less than controls.

The Virginia station investigated beta-adrenergic agonist control of muscle fiber type. In these studies, myosin heavy chain (MyHC) IIX-and IIB-null mice were used to test our overall hypothesis that a portion of the 'beta-agonist' growth response is due to exaggerated growth of 1m muscle fibers. Four-wk-old mice were administered 20 ppm clenbuterol in the drinking water for 2 wks. Clenbuterol feeding increased the size of all four muscle fiber types (I, IIA, IIX and IIB) in gastrocnemius muscle of wild type mice, but only type I and lIA fibers in null mice. Furthermore, clenbuterol caused fast- contracting muscles to contain even greater amounts type IIX and IIB MyHC than muscle in wild type mice; but the reverse was observed in null mice. Paralleling the changes of MyHC composition, clenbuterol increased LDH activity in fast-contracting muscles (gastrocnemius and TA) in wild type mice, but decreased in fast muscles of null mice. In soleus muscles, which normally contain no IIX or lIB fibers, the hypertrophic effect of clenbuterol was not observed in wild type and null mice. However, clenbuterol feeding resulted in the detection of type IIX and 1m fibers in the soleus. The cross-sectional area of existing fibers (type I and IIA), however, were significantly larger in null mice. In contrast, clenbuterol-induced muscle hypertrophy was blunted in fast muscles (gastrocnemius, tibialis anterior and extensor digitorium longum) of IIX-or IIB -null mice. Myostatin knockout induced muscle hypertrophy was blunted in mice lacking 1m fibers, as indicated by reduced body weight, gasstrocnemius mass, and total fiber number in gastrocnemius. Data presented herein support the hypothesis that the presence of IIX and IIB muscle fibers is essential for clenbuterol- induced and myostatin knockout-induced skeletal muscle hypertrophy.

They also examined calcium modulation of AMPKactivation in muscle. Activity of AMP-activated protein kinase (AMPK) is regulated by phosphorylation of Thr172 in - subunit by AMPK kinases. Ca2+-regulated Ca2+-calmodulin-dependent kinase kinase (CaMKK) has recently been identified as upstream regulator of AMPK. Cytosolic Ca2+ signals are dynamic and transient, and the Ca2+ frequency is decoded and transduced by CaMKK and its primary downstream substrates, CaMKs. To understand further the specific roles of various types of Ca2+ frequency on AMPK activity, they administered 5-amino-4-imidazolecarboxamide ribonucleoside (AICAR, a known AMPK activator), caffeine (Ca2+ releasing agent) and dantrolene (ryanodine receptor stabilizer) or in combination to mice for 10 d. To characterize further the function of Ca2+ oscillation on AMPK activity, they also simulated different Ca2+ frequencies in C2C12 myotubes by switching media between caffeine-containing media (for 20 min) and dantrolene- containing media (for 3 min). To define the function of Ca2+ signaling molecules in this pathway, CaMKK was knocked down with siRNA, and CaMKlI activity was inhibited by KN93. We found that cytosolic Ca2+controls AMPK activity in a biphasic manner. In particular, low Ca2+ frequencies have a positive effect on AICAR-induced AMPK activity, whereas continuous Ca2+ stimulation decreases AICAR-induced AMPK activity. The negative effect of chronic Ca2+ on AMPK activity is partly through the CaMKK-CaMKII signaling cascade. These data show different cytosolic Ca2+ waves elucidate distinctly different responses in muscle cells and suggests that inhibition of AMPK by Ca2+is mediated in part by the CAMK signaling cascade.

The Washington station has previously shown that (even in the presence of numerous regulators) mature adipocytes possess ability to return to proliferative-competent progeny cells and begin to undergo population expansion. The overall mechanism of conversion of a lipid-filled adipocyte into a cell resembling an adipofibroblast seems to be species dependent, and involves different mechanisms to handle the cellular lipid load prior to proliferation (numerous studies in progress). Moreover, while some progeny cells possess ability to re-differentiate to form lipid-filled adipocytes, the intracellular markers commonly associated with stromal vascular cell or 3T3-L1 cell differentiation are not (all) detected in the present cell system. In addition, commonly used "conversion" mixtures (like DMI) are not required for re-differentiation of these cells--reflecting more of an in vivo system. As some of the proliferative-competent cells are reluctant to re-differentiate, we speculate that some progeny cells are capable of forming other cell types (stem cells; studies in progress). Methods to continue assessment of the physical conversion of mature adipocytes to form progeny cells are being developed. Also, they are in the process of assessing the microRNA status of progeny cultures, in a manner similar to our examination of adipose tissue in vivo. Finally, they (along with Canadian researchers) are hoping to develop NAPPA methodology to assess the association between gene and protein expression in both cells and tissues of the bovine. Results: Cell handling, isolation, purification and propagation methods are established. Protein and gene association studies have been conducted or designed. Impact Statement: This cell system was (a few years ago) considered awkward, not interpretable, and passé. However, just recently, this research area has exploded, and many participants are quite active in conducting research designs. They (perhaps) have, however, the only "purified" cell system whereby data are being reported. They believe that use of this system will (in part) provide new insight into adipose depot formation/regulation/metabolism in meat animals (among numerous other areas and impacts).

The Wyoming station continues to focus on fetal skeletal muscle development. Enhancing skeletal muscle growth is crucial for animal agriculture, since skeletal muscle provides meat for consumption. An increasing body of evidence shows that the level of maternal nutrition alters fetal skeletal muscle development with long-term effects on offspring growth and performance. Fetal skeletal muscle development mainly involves myogenesis (muscle cell development), but also involves adipogenesis (adipocyte development) and fibrogenesis (fibroblast development). All these tissues derive from mesenchymal stem cells (MSCs). Shifting the commitment of MSCs from myogenesis to adipogenesis increases intramuscular fat (marbling) improving the quality grade of meats. Results from our laboratory indicate that Wnt/-catenin signaling regulates MSC differentiation. Wnt/-catenin up-regulation promotes myogenesis and down-regulation enhances adipogenesis. AMP-activated protein kinase (AMPK) enhances myogenesis at least partially through intensifying -catenin mediated up-regulation of myogenesis. Inhibition of AMPK promotes adipogenesis in fetal muscle, which should increase marbling. Objective 2. Determine molecular mechanisms that control gene expression in skeletal muscle.

The Arizona station examines the role of environmental influences on skeletal muscle growth, metabolism and physiology. Heat-stressed cattle also exhibit alterations in glucose metabolism by increasing glucose disposal (rate of glucose entry into cells). Evidence indicates that beef and lactating dairy cattle experiencing heat stress undergo dramatic changes in energy and nutrient metabolism that may affect both their heat tolerance and production parameters, such as lean tissue accretion or milk yield. Because a large proportion of an animal’s mass is comprised of skeletal muscle, any alteration in skeletal muscle metabolism and function can have a profound impact on whole-animal energy metabolism and nutrient homeostasis especially during periods of stress. We have initiated a series of studies aimed at understanding how hyperthermia influences the set points of several metabolic pathways within skeletal muscle. One of their first studies examined the effect of heat strain on skeletal muscle during beef cattle adaptation to chronic heat stress conditions using microarray analysis. Interrogation of the microarray data by pathway analysis has revealed dramatic changes in the skeletal muscle transcriptional profile relating to mitochondrial function and insulin action. It appears that during heat stress bovine skeletal muscle experiences mitochondrial dysfunction leading to impaired cellular energy status. This may have broad implications for the reduced growth and heat intolerance seen during heat stress especially if skeletal muscle is not able to make necessary contributions to whole-body energy homeostasis. Recently, they have begun similar studies using rodents to establish experimental models that can be applied to both animal agriculture and the biomedical sector. These ongoing studies clearly demonstrate that exposure to a heat load induces the differential expression of several energy metabolism genes based on muscle type (soleus versus TA). Moreover, changes in gene expression are consistent with impaired cellular energy status, perhaps due to mitochondrial dysfunction, in oxidative compared to glycolytic muscle fibers during hyperthermia. Taken together, their studies in ruminants and rodents show that heat stress alters skeletal muscle gene expression in a manner that affects skeletal muscle metabolism and function, which may be important for whole-body metabolism and overall physiological adaptation to hyperthermia.

The Florida station is pursuing the identification of signaling events that regulate lineage decisions and growth kinetics of bovine muscle stem cells. Early work by this group revealed that bone morphogenetic protein 6 (BMP6) is produced by mature muscle fibers. As such, the effects of BMP6 were examined in primary cultures of bovine satellite cells isolated from young (< d7) Holstein bull calves. Satellite cells (24 hr post-seeding) were cultured for 48 hours in media supplemented with 50 ng/ml of BMP6. Cells were fixed and immunostained for Pax7 and Myf5 expression. Results indicate that BMP6 does not alter expression of the lineage markers or the percentage of muscle stem (Pax7) or progenitor cells (Pax7:Myf5). Importantly, satellite cells do not experience transdifferentiation to the osteogenic lineage. Satellite cells and C2C12 myoblasts were treated with BMP6 (50 ng/ml) for 48 hours prior to fixation and colorimetric detection of alkaline phosphatase (AP), an enzymatic marker of bone cells. Virtually all C2C12 myoblasts express abundant amounts of the bone enzyme. By contrast, no AP expressing satellite cells were observed. Thus, BMP6 does not appear to affect myogenic lineage decisions of satellite cells. Additionally, treatment of satellite cells with BMP6 does not affect proliferation rates. This is in contrast to mouse C2C12 myoblasts which are mitotic in response to BMP6. To examine the effect of BMP6 on differentiation, satellite cells were cultured in growth media for 3 days followed by treatment with BMP6 in 2% horse serum supplemented media for 48 hours. Cells were fixed and immunostained for myosin heavy chain and myofiber formation was evaluated. A fusion index was calculated as the numbers of nuclei in a myofiber divided by the total number of nuclei. A muscle fiber is defined as a myosin expressing cell with 3 or more nuclei. Results demonstrate that BMP6 inhibits fiber formation and muscle gene expression by 40% when compared to controls. By analogy, treatment of C2C12 myoblasts with BMP6 completely eliminates the myogenic differentiation program. The blunted response of primary satellite cells is not due to an inability to respond to the growth factor. Satellite cells transfected with a BMP response element reporter gene elicited a 4-fold increase in luciferase activity following treatment with BMP6. From these results, we conclude that BMP6 is produced by skeletal muscle where is acts as a paracrine inhibitor of satellite cell differentiation.

The Illinois station has studied gene expression profiling of clenbuterol-treated myostatin null mice. They reported previously that myostatin null mice respond to clenbuterol treatment with increases in body and muscle weights and reduced carcass fat content. Therefore, they concluded that the effects of the myostatin null mutation and clenbuterol treatment were additive, and hypothesized the mechanisms of the two effects did not overlap. They were interested in determining specific pathways activated or inhibited by each effect, and to that end, performed a microarray analysis on these animals. After two weeks of clenbuterol treatment, however, the effect of treatment on gene expression was minimal. It is possible that changes in gene expression occur as an early effect of clenbuterol treatment and do not persist throughout treatment. There were numerous differences, however, between myostatin null and wild type mice including increased expression of several developmental genes similar to previous reports. Expression of genes involved in muscle hypertrophy (Igf2, Meg3, Il15) were increased in myostatin null compared to wild type mice. Furthermore, expression of several genes (Fabp3, CD36, Lpl and Vldlr) involved in fatty acid transport in muscle was reduced in myostatin null mice compared with wild type. They also noted increased expression of Gatm and Gamt, two enzymes involved in creatine synthesis, in myostatin null compared to wild type mice. Increased expression of Gamt was confirmed by RT-PCR. Furthermore, muscle creatine content and creatine kinase activity was increased in myostatin null mice compared with wild type. Alterations in fatty acid transport and creatine synthesis, however, may be secondary to changes in fiber type as myostatin null mice exhibited reduced expression of slow myosin heavy chain isoform genes compared with wild type mice.

The Indiana station investigated molecular signatures that define satellite cell heterogeneity in fast and slow muscles. Efficient meat production relies on optimized muscle differentiation and growth, processes mediated by satellite cells. Phenotypic heterogeneities have been documented in satellite cells derived from slow and fast muscles. However, the intrinsic molecular mechanisms are unknown due to technical difficulties in isolating fiber type-specific satellite cells. They have initiated a project to explore the intrinsic molecular mechanisms that program the developmental fate of satellite cells and restrict them to a specific muscle fiber type. They have established unique mouse models that express cyan and red fluorescent proteins in the type I slow and type IIa fast fibers, respectively. These mice provide an unprecedented opportunity to isolate fresh satellite cells from these specific cohorts of fibers and investigate their unique gene expression patterns by high throughput ‘microarray’ or ‘illumina array’ analysis. This project will form a foundation for our long-term goal of understanding the molecular regulation of muscle development and growth. It will also address whether satellite cells are pre-determined to become a specific fiber type and, if so, what are the molecular determinants. Most importantly, this knowledge may lead to novel strategies for regulating muscle fiber type that affects lean growth efficiency and meat quality, hence maintaining the sustainability and competitiveness of US meat industry.

Current efforts at the Texas Station focus on a funded project for functional characterization of genetic mechanisms associated with beef tenderness and electrical stimulation of carcasses. This research continues to utilize a unique population of cattle population known as the McGregor Genomics Herd to investigate gene expression networks that are important in skeletal muscle and result from inheritance of specific Bos taurus or Bos indicus alleles. This project integrates genome-wide SNP data, microarray, quantitative realtime RT-PCR, and protein analyses of skeletal muscle from steers to characterize the Brahman influence on carcass traits and meat characteristics. Expression profiles will be correlated to extensive phenotype, SNP and QTL data from this population of animals for identification of gene networks. The group continues to examine the association of alpha-actinin-3 expression in skeletal with feed efficiency, in collaboration with Min Du of Wyoming. Messenger RNA quantity was measured for several mitochondrial respiratory subunit genes, but no association of gene expression (in liver or muscle) was found to correlate with efficiency status.

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

The Arizona station investigates myofibrillar protein turnover. Myofibrillar proteins must be removed from the myofibril before they can be turned over metabolically in functioning muscle cells. It is uncertain how this removal is done without disrupting the contractile function of the myofibril. It has been proposed that the calpains could remove the outer layer of filaments from myofibrils as a first step in myofibrillar protein turnover. Several studies have found that myofilaments can be removed from myofibrils by trituration in the presence of ATP. These easily releasable myofilaments (ERMs) were proposed to be intermediates in myofibrillar protein turnover. It was unclear, however, whether the ERMs were an identifiable entity in muscle, or whether additional trituration would remove more myofilaments until the myofibril was gone, and whether calpains could release ERMs from intact myofibrils. The present study shows that few ERMs could be obtained from the residue after the first removal of ERMs, and yield of ERMs from well washed myofibrils was reduced, probably because some ERMs had been removed during washing. Mild calpain treatment of myofibrils released filaments that had a polypeptide composition and that were ultrastructurally similar to ERMs. The yield of calpain-released ERMs was 2-3-fold greater than the normal yield. Hence, ERMs are an identifiable entity in myofibrils and calpain releases filaments that are similar to ERMs. The role of ERMs in myofibrillar protein turnover is unclear because only filaments on the surface of the myofibril would turnover, and changes in myofibrillar protein isoforms during development could not occur by the ERM mechanism.

The Indiana station also studies the myofibril, a dynamic structure beyond its ability to contract. Proteins (structural, contractile, and regulatory) associate and disassociate within this supramolecular structure, and this process is involved in formation, turnover and transformation of the myofibril. There are two extremes in terms of modeling protein dynamics within the myofibril. One is complete proteolysis of myofibrillar proteins likely via the calpain/proteosome system and the other is non- protease dependent release of myofibrillar proteins followed by proteolysis by the calpain/proteosome system. The second extreme is dependent upon the binding constant of the specific protein within the myofilament lattice that in turn is dependent on the association and dissociation rate constants. If the dissociation rate of a particular protein is slower than its turnover rate then proteolysis is required to increase its dissociation rate from the myofibril for subsequent protein turnover. Measurement of troponin dissociation kinetics in vitro show that it is not likely rate limiting for turnover (Yang et al., Biophys. J. 97:183). The slowest Tn dissociation rate had a t1/2 of about 1 day, well within the 3 - 5 day t1/2 observed in protein turnover studies. This comparison suggests that, at least for troponin, turnover does not require disruption of myofibril structure. Other proteins such as myosin, which have longer t1/2, may have much slower dissociation kinetics because of their multiple protein interactions within the sarcomere.

One area of interest is in vitro models for studying myosin dissociation kinetics. Measurement of the dissociation kinetics of myosin in vitro is complicated because there are two major protein interactions involved. There are acto-myosin interactions that involve the motor domain (Subfragment 1 or S1) and myosin interactions with itself and other proteins within the thick filament that involve the rod domain. In the presence of ATP or ATP analogues and absence of calcium, the myofibrils are “relaxed” and thus the acto-myosin interaction is minimal. Under these conditions (similar to in vivo relaxed muscle), the primary determinant of myosin dissociation involves its interactions within the thick filament. Preliminary in vitro studies using various ATP analogues showed that, even in the absence of calcium, the sarcomeres shortened and that there was slow hydrolysis of the analogues. Polyphosphates, particularly tripolyphosphate (TPP) and pryophosphate (PPi) have been shown to dissociate acto- myosin but their hydrolysis has not been well characterized. To characterize the ability of myosin to hydrolyze these polyphosphates, studies were done using isolated S1 from bovine fast (cutaneous trunci) and slow (masseter) myosin. S1 hydrolyzed TPP but the rate was 16 – 32% slower than when ATP was the substrate. Fast S1 hydrolyzed both substrates faster compared to slow S1 and the difference between the isoforms was greater with TPP as the substrate. The Vmax was 0.937 and 4.99 nmole Pi/mg S1 protein/min while the Km was 0.380 and 0.898 mM TPP for slow and fast S1, respectively. Pyrophosphate was strong inhibitor of TPPase activity with a Ki of 88.4 and 8.33 µM PPi for fast and slow S1 isoforms, respectively. Pyrophosphate was not hydrolyzed by either isoform. Addition of TPP in the presence of calcium to myofibrils did not result in sarcomere shortening even though TPP was hydrolyzed. These studies suggest that TPP and/or PPi are good analogues to use to give relaxed, unshortened myofibrils to study myosin dissociation kinetics. To further refine conditions that minimize acto-myosin interactions, the effects of NaCl and PPi on acto-myosin interactions was studied using pyrene actin fluorescence. Both NaCl and MgPPi increased myosin S1 dissociation rate. When salt concentration increased from 0.1 to 1.0 M, the dissociation rate of S1 from bovine fast and slow muscle increased 78 and 38 fold, respectively. MgPPi had an even greater effect on S1 dissociation from actin with the dissociation rate being about 100 fold faster. Unlike salt, MgPPi had no apparent difference in its ability to dissociate slow or fast S1 isoforms from actin with the Kd being 6 – 8 µM. In summary, these studies suggest that using PPi and potentially TPP at 2 – 10 mM minimizes acto-myosin interactions. These conditions can be used for future studies on myosin dissociation kinetics that focus primarily in myosin inter- protein interactions within the thick filament.

The North Carolina station has investigated the identification of poultry meat from pork and beef on the baisi of the titin PEVK region by PCR. The possibility of distinguishing three kinds of meat species (chicken, porcine and bovine) alone or in two-component meat mixtures was investigated using the polymerase chain reaction. The contribution of each component in the meat mixtures ranged from 1 to 100%. The identification of meat was performed on the basis of the sequence coding the PEVK region of titin and by employing polymerase chain reaction. DNA was isolated from raw, fresh and chilled meat. The data presented in this study suggest that it is possible to detect chicken meat, pork and beef in meat mixtures on the basis of PEVK region by using PCR.

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, cell growth and 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 is focused on development of proteomic methods for characterization of SR proteins. Two approaches are being investigated. The first involves printed protein microarrays for multiplexed profiling and investigation of protein-protein interactions. The second involves development of 2-dimensional electrophoretic methods for characterization of the constituents of this membrane protein fraction. These methods are applied in concert to investigate the calcium regulatory system in skeletal muscle and related abnormalities in meat animals. Results: Two-dimensional electrophoretic separations of muscle membrane proteins have been utilized. The hydrophobic nature of SR membrane proteins presents strong challenges to characterize and study the physiological function of the proteins. Protein hydrophobicity and size contribute to limited resolution in traditional 2-D electrophoretic methods. We have developed a non-denaturing electrophoresis system based on solublization of membrane proteins with other surfactants. Selective dissection of membrane protein complexes has been achieved using various surfactants. Most recently, the non-denaturing 2-D electrophoresis separation protocol was applied to the investigation of mitochondrial membrane proteins. Eight spots were excised from 2-D gels and identified by LC-MS/MS analysis. This electrophoresis system is unique in the kinds of information it can provide. Specifically, it has been shown to resolve very high molecular weight proteins (approx 1.5 MDa) and define constituent proteins of large (native) complexes.

Microarray screening of phage display antibodies (SR membrane protein targets) has been used in work being conducted with Dr Tommie McCarthy at University College Cork (Ireland) to analyze libraries of SR-specific monoclonal antibodies produced in his lab by the phage display technique. A selected group of recombinant protein fragments and synthetic peptides were spotted on to expoy-derivatized slides and examined for antibody recognition from a large set of clones. The microarray approach facilitates identification of antibody specificity and potential cross-reaction. However, preliminary results using a cassette of peptides and clones provided by Dr McCarthy were unsuccessful in that the response was weak and highly variable.

Finally, Work has also been started to determine the level of carnosine and other important bioactive peptides in bovine skeletal muscle tissue. This HPLC-MS/MS approach has shown that carnosine level varies with muscle fiber type. The goal is to develop a rapid method capable of bioactive peptide level determination from very small tissue samples, perhaps biopsy.

The Wisconsin station contributed to the annotation of the bovine genome by working on the myofibrillar proteins. GLEAN sequences were initially Blasted against known human and mouse genes for identification. Subsequently the mRNA reference sequences of other known human and mouse myofibrillar protein genes were blasted against the bovine genomic sequence. Genes identified included myosin heavy chains ID/X (Myh1), IIA (Myh2), embryonic (Myh3), IIB (Myh4), cardiac alpha (Myh6), I- cardiac beta/slow (Myh7); myosin light chains alkali (Myl1), MLC2s (slow/cardiac) (Myl2), embryonic/atrial (Myl4); skeletal alpha actin (Acta1), troponin T slow (Tnnt1), troponin I slow (Tnni1), fast (Tnni2) cardiac (Tnni3), troponin C fast (Tnnc2), skeletal alpha actinin (Actn2), skeletal fast alpha actinin (Actn3), titin (Ttn) and nebulin (Neb). Surprisingly the sequences for the troponin C slow/cardiac (Tnnc1) and troponin T cardiac (Tnnt2) genes have not yet been found in the Btau_4.0 build. It should be noted that GenBank sequences for isoforms of myosin heavy and light chains include many incorrect annotations because of their sequence similarities and historically varied names. Another project focused on characterization of a mutation that affects alternative splicing of titin. A rat autosomal dominant mutation that dramatically alters the alternative splicing pattern of titin has been described (Greaser et al. J Mol. Cell. Cardiol. 44:983, 2008). The mutation has been mapped to rat chromosome 1, and the 95,000 bp deletion removes exons 2 through 14 plus the 3’ UTR for a previously uncharacterized splicing factor. Exon arrays have identified more than 60 genes with altered splicing patterns in the heart, and these genes have a large variety of functions (nearly 3/4ths are involved in signal transduction, metabolism, and transcription/translation). A point mutation in the same gene in a human family with dilated cardiomyopathy has been identified, and these individuals also show the same sudden death and development of fibrosis as our rat model. The rat mutation also shows changes in skeletal muscle. The mutation model should provide a useful system to test hypotheses regarding titin’s role in stretch signaling.

Previous studies have indicated that the giant protein titin can be phosphorylated, and that phosphorylation alters the resting tension of skinned cells. Protein kinase C (PKC) regulates contractility of muscle cells by phosphorylating several thin- and thick- filament proteins. Sarcomeres also contain titin, and it is unknown whether titin can be phosphorylated by PKC and whether it affects passive tension. The purpose of this study was to examine the effect of PKC on titin phosphorylation and titin-based passive tension. Phosphorylation assays with PKCalpha revealed that titin is phosphorylated in skinned myocardial tissues; this effect is exacerbated by pretreating with protein phosphatase 1. In vitro phosphorylation of recombinant protein representing titin’s spring elements showed that PKC alpha targets the proline – glutamate – valine –lysine (PEVK) spring element. Furthermore, mass spectrometry in combination with site- directed mutagenesis identified 2 highly conserved sites in the PEVK region that are phosphorylated by PKC alpha (S11878 and S12022). When these 2 sites are mutated to alanine, phosphorylation is effectively abolished. Mechanical experiments with skinned left ventricular myocardium revealed that PKC alpha significantly increases titin-based passive tension, an effect that is reversed by protein phosphatase 1. Single molecule force-extension curves show that PKC decreases the PEVK persistence length (from 1.20 nm to 0.55 nm), without altering the contour length, and, using a serially-linked wormlike chain model, it was shown that this increases titin-based passive force with a sarcomere length dependence that is similar to that measured in skinned myocardium after PKC alpha phosphorylation. Thus PKC phosphorylation of titin is a novel and conserved pathway that links myocardial signaling and myocardial stiffness.

Finally, computer program that uses non-linear regression techniques to fit mathematical functions to densitometry profiles of protein gels has been developed. This allows for improved quantification of gels with partially overlapping and potentially asymmetric protein bands. The program can also be used to analyze immunoblots with closely spaced bands. GelBandFitter was developed in Matlab and the source code and/or a Windows executable file can be downloaded at no cost to academic users from http://www.gelbandfitter.org.

IMPACT STATEMENTS

• Results of a study by the Hawaii station show that myostatin activity is suppressed by a recombinant pig ActRIIB-ECD produced in P. pastoris. The recombinant protein would be a useful resource in future studies investigating whether skeletal muscle growth of pigs could be improved by administration of pig ActRIIB- ECD.

• Differential function of the glycosaminoglycan chains in syndecan-4 and glypican-1 in terms of regulating satellite cell proliferation and differentiation. • Demonstration that the N-glycosylation chains attached to the core protein of syndecan-1 and glypican-1 have a biological function during the differentiation of satellite cells. • The Ohio station successfully completed site directed mutagenesis for syndecan- 4 and glypican-1 creating a series of clones lacking both GAG and N-glycosylation attachment sites. The role of proteoglycans in the regulation of muscle growth is not well understood at this time. It is clear that muscle cells especially the satellite cells are responsive to growth factors like FGF2 and TGF-β. The regulation of muscle cell responsiveness to each of these growth factors is critical in the muscle growth and regeneration processes. The regulation of muscle growth especially the breast which is the most economically valuable part of the bird carcass is on importance to the poultry industry. For example for the turkey industry a 1% increase in breast muscle yield from a 40 lb bird per million birds processed results in an increase in revenues estimated to be $704,000 not including improved feed conversation in the calculation. • Mouse models generated by the Indiana station provide invaluable tools to examine how Notch function in the regulation of satellite cell function. These studies are expected to reveal critical information regarding the molecular control of satellite cell function and muscle differentiation that may lead to novel strategies to enhance muscle growth, hypertrophy and meat production in farm animals. These mouse models will provide invaluable tools to examine how Notch function in the regulation of satellite cell function. Together, they expect these studies to reveal critical information regarding the molecular control of satellite cell function and muscle differentiation. Such knowledge may lead to novel strategies to enhance muscle growth, hypertrophy and meat production in farm animals. Publications: The group reported 82 refereed publications, 22 abstracts, 1 book, 6 book chapters, and 9 theses for this period.

Refereed Publications

Arizona Alexander LS, A Mahajan, J Odle, KL Flann, RP Rhoads, and CH Stahl. 2009. Dietary P restriction decreases stem cell proliferation and subsequent growth potential in the neonatal pig. J. Nutr. (submitted).

O’Brien MD, RP Rhoads, SR Sanders, GC Duff, LH Baumgard. 2009. Metabolic adaptations to heat stress in growing cattle. Domest. Anim. Endocrinol. doi:10.1016/j.domaniend.2009.08.005.

Rhoads RP, ME Fernyhough, S Velleman, DC McFarland, X Liu, GJ Hausman and MV Dodson. 2008. Invited Review: Extrinsic regulation of domestic animal-derived myogenic satellite cells II. Domest. Anim. Endocrinol. 36:111-26.

Rhoads RP, RM Johnson, CR Rathbone, X Liu, C Temm-Grove, SM Sheehan, JB Hoying and RE Allen. 2008. Satellite cell mediated angiogenesis coincides with a functional hypoxia-inducible factor (HIF) pathway. Am. J. Physiol. Cell Physiol. 296:C1321-8.

Neti, G., Novak, S.M., Thompson, V.F., and Goll, D.E. (2009) Properties of easily releasable myofilaments: are they the first step in myofibrillar protein turnover? Am. J. Physiol. Cell Physiol. 296, 1383-1390.

Tatsumi R, Sankoda Y, Anderson J, Sato Y, Mizunoya W, Shimizu N, Suzuki T, Yamada M, Rhoads RP, Ikeuchi Y and Allen RE. 2009. Possible implication of satellite cells in regenerative motoneuritogenesis: HGF up-regulates neural chemorepellent Sema3A during myogenic differentiation. Am. J. Physiol. Cell Physiol. 297:C238-52.

Hawaii Lee S.B., Y.S. Kim, M.Y. Oh, I.H. Jeong, K.B. Seong, and H.J. Jin. 2009. Improving fish growth by treatment with fish myostatin prodomain (Paralichthys olivaceus) expressed in soluble forms in E. coli system. Aquaculture (submitted). Li Z., B. Zhao, Y.S. Kim, C.Y. Hu and J. Yang. 2009. Administration of a mutated myostatin propeptide to neonatal mice significantly enhances skeletal muscle growth. Mol. Reprod. Dev. 76:1685-1694.

Illinois Boler, D.D., Gabriel, S.R., Yang, H., Balsbaugh, R., Mahan, D.C., Brewer, M.S., McKeith, F.K. & Killefer, J. (2009) Effect of different dietary levels of natural-source vitamin E in grow-finish pigs on pork quality and shelf life. Meat Sci. 83(4), 723-730.

Boler, D.D., Holmer, S.F., McKeith, F.K., Killefer, J., VanOverbeke, D.L., Hilton, G.G., Delmore, R.J., Beckett, J.L., Brooks, J.C. & Miller, R.K. (2009) Effects of feeding zilpaterol hydrochloride for 20 to 40 days on carcass cutability and subprimal yield of calf-fed Holstein steers. J. Anim. Sci. (in press).

Carr, S.N., Hamilton, D.N., Miller, K.D., Schroeder, A.L., Fernández-Dueñas, D., Killefer, J., Ellis, M. & McKeith, F.K. (2009) The effect of ractopamine hydrochloride (Paylean®) on lean carcass yields and pork quality characteristics of heavy pigs fed normal and amino acid fortified diets. Meat Sci. 81(3), 533-539.

Dilger, A.C., Gabriel, S.R., Kutzler, L.W., McKeith, F.K. & Killefer, J. (2009) The myostatin null mutation and clenbuterol administration elicit additive effects in mice. Animal (in press).

Gooding, J.P., Holmer, S.F., Carr, S.N., Rincker, P.J., Carr, T.R., Brewer, M.S., McKeith, F.K. & Killefer, J. (2009) Characterization of striping in fresh, enhanced pork loins. Meat Sci. 81(2), 364-371.

Holmer, S.F., Fernandez-Duenas, D.M., Scramlin, S.M., Souza, C.M., McKeith, F.K., Killefer, J., Delmore, R.J., Beckett, J.L., Lawrence, T.E. & VanOverbeke, D.L. (2009) The effect of zilpaterol hydrochloride on meat quality of calf-fed Holstein steers. J. Anim. Sci. (in press).

Holmer, S.F., Homm, J.W., Berger, L.L., Brewer, M.S., McKeith, F.K. & Killefer, J. (2009) Realimentation of Cull Beef Cows. I. Live Performance, Carcass Traits, and Muscle Characteristics. J. Muscle Foods 20(3), 293-306.

Holmer, S.F., Homm, J.W., Berger, L.L., Stetzer, A.J., Brewer, M.S., McKeith, F.K. & Killefer, J. (2009) Realimentation of Cull Beef Cows. II. Meat Quality of Muscles from the Chuck, Loin and Round in Response to Diet and Enhancement. J. Muscle Foods 20(3), 307-324. Holmer, S.F., Kutzler, L.W., McKeith, F.K. & Killefer, J. (2009) Sodium citrate as a replacement for sodium chloride in a brine solution when evaluated in cows of different backfat thickness. Meat Sci. 81(2), 349-356.

Holmer, S.F., McKeith, R.O., Boler, D.D., Dilger, A.C., Eggert, J.M., Petry, D.B., McKeith, F.K., Jones, K.L. & Killefer, J. (2009) The effect of pH on shelf-life of pork during aging and simulated retail display. Meat Sci. 82(1), 86-93.

Myers, A.J., Scramlin, S.M., Dilger, A.C., Souza, C.M., McKeith, F.K. & Killefer, J. (2009) Contribution of lean, fat, muscle color and degree of doneness to pork and beef species flavor. Meat Sci. 82(1), 59-63.

Scramlin, S.M., Carr, S.N., Parks, C.W., Fernandez-Dueñas, D.M., Leick, C.M., McKeith, F.K. & Killefer, J. (2008) Effect of ractopamine level, gender, and duration of ractopamine on belly and bacon quality traits. Meat Sci. 80(4), 1218-1221.

Indiana Yang, Z., M. Yamazaki, Q. Shen, and D.R. Swartz. 2009. Differences between cardiac and skeletal troponin interaction with the thin filament probed by Tn exchange in skeletal myofibrils. Biophys. J. 97:183-194.

Shen, Q.W. and D.R. Swartz. 2009. Influence of salt and pyrophosphate on bovine fast and slow myosin S1 dissociation from actin. Meat Sci. (in press).

North Carolina Nierobisz L. S., J. V. Felts, and P. E. Mozdziak. 2009. Apoptosis and macrophage infiltration occur simultaneously and present a potential sign of muscle injury in skeletal muscle of nutritionally compromised, early post-hatch turkeys. Comp. Biochem. Physiol. B. 153:61-65.

Spychaj A., P. E. Mozdziak, E. Pospiech. 2009. Identification of poultry meat from pork and beef on the basis of the titin PEVK region using PCR. J. Muscle Foods 20:341-351.

Ohio Li, X., and Velleman, S.G. 2009. Effect of transforming growth factor-1 on embryonic and posthatch muscle growth and development in normal and low score normal chicken. Poult. Sci. 88:265-275. Li, X., and Velleman, S.G. 2009. Effect of transforming growth factor-1 on decorin expression and muscle morphology during chicken embryonic and posthatch growth and development. Poult. Sci. 88:387-397.

Rhoads, R., Fernyhough, M., Liu, X., McFarland, D.C., Velleman, S.G., Hausman, G.J., and Dodson, M.V. 2009. Invited Review: Extrinsic regulation of domestic animal- derived myogenic satellite cells II. Dom. Anim. Endocrinol. 36:111-126.

Shin, J., Velleman, S.G., Latshaw, J.D., Wick, M.P., Suh, Y., and Lee, K. 2009. The ontogeny of delta-like protein 1 mRNA expression during muscle development and regeneration: Comparison between broiler and leghorn chicken. Poult. Sci. 88:1427-1437.

Li, X., McFarland, D.C., and Velleman, S.G. 2009. Transforming Growth Factor-β1 Induces Satellite Cell Apoptosis by β1 Integrin-Mediated Focal Adhesion Kinase Activation. Poult. Sci. 88:1725-1734.

Song, Y., Nestor, K.E., McFarland, D.C., and Velleman, S.G. 2009. Effect of glypican-1 covalently attached chains on turkey myogenic satellite cell proliferation, differentiation, and fibroblast growth factor 2. Poult. Sci. (In press).

South Dakota McFarland, Douglas C. and J. E. Pesall. 2008. Phospho-MAPK as a marker of myogenic satellite cell responsiveness to growth factors. Comp. Biochem. Physiol. 149B: 463-467.

Li, X., D. C. McFarland, and S. G. Velleman. 2008. Extracellular Matrix Proteoglycan Decorin-Mediated Myogenic Satellite Cell Responsiveness to Transforming Growth Factor-β during Satellite Cell Proliferation and Differentiation. Domest. Anim. Endocrinol. 35:263-273.

Zhang, X., K. E. Nestor, D. C. McFarland, and S. G. Velleman. 2008. The role of syndecan-4 and attached glycosaminoglycan chains on myogenic satellite cell growth. Matrix Biol. 27:619-630.

Velleman, S. G., X. Li, C. S. Coy, and D. C. McFarland. 2008. The Effect of Fibroblast Growth Factor 2 on the in vitro expression of Syndecan-4 and Glypican-1 in Turkey Satellite Cells. Poult. Sci. 87:1834-1840.

Li, X., D. C. McFarland, and S. G. Velleman. 2008. Effect of Smad3-Mediated Transforming Growth Factor-β1 Signaling on Satellite Cell Proliferation and Differentiation in chickens. Poult. Sci. 87:1823-1833. Li, X., D. C. McFarland, and S. G. Velleman. Transforming Growth Factor-β1 Induced Satellite Cell Apoptosis in Chickens is Associated with β1 Integin-Mediated Focal Adhesion Kinase Activation. Poult. Sci. 88:1725-1734.

Rhoads, R. P., M. E. Fernyhough, X. Liu, D. C. McFarland, S. G. Velleman, G. J. Hausman, and M. V. Dodson. 2009. Invited Review: Extrinsic Regulation of Domestic Animal-derived Myogenic Satellite Cells II. Domest. Anim. Endocrinol. 36:111-126.

Velleman, Sandra G., Xiangfeng Zhang, Cynthia S. Coy, Yan Song, and Douglas C. McFarland. Changes in Satellite Cell Proliferation and Differentiation during Turkey Muscle Development. (submitted).

Song, Y., K. E. Nestor, D. C. McFarland, and S. G. Velleman. Effect of Glypican-1 Covalently Attached Chains on Turkey Myogenic Satellite Cell Proliferation, Differentiation, and Fibroblast Growth Factor 2 Responsiveness. Poult. Sci. (In Press).

Dodson, M.V., M. Benson, L. L. Guan, M. E. Fernyhough, P. S Mir¢, L. Bucci, D. C. McFarland, J. Novakofski, J. M. Reecy, K. M. Ajuwon, D. P. Thompson, G. J. Hausman, W. G. Bergen, and Z. Jiang. Formation of an interdisciplinary team: A win—win situation during depressed economic times. (submitted).

Song, Yan, Douglas C. McFarland, Sandra G. Velleman. Effect of syndecan-4 covalently attached chains on turkey satellite cell proliferation, differentiation, and responsiveness to fibroblast growth factor 2. (submitted)

Texas Almeida, L. M., M. E. J. Amaral, I. T. Silva, W. A. Silva, Jr., P. K. Riggs, and C. M. Carareto. 2008. Report of a chimeric origin of transposable elements in a bovine-coding gene. Genet. Mol. Res. 7: 107-116.

Amaral, M. E., J. R. Grant, P. K. Riggs, N. B. Stafuzza, E. A. Filho, T. Goldammer, R. Weikard, R. M. Brunner, K. J. Kochan, A. J. Greco, J. Jeong, Z. Cai, G. Lin, A. Prasad, S. Kumar, G. P. Saradhi, B. Mathew, M. A. Kumar, M. N. Miziara, P. Mariani, A. R. Caetano, S. R. Galvao, M. S. Tantia, R. K. Vijh, B. Mishra, S. T. Kumar, V. A. Pelai, A. M. Santana, L. C. Fornitano, B. C. Jones, H. Tonhati, S. Moore, P. Stothard, and J. E. Womack. 2008. A first generation whole genome RH map of the river buffalo with comparison to domestic cattle. BMC Genomics 9: 631. Kochan, K. J., M. E. Amaral, R. Agarwala, A. A. Schaffer, and P. K. Riggs. 2008. Application of dissociation curve analysis to radiation hybrid panel marker scoring: Generation of a map of river buffalo (B. bubalis) chromosome 20. BMC Genomics 9: 544.

Bovine Genome Consortium. 2009. The genome sequence of Taurine cattle: A window to ruminant biology and evolution. Science 324: 522-528.

Kochan, K. J., R. N. Vaughn, T. S. Amen, C. A. Abbey, J. O. Sanders, D. K. Lunt, A. D. Herring, J. E. Sawyer, C. A. Gill, and P. K. Riggs. 2009. Expression of mitochondrial respiratory complex genes in liver tissue of cattle with different feed efficiency phenotypes. Proc. Assoc. Advmt. Anim. Breed. Genet. 18:175-178.

Satterfield, M. C., G. Song, K. J. Kochan, P. K. Riggs, R. M. Simmons, C. G. Elsik, D. L. Adelson, F. W. Bazer, H. Zhou, and T. E. Spencer. 2009. Discovery of candidate genes and pathways in the endometrium regulating ovine blastocyst growth and conceptus elongation. Physiol. Genomics 39:85-99.

Virginia Park, S., T.L. Scheffler, AM. Gunawan, H. Shi, C. Zeng, K.M. Hannon, A.L. Grant and D.E. Gerrard. 2009. Chronic elevated calcium blocks AMPK-induced GLUT-4 expression in skeletal muscle. Amer. J. Physiol. 296: C106-15.

Iglay, H.B., J.W. Apolzan, D.E. Gerrard, J.K. Eash, J.C. Anderson and W.W. Campbell. 2009. Moderately increased protein intake predominately from egg sources does not influence whole body, regional, or muscle composition responses to resistance training in older people. J. Nutr. Health Aging 13: 108-14.

Weaver, AD., B. Bowker, A.L. Grant and D.E. Gerrard. 2009. Sarcomere length influences mu-calpain-mediated proteolysis of bovine myofibrils. J. Anim. Sci. 87:2096- 103.

Shi, H., J.M. Pleitner, J.M. Scheffler, C. Zeng K.M. Hannon, AL. Grant and D.E. Gerrard. 2009. Mitogen-activated protein kinase signaling is necessary for the maintenance of muscle mass. Am. J. Physiol. 296:C1040-8.

Gunn, J.P., AD. Weaver, R.P. Lemenager, D.E. Gerrard, M.E. Claeys and S. L. Lake. 2009. Effects of dietary fat and crude protein on feedlot performance, carcass characteristics, circulating plasma metabolites, and meat quality in steers fed differing levels of distiller's dried grains with solubles. J. Anim. Sci. 87:2882-90. Park, S.K, A M. Gunawan, T. L. Scheffler, A. L. Grant and D. E. Gerrard. 2009. Myosin heavy chain isoform content and energy metabolism can be uncoupled in pig skeletal muscle J. Anim. Sci. 87:522-31.

Park, S.K., T. L. Sheffler, M. E. Spurlock, A L. Grant and D. E. Gerrard. 2009. Chronic activation of 5'-AMP-activated protein kinase changes myosin heavy chain expression in growing pigs J. Anim. Sci. 87(10):3124-33.

Gunn, P.J., R.P. Lemenager, M.E. Claeys, D.E. Gerrard, D.R. Buckmaster And S.L. Lake. 2009. Glycerin improves feedlot performance of early weaned steer consuming a distiller's grain-based diet. J. Anim. Sci. 87(9):2882-90.

Depreux, F.F.S, AL. Grant, C.A Bidwell and D.E. Gerrard. Molecular cloning and characterization of porcine calcineurin-alpha subunit delta isoform expression in skeletal muscle. J. Anim. Sci. (accepted).

Washington Fernyhough, M.E., L.R. Bucci, J. Feliciano and M.V. Dodson. (being finalized for submittal). The effects of nutritional supplements on the proliferation and differentiation of muscle-derived satellite cells in vitro. J. Nutr.

Dodson, M.V., G.J. Hausman and M.E. Fernyhough. (in preparation). Twenty-five years in the field of skeletal muscle stem cell research and lessons learned leading to the next twenty-five years. J. Anim. Sc.

Dodson, M.V. G.J. Hausman, S.P. Poulos, P. Mir, M.E. Fernyhough and Z. Jiang. (invited; in preparation). Glutony 101: Adipose depots are different in terms of what they eat, make and secrete. Obesity Rev.

Dodson, M.V. (submitted). Where have our animal science graduates gone? J. Anim. Sci.

Dodson, M.V., L.L. Guan, M.E. Fernyhough, P.S. Mir, L. Bucci, D.C. McFarland, J. Novakofsky, J.M. Reecy, K.M Ajuwon, D.P. Thompson, G.J. Hausman, W.G. Bergen, M. Benson and Z. Jiang. (submitted). Formation of an interdisciplinary research team: A win-win situation during depressed economic times. J. Anim. Sci.

Zhao, Y.M., U. Basu, M. Zhou, M.V. Dodson, J.A. Basarb and L.L. Guan. (submitted). Proteome analysis of subcutaneous fat tissues from beef cattle with different backfat thickness and breeds. BMC Proteome Science Jin, W., M.V. Dodson, S.S. Moore and L.L. Guan. (submitted). Characterization of microRNA expression in bovine subcutaneous fat tissues: A potential regulatory mechanism of subcutaneous adipose tissue development. BMC Mol. Biol.

Chen, J., M.V. Dodson and Z. Jiang. (accepted with minor revision; being revised). Cellular and molecular comparison of redifferentiation of intramuscular- and subcutaneous- adipocyte derived progeny cells. Domest. Anim. Endocrinol.

He, M.L., R. Sharma, P. Mir, E. Okine and M.V. Dodson. 2009. Feed withdrawl regimens to study retroperitoneal and inguinal adipocyte number and diameter and their association with glucose tolerance in rats. Nutr. Res. (in press).

Dodson, M.V., Z. Jiang, J. Chen, G.J. Hausman, L.L. Guan, J. Novakofski, D. Thompson, C. Lorenzen, M.E. Fernyhough, P. Mir and J. Reecy. (in press). Allied industry approaches to alter intramuscular fat content and composition in beef animals. J. Food Sci.

Su, S.Y., M.V. Dodson, X.-B. Li, Q.F. Li, H.W. Wang and Z. Xie. 2009. The effects of dietary betaine supplementation on fatty liver performance, serum parameters, histological changes, methylation status and the mRNA expression level of spot14α in Landes goose fatty liver. Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 154:308- 31424.

Dodson, M.V. 2009. Citation analysis: Maintenance of h-index and use of e-index. Biochem. Biophys.l Res. Comm. 387:625-626.

Rhoads, R., M.E. Fernyhough, S. Velleman, D.C. McFarland, X. Liu, G.J. Hausman and M.V. Dodson. 2009. Invited Review: Extrinsic regulation of domestic animal-derived myogenic satellite cells II. Domest. Anim. Endocrinol. 36:111-126.

Hausman, G.J., M.V. Dodson, K. Ajuwon, M. Azain, K. Barnes, L.L. Guan, Z. Jiang, S. Poulos, R.D. Sainz, S. Smith, M. Spurlock, J. Novakofski, M.E. Fernyhough and W.G. Bergen. 2009. 8th Board Invited Review: The biology and regulation of preadipocytes and adipocytes in meat animals. J. Anim. Sci. 87:1218-1246.

Chen, J., M. Guridi, M.E. Fernyhough, Z. Jiang, L.L. Guan, G.J. Hausman and M.V. Dodson. 2009. Initial differences in lipid processing leading to pig- and beef-derived mature adipocyte dedifferentiation. Basic and Applied Myology 19(5):243-246. Chen, J., M. Guridi, M.E. Fernyhough, Z. Jiang, L.L. Guan, G,J, Hausman and M.V. Dodson. 2009. Clonal mature adipocyte production of proliferative-competent daughter cells requires lipid export prior to cell division. International Journal of Stem Cells 2:76- 79.

Wang, X., C. Xue, X. Wang, H. Liu, Y. Xu, R. Zhao, Z. Jiang, M.V. Dodson and J. Chen. 2009. Differential display of expressed genes reveals a novel function of SFRS18 in regulation of intramuscular fat deposition. International Journal of Biological Sciences 5(1):28-33.

Wisconsin Bovine Genome Sequencing and Analysis Consortium. (240 authors) 2009. The genome sequence of taurine cattle: a window to ruminant biology and evolution. Science 324(5926):522-8.

Mitov,M.I., M.L Greaser, and K.S. Campbell. 2009. GelBandFitter - A computer program for analysis of closely spaced electrophoretic and immunoblotted bands. Electrophoresis 30:848-851.

Hidalgo C, Hudson B, Bogomolovas J, Zhu Y, Anderson B, Greaser M, Labeit S, Granzier H. 2009. PKC phosphorylation of titin's PEVK element. A novel and conserved pathway for modulating myocardial stiffness. Circ. Res. 105:631-638.

Wyoming Yan, X., W. Xu, M. J. Zhu, J. F. Tong, S. P. Ford, P. W. Nathanielsz, and M. Du. (2009). Up-regulation of TLR4/NF-κB signaling is associated with enhanced adipogenesis and insulin resistance in fetal skeletal muscle of obese sheep at late gestation. Endocrinology, In press.

Du, M., J. F. Tong, J. X. Zhao, K. R. Underwood, M. J. Zhu, S. P. Ford, and P. W. Nathanielsz. (2009). Fetal programming of skeletal muscle development in ruminant animals. J. Anim. Sci., In press.

Du, M., X. Yan, J. F. Tong, J. X. Zhao, and M. J. Zhu. (2009). Maternal obesity, inflammation and fetal skeletal muscle development. Biol. Reprod., In press.

Tong, J. F., K.R. Underwood, X. Yan, M. J. Zhu, and M. Du. (2009). AMP-activated protein kinase enhances the expression of muscle-specific ubiquitin ligases despite its activation of IGF-1/Akt signaling in C2C12 myotubes. J. Cell. Biochem., 108: 458-468. Tong, J. F., X. Yan, M. J. Zhu, S. P. Ford, P. W. Nathanielsz, and M. Du. (2009). Maternal obesity down-regulates myogenesis and β-catenin signaling in fetal skeletal muscle. Am. J. Physiol.-Endocrinol. .Metabol., 296: E917-924.

Abstracts Arizona Alexander LS, A Mahajan, KL Flann, RP Rhoads, J Odle, and CH Stahl. 2009. Effects of P deficiency on growth and tissue specific stem cell proliferation in neonatal pigs. FASEB J. 23:33.4.

Bernabucci U, N Lacetera, LH Baumgard, RP Rhoads, B Ronchi and A Nardone. 2009. Metabolic and hormonal adaptations to heat stress in ruminants. XIth International Symposium on Ruminant Physiology.

Flann KL, CR Rathbone, RP Rhoads and RE Allen. 2009. The Role of Hepatocyte Growth Factor in Satellite Cell Mediated Angiogenesis. FASEB J. 23:467.3.

Sanders SR, LC Cole, KL Flann, LH Baumgard and RP Rhoads. 2009. Effects of acute heat stress on skeletal muscle gene expression associated with energy metabolism in rats. FASEB J. FASEB J. 23:598.7.

Hawaii Kim K.H., Y.S. Kim. 2009. Skeletal muscle hypertrophic effect of clenbuterol is additive to the hypertrophic effect of myostatin suppression. FASEB J. 23:600.26 (Abstract).

Texas Abel E. L., Angel J. M., Meyer S., Coyen T., Riggs P. K., Elizondo L., DiGiovanni J. 2008. Gsta4 is a mouse skin tumor promotion susceptibility gene that maps to promotion susceptibility locus 1.2 (Psl1.2) on chromosome 9. 3rd Frontiers in Cancer Research and 8th International Skin Carcinogenesis Conferences October 4-7, 2008 Austin, MN.

Gill C. A., T. S. Amen, R. R. Funkhouser, K. L. Nicholson, C. R. Boldt, L. L. Hulsman, S. M. Vitanza, M. A. Wegenhoft , K.R. Wunderlich, C.A. Abbey, J. Choi, C. M. Dickens, A. D. Herring, P. K. Riggs, J. E. Sawyer, J. W. Savell, R. K.Miller, D. K. Lunt, J. O. Sanders. 2009. Identification Of QTL For Behavior, Feed Efficiency And Carcass Traits In Bos indicus Influenced Beef. Plant & Animal Genomes XVII Conference, Jan. 10-14, San Diego, CA, Abstract W089. Amaral, M. E. J., J. R. Grant, P. Stothard, P. K. Riggs, G. Lin, T. Goldammer, S. Kumar, P. Mariani, A. R. Caetano, H. Tonhati, M. S. Tantia, S. Moore, and J. E. Womack. 2009. The River Buffalo Genome Mapping Initiative: An International Effort To Construct A First Generation Whole Genome Radiation Hybrid Map For The River Buffalo. Plant & Animal Genomes XVII Conference, Jan. 10-14, San Diego, CA, Abstract W098.

Vaughn R. N., K. J. Kochan, T. S.Amen, C. A.Abbey, C. A. Gill, J. O. Sanders A. D.Herring, D. K. Lunt, J. E. Sawyer, and P. K Riggs. 2009. Association Of α-Actinin 3 Gene Expression With Bovine Feed Efficiency. Plant & Animal Genomes XVII Conference, Jan. 10-14, San Diego, CA, Abstract P471.

Shields J. E., K. J. Kochan, J. Jeong, C. A. Abbey, T. Raudsepp, and P. K. Riggs. 2009. Preliminary Characterization Of A Novel Equine Gene Potentially Associated With Spermatogenesis. Plant & Animal Genomes XVII Conference, Jan. 10-14, San Diego, CA, Abstract P545.

Amaral M. E. J., J. R.Grant, P. K. Riggs, N. B. Stafuzza, E. A. Rodrigues Filho, T. Goldammer R. Weikard, R.M. Brunner, K.J. Kochan, A.J. Greco, J. Jeong, Z. Cai, G. Lin, A. Prasad. S. Kumar, G.P. Saradhi, B. Mathew, M.A. Kumar, M. N. Miziara, P. Mariani, A. R. Caetano, S. R. Galvao, M. S. Tantia, V. A. Pelai, A. M. Santana, L. C. Fornitano, B. C. Jones, H. Tonhati, S. Moore, P. Stothard, and J. E. Womack. 2009. A First Generation Whole Genome RH Map Of The River Buffalo With Comparison To Domestic Cattle. Plant & Animal Genomes XVII Conference, Jan. 10-14, San Diego, CA, Abstract P589.

Shields, J. E., K. J. Kochan, J. Jeong, C. A. Abbey, T. Raudsepp, and P. K. Riggs. 2009. Initial characterization of a gene abundantly expressed in stallion testis. J Equine Vet Sci 29: 324-325

Loux S. C., P. K. Riggs, D. D. Varner, T. H. Welsh, Jr., and N. H. Ing. Identification of Novel Factors Implicated in Glucocorticoid- and Stress-Related Cases of Subfertility in Stallions. 2009. AgriLife Conference, College Station, TX, Jan. 12-15.

Vitanza S. M., C. A. Abbey, K. J. Kochan, B. R. Lindsey, P. K. Riggs, and C. A. Gill. 2009. Expression of candidate genes for horn growth in early bovine development. Texas A&M University, Student Research Week, College Station, TX. Mar. 27.

Butler J. L., P.K. Riggs, and P.J. Holman. 2009. STAC3 found upregulated at tick attachment site in calves resistant to Amblyomma americanum infestation. Texas Genetics Society Annual Meeting. April 2 - 4, Austin, TX. Wyoming Yan, X., W. Xu, J. F. Tong, M. J. Zhu, S. P. Ford, and M. Du. (2009). Maternal obesity affects adipogenesis and myogenesis in fetal sheep muscle at late gestation, Western Region COBRE-INBRE Scientific Conference, Big Sky, Montana, Sept. 17-18, 2009.

K. R. Underwood, J. F. Tong, X. Yan, L. Nicodemus, P. L. Price, W. J. Means, B. W. Hess, and M. Du. (2009). Maternal protein supplementation at a crucial stage for muscle and fat development diverts adipogenesis to myogenesis in beef steers while not affecting tenderness of longissimus steaks. American Meat Science Association 2009 Reciprocal Meat Conference, June 21-24, Rogers, Arkansas.

Long, N. M., M. J. Zhu, M. Du, P. W. Nathanielsz, and S. P. Ford. (2009). Effects of maternal obesity and high nutrient intake on fetal adiposity and associated fatty acid and glucose transporter expression. Society for the Study of Reproduction 42nd Annual Meeting, Pittsburgh, PA. July 18-22, 2009.

Yan, X. Jun F. Tong, Mei J. Zhu, Stephen P. Ford, Peter W. Nathanielsz, and Min Du. (2009). Maternal obesity induces inflammation and adipogenesis in late gestation fetal sheep muscle. American Diabetes Association 69th Annual Meeting, June 5-9, New Orleans, Lousiana.

Tong, J. F., X. Yan, J. X. Zhao, and M. Du. (2009). Metformin inhibits adipogenesis in fetal muscle of dam receiving high energy diet. American Society of Animal Scientists Annual Meeting, Montreal, Canada. July 12 to 16, 2009.

Yan, X., W. Xu, J. F. Tong, M. J. Zhu, S. P. Ford, and M. Du. (2009). Maternal obesity affects adipogenesis and myogenesis in fetal sheep muscle at late gestation. American Society of Animal Scientists Annual Meeting, Montreal, Canada. July 12 to 16, 2009.

Zhao, J., X. Yan, J. F. Tong, M. J. Zhu, and M. Du. (2009). AMP-activated protein kinase γ3 subunit mutant in RN- pigs inhibits adipogenesis. American Society of Animal Scientists Annual Meeting, Montreal, Canada. July 12 to 16, 2009.

Book Wyoming Du, M., and R. J. McCormick. (2008). Applied Muscle Biology and Meat Science. CRC press, Boca Raton, FL. Book chapter

Indiana Swartz, D.R.*, M.L. Greaser and M.A. Cantino. 2009. Muscle Structure and Function. In “Applied Muscle Biology and Meat Science”, Ed. by M. Du and R. McCormick. CRC Press, Boca Raton, FL. pp. 1 – 45.

Texas Riggs P. K., and Gill C. A. 2009. Molecular mapping and marker assisted breeding for meat quality. In Applied Muscle Biology and Meat Science, Du M, ed. CRC Press, Boca Raton, pp288-310.

Washington Basu, U., L.L. Guan, M. Taniguchi, Y. Zhao and M.V. Dodson. 2010. Application of 'omics' technologies on improvement of meat quality. In Nutritional Biochemistry: Genomics, Metabolomics and Food Supply. Nova Science Publishers, Inc. Hauppauge, NY (finished; to appear the first quarter of 2010) [Book Chapter]

Wisconsin Swartz, D.R., M.L. Greaser, and M.A. Cantino. 2009. Muscle structure and function. In: Applied Muscle Biology and Meat Science, CRC Press, Boca Raton, Florida, pp. 1-30.

Greaser, M.L. and C. M. Warren. 2009. Efficient electroblotting of very large proteins using a vertical agarose electrophoresis system. Methods Mol Biol. 536:221-227.

Greaser, M.L. 2009. Carcass Composition and Quality: Postmortem. In: Encyclopedia of Animal Science, W.G. Pond & A.W. Bell, Eds., Marcel Dekker, New York, 2nd Ed.

Greaser, M.L. 2009. Carcass Composition and Quality: Genetic Influence. In: Encyclopedia of Animal Science, W.G. Pond & A.W. Bell, Eds., Marcel Dekker, New York, 2nd Ed.

Theses

Illinois Dilger, A.C (2009) Altering Muscle Hypertrophy and Fat Accumulation in Myostatin Null Mice. Ph.D. Dissertation, University of Illinois. Fernández-Dueñas, D.M. (2009) Impact of Oxidized Corn Oil and Synthetic Antioxidant on Swine Performance, Antioxidant Status of Tissues, Pork Quality and Shelf Life Evaluation. Ph.D. Dissertation, University of Illinois.

Gabriel, S.R. (2009) Gene Expression Profiling in Myostatin Null Mice. M.S. Thesis, University of Illinois.

Holmer, S.F. (2009) Factors Affecting the Textural Properties of Pork. Ph.D. Dissertation, University of Illinois.

Scramlin, S.M. (2009) Bacon Production: Evaluating Potential Processing and Management Practices to Improve Product Quality of Industrial Sliced Bacon Ph.D. Dissertation, University of Illinois.

Souza, C.M. (2009) The effect of high pressure processing on pork quality, shelf life, palatability, and further processed products. M.S. Thesis, University of Illinois.

Varnold, K.A. (2009) The Effects of Feeding Pigs Diets Containing Distillers Dried Grains with Solubles and Conjugated Linoleic Acid on Pork Fat Quality M.S. Thesis, University of Illinois.

Indiana Yamazaki, Marie. Tripolyphosphatase activity of bovine fast and slow myosin. M.S.

Texas Caldwell L. C. “The influence of breed and temperament on circulating concentrations of insulin-like growth factor-I (IGF-I) and its relationship to feed efficiency in beef cattle.” Master of Science Thesis, Texas A&M University, May 2009.