Brief Summary of Minutes of Annual Meeting

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