Brief Summary of Minutes of Annual Meeting

Objective 1:  Characterize the signal transduction pathways that regulate skeletal muscle growth and metabolism including the influence of endogenous growth factors and various production practices.

Alabama station:     1.  Impact of in ovo thermal manipulation on broiler chicken muscle development, growth, and satellite cell activity. Completed the live animal growth performance and carcass yield data collection portion of the project. The cryohistology and immunofluorescence analysis portions of the project are ongoing.   2.  Effects of dietary amino acid density on growth performance, satellite cell activity, collagen gene expression, and the incidence of wooden breast. Completed the live animal growth performance, carcass yield data collection, and sample collection portions of the project. The cryohistology and immunofluorescence analysis portions of the project are ongoing.  Arkansas station: 1. Defining the role of leucine in skeletal muscle energy metabolism (J. Baum). This study demonstrated that leucine regulates skeletal muscle bioenergetics via SIRT1 and the mammalian target of rapamycin (mTOR) under conditions of metabolic stress in a murine in vitro muscle cell model (C2C12 cells). 2. Regulation of muscle mitochondrial function, biogenesis, bioenergetics and dynamics by orexin (S. Dridi).We found, using several molecular techniques, that orexin system is expressed and secreted in avian muscle. In vitro administration of recombinant orexin regulates the expression of prepro-orexin and its related receptors ORXR1 and ORXR2 in quail myoblast cells. Recombinant orexin regulates mitochondrial dynamics (fusion and fission), biogenesis, function and bioenergetics. 3. Identification of downstream cascades employed by 25-hydroxycholecalciferol [25(OH)D3] to enhance broiler breast muscle growth (S. Dridi) We found that supplementation of 25(OH)D3 in diet enhanced breast muscle yield in broiler (meat-type) chickens. 25(OH)D3 supplementation increased the fractional rate of protein synthesis. Molecular analyses revealed that breast muscle from chickens fed the 25(OH)D3 expressed higher concentration of VDR, phospho mTOR, and phosphor S6K. Mechanistic and functional in vitro studies showed that 25(OH)D3 induce avian muscle proliferation via mTOR-S6K pathways. 4. Protein degradation and fat metabolism profile in breast muscle with white striping (S. Dridi). We found that fractional breakdown rate was significantly higher in broiler breast muscle with severe white striping compared to normal counterparts. We also found that the expression of lipogenic genes (FAS, ACC) was down regulated in breast with white striping compared to normal ones. 5. Improvement of Broiler Muscle Health through Selection for Hyperplastic Growth (N. Anthony) We have developed research lines for 4-day breast yield in broiler chickens. We also developed research lines through selection for pale, soft, and exudative like (PSE-like) meat in broilers.  Florida station: Determining the role of Bos indicus genetics on muscle growth and metabolism. Longissimus muscle samples were collected from 36 steers representing a continuum of Angus and Brahman genetics (0% Anugs/100% Brahman to 100% Angus/0% Brahman). Fiber cross-sectional area and enzyme analyses revealed that steers with high percentage of Brahman had larger 2x fibers and greater citrate synthase activity (mitochondrial marker).  Hawaii station:  Myostatin (MSTN) inhibitory region of fish (Paralichthys olivaceus) myostatin-1 propeptide. A MBP-fused flatfish MSTN1pro region consisting of residues 45-100 had the same MSTN-inhibitory potency as the full sequence flatfish MSTN1pro (residues 23-265), indicating that the region of flatfish MSTN1pro consisting of residues 45-100 is sufficient to maintain the full MSTN-inhibitory capacity. Results also indicate that residues 45-65 of flatfish MSTN1pro is essential for MSTN inhibition. In conclusion, current study show that like the mammalian MSTNpro, the MSTN-inhibitory region of flatfish MSTN1pro resides near its N-terminus, and imply that smaller sizes of MSTNpro can be effectively used in various applications designed for MSTN inhibition.  Idaho station: 1. Implemented a research trial on 48 beef cattle (REVLOR-XS implants), examining the effects of rumen protected histidine supplementation at 2 doses for final 50d pre-harvest on growth, FCR, carcass yield and quality. Performed T-Bar, color and color stability assessment on longissimus thoracis and gluteus medius Completing taste panel, HPLC metabolite analyses and statistical analyses on trial. This will help discern whether dietary histidine availability is limiting growth of finishing steers on an aggressive implant strategy and standard finishing rations in the NW United States. One MS student currently assigned and trained on project with expected completion date of June 2017.  2.  Completing an additional aquaculture feeding trial in sablefish (Anoplopoma fimbria) examining the influence of rearing temperature and dietary composition on growth traits and temporal expression of myogenic and metabolic genes in both white and red skeletal muscle. This comprises the final research trial for an MS student with projected completion of December 2017.   Illinois station:  Interaction of IGF2 and Myostatin in the Regulation of Muscle Growth and Development. Cloned sows, heterozygous for an engineered mutation in myostatin, were bred to commercial boars to produce an F1 generation with differential IGF2 and myostatin alleles. This F1 generation was interbred to produce pigs with zero, one, or two mutated myostatin alleles and either a paternal A or a paternal G IGF2 allele. However, matings between F1 individuals (F1 x F1) or matings between F0 and F1 individuals (F0 x F1) resulted in approximately 36% and 25% piglet viability, respectively, and 100% of myostatin homozygous mutant (MKO) pigs were non-viable.  Viability of piglets lacking mutant myostatin alleles (wild type) was related to the percentage of the genome derived from clones. We hypothesize that this relationship between F0 genome proportion and viability can be explained by genome-wide epigenetic variation that is stably inherited from the F0 ancestor, a potential example of transgenerational epigenetic inheritance. When analyzed at birth, muscle weight, as a percentage of body weight, was increased in MKO pigs compared with wild type.  Indiana station: 1. Discovered a role of miR-133a in muscle mitochondria biogenesis and exercise capacity (Nie et al, 2016, FASEB J). 2. Reported the role of Pten in muscle hypertrophy and satellite cell homeostasis (Yue et al, 2016, Cell Reports).  Iowa station:  Effect of quercetin treatment on dystrophic skeletal muscle and heart. We completed our biochemical and histological evaluation of dystrophic diaphragms and soleus muscles treated with quercetin. We found that following 12 months of treatment quercetin-driven pathways were insensitive to continued quercetin treatment. In the heart our biochemical and histological data support continued efficacy of quercetin treatment for the duration of the study period. In addition, careful review of functional data support quercetin as a therapeutic intervention.  Future efforts are geared toward identifying differences in heart and skeletal muscle that will allow efficacy in one tissue but not another so that our strategy can be refined to maximize therapeutic effects. We have also begun to explore the role of autophagy in dystrophic muscle. Our preliminary data point toward increased autophagic signaling but suppressed degradation of autophagosomes.  Mississippi station:  The effects of different dietary lysine levels on the plasma concentrations of amino acids, some metabolites and hormones, and on the skeletal muscle gene expression profile were studied with Large White and Landrace cross-bred finishing pigs.  Increasing dietary lysine concentration (from 0.43% to 0.98%) linearly increased the carcass dressing percentage, the ham weights, and the total lean cut weight of the late stage finishing pigs (Wang et al., 2015c). (2) Dietary lysine can affect the plasma concentrations of 13 amino acids in late-stage finishing pigs with threonine, histidine, phenylalanine, isoleucine, valine, arginine and citrulline being decreased with the lysine-adequate diet but not further decreased with the lysine-excess diet when compared to the lysine-deficient diet. Among these amino acids, arginine was decreased in the greatest proportion. It was suggested that the skeletal-muscle growth of late-stage finishing pigs may be further increased with a lysine-excess diet if the plasma concentrations of theses 7 amino acids, primarily arginine, can be increased through dietary supplementation (Regmi et al., 2015, 2016).   New Jersey station: 1. Effects of dietary methionine restriction on muscle protein synthesis. Completed sample collection from study investigating the effects of dietary sulfur amino acid restriction on muscle protein synthesis in mice. Completed measurement of protein synthesis in subcellular (sarcoplasmic, myofibrillar, mitochondrial) fractions of skeletal muscle. Manuscript including these data is currently under preparation. These data were included in the preliminary data set of a recently funded NIH grant proposal (DK109714, listed under Grants).  2. Effects of exercise training on signaling pathways regulating proteostasis and skeletal muscle metabolism in Standardbred horses.  Completed sample collection from study investigating the regulation of the Unfolded Protein Response by exercise in the skeletal muscle of Standardbred horses. Completed sample collection from study investigating the impact of exercise training on postexercise changes in the skeletal muscle metabolome in Standardbred horses. Received metabolite summary from Metabolon. To my knowledge, this is the first global view of skeletal muscle metabolism in the Standardbred horse. Further analyses and additional method/technique development to evaluate gene expression is ongoing.  Washington station: We have been focused on the impacts of maternal nutrition on the early development of brown adipocytes, and on which shares a common pool of progenitor cells with myogenic cells. We also explored the role of AMPK in myogenesis and muscle regeneration. We found that AMPK activity is required for both brown adipogenesis and myogenesis. Drugs such as metformin activate AMPK, which promotes brown adipocyte development during fetal and neonatal development. In addition, AMPK activation facilitates muscle regeneration following injury.

Objective 2: Characterize the cellular and molecular basis of myogenesis

Arkansas station: 1. Muscle mitochondrial biosynthesis in over-nutrition environment for cell culture (Y. Huang) Preliminary studies were conducted using a murine in vitro myoblasts model (C₂C₁₂) to detect the change of mitochondria biosynthesis and function caused by n-3 fatty acid (EPA and DHA) treatment. EPA and DHA were added into culture media to mimic the maternal over-nutrition during gestation.  Data show fatty acids treatment limits the formation of myotubes, as well as the marker genes expression of myogenesis. Genes expression related to adipogenesis are upregulated by fatty acids treatment. Mitochondrial biosynthesis is inhibited by fatty acids, and it is confirmed by analyzing mitochondrial respiration, which shows fatty acids treatment decreases the oxygen consumption rate. Peroxisomes biosynthesis genes were upregulated by fatty acids. 2. Effects of maternal over-nutrition on fetal mitochondrial biosynthesis characteristics in mouse model (Y. Huang) With the preliminary data of C₂C₁₂ cell culture study, we hypothesize that mitochondria play an important role in fetal skeletal growth programmed by maternal nutrition. High energy diet with 140% energy is given to female mice to induce maternal over-nutrition. Fetal and offspring skeletal samples will be harvested in different life stages and target genes and proteins related with muscle growth and development, mitochondria and peroxisomes biosynthesis, and mitochondrial activity will be analyzed. 3. Gene edition technology in transgenic beef production (Y. Huang)  Galactose-α-1,3-galactose (Alpha-Gal) is a mammalian carbohydrate compound that present in vertebrate animals except humans or Old World monkeys. Alpha-Gal is synthesized by a glycosylation enzyme α-1,3-Galactosyltransferase (GGTA1). Genetically knocking out GGTA1 in pig provides protection of xenotransplantation from hyperacute rejection. We have designed target gRNAs for bovine GGTA1 gene. Next step is using CRISPR-Cas9 to edit GGTA1 gene in bovine primary cell culture. Future studies include making GGTA1 KO embryo and embryo transplantation. 4. Nutrient modulation of the mammalian target of rapamycin (mTOR) to improve muscle function and growth (J. Baum). Leucine is a key nutrient for stimulation of translation initiation in muscle that has undergone metabolic stress. Leucine regulates mitochondria biogenesis via the mammalian target of rapamycin (mTOR).   Connecticut station:  Effects of poor maternal nutrition during gestation on fetal muscle development. Completed sample collection on study investigating the effects of poor maternal nutrition during gestation on muscle development in sheep. Pregnant ewes were fed 100% (CON), 60% (RES), or 140% (OVER) of NRC requirements for pregnant ewes from d 30 of gestation until necropsy at d 45, d90, d135 or parturition.  Offspring were necropsied and the semitendinosus, longissimus dorsi, and triceps brachii were collected.  Completed immunohistochemistry of fetal muscle samples at d45, d90, d135, and birth, determined muscle fiber CSA and the number of primary and secondary fibers at d90.  Completed PCR arrays identifying changes in circulating inflammatory factors that are affected by poor maternal nutrition in the ewe and offspring.    Idaho station:  Designed and implemented an in vitro trial relating to the effects of anthocyanidins on myogenic differentiation and antioxidant defense using differentiated and undifferentiated primary myoblasts isolated from Oncorhynchus mykiss (rainbow trout). This was the final component of a PhD student that completed his program in December 2015.  Indiana station: 1. Determined the molecular mechanisms undying the role of hypoxia in myogenesis (Wang et al, 2015 JBC). 2. Identified brain expressed x-linked gene 1 (Bex1) as a new regulator of myoblast fusion (Jiang et al, 2016, Dev Biol).  3. Investigated the role of liver kinase B1 (Lkb1) in regulating Pax7 in myoblasts (Shan et al, 2016, In J Biochem Cell Biol). 4. Elucidated stage-specific functions of Notch signaling in myogenesis (Bi et al, 2016, Elife). Kansas station: 1. Fetal myoblasts and neonatal satellite cells exhibit divergent cellular kinetics when treated with a porcine plasma product in vitro. a. Myoblasts harvested from 60-d of gestation fetuses treated with a plasma product in vitro exhibited: increased proliferation and stem cell commitment, and no effect on myotube enlargement of PI3K signaling. b. Satellite cells from neonatal piglets treated with a plasma product in vitro exhibited: decreased proliferation rate and increased differentiation, larger myotubes, and increased phosphorylation of AKT, 4EBP, and SK6.   Mississippi station: 1. Effects of melatonin supplementation during gestation on fetal and neonatal muscle development in bovine offspring. a. Completed sample collection on study investigating the effects of melatonin supplementation on fetal bovine muscle Development. Pregnant heifers and cows were supplemented with (n = 29) or without (n = 28) melatonin delivered via a melatonin implant at 180, 210, and 240 days of gestation. At 240 days of gestation a subset of heifers were subjected to cesarean section to collect the developing fetus. These offspring were necropsied and the semitendinosus and longissimus dorsi muscles were collected for subsequent analysis. b. Immunohistochemistry of fetal muscle samples to determine muscle fiber CSA and fiber type distribution is underway. c. Quantitative PCR for expression of mRNA and miRNA involved in muscle growth and development are underway.   Ohio station:  1. Effect of Thermal Stress and Growth Selection on Satellite Cell Proliferation and Differentiation in Turkeys. a. Poultry selected for growth have an inefficient thermoregulatory system and are more sensitive to temperature extremes. b. Satellite cells are precursors to skeletal muscle and mediate all posthatch muscle growth. Their physiological functions are affected by temperature.  c. The objective of the current study was to determine how temperature affects satellite cells isolated from the pectoralis major (p. major) muscle (breast muscle) of turkeys selected for increased 16 wk body weight (F line) in comparison to a Randombred Control line (RBC2) from which the F line originated.  d. Pectoralis major muscle satellite cells were thermally challenged by culturing between 33°C and 43°C to analyze the effects of cold and heat on proliferation and differentiation as compared to control temperature of 38 °C.  e. Expression levels of myogenic regulatory factors: myogenic differentiation factor 1 (MYOD1) and myogenin (MYOG) were quantified by quantitative polymerase chain reaction (qPCR). At all sampling times, proliferation increased at a linear rate across temperature in both the RBC2 and F lines. f.  Differentiation also increased at a linear rate across temperature from 33 to 41 °C at all sampling times in both the F and RBC2 lines. g. Satellite cells isolated from F line turkeys were more sensitive to both hot and cold temperatures as proliferation and differentiation increased to a greater extent across temperature (33 to 43 °C) when compared with the RBC2 line.  h. Expression of MYOD1 and MYOG increased as temperatures increased from 33 to 41 °C at all sampling times in both the F and RBC2 lines.  i.  These results demonstrate that satellite cell function is sensitive to both cold and hot temperatures and p. major muscle satellite cells from F line turkeys are more sensitive to temperature extremes than RBC2 satellite cells. Wyoming station:  1. Effects of maternal obesity (MO) during gestation on fetal muscle function and development. a.Completed animal experiments. Ewes were fed 150% National Research Council (NRC) from 60 days before conception and through pregnancy and control ewes fed 100% NRC recommendations. Offspring necropsy occurred at gestation day 135 (d135), heart muscle (left ventricle, right ventricle, left atria, right atria and septum) and skeletal muscle (semitendinosus, longissimus dorsi, soleus, extensor digitorum longus, tibialis anterior and gastrocnemius) were collected. These tissues are liquid nitrogen snap-frozen and stored at -80 degree and paraffin embedded for future uses. b. Completed myofilament protein analysis in heart muscle, Titin isoform ratios of different heart champers have been analyzed between control and obese ewes and between control and MO offspring at gestation day 135. c. Completed contractility analysis with cardiomyocytes at d135, during the necropsy, we took fresh heart to isolate cardiomyocytes to determine whether maternal obesity affects affspring heart contractile function.

Objective 3: Characterize mechanisms of protein assembly and degradation in skeletal muscle. 

Utah station:  1. Determination of mechanism through which decreased plane of nutrition in second trimester alters end-product quality of offspring in beef cattle.  a. Samples were collected from offspring of mother cows that either maintained BCS during the second trimester (MAIN) or from cows that dropped one BCS during the second trimester of pregnancy. Samples were collected from the longissimus dorsi at weaning, prior to beginning the feedlot phase and immediately following harvest. b. Completed miRNA analysis of samples from the beginning of the feedlot phase and immediately following harvest. Ten different miRNA were analyzed using qRT-PCR methods.  2. Gained insight into the molecular mechanism responsible for development of beef tenderness during aging. a. Samples were collected from the longissimus dorsi of steaks that had been aged for 14 days. Samples were then analyzed for tenderness with WBSF methods. b. Protein expression of HSPβ1 and HSP70 were analyzed in the most tender (n=24) samples and compared to the least tender samples (n=24).    Wyoming station:  Role of RBM20 in the regulation of muscle gene splicing in muscle structure, function and metabolism. a. Completed insulin regulation in myofilament protein titin isoform transition. This work has been done in both rat model and in vitro cultured cell model. The manuscript is in preparation. b. Completed next generation sequencing analysis on two skeletal muscle type longissimus dorsi and tibialis anterior between wild type and RBM20 knockout rats. This work is to determine whether RBM20 deficiency causes gene differential expression and splice form variation between these two muscle types and between wild type and Rbm20 knockout.