New Insight into the Effects of Small Heat Shock Proteins on Callipyge Lamb Meat Tenderness

Callipyge lambs are a type of sheep that are genetically known to produce tough meat. High expression of calpastatin, which inhibits proteolytic activity of µ-calpain, has been identified as the main factor behind the toughness of callipyge lamb meat. Another group of proteins called small heat shock proteins (sHSP) has recently been suggested for its possible involvement in tenderness development of meat, where up-regulation of sHSP may be associated with toughness. However, the role of sHSP in meat tenderization of callipyge lambs has never been investigated; therefore, the objective of this study is to determine possible involvement of sHSP in myofibrillar protein degradation of callipyge lambs during post-mortem aging. A total of 17 lambs from four different genotypes were slaughtered. Muscle samples from M. longissmus dorsi were collected at 3, 6, and 9 days post-mortem for protein extraction. Western blots were performed to determine the extent of degradation of myofibrillar proteins, such as desmin and troponin-T, µ-calpain autolysis, and sHSP 20, 27, and 70. The qualitative results showed that meat samples from callipyge lambs had less myofibrillar protein degradation and µ-calpain autolysis than samples from normal lambs. Furthermore, more intact sHSP were found in the samples from callipyge lambs throughout whole-aging periods than the samples in normal lambs. These observations suggest that up-regulation of sHSP could be related to less extent of myofibrillar protein degradation of muscles from callipyge lambs. Future studies to determine the exact mechanisms by which sHSP protect muscle structure from proteolysis should be warranted

Identification of Genes Directly Responding to DLK1 Signaling in Callipyge Sheep

Background In food animal agriculture, there is a need to identify the mechanisms that can improve the efficiency of muscle growth and protein accretion. Callipyge sheep provide excellent machinery since the up-regulation of DLK1 and RTL1 results in extreme postnatal muscle hypertrophy in distinct muscles. The aim of this study is to distinguish the genes that directly respond to DLK1 and RTL1 signaling from the genes that change as the result of muscle specific effects. Results The quantitative PCR results indicated that DLK1 expression was significantly increased in hypertrophied muscles but not in non-hypertrophied muscles. However, RTL1 was up-regulated in both hypertrophied and non-hypertrophied muscles. Five genes, including PARK7, DNTTIP1, SLC22A3, METTL21E and PDE4D, were consistently co-expressed with DLK1, and therefore were possible transcriptional target genes responding to DLK1 signaling. Treatment of myoblast and myotubes with DLK1 protein induced an average of 1.6-fold and 1.4-fold increase in Dnttip1 and Pde4d expression respectively. Myh4 expression was significantly elevated in DLK1-treated myotubes, whereas the expression of Mettl21e was significantly increased in the DLK1-treated myoblasts but reduced in DLK1-treated myotubes. DLK1 treatment had no impact on Park7 expression. In addition, Park7 and Dnttip1 increased Myh4 and decreased Myh7 promoter activity, resemble to the effects of Dlk1. In contrast, expression of Mettl21e increased Myh7 and decreased Myh4 luciferase activity. Conclusion The study provided additional supports that RTL1 alone was insufficient to induce muscle hypertrophy and concluded that DLK1 was likely the primary effector of the hypertrophy phenotype. The results also suggested that DNTTIP1 and PDE4D were secondary effector genes responding to DLK1 signaling resulting in muscle fiber switch and muscular hypertrophy in callipyge lamb

The Imprinted Retrotransposon-Like Gene PEG11 (RTL1) Is Expressed as a Full-Length Protein in Skeletal Muscle from Callipyge Sheep

peer-reviewedMembers of the Ty3-Gypsy retrotransposon family are rare in mammalian genomes despite their abundance in invertebrates and some vertebrates. These elements contain a gag-pol-like structure characteristic of retroviruses but have lost their ability to retrotranspose into the mammalian genome and are thought to be inactive relics of ancient retrotransposition events. One of these retrotransposon-like elements, PEG11 (also called RTL1) is located at the distal end of ovine chromosome 18 within an imprinted gene cluster that is highly conserved in placental mammals. The region contains several conserved imprinted genes including BEGAIN, DLK1, DAT, GTL2 (MEG3), PEG11 (RTL1), PEG11as, MEG8, MIRG and DIO3. An intergenic point mutation between DLK1 and GTL2 causes muscle hypertrophy in callipyge sheep and is associated with large changes in expression of the genes linked in cis between DLK1 and MEG8. It has been suggested that over-expression of DLK1 is the effector of the callipyge phenotype; however, PEG11 gene expression is also strongly correlated with the emergence of the muscling phenotype as a function of genotype, muscle type and developmental stage. To date, there has been no direct evidence that PEG11 encodes a protein, especially as its anti-sense transcript (PEG11as) contains six miRNA that cause cleavage of the PEG11 transcript. Using immunological and mass spectrometry approaches we have directly identified the full-length PEG11 protein from postnatal nuclear preparations of callipyge skeletal muscle and conclude that its over-expression may be involved in inducing muscle hypertrophy. The developmental expression pattern of the PEG11 gene is consistent with the callipyge mutation causing recapitulation of the normal fetal-like gene expression program during postnatal development. Analysis of the PEG11 sequence indicates strong conservation of the regions encoding the antisense microRNA and in at least two cases these correspond with structural or functional domains of the protein suggesting co-evolution of the sense and antisense genes

A gene network switch enhances the oxidative capacity of ovine skeletal muscle during late fetal development

Background: The developmental transition between the late fetus and a newborn animal is associated with profound changes in skeletal muscle function as it adapts to the new physiological demands of locomotion and postural support against gravity. The mechanisms underpinning this adaption process are unclear but are likely to be initiated by changes in hormone levels. We tested the hypothesis that this developmental transition is associated with large coordinated changes in the transcription of skeletal muscle genes.Results: Using an ovine model, transcriptional profiling was performed on Longissimus dorsi skeletal muscle taken at three fetal developmental time points (80, 100 and 120 d of fetal development) and two postnatal time points, one approximately 3 days postpartum and a second at 3 months of age. The developmental time course was dominated by large changes in expression of 2,471 genes during the interval between late fetal development (120 d fetal development) and 1-3 days postpartum. Analysis of the functions of genes that were uniquely up-regulated in this interval showed strong enrichment for oxidative metabolism and the tricarboxylic acid cycle indicating enhanced mitochondrial activity. Histological examination of tissues from these developmental time points directly confirmed a marked increase in mitochondrial activity between the late fetal and early postnatal samples. The promoters of genes that were up-regulated during this fetal to neonatal transition were enriched for estrogen receptor 1 and estrogen related receptor alpha cis-regulatory motifs. The genes down-regulated during this interval highlighted de-emphasis of an array of functions including Wnt signaling, cell adhesion and differentiation. There were also changes in gene expression prior to this late fetal - postnatal transition and between the two postnatal time points. The former genes were enriched for functions involving the extracellular matrix and immune response while the latter principally involved functions associated with transcriptional regulation of metabolic processes.Conclusions: It is concluded that during late skeletal muscle development there are substantial and coordinated changes in the transcription of a large number of genes many of which are probably triggered by increased estrogen levels. These changes probably underpin the adaption of muscle to new physiological demands in the postnatal environment

The Imprinted Retrotransposon-Like Gene PEG11 (RTL1) Is Expressed as a Full-Length Protein in Skeletal Muscle from Callipyge Sheep

Members of the Ty3-Gypsy retrotransposon family are rare in mammalian genomes despite their abundance in invertebrates and some vertebrates. These elements contain a gag-pol-like structure characteristic of retroviruses but have lost their ability to retrotranspose into the mammalian genome and are thought to be inactive relics of ancient retrotransposition events. One of these retrotransposon-like elements, PEG11 (also called RTL1) is located at the distal end of ovine chromosome 18 within an imprinted gene cluster that is highly conserved in placental mammals. The region contains several conserved imprinted genes including BEGAIN, DLK1, DAT, GTL2 (MEG3), PEG11 (RTL1), PEG11as, MEG8, MIRG and DIO3. An intergenic point mutation between DLK1 and GTL2 causes muscle hypertrophy in callipyge sheep and is associated with large changes in expression of the genes linked in cis between DLK1 and MEG8. It has been suggested that over-expression of DLK1 is the effector of the callipyge phenotype; however, PEG11 gene expression is also strongly correlated with the emergence of the muscling phenotype as a function of genotype, muscle type and developmental stage. To date, there has been no direct evidence that PEG11 encodes a protein, especially as its anti-sense transcript (PEG11as) contains six miRNA that cause cleavage of the PEG11 transcript. Using immunological and mass spectrometry approaches we have directly identified the full-length PEG11 protein from postnatal nuclear preparations of callipyge skeletal muscle and conclude that its over-expression may be involved in inducing muscle hypertrophy. The developmental expression pattern of the PEG11 gene is consistent with the callipyge mutation causing recapitulation of the normal fetal-like gene expression program during postnatal development. Analysis of the PEG11 sequence indicates strong conservation of the regions encoding the antisense microRNA and in at least two cases these correspond with structural or functional domains of the protein suggesting co-evolution of the sense and antisense genes

Effect of DLK1 and RTL1 but Not MEG3 or MEG8 on Muscle Gene Expression in Callipyge Lambs

Callipyge sheep exhibit extreme postnatal muscle hypertrophy in the loin and hindquarters as a result of a single nucleotide polymorphism (SNP) in the imprinted DLK1-DIO3 domain on ovine chromosome 18. The callipyge SNP up-regulates the expression of surrounding transcripts when inherited in cis without altering their allele-specific imprinting status. The callipyge phenotype exhibits polar overdominant inheritance since only paternal heterozygous animals have muscle hypertrophy. Two studies were conducted profiling gene expression in lamb muscles to determine the down-stream effects of over-expression of paternal allele-specificDLK1 and RTL1 as well as maternal allele-specific MEG3, RTL1AS and MEG8, using Affymetrix bovine expression arrays. A total of 375 transcripts were differentially expressed in callipyge muscle and 25 transcripts were subsequently validated by quantitative PCR. The muscle-specific expression patterns of most genes were similar to DLK1 and included genes that are transcriptional repressors or affect feedback mechanisms in β-adrenergic and growth factor signaling pathways. One gene, phosphodiesterase 7A had an expression pattern similar to RTL1 expression indicating a biological activity for RTL1 in muscle. Only transcripts that localize to the DLK1-DIO3 domain were affected by inheritance of a maternal callipyge allele. Callipyge sheep are a unique model to study over expression of both paternal allele-specific genes and maternal allele-specific non-coding RNA with an accessible and nonlethal phenotype. This study has identified a number of genes that are regulated by DLK1 and RTL1expression and exert control on postnatal skeletal muscle growth. The genes identified in this model are primary candidates for naturally regulating postnatal muscle growth in all meat animal species, and may serve as targets to ameliorate muscle atrophy conditions including myopathic diseases and age-related sarcopenia

Dlk1 Is Necessary for Proper Skeletal Muscle Development and Regeneration

Delta-like 1homolog (Dlk1) is an imprinted gene encoding a transmembrane protein whose increased expression has been associated with muscle hypertrophy in animal models. However, the mechanisms by which Dlk1 regulates skeletal muscle plasticity remain unknown. Here we combine conditional gene knockout and over-expression analyses to investigate the role of Dlk1 in mouse muscle development, regeneration and myogenic stem cells (satellite cells). Genetic ablation of Dlk1 in the myogenic lineage resulted in reduced body weight and skeletal muscle mass due to reductions in myofiber numbers and myosin heavy chain IIB gene expression. In addition, muscle-specific Dlk1 ablation led to postnatal growth retardation and impaired muscle regeneration, associated with augmented myogenic inhibitory signaling mediated by NF-κB and inflammatory cytokines. To examine the role of Dlk1 in satellite cells, we analyzed the proliferation, self-renewal and differentiation of satellite cells cultured on their native host myofibers. We showed that ablation of Dlk1 inhibits the expression of the myogenic regulatory transcription factor MyoD, and facilitated the self-renewal of activated satellite cells. Conversely, Dlk1 over-expression inhibited the proliferation and enhanced differentiation of cultured myoblasts. As Dlk1 is expressed at low levels in satellite cells but its expression rapidly increases upon myogenic differentiation in vitro and in regenerating muscles in vivo, our results suggest a model in which Dlk1 expressed by nascent or regenerating myofibers non-cell autonomously promotes the differentiation of their neighbor satellite cells and therefore leads to muscle hypertrophy

Gene expression profiling of hypertrophied muscles in callipyge sheep

Callipyge sheep exhibit extreme postnatal hypertrophy in the loin and hindquarters as a result of single nucleotide polymorphism (SNP) in the imprinted DLK1-MEG3 gene cluster on ovine chromosome 18. The callipyge SNP up-regulates the expression of nearby imprinted transcripts when inherited in cis. This thesis contains phenotypic measurements of lambs possessing a callipyge allele from 10 days of age to 200 days of age. These animals were used in three studies designed to profile gene expression in lamb muscles to determine down-stream effects of paternal allele specific and maternal allele specific transcripts. Affymetrix Bovine Expression Arrays and quantitative PCR were used to discover transcripts differentially expressed as a result of the callipyge mutation. To determine the transcripts affected during callipyge muscle hypertrophy, two hypertrophied muscles (longissimus dorsi and semimembranosus) from callipyge (+/CPat ; n=8) and wild-type lambs (+/+; n=8) were compared across four ages that encompass the postnatal muscle hypertrophy development. Analysis methods by MAS5 and RMA identified 149 to 378 transcripts, respectively, that were differentially expressed in callipyge muscles. These included metabolic enzymes, apoptotic factors, proteins involved in regulating common signaling pathways, and transcription factors. Thirty-three novel transcripts were validated by qPCR to be differentially expressed between callipyge and normal muscles across additional ages. The third experiment aimed at detecting differences in genes expression affected by the presence of a maternal callipyge allele (CMat/+ and C/C). These lambs were contrasted against lambs with a wild-type maternal allele (+/ CPat and +/+). The microarray and qPCR data only validated significant differences in expression of MEG3 and MEG8, both genes within the callipyge imprinted cluster. These data indicate that the up-regulation of MEG3 and MEG8 does not influence gene expression of any transcripts on the bovine expression array which are not located near the callipyge mutation. High levels of DLK1 and RTL1 in the paternal heterozygous lambs are enhancing factors involved in existing muscle growth mechanisms including the AKT/mTOR signaling pathway

Park7 Expression Influences Myotube Size and Myosin Expression in Muscle

<div><p>Callipyge sheep exhibit postnatal muscle hypertrophy due to the up-regulation of <i>DLK1</i> and/or <i>RTL1</i>. The up-regulation of <i>PARK7</i> was identified in hypertrophied muscles by microarray analysis and further validated by quantitative PCR. The expression of PARK7 in hypertrophied muscle of callipyge lambs was confirmed to be up-regulated at the protein level. PARK7 was previously identified to positively regulate PI3K/AKT pathway by suppressing the phosphatase activity of PTEN in mouse fibroblasts. The purpose of this study was to investigate the effects of PARK7 in muscle growth and protein accretion in response to IGF1. Primary myoblasts isolated from <i>Park7</i> (+/+) and <i>Park7</i> (−/−) mice were used to examine the effect of differential expression of <i>Park7</i>. The <i>Park7</i> (+/+) myotubes had significantly larger diameters and more total sarcomeric myosin expression than <i>Park7</i> (−/−) myotubes. IGF1 treatment increased the mRNA abundance of <i>Myh4, Myh7 and Myh8</i> between 20-40% in <i>Park7</i> (+/+) myotubes relative to <i>Park7</i> (−<i>/</i>−). The level of AKT phosphorylation was increased in <i>Park7</i> (+/+) myotubes at all levels of IGF1 supplementation. After removal of IGF1, the <i>Park7</i> (+/+) myotubes maintained higher AKT phosphorylation through 3 hours. PARK7 positively regulates the PI3K/AKT pathway by inhibition of PTEN phosphatase activity in skeletal muscle. The increased PARK7 expression can increase protein synthesis and result in myotube hypertrophy. These results support the hypothesis that elevated expression of <i>PARK7</i> in callipyge muscle would increase levels of AKT activity to cause hypertrophy in response to the normal IGF1 signaling in rapidly growing lambs. Increasing expression of PARK7 could be a novel mechanism to increase protein accretion and muscle growth in livestock or help improve muscle mass with disease or aging.</p></div

PARK7 protein expression in <i>vastus lateralis</i>.

<p>PARK7 protein expression was not detectable in <i>vastus lateralis</i> in <i>Park7</i> (−/−) mice in two replicated experiments of two animals per genotype. α-Tubulin was used as a control to show equal protein loading.</p