Expression of myostatin gene in regenerating skeletal muscle of the rat and its localization

The expression of myostatin mRNA was examined in regenerating skeletal muscle of the rat. Skeletal muscle regeneration was induced by injecting bupivacaine or hypertonic saline solution into the femoral muscle, and the tissues were collected 48 h after the treatment. In situ hybridization analysis revealed that the cells positive for myostatin message were localized in the regenerating area of the bupivacaine-treated tissues, where a numerous number of mononucleated cells were present. The myostatin-positive mononucleated cells contained both myogenic and nonmyogenic cells, as revealed by immunohistochemical staining for desmin and vimentin. Bupivacaine treatment to the testes resulted in no myostatin message expression in the testicular vimentin-positive cells, suggesting that the expression of myostatin message in vimentin-positive cells is a skeletal muscle-specific phenomenon. Furthermore, crushed muscle extract prepared from regenerating skeletal muscle had induced myostatin mRNA expression in skeletal muscle-derived fibroblasts in a dose-dependent manner. These results indicated that myostatin is expressed during skeletal muscle regeneration both in myogenic and nonmyogenic cells, and suggested that some factor(s) capable of inducing myostatin expression in fibroblasts are present in regenerating skeletal muscle.     

Differential DNA methylation with age displays both common and dynamic features across human tissues that are influenced by CpG landscape

Background

DNA methylation is an epigenetic modification that changes with age in human tissues, although the mechanisms and specificity of this process are still poorly understood. We compared CpG methylation changes with age across 283 human blood, brain, kidney, and skeletal muscle samples using methylation arrays to identify tissue-specific age effects.

Results

We found age-associated CpGs (ageCGs) that are both tissue-specific and common across tissues. Tissue-specific ageCGs are frequently located outside CpG islands with decreased methylation, and common ageCGs show the opposite trend. AgeCGs are significantly associated with poorly expressed genes, but those with decreasing methylation are linked with higher tissue-specific expression levels compared with increasing methylation. Therefore, tissue-specific gene expression may protect against common age-dependent methylation. Distinguished from other tissues, skeletal muscle ageCGs are more associated with expression, enriched near genes related to myofiber contraction, and closer to muscle-specific CTCF binding sites. Kidney-specific ageCGs are more increasingly methylated compared to other tissues as measured by affiliation with kidney-specific expressed genes. Underlying chromatin features also mark common and tissue-specific age effects reflective of poised and active chromatin states, respectively. In contrast with decreasingly methylated ageCGs, increasingly methylated ageCGs are also generally further from CTCF binding sites and enriched within lamina associated domains.

Conclusions

Our data identified common and tissue-specific DNA methylation changes with age that are reflective of CpG landscape and suggests both common and unique alterations within human tissues. Our findings also indicate that a simple epigenetic drift model is insufficient to explain all age-related changes in DNA methylation.     

Urokinase is required for the formation of mactinin,an alpha-actinin fragment that promotes monocyte/macrophage maturation

Myostatin is a recently discovered gene that inhibits muscle growth. In the present study, we characterized the myostatin locus and its expression in channel catfish (Ictalurus punctatus). The genomic DNA and cDNA encoding the channel catfish myostatin were cloned and sequenced. The myostatin gene has three exons encoding a protein of 389 amino acids. Comparison of the genomic sequences with those of the cDNA revealed that the myostatin cDNA was 1673 base pair (bp) long with a 5'-untranslated region (UTR) and 3'-UTR of 180 and 323 bp, respectively. The deduced amino acid sequences of the catfish myostatin is highly conserved with those of other organisms. The myostatin locus is highly polymorphic in channel catfish because of the presence of several microsatellites and single nucleotide polymorphic sites. The myostatin gene was expressed in various tissues and developmental stages at differential levels, suggesting complex regulation of this gene and perhaps roles for myostatin in addition to those originally suggested.     

Acute exercise remodels promoter methylation in human skeletal muscle

DNA methylation is a covalent biochemical modification controlling chromatin structure and gene expression. Exercise elicits gene expression changes that trigger structural and metabolic adaptations in skeletal muscle. We determined whether DNA methylation plays a role in exercise-induced gene expression. Whole genome methylation was decreased in?skeletal muscle biopsies obtained from healthy sedentary men and women after acute exercise. Exercise induced a dose-dependent expression of PGC-1α, PDK4, and PPAR-δ, together with a marked hypomethylation on each respective promoter. Similarly, promoter methylation of PGC-1α, PDK4, and PPAR-δ was markedly decreased in mouse soleus muscles 45?min after ex?vivo contraction. In L6 myotubes, caffeine exposure induced gene hypomethylation in parallel with an increase in the respective mRNA content. Collectively, our results provide evidence that acute gene activation is associated with a dynamic change in DNA methylation in skeletal muscle and suggest that DNA hypomethylation is an early event in contraction-induced gene activation.     

Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin

Mutations in the myostatin gene are associated with hypermuscularity, suggesting that myostatin inhibits skeletal muscle growth. We postulated that increased tissue-specific expression of myostatin protein in skeletal muscle would induce muscle loss. To investigate this hypothesis, we generated transgenic mice that overexpress myostatin protein selectively in the skeletal muscle, with or without ancillary expression in the heart, utilizing cDNA constructs in which a wild-type (MCK/Mst) or mutated muscle creatine kinase (MCK-3E/Mst) promoter was placed upstream of mouse myostatin cDNA. Transgenic mice harboring these MCK promoters linked to enhanced green fluorescent protein (EGFP) expressed the reporter protein only in skeletal and cardiac muscles (MCK) or in skeletal muscle alone (MCK-3E). Seven-week-old animals were genotyped by PCR of tail DNA or by Southern blot analysis of liver DNA. Myostatin mRNA and protein, measured by RT-PCR and Western blot, respectively, were significantly higher in gastrocnemius, quadriceps, and tibialis anterior of MCK/Mst-transgenic mice compared with wild-type mice. Male MCK/Mst-transgenic mice had 18-24% lower hind- and forelimb muscle weight and 18% reduction in quadriceps and gastrocnemius fiber cross-sectional area and myonuclear number (immunohistochemistry) than wild-type male mice. Male transgenic mice with mutated MCK-3E promoter showed similar effects on muscle mass. However, female transgenic mice with either type of MCK promoter did not differ from wild-type controls in either body weight or skeletal muscle mass. In conclusion, increased expression of myostatin in skeletal muscle is associated with lower muscle mass and decreased fiber size and myonuclear number, decreased cardiac muscle mass, and increased fat mass in male mice, consistent with its role as an inhibitor of skeletal muscle mass. The mechanism of gender specificity remains to be clarified.     

Molecular characterization of myostatin in black seabream, Acanthopagrus schlegelii.

Myostatin is a negative regulator of skeletal muscle growth and has a potential application in aquaculture. The black seabream myostatin gene was cloned and sequenced. It had three exons encoding a protein of 382 amino acids. A 90 bp 5'-untranslated region (UTR) and a 536 bp 3'-UTR were obtained by RACE. Four microsatellite sequences, a (CAG)9, a (TC)12, a (CA)16 repeat and an "imperfect" (CA)25 microsatellite, were found in the myostatin. Two introns were 329 and 742 bp in length, respectively. The deduced amino acid sequence of the myostatin had a putative amino terminal signal sequence, a TGF-beta propeptide domain, a RXXR proteolytic processing site, a TGF-beta domain, and 12 conserved cysteine residues. The myostatin gene was expressed in four of the examined ten tissues and organs. The expression of myostatin was the strongest in the skeletal muscle and brain, intermediate in the eye, and low in the heart.     

Characterization of gene expression in double-muscled and normal-muscled bovine embryos

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth. Cattle with mutations that inactivate myostatin exhibit a remarkable increase in mass of skeletal muscle called double muscling that is accompanied by an equally remarkable decrease in carcass fat. Although a mouse knockout model has been created which results in mice with a 200% increase in skeletal muscle mass, molecular mechanisms whereby myostatin regulates skeletal muscle and fat mass are not fully understood. Using suppressive subtractive hybridization, genes that were differentially expressed in double-muscled vs. normal-muscled cattle embryos were identified. Genetic variation at other loci was minimized by using embryonic samples collected from related Piedmontese x Angus dams or Belgian Blue x Hereford dams bred to a single sire of the same breed composition. Embryos were collected at 31-33 days of gestation, which is 2-4 days after high-level expression of myostatin in the developing bovine embryo. The suppressive subtraction resulted in 30 clones that were potentially differentially expressed, 19 of which were confirmed by macroarray analysis. Several of these genes have biological functions that suggest that they are directly involved in myostatin's regulation of skeletal muscle development. Furthermore, several of these genes map to quantitative trait loci known to interact with variation in the myostatin gene.     

Myostatin from the heart: local and systemic actions in cardiac failure and muscle wasting

A significant proportion of heart failure patients develop skeletal muscle wasting and cardiac cachexia, which is associated with a very poor prognosis. Recently, myostatin, a cytokine from the transforming growth factor-β (TGF-β) family and a known strong inhibitor of skeletal muscle growth, has been identified as a direct mediator of skeletal muscle atrophy in mice with heart failure. Myostatin is mainly expressed in skeletal muscle, although basal expression is also detectable in heart and adipose tissue. During pathological loading of the heart, the myocardium produces and secretes myostatin into the circulation where it inhibits skeletal muscle growth. Thus, genetic elimination of myostatin from the heart reduces skeletal muscle atrophy in mice with heart failure, whereas transgenic overexpression of myostatin in the heart is capable of inducing muscle wasting. In addition to its endocrine action on skeletal muscle, cardiac myostatin production also modestly inhibits cardiomyocyte growth under certain circumstances, as well as induces cardiac fibrosis and alterations in ventricular function. Interestingly, heart failure patients show elevated myostatin levels in their serum. To therapeutically influence skeletal muscle wasting, direct inhibition of myostatin was shown to positively impact skeletal muscle mass in heart failure, suggesting a promising strategy for the treatment of cardiac cachexia in the future.     

The pattern of DNA methylation in the delta-crystallin genes in transdifferentiating neural retina cultures

Terminally differentiated lens fibre cells are formed in the vertebrate lens throughout life. Lens fibre cells may also be obtained by an in vitro process termed transdifferentiation, from certain tissues of different developmental origin from lens, such as embryo neural retina. delta-Crystallin is the major protein in the chick embryo lens fibre cells, and also in transdifferentiated lens cells obtained from cultured embryonic neural retina. Lens crystallin proteins and mRNA are present at low levels in the intact embryonic neural retina but are no longer detectable in the early stages of neural retina cell culture. However, levels rise steeply in the later stages and crystallins become the major products in terminally transdifferentiating neural retina cultures. We have used this system to test the hypothesis that the patterns of DNA methylation in particular genes are correlated with gene expression. A number of developmentally regulated genes have been found to be undermethylated in tissues where they are expressed, and methylated in tissues where they are not. However this correspondence does not always hold true. Eight-day-old embryonic neural retina was cultured for the period of time during which crystallin gene expression increases 100-fold. DNA methylation in the delta-crystallin gene region was analysed at several stages of cell culture by using the restriction endonucleases HpaII and MspI which cleave at the sequence CCGG. The former enzyme cannot cleave internally methylated cytosine (CmCGG) while the latter cannot cleave externally methylated cytosine (mCCGG). We detect no change in the methylation of CCGG sites within the delta-crystallin gene regions during transdifferentiation. Since dramatic changes in delta-crystallin gene expression occur during this process we conclude that large scale alterations in the pattern of DNA methylation are not a necessary accompaniment to changes in gene activity.     

The regulation of LncRNA GTL2 expression by DNA methylation during sheep skeletal muscle development

DNA methylation has crucial roles in regulating the expression of genes involved in skeletal muscle development. However, the DNA methylation pattern of lncRNA during sheep skeletal muscle development remains unclear. This study investigated previous WGBS and LncRNA data in skeletal muscle of sheep (fetus and adult). We then focused on LncRNA GTL2, which is differentially expressed in skeletal muscle and has multiple DMRs. We found that the expression level of GTL2 decreased with age. GTL2 DMRs methylation levels were significantly higher in adult muscle than in fetal muscle. After 5AZA treatment, GTL2 expression was significantly increased in a dose-dependent manner.The dCas9-DNMT3A-sgRNA significantly reduced the expression level of GTL2 in cells, but increased GTL2 DMR methylation levels. The above studies indicate that dCas9-DNMT3A can effectively increase the methylation level in the DMR region of GTL2, the expression level of GTL2 is regulated by DNA methylation during muscle development.     

The two myostatin genes of Atlantic salmon (Salmo salar) are expressed in a variety of tissues.

Two myostatin isoforms were identified in Atlantic salmon (Salmo salar) by RT-PCR, and genomic sequences encoding this negative muscle growth factor were for the first time isolated from a nonmammalian species. Salmon myostatin isoform I is transcribed in white skeletal muscle as a 2346-nucleotide mRNA species that encodes a precursor protein of 373 amino acids. Salmon myostatin I shows 93% sequence identity with isoform II which was isolated from white muscle as a partial cDNA sequence of 1409 nucleotides. In contrast to the restricted gene expression of myostatin in mammals, salmon myostatin I and II mRNAs were identified by RT-PCR in multiple tissues, including white muscle, intestine, brain, gills, tongue and eye. In addition, isoform I mRNA was found in red skeletal muscle, heart, spleen, and ovarian tissue. Using polyclonal antibodies against both isoforms, a 55-kDa precursor protein was detected by Western blot analysis in the red and white skeletal muscle, heart, intestine, and brain. Immunoreactive peptides of 35-40 kDa were identified in the gills, tongue, spleen, and head kidney, while the 25-kDa mature myostatin was found in the eye and serum, and in vitro expressed in rabbit reticulocyte lysate. Salmon myostatin was immunohistochemically localized in the sarcoplasma of red and white muscle fibres, in intestinal epithelial cells, at the basis of the branchial primary lamellae, and in odontoblasts and ameloblasts of the tongue teeth. The results indicate that the role of fish myostatin may not be restricted to muscle growth regulation, but may have additional functions similar to the growth/differentiation factor-11 in mammals.     

Extensive tissue-specific variation of allelic methylation in the Igf2 gene during mouse fetal development: relation to expression and imprinting

The imprinted Igf2 gene is active only on the paternal allele in most tissues. Its imprinting involves a cis-acting imprinting-control region (ICR) located upstream of the neighboring and maternally expressed H19 gene. It is thought that differential methylation of the parental alleles at the ICR is crucial for parental imprinting of both genes. Differentially methylated regions (DMRs) have also been identified within the Igf2 gene and their differential methylation is thought to be established during early development. To gain further insight into the function of these DMRs, we performed a quantitative analysis of their allelic methylation levels in different tissues during fetal development and the postnatal period in the mouse. Surprisingly, we found that the methylation levels of Igf2 DMRs vary extensively during fetal development, mostly on the expressed paternal allele. In particular, in skeletal muscle, differential allelic methylation in both DMR 1 and DMR 2 occurs only after birth, whereas correct paternal monoallelic expression is always observed, including in the embryonic stages. This suggests that differential methylation in the DMR 1 and DMR 2 of the Igf2 gene is dispensable for its imprinting in skeletal muscle. Furthermore, progressive methylation of the Igf2 paternal allele appears to be correlated with concomitant postnatal down-regulation and silencing of the gene. We discuss possible relations between Igf2 allelic methylation and expression during fetal development.     

Epigenetic regulation of the Igf2/H19 gene cluster

Igf2 (insulin‐like growth factor 2) and H19 genes are imprinted in mammals; they are expressed unevenly from the two parental alleles. Igf2 is a growth factor expressed in most normal tissues, solely from the paternal allele. H19 gene is transcribed (but not translated to a protein) from the maternal allele. Igf2 protein is a growth factor particularly important during pregnancy, where it promotes both foetal and placental growth and also nutrient transfer from mother to offspring via the placenta. This article reviews epigenetic regulation of the Igf2/H19 gene‐cluster that leads to parent‐specific expression, with current models including parental allele‐specific DNA methylation and chromatin modifications, DNA‐binding of insulator proteins (CTCFs) and three‐dimensional partitioning of DNA in the nucleus. It is emphasized that key genomic features are conserved among mammals and have been functionally tested in mouse. ‘The enhancer competition model’, ‘the boundary model’ and ‘the chromatin‐loop model’ are three models based on differential methylation as the epigenetic mark responsible for the imprinted expression pattern. Pathways are discussed that can account for allelic methylation differences; there is a recent study that contradicts the previously accepted fact that biallelic expression is accompanied with loss of differential methylation pattern.