Regulation Of Skeletal Muscle Development Research Articles

MiR-203b: a novel regulator of MyoD expression in tilapia skeletal muscle

MyoD is one of the helix-loop-helix proteins regulating muscle-specific gene expression in tilapia. Tight regulation of the MyoD protein level is necessary for the precise regulation of skeletal muscle development. MicroRNAs (miRNAs) are a class of regulatory RNAs that post-transcriptionally regulate gene expression. An increasing amount of evidence has suggested that miRNAs play an important role in regulating skeletal muscle development. We reasoned that MyoD expression may be regulated by miRNAs. Predictions from bioinformatics have identified a putative miR-203b target site in the 3'-UTR of the MyoD gene. Interestingly, miR-203b expression is negatively correlated with MyoD expression, whereas miR-203b suppression leads to a significant increase in MyoD expression, thereby activating MyoD downstream genes. A 3'-UTR luciferase reporter assay further verifies the direct interaction between miR-203b and MyoD. Taken together, our results reveal a novel molecular mechanism in which miRNA participates in transcriptional circuits that regulate gene expression in tilapia skeletal muscle.

Peroxisome Proliferator-activated Receptor γ (PPARγ) Mediates a Ski Oncogene-induced Shift from Glycolysis to Oxidative Energy Metabolism

Overexpression of the Ski oncogene induces oncogenic transformation of chicken embryo fibroblasts (CEFs). However, unlike most other oncogene-transformed cells, Ski-transformed CEFs (Ski-CEFs) do not display the classical Warburg effect. On the contrary, Ski transformation reduced lactate production and glucose utilization in CEFs. Compared with CEFs, Ski-CEFs exhibited enhanced TCA cycle activity, fatty acid catabolism through β-oxidation, glutamate oxidation, oxygen consumption, as well as increased numbers and mass of mitochondria. Interestingly, expression of PPARγ, a key transcription factor that regulates adipogenesis and lipid metabolism, was dramatically elevated at both the mRNA and protein levels in Ski-CEFs. Accordingly, PPARγ target genes that are involved in lipid uptake, transport, and oxidation were also markedly up-regulated by Ski. Knocking down PPARγ in Ski-CEFs by RNA interference reversed the elevated expression of these PPARγ target genes, as well as the shift to oxidative metabolism and the increased mitochondrial biogenesis. Moreover, we found that Ski co-immunoprecipitates with PPARγ and co-activates PPARγ-driven transcription.

Effect of an exon 1 mutation in the myostatin gene on the growth traits of the Bian chicken

Myostatin (MSTN), or growth and differentiation factor 8 (GDF8), is a member of the transforming growth factor (TGF)-β superfamily. This family functions as a negative regulator of skeletal muscle development and growth in mammals. Single-nucleotide polymorphisms in exon 1 of the Bian chicken myostatin gene were detected by polymerase chain reaction-restriction fragment length polymorphism. A mutation (c.234G>A) in exon 1 was found. Female Bian chickens of genotypes AA and GA had significantly higher body weights than those of genotype GG (P < 0.05 or P < 0.01) from 6 to 18 weeks of age. These results suggested that the mutation c.234G>A in exon 1 could be used as a genetic marker for Bian chicken growth traits.

Formoterol treatment downregulates the myostatin system in skeletal muscle of cachectic tumour-bearing rats

Cachexia is a common systemic manifestation. Additionally, myostatin is known to be a negative regulator of skeletal muscle development. The present study aimed to investigate whether formoterol down-regulates the myostatin system in skeletal muscle of tumour-bearing rats. Real-time PCR and Western blotting were used for the analysis. Results showed that rats bearing the Yoshida AH-130 ascites hepatoma, a cachexia-inducing tumour, exhibited marked muscle wasting that affected the mass of the muscles studied. The cachectic animals exhibited a significant increase in the mRNA levels of the myostatin receptor (ActIIB) in gastrocnemius muscles. Notably, the expression of the various forms of follistatin, a protein with the opposite effects to those of myostatin, was significantly reduced as a result of the implantation of the tumour. When the animals were treated with formoterol, a β-agonist with anti-cachectic potential, increases in skeletal muscle weights were observed. The β-agonist significantly increased levels of various follistatin isoforms and significantly decreased the expression levels of the myostatin receptor. In addition, formoterol treatment resulted in a significant decrease of the myostatin protein content of the gastrocnemius muscle. In conclusion, the results presented indicate that certain anabolic actions of formoterol on the skeletal muscle of cachectic animals may be mediated via the myostatin system.

Molecular characterization, expression pattern, and association analysis with carcass traits of the porcine SHIP2 gene

Src homology 2-containing inositol 5-phosphatase 2 (SHIP2) has been identified as 5'-inositol phosphatase that hydrolyzes PI(3,4,5)P(3) to PI(3,4)P(2), which negatively regulates insulin-induced Akt signaling in skeletal muscle. In this study, we obtained a 3,795-bp mRNA sequence of porcine SHIP2 that included the full coding region for a protein of 1,264 amino acids. With the use of comparative mapping, we mapped this gene to SSC9 p23-24, where many QTLs affect average backfat thickness, average daily weight gain (birth-10 weeks), adipocyte number, belly fat area, and mid-back fat traits. As a candidate gene for carcass traits, a novel single nucleotide polymorphism in intron 21 (A > G) was detected by PCR-RFLP. The results showed that the AA genotype had higher skin percentage, shoulder fat thickness, and m. longissimus dorsi width, but lower m. longissimus dorsi height compared with the genotype GG (P < 0.05), and that allele G appeared to be associated with an increase in the growth trait. SHIP2 was expressed abundantly in skeletal muscle tissue and was transcriptionally decreased during the proliferative phase, but increased in the intermediate stages of muscle differentiation. Analysis of the porcine SHIP2 promoter sequence demonstrated that the E2F element is involved in downregulating SHIP2 mRNA expression in proliferating myoblasts. Using RNAi, we found that the MyoD transcription factor played a role in upregulating SHIP2 expression in differentiating myotubes. In summary, we suggest that SHIP2 might play a role in the regulation of skeletal muscle development in pigs.

어류 유래 마이오스타틴 프로도메인 단백질에 의한 시마연어(Oncorhychus masou) 성장효과

성장과 분화를 조절하는 인자인 myostatin은 포유류에서 주로 골격근에 분포하며 근육성장을 억제하는 것으로 알려져 있다. Myostatin은 포유류에서뿐만 아니라 어류에 있어서도 그 기능이 유사하며 본 연구에서는 넙치와 조피볼락 유래 재조합 myostatin 단백질을 생산하여 시마연어에 침지방법을 통해 처리하였다. 처리 결과 시마연어의 무게와 생화학 분석에서는 유의성이 나타날 정도의 증가는 없었지만 근(muscle) 조직학적 분석에서 넙치와 조피볼락 유래 재조합 myostatin prodomain에 의해 12주째에는 세포의 수가 증가하는 hyperplasia가 일어났으며 22주째에는 조피볼락 유래의 재조합 myostatin prodomain을 처리한 군에서만 hypertrophy가 일어났다. 결론적으로 어류 유래 재조합 myostatin prodomain이 시마연어 근육성장 시 hyperplasia와 hypertrophy가 순차적으로 유도되는 것으로 확인되었다. Myostatin (MSTN) belongs to the transforming growth factor-<TEX>${\beta}$</TEX> superfamily or growth and differentiation factor 8 (GDF-8), and functions as a negative regulator of skeletal muscle development and growth. Previous studies in mammals have suggested that myostatin knock-out increased muscle mass and decreased fat content compared to those of the wide type. Recently, several studies on myostatin have beenconducted on the block myostatin signal pathway with myostatin antagonists and the MSTN regulation with RNAi to control myostatin function. This study was performed to analyze growth and muscle alteration of Oncorhychus masou by treatment with recombinant myostatin prodomains derived from fish. We designed myostatin prodomains derived from P. olivaceus (pMALc2x-poMSTNpro) and S. schlegeli (pMALc2x-sMSTNpro) in a pMALc2x expression vector, and then purified the recombinant proteins using affinity chromatography. The purified recombinant proteins were treated in O. masou through an immersion method. Recombinant protein treated groups did not show a significant difference in weight, protein, or lipid composition compared to the control. However, there was a difference in the average number and area for histological analyses in the muscle fiber. At twelve and twenty-two weeks from the initial treatment, there were differences in averagefiber number and area between the 0.05 mg/l treated-group and the control, but the numbers were similar to those of the control during the same time period. At twelve weeks, however, 0.2 mg/l treated-group had an increase in average fiber number and decrease in average fiber area compared to the control. At twenty-two weeks, the pMALc2x-sMSTNpro 0.2 mg/l treated-group was induced and showed a decrease in average fiber number and increase in average fiber area. The results between twelve and twenty-two weeks showed that the fiber numbers had decreased, whereas average fiberarea had increased due to sMSTNpro. It is understood that the sMSTNpro induced only hyperplasia at twelve weeks, after which it induced hypertrophy. Recombinant myostatin prodomains derived from fish may induce hyperplasia and hypertrophy in O. masou depending upon the time that has elapsed.

Identification of Extracellular Matrix and Cell Adhesion Molecule Genes Associated with Muscle Development in Pigs

Extracellular matrix (ECM) and cell adhesion molecule (CAM) genes are involved in the regulation of skeletal muscle development; however, their roles in skeletal muscle development in pigs are still poorly understood. 65 days postcopulation (dpc) is a critical time point in pig development. Therefore, we analyzed expression of ECM and CAM genes in the longissimus dorsi muscles at 65 dpc from Landrace (lean-type: L65), Tongcheng (obese-type: T65), and Wuzhishan pigs (miniature-type: W65) using microarray technology. A total of 35 genes were differently expressed between the breeds, and of them, 18, 18, and 20 genes, were observed in the comparisons of L65 versus T65, L65 versus W65, and T65 versus W65 (L65/T65, L65/W65, and T65/W65), respectively. In L65/T65, differently expressed genes were widely distributed, whereas in L65/W65 and T65/W65, they mostly focused on the genes encoding CAMs and ECMs proteins. Moreover, the largest number of up-regulated genes involved in skeletal muscle development was detected in L65, a moderate number in W65, and the smallest number was in T65. Cluster analysis suggested that T65 showed a more similar expression pattern to L65 than W65. In addition, we validated that five genes from microarray data were more highly expressed in the prenatal as compared to postnatal periods in Landrace and Tongcheng pigs and showed a greater range of high-level expression during gestation in Landrace than Tongcheng pigs. Our data indicated that ECM and CAM genes are differently expressed among the three breeds, and more complicated molecular events involving CAMs and ECMs were observed in Wuzhishan pigs. This study advances our knowledge of the molecular basis of phenotypic variation and provides a helpful resource for the identification of candidate genes associated with meat production traits in pigs.

Blocking the Myostatin Signal With a Dominant Negative Receptor Improves the Success of Human Myoblast Transplantation in Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a recessive disease caused by a dystrophin gene mutation. Myoblast transplantation permits to introduce the dystrophin gene in dystrophic muscle fibers. However, the success of this approach is reduced by the short duration of the regeneration following the transplantation, which reduces the number of hybrid fibers. Myostatin (MSTN) is a negative regulator of skeletal muscle development and responsible for limiting regeneration. It binds with high affinity to the activin type IIB receptor (ActRIIB). Our aim was to verify whether the success of the myoblast transplantation is enhanced by blocking the MSTN signal with expression of a dominant negative mutant of ActRIIB (dnActRIIB). In vitro, blocking MSTN activity with a lentivirus carrying dnActRIIB increased proliferation and fusion of human myoblasts because MSTN regulates the expression of several myogenic regulatory factors. In vivo, myoblasts infected with the dnActRIIB lentivirus were transplanted in immunodeficient dystrophic mice. Dystrophin immunostaining of tibialis anterior (TA) cross-sections of these mice 1 month post-transplantation revealed more human dystrophin-positive myofibers following the transplantation of dnActRIIB myoblasts than of control myoblasts. Thus, blocking the MSTN signal with dnActRIIB improved the success of myoblast transplantation by increasing the myoblast proliferation and fusion and changed the expression of myogenic regulatory factors.

Myostatin-2 gene structure and polymorphism of the promoter and first intron in the marine fish Sparus aurata: evidence for DNA duplications and/or translocations

BackgroundMyostatin (MSTN) is a member of the transforming growth factor-ß superfamily that functions as a negative regulator of skeletal muscle development and growth in mammals. Fish express at least two genes for MSTN: MSTN-1 and MSTN-2. To date, MSTN-2 promoters have been cloned only from salmonids and zebrafish.ResultsHere we described the cloning and sequence analysis of MSTN-2 gene and its 5' flanking region in the marine fish Sparus aurata (saMSTN-2). We demonstrate the existence of three alleles of the promoter and three alleles of the first intron. Sequence comparison of the promoter region in the three alleles revealed that although the sequences of the first 1050 bp upstream of the translation start site are almost identical in the three alleles, a substantial sequence divergence is seen further upstream. Careful sequence analysis of the region upstream of the first 1050 bp in the three alleles identified several elements that appear to be repeated in some or all sequences, at different positions. This suggests that the promoter region of saMSTN-2 has been subjected to various chromosomal rearrangements during the course of evolution, reflecting either insertion or deletion events. Screening of several genomic DNA collections indicated differences in allele frequency, with allele 'b' being the most abundant, followed by allele 'c', whereas allele 'a' is relatively rare. Sequence analysis of saMSTN-2 gene also revealed polymorphism in the first intron, identifying three alleles. The length difference in alleles '1R' and '2R' of the first intron is due to the presence of one or two copies of a repeated block of approximately 150 bp, located at the 5' end of the first intron. The third allele, '4R', has an additional insertion of 323 bp located 116 bp upstream of the 3' end of the first intron. Analysis of several DNA collections showed that the '2R' allele is the most common, followed by the '4R' allele, whereas the '1R' allele is relatively rare. Progeny analysis of a full-sib family showed a Mendelian mode of inheritance of the two genetic loci. No clear association was found between the two genetic markers and growth rate.ConclusionThese results show for the first time a substantial degree of polymorphism in both the promoter and first intron of MSTN-2 gene in a perciform fish species which points to chromosomal rearrangements that took place during evolution.

Mammalian target of rapamycin regulates miRNA-1 and follistatin in skeletal myogenesis.

Mammalian target of rapamycin (mTOR) has emerged as a key regulator of skeletal muscle development by governing distinct stages of myogenesis, but the molecular pathways downstream of mTOR are not fully understood. In this study, we report that expression of the muscle-specific micro-RNA (miRNA) miR-1 is regulated by mTOR both in differentiating myoblasts and in mouse regenerating skeletal muscle. We have found that mTOR controls MyoD-dependent transcription of miR-1 through its upstream enhancer, most likely by regulating MyoD protein stability. Moreover, a functional pathway downstream of mTOR and miR-1 is delineated, in which miR-1 suppression of histone deacetylase 4 (HDAC4) results in production of follistatin and subsequent myocyte fusion. Collective evidence strongly suggests that follistatin is the long-sought mTOR-regulated fusion factor. In summary, our findings unravel for the first time a link between mTOR and miRNA biogenesis and identify an mTOR-miR-1-HDAC4-follistatin pathway that regulates myocyte fusion during myoblast differentiation in vitro and skeletal muscle regeneration in vivo.

Effect of Myostatin (MSTN) g+6223G&amp;gt;A on Production and Carcass Traits in New Zealand Romney Sheep

Myostatin, which is also known as growth and differentiation factor 8 (GDF8), has been reported to act as a negative regulator of skeletal muscle development. Variation in the myostatin gene (MSTN) has been associated with variation in muscularity in certain meaty sheep breeds. Polymerase Chain Reaction-Single Strand Conformational Polymorphism (PCR-SSCP) analysis was used to investigate allelic variation in the previously described g+6223G>A single-nucleotide polymorphism (SNP) in the 3' untranslated region (3'UTR) of MSTN. The sheep studied were 79 New Zealand (NZ) Romney lambs derived from a single sire heterozyous for g+6223G>A, which is in itself notable as this polymorphism has not been described previously in this breed. Allelic variation was observed to be associated with an abnormal gender ratio (p = 0.046) in the progeny. The presence of allele A was observed to have an effect (p<0.05) on birth weight, mean loin yield, proportion yield loin and total muscle yield. Allelic variation did not significantly affect mean shoulder yield, leg yield, proportion yield shoulder and proportion yield leg. This preliminary result suggests that while the A allele at MSTN g+6223 appears to improve some valuable traits in NZ Romney sheep, further research is required to understand if and how it may affect other traits.

MyoD control of SKIP expression during pig skeletal muscle development

Skeletal muscle and kidney enriched inositol phosphatase (SKIP) was identified as a 5'-inositol phosphatase that hydrolyzes PI(3,4,5)P3 to PI(3,4)P2 that negatively regulates insulin-induced phosphatidylinositol 3-kinase signaling in skeletal muscle. In this study, we obtained a 1575-bp mRNA sequence of porcine SKIP that included the full coding region encoding a protein of 450 amino acids. With the use of comparative mapping, we mapped this gene to SSC12 q1.3, where many QTLs affect Backfat thickness at 10th rib, carcass yield, the number of muscle fibers, and ham weight traits. As a candidate gene for growth and carcass traits, a novel single nucleotide polymorphism in exon 12 (G>A) was detected by PCR-RFLP. The results showed that the GG genotype had higher skin percentage (SP), carcass length to first spondyle (CL1), carcass length to first rib (CL2), but lower intramuscular fat (IMF) as compared with genotype AG (P<0.05), and allele G seemed to be associated with an increase in the growth trait. Porcine SKIP was expressed abundantly in skeletal muscle tissue and was transcriptionally upregulated during skeletal muscle differentiation. Analysis of the porcine SKIP promoter sequence demonstrated that MyoD was involved in regulating SKIP mRNA expression in myotubes, partly via the cis-acting elements in SKIP promoter. In summary, we suggested that SKIP might play a role in the regulation of skeletal muscle development in pigs.

Enhanced Muscle Growth by Plasmid-Mediated Delivery of Myostatin Propeptide

Myostatin is a member of the transforming growth factor beta (TGF-β) superfamily that functions as a negative regulator of skeletal muscle development and growth. Myostatin blockade therefore offers a strategy for promoting muscle growth in livestock production without resorting to genetic manipulation. In this report, we examined the effect of myostatin inhibition by plasmid-mediated delivery of a mutant myostatin propeptide (MProD76A), a natural inhibitor of myostatin, on the growth performance of mice. A significant increase in skeletal muscle mass was observed after a single intramuscular injection of naked plasmid DNA encoding MProD76A into mice. Enhanced muscle growth occurred because of myofiber hypertrophy, but no cardiac muscle hypertrophy and organomegaly was observed in the mice after myostatin inhibition by plasmid-mediated MProD76A delivery. These results demonstrate a promising approach to enhancing muscle growth that warrants further investigation in domestic animals.

Inferring the Transcriptional Landscape of Bovine Skeletal Muscle by Integrating Co-Expression Networks

BackgroundDespite modern technologies and novel computational approaches, decoding causal transcriptional regulation remains challenging. This is particularly true for less well studied organisms and when only gene expression data is available. In muscle a small number of well characterised transcription factors are proposed to regulate development. Therefore, muscle appears to be a tractable system for proposing new computational approaches.Methodology/Principal FindingsHere we report a simple algorithm that asks “which transcriptional regulator has the highest average absolute co-expression correlation to the genes in a co-expression module?” It correctly infers a number of known causal regulators of fundamental biological processes, including cell cycle activity (E2F1), glycolysis (HLF), mitochondrial transcription (TFB2M), adipogenesis (PIAS1), neuronal development (TLX3), immune function (IRF1) and vasculogenesis (SOX17), within a skeletal muscle context. However, none of the canonical pro-myogenic transcription factors (MYOD1, MYOG, MYF5, MYF6 and MEF2C) were linked to muscle structural gene expression modules. Co-expression values were computed using developing bovine muscle from 60 days post conception (early foetal) to 30 months post natal (adulthood) for two breeds of cattle, in addition to a nutritional comparison with a third breed. A number of transcriptional landscapes were constructed and integrated into an always correlated landscape. One notable feature was a ‘metabolic axis’ formed from glycolysis genes at one end, nuclear-encoded mitochondrial protein genes at the other, and centrally tethered by mitochondrially-encoded mitochondrial protein genes.Conclusions/SignificanceThe new module-to-regulator algorithm complements our recently described Regulatory Impact Factor analysis. Together with a simple examination of a co-expression module's contents, these three gene expression approaches are starting to illuminate the in vivo transcriptional regulation of skeletal muscle development.

Suppression of myostatin with vector-based RNA interference causes a double-muscle effect in transgenic zebrafish

Myostatin belongs to the transforming growth factor (TGF)-β superfamily and is a potent negative regulator of skeletal muscle development and growth. We utilized microinjection of an antisense RNA-expressing vector to establish a hereditarily stable myostatin gene knockdown zebrafish strain with a double-muscle phenotype. Real-time PCR and immunostaining revealed that the myostatin messenger (m)RNA and protein levels in homozygous transgenic zebrafish were 33% and 26% those of the non-transgenic controls, respectively. Also, the mRNA levels of myogenic regulatory factor markers such as MyoD, myogenin, Mrf4, and Myf5 were dramatically elevated in myostatin-suppressed transgenic fish compared to the non-transgenic controls. Although there was no significant difference in body length, homozygous transgenic zebrafish were 45% heavier than non-transgenic controls. Histochemical analysis showed that the cross-sectional area of the muscle fiber of homozygous transgenic fish was twice as large as that of non-transgenic controls. This is the first model zebrafish with a hereditarily stable myostatin-suppressed genotype and a double-muscle phenotype.

Characterization and functional analysis of the 5′ flanking region of Sparus aurata myostatin-1 gene

Myostatin (MSTN) is a member of the transforming growth factor-β superfamily that functions as a negative regulator of skeletal muscle development and growth in mammals. Although several MSTN promoters were described in fish, no functional analysis was reported so far. Here, the 5′ flanking region (1372 bp) of the MSTN-1 gene of the marine fish Sparus aurata (saMSTN-1) was cloned, sequenced and characterized. It contains two consensus sequences for TATA box (TATAA), a CAAT box, ten putative E-boxes known as binding sites to myogenic basic helix–loop–helix transcription factors (TFs) and two putative binding sites to TF Myocyte enhancing factor-2 (MEF2). In addition, it has several putative binding sites to TF Pit-1a and several response elements to nuclear receptors (GRE, ERE, PRE, ARE, TRE, RARE and PPARRE) and cAMP-response elements. Transcriptional activity of five genomic fragments (truncated at their upstream region) of 372, 941, 972, 1113 and 1355 bp was studied in vitro, using transient transfection in A204 cells. All constructs directed luciferase activity, with the highest activity obtained by the 1113 bp fragment. These experiments show that all five genomic fragments are functional MSTN promoters and differences in promoter activity might be due to presence of enhancers and/or repressor sequences, regulating MSTN promoter activity.

Clenbuterol increases muscle fiber size and GATA‐2 protein in rat skeletal muscle in utero

Certain beta(2)-adrenoceptor agonists, such as clenbuterol, are known to elicit a muscle-specific anabolism or hypertrophy in both normal and catabolic muscle in a wide variety of species. However, the underlying mechanism(s) of the beta(2)-agonist-induced anabolism remains unclear. This study aimed to determine the effects of clenbuterol administration in utero on skeletal muscle and to examine the underlying molecular mechanisms. Pregnant rats were fed clenbuterol (2 mg/kg diet) from Day 4 of gestation (4 dg) until weanling and fetal samples were taken from 13.5, 15.5, 17.5, and 19.5 dg and from 1d neonatal pups. Muscles were analyzed for total DNA, RNA and protein and sections examined morphologically for changes in muscle development. Western and immunohistochemical analyses were performed to identify changes in known myogenic signaling proteins. Clenbuterol increased the size of both fast and slow fibers in utero which was associated with a decreased DNA:protein ratio (28%) and an increased RNA:DNA ratio (36%). Additionally, drug treatment in utero induced a decrease in the fast:slow fiber ratio (38%). These myogenic changes were correlated with an increase in the GATA-2 hypertrophic transcription factor at both 17.5 dg (by 250%) and 19.5 dg (by 40%) in fetuses from clenbuterol treated dams. In addition, drug treatment resulted in increased membrane association of PKC-micro at 17.5 dg (325%) and increased PKC-alpha cytosolic abundance (40%) and PKC-theta membrane abundance at 19.5 dg (250%). These results are the first demonstration that beta(2)-agonists such as clenbuterol may act through upregulating the GATA-2 transcription factor and implicate certain PKC isoforms in the drug-induced regulation of skeletal muscle development.

Molecular Cloning and Expression Analysis of the Myostatin Gene in Sea Perch (Lateolabrax japonicus)

Myostatin (MSTN) is a member of the transforming growth factor-beta (TGF-beta) superfamily that functions as a negative regulator of skeletal muscle development and growth in mammals. However, few reports are available about the structure and function of MSTN in teleost. Here, the MSTN gene was cloned from sea perch (Lateolabrax japonicus) by homology cloning and genomic walking. In the 4873-bp genomic sequence, three exons, two introns, and 5' and 3' flanking sequences were identified. The sea perch MSTN gene encodes a 374-amino acid protein, including a signal peptide, conserved cysteine residues, and a RXXR proteolytic cleavage domain. Expression analysis of MSTN revealed that MSTN was highly expressed in eyes, brain, and muscle; intermediately in intestine; and weakly in gill, spleen, liver, and heart. It was demonstrated that MSTN mRNA was highly expressed in embryonic stem cell line (LJES1), but it was undetectable in several types of somatic cell lines from sea perch, including fibroblast-like cell, epithelioid cell, and lymphocyte-like cell. Further, it was demonstrated that the 5' flanking region of the MSTN gene can drive the expression of green fluorescent protein (GFP) reporter gene in LJES1 cells and transgenic zebrafish (Danio rerio). This is the first report on the expression profile of MSTN gene in various types of cell cultures.

Analysis of a polymorphism within a TATA box of horse myostatin gene promoter in different breeds

Myostatin or growth and differentiation factor 8 (MSTN or GDF8, respectively) is a member of the transforming growth factor-β superfamily that acts as a negative regulator of skeletal muscle development and growth. The myostatin encoding gene (MSTN) and the 5’ regulative region in different species has been investigated. The MSTN gene consists of three exons and two intronic regions. In cattle, the gene has well-characterized mutations determining phenotypes with increased muscle mass. In a previous study, by alignment of nucleotide sequences of PCR products of horse MSTN gene, we identified some SNPs of which two T=C transitions in the promoter of the gene. The aim of this work is to study in different horse breeds the frequency of the polymorphism located 516 nt upstream of the ATG start codon. This SNP is within a TATA box sequence (NATAAAA, where N= T or C) and the comparative sequence analysis of mammals myostatin gene promoters reveals that this TATA-box motive is conserved in position and sequence among some species including cattle, sheep and goat. The point mutation is not recognised by restriction enzymes, then the genotyping was done by restriction site insertion-PCR. The primer pair was designed on the basis of the obtained nucleotide sequences. The reverse primer was modified and designed with a single nucleotide mismatched with respect to target sequence to introduces an SspI artificial restriction site when the T nucleotide occurred. The PCR products of 203 bp were digested and the resulting fragments were resolved on 3.5% agarose gel. Two hundred and six samples of DNA belonging to 18 horse breeds (Bardigiano, 20; Breton, 5; Criollo, 10; Esperia Pony, 4; Haflinger, 9; Italian Heavy Draught Horse, 24; Italian Saddle, 21; Italian Trotter, 16; Lipizzan, 11; Maremmano, 13; Murgese, 12; Norico, 10; Purebred Spanish Horse, 10; Salernitano, 12; San Fratellano, 3; Tolfetano, 7; Thoroughbred, 11 and Ventasso Horse, 8) were genotyped. The T allele occurs in all the breeds. The C allele, which eliminates TATA box motif, was detected in 11 breeds and its value frequency ranged from 0.006 (Thoroughbred) to 0.400 (Norico). The homozygous C/C genotype was detected in Haflinger, Bardigiano, Norico and Tolfetano breeds. The presence of the polymorphism within a conserved region of promoter of horse myostatin gene could affect expression gene. This marker could be used for association studies with performance traits in horses.

The ABCs of IGF-I isoforms: impact on muscle hypertrophy and implications for repair

Insulin-like growth factor I (IGF-I) plays a critical role in the growth and development of many tissues in the body. It is a key regulator of skeletal muscle development, and continues to enhance the ability for muscle to grow and undergo repair throughout life. Although the focus of research has been on the molecular actions and physiological impact of IGF-I, there has also been a growing undercurrent of studies geared toward the characterization of additional potentially active peptides produced by the igf1 gene. Alternative splicing of the gene results in multiple isoforms that retain the identical sequence for mature IGF-I, but also give rise to divergent C-terminal peptides. The peptides might modulate the actions, stability, or bioavailability of IGF-I, or they might have independent activity. These possibilities have gained the attention of the skeletal muscle field, where novel actions of IGF-I could have significant impact on muscle mass, strength, and repair.