Discovery of a Mammalian Splice Variant of Myostatin That Stimulates Myogenesis

Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C₂C₁₂ myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C₂C₁₂ myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over-expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P<0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product.     

Molecular Cloning and Characterization of the Myostatin Gene in Croceine Croaker, Pseudosciaena crocea

Myostatin (MSTN) is a negative regulator of skeletal muscle mass and has a potential application in aquaculture. We reported the characterization of the myostatin gene and its expression in the croceine croaker, Pseudosciaena crocea. The myostatin gene had three exons encoding 376 amino acids. The cDNA was 1,906 bp long with a 5′-UTR and 3′-UTR of 108 bp and 667 bp, respectively. A microsatellite sequence, CA₃₀ and CA₂₆ separated by TA, existed in the 3′-UTR. Intron I and II were 343 bp and 758 bp in length, respectively. The deduced amino acid sequence was highly conserved, and had more than 90% identical to shi drum, gilthead seabream, striped sea-bass, white perch, and white bass proteins. The myostatin of croceine croaker had a putative amino terminal signal sequence (residues 1–22), a transforming growth factor-beta (TGF-β) propeptide domain (residues 41–256), a RXXR proteolytic processing site (RARR, residues 264–267, matching the RXXR consensus site), and a TGF-β domain (residues 282–376). There were 13 conserved cysteine residues in croceine croaker myostatin, nine of which are common to all TGF-β superfamily members. The most conserved region of vertebrate myostatins is the TGF-β domain, which was the mature bioactive domain of the myostatin protein. The myostatin gene was expressed not only in the skeletal muscle, but also in the other tissues.     

IGF and myostatin pathways are respectively induced during the earlier and the later stages of skeletal muscle hypertrophy induced by clenbuterol,a β2‐adrenergic agonist

Clenbuterol, a β₂‐adrenergic agonist, increases the hypertrophy of skeletal muscle. Insulin‐like growth factor (IGF) is reported to work as a potent positive regulator in the clenbuterol‐induced hypertrophy of skeletal muscles. However, the precise regulatory mechanism for the hypertrophy of skeletal muscle induced by clenbuterol is unknown. Myostatin, a member of the TGFβ super family, is a negative regulator of muscle growth. The aim of the present study is to elucidate the function of myostatin and IGF in the hypertrophy of rat masseter muscle induced by clenbuterol. To investigate the function of myostatin and IGF in regulatory mechanism for the clenbuterol‐induced hypertrophy of skeletal muscles, we analysed the expression of myostatin and phosphorylation levels of myostatin and IGF signaling components in the masseter muscle of rat to which clenbuterol was orally administered for 21 days. Hypertrophy of the rat masseter muscle was induced between 3 and 14 days of oral administration of clenbuterol and was terminated at 21 days. The expression of myostatin and the phosphorylation of smad2/3 were elevated at 21 days. The phosphorylation of IGF receptor 1 (IGFR1) and akt1 was elevated at 3 and 7 days. These results suggest that myostatin functions as a negative regulator in the later stages in the hypertrophy of rat masseter muscle induced by clenbuterol, whereas IGF works as a positive regulator in the earlier stages. Copyright © 2012 John Wiley & Sons, Ltd.     

Administration of a mutated myostatin propeptide to neonatal mice significantly enhances skeletal muscle growth

Myostatin is a dominant inhibitor of skeletal muscle development and growth. As transgenic over‐expression of myostatin propeptide dramatically enhanced muscle mass, we hypothesized that administration of myostatin propeptide will increase muscle growth. In this study, the wild‐type form of porcine myostatin propeptide and its mutated form at the cleavage site of metalloproteinases of BMP‐1/TLD family were produced from insect cells. In vitro A204 cells reporter assays showed that both wild‐type and the mutated propeptides depressed myostatin activity. The recombinant propeptides at four‐fold myostatin concentration can effectively block myostatin function during co‐incubation with A204 cells. In particular, the mutated propeptide appeared much more effective than wild‐type propeptide over a long period during the in vitro co‐incubation. Administration of the mutated propeptide to neonatal mice at the age of 11 and 18 days was tested and showed significant increase in growth performance by 11–15% from the age of 25 to 57 days (P < 0.05). The major skeletal muscles of mice that were injected with mutated propeptide were 13.5–24.8% heavier than the control group (P < 0.05) as a result of muscle fiber hypertrophy. In conclusion, administration of the mutated myostatin propeptide during the neonatal period is an effective way for promoting muscle growth. Mol. Reprod. Dev. 77: 76–82, 2010. © 2009 Wiley‐Liss, Inc.     

Tertiary Windowing to Detect Positive Diversifying Selection

As a protein-encoding gene evolves, different selective pressures act on the gene temporally and spatially. An examination of the ratio of nonsynonymous-to-synonymous nucleotide substitution rate ratios (K_a/K_s) has proven to be a valuable method to examine selective pressures on protein encoding genes, including detecting positive diversifying selection. To gain power over averaging all sites in a gene together, examination of sites in primary sequence windows has frequently been employed. However, selection acts on folded proteins and sites that are close in tertiary space may not be close in primary sequence. A new method for the examination of K_a/K_s ratios based upon windows in tertiary structure is introduced and applied to the leptin gene family in mammals. Tertiary sequence windowing detects new sites under positive diversifying selection and detects positive diversifying selection with a more significant signal along various branches of the leptin gene family tree.Reviewing Editor: Dr. Rasmus Nielsen     

The single nucleotide polymorphisms of the chicken myostatin gene are associated with skeletal muscle and adipose growth

Myostatin, a new member of the TGF-ß superfamily, is predominantly expressed in skeletal muscle cells and functions as a negative regulator of skeletal muscle growth in animals. Recently, we have reported three single nucleotide polymorphisms (SNPs) in the chicken myostatin gene. Herein, we investigate the association of those SNPs with the production traits in a F₂ chicken line derived from Broilers crossing to Silky with the least square analysis. The results show that the BB and AA genotypes are strongly associated with abdominal fat weight (AFW), abdominal fat percentage (AFP), and birth weight (BW) (P < 0.05). Breast muscle percentage (BMP) of the AA type is higher than that of the AB type. The breast muscle weight and breast muscle percentages of F₂ individuals have significant difference between CC and DD genotypes (P < 0.05). Breast muscle weight (BMW) of EF birds is higher than that of EE birds (P < 0.05). In this report, we present the first genetic evidence to show that chicken myostatin not only plays an important role in controlling skeletal muscle growth and differentiation, but also may be involved in regulation of adipose growth in chicken.     

Preliminary Investigation into a Potential Role for Myostatin and Its Receptor (ActRIIB) in Lean and Obese Horses and Ponies

Obesity is a widespread problem across the leisure population of horses and ponies in industrialised nations. Skeletal muscle is a major contributor to whole body resting energy requirements and communicates with other tissues through the secretion of myokines into the circulation. Myostatin, a myokine and negative regulator of skeletal muscle mass, has been implicated in obesity development in other species. This study evaluated gene and protein expression of myostatin and its receptor, ActRIIB in adipose tissues and skeletal muscles and serum myostatin concentrations in six lean and six obese animals to explore putative associations between these factors and obesity in horses and ponies. Myostatin mRNA expression was increased while ActRIIB mRNA was decreased in skeletal muscles of obese animals but these differences were absent at the protein level. Myostatin mRNA was increased in crest fat of obese animals but neither myostatin nor ActRIIB proteins were detected in this tissue. Mean circulating myostatin concentrations were significantly higher in obese than in lean groups; 4.98 ng/ml (±2.71) and 9.00 ng/ml (±2.04) for the lean and obese groups, respectively. In addition, there was a significant positive association between these levels and myostatin gene expression in skeletal muscles (average R² = 0.58; p<0.05). Together, these results provide further basis for the speculation that myostatin and its receptor may play a role in obesity in horses and ponies.     

Single nucleotide polymorphisms in the upstream regulatory region alter the expression of myostatin

The expression of the gene encoding myostatin (MSTN), the product of which is a negative regulator of skeletal muscle growth and development in mammals, is regulated by many cis-regulatory elements, including enhancer box (E-box) motifs. While E-box motif mutants of MSTN exhibit altered expression of myostatin in many animal models, the phenotypes of these mutations in chicken are not investigated. In this study, we cloned and sequenced the full encoded DNA sequence of MSTN gene and its upstream promoter region in Wenshang Luhua chicken breed. After analysis of the sequence, 13 E-box motifs were identified in the MSTN promoter region, which were denoted by E1 to E13 according to their positions in the region. Although many single nucleotide polymorphisms (SNPs) were revealed in the MSTN promoter region, only two SNPs were in the E-boxes, i.e., the first nucleotide of the E3 and the fifth nucleotide of E4. The effects of these two polymorphisms on the expression of MSTN gene were explored both with MSTN-GFP reporter constructs in vitro and real-time PCR in vivo. The results suggested that the E-boxes in the chicken MSTN promoter region are involved in the regulation of myostatin expression and the polymorphisms in E3 and E4 altered the expression of myostatin.     

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.     

Sleeping Beauty-mediated knockdown of sheep myostatin by RNA interference

Myostatin is a negative regulator of skeletal muscle growth. Myostatin dysfunction therefore offers a strategy for promoting animal muscle growth in livestock production. Knockdown of myostatin was achieved by combining RNA interference and the Sleeping Beauty (SB) transposon system in sheep cells. Four targeting sites of sheep myostatin were designed and measured for myostatin silencing in sheep fetal fibroblasts by real-time PCR. The sh3 construct induced significant decrease of myostatin gene expression by 90% (P < 0.05). Myostatin silencing induced by SB-mediated sh3 was further tested in stably transfected cells. SB transposition increased the integration frequency of genes into sheep genomes and mediated a more efficient myostatin knockdown than random integration of sh3. We suggest that SB-mediated shRNA provides a novel potential tool for gene knockdown in the donor cells of animal cloning.     

Haploinsufficiency of myostatin protects against aging‐related declines in muscle function and enhances the longevity of mice

The molecular mechanisms behind aging-related declines in muscle function are not well understood, but the growth factor myostatin (MSTN) appears to play an important role in this process. Additionally, epidemiological studies have identified a positive correlation between skeletal muscle mass and longevity. Given the role of myostatin in regulating muscle size, and the correlation between muscle mass and longevity, we tested the hypotheses that the deficiency of myostatin would protect oldest-old mice (28–30 months old) from an aging-related loss in muscle size and contractility, and would extend the maximum lifespan of mice. We found that MSTN^+/− and MSTN^−/− mice were protected from aging-related declines in muscle mass and contractility. While no differences were detected between MSTN^+/+ and MSTN^−/− mice, MSTN^+/− mice had an approximately 15% increase in maximal lifespan. These results suggest that targeting myostatin may protect against aging-related changes in skeletal muscle and contribute to enhanced longevity.     

军曹鱼肌生成抑制素基因的克隆与序列分析

肌生成抑制素(Myostation,MSTN)是一种骨骼肌生长的负调控因子,其生物功能主要是抑制骨骼肌的生长。肌生成抑制素的活性降低或丧失,可使肌肉与其他组织的比例大大提高,因此在动物育种和医疗上有很大的潜在应用价值。目前包括鱼类在内的20多种脊椎动物的MSTN cDNA已经得到克隆和测序。本实验依据已知的鱼MSTN cDNA的保守区域设计一对特异引物,利用PCR技术分别从军曹鱼基因组中扩增出一个约1000bp的特异片段和300bp片段,所得目的片段回收纯化,将其酶切产物连接到pMDl8-T克隆载体上,转化入JM109感受态细胞中,挑取阳性克隆进行转化子鉴定,其质粒测序结果与文献报道的一致,证明成功地克隆了军曹鱼肌生成抑制素基因。     

MiR-27b promotes sheep skeletal muscle satellite cell proliferation by targeting <Emphasis Type="Italic">myostatin</Emphasis> gene

To investigate the role of miR-27b in sheep skeletal muscle development, here we first cloned the sequence of sheep pre-miR-27b, then further investigated its expression pattern in sheep skeletal muscle in vivo, the relationship of miR-27b expression and sheep skeletal muscle satellite cell proliferation and differentiation in vitro, and then finally confirmed its target gene during this development process. MiR-27b sequence, especially its mature sequence, was conservative among different species. MiR-27b highly expressed in sheep skeletal muscle than other tissues. In skeletal muscle of Suffolk and Bashbay sheep, miR-27b was upregulated during foetal period and downregulated during postnatal period significantly (\(P{<}0.01\)), but it still kept a relatively higher expression level in skeletal muscle of postnatal Suffolk sheep than Bashbay. There is a potential target site of miR-27b on \(3^\prime \)-UTR of sheep myostatin (MSTN) mRNA, and the double luciferase reporter assay proved that miR-27b could successfully bind on this site. When sheep satellite cells were in the proliferation status, miR-27b was upregulated and MSTN was downregulated significantly (\(P{<}0.01\)). When miR-27b mimics was transfected into sheep satellite cells, the cell proliferation was promoted and the protein level of MSTN was significantly downregulated (\(P{<}0.01\)). Moreover, miR-27b regulated its target gene MSTN by translation repression at an early step, and followed by inducing mRNA degradation in sheep satellite cells. Based on these results, we confirm that miR-27b could promote sheep skeletal muscle satellite cell proliferation by targeting MSTN and suppressing its expression.     

ATP-dependent potassium channels of muscle cells: Their properties,regulation, and possible functions

ATP-dependent potassium channels are present at high density in the membranes of heart, skeletal, and smooth muscle and have a lowP _open at physiological [ATP]_i. The unitary conductance is 15–20 pS at physiological [K⁺] _o , and the channels are highly selective for K⁺. Certain sulfonylureas are specific blockers, and some K channel openers may also act through these channels. K_ATP channels are probably regulated through the binding of ATP, which may in turn be regulated through changes in the ADP/ATP ratio or in pH_i. There is some evidence for control through G-proteins. The channels have complex kinetics, with multiple open and closed states. The main effect of ATP is to increase occupancy of long-lived closed states. The channels may have a role in the control of excitability and probably act as a route for K⁺ loss from muscle during activity. In arterial smooth muscle they may act as targets for vasodilators.     

Stable suppression of myostatin gene expression in goat fetal fibroblast cells by lentiviral vector‐mediated RNAi

Myostatin (MSTN) is a secreted growth factor that negatively regulates skeletal muscle mass, and therefore, strategies to block myostatin‐signaling pathway have been extensively pursued to increase the muscle mass in livestock. Here, we report a lentiviral vector‐based delivery of shRNA to disrupt myostatin expression into goat fetal fibroblasts (GFFs) that were commonly used as karyoplast donors in somatic‐cell nuclear transfer (SCNT) studies. Sh‐RNA positive cells were screened by puromycin selection. Using real‐time polymerase chain reaction (PCR), we demonstrated efficient knockdown of endogenous myostatin mRNA with 64% down‐regulation in sh2 shRNA‐treated GFF cells compared to GFF cells treated by control lentivirus without shRNA. Moreover, we have also demonstrated both the induction of interferon response and the expression of genes regulating myogenesis in GFF cells. The results indicate that myostatin‐targeting siRNA produced endogenously could efficiently down‐regulate myostatin expression. Therefore, targeted knockdown of the MSTN gene using lentivirus‐mediated shRNA transgenics would facilitate customized cell engineering, allowing potential use in the establishment of stable cell lines to produce genetically engineered animals. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:452–459, 2015