Growth/differentiation factor 8 (GDF8), or myostatin, negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-β. Using a combination of small-angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or "triggered" state where the prodomain remains in complex with the mature domain. However, these states are not reversible, indicating the latent GDF8 is "spring-loaded." Structural analysis shows that the prodomain:GDF8 complex adopts an "open" configuration, distinct from the latency state of TGF-β and more similar to the open state of Activin A and BMP9 (nonlatent complexes). We determined that GDF8 maintains similar features for latency, including the alpha-1 helix and fastener elements, and identified a series of mutations in the prodomain of GDF8 that alleviate latency, including I56E, which does not require activation by the protease Tolloid. In vivo, active GDF8 variants were potent negative regulators of muscle mass, compared with WT GDF8. Collectively, these results help characterize the latency and activation mechanisms of GDF8.

Recently, several promising treatments have emerged for neuromuscular disorders, highlighting the need for robust biomarkers for monitoring therapeutic efficacy and maintenance of the therapeutic effect. Several studies have proposed circulating and tissue biomarkers, but none of them has been validated to monitor acute and long-term drug response. We previously described how the myostatin (MSTN) level is naturally downregulated in several neuromuscular diseases, including Duchenne muscular dystrophy (DMD). Here, we show that the dystrophin-deficient Golden Retriever muscular dystrophy (GRMD) dog model also presents an intrinsic loss of Mstn production in muscle. The abnormally low levels of Mstn observed in the GRMD dog puppies at 2 months were partially rescued at both mRNA and protein level after adeno-associated virus (AAV)-microdystrophin treatment in a dose-dependent manner. These results show that circulating Mstn is a robust and reliable quantitative biomarker, capable of measuring a therapeutic response to pharmaco-gene therapy in real time in the neuromuscular system, as well as a quantitative means for non-invasive follow-up of a therapeutic effect. Moreover, a 2-year follow-up also suggests that Mstn could be a longitudinal monitoring tool to follow maintenance or decrease of the therapeutic effect.

Keywords: Duchenne muscular dystrophy (DMD); GDF8; biomarker; clinical trials; myostatin; neuromuscular disorders; therapy.

Targeting the signaling pathway of growth differentiation factor 8 (GDF8), also known as myostatin, has been regarded as a promising strategy to increase muscle mass in the elderly and in patients. Accumulating evidence in animal models and clinical trials has indicated that a rational approach is to inhibit a limited number of transforming growth factor β (TGF-β) family ligands, including GDF8 and activin A, without affecting other members. Here, we focused on one of the endogenous antagonists against TGF-β family ligands, follistatin-like 3 (FSTL3), which mainly binds and neutralizes activins, GDF8, and GDF11. Although bivalent human FSTL3 Fc-fusion protein was rapidly cleared from mouse circulation similar to follistatin (FST)-Fc, monovalent FSTL3-Fc (mono-FSTL3-Fc) generated with the knobs-into-holes technology exhibited longer serum half-life. Systemic administration of mono-FSTL3-Fc in mice induced muscle fiber hypertrophy and increased muscle mass in vivo. Our results indicate that the monovalent FSTL3-based therapy overcomes the difficulties of current anti-GDF8 therapies.

Keywords: Human metabolism; Musculoskeletal medicine; Physiology.

Clinical investigations have demonstrated that polycystic ovary syndrome (PCOS) is often accompanied by insulin resistance (IR) in more than 70% of women with PCOS. However, the etiology of PCOS with IR remains to be characterized. Growth differentiation factor 8 (GDF8) is an intraovarian factor that plays a vital role in the regulation of follicle development and ovulation. Previous studies have reported that GDF8 is a pathogenic factor in glucose metabolism disorder in IR patients. To date, the role of GDF8 on glucose metabolism of granulosa cell in PCOS patients remains to be determined. In the current study, we demonstrated that the expression and accumulation of GDF8 in human granulosa-lutein (hGL) cells and follicular fluid from PCOS patients were higher compared with those of non-PCOS women. GDF8 treatment caused glucose metabolism defects in hGL cells. Transcriptome sequencing results showed that SERPINE1 mediated GDF8-induced impairment of hGL glucose metabolism defects. Using pharmacological and small interfering RNA (siRNA)-mediated knockdown approaches, we demonstrated that GDF8 upregulated the expression of SERPINE1 via the ALK5-mediated SMAD2/3-SMAD4 signaling pathway. Interestingly, the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway was also activated with GDF8 treatment but did not participate in the effect of GDF8 on SERPINE1 expression. Our results also showed that TP53 was required for the GDF8-stimulated increase in SERPINE1 expression. Importantly, our study demonstrated that SB-431542 treatment significantly improved DHEA-induced PCOS-like ovaries. These findings support a potential role for GDF8 in metabolic disorders in PCOS.

Keywords: glucose metabolism; growth differentiation factor 8; human granulosa-lutein cells; polycystic ovary syndrome.

Growth differentiation factor 8 (GDF8) is a member of the transforming growth factor-β family and a physiological regulator. According to recent studies, GDF8 can be detected in follicular fluid and the uterus, suggesting that GDF8 may affect preimplantation embryonic development and act in a paracrine manner to improve the success of late-blastocyst implantation in vivo. We investigated the effect of GDF8 supplementation during in vitro culture (IVC) of porcine embryos derived from in vitro fertilization (IVF) and parthenogenetic activation (PA) on cleavage, blastocyst formation rate, and total cell number and analysed gene transcription levels and cell linage specification in the resulting blastocysts. First, the concentration of GDF8 in porcine oviductal fluid was determined to be 139.8 pg/mL. Then, 0, 0.2, 2, or 20 ng/mL GDF8 was added to embryos throughout the entire IVC period. Our results showed that supplementation with GDF8 during porcine preimplantation embryo IVC enhanced blastocyst formation and total cell number and altered the transcriptional patterns of genes that regulate pluripotency and cavitation. Furthermore, using differential immunostaining, we demonstrated that supplementation with GDF8 enhanced the expression of the genuine inner cell mass (ICM) marker SOX2 and the ICM/trophectoderm ratio, improving IVF blastocyst quality. In conclusion, for the first time, we demonstrated the presence of the in vivo oviductal factor GDF8 in oviductal fluid. Furthermore, we found that GDF8 supplementation at 0.2 ng/mL increased the blastocyst total cell number and ICM/trophectoderm ratio by inducing the transcription of genes involved in developmental competence and the expression of genuine ICM marker SOX2 during porcine IVF embryo development in vitro.

The growth and differentiation factor Myostatin (MSTN, also known as GDF8) negatively regulates skeletal muscle development and growth in vertebrates. Most fish genomes contain two or more mstn genes, which are expressed in muscle and other tissues. Yet, in the genome of Nile tilapia (Oreochromis niloticus), which is one of the world's most important aquaculture fish species, only one mstn gene has previously been identified. Here, we identify a second mstn gene in Nile tilapia. We show that it clusters phylogenetically with other piscine mstn2 genes and that it shares chromosomal synteny with the human and zebrafish orthologs. We further show that mstn2 is not expressed in red or white muscles of Nile tilapia, but rather that its main site of expression is the brain. To determine which physiological functions are correlated with mstn expression, adult Nile tilapia were exposed to various environmental conditions and their effect on mstn1 and mstn2 expression in the brain and muscles was measured using real-time PCR. We found that the centrally- and muscle-expressed mstn genes differ in their responsiveness to diverse challenges, suggesting differential gene- and tissue-specific regulation of their expression. Metabolic and stress marker analyses showed that the altered mstn expression is not regulated by classical stress response. Taken together, our findings expand the understanding of the MSTN system in Nile tilapia and provide evolutionary insight into its function.

Growth factors of the TGFβ superfamily play key roles in regulating neuronal and muscle function. Myostatin (or GDF8) and GDF11 are potent negative regulators of skeletal muscle mass. However, expression of myostatin and its cognate receptors in other tissues, including brain and peripheral nerves, suggests a potential wider biological role. Here, we show that Myoglianin (MYO), the Drosophila homolog of myostatin and GDF11, regulates not only body weight and muscle size, but also inhibits neuromuscular synapse strength and composition in a Smad2-dependent manner. Both myostatin and GDF11 affected synapse formation in isolated rat cortical neuron cultures, suggesting an effect on synaptogenesis beyond neuromuscular junctions. We also show that MYO acts in vivo to inhibit synaptic transmission between neurons in the escape response neural circuit of adult flies. Thus, these anti-myogenic proteins act as important inhibitors of synapse function and neuronal growth.

Circulating polypeptides and proteins have been implicated in reversing or accelerating aging phenotypes, including growth/differentiation factor 8 (GDF8), GDF11, eotaxin, and oxytocin. These proteoforms, which are defined as the protein products arising from a single gene due to alternative splicing and PTMs, have been challenging to study. Both GDF8 and GDF11 have known antagonists such as follistatin (FST), and WAP, Kazal, immunoglobulin, Kunitz, and NTR domain-containing proteins 1 and 2 (WFIKKN1, WFIKKN2). We developed a novel multiplexed SRM assay using LC-MS/MS to measure five proteins related to GDF8 and GDF11 signaling, and in addition, eotaxin, and oxytocin. Eighteen peptides consisting of 54 transitions were monitored and validated in pooled human plasma. In 24 adults, the mean (SD) concentrations (ng/mL) were as follows: GDF8 propeptide, 11.0 (2.4); GDF8 mature protein, 25.7 (8.0); GDF11 propeptide, 21.3 (10.9); GDF11 mature protein, 16.5 (12.4); FST, 29.8 (7.1); FST cleavage form FST303, 96.4 (69.2); WFIKKN1, 38.3 (8.3); WFIKKN2, 32.2 (10.5); oxytocin, 1.9 (0.9); and eotaxin, 2.3 (0.5). This novel multiplexed SRM assay should facilitate the study of the relationships of these proteoforms with major aging phenotypes.

Myostatin (also called as growth and differentiation factor 8 or GDF8), a member of the transforming growth factor β (TGF-β) superfamily of secreted differentiation and growth factors, is a potent inhibitor of skeletal muscle mass in mammals. Although myostatin also plays pivotal roles in cardiac growth and metabolism, postnatal glucose metabolism and adipogenesis, little information is available for myostatin function in the adult central nervous system (CNS). We, thus, investigated myostatin expression in the adult rat CNS using immunohistochemistry. Myostatin was intensely expressed in most neurons and their axons. Furthermore, we found that oligodendrocytes, astrocytes and ependymal cells also express myostatin protein. These data indicate that myostatin is widely expressed throughout the adult CNS, and its abundant expression in the adult brain suggests the idea that myostatin plays important roles in the CNS.

Myostatin (MSTN), also named growth differentiation factor 8 (GDF8), is a transforming growth factor-β (TGF-β) family member with a key role in the negative regulation of skeletal muscle growth. However, its role in ovarian folliculogenesis remains unclear. To provide us with a basis for understanding this role, we cloned MSTN and examined its expression patterns in water buffalo (Bubalus bubalis). The complete ORF of the water buffalo MSTN gene is 1,128 nucleotides, which encode a 375 amino acid protein and sharing 99% identity at the deducted amino acid level with that of Bos taurus. Protein sequence analysis showed that MSTN is a weakly acerbic extracellular protein, consisting of signal peptides at 18-19 sites, a TGF-β propeptide, and a TGF-β domain. RT-PCR analyses demonstrated that water buffalo MSTN was expressed in multiple tissues but not limited to muscle. Immunohistochemistry staining confirmed the presence of MSTN in oocytes and granulosal cells. To our knowledge, this is the first study to confirm the expression of MSTN in the water buffalo ovary, suggesting an additional role of MSTN in water buffalo folliculogenesis, along with its role in skeletal muscle growth regulation. Further study of the regulatory mechanism of MSTN in water buffalo reproduction is warranted.

BACKGROUND

MSTN, myostatin; ORF, open reading frame.

Wnt10a is a member of the WNT family. Although deficiency of this gene causes symptoms related to teeth, hair, nails, and skin, we recently demonstrated a new phenotype of Wnt10a knockout (KO) mice involving bone and fat. The in vivo effect of the Wnt10a gene on bone and fat is unclear, and the relationship between bone/fat and muscle in Wnt10a signaling is also interesting. We aimed to evaluate the tissue changes in Wnt10a KO mice compared to wild-type mice and show the findings as a starting point to unravel the underlying mechanisms of in vivo interactions involving Wnt10a in bone, fat and muscle. Trabecular bone loss in the lower limbs of Wnt10a mice and decreased bone mineralization were observed. The adipose tissue in bone marrow was also decreased, and adipocyte differentiation was reduced. The body fat mass in Wnt10a KO mice was decreased, and white adipocytes in subcutaneous fat were converted to beige adipocytes. The muscle weight of the lower limbs was not decreased despite trabecular bone loss, but Gdf8/myostatin expression was reduced in the subcutaneous fat and gastrocnemius muscles of Wnt10a KO mice. Thus, in vivo deletion of Wnt10a inhibited osteogenic activity, promoted beige adipogenesis of white adipocytes and maintained muscle mass. These results suggest that regulation of Gdf8 by Wnt10a may help maintain the muscle mass of Wnt10a KO mice. This study was the first to histologically evaluate the bone, fat and muscle phenotypes of Wnt10a KO mice. The results of this study, which were obtained by investigating the three tissues together, could influence the understanding of in vivo interactions involving the Wnt10a gene.

Myotubular myopathy, also called X-linked centronuclear myopathy (XL-CNM), is a severe congenital disease targeted for therapeutic trials. To date, biomarkers to monitor disease progression and therapy efficacy are lacking. The Mtm1 ^-/y mouse is a faithful model for XL-CNM, due to myotubularin 1 (MTM1) loss-of-function mutations. Using both an unbiased approach (RNA sequencing [RNA-seq]) and a directed approach (qRT-PCR and protein level), we identified decreased Mstn levels in Mtm1 ^-/y muscle, leading to low levels of myostatin in muscle and plasma. Myostatin (Mstn or growth differentiation factor 8 [Gdf8]) is a protein released by myocytes and inhibiting muscle growth and differentiation. Decreasing Dnm2 by genetic cross with Dnm2 ^+/- mice or by antisense oligonucleotides blocked or postponed disease progression and resulted in an increase in circulating myostatin. In addition, plasma myostatin levels inversely correlated with disease severity and with Dnm2 mRNA levels in muscles. Altered Mstn levels were associated with a generalized disruption of the myostatin pathway. Importantly, in two different forms of CNMs we identified reduced circulating myostatin levels in plasma from patients. This provides evidence of a blood-based biomarker that may be used to monitor disease state in XL-CNM mice and patients and supports monitoring circulating myostatin during clinical trials for myotubular myopathy.

Keywords: GDF8; MSTN; antisense oligonucleotides; biomarker; centronuclear myopathies; dynamin; myotubular myopathy; therapy.

Sheep chromosome 2q (OAR2q), which is homologous with human chromosome 2q (HSA2q), and cattle chromosome 2 (BTA2), is known to contain several loci contributing to carcass traits. However, the chromosomal rearrangements differentiating these chromosomes among the three species have not yet been determined and thus precise correspondences between the locations of sheep and human genes are not known. Twenty-six genes from HSA2q (2q21.1-->2q36) have been assigned to OAR2q by genetic linkage mapping to refine this area of the sheep genome. Seventy-six genes were initially selected from HSA2q. Sixty-eight percent of the PCR primer sets designed for these genes amplified successfully in sheep, and 34% amplified polymorphic products. Part of the proximal arm of OAR2q was found to be inverted compared with HSA2q. The breakpoint has been localised near the growth differentiation factor 8 gene (GDF8), spanning 380 kb between the positions of the hypothetical protein (FLJ20160) (HSA2:191008944-191075046) and glutaminase (GLS) (HSA2:191453847-191538510) (Build36.1).

The Wiedemann-Beckwith syndrome (WBS) was first described in 1963 as a group of anomalies involving primarily macrosomia, macroglossia, and omphalocele. Histologic studies of WBS show nesidioblastosis of the pancreas, adrenocortical cytomegaly, and persistent metanephric blastema of the kidney. Multiple lines of evidence indicate that the human 11p15.5 region is the locus of abnormality in WBS. Insulin-like growth factor II (IGF-2) frequently has been considered a candidate gene, and expression of IGF-2 is known to be significantly delayed in fetal skeletal muscle of double-muscle (DM) cattle. Other candidate genes recently have been proposed for WBS. A number of recessive alleles in the bovine myostatin gene (GDF8, mapped to bovine chromosome 2 and apparently orthologous to the human 2q22 region) have been shown to be responsible for DM. Recently the first human case of deficient GDF8 function has been reported, confirming the importance of this gene. Bovine IGF-2 has been sequenced and localized to chromosome 25. The primary purpose of this study was to compare and contrast histologic findings in DM and WBS. Immunohistochemical staining confirms changes similar to nesidioblastosis in the pancreas. Other dysplastic changes of a cystic nature are seen in the adrenal. The renal histology of DM fetuses did not appear significantly different than controls.

In animal breeding, microsatellite marker plays an important role in constructing genetic maps, QTL mapping and function analysis of structural genes. Myostatin, also known as GDF8, is a negative regulator of skeletal muscle mass and, in swine, it is evidenced to be related to birth weight and average daily gain from 60 kg to 100 kg of body weight. In present study, by subcloning and sequencing,we identified a novel microsatellite marker which is useful for fine QTL mapping for meat traits. A BAC clone containing porcine MSTN was extracted and digested with EcoR I to recover the fragment of>> 4 kb for subcloning in pGEM-3zf (+). Sequencing and alignment results showed that this subcloned fragment was not from porcine MSTN, but included a tandem repeat of (TG) 13, which is a novel microsatellite marker (GenBank accession number: AF454400) flanking MSTN. To exclude its vector origin we designed specific primers flanking this marker and successfully amplified this fragment from porcine genome. Through a pedigree analysis of a double-muscled Yorshire strain, we found that it is inherited in a co-dominant manner. We also checked the gene frequencies of this locus in 381 unrelated individuals of 7 pig breeds, namely Laiwu,Landrace, Yorkshire,Duroc, Peterian, Min and Erhualian. Only two alleles were detected, the repeating number of which are 13 (allele A) and 19 (allele B) respectively, which indicated that it is a low poly morphic microsatellite marker. In addition, the frequencies of the two alleles are different between the two types of pig breeds, while allele A is dominant in Chinese local breeds, allele B is dominant in imported breeds. Alignment with AY208121 indicate that this locus is located 42 kb downstream of porcine MSTN. We speculate that this microsatellite DNA is an important marker both in fine QTL mapping for meat traits and in the expression study of porcine MSTN.

Myostatin (MSTN) gene, also known as growth differentiation factor 8 (GDF8), is a member of the transforming growth factor-beta super-family and plays a negative role in muscle development. It acts as key points during pre- and post-natal life of amniotes that ultimately determine the overall muscle mass of animals. There are several studies that concentrate on the effect of a 5 bp insertion/deletion (indel) within the 5' untranslated region (5' UTR) of goat MSTN gene in goats. However, almost all sample sizes were below 150 individuals. Only in Boer goats, the sample sizes reached 482. Hence, whether the 5 bp indel was still associated with the growth traits of goats in large sample sizes which were more reliable is not clear. To find an effective and dependable DNA marker for goat rearing, we first enlarged the sample sizes (n = 1074, Shaanbei White Cashmere goat) which would enhance the robustness of the analysis and did the association analyses between the 5 bp indel and growth traits. Results uncovered that the 5 bp indel was significantly related to body height, height at hip cross, and chest width index (p < 0.05). In addition, individuals with DD genotype had a superior growing performance than those with the ID genotype. These findings suggested that the 5 bp indel in MSTN gene are significantly associated with growth traits and the specific genotype might be promising for maker-assisted selection (MAS) of goats.

Myostatin (or growth/differentiation factor 8 [GDF8]) is a member of the transforming growth factor β (TGF-β) superfamily of growth factors and negatively regulates skeletal muscle growth. Its dysregulation is implicated in muscle wasting diseases. SRK-015 is a clinical-stage monoclonal antibody that prevents extracellular proteolytic activation of pro- and latent myostatin. Here, we used integrated structural and biochemical approaches to elucidate the molecular mechanism of an antibody-mediated neutralization of pro-myostatin activation. The crystal structure of pro-myostatin in complex with 29H4-16 Fab, a high-affinity variant of SRK-015, at 2.79 Å resolution revealed that the antibody binds to a conformational epitope in the arm region of the prodomain distant from the proteolytic cleavage sites. This epitope is highly sequence divergent, sharing only limited similarity to other closely related members of the TGF-β superfamily. Hydrogen/deuterium exchange-MS experiments indicated that antibody binding induces conformational changes in pro- and latent myostatin that span the arm region, the loops contiguous to the protease cleavage sites, and the latency-associated structural elements. Moreover, negative-stain EM with full-length antibodies disclosed a stable, ring-like antigen-antibody structure in which the two Fab arms of a single antibody occupy the two arm regions of the prodomain in the pro- and latent myostatin homodimers, suggesting a 1:1 (antibody:myostatin homodimer) binding stoichiometry. These results suggest that SRK-015 binding stabilizes the latent conformation and limits the accessibility of protease cleavage sites within the prodomain. These findings shed light on approaches that specifically block the extracellular activation of growth factors by targeting their precursor forms.

Directed differentiation of stem cells plays a vital role in cell replacement therapy. Many activators and inhibitors targeting different signaling pathways have been identified to contribute to each step of differentiation. Most studies relied on empirically optimizing the combinations of the aforementioned factors for each step to optimize the efficiency of differentiation, which are time-consuming and nonsystematic. Design-of-experiment (DOE) is a powerful strategy to identify the critical combinations from multiple factors systematically. However, it is prohibitively complicated for typical laboratories, given a large number of potential combinations. Here, we develop a multilayer polymethyl methacrylate-based, reusable microfluidic chip to directly facilitate the DOE in the differentiation of stem cells. The chip consists of an inlet layer and multiple disperse layers. Different solutions are injected simultaneously to the chip through the inlet layer. Subsequently, the channels in the disperse layers split and recombine the flow streams to generate solution combinations based on hard-wired DOE designs. We demonstrated that it is in quantitative agreement with the designs using fluorescent dyes. Moreover, we constructed a human-induced pluripotent stem reporter cell line to improve the consistency of the cellular state measurements and use the chip to identify critical factors for cell differentiation to definitive endoderm (DE). We found that the differentiation efficiencies under various factor combinations are significantly different, and CHIR99201 and GDF8 are the most critical factors for differentiation to DE. Our method is potentially applicable to the optimization of factor combinations for multi-step stem cell differentiation and combinatorial drug screening.

Heparin and heparan sulfate (HS) are highly sulfated polysaccharides covalently bound to cell surface proteins, which directly interact with many extracellular proteins, including the transforming growth factor-β (TGFβ) family ligand antagonist, follistatin 288 (FS288). Follistatin neutralizes the TGFβ ligands, myostatin and activin A, by forming a nearly irreversible non-signaling complex by surrounding the ligand and preventing interaction with TGFβ receptors. The FS288-ligand complex has higher affinity than unbound FS288 for heparin/HS, which accelerates ligand internalization and lysosomal degradation; however, limited information is available for how FS288 interactions with heparin affect ligand binding. Using surface plasmon resonance (SPR) we show that preincubation of FS288 with heparin/HS significantly decreased the association kinetics for both myostatin and activin A with seemingly no effect on the dissociation rate. This observation is dependent on the heparin/HS chain length where small chain lengths less than degree of polymerization 10 (dp10) did not alter association rates but chain lengths >dp10 decreased association rates. In an attempt to understand the mechanism for this observation, we uncovered that heparin induced dimerization of follistatin. Consistent with our SPR results, we found that dimerization only occurs with heparin molecules >dp10. Small-angle X-ray scattering of the FS288 heparin complex supports that FS288 adopts a dimeric configuration that is similar to the FS288 dimer in the ligand-bound state. These results indicate that heparin mediates dimerization of FS288 in a chain-length-dependent manner that reduces the ligand association rate, but not the dissociation rate or antagonistic activity of FS288.

Keywords: Follistatin; activin; heparan sulfate; heparin; myostatin (GDF8); transforming growth factor beta.

Myostatin (MSTN) gene, also known as growth and differentiation factor 8 (GDF8) gene, is a negative regulator of skeletal muscle growth and development, especially the number, size and type of muscle fibers. Its mutations contribute to the double-muscling (DBM) phenomenon which significantly increases the muscle mass. Hence, variations within MSTN/GDF8 gene receive so much attention in several kinds of species such as bovines, poultries, goats, sheep, horses. A 5-base pairs (bp) indel in the 5' untranslated region (5'UTR) of goat MSTN/GDF8 was verified to be significantly associated with growth traits except Inner Mongolia White Cashmere (IMWC) goats. Given that almost all sample sizes were below 150, we enlarged sample sizes to more than 500 to uncover the association between the 5-bp indel and growth traits in IMWC goats. Only two genotypes (deletion/deletion (DD) and insertion/deletion (ID)) were found, and DD genotypes were dominant genotypes. The detected locus displayed low genetic diversity (PIC = 0.090). Interestingly, the association analyses revealed that the 5-bp indel had a significant effect on the chest depth (p = 0.003), and DD genotypes were dominant genotypes. Hinted that the 5-bp indel could act as an effective marker in molecular marker-assisted selection (MAS) processes for selection of excellent goat individuals.

The myostatin gene, known as Growth Differentiation Factor 8 (GDF8), located at chromosome 2 (BTA2) in cattle, is specifically expressed during embryo development and in the adult skeletal muscle. Molecular analysis of this gene reveals the presence of three exons and two introns. Several cattle breeds, such as Piedmontese, Belgian Blue, Blond'Aquitaine, among others, show polymorphisms in this gene, which are directly related to double muscling phenotype. Piedmontese cattle shows a nucleotide transition G ->> A (G938A) at exon 3, resulting in the substitution of cysteine to tyrosine, leading to a protein structure change, which determines myostatin inactivation and consequent muscular hypertrophy. The objective of this work was to implant the polymorphism G938A, naturally existent in Piedmontese breed, into in vitro propagated foetal myoblasts, from Nellore cattle. Single strand DNA (ssDNA) oligonucleotides were used to direct the same nucleotidic transition (G938A) to exon 3. Two transfection protocols (cationic lipid solution and electroporation) were tested and, 48 hours after transfection, RNA and DNA were extracted from myoblasts. Reverse transcription and polymerase chain reaction (PCR) were performed, using primers flanking the mutation region. The PCR products were cloned and analyzed by DNA sequencing, and it was possible to detect the nucleotidic CT transition at position 938, in the electroporated myoblasts. The existence of a positive signal in the transfection indicates that ssDNA oligonucleotides can be used to introduce this point mutation in specific functional gene sites.

Tumor necrosis factor alpha (TNF-α) plays a critical role in the progression of inflammation and affects the cells of the synovial membrane. Another key factor in the progression of rheumatoid inflammation is interleukin-6 (IL-6). Both TNF-α and IL-6 promote the proliferation of synovial membrane cells thus stimulating the production of matrix metalloproteinases and other cytotoxins and leading towards bone erosion and destruction of the cartilage. Growth differentiation factor-11 (GDF11) and growth differentiation factor-8 (GDF8) which is also known as myostatin are members of the transforming growth factor-β family and could be used as antagonists to inflammatory responses which are associated with rheumatoid arthritis. In the current study, to elucidate the evolutionary relationships of GDF11 with its homologs from other closely related organisms, a comprehensive phylogenetic analysis was performed. From the phylogram, it was revealed that the clade of Primates that belong to superorder Euarchontoglires showed close evolutionary relationships with order Cetartiodactyla of the Laurasiatheria superorder. Fifty tetrapeptides were devised from conserved regions of GDF11 which served as ligands in protein-ligand docking against TNF-α and IL-6 followed by drug scanning and ADMET profiling of best selected ligands. The peptides SAGP showed strong interactions with IL-6, and peptides AFDP and AGPC showed strong interactions with TNF-α, and all three peptides fulfilled all the pharmacokinetic parameters which are important for bioavailability. The potential of GDF8 as an antagonist to TNF-α and IL-6 was also explored using a protein-protein docking approach. The binding patterns of GDF8 with TNF-α and IL-6 showed that GDF8 could be used as a potential inhibitor of TNF-α and IL-6 to treat rheumatoid arthritis.

Growth differentiation factor 8 (GDF8), a.k.a. myostatin, is a member of the larger TGFβ superfamily of signaling ligands. GDF8 has been well characterized as a negative regulator of muscle mass. After synthesis, GDF8 is held latent by a noncovalent complex between the N-terminal prodomain and the signaling ligand. Activation of latent GDF8 requires proteolytic cleavage of the prodomain at residue D99 by a member of the tolloid family of metalloproteases. While tolloid proteases cleave multiple substrates, they lack a conserved consensus sequence. Here we investigate the tolloid cleavage site of the GDF8 prodomain to determine what residues contribute to tolloid recognition and subsequent proteolysis. Using sequential alanine mutations, we identified several residues adjacent to the scissile bond, including Y94, that when mutated, abolish tolloid-mediated activation of latent GDF8. Using the astacin domain of Tll1 (Tolloid Like 1) we determined that prodomain mutants were more resistant to proteolysis. Purified latent complexes harboring the prodomain mutations, D92A and Y94A, impeded activation by tolloid but could be fully activated under acidic conditions. Finally, we show that co-expression of GDF8 WT with prodomain mutants that were tolloid resistant, suppressed GDF8 activity. Taken together our data demonstrate that residues towards the N-terminus of the scissile bond are important for tolloid-mediated activation of GDF8 and that tolloid-resistant version of the GDF8 prodomain can function dominant negative to WT GDF8.

Keywords: GDF8; Latency; Procomplex; Tolloid; myogenesis; transforming growth factors.