Enhanced Muscle Fibers of Epinephelus coioides by Myostatin Autologous Nucleic Acid Vaccine.

Epinephelus coioides is a fish species with high economic value due to its delicious meat, high protein content, and rich fatty acid nutrition. It has become a high-economic fish in southern parts of China and some other Southeast Asian countries. In this study, the myostatin nucleic acid vaccine was constructed and used to immunize E. coioides. The results from body length and weight measurements indicated the myostatin nucleic acid vaccine promoted E. coioides growth performance by increasing muscle fiber size. The results from RT-qPCR analysis showed that myostatin nucleic acid vaccine upregulated the expression of myod, myog and p21 mRNA, downregulated the expression of smad3 and mrf4 mRNA. This preliminary study is the first report that explored the role of myostatin in E. coioides and showed positive effects of autologous nucleic acid vaccine on the muscle growth of E. coioides. Further experiments with increased numbers of animals and different doses are needed for its application to E. coiodes aquaculture production.

Preclinical Safety Assessment and Toxicokinetics of Apitegromab, an Antibody Targeting Proforms of Myostatin for the Treatment of Muscle-Atrophying Disease.

Myostatin is a negative regulator of skeletal muscle and has become a therapeutic target for muscle atrophying disorders. Although previous inhibitors of myostatin offered promising preclinical data, these therapies demonstrated a lack of specificity toward myostatin signaling and have shown limited success in the clinic. Apitegromab is a fully human, monoclonal antibody that binds to human promyostatin and latent myostatin with a high degree of specificity, without binding mature myostatin and other closely related growth factors. To support the clinical development of apitegromab, we present data from a comprehensive preclinical assessment of its pharmacology, pharmacokinetics, and safety across multiple species. In vitro studies confirmed the ability of apitegromab to inhibit the activation of promyostatin. Toxicology studies in monkeys for 4 weeks and in adult rats for up to 26 weeks showed that weekly intravenous administration of apitegromab achieved sustained serum exposure and target engagement and was well-tolerated, with no treatment-related adverse findings at the highest doses tested of up to 100 mg/kg and 300 mg/kg in monkeys and rats, respectively. Additionally, results from an 8-week juvenile rat study showed no adverse effects on any endpoint, including neurodevelopmental, motor, and reproductive outcomes at 300 mg/kg administered weekly IV. In summary, the nonclinical pharmacology, pharmacokinetic, and toxicology data demonstrate that apitegromab is a selective inhibitor of proforms of myostatin that does not exhibit toxicities observed with other myostatin pathway inhibitors. These data support the conduct of ongoing clinical studies of apitegromab in adult and pediatric patients with spinal muscular atrophy (SMA).

Novel myostatin-specific antibody enhances muscle strength in muscle disease models.

Myostatin, a member of the transforming growth factor-ß superfamily, is an attractive target for muscle disease therapy because of its role as a negative regulator of muscle growth and strength. Here, we describe a novel antibody therapeutic approach that maximizes the potential of myostatin-targeted therapy. We generated an antibody, GYM329, that specifically binds the latent form of myostatin and inhibits its activation. Additionally, via "sweeping antibody technology", GYM329 reduces or "sweeps" myostatin in the muscle and plasma. Compared with conventional anti-myostatin agents, GYM329 and its surrogate antibody exhibit superior muscle strength-improvement effects in three different mouse disease models. We also demonstrate that the superior efficacy of GYM329 is due to its myostatin specificity and sweeping capability. Furthermore, we show that a GYM329 surrogate increases muscle mass in normal cynomolgus monkeys without any obvious toxicity. Our findings indicate the potential of GYM329 to improve muscle strength in patients with muscular disorders.

Structural basis of specific inhibition of extracellular activation of pro- or latent myostatin by the monoclonal antibody SRK-015.

Myostatin (or growth/differentiation factor 8 (GDF8)) is a member of the transforming growth factor ß superfamily of growth factors and negatively regulates skeletal muscle growth. Its dysregulation is implicated in muscle wasting diseases. SRK-015 is a clinical-stage mAb that prevents extracellular proteolytic activation of pro- and latent myostatin. Here we used integrated structural and biochemical approaches to elucidate the molecular mechanism of 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, having only limited similarity to other closely related members of the transforming growth factor ß 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.

The roles of two myostatins and immune effects after inhibition in Qi river crucian carp (Carassius auratus).

Myostatin, through type I receptor (kinase 4, 5, ALK4/5), functions to participate in the immune system and negatively regulate muscle growth in mammals. However, the role of myostatin (mstn) in the immune system of teleosts is largely unknown. In a previous study, we cloned the mstn1 cDNA encoding myostatin in Qi river crucian carp (Carassius auratus). In the present study, we have cloned mstn2 cDNA, which was characterized and analyzed together with mstn1. Tissue distribution analysis showed that both mstn genes are expressed in numerous tissues, with mstn1 dominantly expressed in the muscle and brain, whereas mstn2 is mainly expressed in the brain. During embryogenesis, mstn1 and mstn2 exhibit different expression patterns. Both mstn1 and mstn2 expression increased stepwise in the brain at different developmental stages. Furthermore, both genes are differentially regulated during different periods of fasting/re-feeding. Following the exposure of C. auratus to polyI:C, lipopolysaccharide (LPS), and Aeromonas hydrophila, both genes were upregulated in different tissues, which indicated that they might be involved in the immune response against pathogenic invasion. Blocking the Mstn signal pathway with SB-431542 (a chemical inhibitor of ALK4/5) resulted in significantly increased body length and weight. However, the mortality of SB-431542-treated fish was higher after A. hydrophila challenge. Moreover, decreased expression of lysozymes (lyz), complement component 3 (c3), ß-defensin 3 (defb3), and interferon γ (ifnγ) were exhibited in treated fish, compared with the controls. Furthermore, the expression of nf-κb1, three pro-inflammatory cytokines (il1ß, il6, and tnfα), and inflammatory cytokines (il8 and il10) were significantly increased in both the SB-431542-treated group and the control after A. hydrophila infection, suggesting that the NF-κB pathway was not suppressed in the SB-431542-treated fish. Taken together, our data suggest that both mstn1 and mstn2 play important roles in early body development, muscle growth, and the immune system by acting downstream of the NF-κB signal pathway.

Biological scaffold-mediated delivery of myostatin inhibitor promotes a regenerative immune response in an animal model of Duchenne muscular dystrophy.

Recent studies have reported that the immune system significantly mediates skeletal muscle repair and regeneration. Additionally, biological scaffolds have been shown to play a role in polarizing the immune microenvironment toward pro-myogenic outcomes. Moreover, myostatin inhibitors are known to promote muscle regeneration and ameliorate fibrosis in animal models of Duchenne muscular dystrophy (DMD), a human disease characterized by chronic muscle degeneration. Biological scaffolds and myostatin inhibition can potentially influence immune-mediated regeneration in the dystrophic environment, but have not been evaluated together. Toward this end, here we created an injectable biological scaffold composed of hyaluronic acid and processed skeletal muscle extracellular matrix. This material formed a cytocompatible hydrogel at physiological temperatures in vitro When injected subfascially above the tibialis anterior muscles of both WT and dystrophic mdx-5Cv mice, a murine model of DMD, the hydrogel spreads across the entire muscle before completely degrading at 3 weeks in vivo We found that the hydrogel is associated with CD206+ pro-regenerative macrophage polarization and elevated anti-inflammatory cytokine expression in both WT and dystrophic mice. Co-injection of both hydrogel and myostatin inhibitor significantly increased FoxP3+ regulatory T cell modulation and Foxp3 gene expression in the scaffold immune microenvironment. Finally, delivery of myostatin inhibitor with the hydrogel increased its bioactivity in vivo, and transplantation of immortalized human myoblasts with the hydrogel promoted their survival in vivo This study identifies a key role for biological scaffolds and myostatin inhibitors in modulating a pro-regenerative immune microenvironment in dystrophic muscle.

A mouse anti-myostatin antibody increases muscle mass and improves muscle strength and contractility in the mdx mouse model of Duchenne muscular dystrophy and its humanized equivalent, domagrozumab (PF-06252616), increases muscle volume in cynomolgus monkeys.

BACKGROUND: The treatments currently approved for Duchenne muscular dystrophy (DMD), a progressive skeletal muscle wasting disease, address the needs of only a small proportion of patients resulting in an urgent need for therapies that benefit all patients regardless of the underlying mutation. Myostatin is a member of the transforming growth factor-ß (TGF-ß) family of ligands and is a negative regulator of skeletal muscle mass. Loss of myostatin has been shown to increase muscle mass and improve muscle function in both normal and dystrophic mice. Therefore, myostatin blockade via a specific antibody could ameliorate the muscle weakness in DMD patients by increasing skeletal muscle mass and function, thereby reducing patients' functional decline. METHODS: A murine anti-myostatin antibody, mRK35, and its humanized analog, domagrozumab, were developed and their ability to inhibit several TGB-ß ligands was measured using a cell-based Smad-activity reporter system. Normal and mdx mice were treated with mRK35 to examine the antibody's effect on body weight, lean mass, muscle weights, grip strength, ex vivo force production, and fiber size. The humanized analog (domagrozumab) was tested in non-human primates (NHPs) for changes in skeletal muscle mass and volume as well as target engagement via modulation of circulating myostatin. RESULTS: Both the murine and human antibodies are specific and potent inhibitors of myostatin and GDF11. mRK35 is able to increase body weight, lean mass, and muscle weights in normal mice. In mdx mice, mRK35 significantly increased body weight, muscle weights, grip strength, and ex vivo force production in the extensor digitorum longus (EDL) muscle. Further, tibialis anterior (TA) fiber size was significantly increased. NHPs treated with domagrozumab demonstrated a dose-dependent increase in lean mass and muscle volume and exhibited increased circulating levels of myostatin demonstrating target engagement. CONCLUSIONS: We demonstrated that the potent anti-myostatin antibody mRK35 and its clinical analog, domagrozumab, were able to induce muscle anabolic activity in both rodents, including the mdx mouse model of DMD, and non-human primates. A Phase 2, potentially registrational, clinical study with domagrozumab in DMD patients is currently underway.

Cardiokines as Modulators of Stress-Induced Cardiac Disorders.

Almost 30 years ago, the protein, atrial natriuretic peptide, was identified as a heart-secreted hormone that provides a peripheral signal from the myocardium that communicates to the rest of the organism to modify blood pressure and volume under conditions of heart failure. Since then, additional peripheral factors secreted by the heart, termed cardiokines, have been identified and shown to coordinate this interorgan cross talk. In addition to this interorgan communication, cardiokines also act in an autocrine/paracrine manner to play a role in intercellular communication within the myocardium. This review focuses on the roles of newly emerging cardiokines that are mainly increased in stress-induced cardiac diseases. The potential of these cardiokines as clinical biomarkers for diagnosis and prognosis of cardiac disorders is also discussed.

INVITED REVIEW: Inhibitors of myostatin as methods of enhancing muscle growth and development.

With the increasing demand for affordable, high-quality meat, livestock and poultry producers must continually find ways to maximize muscle growth in their animals without compromising palatability of the meat products. Muscle mass relies on myoblast proliferation during prenatal or prehatch stages and fiber hypertrophy through protein synthesis and nuclei donation by satellite cells after birth or hatch. Therefore, understanding the cellular and molecular mechanisms of myogenesis and muscle development is of great interest. Myostatin is a well-known negative regulator of muscle growth and development that inhibits proliferation and differentiation in myogenic cells as well as protein synthesis in existing muscle fibers. In this review, various inhibitors of myostatin activity or signaling are examined that may be used in animal agriculture for enhancing muscle growth. Myostatin inhibitors are relevant as potential therapies for muscle-wasting diseases and muscle weakness in humans and animals. Currently, there are no commercial myostatin inhibitors for agriculture or biomedical purposes because the safest and most effective option has yet to be identified. Further investigation of myostatin inhibitors and administration strategies may revolutionize animal production and the medical field.

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody.

Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted anti-myostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall "humanness" was increased for both the light and heavy chain variable regions.

Oral administration of myostatin-specific recombinant Saccharomyces cerevisiae vaccine in rabbit.

Yeast is considered as a simple and cost-effective host for protein expression, and our previous studies have proved that Saccharomyces cerevisiae can deliver recombinant protein and DNA into mouse dendritic cells and can further induce immune responses as novel vaccines. In order to know whether similar immune responses can be induced in rabbit by oral administration of such recombinant S. cerevisiae vaccine, we orally fed the rabbits with heat-inactivated myostatin-recombinant S. cerevisiae for 5 weeks, and then myostatin-specific antibody in serum was detected successfully by western blotting and ELISA assay. The rabbits treated with myostatin-recombinant S. cerevisiae vaccine grew faster and their muscles were much heavier than that of the control group. As a common experimental animal and a meat livestock with great economic value, rabbit was proved to be the second animal species that have been successfully orally immunized by recombinant S. cerevisiae vaccine after mice.

Anti-myostatin antibody increases muscle mass and strength and improves insulin sensitivity in old mice.

Sarcopenia, or skeletal muscle atrophy, is a debilitating comorbidity of many physiological and pathophysiological processes, including normal aging. There are no approved therapies for sarcopenia, but the antihypertrophic myokine myostatin is a potential therapeutic target. Here, we show that treatment of young and old mice with an anti-myostatin antibody (ATA 842) for 4 wk increased muscle mass and muscle strength in both groups. Furthermore, ATA 842 treatment also increased insulin-stimulated whole body glucose metabolism in old mice, which could be attributed to increased insulin-stimulated skeletal muscle glucose uptake as measured by a hyperinsulinemic-euglycemic clamp. Taken together, these studies provide support for pharmacological inhibition of myostatin as a potential therapeutic approach for age-related sarcopenia and metabolic disease.

TALENs-mediated gene disruption of myostatin produces a larger phenotype of medaka with an apparently compromised immune system.

Although myostatin, a suppressor of skeletal muscle development and growth, has been well studied in mammals, its function in fish remains unclear. In this study, we used a popular genome editing tool with high efficiency and target specificity (TALENs; transcription activator-like effector nucleases) to mutate the genome sequence of myostatin (MSTN) in medaka (Oryzias latipes). After the TALEN pair targeting OlMyostatin was injected into fertilized medaka eggs, mutant G0 fish carrying different TALENs-induced frameshifts in the OlMSTN coding sequence were mated together in order to transmit the mutant sequences to the F1 generation. Two F1 mutants with frameshifted myostatin alleles were then mated to produce the F2 generation, and these F2 OlMSTN null (MSTN(-/-)) medaka were evaluated for growth performance. The F2 fish showed significantly increased body length and weight compared to the wild type fish at the juvenile and post-juvenile stages. At the post-juvenile stage, the average body weight of the MSTN(-/-) medaka was ∼25% greater than the wild type. However, we also found that when the F3 generation were challenged with red spotted grouper nervous necrosis virus (RGNNV), the expression levels of the interferon-stimulated genes were lower than in the wild type, and the virus copy number was maintained at a high level. We therefore conclude that although the MSTN(-/-) medaka had a larger phenotype, their immune system appeared to be at least partially suppressed or undeveloped.

Myostatin inhibitors in sports drug testing: Detection of myostatin-neutralizing antibodies in plasma/serum by affinity purification and Western blotting.

PURPOSE: Myostatin is a key regulator of skeletal muscle growth and inhibition of its signaling pathway results in an increased muscle mass and function. The aim of this study was to develop a qualitative detection assay for myostatin-neutralizing antibodies for doping control purposes by using immunological approaches. EXPERIMENTAL DESIGN: To detect different types of myostatin-neutralizing antibodies irrespective of their amino acid sequence, an immunological assay specific for antibodies directed against myostatin and having a human Fc domain was established. Affinity purification and Western blotting strategies were combined to allow extracting and identifying relevant analytes from 200 µL of plasma/serum in a non-targeted approach. The assay was characterized regarding specificity, linearity, precision, robustness, and recovery. RESULTS: The assay was found to be highly specific, robust, and linear from 0.1 to 1 µg/mL. The precision was successfully specified at three different concentrations and the recovery of the affinity purification was 58%. CONCLUSIONS AND CLINICAL RELEVANCE: Within this study, an immunological detection assay for myostatin-neutralizing antibodies present in plasma/serum specimens was developed and successfully characterized. The presented approach can easily be modified to include other therapeutic antibodies and serves as proof-of-concept for the detection of antibody-based myostatin inhibitors in doping control samples.

Myostatin antibody (LY2495655) in older weak fallers: a proof-of-concept, randomised, phase 2 trial.

BACKGROUND: Myostatin inhibits skeletal muscle growth. The humanised monoclonal antibody LY2495655 (LY) binds and neutralises myostatin. We aimed to test whether LY increases appendicular lean body mass (aLBM) and improves physical performance in older individuals who have had recent falls and low muscle strength and power. METHODS: In this proof-of-concept, randomised, placebo-controlled, double-blind, parallel, multicentre, phase 2 study, we recruited patients aged 75 years or older who had fallen in the past year from 21 investigator sites across Argentina, Australia, France, Germany, Sweden, and the USA. Eligible patients had low performance on hand grip strength and chair rise tests, tested with the procedure described by Guralnik and colleagues. Participants were stratified by country, age, hand grip strength, and performance on the chair rise test, and were randomly assigned (1:1) by a computer-generated random sequence to receive subcutaneous injections of placebo or 315 mg LY at weeks 0 (randomisation visit), 4, 8, 12, 16, and 20, followed by 16 weeks observation. The primary outcome was change in aLBM from baseline to 24 weeks. We measured physical performance as secondary outcomes (four-step stair climbing time, usual gait speed, and time to rise five times from a chair without arms, or with arms for participants unable to do it without arms) and exploratory outcomes (12-step stair climbing test, 6-min walking distance, fast gait speed, hand grip strength, and isometric leg extension strength). Efficacy analyses included all randomly assigned patients who received at least one dose and had a baseline and at least one subsequent measure. The primary analysis and all other tests of treatment effect (except physical performance tests) were done at a two-sided alpha level of 0·05. Tests of treatment effect on physical performance tests were done at a pre-specified two-sided alpha level of 0·1. This trial is registered with ClinicalTrials.gov, number NCT01604408. FINDINGS: Between June 19, 2012, and Dec 12, 2013, we screened 365 patients. 99 were randomly assigned to receive placebo and 102 to receive LY. Treatment was completed in 85 (86%) of patients given placebo and in 82 (80%) given LY. At 24 weeks, the least-squares mean change in aLBM was -0·123 kg (95% CI -0·287 to 0·040) in the placebo group and 0·303 kg (0·135 to 0·470) in the LY group, a difference of 0·43 kg (95% CI 0·192 to 0·660; p<0·0001). Stair climbing time (four-step and 12-step tests), chair rise with arms, and fast gait speed improved significantly from baseline to week 24 with differences between LY and placebo of respectively -0·46 s (p=0·093), -1·28 s (p=0·011), -4·15 s (p=0·054), and 0·05 m/s (p=0·088). No effect was detected for other performance-based measures. Injection site reactions were recorded in nine (9%) patients given placebo and in 31 (30%) patients given LY (p<0·0001), and were generally mild, and led to treatment discontinuation in two patients given LY. INTERPRETATION: Our findings show LY treatment increases lean mass and might improve functional measures of muscle power. Although additional studies are needed to confirm these results, our data suggest LY should be tested for its potential ability to reduce the risk of falls or physical dependency in older weak fallers. FUNDING: Eli Lilly and Company.

The administration of Fructus Schisandrae attenuates dexamethasone-induced muscle atrophy in mice.

In the present study, we aimed to determine whether ethanol extracts of Fructus Schisandrae (FS), the dried fruit of Schizandra chinensis Baillon, mitigates the development of dexamethasone-induced muscle atrophy. Adult SPF/VAT outbred CrljOri:CD1 (ICR) mice were either treated with dexamethasone to induce muscle atrophy. Some mice were treated with various concentrations of FS or oxymetholone, a 17α-alkylated anabolic-androgenic steroid. Muscle thickness and weight, calf muscle strength, and serum creatine and creatine kinase (CK) levels were then measured. The administration of FS attenuated the decrease in calf thickness, gastrocnemius muscle thickness, muscle strength and weight, fiber diameter and serum lactate dehydrogenase levels in the gastrocnemius muscle bundles which was induced by dexamethasone in a dose-dependent manner. Treatment with FS also prevented the dexamethasone-induced increase in serum creatine and creatine kinase levels, histopathological muscle fiber microvacuolation and fibrosis, and the immunoreactivity of muscle fibers for nitrotyrosine, 4-hydroxynonenal, inducible nitric oxide synthase and myostatin. In addition, the destruction of the gastrocnemius antioxidant defense system was also inhibited by the administration of FS in a dose-dependent manner. FS downregulated the mRNA expression of atrogin-1 and muscle ring-finger protein-1 (involved in muscle protein degradation), myostatin (a potent negative regulator of muscle growth) and sirtuin 1 (a representative inhibitor of muscle regeneration), but upregulated the mRNA expression of phosphatidylinositol 3-kinase, Akt1, adenosine A1 receptor and transient receptor potential cation channel subfamily V member 4, involved in muscle growth and the activation of protein synthesis. The overall effects of treatment with 500 mg/kg FS were comparable to those observed following treatment with 50 mg/kg oxymetholone. The results from the present study support the hypothesis that FS has a favorable ameliorating effect on muscle atrophy induced by dexamethasone, by exerting anti-inflammatory and antioxidant effects on muscle fibers, which may be due to an increase in protein synthesis and a decrease in protein degradation.

Myostatin Neutralization Results in Preservation of Muscle Mass and Strength in Preclinical Models of Tumor-Induced Muscle Wasting.

Skeletal muscle wasting occurs in a great majority of cancer patients with advanced disease and is associated with a poor prognosis and decreased survival. Myostatin functions as a negative regulator of skeletal muscle mass and has recently become a therapeutic target for reducing the loss of skeletal muscle and strength associated with clinical myopathies. We generated neutralizing antibodies to myostatin to test their potential use as therapeutic agents to attenuate the skeletal muscle wasting due to cancer. We show that our neutralizing antimyostatin antibodies significantly increase body weight, skeletal muscle mass, and strength in non-tumor-bearing mice with a concomitant increase in mean myofiber area. The administration of these neutralizing antibodies in two preclinical models of cancer-induced muscle wasting (C26 colon adenocarcinoma and PC3 prostate carcinoma) resulted in a significant attenuation of the loss of muscle mass and strength with no effect on tumor growth. We also show that the skeletal muscle mass- and strength-preserving effect of the antibodies is not affected by the coadministration of gemcitabine, a common chemotherapeutic agent, in both non-tumor-bearing mice and mice bearing C26 tumors. In addition, we show that myostatin neutralization with these antibodies results in the preservation of skeletal muscle mass following reduced caloric intake, a common comorbidity associated with advanced cancer. Our findings support the use of neutralizing antimyostatin antibodies as potential therapeutics for cancer-induced muscle wasting.

Expression of Myostatin in Intrauterine Growth Restriction and Preeclampsia Complicated Pregnancies and Alterations to Cytokine Production by First-Trimester Placental Explants Following Myostatin Treatment.

Preeclampsia (PE) and intrauterine growth restriction (IUGR) are major obstetric health problems. Higher levels of T-helper (Th) 1 (proinflammatory) cytokines have been observed in pregnancies complicated with PE and IUGR; this is in contrast to the predominant Th2 (anti-inflammatory) cytokine environment found in uncomplicated pregnancies. Myostatin is best known as a negative regulator of muscle development and reportedly has a role in fat deposition, glucose metabolism, and cytokine modulation (outside the placenta). Myostatin concentrations in plasma and protein expression in placental tissue are significantly higher in women with PE. Expression of myostatin in IUGR and PE-IUGR and the effect of this protein on the cytokine production from the placenta is unknown. In the current study, significant differences were identified in the expression of myostatin in pregnancies complicated with IUGR, PE, and PE with IUGR. Furthermore, cytokine production by first-trimester placental tissues was altered following myostatin treatment.

A prepared anti-MSTN polyclonal antibody reverses insulin resistance of diet-induced obese rats via regulation of PI3K/Akt/mTOR&FoxO1 signal pathways.

Suppression of myostatin (MSTN) is associated with skeletal muscle atrophy and insulin resistance. However, the mechanisms by which MSTN regulates insulin resistance are not well known. We have explored the signaling pathways through which MSTN regulates insulin resistance in diet-induced obese rats using a polyclonal antibody for MSTN. The anti-MSTN polyclonal antibody significantly improved insulin resistance and whole-body insulin sensitivity, decreased MSTN protein expression in muscle samples by 39% in diet-induced obese rats. Furthermore, the anti-MSTN polyclonal antibody significantly enhanced PI3K activity (140%), Akt phosphorylation (86%), GLUT4 protein expression (23%), the phosphorylation of mTOR (21%), and inhibited the phosphorylation of FoxO1 (57%), but did not affect the phosphorylation of GSK-3ß. Thus, suppression of MSTN by the anti-MSTN polyclonal antibody reverses insulin resistance of diet-induced obesity via MSTN/PI3K/Akt/mTOR and MSTN/PI3K/Akt/FoxO1 signaling pathways.

Construction, expression and characterization of a single chain variable fragment antibody against human myostatin.

Myostatin plays negative roles in muscle development. To block the inhibitory effects of myostatin on myogenesis, a 759 bp single chain variable fragment antibody (scFv) against myostatin was constructed and expressed in Escherichia coli. ELISA detection showed that the scFv could bind to myostatin, and change of the scFv N-terminal peptides decreased its binding affinity. MTT assay and cell morphology demonstrated that the cell number and viability of the C2C12 myoblast were enhanced by the scFv. Meanwhile, the scFv significantly inhibited the myostatin-induced expression of cyclin-dependent kinase inhibitor p21 and Smad binding element-luciferase activity. H2O2 increased the expression of Muscle RING Finger 1 (MuRF1) and Muscle Atrophy F-box (MAFbx) in myoblasts as well as myostatin and MuRF1 in myotubes, and the scFv significantly decreased the H2O2-elevated expression of these genes. Conclusively, the scFv we developed could antagonize the inhibitory effects of myostatin on myogenesis through Smad pathway and regulation of p21, MuRF1 and MAFbx gene expression. The scFv may have application in the therapy of muscular dystrophy and improvement of animal meat production.