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Identification of the minimum region of flatfish myostatin propeptide (Pep45-65) for myostatin inhibition and its potential to enhance muscle growth and performance in animals.

ABSTRACT: Myostatin (MSTN) negatively regulates skeletal muscle growth, and its activity is inhibited by the binding of MSTN propeptide (MSTNpro), the N-terminal domain of proMSTN that is proteolytically cleaved from the proMSTN. Partial sequences from the N-terminal side of MSTNpro have shown to be sufficient to inhibit MSTN activity. In this study, to determine the minimum size of flatfish MSTNpro for MSTN inhibition, various truncated forms of flatfish MSTNpro with N-terminal maltose binding protein (MBP) fusion were expressed in E. coli and purified. MSTNpro regions consisting of residues 45-68, -69, and -70 with MBP fusion suppressed MSTN activity with a potency comparable to that of full-sequence flatfish MSTNpro in a pGL3-(CAGA)12-luciferase reporter assay. Even though the MSTN-inhibitory potency was about 1,000-fold lower, the flatfish MSTNpro region containing residues 45-65 (MBP-Pro45-65) showed MSTN-inhibitory capacity but not the MBP-Pro45-64, indicating that the region 45-65 is the minimum domain required for MSTN binding and suppression of its activity. To examine the in vivo effect of MBP-fused, truncated flatfish MSTNpro, MBP-Pro45-70-His6 (20 mg/kg body wt) was subcutaneously injected 5 times for 14 days in mice. Body wt gain and bone mass were not affected by the administration. Grip strength and swimming time were significantly enhanced at 7 d after the administration. At 14 d, the effect on grip strength disappeared, and the extent of the effect on swimming time significantly diminished. The presence of antibody against MBP-Pro45-70-His6 was observed at both 7 and 14 d after the administration with the titer value at 14 d being much greater than that at 7 d, suggesting that antibodies against MBP-Pro45-70-His6 neutralized the MSTN-inhibitory effect of MBP-Pro45-70-His6. We, thus, examined the MSTN-inhibitory capacity and in vivo effect of flatfish MSTNpro region 45-65 peptide (Pep45-65-NH2), which was predicted to have no immunogenicity in silico analysis. Pep45-65-NH2 suppressed MSTN activity with a potency similar to that of MBP-Pro45-65 but did not suppress GDF11, or activin A. Pep45-65-NH2 blocked MSTN-induced Smad2 phosphorylation in HepG2 cells. The administration of Pep45-65 (20 mg/kg body wt, 5 times for 2 weeks) increased the body wt gain with a greater gain at 14 d than at 7 d and muscle wt. Grip strength and swimming time were also significantly enhanced by the administration. Antibody titer against Pep45-65 was not detected. In conclusion, current results indicate that MSTN-inhibitory proteins with heterologous fusion partner may not be effective in suppressing MSTN activity in vivo due to an immune response against the proteins. Current results also show that the region of flatfish MSTNpro consisting of 45-65 (Pep45-65) can suppress mouse MSTN activity and increase muscle mass and function without invoking an immune response, implying that Pep45-65 would be a potential agent to enhance skeletal muscle growth and function in animals or to treat muscle atrophy caused by various clinical conditions.

SUBMITTER: Kim JH

PROVIDER: S-EPMC6472743 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Identification of the minimum region of flatfish myostatin propeptide (Pep45-65) for myostatin inhibition and its potential to enhance muscle growth and performance in animals.

Kim Jeong Hwan JH Kim Jeong Han JH Sutikno Lisa Andriani LA Lee Sang Beum SB Jin Deuk-Hee DH Hong Yong-Ki YK Kim Yong Soo YS Jin Hyung-Joo HJ

PloS one 20190418 4

Myostatin (MSTN) negatively regulates skeletal muscle growth, and its activity is inhibited by the binding of MSTN propeptide (MSTNpro), the N-terminal domain of proMSTN that is proteolytically cleaved from the proMSTN. Partial sequences from the N-terminal side of MSTNpro have shown to be sufficient to inhibit MSTN activity. In this study, to determine the minimum size of flatfish MSTNpro for MSTN inhibition, various truncated forms of flatfish MSTNpro with N-terminal maltose binding protein (M ...[more]

PMID: 30998775

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Project description:Eccentric contractions produce a significant degree of inflammation and muscle injury that may increase the expression of myostatin. Due to its anti- oxidant and anti-flammatory effects, circulating 17-? estradiol (E2) may attenuate myostatin expression. Eight males and eight females performed 7 sets of 10 reps of eccentric contractions of the knee extensors at 150% 1-RM. Each female performed the eccentric exercise bout on a day that fell within her mid-luteal phase (d 21-23 of her 28-d cycle). Blood and muscle samples were obtained before and 6 and 24 h after exercise, while additional blood samples were obtained at 48 and 72 h after exercise. Serum E2 and myostatin LAP/propeptide (LAP/pro) levels were determined with ELISA, and myostatin mRNA expression determined using RT-PCR. Data were analyzed with two-way ANOVA and bivariate correlations (p < 0.05). Females had greater levels of serum E2 throughout the 72- h sampling period (p < 0.05). While males had greater body mass and fat-free mass, neither was correlated to the pre-exercise levels of myostatin mRNA and LAP/pro for either gender (p > 0.05). Compared to pre-exercise, males had significant increases (p < 0.05) in LAP/propetide and mRNA of 78% and 28%, respectively, at 24 h post-exercise, whereas females underwent respective decreases of 10% and 21%. E2 and LAP/propeptide were correlated at 6 h (r = -0.804, p = 0.016) and 24 h post- exercise (r = -0.841, p = 0.009) in males, whereas in females E2 levels were correlated to myostatin mRNA at 6 h (r =0.739, p = 0.036) and 24 h (r = 0.813, p = 0.014) post-exercise and LAP/propeptide at 6 h (r = 0.713, p = 0.047) and 24 h (r = 0.735, p = 0.038). In females, myostatin mRNA expression and serum LAP/propeptide levels do not appear to be significantly up-regulated following eccentric exercise, and may be due to higher levels of circulating E2. Key PointsThe pre-exercise levels of myostatin mRNA and propeptide were not significantly different between genders, and even though the total body mass and fat-free mass of males were significantly greater than females, neither was correlated to myostatin mRNA or LAP/propeptide.Myostatin mRNA expression in females is less than in males 24 h after a single bout of eccentric exercise.Myostatin LAP/propeptide levels in females are lower in females than in males 24 h after a single bout of eccentric exercise, thereby suggesting a gender-specific mechanism in which females may be less responsive to eccentric exercise than males.Myostatin mRNA expression in females is attenuated, possibly due to inhibition in myostatin signaling, and appears to be more related to the presence of a higher level of circulating E2 rather than body composition.Due to their higher level of E2, females seem to be less susceptible to the mechanism by which eccentric exercise apparently up-regulates myostatin mRNA expression in males.

Project description:BackgroundGrowth differentiation factor 11 (GDF11) is a member of the transforming growth factor β superfamily. The GDF11 propeptide, which is derived from the GDF11 precursor protein, blocks the activity of GDF11 and its homolog, myostatin, which are both potent inhibitors of muscle growth. Thus, treatment with GDF11 propeptide may be a potential therapeutic strategy for diseases associated with muscle atrophy like sarcopenia and the muscular dystrophies. Here, we evaluate the impact of GDF11 propeptide-Fc (GDF11PRO-Fc) gene delivery on skeletal muscle in normal and dystrophic adult mice.MethodsA pull-down assay was used to obtain physical confirmation of a protein-protein interaction between GDF11PRO-Fc and GDF11 or myostatin. Next, differentiated C2C12 myotubes were treated with AAV6-GDF11PRO-Fc and challenged with GDF11 or myostatin to determine if GDF11PRO-Fc could block GDF11/myostatin-induced myotube atrophy. Localized expression of GDF11PRO-Fc was evaluated via a unilateral intramuscular injection of AAV9-GDF11PRO-Fc into the hindlimb of C57BL/6J mice. In mdx mice, intravenous injection of AAV9-GDF11PRO-Fc was used to achieve systemic expression. The impact of GDF11PRO-Fc on muscle mass, function, and pathological features were assessed.ResultsGDF11PRO-Fc was observed to bind both GDF11 and myostatin. In C2C12 myotubes, expression of GDF11PRO-Fc was able to mitigate GDF11/myostatin-induced atrophy. Following intramuscular injection in C57BL/6J mice, increased grip strength and localized muscle hypertrophy were observed in the injected hindlimb after 10 weeks. In mdx mice, systemic expression of GDF11PRO-Fc resulted in skeletal muscle hypertrophy without a significant change in cardiac mass after 12 weeks. In addition, grip strength and rotarod latency time were improved. Intramuscular fibrosis was also reduced in treated mdx mice; however, there was no change seen in central nucleation, membrane permeability to serum IgG or serum creatine kinase levels.ConclusionsGDF11PRO-Fc induces skeletal muscle hypertrophy and improvements in muscle strength via inhibition of GDF11/myostatin signaling. However, GDF11PRO-Fc does not significantly improve the dystrophic pathology in mdx mice.

Project description:Skeletal muscle growth and maintenance are essential for human health. One of the muscle regulatory genes, namely myostatin, a member of transforming growth factor-beta, plays a dominant role in the genetic control of muscle mass. Myostatin is synthesized as a precursor protein, which generates the N-terminal propeptide and the C-terminal mature myostatin peptide by a post-translational cleavage event. Previously, transgenic over-expression of myostatin propeptide in skeletal muscle results in significant muscle growth in early stages of development. The objectives of present study were to further characterize muscle growth in later stages of life and to identify genes and their expression patterns that are responsible for adult muscle build-up by myostatin propeptide.Immunohistochemical staining with an antibody to the N-terminus indicates a high level of myostatin propeptide present in the muscles of transgenic mice while there were no apparent differences in myostatin protein distribution in the muscle fibers between the transgenic and wild-type mice. Main individual muscles increased by 76-152% in the transgenic mice over their wild-type littermate mice at 12 months of age. A large number of nuclei were localized in the central and basal lamina of the myofibers in the transgenic mice as the number of nuclei per fiber and 100 microm(2) area was significantly higher in transgenic mice than wild-type mice. By systemic comparisons of global mRNA expression patterns between transgenic mice and wild-type littermates using microarray and qRT-PCR techniques, we have identified distinct gene expression patterns to support adult muscle build-up by myostatin propeptide, which are comprised of enhanced expressions of myogenic regulatory factors and extracelullar matrix components, and differentially down-regulated expressions of genes related to protein degradation and mitochondrial ATP synthesis.The results present a coordinated pattern of gene expressions for reduced energy utilization during muscle build-up in adult stage. Enhanced muscle buildup by myostatin propeptide is sustained by reduced ATP synthesis as a result of a decreased activity of protein degradation. Myostatin propeptide may have a therapeutic application to the treatment of clinical muscle wasting problems by depressing myostatin activity.

Dataset Information

Identification of the minimum region of flatfish myostatin propeptide (Pep45-65) for myostatin inhibition and its potential to enhance muscle growth and performance in animals.

Publications

Identification of the minimum region of flatfish myostatin propeptide (Pep45-65) for myostatin inhibition and its potential to enhance muscle growth and performance in animals.

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