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-...

Identification of Molecules from Coffee Silverskin That Suppresses Myostatin Activity and Improves Muscle Mass and Strength in Mice

“…The free fatty acid concentration in serum was tended to decrease by the administration of CSE, indicating an active utilization of blood free fatty acids by CSE administration. This result is in agreement with other studies that also reported a decrease in blood free fatty acid level by the administration of MSTN-inhibitory compounds in mice fed either a regular [ 32 ] or high-fat diet [ 33 ]. However, the levels of blood glucose and triglyceride were not affected by the administration of CSE in contrast to the results of the above reports [ 17 , 31 ], where decreases in blood glucose and triglyceride were observed by MSTN-inhibitory compound administration.…”

“…The effects of the coffee silverskin ethanol extracts (CSE; mixture of various varieties) on MSTN-, GDF11-, and Activin A-induced Smad3 phosphorylation were examined by the Western blot analysis following the procedure previously described [ 32 ]. HepG2 cells were seeded in 6-well plates at 2 × 10 5 cells per well and grown in DMEM containing 10% FBS and 1% penicillin/streptomycin at 37 °C with 5% CO 2 for 24 h. Cells were then switched to serum-free DMEM for 4 h, followed by treatment with 10-nM MSTN (R&D Systems, Minneapolis, MN, USA), 10-nM MSTN plus CSE (50 µg/mL), 10-nM GDF11 (R&D Systems, Minneapolis, MN, USA), 10-nM GDF11 plus CSE (50 µg /mL), 10-nM Activin A (R&D Systems, Minneapolis, MN, USA), and 10-nM Activin A plus CSE (50 µg /mL) for 30 min.…”

Myostatin regulates fatty acid desaturation and fat deposition through MEF2C/miR222/SCD5 cascade in pigs

“…A pluripotent cell line C3H10T1/2 can be induced by the glucocorticoid dexamethasone to form adipocytes, and this process can be reinstated by replacing dexamethasone with recombinant MSTN 13 . In comparison, adipogenesis was not induced by the treatment with MSTN in 3T3-L1 cells, which is an adipocyte lineage-committed cell line believed to be further differentiated than C3H10T1/2 cells 4,14,15 . A possible explanation is that MSTN activates SMAD4 to enhance miR124-3p to repress glucocorticoid receptor, resulting in the inhibition of adipogenic differentiation of 3T3-L1 cells 16 .…”

“…Functional blockade of MSTN results in the double muscling trait in animals, which features overgrown skeletal muscle and reduced fat mass. Owning to this advantage, MSTN has been a prime genetic target for the development of high-leanness animal cultivars in livestock, poultry and fishery industries, via multifaceted approaches like ectopically expressed MSTN propeptide, dominant negative competitor, RNA interference, gene targeting and gene editing [1][2][3][4] . Among these methods, programmable nuclease-mediated gene editing proved to be robust to introduce random indels into the coding sequence of MSTN, thus disrupting its frame readability.…”

Dietary Antioxidant Supplementation Promotes Growth in Senegalese Sole Postlarvae

“…In addition to myod2 and mymk , the inclusion of antioxidants also modified the expression of mstn1 in postlarvae. In mammals, mstn gene is known to act as potent regulator of muscle growth, and a mstn -knockout fish highly increased muscle mass ( de Santis et al, 2012 ; Kim J. et al, 2019 ; Kim J. H. et al, 2019 ). Contrarily to mammals, mstn transcription in fish species is ubiquitous expressed indicating an involvement in other physiological mechanisms as well as in skeletal muscle growth regulation ( Campos et al, 2010 ; Li et al, 2012 ; Canada et al, 2016 ).…”