Study Of Myostatin-propeptide-enriched Exosomes In Duchenne Muscular Dystrophy

Objective: Duchenne Muscular Dystropy(DMD)is a rare and lethal muscle-wasting disease with an incidence rate of 1 in 3500 newborn boys,caused by frame-disrupting mutations in the DMD gene and resulted in the loss of functional dystrophin protein.The clinical manisfestations of DMD patiens show progressive muscle-wasting and most of patients die of respiratory failure and cardiomyopathy at later stages.Currently,there is no effective treatment available in the clinic.Numerous therapeutic strategies are under investigation with the aim to alleviate sympotoms or restore the expression of functional dystrophin.Myostatin is a growth differentiation factor and functions as an inhibitor for myogenesis.Therefore,myostatin is one of the most important targets for treating DMD.It was shown that myostatin propeptide can inhibit myostatin and thus deregulate the inhibitory effect of myostatin on muscle regeneration and differentiation.It is well-recognized that peptides are unstable in serum with a short half-life,in our previous studies,we demonstrated that exosomes,nanovesicles secreted by most mammalian cells,can be used as delivery vehicles for protein and nucleic acids and also protect nucleci acids from degradation.Therefore,in our current study,we aim to investigate the potential and feasibility of exosomes as delivery vehicles for myostatin-propetides in mdx mice and wish to open a new avenue for myostatin-propeptide delivery and a new approach for treating DMD.Methods: The inhibitory domain of myostatin is located in the region between amino acids 42 and 115 of the propetide and the target gene was obtained by RT-PCR from skeletal muscle followed by insertion into the lenti-vector pm.CD63.e GFP.Murine NIH3T3 cells stably expressing CD63-propeptide(CD63-Pro)was established by lentivirus transduction and the expression of CD63-Pro was confirmed by Western Blot.Exosomes were harvested by ultracenfugation and ultrafiltration and confirmed by Western Blot and transmission electron microscopy(TEM).To verify the binding ability of CD63-Pro-enirched exosomes(EXOmyo-pro)with myostatin in the serum,we intravenously administered control exosomes(EXO)or EXOmyo-pro(10 mg/kg)into C57BL/6 mice and harvested serum after circulating for 24 h.Western Blot was used to examine the level of myostatin in the serum.The inhibitory effect of EXOmyo-pro on mysotatin was investigated in muscle cells,cardiotoxin(CTX)–injured C57BL/6 and dystrophin-deficient mdx mice at different dosing regimens.Histological staining and immunohistochemistry were used to examine the pathological,morphological changes and muscle regeneration,differentiation.Myo D and Myo G were measured with Western Blot to reflect the influence of EXOmyo-pro on muscle differentiation.In addition,body weight,muscle mass,grip strength and muscle sectional area were meansured to reflect the functional impact of EXOmyo-pro on mice.Results: We successfully established the CD63-Pro-expressing NIH3T3 cell line and Western Blot confirmed the expression of CD63-Pro in cells and exosomes derived from CD63-Pro-expressing cells.TEM and Size potential results showed that the expression of CD63-Pro does not alter the size and morphology of exosomes.The binding assay showed that EXOmyo-pro efficiently bound to myostain in serum and thus significantly decreased the level of myostatin in the serum.Compared to EXO,the level of Myo G expression was increased in C2C12 muscle cells treated with EXOmyo-pro,suggesting that EXOmyo-pro can promote muscle differentiation.Examination of EXOmyo-pro in CTX-injured C57BL/6 mice demonstrated increased muscle mass,enlarged muscle cross-sectional area and increased number of regenerating myofibres in tiblialis antierior(TA)treated with EXOmyo-pro,compared to EXO,indicating that EXOmyo-pro can promote muscle regeneration and repair.The dosing optimization study showed no improvement achieved in mdx mice treated with EXOmyo-pro at the dose of 10 mg/kg/week for 3 weeks,compared to EXO.Significant improvement was achievevd in muscle mass,body weight and levels of Myo D and Myo G expression in mdx mice treated with compared to EXO,when the dose was increased to 20 mg/kg/week for 3 weeks,suggesting there is a threshold concentration for EXOmyo-pro.Therefore 20 mg/kg was used as the tested dose for subsequent experiements.Long-term repeated administration of EXOmyo-pro resulted in cummulative beneficial effects in mdx mice demonstrated by increased body weight,muscle mass and levels of Myo D and Myo G expression.Conclusion: Our study demonstrates that exosomes can be used as delivery vehicles for myostatin-propeptide and result in effective binding of serum myostatin and deregulate the inhibition of myostatin on muscle growth and differentiation.