Preliminary Study On The Role Of ARF6 And Its Regulator And Effector In The Differentiation And Fusion Of Myoblasts

There are about 600 skeletal muscles in the human body,which play an important role in mechanical movement and energy metabolism and are an important organ of the body.Skeletal muscle is composed of muscle fibers arranged in bundles,and each muscle fiber is a syncytia formed by the fusion of mononuclear myoblasts.Satellite cells which are situated between sarcolemma of muscle fibers and the basement membranes,are skeletal muscle stem cells.Satellite cells are activated after muscle damage,proliferate and differentiate into myoblasts.Myoblast fusion is a set of well-orchestrated and multi-step specific cellular events that play an important role in the formation and repair of skeletal muscle after injury.Although skeletal muscle has a strong ability to repair damage,when a person suffers from severe skeletal muscle progressive muscular dystrophy disease or the body encounters a large area of muscle damage that exceeds its self-repair ability,the muscle regeneration ability will gradually decrease or even disappear.Duchenne muscular dystrophy(DMD)is an X-linked recessive genetic rare disease.Due to the mutation of the disease-causing gene DMD which leads the loss of the dystrophin function,and the skeletal muscle is progressively damaged and the damage cannot be repaired,leading to muscle dystrophy and eventually the patient is died of respiratory failure.There is no effective treatment currently.Recent studies have shown that allograft of myoblast from the donor or implantation of gene-edited myoblasts from DMD patients brings hope to the patients.The activated satellite cells cultured in vitro and injected into the muscle of the patient can fuse with the muscle fibers of the patient and express normal functional dystrophin.Myoblast fusion is an important part of myoblast therapy,so it is important to understand the mechanism of myoblast fusion.The small GTPase ARF6(ADP ribosylation factor 6)is a membrane associated protein of Ras superfamily,usually in a state of binding with GDP.After receiving GTP provided by Guanine nucleotide-exchange factors(GEF)to replace GDP,the ARF6-GDP becomes ARF6-GTP and is activated.It participates in many important life processes through its activation of downstream molecules.Studies have found that ARF6 is necessary for myotube formation and plays a role in both primary and secondary fusion of myoblasts.Activated ARF6 can activate PLD1 and together with phospholipases D1(PLD1)activate phosphatidylinositol 4-phosphate 5-kinase,regulate the production of PIP2 and thus regulate myoblast fusion.The preliminary results of our laboratory show that PLD1 mainly promotes the formation of mature myotubes during the secondary fusion process,while ARF6,as the upstream factor of PLD1,promotes both the primary and secondary fusion of myoblasts,indicating that PLD1 was not the only ARF6 effector in myoblast fusion.In addition to PLD1,what other ARF6 regulatory factors and effector molecules are involved in the myoblast fusion process promoted by ARF6?In this study,using the combination of GEF and ARF6,using mutants expressing ARF6 activating and non-activating proteins,the GST-Pull down experiment was used to collect and compare the ARF6 binding proteins of monocytes and differentiated and fused myoblasts,looking for ARF6 regulatory factors and Effectors,hope that the findings of the study will provide a new theoretical basis for the treatment of muscular dystrophy and other skeletal muscle diseases.Objectives:1.Study the role of ARF6 in the process of primary myoblast fusion.2.Looking for the regulatory factors and effectors of ARF6 in the process of myoblast differentiation and fusion.Methods:1.Use immunofluorescence staining and Western blot to detect the expression of Myosin and Myogenin during primary myoblast differentiation and fusion after knocking down ARF6.2.Several mutant plasmids of ARF6(ARF6-WT-FLAG,ARF6-Q67L-FLAG,ARF6-T27N-FLAG)induced by IPTG were expressed in E.coli,purified and digested.3.Use the purified ARF6 mutant protein to pull down/IP the protein sample during the differentiation and fusion process of primary myoblasts.Results:1.Four days after infection of ARF6^flox/flox primary myoblasts with Adeno-Cre,the expression of ARF6 decreased to 40%..2.Knockdown of ARF6 will inhibit the fusion of primary myoblasts,reduce the fusion index of primary fusion and secondary fusion,and mainly inhibit the generation of mature myotubes.And Western blot results also showed that knocking down ARF6would inhibit the fusion of primary myoblasts,and the expression of Myosin was reduced during the differentiation of day 2 and day 4,and there was no significant difference in the expression of myogenin.3.After mass spectrometry analysis and Western blot detection,we purified the ARF6-Q67L-FLAG protein.Conclusions:1.ARF6 is involved in regulating the differentiation and fusion of myoblasts.2.Knocking down ARF6 inhibits myoblast fusion.The formation of mature myotubules was inhibited in early and late differentiation.3.No new regulators and effectors of ARF6 in the process of myoblast differentiation and fusion have been found.