Investigation Of The Physiological Function And Mechanism Of BAMBI In The Development Of Skeletal Muscle

The transmembrane inhibitory protein of bone morphogenetic protein and activin（BAMBI）is called a pseudo-receptor because it is similar to the extracellular structure of the TGFβfamily of type I receptors but lacks an intracellular signaling domain.At present,BAMBI has been reported in many fields,such as the physiology of porcine ovarian granulosa cells,immune cell inflammatory response,liver tumorigenesis and development,adipocyte proliferation and differentiation.In addition,BAMBI interacts with the classical WNT/β-catenin signaling pathway.The development of skeletal muscle is highly regulated by TGFβand WNT signaling pathways.In the adult animals,the activation,proliferation,differentiation and fusion processes of muscle satellite stem cells（SCs）are regulated by signaling pathways.Therefore,it is speculated that BAMBI may play an important role in the development of skeletal muscle.The expression of BAMBI is reduced by si RNA in C2C12 myoblasts,which inhibits the myogenic differentiation of C2C12.However,si RNA utility is limited by the inherent incompleteness of protein depletion and has a confounding off-target effects.The biological function in skeletal muscle is still unknown and needs further investigation.In this experiment,we constructed BAMBI deletion and overexpression on C2C12 myoblasts and induced C2C12 myogenic differentiation.The effects of BAMBI on myoblasts were studied by Western blot,RT-qPCR,RNA-seq and immunofluorescence.In addition,we further explored of the biological function of BAMBI in adulthood and development of mouse skeletal muscle.By immunofluorescence staining and isolation of single muscle fibers showed that BAMBI expression was inconsistent in different muscle fiber types.At the same time,by Ba Cl₂inducing mouse skeletal muscle injury model,BAMBI expressed in different locations of muscle cell during injury repair.This study also provides a theoretical basis for the improvement of meat quality in livestock by regulating the expression of BAMBI gene.The results obtained are as follows:1.BAMBI expression was highest in the early stage of C2C12 cell differentiation,and gradually decreased with differentiation.The myoblast cell line of BAMBI knockout in C2C12 was constructed by CRISPR Cas9.After knockout BAMBI,the growth viability of the cells was not affected.After induction differentiation,knockdown of BAMBI significantly inhibited the myogenic differentiation of C2C12 myoblasts.2.Establishment overexpression BAMBI myoblast cell line in C2C12 myoblasts by retroviral packaging of BAMBI,overexpression of BAMBI did not affect the growth viability of C2C12 myoblasts,and significantly increased the m RNA and protein expression of myogenic-related myogenic genes after induction of differentiation.3.By RT-qPCR,BAMBI mainly expressed in skeletal muscle,and it was the lowest expression in white fat.It was found that BAMBI was mainly expressed in TA in four skeletal muscle types,followed by EDL and Gas.BAMBI expression was the lowest in Sol.4.By extracting the cytoplasm and membrane proteins of TA tissue.BAMBI was mainly expressed in cytoplasm of adult skeletal muscle.5.By comparing the expression of BAMBI in developing mice（2 weeks）with adult mice（10 weeks）,we found that BAMBI expression was low in developing skeletal muscle,and BAMBI expression gradually increased with the growth and development of mice.At the same time found that BAMBI protein is mainly enriched in the cell membrane in development of skeletal muscle.6.Immunofluorescence staining in different muscle tissues showed that BAMBI was mainly co-localized with IIX muscle fibers,a small part was expressed in IIA and IIB,and almost no expression in type I muscle fibers.7.Induction of skeletal muscle injury by injection of Ba Cl₂confirmed that BAMBI was mainly expressed on the membrane of new muscle fibers at 5dpi.BAMBI returned to the muscle cytoplasm with the regeneration of muscle tissue.8.By HSA and Pax7 driving,the deletion of BAMBI leads to activation of satellite cell.BAMBI is downstream target of HIF2α.In summary,knockout or overexpression BAMBI in C2C12 myoblasts did not affect cell viability.BAMBI plays a positive regulatory role in the differentiation of C2C12myoblasts.BAMBI expression is gradually enhanced with skeletal muscle development.BAMBI highly expressed in TA tissue of skeletal muscle tissue and localized in IIX muscle fibers.In addition,during muscle injury,BAMBI shuttled from the cytoplasm to the cell membrane to perform its function.The knockout BAMBI results to activation of satellite cells on skeletal muscle and satellite cell.The above results provide a theoretical basis for targeting the BAMBI gene in pig meat quality improvement.