The Mechanism Of Pax3/Pax7 Determining The Types Of Skeletal Muscle Fibers And Its Regulation By Vitamin D₃

Skeletal muscle fiber type composition is closely related to meat quality.Both postnatal nutrition regulation of muscle fiber types and type of transformation depend on the differentiation of satellite cells(SCs).SCs express myogenic regulatory factors,Pax3/Pax7,whose characteristics of the inherent differences in DNA binding lead to different myogenic fate.Previous studies found that vitamin D3 could promote the increase of the.proportion of fast.muscle fibers and.cross-sectional area,which was related to the rising number of SCs in fast muscle fibers,however,its molecular mechanism is still unclear.This study assumes that Pax3/Pax7 plays a decisive role in fast muscle fiber formation promoted by vitamin D3.The molecular mechanism of Pax3/Pax7 in the differentiation of different types of muscle fibers was determined by using molecular cell biotechnology,gene knockout mice and vitamin D3 nutritional model mice.This research would lay a theoretical foundation for further understanding developmental rule of muscle fibers and establishing nutritional regulation strategy.In this paper,four experiments were designed and their results were obtained as follows:Experiment 1.The distinct proliferation and differentiation between Pax3+ SCs and Pax7+ SCsIn this study,after sorting the SCs from C57BL/6 mice by flow cytometry,the proliferation and differentiation of Pax3+ SCs and Pax7+ SCs were analyzed by immunofluorescence and Western Blotting.The results showed that Pax3+ SCs maintained a longer self-renewal and proliferative capacity than Pax7+ ones.On the contrary,Pax7+ SCs were more likely to differentiate.After differentiation,Pax3+ SCs were differentiated into myotubes with rhythmical contraction which were mainly slow muscle fibers.Pax7+ SCs were divided into myotubes with short-term rapid contraction which were mainly fast muscle fibers.Conclusion:Pax3 maintains longer sternness of SCs and regulates slow muscle formation,and Pax7 regulates fast muscle formation.Experiment 2.Zacl/GPR39 phosphorylating CaMK-? contributes to the distinct roles of Pax3 and Pax7 in myogenic progressionPax3 and Pax7's interacting proteins were identified by Co-immunoprecipitation(CoIP)in C2C12 myoblasts,and the molecular mechanism of Pax3/Pax7's determination on the proliferation and differentiation of muscle fibers was investigated by in vivo transplantation and gait analysis.The results showed that both Zacl and GPR39 were upregulated by the overexpression of Pax7,whereas Pax3 did not induce Zac1 and GPR39 expression.CoIP confirmed that Zacl interacted directly with Pax7,Pax7-regulated GPR39 expression was achieved by activating Zacl.The silencing of Zac1 in the C2C12 transfected with Pax7 RV revealed that the Zac1/GPR39 system could facilitate the differentiation and lead to the production of fast muscle fibers.Gait analysis showed that transplantation of GFP-labeled Pax7 RV/siZacl transfected cells into CTX-injured mdx mice resulted in delayed muscle function repair.After the second injury,the cells presented to be able to maintain longer regeneration capability.Molecular mechanism studies have shown that the Zacl/GPR39 system is associated with different myogenic functions of Pax3 and Pax7.Pax7 activates the Zacl/GPR39 system,mediating the phosphorylation of CaMK-? and the dephosphorylation of p-ERK1/2,which inhibits ?-catenin expression and promotes the formation of fast muscle fibers.Cells lacking Zacl/GPR39 system tend to retain sternness and form slow muscle fibers after induced differentiation.Conclusion:Pax7 activates Zacl/GPR39 system,which phosphorylates CaMK-? and inhibits ?-catenin,promotes the formation of fast muscle fibers.Experiment 3.Pcbpl regulates cellular differentiation through modulation of miRNA processing in skeletal muscleThe poly(C)-binding proteinl(Pcbp1)has been reported as a component of the miRNA processing pathway to regulate the biogenesis of miRNAs.However,the biological function and processing pathway of this protein remain largely undetermined.The Pcbplf/f knockout mice were established for the first time.To explore the new function of Pcbpl involved in miRNA maturation and the molecular mechanisms in regulating skeletal muscle development and its cells' proliferation and differentiation,qPCR and Northern Blotting were used to detect the expression of miRNA and Pax3/Pax7.Immunofluorescence staining was used to observe the changes of MyHC-I and MyHC-II.CoIP identified miRNA processing and mature components were interacting with Pcbp1.The results showed that Pcbpl interacted directly with Ago2 and other miRNA pathway components to modulate the processing of muscle-specific miR-1,miR-133 and miR-206.Pcbp1f/f mice demonstrated early embryonic lethality,indicating that Pcbp1 was indispensable for animal development.Pcbp1f/f mice were observed to have reduced number of SCs on the muscle fibers In addition,their Pax3 expression was decreased and Pax7 expression.was upregulated,presented as muscle growth defect and fast muscle increase.In vivo miR-1,mi.R-133 and miR-206 expression was also.reduced,but miR208,related to the regulation of muscle fiber type,showed no significant differenee.Conclusion:the type of muscle fiber in Pcbplf/f mice is not regulated:by miRNA.The up-regulation of Pax7 induced by Pcbpl might contribute to promoting fast muscle fibers formation.Experiment 4.The pathway of vitamin D3 modulates skeletal muscle fiber types The objective of this experiment is to explore whether fast muscle fiber atrophy and proportion decrease is related to Pax3/Pax7 SCs content.Vitamin D3 nutritional model in mice(VD3+/VD3-)was established,Radioimmunoassay was used to detect thyroid hormone levels.In addition,in vivo overexpression of GPR39 was achieved by electroporation,and shh signal pathway related protein expression was probed by Western Blotting;immunofluorescence staining was applied before single muscle fibers counting.After CTX injury,HE staining and frozen sections were used to identify the regeneration of the mice.qPCR and Western Blotting were used to detect the expression of Pcbp1,Pax3/Pax7 and related genes.The results showed that vitamin D3 increased the concentration of thyroid hormone T3.Vitamin D3 upregulated GPR39 expression.whereas the expression of Gli2 and DI03 in shh signaling pathway was decreased.In vivo overexpression of GPR39 in VD3+ mouse resulted in the increase of Gli2 and DIO3 expression.Compared with group VD3+/Hypo-,we found that the content of satellite cells on the single muscle fibers was reduced and the repair was delayed after injury in group hypothyroid Hypo-..Western Blotting indicated the expression of Pcbpl protein was significantly increased.The results of qPCR showed a significant increase in the expression of Pax3 and genes regarding slow muscle protein(MYH7,Tnni1 and Myoglobin),whereas the expression of Pax7 and MYH genes(MYH1 and MYH2)regulating fast muscle protein was decreased.Conclusion:vitamin D3 could promote the formation of fast muscle,the underlying mechanism is that VD3 blocks the shh signaling pathway to;ncrease the concentration of thyroid hormone T3,resulting in decreased Pcbpl and increased Pax7 expression,which promotes differentiation to the fast muscle fibers.In summary,the Pax3+ SCs and Pax7+ SCs have distinct proliferation and differentiation.Pax3 maintains longer sternness of SCs and regulates slow muscle formation,and Pax7 regulates fast muscle formation.Pax7 activates Zacl/GPR39 system,which phosphorylates CaMK-? and inhibits ?-catenin,promotes the formation of fast muscle fibers.Vitamin D3 block the shh signaling pathway to increase the concentration of thyroid hormone T3,resulting in decreased Pcbpl and increased Pax7 expression,which promotes differentiation to the fast muscle fibers.