Functional Study Of Atp11a And Tmem30a In Skeletal Muscel Regeneration

Background and purpose:Muscle stem cells are a class of undifferentiated monocytes that can complete self-renewal.They are normally in a resting state.When the body's muscle tissue is lost,muscle stem cells are activated in order to rapidly proliferate and differentiate to form new muscle fibers,which can repair of damaged muscle tissue.Phosphatidylserine?PS?is asymmetrically distributed in the plasma membrane of eukaryotic cells.P4-ATPase transports PS from the outer to the inner leaflet of the membrane bilayer to maintain PS asymmetry.This is crucial for the biological activity of the cell.P4-ATPases require interaction with a?-subunit TMEM30A to function properly.Previous studies have shown that ATP11A and TMEM30A are molecular switches of myotube formation,but the mechanism of ATP11A and TMEM30A during muscle regeneration has not been explored,and the detailed cellular functions of Atp11a protein are poorly understood.Mutations of L127P,L127V,I344F,and E897K in ATP8B1 are known to cause serious human diseases.We matched these mutations to the equivalent sites of human ATP11A,which are I80P,I80V,Y300F and D913K respectively,and investigated the roles of these mutation residues in ATP11A.Methods:Tmem30a^LoxP/LoxP mice were crossed with Pax7-Cre^ER mice to obtain mice that specific knockout Tmem30a in muscle stem cells.The mice were screened by genotyping.By weighting cKO mouse,the effects on muscle development of knocking out Tmem30a in muscle stem cells were excluded.Hematoxylin and eosin?HE?staining to observe muscle pathological changes,quantitative analysis of the number and size of new muscle fibers.The expression of ATP11A protein and its variants in cells was detected by Western Blot technology.Immunocytochemistry was used to determine the localization of ATP11A protein and its variants in cells.Flow cytometry was used to analyze the flippase activity of ATP11A protein and its variants.Results:This study found that the specific knockout Tmem30a in muscle stem cells did not affect the normal development of muscles,but the number and size of muscle stem cells in cKO mice were significantly fall behind WT mice in 21 days after muscle injury.In the study of ATP11A,Western Blot analysis results showed that expression of Y300F variant was reduced about by 40%compared to WT.The Immunocytochemical results showed that when co-expressed with TMEM30A,ATP11A protein was mainly located in Golgi,and variants of Y300F and D913K could not be correctly located in Golgi.Flow cytometry results revealed that variants of Y300F and D913K resulted in reduced PS internalization in plasma membrane.Conclusion:Deletion of Tmem30a in muscle stem cells slows down muscle regeneration,indicating that Tmem30a plays an important role in muscle regeneration.Y300F mutation of ATP11A could cause a decrease in ATP11A intracellular content,and variants of Y300F and D913K affected the subcellular localization and flippase activity of ATP11A protein.This indicates that the Y300 and D913 residues play an important role in the normal function of the ATP11A protein.