Tudor-SN Protein,as A Bridging Molecule Of MTOR-YAP Signaling Pathway,Participates In The Regulation Of Neonatal Mice Cardiomyocytes Proliferation

Objective: In recent years,with the acceleration of aging socity,cardiovascular diseases have been severely threatening people's health.The morbidity and mortality caused by cardiovascular diseases have been increasing year by year,and the patients gradually tend to be younger.Cardiovascular diseases have become an important public health problem in China and even worldwide.During cardiac development,the cardiomyocytes gradually exit the cell cycle and then enter the terminal differentiation stage,causing the ability of proliferation and regeneration of cardiomyocytes gradually lost.Although studies have reported that mature cardiomyocytes have the ability to proliferate,comparing with other tissues like liver,kidney and spleen,the limited ability of proliferation making it diffcult to reapir the damage and restore contractile function following injury.Therefore,to study the proliferation characteristics of cardiomyocytes and to delay their withdrawal from cell cycle provide an important strategy for further study on regeneration after injury.Tudor-SN(Tudor staphylococcal nuclease)protein is a highly conserved and ubiquitously expressed multifunctional protein,which is involved in gene transcriptional regulation,pre-m RNA splicing,DNA damage repair,and stress granule assembly.Our previous study has found that Tudor-SN protein,as a cell cycle regulator,participates in the regulation of cell cycle,and its expression very low or undetectable in terminally differentiated tissues such as the heart.However,the correlation between Tudor-SN protein and cardiomyocyte proliferation has not been reported.Therefore,the purpose of this study is to investigate the mechanism of the low expression of Tudor-SN protein in mature myocardia and the effect of Tudor-SN protein on cardiomyocytes proliferation and cardiac function.Methods: This study is divided into three parts: Part I:(1)The western blot(WB),immunohistochemical(IHC)staining,real-time puantitative polymerase chain reaction(q RT-PCR),polysome profile were used to compare the expression of Tudor-SN protein in the myocardia from neonatal mice(1 day old,1d)and adult mice(8 weeks old,8w).(2)The perdication from data base of transcription start sites(DBTSS),5' rapid amplification of c DNA ends(RACE)assay were used to identify the transcription start site of(TSS)the Tudor-SN gene,so as to determine whether the Tudor-SN gene contained terminal oligo-pyrimidine sequence(TOP)in 5' untranslated region(5'UTR).The luciferase assay,WB and q RT-PCR were used to analyze whether the Tudor-SN was a TOP gene.(4)Gene profile analyzed the expression of m RNA in 1d and 8w myocardia.The q RT-PCR and WB were used to detect the activity of mTORC1 signaling pathway in adult myocardia.Part II:(1)The immunofluroscence(IF)staining was used to compare the proliferation characteristic of cardiomyocytes at different age from global Tudor-SN transgenic(Tg)mice and wild-type(WT)mice.(2)The primary cardiomyocytes were obtained from 7d and 21 d WT and Tg-mice,and the number cardiomyocytes was statistically analyzed by cell count.Subsequently,nucleus was stained with DAPI and the ratio of monocleate cells,binucleate cells and multinucleate cells were statistically analyzed.(3)The ratio of heart weight to body weight and H&E staining were used to compare the caridac size of WT and Tg-mice at different ages.(4)The echocardiography was used to analyze the cardiac function of adult male and female WT and Tg-mice at different ages in physiologic condition or in pathologic condition after induced cardiac injury.Part III :(1)The MS was used to analyze the proteins that associated with Tudor-SN in H9c2 cells and involved in cell proliferation;(2)The co-immunoprecipitation(Co-IP),WB,IF,q RT-PCR were used to determine the interaction between Tudor-SN and YAP,whether Tudor-SN affects the localization of YAP protein and the expression of YAP downstream target genes that involved in cell proliferation.(3)The Co-IP was used to analyze the interaction of Tudor-SN protein and Ipo5 protein in H9c2 cells.(4)The Co-IP,IF and WB were used to detect the interaction between YAP protein and Ipo5 protein in H9c2 cells,whether Tudor-SN affects the interactioin between YAP and Ipo5 protein,and the effect of nuclear localization of YAP protein after knocked down Ipo5 protein.Results: Part I :(1)The WB and IHC staining showed that Tudor-SN highly expressed in neonatal mice myocardia and barely expressed in adult mice.The q RT-PCR indicated that there was no significant difference at m RNA level of Tudor-SN between 1d and 8w mice myocardia.Polysome profile analysis showed that the Tudor-SN m RNA loaded with polysome in adult myocardia was significantly reduced compared with neonatal myocardia.(2)The predication from DBTSS and 5' RACE assay showed the 5' TOP motif of Tudor-SN m RNA both in human,mouse and rat.(3)The WB showed the expression of Tudor-SN gradually decreased when the mTORC1 activity was inhibited in H9c2 cells,and the decreased expression of Tudor-SN was not caused by the protein degradation.q RT-PCR showed there was no change of Tudor-SN m RNA during this process.Conversely,the expression of Tudor-SN increased when activated mTORC1,but the m RNA expression level also did not change.(4)The q RT-PCR showed that the expression of many upstream genes of mTORC1 in adult myocardia was decreased compared with neonatal myocardia,including growth factor,integrin and extracellular matrix,et al.WB showed that the protein phosphorylation level which indicating the activity of mTORC1 signaling pathway was significantly decreased in adult myocardia.Part II :(1)IF staining results showed that cardiomyocytes gradually lose the ability of cell proliferation around 7d postnatal and exit cell cycle at 14 d postnatal.In the Tg-mice,the highly expressed Tudor-SN promoted the cardiomyocytes proliferation at 1d and 7d,and extended the proliferative window up to 21 d postnatal.However,there was no significant difference in 28 d postnatal and adult myocardia between Tg and WT-mice.(2)The total number of cardiomyocytes was increased in the Tg-mice at 7d and 21 d postnatal.It was accompanied by an increase in the percentage of mononucleated cardiomyocytes and a decrease in the percentage of binucleated cardiomyocytes.(3)The heart to body weight ratio illustrated that the Tg-mice at 21 d,28d and 8w,have relatively “larger” heart.Th H&E staining showed that the left ventricle wall of Tg-mice of 21 d and 8w mice were much thicker than the WT-mice.(4)The echocardiography showed that the adult male Tg-mice exhibited enhanced cardiac function in all the different ages,which is the same as in the female Tg-mice.In the heart failure model,the cardiac function of WT-mice was obviously damaged,but no significant effect was observed in Tg-mice.Part III :(1)The MS revealed that Tudor-SN was co-purified with number of proteins involved in cell proliferation,including YAP protein.(2)The Co-IP and IF showed that Tudor-SN and YAP protein interacted with each other and Tudor-SN protein could affect the translocation of YAP protein in H9c2 cells.The q RT-PCR showed that Tudor-SN protmoted the genes expression of YAP downstram targets which related to cell proliferation.(3)The Co-IP showed that Tudor-SN interacted with Ipo5 protein in H9c2 cells.(4)The Co-IP and IF showed that YAP interacted with Ipo5 protein and Tudor-SN affected the interaction of YAP and Ipo5 protein in H9c2 cells.And the localization of YAP changed after knocked down Ipo5 protein.Conclusion:(1)As a novel TOP gene,the expression of Tudor-SN was regulated by mTORC1 signaling pathway at translation level.(2)The low expression of Tudor-SN protein in adult mice mayocarida was due to the inactivition of mTORC1 pathway.(3)In Tg-mice,the overexpression of Tudor-SN promoted and prolonged neonatal cardiomyocytes proliferation and improved adult heart function.(4)In cardiomyocytes,Tudor-SN affected the nuclear translocation of YAP protein by influencing the interaction of YAP and Ipo5 protein,thus affected the proliferation of cardiomyocytes.