The Effect And Mechanism Of YAP1 Alternative Splicing On Proliferation Of Cardiomyocytes

BACKGROUNDIn recent years,great progress has been made in cardiovascular interventional diagnosis and treatment technology.The improvement and popularization of emergency PCI technology has made the mortality rate of acute myocardial infarction decrease significantly.However,cardiovascular disease is still one of the main causes of human death.Emergency PCI technology makes occluded blood vessels reopen earlier and saves some myocardial cells in infarcted area,but there is still no way to deal with necrotic myocardial cells.These myocardial cells are often replaced by fibrous tissue,resulting in ventricular remodeling and further heart failure.Therefore,promoting myocardial cell regeneration and replacing necrotic myocardial tissue with regenerated myocardial cells rather than fibrous tissue has been an important research direction of myocardial infarction.Hippo signaling pathway was first discovered in Drosophila in 1995.It is a highly conserved signaling pathway.YAP1 is an important downstream effector molecule of Hippo signaling pathway.Studies have confirmed that this pathway plays a key role in cardiomyocyte proliferation and cardiac size regulation.Inactivation of the Hippo pathway or its downstream target can promote cardiomyocyte regeneration.However,the mechanism underlying the regulation of Hippo-YAP1 pathway remains unclear.YAP1 has eight different alternative splicing subtypes.Different alternative splicing subtypes may have different or even antagonistic functions.Through the study of YAP1 specific subtype knockout mice,we can effectively study the function of different splicing subtypes of YAP1 and the mechanism of variable splicing regulation,which will conducive to understanding of the role and regulation mechanism of Hippo-YAP1 pathway more deeply.It will provide new research directions for myocardial regeneration and myocardial repair of infarcted myocardium.METHODSPart I: Identification and expression detection of major alternative splicing subtypes of YAP11.HCT116,MDA-MB-231,A549,H157,786-O,293 T,Hela and other cell lines were cultured,and RNA was extracted to detect the expression of 8 alternative splicing subtypes of YAP1 in the above cell lines.2.Extraction of tissue RNA,detecting the changes of YAP1 splicing isoforms in ICM and DCM.3.Tissue RNA was extracted to detect the expression of YAP1 alternative splicing subtypes in human,rhesus monkey,rat,mouse,zebrafish and other heart tissues.Part Ⅱ:Study on phenotype of YAP1 Exon6 knockout mice1.The obtained loxP floxed mice were hybridized with DAPP-Cre transgenic mice to produce YAP1 Exon6 mutant heterozygous mice.Homozygote knockout mice were obtained by further hybridization.2.The mouse genotype was identified by genotype identification of tail and DNA identification of heart tissue.3.The growth and development of different genotypes of mice were observed.Orbital blood was taken to detect the changes of blood routine.Heart changes were observed by HE staining of heart tissue of newborn mice.4.The myocardial proliferation of YAP1 Exon6 knockout mice was observed by immunohistochemistry and immunofluorescence staining.5.The expression of PCNA in myocardium of neonatal mice with YAP1 Exon6 gene knockout was detected by Western blotting.Part Ⅲ: Analysis of cardiac function and myocardial regeneration in YAP1 Exon 6 knockout adult mice1.RNA was extracted from left ventricular myocardium of adult mice.The expression of ANP,BNP,MYH6,MYH7 and other cardiac function markers in YAP1 Exon 6 knockout adult mice was detected by qRT-PCR.2.Cardiac echocardiography was performed in adult mice,and the changes of cardiac echocardiographic parameters in YAP1 Exon 6 knockout adult mice were analyzed.3.HE staining of adult mouse hearts further clarified the changes of ventricular wall thickness and myocardial cell size.4.Ki67 and PCNA immunofluorescence assays were used to evaluate the myocardial proliferation of YAP1 Exon6 knockout mice.5.The PCNA protein imprinting assay of YAP1 Exon6 myocardial tissue-specific knockout mice was used to evaluate the changes of myocardial proliferation markers during myocardial tissue-specific knockout.6.The myocardial regeneration ability of YAP1 Exon6 knockout adult mice was measured in acute myocardial infarction model.Part Ⅳ: Study on the YAP1 splicing mechanism1.Overexpression of YAP1 variable splicing subtypes was used to detect the expression of YAP1 protein in four different alternative splicing subtypes at the same RNA level.2.The expression of YAP1 protein in YAP1 Exon6 knockout mice was detected by Western blotting.3.The changes of some validation factors,adhesion factors and downstream genes in YAP1 Exon6 knockout mice were detected by qRT-PCR.RESULTSPart I: Exon6 skipping is the main form of YAP1 alternative splicingBy detecting the expression of YAP1-1α、YAP1-1β、YAP1-1γ、YAP1-1δ、YAP1-2α、YAP1-2β、YAP1-2γ and YAP1-2δ in different cell lines,it was found that Exon 6 skipping was the main form of YAP1 alternative splicing,and YAP1-α and YAP1-γ were the two main alternative splicing subtypes of YAP1.The expression of these two alternative splicing subtypes will change in human cardiomyopathy such as ICM and DCM.These two alternative splicing subtypes have different physiological and pathological functions.The expression of YAP1 subtypes in human,rhesus monkey,rat,mouse and zebrafish heart tissues was detected.It was found that they were highly species conservative.Part Ⅱ: Proliferation of cardiomyocytes in neonatal mice with YAP1 Exon6 knockoutYAP1 Exon6 knockout mice were prepared by CRISPR-Cas9 system,and genotype was identified by genotype identification.By weight measurement and analysis of 4w,8W and 20 w,it was found that YAP1 Exon6 knockout mice would lose weight.No significant statistical difference was found in routine blood analysis due to YAP1 Exon6 knockout.HE staining of the heart of neonatal mice showed that the thickness of ventricular wall increased significantly in YAP1 Exon6 knockout mice.PCNA immunohistochemistry and Ki67 immunofluorescence staining in cardiac muscle tissue of neonatal mice suggested that YAP1 Exon6 knockout mice had more active proliferative behavior.PCNA and Cyclin D2 protein imprinting experiments in myocardial tissue further confirmed that YAP1 Exon6 knockout could lead to cell proliferation activity.Mason staining of myocardial tissue suggested that YAP1 Exon6 knockout could also lead to the increase of fibrinogen in myocardial tissue.Part Ⅲ:YAP1 Exon6 gene knockout induced ventricular wall thickening and deterioration of cardiac function in mice,and enhanced myocardial regenerationThe results of echocardiography in adult myocardial mice showed that the deletion of YAP1 Exon6 gene could result in significant thickening of ventricular wall,shrinkage of ventricular chamber and decrease of EF% in adult mice.HE staining further clarified the effect of YAP1 Exon6 gene knockout on ventricular wall thickening in adult mice.At the same time,statistical analysis of cross-sectional area of myocardial cells showed that YAP1 Exon6 gene knockout did not increase the size of myocardial cells,and the increase of ventricular wall thickness was due to the increase of the number of myocardial cells.Ki67 and PCNA staining in adult mice suggested that YAP1 Exon6 knockout mice had more active proliferative behavior.The detection of ANP and BNP in adult mouse cardiomyocytes suggested that YAP1 Exon6 gene knockout could lead to deterioration of cardiac function in adult mice.Western blot analysis of PCNA in myocardial tissue of muscle tissue-specific knockout mice showed that YAP1 Exon6 knockout could increase the proliferation activity of myocardial tissue after excluding the effects of other tissues.Myocardial infarction model suggested that YAP1 Exon6 knockout mice had stronger regeneration ability of myocardial cells and significantly reduced the area of fibrotic scar in infarcted area.Part Ⅳ:Knockout of YAP1 Exon6 gene results in a decrease in the expression of YAP1 proteinBy overexpressing different alternative splicing subtypes,it was found that the expression of YPA1 subtype with Exon 6 was significantly higher than that without Exon 6 at similar levels of over-expression of different subtypes.After extracting protein from myocardial tissue of adult mice with YAP1 Exon6 gene knockout,Western blot experiments were carried out.The results showed that YAP1 Exon6 gene knockout resulted in a significant decrease in the expression of YAP1 protein.There was no significant difference in qRT-PCR expression of CRP,IL1,IL6,MCP1,CDH1,CDH2,CDH5,ITGB and other downstream target genes such as Her1,CYCD1,Hes1,CTGF,IGFBP3,LMNB and CYR6 in adult mice with YAP1 Exon6 gene knockout.CONCLUSION1.Exon6 skipping is the main form of YAP1 alternative splicing.Different splicing subtypes of YAP1 have different functions.2.YAP1 Exon6 knockout can increase the thickness of ventricular wall and actively proliferate myocardial cells in neonatal mice.3.Knockout of YAP1 Exon6 can result in more active cell proliferation,thickening of ventricular wall and decreased cardiac function in adult mice.4.YAP1 Exon6 knockout mice had stronger myocardial regeneration ability after myocardial infarction.5.The protein expression of YAP1 gene without Exon6 was lower,but the effect of promoting cell proliferation was stronger.