| Background:The heart is a very sensitive organ to force.Biomechanical stimulation plays an important role in the process of cardiac development and pathological remodeling.In recent years,a large number of studies have confirmed that Hippo pathway is the key signal pathway to sense biological power.Hippo pathway is the first signal pathway found in Drosophila melanogaster,which can maintain the balance between cell proliferation and apoptosis,and control organ size.Its biological effect is mainly through the regulation of its downstream transcription factor yap.Myocardial hypertrophy is a pathological process in the progress of heart failure.The pressure load caused by hypertension or aortic stenosis can cause compensatory myocardial hypertrophy to maintain ventricular wall tension and cardiac output and meet the needs of various organs of the human body.However,the long-term excessive pressure load of the heart can lead to the death of myocardial cells,and because the regeneration ability of adult cardiac myocytes is limited,it will eventually lead to death It can lead to heart failure.Therefore,it is very important to further study the role of Yap in the process of pressure overload induced cardiac hypertrophy,which will provide a new molecular theoretical basis for clinical treatment of heart failure.Purpose:Objective to investigate the role of Hippo downstream transcription factor Yap in the occurrence and development of pressure overload induced cardiac hypertrophy.Method:At the animal level:1.Twenty SPF 8-week male C57BL/6J mice were randomly divided into two groups: sham group and AC-28 days group.After 28 days,the cardiac function of mice was detected by color Doppler ultrasound;the mice and their dissected hearts were weighed;the m RNA expression levels of cardiac fibrosis and cardiac hypertrophy in each group were detected by realtime q PCR;the cardiac fibrosis in each group was detected by Masson staining.2.Twenty four SPF grade 8-week male C57BL/6 mice were randomly divided into four groups: sham group,AC 1-day group,AC 3-days group and AC 7-days group,with 6 mice in each group.Realtime q PCR was used to detect the m RNA expression levels of FN1,COL1A2,CTGF,Cyr61 and ANKRD1;Western blot was used to detect the protein expression levels of Yap,TAZ,FN1,COL1A2,CTGF,Cyr61 and ANKRD1;immunohistochemistry was used to detect the expression levels of yap.3.Twenty four SPF grade 8-week male C57BL/6 mice were randomly divided into three groups: sham group,AC 28 day group,AC 28 day plus atorvastatin group,with 8 mice in each group.After 28 days,the cardiac function of mice was detected by color Doppler ultrasound;the mice and their dissected hearts were weighed and photographed;the m RNA expression levels of NPPA,NPPB,ANKRD1,CTGF and COL3A1 in the heart tissue of mice in each group were detected by realtime q PCR;Western blot was used The expression levels of Yap,TAZ,FN1,COL1A2 and α-SMA protein in the heart tissue of mice in each group were detected;the fibrosis of the heart tissue of mice in each group was detected by Masson staining;the cross-sectional area of the heart was detected by immunohistochemical staining;the m RNA expression of related genes in the heart tissue of mice in each group was detected by RNA sequencing At the cellular level1.The cardiomyocytes of neonatal SD rats were stimulated with phenylephrine(PE)to induce cell hypertrophy and establish cell model.Cardiomyocytes were pretreated with atorvastatin for 24 hours,and then with PE for 30 hours.The expression of NPPA and Myh7 m RNA was detected by realtime q PCR,and the expression of α-actin,F-actin and Yap was detected by immunofluorescence.2.Cardiomyocytes of 24 mice were pretreated with siyap / TAZ small molecule inhibitor and then treated with PE for 30 hours.The expression levels of NPPA and Myh7 m RNA were detected by realtime q PCR.Result At animal level1.abdominal aortic constriction(AC)can induce myocardial hypertrophy,myocardial fibrosis and heart failure(1)Compared with the sham group,the weight of the mice in AC 28 day group decreased significantly(P < 0.05).(2)Compared with the sham group,the ratio of heart to weight in AC 28 day group was significantly higher(P < 0.05).(3)The results of realtime q PCR showed that the m RNA expression levels of FN1,COL1A2 and COL3A1 in left ventricular tissue of AC 28 day group were significantly higher than that of sham group(P < 0.05);NPPA and NPPB m RNA expression of cardiac failure related genes were significantly increased(P < 0.05);Masson staining showed that the expression level of NPPA and NPPB m RNA in AC28 day group was significantly higher(P < 0.05);Masson staining showed that the expression level of NPPA and NPPB m RNA in cardiac hypertrophy and heart failure was significantly increased(P < 0.05);Masson staining The fibrosis area of left ventricular tissue in 28 days group increased significantly.(4)The results of echocardiography showed that compared with the sham group,the AC 28 day group had cardiac dysfunction,ventricular enlargement and wall thickening.2.AC can induce Yap activation and up regulate the expression of genes and proteins related to myocardial hypertrophy and fibrosis(1)Western blot showed that compared with sham group,yap and its classical downstream genes CTGF,Cyr61 and ANKRD1 were significantly increased in AC group(P < 0.05),and FN1 and col1 fibrotic related proteins in AC group were significantly increased(P < 0.05).(2)The results of realtime q PCR showed that compared with sham group,the m RNA levels of FN1 and COL1A2 were significantly increased in AC group(P <0.05),CTGF,Cyr61 and ANKRD1(P < 0.05),NPPA and NPPB were significantly increased in AC group(P < 0.05).(3)The results of immunohistochemistry showed that compared with sham group,Yap expression in heart tissue of AC group was significantly increased.3.Atorvastatin inhibited the activation of Yap,lowered the expression of fibrosis related genes and proteins,reduced myocardial hypertrophy and improved cardiac function.(1)Western blot showed that yap/taz,FN1,COL1 and α-SMA in left ventricular tissue of AC 28 day group were significantly up regulated(P<0.05),and YAP,TAZ,FN1,COL1 and α-SMA in left ventricular tissue of the Atorvastatin treated group were significantly lower than those in AC 28 day group(P<0.05).(2)The results of realtime q PCR showed that compared with sham group,the m RNA level of the fibrosis related genes in left ventricular tissue of AC 28 day group was significantly increased(P<0.05),CTGF and ANKRD1,the classical downstream genes of Yap were significantly up regulated in AC28 days group,the level of COL3A1 m RNA in left ventricular tissue fibrosis related genes in atorvastatin group was significantly decreased(P<0.05),while CTGF and ANKRD1 were significantly up regulated(P<0.05),and NPPA and NPPB were significantly lower in myocardial hypertrophy and heart failure(P<0.05)(3)The results of Masson staining showed that compared with the sham group,the area of left ventricular fibrosis in AC 28 day group was significantly increased,and that of the rats in the atorvastatin treatment group was significantly lower than that of AC 28 day group.(4)The results of immunohistochemistry showed that the cross-sectional area of the AC-28 day group was significantly increased compared with the sham group,and the cross-sectional area of the heart of the mice treated with atorvastatin was significantly reduced compared with the AC-28 day group.(5)RNA sequencing showed that atorvastatin could reverse most of the AC induced genes,including fibrosis related genes,myocardial hypertrophy and heart failure related genes.At the cellular level1.Atorvastatin can reduce PE induced cardiomyocyte hypertrophy by inhibiting Yap(1)Compared with the control group(NT),the expression of NPPA and MYH7 gene m RNA in myocardial cells was significantly increased in PE treatment group(P< 0.05),and the cardiomyocytes were hypertrophic.The expression of NPPA and MYH7 in atorvastatin treatment group or yap/taz group could be decreased(P < 0.05),and that of atorvastatin treatment group decreased.2.PE activates Yap by promoting F-actin aggregation(1)Compared with the control group(NT),F-actin aggregation and Yap entry increased in PE treatment group.Conclusion(1)Yap plays an important role in the development of cardiac hypertrophy induced by stress.(2)Atorvastatin has certain therapeutic effects on the myocardial hypertrophy,myocardial fibrosis and heart failure induced by stress.The mechanism may be based on the inhibition of atorvastatin on yap and its target genes. |
PDF Full Text Request