The Effects And Mechanism Of YAP On Myocardial Injury In Diabetic Cardiomyopathy

BackgroundDiabetic cardiomyopathy(DCM)is a cardiovascular complication of diabetes.It means that in the absence of hypertension and coronary artery diseases,the structure and function of the heart are damaged,mainly manifested as heart enlargement and decreased myocardial diastolic and systolic functions.Myocardial fibrosis is one of the main pathological changes in diabetic cardiomyopathy.But the specific pathogenesis of DCM is still unclear and remains to be studied.In recent years,studies have shown that the important effector molecule in the Hippo pathway,Yes-associated protein(YAP),is closely related to the pathological process of cardiovascular diseases.However,in the current research on diabetic cardiomyopathy,the effect and significance of YAP on myocardial injury are still unclear.Studies have shown that YAP can promote the expression of CTGF and Fibronectin proteins,thereby affecting the process of fibrosis.Exploring the role and influence of YAP on myocardial injury in diabetic cardiomyopathy may provide new ideas and targets for the treatment of diabetic cardiomyopathy.Objective1.To construct diabetic cardiomyopathy rat model,detect the change of yes-associated protein(YAP)expression in diabetic cardiomyopathy rats and the pathological changes in diabetic cardiomyopathy rats.2.To constructed the lenti virus vector of YAP gene silencing and observe the effect of YAP gene silencing on myocardial fibrosis and cardiac function in diabetic cardiomyopathy rats.3.To detect the change of YAP expression and fibrosis-related proteins in primary myocardial fibroblasts under high glucose stimulation and investigate the mechanism of YAP on myocardial injury in diabetic cardiomyopathy.Method1.Construction of lentivirus-packed YAP interfering RNA:the related gene sequences of YAP small interference in rats were searched according to the literature.Short hairpin RNA(shRNA)fragments were constructed corresponding to Yes-associated protein(YAP)which were encapsulated with recombinant lentivirus to form an lentivirus-coated YAP shRNA fragment by Genechem.Scramble-shRNA served as a negative.control.2.Animal model establishment:60 male 8-week-old Wistar rats.(200 ±15 g)were selected for the experiment.After a week of adaptive feeding,the rats were randomly divided into the normal control group(n=15)and the diabetes model group(n=45).Rats in the diabetes model group were first injected with streptozotocin(65mg/kg)diluted with citric acid buffer solution for a single large dose of intrauterine injection to establish the diabetes model.After a week of modeling,the rats that the blood glucose>11.1 mmol/L and accompanied with typical clinical symptoms of polydipsia,polyphagia and polyuria were considered as diabetic rat models.Twelve weeks after the establishment of the diabetic rat model,echocardiography was used to detect cardiac function in rats,the results showed that the cardiac was enlarged and systolic function decreased,which was considered as a successful diabetic cardiomyopathy model.Then the diabetic model group rats were divided into DCM group(n=15),LV-YAP-shRNA group(n=15)and LV-SC-RNA group(n=15).Rats in the LV-SC-RNA group and the LV-YAP-shRNA group were injected with 1×10^6 PFU(dissolved in 100μl normal saline)per rat of recombinant lentivirus containing Scramble-shRNA or YAP-shRNA via the tail vein.3 rats were randomly selected from each group at 2 weeks after virus injection to test the virus transfection efficiency.After virus injection for 4 weeks,ultrasonic examination was performed under anesthesia and the samples were sacrificed.3.Cardiac function detection:cardiac function was measured in all rats 4 weeks after lentivirus injection.After anesthesia,VEVO770 imaging system was used to evaluate the cardiac function of rats.4.General condition measurement of rats:blood glucose,body weight,heart weight and blood pressure were measured.5.Immunohistochemical staining:routine pathological sections of 4.5μm thick myocardial tissue were used for immunohistochemical staining to evaluate the expression of proteins such as Collagen I,Collagen III,CTGF,Fibronectin and PAI-1 in myocardial tissue.6.Evaluation of collagen deposition in rat cardiac interstitium by Masson trichrome staining.7.Myocardial injury was assessed by routine HE staining.8.Extraction and culture of primary cardiac fibroblasts:the hearts of Wistar rats aged 1-3 days were firstly extracted and separated and cut into pieces using collagenase to make a single-cell suspension,cardiac fibroblasts were isolated from cardiac myocytes according to the difference in cell adherent velocity and cultured for subsequent cell experiments.9.Follow-up experiments on cardiac fibroblasts:cardiac fibroblasts were transfected with YAP-shRNA and scramble-shRNA,and then cultured under normal or high glucose conditions for 48h.10.Western blot:Western blot was used to detect the expression of YAP and fibrosis-related indicators in the total protein of rat myocardial tissue and fibroblasts.11.Real-time PCR:total RNA of myocardial tissue and cardiac fibroblasts was extracted,and mRNA expression levels of YAP,collagen Ⅰ,collagen Ⅲ and PAI-1 were detected.12.Co-IP:to detect the binding of YAP and TEAD in primary cardiac fibroblasts.13.Ch-IP:to detect the relationship between YAP and CTGF.14.Statistical analysis:All data are expressed as the mean ± standard deviation.statistical analyses were evaluated using of GraphPad Prism 7.0 software(GraphPad Software,La Jolla,USA).Intergroup data were compared by one-way ANOVA.A value of P<0.05 was considered statistically significant.Results1.YAP expression was significantly increased in myocardium of diabetic cardiomyopathy rats.YAP gene silencing can down-regulate YAP expression in diabetic cardiomyopathy rats.2.In the diabetic cardiomyopathy rats,myocardium fibrosis was obviously aggravated and cardiac function was decreased.YAP gene silencing can improve myocardial fibrosis and improve cardiac function in diabetic cardiomyopathy rats.3.The expression level of YAP was significantly increased in the primary cardiac fibroblasts stimulated by high glucose,and the expression of fibrosis-related indicators could be down-regulated by YAP gene silencing.4.YAP can be involved in the pathological damage process of diabetic cardiomyopathy by binding to TEAD and affecting CTGF at chromatin level.ConclusionIn diabetic cardiomyopathy rats,the expression of YAP increased under the stimulation of high glucose.The increased expression of YAP was involved in the impairment of cardiac function and increased myocardial fibrosis in diabetic cardiomyopathy rats.YAP interfering RNA can effectively reduce the expression of YAP in the myocardium and the degree of myocardial fibrosis of diabetic cardiomyopathy rats,thus improving the cardiac function of diabetic cardiomyopathy rats.This may be related to the regulation of gene expression of CTGF by YAP through binding to TEAD.This discovery provides new ideas for the future treatment of diabetic cardiomyopathy.