Role Of Yes-associated Protein In Activation Of Cardiac Fibroblasts Induced By Diabetic Cardiomyopathy

Background:Diabetic cardiomyopathy is a special cardiomyopathy that is independent of risk factors of coronary disease,hypertension and other cardiac disease.Diabetic cardiomyopathy is associated with structural and functional abnormalities that eventually lead to left ventricular hypertrophy,diastolic and systolic dysfunction as well as heart failure.Diabetic cardiomyopathy,serving as a common and serious complication,is the leading cause of high disability and mortality of diabetes mellitus.It is pathologically characterized by myocardial hypertrophy,interstitial fibrosis and vascular basement membrane thickening.It has been reported recently that cardiac fibrosis,which characterized by excessive deposition of extracellular matrix proteins,plays a pivotal role in the development and prognosis of heart failure in DCM.However,there is no effective antifibrotic treatment strategy available.Studies showed that the proliferation and conversion of CFs is the main resource and cause of fibrosis.Recently,many studies have shown that YES-associated protein(YAP)plays a significtant role in the development of cardiovascular system.In addition,YAP also have an effect on cardiovascular diseases.In myocardial infarction mice with heart-specific inactivation of YAP,the myocardial injury was aggravated due to the lack of fibrosis response.Conversely,mice with heart-specific overexpression of YAP increased the regeneration of cardiomyocytes,decreased infarction size and improved cardiac function.Separate from that,YAP also participates in the progress of artery injury and atherosclerosis.In artery injury mice,YAP was increased in vascular smooth muscle cells(VSMC)and promoted VSMC proliferation and migration by switch to secretory phenotype from contractile phenotype.Inhibition of YAP reduced injury-related pro-fibrotic gene expression,and blocked increased VSMC proliferation and migration,improved intimal hyperplasia.These findings suggest that YAP have a pro-fibrotic effect on cardiomyocytes and VSMC,as well as regulated myocardial infarction and atherosclerosis.However,it is not clear whether the YAP promotes cardiac fibroblast proliferation or the transition of these fibroblasts to a myofibroblast phenotype in diabetic cardiomyopathy.Objective:To investigate the effect and mechanism of Yes-associated protein on the influence of cardiac fibroblasts proliferation and activation induced by diabetes.Methods:In vivo experiment: the model of type II diabetes mellitus was established by high energy food and STZ intraperitoneal injection.The interstitial fibrosis of heart was detected by sirius red staining and masson staining.The protein expression level of Collagen I and collagen III was detected by immunohistochemical staining and western blot.The protein expression level of smooth muscle α-actin,YAP and connective growth factor was detected by western blot.In vitro experiment: the CFs were isolated and extracted from newborn SD rats.CFs were treated with D-glucose of concentration 40 mmol/L.The relative expression level of YAP,αSMA,collagen I,collagen III and connective tissue growth factor(CTGF)were detected by Western blot at the time points 24 hours,respectively.The cells were divided into normal glucose control group(5.5 mmol/L D-glucose),high glucose group(40mmol/L D-glucose),high glucose +0.5μmol/L verteporfin group,and high glucose +0.5μmol/L verteporfin group.VP is an inhibitor of YAP.,which blockade the interaction between YAP and TEAD.Immunofluorescence of vimentin was used to identify CFs.Immunofluorescence of Ki-67 was used to detect the proliferation activities of CFs in various groups.Western blot was used to detect the levels of α-SMA,collagen Ⅰ and collagen Ⅲ,YAP and CTGF proteins in the CFs in various groups.Results:In vivo:(1)Both the expression of YAP and the deposition of collagen protein(cardiac fibrosis)have increased in T2 DM heart.(2)The levels of α-SMA increased in diabetic heart,suggesting myofibroblast transdifferentiation take part in the development of cardiac fibrosis in diabetic cardiomyopathy.In vitro:(1)High glucose can stimulate myofibroblast transdifferentiation.(2)High concentrations of glucose contributed to the YAP and CTGF increased in CFs,and inhibition of YAP-TEAD interaction by verteporfin inhibited high-glucose-mediated CFs transdifferentiation.(3)In CFs,CTGF is a direct target of YAP,and YAP inhibitors inhibit HG-induced CFs differentiation by regulating CTGF expression.Conclusions:These findings suggest a potential therapeutic role for YAP in the regulation of CMFs transdifferentiation in response to high glucose induced by diabetes and indicate a comprehensive pathway driving CMFs formation in conjunction with TEAD and CTGF.