The Effect Of Deubiquitinase USP7 On Regulating Right Heart Fibrosis Induced By Pulmonary Hypertension By Stabilizing CTGF

【Background】Dilated cardiomyopathy is the main complication and cause of death in patients with pulmonary artery hypertension.Myocardial fibrosis is the main pathological mechanism of dilated cardiomyopathy.The activation,differentiation and secretion of extracellular matrix of cardiac fibroblasts involves the formation of myocardial fibrosis.Studies have shown that connective tissue growth factor(CTGF)is an important fibrosis regulator of various organs.In liver fibrosis,CTGF has a wide range of functions,involving the proliferation,differentiation,adhesion,migration,and extracellular matrix release of hepatic stellate cells.CTGF is not only mediated by TGF-β in renal fibrosis,but can also activate the Wnt pathway to enhance collagen expression.In the myocardial fibrosis,CTGF acts as an mediator can induce the expression of TGF-β through positive feedback upwardly,and it also can activate myocardial fibroblasts downwardly.But it’s unkown about stability regulation of it.In the early stage,we found that there was obvious fibrosis in the model of pulmonary artery constriction(PAC),in which the expression of ubiquitin-specific protease 7(USP7)was increased.In the studies of fibroblasts,we found there is an interaction between USP7 and CTGF.USP7 regulates the ubiquitination level of CTGF and regulates the proliferation of fibroblasts.This purpose of this study to explore the mechanism of USP7 may regulating myocardial fibrosis by stabilizing CTGF protein.【Methods】1.The right heart fibrosis model under right heart pressure overload is established by pulmonary artery contraction(PAC).The experiment was divided into two groups:WT Sham group and WT PAC group.Masson staining,was used to detect the changes of cardiac collagen fibers.Western blot was used to detect the expression USP7.2.The neonatal rat primary cardiac fibroblasts(NRCFs)were extracted and stimulated by TGF-β to construct myocardial fibrosis model in vitro.The experiment was divided into two groups: Blank and TGF-β.Using western blot to detect the expression of USP7 and fibrosis markers(Collagen Ⅲ、CTGF、α-SMA).3.LC-MS/MS analysis was used to investigate the correlation between USP7 and CTGF.4.Using co-immunoprecipitation to detect the interaction between USP7 and CTGF.And study the ubiquitination level regulation of CTGF by USP7.5.Adding cycloheximide(CHX)to NRCFs inhibits protein synthesis while inhibiting the effect of USP7.The experiment was divided into two groups,DMSO+CHX and P5091+CHX.Using western blot to detect the expression of CTGF and explore the stabilizing effect of USP7 on CTGF.6.In myocardial fibrosis model in vitro,using different treatments to inhibit the effect of USP7(P5091 inhibits the activity of USP7,si RNA interferes with the expression of USP7).MTS assay was used to detect the viability of NRCFs.Using western blot to detect the expression of CTGF and investigate the regulatory effect of USP7 on the proliferation of cardiac fibroblasts and CTGF.【Results】1.The expression of USP7 in right heart fibrosis tissue increased: the right heart of mice with PAC model developed obvious fibrosis.The heart tissue was taken out on the 3rd and 7th days after the establishment of the mouse PAC model,and the expression of USP7 protein was detected by Western blot after the removal of the atrium and left ventricular wall.The results showed that USP7 was significantly increased in the model group.2.USP7 has more expression in the myocardial fibrosis model in vitro: the NRCFs were extracted,and Western blot shows that the expression of USP7 protein was significantly increased under the stimulation of TGF-β.3.Interaction between USP7 and CTGF: LC-MS/MS analysis showed that USP7 interacted with CTGF.4.USP7 stabilizes the expression of CTGF: Western blot results show that inhibiting USP7 can accelerate the degradation of CTGF.5.USP7 regulates the ubiquitination of CTGF: Co-IP results showed that USP7 and CTGF can bind to each other,and inhibiting or knocking down USP7 can increase the ubiquitination level of CTGF.6.USP7 affects the proliferation of fibroblasts: MTS results showed that inhibiting or knocking down USP7 can decrease the viability of fibroblasts.Western blot results show that inhibiting or knocking down USP7 can decrease the expression of CTGF.【Conclusion】1.USP7 is increased in the myocardium of the right heart of mice after PAC.2.USP7 may regulate the fibrosis process of right ventricular myocardium through ubiquitination degradation of CTGF.