Deubiquitinase BRCC36 Protects Heart Against Chronic Pressure Overload-induced Cardiac Remodeling In Mice And Cardioprotective Effects Of Triptolide

The abnormal persistent high on pressure of vascular system in body such as hypertension could cause cardiac over-stress,resulting in deterioration of cardiac structure and pump function,such as the lack of effective and timely treatment intervention,will eventually lead to the occurrence of heart failure,also can be accompanied with malignant arrhythmia,coronary heart disease and other complications at the same time.The cardiac fibroblasts could proliferate and transform into myofibroblast under the action of chronic pressure overload,and synthesize a large number of extracellular matrix(ECM)such as type ?/? collagen,which deposits in the interstitial space of heart tissue,could increase cardiac wall stiffness,decreased myocardial compliance and impair normal ventricular diastolic function.In addition,excessive deposition of extracellular matrix around the coronary artery also affect the normal exchange of oxygen and metabolites,causing myocardial ischemia and energy metabolism disorders,will increase myocardial cell hypertrophy and even apoptosis.As the main constituent cells of heart tissue,the biological function changes of cardiomyocytes and cardiac fibroblasts are the main pathophysiological basis of the heart remodeling under the chronic pressure overload.The study on the key signaling pathways and related regulatory molecules in the process of cardiac remodeling have always been the focus of the study all over the world.It is of great clinical significance in the development of new methods of diagnosis and treatment of hypertension.In the preliminary research work,we have analyzed the cardioprotective effects of atrial natriuretic peptide(ANP).We found that the ANP can promote the transformation of intracellular GTP to cGMP and activate ANP-cGMP-PKG signaling pathway by binding its cell membrane type A receptor(APR-A),which can antagonize the cardiac remodeling mediated by hyperactivity of TGF-?1 signaling pathway under the chronic pressure overload.By 2D electrophoresis and mass spectrometry analysis,it was found that the Smad3 which is main signal molecule of transforming growth factor-?1(TGF-?1)in the cells enhances binding to deubiquitinase BRCC36 under the action of cGMP.Subsequently,deubiquitinase BRCC36 withholding Srmad3 with the cytoskeleton protein ?2-tubulin together and prevents Smad3 translocated to the nucleus to regulate target gene,thereby inhibiting TGF-?1-mediated fibrosis.However,whether BRCC36 can interact directly with Smad3,as well as the specific form of action and the corresponding biological effects,lack of research reports at home and abroad.The deubiquitinase BRCC36 is a newly discovered intracellular active substance in 2003 and belongs to the metallopeptidase JAMM/MPN+ family,which can specifically hydrolyze the ubiquitin chain formed by the K63 site-linked,thereby reducing the ubiquitination of substrate.BRCC36 is constitutively expressed in a variety of eukaryotic organisms and can participate in a number of cellular biology processes such as cell signaling and DNA damage repair processes.However,BRCC36 is closely related to the profibrotic TGF-?1/Smad3 signaling pathway,whether it can play a regulatory role in cardiac remodeling under the chronic pressure overload is not known and needs further experimental study.Triptolide(TPL)is a diterpene lactone that is chemically extracted from the roots of Tripterygium wilfordii Hook F.TPL has a significant immune regulation and anti-inflammatory effects,is widely used in the treatment of nephrotic syndrome,systemic lupus erythematosus and other chronic immune system diseases in clinical.Based on the anti-inflammatory and anti-proliferative characteristics and the importance of inflammatory response and fibrosis in the process of cardiac remodeling,we investigated the effects of triptolide on chronic pressure overload induced cardiac remodeling and potential mechanisms.For this reason,we used a model of chronic pressure overload induced cardiac remodeling in C57BL/6 male mice by transverse aortic constriction which is internationally recognized.To detect the expression of BRCC36,TGF-?1 and related cytokines in the heart tissue.And then construct a transgenic mouse with systemic expression of BRCC36,to investigate the effect of BRCC36 on cardiac remodeling under the chronic pressure overload and its possible mechanism.Followed by primary culture cardiac fibroblasts of C57BL/6 mice and infect recombinant adenovirus that can regulate the expression of intracellular BRCC36,investigate the interaction and possible links of BRCC36 and TGF-?1/Smad3 signaling pathway directly at the cellular level.By translating Smad3 plasmids containing different domains into 293T cells,to further clarified that BRCC36 was a functional deubiquitinase of Smad3 that specifically hydrolyze the ubiquitin chain formed by the K63 site-linked and the possible structural regions of Smad3 when interacts with BRCC36.Finally,the effects of triptolide on cardiac remodeling under the chronic pressure overload and the effect on BRCC36 expression were investigated in vivo level.The research will be described in five parts as follows:Part ?The expression change of BRCC36,TGF-?1 and related cytokines in chronic pressure overload induced cardiac remodelingObjective:To observe the dynamic changes of BRCC36 and TGF-?1-related cytokines during chronic pressure overload induced cardiac remodeling.Methods:C57BL/6 wild male mice(10-12 weeks old)were subjected to TAC operation or sham operation for 2 weeks,4 weeks and 8 weeks respectively,using transthoracic cardiac echocardiographic to assess the success of model.Detecting the cTGF,PAI-1,Periostin mRNA expression by qPCR,detecting the expression of BRCC36,TGF-?1,total Smad3(t-Smad3),phosphorylated Smad3(p-Smad3)and apoptosis-related proteins by Western blot.Immunohistochemical staining,Masson staining and TUNEL staining were used to observe the pathological changes of cardiac tissue.Results:The cardiac remodeling was observed after TAC operation,and a number of functional indicators worsened,suggesting that cardiac remodeling model was established.Masson staining showed increased myocardial collagen deposition.HE staining showed increased in cardiac apoptotic cells and cardiomyocyte hypertrophy.Western blot showed that the expression of BRCC36 was down-regulated in TAC,the expression of TGF-?1 and p-Smad3 were significantly increased,but the level of t-Smad3 was not changed.The expression of pro-apoptotic protein Puma,Bad were up-regulated,and the expression of pro-survival protein Bcl-2 and Mcl-1 were decreased.These phenomena are more pronounced with the prolongation of the pressure load.The target gene of TGF-?1/Smad3 signaling pathway such as cTGF,PAI-1,Periostin and a-SMA were up-regulated after TAC operation.Conclusion:Excessive activation of TGF-?1/Smad3 signaling pathway is an important factor during chronic pressure overload induced cardiac remodeling.It was also confirmed that the expression of BRCC36 was significantly changed,suggesting that BRCC36 may be involved in chronic pressure overload induced cardiac remodeling and is closely related to TGF-?1/Smad3 signaling pathway.Part ?Study on the role of BRCC36 involved in chronic pressure overload induced cardiac remodelingObjective:To clarify the effect of BRCC36 in chronic pressure overload induced cardiac remodeling.Methods:Generated the transgenic mice with systemic overexpression of BRCC36.TAC was used to construct the chronic pressure overload model.Transthoracic cardiac echocardiographic to assess the heart structure and function change after 8 weeks of TAC operation.Immunohistochemical staining,Masson staining and TUNEL apoptosis staining were used to observe the pathological changes of cardiac tissue.Detecting the cTGF,PAI-1,Periostin mRNA expression by qPCR,detecting the expression of BRCC36,TGF-?1,total Smad3(t-Smad3),phosphorylated Smad3(p-Smad3)and apoptosis-related proteins by Western blot,detecting the K63 site-linked special ubiquitination levels of Smad3 by immunoprecipitation.Results:Systemic overexpression of BRCC36 was successfully constructed and the expression of BRCC36 was significantly overexpressed in cardiac tissue.Compared with TAC wild mice,overexpression of BRCC36 was able to relieve chronic pressure overload induced left ventricular concentric pachynsis,improve ventricular systolic and diastolic function.Masson staining showed that the cardiac collagen deposition was alleviated,and the reduction of type I and ? collagen secreted by cardiac fibroblasts were dominant.HE staining showed cardiomyocyte hypertrophy was decreased,the damage of the normal structure of myocardial fibers decreased.Western blot analysis showed that overexpression of BRCC36 in the heart did not affect the expression of TGF-?1 and t-Smad3 induced by chronic pressure overload,but could decrease the level of p-Smad3 and inhibit the downstream of TGF-?1/Smad3 pathway.Meanwhile,the K63 site-linked ubiquitination level of Smad3 was decrease.In addition,the overexpression of BRCC36 in the heart can inhibit the expression of pro-apoptotic protein Puma and Bad,and enhance the expression of pro-survival protein Bcl-2 and Mcl-1.Conclusion:BRCC36 has a protective effect on chronic pressure overload induced cardiac remodeling,which may be related to inhibition of TGF-?1/Smad3 signaling pathway activation and promotion of cardiomyocyte survival and reduction of apoptosis.The regulation of BRCC36 on Smad3 was associated with changes of Smad3 K63 site-linked ubiquitination level.Part ?Study on the mechanism of BRCC36 in the regulation of TGF-?1/Smad3 signaling pathwayObjective:To investigate the specific mechanism of BRCC36 in the regulation of TGF-?1/Smad3 signaling pathway and its biological effects.Methods:The recombinant adenoviruses with reduced-expression/overpexression of BRCC36 were constructed,followed by infection of primary cultured adult mouse cardiac fibroblasts in vitro.Detecting the change on phosphorylation of Smad3 Ser423/425 site,the Smad3 translocation into the nucleus,target gene expression induced by TGF-?1 using Western blot,qPCR,immunohistochemistry,immunofluorescence staining.Detecting the K63 site-linked ubiquitination of Smad3 by immunoprecipitation.Detecting the proliferation of CFs by CCK-8.Results:TGF-?1 significantly induced the activation of TGF-?1/Smad3 signaling pathway.Smad3 phosphorylation was the strongest at 30 min point after administration.TGF-?1 can feedback to notably induce BRCC36 expression after 12 hours stimulation.Compared with cardiac fibroblasts transfected with control adenovirus,overexpression of BRCC36 inhibited TGF-?1-induced phosphorylation of Smad3 and reduced the K63 site-linked ubiquitination level of Smad3.Immunofluorescence showed that overexpression of BRCC36 inhibited Smad3 translocation into the nucleus.The expression of cTGF,PAI-1,Periostin and a-SMA mRNA were down-regulated,and the proliferation of CFs induced by TGF-?1 was also inhibited to some extent.Conclusion:BRCC36 inhibits TGF-?1-induced phosphorylation and nuclear translocation of Smad3 by reducing the K63 site-linked ubiquitination level of Smad3,resulting in inhibiting the activation of TGF-?1/Smad3 signaling pathway.The proliferation of cardiac fibroblasts and phenotypic transformation and extracellular matrix gene expression induced by TGF-?1 were also inhibited.BRCC36 expression can be induced by TGF-?1,which could negative regulation of TGF-?1/Smad3 signaling pathway activation.Part ?Study on the mechanism of interaction between BRCC36 and Smad3Objective:To clarify the possible structural regions of Smad3 interacting with BRCC36.Methods:Smad3 plasmids containing full length and different domains were co-transfected into 293T cells with BRCC36 plasmids respectively.The possible domains of interaction between BRCC36 and Smad3 were identified by immunoprecipitation method.Results:Immunoprecipitation showed that the BRCC36 signal band was detected only in the precipitation containing MH2 domain of Smad3 fragment.Ubiquitination analysis showed that BRCC36 significantly reduced the K63 site-linked ubiquitination level of Smad3 fragment only containing the MH2 domain.Conclusion:BRCC36 can hydrolyze the ubiquitin chain of Smad3 formed by the K63 site-linked through interacting with MH2 domain of Smad3 directly.Part ?The effect of triptolide on cardiac remodeling and BRCC36 expressionObjective:To observe the effect of triptolide on cardiac remodeling induced by chronic pressure overload and whether it is related to the regulation of BRCC36 expression.Methods:C57BL/6 mice were subjected to TAC operation or sham operation for six weeks,then randomly divided into the sham with vehicle group,TAC with vehicle group,TAC with low dose triptolide treatment group(20 ug/kg/day)and TAC with high dose triptolide treatment group(100ug/Kg/day)respectively for additional six weeks.The effect of triptolide on cardiac remodeling induced by chronic pressure overload were evaluated by immunohistochemical staining,Sirius red staining,qPCR and western blot.Results:Compared with the TAC+vehicle group,triptolide could improve cardiac structural and cardiac function under the chronic pressure overload;inhibit profibrotic factor TGF-?1 overactivation-mediated cardiac remodeling;relieve Type ?/? collagen deposition in heart tissue and cardiomyocyte hypertrophy;reduce the expression of NLRP3 inflammasome,down-regulated the expression of inflammatory mediators and inflammatory cell infiltration in the heart tissue;up-regulated the expression of the BRCC36 under the chronic pressure overload.Conclusion:Triptolide has a protective effect on cardiac remodeling induced by chronic pressure overload.The mechanism could be related to the inhibition of NLRP3 inflammasome expression and the inhibition of TGF-?1/Smad3 signaling pathway.The inhibitory effect of triptolide on TGF-?1/Smad3 signaling pathway was associated with up-regulation of BRCC36,which was closely related to the negative regulation of TGF-?1/Smad3 signaling pathway.