The Role Of Deacetylase SIRT2 And Septin4 In Myocardial And Vascular Injury

Objective:Part I:Long-term poor control of hypertension leads to changes of cardiac structure and function,which is called hypertensive heart disease.It is mainly manifested by left ventricular diastolic dysfunction,left ventricular hypertrophy and progressive myocardial systolic dysfunction.Epidemiological studies have shown that70%of heart failure is caused by hypertension.Angiotensin II（Ang II）is the most important vasoactive peptide in the development of hypertension.On one hand,AngII can stimulate vascular contraction,which promotes the secretion of aldosterone and elevated blood pressure and increases cardiac afterload and myocardial injury;On the other hand,AngII can induce myocardial hypertrophy and fibrosis,which induces inflammation,oxidative stress and myocardial injury.In recent years,it has been found that posttranslational modification of protein is closely related to cardiovascular diseases^[1-3].Acetylation is one of the most important modifications in protein post-translational modification.Therefore,acetylation has gradually become the focus of cardiovascular diseases.However,the role of deacetylase in hypertensive myocardial injury is still unclear.Therefore,it is possible to elucidate the mechanism of deacetylase in hypertensive myocardial injury to provide new targets for the treatment of hypertension.SIRT2 plays an important role in lipid metabolism,glycometabolism,inflammatory response and oxidative stress^[4-13].SIRT2 can be used to modify FOXO1 by deacetylation and takes participate in the lipid metabolism^[4-5].PEPCK1 is an important enzyme in sugar metabolism,and SIRT2 can modify PEPCK1 by deacetylation and then regulates its ubiquitination to participate in the glucose metabolism response^[6-7].SIRT2 also plays an important role in suppressing inflammatory reaction and oxidative stress.SIRT2 is able to resist inflammation in brain tissue by combining and regulating NF-κB and SREBP-2^[8-9].Not only that,SIRT2 can also regulate the oxidative stress response by regulating ACLY and G6PD^[10-11].Recently,studies have shown that SIRT2 knockout aggravates senescent cardiomyopathy and myocardial hypertrophy^[12].Therefore,SIRT2 can play an important role in many biological behaviors,such as metabolism,inflammation and apoptosis.However,the SIRT2 deacetylation in hypertensive myocardial injury is still not known.Our study first knocked down SIRT2 and stimulated H9C2 with AngII,which aims to analyze the effect of SIRT2 on Ang II induced cardiomyocyte injury.We further constructed SIRT2 knockout mice and successfully completed the hypertensive myocardial injury model by Ang II microinjection pump.The purpose is to further explore the role of SIRT2 in AngII induced hypertension and hypertensive myocardial damage.Finally,in order to explore the possible mechanism of SIRT2function,we screened the interaction protein of SIRT2 by mass spectrometry.Part II:Atherosclerosis is the major risk factor of coronary heart disease,cerebral embolism and peripheral vascular disease,which has greatly threatened human health worldwide.Epidemiology studies showed that the prevalence of atherosclerosis is increasing rapidly in the developing countries,especially in China^[14].Vascular endothelial cells injury is one of the key and initial event in the development of atherosclerosis.Among the factors that induce vascular endothelial cells injure,oxidative stress including accumulation of reactive oxygen species（ROS）is the most common and important factor.Therefore,it is essential to explore the mechanism of oxidative stress induced vascular endothelial cells injury which may provide a new strategy in atherosclerosis treatment.PARP1 is a DNA repair enzyme which can be activated as a DNA damage receptor by identifying DNA damaged fragments.In addition,PARP1 is also the cutting substrate of caspases which are the core of cell apoptosis^[15].Numerous studies report that oxidative stress activates PARP-1 via DNA damage which consumes high levels of energy and leads to cell apoptosis^[16-17].Therefore,PARP1 plays a key role in DNA damage repair and cell apoptosis.Recent studies considered that PARP1 is an important factor involved in oxidative stress related cardiovascular diseases,including oxidative stress induced endothelial cell injury^[18].Suppression of PARP1 can significantly alleviate cardiovascular diseases both in vivo and in vitro experiments[19-20].Septin4 is a subtype of Septins family with GTPase activity,which has multiple splicing variants.Septin4 is widely expressed in the eukaryotic cells and mainly located in the chromosome 17q23^[21].Septin4 is considered to be an essential component of the cytoskeleton and involved in many important physiological processes,such as cell transport and apoptosis^[22-23].Septin4 has been considered as a tumor-inhibiting factor by promoting apoptosis of tumor stem cells^[24].Downregulation expression of Septin4 can significantly decrease the expression of DKK and negatively regulate inflammatory response and liver fibrosis^[25].In addition,Septin4 is involved in the Parkinson’s disease by regulating the E3 ubiquitin ligase Nedd4^[26].However,whether Septin4 is involved in cardiovascular diseases,such as oxidative stress inducted endothelial cell injury still unclear.Therefore,our purposes were exploring that whether Septin4 is involved in oxidative stress induced endothelial cell injury by interacting with PARP1.Methods:Part I:H9C2 was treated with 10^-7 mol/L,10^-6 mol/L,10^-5 mol/L AngII for24 h.CCK8 was used to detect cell viability and Western-blot was used to detect the expression of SIRT2,cleaved PARP1 and cleaved Caspase3.Next,we used the SIRT2inhibitor AGK2 inhibited the activity of SIRT2 and used lentiviral shSIRT2 to construct H9C2 stable knockdown of SIRT2 cell lines and giving AngII to induce H9C2 cell injury.And then,we used CCK8 to analysis cell viability of H9C2 cells and detected the apoptosis related protein by western-blot.In addition,we constructed SIRT2 gene knockout mice and used angiotensin II to establish the model of myocardial injury with hypertensive micro injection pump for 14 days.Echocardiography was used to measured heart rate,left ventricular ejection fraction and left ventricular diastolic function of SIRT2 wild-type and SIRT2 knockout mice,respectively.Mouse blood pressure apparatus was used to measured blood pressure and Masson staining was used to analyze mouse heart muscle fibrosis.Western-blot was used to analyze apoptosis,fibrosis related indicators.Finally,mass spectrometry was used to find new substrates for SIRT2 interaction.Part II:We used H₂O₂ as the inducement factor of HUVEC oxidative damage.After12 h treating HUVEC with three concentrations of 150,250,500 and 500μmol/L H₂O₂,the viability of cells was detected by CCK8.Then western-blot was used to analyze apoptosis related genes cleaved PARP1,cleaved Caspase3 and detect the expression of Septin4.Next,we use three Septin4-siRNA fragments to detect the knockdown efficiency of Septin4 in HUVEC and select the best segment and using150 mol/l,250 mol/l and 500 mol/l H₂O₂ to treat HUVEC for 12 h.And CCK8 assay was used to analyze HUVECs viability.Annexin V-fluorescein isothiocyanate（FITC）and PI assay were used to detect cell apoptosis.ROS mediated conversion of non-fluorescent 2,7-DCFH-DA to fluorescent DCFH was used to detect endogenous ROS.Western blot assay was used to detect pathway-related genes cleaved PARP1,cleaved caspase3 and Septin4 expression.In addition,we overexpressed Flag-Septin4and used H₂O₂ to treat HUVEC for 12 h.CCK8 assay was used to analyze HUVECs viability.Annexin V-fluorescein isothiocyanate（FITC）and PI assay were used to detect cell apoptosis.ROS mediated conversion of non-fluorescent 2,7-DCFH-DA to fluorescent DCFH was used to detect endogenous ROS.Western blot assay was used to detect pathway-related genes cleaved PARP1 and cleaved caspase3 expression.Finally,we used co-immunoprecipitation to detect the interaction of Septin4 and PARP1 in physiological status and oxidative stress status.Results:Part I:1,with the increase of Ang II intervention concentration,the activity of H9C2 cells were gradually decreased,while the expression of cleaved PARP1,cleaved caspase-3 and SIRT2 increased gradually.2,after giving AngII stimulation,compared to the normal group,SIRT2 knockdown group cells viability were significantly decreased,while the apoptotic indexes cleaved PARP1 and cleaved caspase-3 were significantly increased.3,Compared to the normal group,giving SIRT2 specific inhibitor AGK2 and AngII stimulation,cells viability were significantly decreased,while apoptotic index cleaved PARP1 and cleaved caspase-3were significantly increased.4,the echocardiogram of mice showed that after Ang II stimulation,compared with SIRT2 wild type mice,SIRT2-KO mice ejection fraction and diastolic function of the mice were significantly decreased.5,Masson staining showed that SIRT2 knockout mice were more fibrotic after AngII stimulation compared with SIRT2 wild type mice.6,Western-blot showed that compared with SIRT2 wild type mice,SIRT2 knockout mice had the higher expression of apoptosis related indicators and fibrosis related indicators after AngII stimulation.7,the blood pressure measurement in mice showed that there was no significant difference in blood pressure between SIRT2 knockout mice and SIRT2 wild type mice after AngII stimulation.8,mass spectrometric analysis showed that SIRT2 was interacted with HSP70,HSP71,HSP90a and HSP90b.Part II:1,HUVECs viability were significantly decreased by treating with H₂O₂ and the expression of Septin4 and apoptosis pathway-related genes cleaved PARP1 and cleaved caspase-3 were significantly increased with the increase of H₂O₂concentration.2,Septin4 knockdown inhibits H₂O₂ induced HUVECs cytotoxicity,apoptosis,ROS production and apoptosis pathway-related genes expression.3,overexpress Septin4 increases H₂O₂ induced HUVECs cytotoxicity,apoptosis,ROS production as well as apoptosis pathway-related genes expression.4,identification Septin4 as a novel PARP1 interacting protein and the interaction is enhanced under oxidative stress.Conclusions:Part I:this study demonstrated that SIRT2 was involved in AngII induced myocardial injury both in vivo and in vitro.AngII can induce the up-regulation of SIRT2 expression in myocardial cells,and knockdown SIRT2 in cardiac myocytes or giving SIRT2 activity inhibitor AGK2 were significantly aggravate AngII induced myocardial injury.In addition,we found that AngII induced mice cardiac dysfunction and myocardial fibrosis which were more serious after SIRT2 knockout.However,SIRT2 knockout did not affect the increase of blood pressure and heart rate,which were induced by AngII.Part II:In conclusion,our study indicates that Setpin4 is involved in oxidative stress induced vascular endothelial cell injury by interacting with PARP1.Suppression of Setpin4 significantly alleviates oxidative stress induced vascular endothelial cell injury.Our study may establish a novel strategy in endothelial injury treatment and suppression of Setpin4 is a potential therapeutic agent.