Study On The Therapeutic Effect And Mechanism Of HDACi In Heart Failure With Preserved Ejection Fractio

BackgroundHeart failure with preserved ejection fraction(HFpEF)accounts for about 50%of the total heart failure patients,and its hospitalization and mortality are similarto heart failure with reduced ejection fraction(HFrEF).The clinical treatment in HFpEF is based on HFrEF treatment-related drugs,to heal complications such as hypertension and diabetes,attenuating the symptoms of heart failure,and there is currently no specific treatment drug for HFpEF.A number of drug studies and clinical trials for HFpEF have been carried out in the past decades,and different degrees of therapeutic effects have been seen in animal experiments,but subsequent clinical trials have all failed.It is currently believed that in the process of drug development in the past,new drugs often play a role in the treatment of HFpEF by attenuating hypertension and diabetes,and no new drug targets have been proposed.In clinical stuff,the improvement of hypertension and diabetes has limited benefit on heart failure symptoms,and some patients with HFpEF are not accompanied by such complications,so the treatment methods are more limited.Therefore,it is particularly important to explore new drug targets.Histone deacetylase(HDAC)was first found to have the function of regulating histone acetylation.With the research deepening,researchers found that HDAC can also regulate the acetylation of other proteins,thereby exerting biological functions.The application of HDACi to inhibit HDAC activity has been proven to have corresponding therapeutic effects in a variety of diseases.Among the HDACi currently being put into clinical treatment,Chidamide has been approved for the treatment of T-cell lymphoma in China,and SAHA has been approved by the U.S.FDA for T-cell lymphoma.In the cardiovascular areas,animal experiments have confirmed that SAHA can improve ischemia-reperfusion injury,cerebral ischemia-reperfusion injury,myocardial hypertrophy,etc.The researchers further explored and found that SAHA may inhibit inflammation,maintain mitochondrial homeostasis,and improve energy supply.Other mechanisms are related,and relevant clinical trials have been gradually carried.MethodsThis study mainly focuses on the therapeutic effect of HDACi on HFpEF,and explores the molecular mechanism through in vivo animal experiments,in vitro cell experiments and cardiomyocyte-specific knockout mouse animal models.The specific contents and methods are as follows:1)Through dietary induction(High-fat diet and L-Name)to construct an animal model of HFpEF,and subcutaneously injected with HDAC inhibitor(SAHA 25 mg/kg/d)for 2 weeks.Functional tests such as exercise distance,ECHO and blood pressure measurement were used to determine the improvement of HFpEF symptoms in mice;2)After HDACi drug treatment for two weeks,the heart tissue of mice was harvested for pathological staining such as HE,PSR,Masson and WGA to observe the morphological changes of cardiomyocytes and heart tissue;3)Obtained mouse heart left ventricular tissue for transcriptome sequencing,further bioinformatics analysis to explore the main signaling pathway changes caused by HDACi drugs in HFpEF,and carried out PCR and Western Blot verification in mouse heart tissue samples;4)Isolated myocardium Cell mitochondria were detected by HDACi on the improvement of mitochondrial function of cardiomyocytes;5)Isolation of rat primary cardiomyocytes,administration of TNF-α to induce myocardial inflammation,administration of HDACi and siRNA to inhibit the expression of HDAC1/2/3,and observation of the expression levels of inflammatory factors Change the situation,parallel CO-IP and ChIP experiments to detect how HDACi regulates to rget gene changes;6)Construct cardiomyocyte-specific knockout HDAC1/2/3 mice,and give HFpEF diet for 7 weeks and 15 weeks,and test the mice functional experiments were performed to determine the effect of HDAC knockout on the phenotype of HFpEF,and myocardial tissue was collected from mice to detect changes in the expression levels of related signaling pathway proteins and mRNAs.Results1、HDACi(SAHA)drug treatment for two weeks could significantly improve the cardiac diastolic dysfunction of HFpEF mice(reduce E/A and E/e ’ratio,increase GLS%),slightly reduced blood pressure and alleviated the impairment of exercise endurance.2、WGA staining showed that HDACi drug treatment did not improve myocardial hypertrophy in HFpEF mice,and the expression of hypertrophy related proteins and genes did not change significantly;PSR and Masson staining suggested that ventricular fibrosis could be improved by HDACi.The detection of fibrotic protein and gene expression found that HDACi drugs inhibited the activation of fibrotic signal pathway in varying degrees.3、RNA seq data and GSEA analysis showed that HDACi may play a therapeutic role by improving the mitochondrial function of cardiomyocytes and inhibiting cardiac inflammation.4、The mitochondria of mouse cardiomyocytes were isolated and the related oxidative respiratory function was detected.It was found that the mitochondrial function of HFpEF central myocytes was damaged,but the therapeutic effect of HDACi was limited;By detecting the expression of inflammatory factor related signal pathway proteins and genes,it was found that HDACi could inhibit HFpEF induced inflammatory activation and reduce NFKb p65 expression in heart tissue.5、In primary rat cardiomyocytes,knockdown of HDAC1/2/3 by HDACi or siRNA can inhibit TNF-α Induced NFκb p65 expression and nuclear translocation,reducing NFKb p65 binds to iNOS gene(NOS2)and inhibits iNOS expression.6、HDAC1/2/3 cardiomyocyte specific knockout can inhibit the progression of HFpEF disease in mice,inhibit the activation of inflammatory factors in cardiac tissue,and improve the diastolic dysfunction of HFpEF.Conclusion5.1.HDACi has therapeutic effect on HFpEF mice,which can reduce blood pressure,alleviate diastolic dysfunction,inhibit ventricular fibrosis and improve myocarditis.5.2.Inhibiting the expression of HDAC1/2/3 can reduce NFKb p65 expression and nuclear translocation in cardiomyocytes,reduce iNOS expression and cardiomyocyte inflammation;Inhibition of HDAC1,HDAC2 or HDAC3 alone does not have the above effect.In clinical drug research and development,it may be necessary to consider a variety of HDAC inhibition in order to achieve therapeutic effect.