Effect Of Histone Methylation Modification Related Proteins On ISO-induced Cardiac Hypertrophy

BackgroundCardiovascular disease is a major problem that is difficult to overcome in the medical area.Myocardial hypertrophy is the inevitable result of the development of various cardiovascular diseases,and it is also one of the most dangerous factors in cardiovascular disease.The early stage of myocardial hypertrophy is an adaptive response,but the long-term myocardial hypertrophy will eventually cause heart failure,and the death rate will be extremely high in the end.The various signal pathways cause hypertrophic growth of the heart,resulting in the transcription system disorders.Accompanied by the development of genomics,the role of epigenetic modification in disease mechanisms has been paid more and more attention.Particularly,the expression of histone methylation-modulating regulatory genes is considered to be related to a variety of pathological signaling pathways,play roles in the development of variety diseases,and it has become a hot topic in recent years.The hotspot of current research are the histone demethylase LSD1 and histone methylase EZH2.LSD1 can demethylate the histone H3 at the 4th lysine(H3K4)and histone H3 at the 4th lysine(H3K9),and show its biological function.In many types of disease,the increase of LSD1 expression was positively correlated with the gain of it's function.Since LSD1 was a key gene for the regulation of H3K4 and H3K9 in normal cells and cancer cells,changes in histone methylation status by altering LSD1 expression may play an important role in the development and progression of certain diseases.EZH2 exerts its function by trimethylation of lysine(H3K27)at position 27 of histone H3.It is shown that the levels of EZH2 and H3K27 trimethylation were increased during the differentiation of cardiac progenitor cells into cardiomyocytes.The stability and functional enhancement of EZH2 can inhibit cardiac hypertrophy.It has been reported that Micro RNA-214 induced cardiac hypertrophy by inhibiting EZH2,but the relationship between EZH2 and cardiac hypertrophy is still unknown.Therefore,it is necessary to further investigate the relationship between histone methylation modification and cardiac hypertrophy signaling pathway.Objective1.To investigate the role of lysine specific demethylase 1(LSD1)in the pathogenesis of pathogenic cardiac hypertrophy induced by isoproterenol(ISO)in order to study the relationship between histone methylation modification and cardiac hypertrophy,and to find new targets for effective clinical drugs for the development of treatment and prevention pathological cardiac hypertrophy.2.To construct specific EZH2 overexpression plasmids and EZH2 catalytic subunit set domain deletion plasmid,assess its expression level in 293 T cells,to provid new tools for the late study of this project,finding the potential roles of myocardial hypertrophy.Methods1.LSD1 inhibition experiment: Healthy male Wistar rats,weighing 140 ~ 180 g,were breeded in SPF-type methods for 48 h.First,24 rats were randomly divided into 4 groups: normal control group(CTRL),ISO treatment group(ISO),LSD1 inhibitor OGL002 group(OGL002)and ISO treatment + LSD1 inhibitor OGL002 group(ISO + OGL002),6 rats per group.ISO treatment group and ISO + inhibitor OGL002 group were injected intraperitoneally 2 mg / kg per day.ISO,CTRL and OGL002 were injected intraperitoneally with the same volume of saline for one week.OGL002 group and ISO + OGL002 group were intraperitoneally injected with LSD1 inhibitor OGL002 solution at a concentration of 50 ?g / kg.The dissolution method was provided by the company.The remaining groups were injected with equal volume of drug dilution for one week.2.LSD1 overexpression experiment: The rats were randomly divided into four groups: normal control group(CTRL),ISO treatment group(ISO),normal rats with LSD1 overexpression virus group(LSD1-OV),and ISO treatment with LSD1 overexpression virus group(ISO+LSD1-OV),and 6 rats in each group.ISO and ISO + LSD1 overexpression virus concentration rats were injected intraperitoneally 2 mg / kg per day.ISO,CTRL and normal rats LSD1 overexpression were injected intraperitoneally with the same volume of saline for one week.Then normal rats LSD1 overexpression virus concentration group and ISO treatment + LSD1 overexpression virus concentration group were given daily ventricular injection of LSD1 overexpression virus,CTRL and ISO treatment group were given intraventricular injection of equal volume of medium for one week.Take the myocardial tissue of the experimental animals from each groups,and the phenotype of cardiac hypertrophy was measured by heart-to-weight ratio and HE staining.Real-time quantitative PCR was used to detect the three hypertrophic factors(MLC-2V,MHC,ANP)and LSD1 m RNA expression.The expression levels of four downstream histones(H3K4me1,H3K4me2,H3K9me1 and H3K9me2)were detected by Western Blot,immunohistochemistry and immunofluorescence.3.EZH2 overexpression and EZH2 catalytic subunit set domain deletion overexpression plasmid construction and identification:The rat EZH2 overexpression plasmid and the SET domain deletion plasmid were constructed by PCR and other gene manipulation methods.The recombinant plasmid was transfected into 293 T cells by Lipofectamine 2000.Real-time quantitative PCR and Western Blot were used to validate EZH2 and H3K27me3 at the level of RNA and protein expression.Results1.LSD1 inhibition experiments(1)Physiological indicators showed that the Bp and HR of rats from the ISO group,the LSD1 inhibitor(OGL002)group and ISO + OGL002 group were significantly lower compared with rats of the control group,and ISO treatment + OGL002 group was the lowest(n = 6,P <0.01).(2)The myocardium phenotype and cardiac output were significantly higher in rats of the ISO group,OGL002 group and ISO + OGL002 group than those of the control group,and the ISO + OGL002 group was the highest in the four groups(n = 6,P <0.05).(3)The results of HE staining showed that the cross-sectional area of cardiac cells in rats of the ISO group,OGL002 group and ISO + OGL002 group were significantly higher than those of the control group.(4)The expression of three hypertrophic factors(MLC-2V,MHC,ANP)in ISO group,OGL002 group and ISO + OGL002 group were significantly higher than those in control group(P <0.05).Hypertrophy factors expression level of the rats from OGL002 group was the highest,and was about 4-5 times of the control group.(n= 6,P <0.05).(5)LSD1 expression level.The results of q PCR,Western Blot,immunohistochemistry and immunofluorescence detection showed that the m RNA and protein expression levels of LSD1 of rats from the ISO group,OGL002 group and ISO + OGL002 group were significantly decreased compared with those from the control group.The m RNA of LSD1 of rats from the control group was about 4 times about those from the ISO and OGL002 group,and 6 times about those from the ISO + OGL002 group.(n = 6,P <0.01).(6)Four downstream histones of LSD1(H3K4me1,H3K4me2,H3K9me1,H3K9me2).Western Blot,immunohistochemistry and immunofluorescence detection showed that the protein expression levels were significantly upregulated in rats from ISO group,OGL002 group and ISO + OGL002 group.The histones expression in ISO + OGL002 group were the highest.2.LSD1 overexpression experiments:(1)Physiological indicators showed that HR and Bp were significantly down-regulated in ISO group compared with control group,HR and Bp were significantly increased in LSD1-OV group and ISO + LSD1-OV group compared with ISO group(n = 6,P <0.01)(2)The myocardial phenotype and cardiac output were significantly higher in rats from the ISO group than those from the control group.The LSD1-OV group and the ISO + LSD1-OV group were significantly lower than those in the ISO group(n = 6,P <0.05)(3)HE staining results showed that the cross-sectional area of the cardiac cells in the rats from the ISO group were significantly higher than those from the control group.The cross-sectional area of the LSD1-OV group and the ISO + LSD1-OV group was significantly lower than that of the ISO group.(3)Three hypertrophic factors(MLC-2V,MHC,ANP)expression were significantly up-regulated in the ISO group compared with the control group.However,compared with the ISO group,the expression of the hypertrophic factors were significantly lower in the LSD1-OV group and the ISO + LSD1-OV group(P <0.05).The expression level of ANP was down-regulated in LSD1-OV group compared with control group(n = 6,P <0.05).(5)LSD1 expression level.q PCR,Western Blot,immunohistochemistry and immunofluorescence detection showed that the m RNA and protein expression levels were significantly downregulated in the ISO group,however,were significantly upregulated in the ISO+LSD1-OV group and the LSD1-OV group compared with the ISO group and the control group,respectively.(n = 6,P <0.01).(6)Four downstream histones of LSD1(H3K4me1,H3K4me2,H3K9me1,H3K9me2).Western Blot,immunohistochemistry and immunofluorescence detection showed that four histones expression levels of ISO group were significantly upregulated,however,were significantly downregulated in the ISO+LSD1-OV group and the LSD1-OV group compared with the ISO group and the control group,respectively.(n = 6,P <0.01).3.EZH2 overexpression plasmid and SET domain deletion plasmid.Western blot and real-time quantitative PCR showed that overexpression of EZH2 plasmid was successfully transfered into 293 T cells.Due to the absence of the SET domain,the expression level of H3K27me3 protein in the overexpression plasmid group of the SET domain was not changed,indicating the importance of the SET domain position for the methylation function of EZH2.Conclusions:1.The study preliminary demonstrated the relationship between histone methylation modification and cardiac hypertrophy.ISO may induce cardiomyocyte hypertrophy by inhibiting LSD1 gene expression and upregulating the expression of hypertrophic factors,while LSD1 overexpression may partially reverse ISO-induced cardiac hypertrophy.The results provide a theoretical basis for using LSD1 as a new target of clinical treatment of myocardial hypertrophy.2.The overexpression plasmid and SET domain deletion plasmid of EZH2 were successfully constructed,which provided a reference for the relationship between protein modification and cardiac hypertrophy signaling pathway in the future.