| Background:Myocardial ischemia reperfusion(MI/R)injury is the major reason acute myocardial infarction(AMI)patients fail to reduce mortality and incidence of heart failure after timely restoration of coronary blood flow,leading to heavy social and medical burden.In recent years,epidemiological surveys showed that the number of patients with AMI increased sharply and exhibited younger trend.Therefore,it is particularly urgent and important to elaborate the precise regulation mechanism of MI/R pathological process in details and propose feasible prevention and treatment strategies to reduce MI/R injury.MI/R injury involves multiple pathological mechanisms,such as oxidative stress,inflammation,imbalance of energy metabolism,calcium overload,imbalance of survival and death signaling pathways,etc.A large number of studies have explained and confirmed how various pathological mechanisms lead to cardiomyocyte damage.However,the proposed interventions targeting these pathological processes have not shown significant myocardial protective effect in clinical treatment.This suggests that we need to explore the regulation of MI/R injury mechanism further to find more crucial regulatory targets for intervention.Histone modification,which belongs to the field of epigenetics,can control the opening and closing of DNA sequence transcription,change gene expression level,maintain cell identity and affect cell functions and fates by modifying histone amino acid sites which bound to DNA,rather than affecting DNA sequence.The trimethylation at 9 lysine site of histone H3(H3K9me3)is involved in the formation of heterochromatin.Showing flexibility and diversity in the silencing pluripotent genes and non-coding regions,and playing important biological roles in embryonic development,cell characteristic maintenance,cell aging and fate regulation.Recent studies have confirmed that with the help of its regulatory enzymes,H3K9me3 is also involved in the occurrence and development of a variety of cardiovascular diseases,such as myocardial hypertrophy,coronary atherosclerosis and myocardial infarction.However,the role of H3K9me3 in MI/R injury has been poorly studied,and influence on regulating MI/R pathological processes remains indistinct.Therefore,this study aims to explore whether H3K9me3 level of cardiomyocytes changes during MI/R injury and find the possible mechanism involved in regulating the pathological process.To promote the research process of histone modification in MI/R injury,and provide a new idea for seeking more effective myocardial protective targets.Aims:1.To determine whether H3K9me3 level of cardiomyocytes changes during MI/R injury.2.To screening the modification enzymes that play a major role in regulating the change of H3K9me3 in MI/R injury.3.To explore the specific regulatory mechanism of H3K9me3 in the pathological process of MI/R injury.4.To test and analyze changes of H3K9me3 in peripheral blood mononuclear cells of AMI patients preliminarily.Methods:1.In order to observe whether the H3K9me3 level changes when MI/R injury occurs,a mouse MI/R injury model was established.The myocardial tissue samples of the Sham operation group,after 3h,6h and 24 h ischemia-reperfusion were taken respectively,and detected the changes of H3K9me3 level by western blot(WB).The myocardial ring of the most obvious change timepoint was confirmed by H3K9me3 immunofluorescence staining.2.In order to screen the modification enzymes mainly involved in regulating H3K9me3 during MI/R injury,we selected the myocardial tissues with the most obvious changes in H3K9me3,MI/R3 h.Using the sham operation group as a reference,the changes of methyltransferase and demethylase responsible for regulating H3K9me3 were detected by real-time quantitative polymerase chain reaction(RT-q PCR).Moreover,the absolute expression levels of methyltransferase,SET domain bifurcated histone lysine methyltransferase 1(SETDB1)and Suppressor of variegation 3-9 homolog 2(SUV39H2)with distinct changes were detected in normal myocardium.The changes of SETDB1 were further verified by WB and tissue immunofluorescence staining of myocardial ring.3.In order to observe whether the changes of H3K9me3 and its methyltransferase SETDB1 in vitro were consistent with those variations in vivo experiments.Primary cardiomyocytes were extracted from C57 neonatal mouse(1-3 days),and the hypoxia/reoxygenation(H/R)injury model was established.WB assay and cell immunofluorescence staining were used to detect the changes of H3K9me3 and SETDB1 in H/R group compared with Normoxia group(control).4.In order to confirm the involvement of SETDB1 and H3K9me3 in H/R injury in vitro experiments,we designed and synthesized small interference sequences(si-NC and siSetdb1)to achieve to reduce the expression of SETDB1 in cardiomyocytes,meanwhile designed and synthesized adenovirus(Ad-Con and Ad-Setdb1)to achieve to increase the expression of SETDB1 in cardiomyocytes as well.After the knockdown or overexpression efficiency was verified,The effects of knockdown or overexpression of SETDB1 on H3K9me3 levels were observed by WB assay and cell immunofluorescence staining.Then the cells were placed in Normoxia or H/R environment and grouped into: si-NC+ Normoxia,si-NC+H/R,si-Setdb1+Normoxia and si-Setdb1+H/R.Ad-Con+Normoxia,Ad-Con+H/R,Ad-Setdb1+Normoxia and Ad-Setdb1+ H/R,CCK8 kit was used to detect cell viability in each group to observe whether knockdown or overexpression of SETDB1 had any effect on cell survival.5.In order to test the involvement of SETDB1 and H3K9me3 in MI/R injury in vivo,we designed and synthesized AAV9-c TNT-sh Setdb1/Zs Green virus(sh-NC and sh-Setdb1)using the small interference sequence confirmed by cell experiments,and expressed it in vivo for one week through intravenous injection of inner canthus to achieve heart-specific knockdown of SETDB1.At the same time,we also achieved SETDB1 overexpression in heart tissue by injecting overexpressed adenovirus(Ad-Con and Ad-Setdb1)into the myocardial sites of the anterior wall of the heart and expressing it in vivo for one week at two injection sites.After the efficiency of SETDB1 knockdown or overexpression in heart tissue and its effect on H3K9me3 level were proved by WB,MI/R injury model of mice was established on this basis,and the mice were divided into: sh-NC+Sham,sh-NC+MI/R,shSetdb1+Sham and sh-Setdb1+MI/R.Ad-Con+Sham,Ad-Con+MI/R,Ad-Setdb1+sham and Ad-Setdb1+MI/R.Echocardiogram indexes,left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),left ventricular end diastolic diameter(LVEDD)and left ventricular end systolic diameter(LVESD)were measured and collected at 24 h after MI/R injury to observe cardiac function of mice in every group.Myocardial ring Terminal deoxynucleotidyl transferase d UTP nick end labeling staining(TUNEL)was used to count the number of apoptotic cells in different groups.5-Triphenyltetrazolium chloride staining(TTC)was used to assess the myocardial infarction area of mice in each group.After 2 weeks of MI/R injury,myocardial tissue slices were taken for masson staining to evaluate the level of myocardial fibrosis.6.In order to explore the potential mechanism of SETDB1 and H3K9me3 involving in cardiomyocyte injury,we provided two groups of primary cardiomyocyte cells si-NC and si-Setdb1,which were verified and qualified for RNA-sequence(RNA-seq).Then carried out some bioinformatics analysis,such as Gene ontology(GO),Kyoto encyclopedia of genes and genomes(KEGG)and Gene set enrichment analysis(GSEA),was conducted to observe the significantly changed differential genes and their functions.At the same time,nuclease targeted cutting and transposition enzyme sequencing analysis(CUT&Tag)was also performed on the two groups to observe whether the signal of chromosome H3K9me3 was changed,and the chromosomal regions with significant changes were personalized annotation and then combined with RAN-seq information for re-analysis.Finally,the most obvious change DNA sequence that maybe play a leading role in mediating myocardial damage was screened out when H3K9me3 decreased.7.In order to verify the sequencing results of Endogenous retroviruses(ERVs)transcriptional activation and screen related nucleic acid sensing molecules,cells were first prepared to verify whether the EVRs transcriptional activation product,double-stranded RNA(ds RNA)accumulated in large amounts in the cytoplasm after the knockout of SETDB1.J2 antibody could be specifically bound to ds RNA for cell immunofluorescence staining(aseptic operation in the whole process,avoiding bacterial contamination to cause experimental statistical error).Subsequently,the small interference sequences of Z-DNA binding protein 1(ZBP1),retinoic acid inducer gene-I(RIG-I),protein kinase R(PKR),Toll-like receptor 3(TLR3)and melanoma differentiation associated gene 5(MDA5),which are related to RNA sensor known to the present research,were designed and synthesized.After efficiency verification,small interference sequences of each molecule were given based on the knock down of SETDB1 and H/R damage,which were grouped as follows: siSetdb1,si-Setdb1+H/R,si-Setdb1+ si-Zbp1+H/R,si-Setdb1+si-Pkr+H/R,si-Setdb1+siTlr3+H/R,si-Setdb1+si-Rig-1+H/R and si-Setdb1+si-Mda5+H/R,and CCK8 was used to detect the cell viability of each group to screen out the most obvious small interference molecule on cell damage recovery.Finally,completed the groups,as follows: siNC+Normoxia,si-NC+H/R,si-Pkr+H/R,si-Setdb1+H/R and si-Setdb1+si-Pkr+H/R,detect the cell viability of each group by CCK8 kit to simply evaluate the recovery degree of cell damage after knockdown SETDB1 by decreasing PKR.8.In order to realize the changes of H3K9me3 in peripheral blood monocytes of AMI patients and its association with clinical indexes,25 peripheral venous blood samples(5ml)were collected from AMI patients and non-coronary heart disease(control)patients in strict accordance with the inclusion criteria and exclusion criteria after the ethical approval of the study protocol.After optimizing centrifugation and other experimental conditions,monocytes were collected using the human peripheral blood monocytes extraction kit.Ten patients in each group were randomly selected for RT-q PCR to observe the expression level of inflammatory genes.The remaining 15 patients in each group were extracted protein and detected the changes of H3K9me3 and SETDB1 levels by WB.Meanwhile,general clinical data,monocyte counts in blood routine examination,levels of inflammatory factor interleukin 6(IL-6),LDH,CK-MB and cardiac troponin I(c Tn I)in AMI patients were collected.Finally,the collected data and experimental results were analyzed statistically and made correlations.Results:1.Compared with the Sham group,H3K9me3 was detected at different MI/R injury time points and showed a trend of decreasing temporarily and then rebounding.The decrease of H3K9me3 was most obvious at MI/R 3h,and tissue immunofluorescence also confirmed that H3K9me3 was overtly lost in the surrounding area of myocardial infarction.2.After detecting the methyltransferases and demethylases regulating H3K9me3 in the sham operation group and MI/R 3h group,it was found that the methyltransferases SETDB1 and SUV39H2 were significantly downregulated at MI/R 3h.After detecting the absolute expression level of the two methylases in heart tissue,it was found that the expression level of SETDB1 were much higher than SUV39H2.In addition,tissue WB and immunofluorescence staining also showed that myocardial SETDB1 decreased at MI/R 3h.These results suggest that the decrease of H3K9me3 in MI/R 3h is primarily caused by the decrease of methyltransferase SETDB1.3.In cell experiments,H/R injury was found to reduce the protein levels of H3K9me3 and SETDB1 in primary cardiomyocytes compared with the Normoxia group,and the fluorescence intensity of both were weakened by cell immunofluorescence staining.These results suggested that H3K9me3 and SETDB1 levels would decrease when cardiomyocytes were damaged,was consistent with the results of in vivo experiments.4.When SETDB1 was knocked down or overexpressed at the cellular level,it was found that H3K9me3 levels also changed accordingly,increasing with the increase of SETDB1 and decreasing with the decrease.The results of CCK8 detection of cell viability showed that knocking down SETDB1 could make H3K9me3 signal lower and aggravate H/R injury.Overexpression of SETDB1 can alleviate cell H/R injury.These results indicate that SETDB1 and H3K9me3 is involved in regulating the H/R injury process of cardiomyocytes.5.After the SETDB1 knockdown or overexpression was performed on the mouse myocardium by using the virus as the carrier,it was observed that H3K9me3 level was also affected correspondingly when the heart tissue was taken to verify virus’ efficiency,which corelated with up or down of SETDB1 expression.After MI/R injury,it was found that SETDB1 downregulation could aggravate MI/R injury,lead to severe cardiac dysfunction,expand the area of myocardial infarction,increase the number of apoptotic cells in the area around infarction and promote myocardial fibrosis.The upregulation of SETDB1 can mitigate MI/R injury,improve cardiac function,restrict the area of myocardial infarction,significantly reduce the number of apoptotic cells in the area surrounding infarction and decrease the level of myocardial fibrosis.These results indicate that SETDB1 and H3K9me3 is participating in the pathological process of MI/R injury.6.CUT&Tag results showed that,compared with the control group,the H3K9me3 signal of the whole chromosome region was significantly decreased by knocking down SETDB1,and the regions of H3K9me3 changed mostly were focused on the intergene and intron.Meanwhile,the RNA sequencing results also confirmed that the number of differentially expressed genes between the two groups was very little.After noting individually in details of the intergenic and intron regions,found that H3K9me3 had the most obvious changes in the long terminal repeats(LTR)element of noncoding region,and the top 20 all belong to the ERVs family by fold change ranking.These results make clear that when SETDB1-H3K9me3 is lost,the ERVs family silencing is weakened and transcription is activated,leading to subsequent cell damage.7.Cell immunofluorescence staining with J2 antibody showed that the level of intracellular ERVs transcription product,ds RNA increased significantly when knocking down SETDB1 and H/R injury compared with solely H/R injury.After interference with five known RNA sensing molecules,found that PKR knockdown could significantly reduce cell damage that aggravated by SETDB1 downregulation.These results manifested that the decrease of SETDB1-H3K9me3 led to the activation of ERV transcription,and a large amount of transcription product ds RNA in the cytoplasm activated the sensing molecule PKR,resulting in cardiomyocytes damage.8.Compared with non-coronary heart disease(control)patients,the peripheral blood monocyte counts of AMI patients was clearly higher,but most of them were in the upper limit of clinical normal reference range.The expression of monocyte inflammatory genes,such as Il-6,Il-1β,Il-18,Tlr2 and Tlr4 were increased,H3K9me3 and SETDB1 expressions were decreased.In addition,the decrease degree of H3K9me3 expression in AMI patients was negatively correlated with the level of IL-6,an indicator of inflammation in body,and troponin I,a marker of myocardial infarction diagnosis.Conclusions:In this study,we demonstrated that decrease of methyltransferase SETDB1 leads to H3K9me3 lost during MI/R injury,and the decline of SETDB1 expression and H3K9me3 modification level results in weakening the inhibition of ERVs and activating transcription,then produces plenty of ds RNA accumulated in the cytoplasm,finally opens downstream damage pathways related to nucleic acid sensing molecule PKR,leads to cell death and participates in the pathological process of MI/R injury.We preliminarily explored a new mechanism of SETDB1 and H3K9me3 involved in regulating MI/R injury,observed and analyzed the changes of H3K9me3 in peripheral blood monocyte of AMI patients,which may provide a new idea for searching for myocardial protective target,diagnostic marker and disease detection to meet clinical great needs. |
PDF Full Text Request