N1-methyladenosine Methylated Map In Ischemic Heart Failure

Background:Ischemic cardiomyopathy,Especially acute myocardial infarction(AMI)seriously threatens human health.In view of the advance of cardiac remodeling and heart failure caused by MI,the current treatments such as medications,percutaneous coronary intervention(PCI),coronary artery bypass grafting(CABG)have achieved significantly results,but there is still a considerable part of myocardium after MI Cell loss,poor ventricular remodeling and deterioration of heart function.It was demonstrated that epigenetics plays an critical role in protecting heart after MI.At present,researches in epigenetics and cardiac remodeling mainly focus on DNA methylation and histone post-translational modification.However,there are few studies inRNA epigenetic post transcriptional modification.Recently,studies have demonstrated that 150 types ofRNA epigenetic post-transcriptional modifications have been discovered,including N6-methyladenosine(m6A),N1-methyladenosine(m1A),N5-methylcytidine(m5C),N4-acetylcytidine(ac4C),etc..Among these post-transcriptional modifications,m6A is the most prevalent modification of mRNA in eukaryotes.Studies have shown that,in mice with heart failure after MI,the fat mass and obesity(FTO)gene of m6A demethylase can improve the cardiac function after ischemia by deregulating m6A,and the methyltransferase like 3(mettl3)gene of m6A methyltransferase can regulate myocardial hypertrophy and promote the deterioration of cardiac function.Therefore,mRNA methylation plays an important role in the heart disease.However,the study of m1A methylation in cardiovascular disease has not been reported.We speculate that similar to the m6A methylation m1A methylation may play an important role in heart disease.Therefore,we performed m1A methylation immunoprecipitation sequencing in human ischemic heart failure myocardial tissue to reveal the m1A methylation map of human heart.We also detected the expression of m1A and its methyltransferases at given times after myocardial infarction in the rat heart.At the same time,the expression of the m1A methyltransferases was detected and the gene level of the methylase Alk B homolog 3(ALKBH3)was screened out.The overall expression level of ALKBH3 gene was significantly up-regulated and continuously up-regulated after myocardial infarction,To further explore the effect of ALKBH3-dependent m1A modification on the heart repair after MI,we injected ALKBH3 inhibitors into mouse to observe the function of the cardioprotection after MI.This study revealed the m1A methylation modification map of the heart of human ischemic heart failure,and conducted a preliminary exploration of the protective effect of m1A modified by the critical enzyme ALKBH3 in the ischemic heart.The resuits of this study will provide potential theoretical and experimental supplements for the treatment of ischemic heart disease via targetingRNA epi-transcriptome.Part ⅠⅡ Changes of m1A in Human ischemic heart failurePurpose:RNA methylation immunoprecipitation sequencing(MeRIP-seq)and data analysis were performed on human ischemic heart tissue to discover the changes in mRNA m1A methylation modification map in human normal and ischemic heart failure hearts.Method:1.MeRIP-sequence:Nanodrop ND-1000 was used to measure theRNA concentration of each sample,and qubit3 was used to quantify and analyze theRNA.Genseq TM m1ARNA IP Kit(genseq)was used for m1ARNA immunoprecipitation.Nebnext~RUltra II directionalRNA library prep kit was used to constructRNA sequencing Library of inputRNA samples and IPRNA samples after immunoprecipitation.The quality of the library was tested by Bioanalyzer 2100.High throughput sequencing was performed on Illumina novaseq 6000 sequencer.After sequencing with Illumina novaseq 6000sequencer,image analysis,base recognition and quality control,the original reads were generated.Go and pathway analysis were performed for the genes encoding differential methylation.2.mRNA sequencing:quantitative quality control,integrity control and library quality enrichment merip sequencing.Ribo zero rRNA removal kits were used to remove rRNAs from totalRNA,and truseq stranded totalRNA library prep kit was used to pretreatRNA to construct sequencing library.Bioanalyzer 2100 was used for quality control and quantification.According to Illumina sequencing instructions,the 10 PM library was denatured into a single stranded DNA molecule and sequenced on Illumina novaseq 6000 sequencer.Result:Through MeRIP-seq sequencing analysis,there is no significant difference between the overall distribution of mRNA m1A and the distribution of m1A peak enrichment in normal and ischemic heart failure hearts.Both are mainly distributed in the coding sequence region,the m1A peak near the start codon is significantly enriched,and the 3’Untranslated regions region is the least enriched.Compared with normal heart tissue,the myocardial mRNA m1A methylation modification of ischemic heart failure has changed significantly.Human heart tissue has specific enrichment of mRNA m1A methylation under both normal and ischemic heart failure states,and the degree of m1A methylation is highly different.GO/KEGG functional enrichment analysis showed that the function of transcripts with significantly different changes in mRNA m1A methylation modification is related to myocardial contractility,oxidative stress,and myocardial fibrosis.The combination analysis of mRNA and mRNA m1A with significantly different changes indicates that there are relatively few parts with significant differences in mRNA expression levels and significant differences in m1A methylation modification in ischemic heart failure.The functional enrichment analysis of significantly differentially expressed mRNA showed that the function of these mRNAs is mainly related to myocardial contraction,myocardial energy metabolism,inflammation,fibrosis and apoptosis.Conclusion:In human ischemic heart failure heart,the methylation profile of myocardial m1A has changed significantly.The transcription enriched by m1A is related to the regulation of key biological processes such as myocardial contractile function,oxidative stress and myocardial fibrosis.Part Ⅱ The effect of ALKBH3 mediated m1A on Rat Myocardial InfarctionPurpose:To detect the modification of m1A and screen the key enzymes that mediate the modification of m1A after MI,and explore the effect of ALKBH3 inhibitors on cardiac repair after MI.Method:1.Detection of enzymes related to m1A and m1A methylation modification:animal experiment groups:sham operation group(n=6)and myocardial infarction group(n=6).We established the SD rat myocardial infarction model,and took the ischemic myocardial tissue 4 hours,1 day,2 days,3 days,7 days,14 days and 28 days after AMI in the sham operation group and the myocardial infarction group for RT-q PCR and Wester blot experiment to detect the expression of m1A methylation-related enzyme genes and target molecular protein.Dot blot experiment detects the expression of m1A in myocardial infarction tissues at the given time points.2.The effect of ALKBH3 inhibit on cardiac repair function after MI:This part of the experimental group:sham operation group,myocardial infarction group and myocardial infarction plus HUHS015 group.We established a Kunming mouse myocardial infarction model.48 hours after modeling,HUHS015 32 mg/kg was subcutaneously injected into the back of the model mouse once a day for 7 consecutive days.Myocardial tissue was taken 28 days after intervention for Masson staining and observing the changes of myocardial fibrosis under microscope,and myocardial fibrosis was quantitatively analyzed by Image J software.Result:1.The expression of m1A in myocardial infarction heart:The expression of m1A in myocardial infarction tissues at given time points showed obvious dynamic changes,and it was highest on the first and second days after acute myocardial infarction,which was statistically significant compared with the control group(P<0.05),the decrease generated on the third day,and the decrease was lower on the 28th day,Compared with the control group,the 3rd,7th,14th and 28th days were statistically significant(P<0.05).The results show that the m1A level changes dynamically after MI,and As time goes on,the overall trend is downward.2.After myocardial infarction,the expression levels of cardiacRNA m1A methylation-related enzymes TRMT6,TRMT61A,ALKBH1,ALKBH3,and BMT2changed dynamically,and Alkbh3 showed a significant regular change,that is,it decreased on the first day after MI,and decreased to the lowest on the second day,which was statistically significant compared with the control group(P<0.05),and began to increase on the third day,which was statistically significant compared with the control group on the14th and 28th day(P<0.05).3.The expression of ALKBH3 protein in MI heart:The expression of ALKBH3 in myocardial infarction tissues at given time points also showed obvious dynamic changes,and it decreased to the lowest after the first and second days after acute myocardial infarction,which was statistically significant compared with the control group(P<0.05),it began to rise on the third day,and compared with the control group on the 7,14 and 28 days,it was statistically significant(P<0.05).The dynamic change of ALKBH3 is negatively correlated with the expression level of m1A.4.After inhibiting the effect of ALKHB3 in the heart of ischemic heart failure,the myocardial tissue was subjected to Masson staining and Image J software to quantitatively analyze myocardial fibrosis.Compared with the myocardial infarction group,the myocardial fibrosis in the myocardial infarction plus HUHS015 group was significantly reduced(P<0.05),which indicates that inhibiting the effect of ALKHB3 can promote heart repair after MI.Conclusion:After myocardial infarction,RNA m1A,ALKBH3 mRNA and ALKBH3protein in the myocardium shown regular changes,and the changes among them are in corresponding regulatory relationship.Inhibiting the activity of m1A demethylase ALKBH3 can improve myocardial fibrosis and promote the repair of heart function.