Study On The Role And Mechanism Of Methyltransferase-like Protein 3 In Lipopolysaccharide-induced Myocardial Injury

Objective:Sepsis is a syndrome of physiological,pathological and biochemical abnormalities caused by various pathogenic microorganisms.At present,there is not a set of scientific and effective treatment methods.Septic cardiomyopathy(SCM)is an important pathological component of severe sepsis and septic shock.In mammals,the innate immune system plays a vital role in protecting the host from infection by participating in the detection of various foreign pathogenic microorganisms.Receptors of the innate immune system can be activated by microbial components,such as LPS,which are key molecules involved in the initiation of sepsis.Methylation of the sixth nitrogen atom on adenosine of the RNA molecule,called n6-methyladenosine(m6A)modification,is a common RNA modification in both m RNA and non-coding RNAs and can affect splicing,translation,stability and epigenetic effects of some non-coding RNAs.A large amount of newly discovered evidence suggests that m6 A modification is involved in a variety of biological processes,especially sepsis and immune disorders.The regulation of m6 A methylation is dynamic,which mainly depends on its three regulatory factors,namely methyltransferase(Writer),demethylase(Erasers)and methylated reading protein(Reader).Among,methyltransferase-like protein 3(METTL3)plays a central role.Therefore,the purpose of this study was to investigate the role and mechanism of METTL3 in lipopolysaccharide-induced myocardial injury.Methods:In this study,the expression changes of various m6A-related enzymes(including methyltransferase demethylase and methylresolution reading protein)were firstly analyzed through GEO database dataset.Western blottin was used to verify the expression of METTL3 in LPS-treated H9C2 cells,and the small interfering RNA of METTL3 was constructed.In vitro,the role of METTL3 in LPS-induced H9C2 cell injury was verified by detecting the changes of inflammatory factors,lactate dehydrogenase activity,creatine kinase activity,apoptosis,mitochondrial membrane potential and intracellular reactive oxygen species of H9C2 cells treated with LPS and si METTL3.In vivo,serum lactate dehydrogenase/creatine kinase-MB/Troponin T and inflammatory factors were detected by ELISA,cardiac echocardiography was used to detect cardiac function in septic rat model,H&E and TUNEL staining were used to observe myocardial tissue damage,and transmission electron microscopy was used to observe mitochondrial changes.Then,transcriptome RNA-seq sequencing analysis was performed on primary cardiomyocytes treated with LPS and LPS+si METTL3 to obtain differentially expressed genes.Database for Annotation,Visualization,and Integrated Discovery(DAVID)conducted GO(Gene Ontology)enrichment analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis was analyzed for different expressed genes.Since m6 A can affect the stability of m RNA,actinomycin D was used to detect the half-life of m RNA.Sequence-based predictors of m6 A modification sites were used to detect potential m6 A modification sites of Myh3 gene.Results:We firstly analyzed the expression changes of various m6A-related enzymes in human samples in the GSE79962 data and the Receiver Operating Characteristic curve of significantly changed m6 A enzymes,showing that METTL3 has a high diagnostic ability in patients with SCM,and may be used as a therapeutic target for SCM.Western blotting confirmed the high expression of METTL3 in LPS-treated H9C2 cells,which was consistent with the above results in human samples.Three small interfering RNAs were used to reduce METTL3 expression,and the one with the best effect was selected for subsequent experimental studies.In vitro,in H9C2 cells treated with LPS and si METTL3,we found that LPS significantly increased the expression level of inflammatory factors,lactate dehydrogenase activity,creatine kinase activity,cell apoptosis,reactive oxygen species,and decreased mitochondrial membrane potential,while reducing METTL3 expression could reverse the above effects.In vivo,ELISA kit showed that LPS significantly increased serum lactate dehydrogenase/creatine kinase isoenzyme/troponin T levels and expression levels of inflammatory factors,and decreased METTL3 expression could also reverse the above changes.Meanwhile,echocardiography,H&E staining,TUNEL staining and superoxide anion fluorescence probe reactive oxygen assay kit showed that the deficiency of METTL3 could improve the cardiac function,cardiac tissue damage,myocardial cell apoptosis and reactive oxygen species levels in l PS-induced sepsis rats,respectively.Transmission electron microscopy(TEM)showed that LPS could significantly damage the mitochondrial structure,including the formation of internal vesicles and the loss of crest,while si METTL3 could significantly improve the mitochondrial damage caused by LPS.In addition,we obtained 213 differential genes(including 101 up-regulated genes and 112 down-regulated genes)through RNA-seq sequencing analysis of transcriptome,and conducted GO enrichment analysis and KEGG pathway analysis of 213 differential genes through DAVID,discovering corresponding main biological processes,cell components,molecular functions and main pathways.We also found that the half-life of Myh3 m RNA was significantly reduced after METTL3 deletion and that Myh3 carries several potential m6 A modification sites.Conclusion:In conclusion,we found that reduced METTL3 reversed LPS-induced myocardial cell and tissue damage and reduced cardiac function,mainly by increasing Myh3 stability.Our study reveals a key role of METTL3-mediated m6 A methylation in septic cardiomyopathy,which may offer a potential mechanism for the therapy of septic cardiomyopathy.