Research On The Mechanism Of WTAP In Regulating Heart Development And Dilated Cardiomyopathy

Background:Abnormal heart development is a key factor in the pathogenesis of dilated cardiomyopathy(DCM).DCM is a cardiac pathological condition characterized by ventricular dilation,thinning of the myocardial wall,and impaired contractile function,ultimately leading to heart failure and arrhythmias.Although the etiology of DCM is complex,increasing evidence suggests that abnormal heart development plays an important role in the pathogenesis of DCM.Normal heart development is a highly precise process involving the precise coordination of various regulatory factors and signaling pathways.During embryonic development,the heart initially forms from the bilateral heart tubes in the mesoderm,followed by key steps such as heart tube ballooning,ventricular differentiation,and morphological changes in the heart wall,ultimately forming a mature heart structure.This complex process is tightly regulated by a series of regulatory factors,including transcription factors,extracellular matrix,cell-cell interactions,and intracellular signaling pathways.When these regulatory mechanisms are disrupted by gene mutations,environmental factors,or other disturbances,it may lead to abnormal heart development,laying the foundation for the occurrence of DCM.In DCM research,various gene mutations related to heart development have been identified,mainly derived from mutations in genes encoding cytoskeletal,sarcomeric,and nuclear envelope proteins.These mutations can lead to pathological changes in cardiomyocytes such as hypertrophy and fibrosis.Genetic regulation plays a key role in this process.Recent studies have shown that genetic regulation of DCM is not limited to gene mutations themselves but also involves complex regulation of gene expression regulatory networks.Additionally,epigenetic regulation of DCM has also attracted widespread attention among researchers.RNA-binding proteins(RBPs)play important regulatory roles posttranscriptionally,primarily involving alternative splicing of mRNA precursors,RNA editing,modifications at the 5’ and 3’ ends,nuclear transport and localization of mRNA,mRNA stability,and translation.Increasing evidence suggests that RBPs are closely related to heart development,function,or disease.Although recent studies have found that proteins related to N6-methyladenosine(m6A)modification play important regulatory roles in embryonic development and various types of heart diseases,our understanding of their specific regulatory mechanisms and potential roles in cardiovascular development and disease remains limited.Previous studies have shown that the m6A methyltransferase WTAP plays a crucial role in embryonic development,but its impact on heart development has not been studied.Therefore,further research is needed to explore the function and regulatory mechanisms of WTAP in heart development to reveal its potential role in the pathogenesis of DCM.In summary,abnormal heart development plays an important role in the pathogenesis of genetic dilated cardiomyopathy.In-depth study of the genetic regulatory mechanisms of DCM,especially the regulatory roles of RNA-binding proteins and m6A modifications,is expected to provide new insights for the prevention and treatment of DCM.Purpose:1)Investigate the impact of Wtap cardiac-specific knockout on mouse heart development;2)Determine whether WTAP regulates heart development and the occurrence of dilated cardiomyopathy(DCM)by interacting with METTL3 and mediating m6A modification;3)Investigate whether WTAP regulates heart development by binding to the promoters of myocyte enhancer factor 2a(Mef2a)and myocyte enhancer factor 2c(Mef2c),thereby modulating their transcriptional activity and influencing heart development.Methods:1)Wtapflox/flox+Myh6-Cre+/-mice were generated using CRISPR/Cas9 technology and subjected to multiple crosses.Echocardiography examinations were performed at different time points to evaluate changes in heart structure and function.Various data including heart weight/body weight ratio,survival time,hematoxylin and eosin staining,transmission electron microscopy,RNA-seq,MeRIP-seq,ATAC-Seq were collected to elucidate the cardiac function,morphology,and microstructural changes in Wtap cardiac-specific knockout(Wtap-CKO)mice.The transcriptional levels,m6A methylation levels,and changes in chromatin accessibility in the hearts of Wtap-CKO mice were determined.2)Wtap-CKO/Mettl3-COE mice were generated and bred to overexpress METTL3 in the hearts of Wtap-CKO mice.The effects on heart function,morphology,and microstructural changes were analyzed to ascertain whether WTAP regulates heart development through the interaction with METTL3 and the m6A modification pathway.3)Through integrated analysis of RNA-Seq and ATAC-seq,combined with techniques such as Western blot,RT-qPCR,Luciferase assay,and ChIP analysis,it was investigated whether WTAP directly promotes the transcription of Mef2a and Mef2c by binding to their promoters,thereby affecting heart development and leading to DCM.Results:Cardiac-specific deletion of Wtap induced dilated cardiomyopathy,heart failure,and neonatal death.Although the lack of WTAP in the heart reduced the protein levels of methyltransferase-like 3(METTL3),cardiac-specific overexpression of METTL3 could not rescue the phenotype in Wtap-CKO mice.Conversely,the absence of WTAP in the heart decreased the chromatin accessibility of the Mef2a and Mef2c promoter regions,resulting in reduced mRNA and protein levels of these genes and downregulation of their target gene expression.Luciferase analysis and ChIP analysis revealed that WTAP directly binds to the Mef2c gene promoter,increasing its promoter luciferase activity and expression.These data suggest that WTAP plays a crucial role in heart development by maintaining the chromatin accessibility of cardiac-specific genes.Conclusion:1)Wtap plays a crucial regulatory role in heart development,and its loss leads to abnormal heart development,resulting in dilated cardiomyopathy,severe heart failure,and neonatal death;2)WTAP is essential for maintaining the protein stability of METTL3,and its regulation of heart development is independent of the METTL3-mediated m6A modification pathway;3)WTAP directly binds to the Mef2c promoter to regulate its transcription,thereby influencing downstream target genes and modulating heart development.