The Role And Mechanism Of RNA Post-transcriptional Regulation In Cardiac Remodeling

Background:The heart undergoes hypertrophic growth in response to pressure overload or other pathological conditions.Hypertrophy initially develops as an adaptive response to physiological and pathological stimuli to adapt to wall tension or increased workload,but pathological hypertrophy generally progresses to heart failure.Cardiac hypertrophy is characterized at the cellular level by an increase in cardiomyocyte size and protein synthesis and reactivation of the fetal gene program.Previous studies have revealed a plenty of signal transduction pathways and genomic/epigenetic regulatory elements of gene expression involved in cardiac hypertrophy.Although RNA post-transcription modification also is one of the key regulatory steps in the process of information transition from genome to protein,its roles in cardiac remodeling are still largely unknown.N6-methyladenosine(m6A)is the most prevalent internal RNA modification that exists in most of eukaryotes.The m6A modification is deposited by the m6A methyltransferase complex composed of methyltransferase like 3(METTL3),methyltransferase like 14(METTL14)and Wilms tumor 1-associating protein(WTAP).The biological functions of m6A modification that control gene expression are achieved by specific recognition and binding by RNA binding proteins,which affects RNA fate by regulating RNA splicing,export,decay,stabilization,and translation.However,the role of mRNA m6A methylation in regulation of cardiac homeostasis is unclear.Alternative splicing(AS)is another important mechanism of post-transcriptional regulation of gene expression,which produces multiple mature mRNA from a single pre-mRNA by selectively removing or retaining exons and/or introns,therefore,increases the diversity of transcriptome and proteome.Up to date,little is known about the AS profile in heart and the roles of AS in cardiac remodeling.So,this present study aims to clarify the role of RNA m6A methylation in cardiac remodeling,and to investigate the AS profile of human cardiac tissue and the dysregulation of AS in cardiac remodeling.Methods:To study the role of RNA m6A methylation in cardiac remodeling,we measured the expression of the three components of the RNA methyltransferase complex in cardiac tissue of HCM patients by Western Blot,as well the level of RNA m6A.We inserted Floxp sequences flanking the fourth exon of the WTAP to construct WTAPFloxp/Floxp mice by Crispr/Cas9 and hybridized with MYH6-creERT2 mice to construct cardiomyocyte-specific WTAP knockout the mouse model induced by tamoxifen.8-week-old mice were injected with tamoxifen(40 mg/kg),and the survival curve was drew at baseline and under pressure overload respectively.The cardiac function of the mice was measured by ejection fraction(EF%)and shortening fraction(FS%)through echocardiography,and cardiac were taken to detect RNA m6A methylation and cardiac hypertrophy marker,cardiac fibrosis,and myocardial cell cross-sectional area.We knocked down and overexpressed WTAP in isolated neonatal rat cardiomyocytes by small interfering RNA(40 mM)and adenovirus(MOI=10),applying hypertrophic stimulation(2%FBS)and measured cardiomyocyte area,RNA m6A methylation and hypertrophy markers.To study the role of AS in cardiac remodeling,total RNAs were extracted from myocardial tissues of patients with HCM and normal donors and subjected to strand-specific RAN sequencing,and the AS events of mRNA were screened and analyzed.The function and pathway enrichment were performed through Gene Ontology(GO)and Pathway enrichment analysis of genes with significant different AS event between HCM and controls.The real-time quantitative PCR was used to verify the expression of differentially alternative spliced genes individually.Results:We found that WTAP,METTL3 and METTL14 of the HCM patients were decreased significantly,suggesting that the level of RNA m6A methylation was also decreased.We measured the level of RNA m6A methylation in HCM patients and found that it was significantly reduced.The survival rate,EF and FS of cardiomyocyte-specific WTAP knockout mouse were significantly reduced compared to the control.WTAP-CKO mice could not develop compensatory hypertrophy and showed a severe reduction in survival rate,EF,FS,the cross-sectional area of myocardial cells,and increasing in cardiac fibrosis and hypertrophy markers upon TAC surgery compared to the other control.Besides,we knockdown the WTAP in neonatal rat cardiomyocytes,founding that the expression of hypertrophy markers was increased.Although the expression of hypertrophy markers increased under the stimulation of hypertrophy,the cell area did not change.And after overexpression of WTAP,the area of cardiomyocytes became larger,but the level of hypertrophy marker was reduced.We totally detected 94990 AS events in 11604 genes,among which 124 were differentially alternative spliced.GO and KEGG Pathway enrichment analysis showed that these genes mainly related to muscle filament sliding,cardiac muscle contraction,hypertrophic cardiomyopathy,dilated cardiomyopathy,calcium signaling.Both RNA sequencing and real-time quantitative PCR showed that although the expression of CAMK2δ remained constant,its two transcripts of mutually exclusive exons,CAMK2δ3 and CAMK2δ9,showed opposite changes in expression.And we also verified this found in myocardial hypertrophic mouse model.Conclusions:Our study determined that WTAP mediated RNA m6A methylation is required for compensatory hypertrophy of cardiomyocytes.Increased RNA methylation leads to compensatory cardiac hypertrophy,while decreased RNA methylation leads to cardiomyocyte dysfunction,indicating the homeostasis of RNA m6A modification is essential to maintain normal structure and function of heart.In this study,we first mapped the alternative splicing profile of the human heart,and confirmed that multiple genes in HCM undergo alternative splicing.We found that even though the total expression of genes did not change,different transcripts of genes may change significantly through alternative splicing.Our research suggests that alternative splicing may play an important role in myocardial hypertrophy,and alternative splicing profile analysis can contribute to identifying disease-related genes and pathways.