Function And Mechanism Of WTAP-mediated M6A Modification In The Regulation Of Cardiac Homeostasis

Background:The mammalian heart may undergo pathological changes and the cardiac homeostasis may be disrupted under physiological and pathological stimulation.The underlying mechanisms for this process remain to be explored.N6-methyladenosine(m6A)methylation is one of the most common internal modifications in mammalian mRNA,and the Wilms’ tumor 1-associating protein(WTAP)is a key regulatory subunit of the RNA m6A methyltransferase complex.Our previous study showed that cardiomyocytes-specific knockout of WTAP resulted in significant cardiac dysfunction with severe left ventricular dilatation,and failure to develop compensated hypertrophy.Knockdown of WTAP prevented cardiomyocyte hypertrophy upon phenylephrine stimulation in vitro,and overexpression of WTAP led to a significant increase in cardiomyocyte size.However,the exact mechanism has not been investigated.In the present study,we investigated the pivotal role and relevant mechanisms of WTAP-mediated m6A modification in cardiac homeostasis.Methods:Quantitative real-time polymerase chain reaction(qPCR)and western blot were used to measure the expression of a particular gene on RNA and protein levels,respectively.The m6A methylation level of total RNA was quantified using the RNA m6A methylation quantification kit.The pattern of m6A modification was determined via methylated RNA immunoprecipitation(MeRIP)followed by high-throughput sequencing and RNAsequencing(RNA-seq),and the functional analysis of differentially expressed or hypomethylated genes was performed.The potential targets of WTAP were identified using integrative analysis and validated by MeRIP-qPCR and qPCR.RNA immunoprecipitation(RIP)-qPCR was employed to verify the interaction between WTAP and its target transcripts.The expression of WTAP in cardiomyocytes or HEK293 cells was manipulated by small interfering RNA or adenovirus.The dual-luciferase reporter and RNA stability analysis were executed to reveal how WTAP regulates the mRNA expression of myosin light chain kinase family member 4(Mylk4).WTAP and MYLK4 were overexpressed in mouse hearts by adeno-associated virus serotype 9 delivery.The cardiac function and structure were examined in vivo by echocardiography.Wheat germ agglutinin staining and Masson staining were used to examine the cross area of cardiomyocytes and the degree of cardiac fibrosis,respectively.Results:We found that the levels of RNA m6A methylation and WTAP protein were both decreased in the myocardial tissue of the patients with hypertrophic cardiomyopathy,as well as in pressure overload stress-induced cardiac hypertrophy in mice.Conjoint analysis of MeRIP-seq and RNA-seq was performed in WTAP conditional knockout and control mice,and combined with previously depicted differentially expressed genes in myocardial tissue of patients with hypertrophic cardiomyopathy as well as pressure overload mice.As a result,we identified five potential downstream targets of WTAP-mediated m6A modification.The analysis of MeRIP-qPCR and qPCR showed that Mylk4 methylation and Mylk4 mRNA levels decreased after WTAP knockout.RIP-qPCR analysis illustrated a direct interaction between WTAP and Mylk4 mRNA in mouse hearts.Dual-luciferase analysis indicated that inhibition of WTAP decreased the expression of Mylk4 in an m6Adependent manner.RNA stability assay suggested that depletion of WTAP decreased the stability of Mylk4 mRNA,thus caused the downregulation of Mylk4 expression.Western blot analysis revealed that the MYLK4 protein level was significantly decreased and phosphorylation of the known MYLK4 substrate,myosin light chain 2-Ser15,was also markedly reduced after WTAP knockout,suggesting that WTAP regulated the expression of MYLK4.Cardiac overexpression of MYLK4 was able to partly alleviate the cardiac dysfunction induced by WTAP deletion.Finally,overexpression of WTAP promoted cardiac hypertrophy and even exacerbated cardiac remodeling and dysfunction induced by transverse aortic constriction.Conclusions:Our results demonstrate that WTAP is a critical regulator of cardiac homeostasis and highlight the importance of WTAP-mediated m6A modification of Mylk4 in the regulation of cardiac remodeling and cardiac homeostasis.Furthermore,targeting MYLK4 may be a promising therapeutic strategy to attenuate pathological cardiac remodeling.Background:Emerging evidence suggests that long noncoding RNAs(lncRNAs)have emerged as critical regulators of various biological processes.However,the exact mechanisms of lncRNAs as mediators in cardiac fibrosis have not been fully elucidated.This study aimed to profile the lncRNA expression pattern in human cardiac fibroblasts(HCFs)with cardiac fibrosis.Methods:We treated HCFs with transforming growth factor-β(TGF-β)to induce their activation.Then,strand-specific RNA-seq was performed,and the data were used to profile,quantify,and classify lncRNAs;and perform functional analysis in HCFs.We study the transformation of HCFs with molecular and cell biology methods.Results:Among all identified lncRNA candidates,176 and 526 lncRNAs were upregulated and downregulated respectively in TGF-β-stimulated HCFs compared with controls.Functional analyses revealed that the target genes of differentially expressed lncRNAs were mainly related to focal adhesion,metabolic pathways,Hippo signaling pathway,PI3K-Akt signaling pathway,regulation of actin cytoskeleton,and hypertrophic cardiomyopathy.As a representative,novel lncRNAs NONHSAG005537 and NONHSAG017620 inhibited the proliferation,migration,invasion,and transformation of HCFs induced by TGF-β.Conclusions:Collectively,our study established the expression signature of lncRNAs in cardiac fibrosis and demonstrated the cardioprotective role of NONHSAG005537 and NONHSAG017620 in the activation of cardiac fibroblasts,providing a promising target for anti-fibrotic therapy.