The Research In The Role And Mechanism Of CNOT3 In Pathological Myocardial Hypertrophy Development And The Research In The Role Of IWS1 In The Early Mouse Embryonic Development

Background and objective:Cardiomyocytes normally exit the cell cycle and begin to differentiate shortly after birth.Therefore,adult hearts,especially after the window of proliferation,generally develop hypertrophy by increasing the size of individual cardiomyocytes to reduce ventricular wall pressure and maintain cardiac function,rather than the number of cardiomyocytes.Hypertrophy initially develops as an adaptive response to pathological and physiological stimuli,but pathological hypertrophy usually progresses into heart failure.Each form of hypertrophy is regulated by different cell signaling pathways.Over the past decade,an increasing number of studies have shown that previously unrecognized mechanisms(including cellular metabolism,proliferation,non-coding RNA,immune response,translation regulation,and epigenetic modification)positively or negatively regulate cardiac hypertrophy.The CCR4-NOT complex is a biologically essential and indispensable conserved multi-subunit complex.It involves in regulating gene expression at many levels.Studies have suggested that CNOT3(the scaffold subunit of the CCR4-NOT complex)is a conservative regulator of heart function in Drosophila and mice.In addition,Genome-Wide Association Study(GWAS)have shown that there is a strong association between single nucleotide polymorphisms in CNOT1 or CNOT3 and the prolongation of human QT intervals.At present,the research on the mechanism of the CNOT3 subunit in myocardial hypertrophy is based on its deadenylation properties,which supervises and controls the mRNA of the autophagy regulatory protein ATG,and finally interacts with P53 to accelerate the death of cardiac myocytes and heart failure.However,the CCR4-NOT complex CNOT3-RIP sequencing results show that CNOT3 binds to nearly 1,000 kinds of mRNA.The underlying mechanism of how to carefully control cardiac function is still elusive,and given the multiple availability of biomolecules and such a wide range of mRNA quality monitoring,it is necessary to further study whether CNOT3 is involved in the hypertrophy mechanism by affecting key molecules in certain signaling pathways.Therefore,this study aims to explore the specific expression of CNOT3 in pathological hypertrophy and its possible regulatory mechanism.It is hoped that it can provide a new research direction for the mechanism of myocardial hypertrophy.Early research found that Iwsl(IWSl,SUPT6 interacting protein)participates in the pluripotency regulatory network of mouse embryonic stem cells with Oct4 as the core factor.Iwsl is necessary for mammals to maintain cell viability and protein expression levels in various tissues in mice have shown significant differences,which may have been involved in early embryonic development.These preliminary studies indicate that Iwsl may have a crucial role in embryonic stem cells,but the role of Iwsl in early embryonic development has not been reported,and it is worth investigating its role and mechanism in more depth.Methods:Quantitative RT-PCR and Western Blot were conducted to detect the expression index of CNOT3 in the control group and hypertrophic group of clinical specimens and samples in TAC mice.Immunohistochemical staining laterally verified the results of TAC mice in statistics.At the same time,CNOT3 specific shRNA and overexpression plasmids were also constructed to detect the relative mRNA and protein levels of Anp,Bnp,Myh6 and Myh7 in neonatal rat cardiomyocytes.Cell-level establishment of the myocardial hypertrophy phenotype in vitro was stimulated with Pgenylephrine(100 μM)and RNAi knockdown Cnot3 was performed to investigate the effect on marker genes expression of myocardial hypertrophy.Immunofluorescence staining for α-ACTININ was used as a statistical reference for calculating the area of neonatal rat cardiomyocytes.Downloaded GSE103629’s CNOT3-RIP-seq data to analyze possible mechanism-related proteins and signaling pathways.The technique of CRISPR-Cas9 was applied to generate a Iwsl knock out mouse embryonic stem cell(mESC)line,and the role of Iwsl in mESC differentiation was evaluated using embryoid body(EB)formation approach.Then,Iwsl expression in cardiomyocytes isolated from hearts at postnatal day 1(P1),7(P7),21(P21),and 56(P56),respectively were determined.Afterwards,the prediction of potential target genes of Iwsl using GeneMANIA,and assessment in their functional enrichment using Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses and gene ontology(GO).Results:The expression of CNOT3 in the control group and hypertrophic cardiomyopathy of clinical specimens showed the significant difference in mRNA and protein level.And the statistical results of immunohistochemistry further verified the reliability of the result.Cardiomyocytes treated with lentivirus which packaged CNOT3-overexpression plasmid increased Anp and Bnp expression,indicating hypertrophy.Compared with PBS-treated cells,the knockdown of CNOT3 under PE stimulation can reverse the hypertrophic phenotype,and the involvement of immunofluorescence for staining the skeleton protein a-ACTININ to calculate the cell area further verified the role of CNOT3.RIP sequencing data was analyzed at www.geneontology.org,and 46 proteins were found to be related to cardiac morphogenesis via biological process.The knockout of Iwsl repressed mesoderm specification in EB formation.In addition,Iwsl highly expressed in cardiomyocytes isolated from P21 hearts,compared to that from P1,P7,and P56 hearts.GeneMANIA analysis predicted 19 target genes of Iwsl which were enriched in biological functions related to redox reactions,C-type leptin receptor signaling pathways,and other physiological processes.Conclusion:Cnot3 is up-regulated in multiple pathological hypertrophic phenotypes across species.Knocking down Cnot3 can reverse the hypertrophic development in vitro.RNA immunoprecipitation experiments show that CNOT3 can directly combine with mRNA of a series of cardiac development genes,which may play a role by regulating mRNA stability.Therefore,Cnot3 may be an important regulatory gene for myocardial hypertrophy.Iwsl is required for mesoderm specification in EB formation which may potentially affect mesoderm-derived organ development.