| Background and Objectives:In adult mammalian hearts,cardiomyocytes renew very slowly,preventing the damaged heart from regenerating cardiomyocytes.In contrast,cardiomyocytes in the embryo or during birth show greater proliferation potential.Reprogramming factors Oct4,Sox2,Klf4 and c-Myc(OSKM)have the ability to reprogram somatic cells,and their short-term expression can make differentiated cardiomyocytes revert to a naive state and promote myocardial regeneration,but the regeneration mechanism has not been clarified.In addition,the oncogene c-Myc can easily lead to the formation of tumors and cannot initiate the proliferation of adult cardiomyocytes.In order to further explore the effects of reprogramming factors on myocardial proliferation,so that we can safely mediate myocardial regeneration,this paper aims to explore the effects of Oct4,Sox2 and Klf4(OSK)on the process of myocardial proliferation.On the other hand,CRISPR/Cas9 technology has been widely used in cardiovascular disease research,and adeno-associated virus(AAV)has become a mainstream delivery tool for gene therapy because of its unique advantages.Combining them together,the CRISPR/Cas9/AAV-based somatic mutagenesis(CASAAV)technique is highly effective in inducing specific gene editing in a wide range of cells,including cardiomyocytes in vivo.Me is1 is a cell cycle blocking gene.With the up-regulation of Meis1 expression during development,cardiomyocytes can exit the cell cycle.Therefore,inhibiting Meisl expression can promote the proliferation of cardiomyocytes.Currently,there are no studies using CRISPR/Cas9 gene editing technology to knockout Meis1 gene to achieve in-situ myocardial proliferation.Therefore,this paper aims to explore the feasibility of in-situ proliferation of mouse cardiomyocytes by inducing specific knockout of Meisl gene based on CASAAV technology.Methods:Part Ⅰ:First,we constructed TetO-FUW-OSK and TetO-FUW-c-Myc lentivirus vectors using TetO-FUW-O-SKKM lentivirus vectors,and carried out lentivirus packaging.Cardiomyocytes of neonatal mice were infected with lentivirus rtTA(Ctrl),OSK,OSKM and c-Myc in vitro.The overexpression levels of target genes and the relative mRNA levels of cell cycle related genes were detected and compared by Real-time PCR.DNA replication marker Edu,cell cycle related marker Ki-67 antigen(K167),phosphorylated histone H3(PH3)and cytokinesis marker Aurora were detected and compared in Myh6-Cre-tdTomato tool mice.Secondly,RNA-Seq was used to analyze differential gene expression in rtTA(Ctrl)group and OSK group,and PCA and Pearson Correlation were used to compare the differential gene expression correlation between the two groups and cardiomyocytes of neonatal mice 1 day after birth(PI)and mice 7 days after birth(P7).GO Term analysis and KEGG analysis enriched the biological functions and signaling pathways related to the up-regulation and down-regulation of OSK group.Finally,mosaic analysis using double markers(MADM)was used to detect and compare cytokinesis levels in mouse cardiomyocytes of MADM-ML-11TG/GT.Part Ⅱ:First,we designed two sgRNA sequences targeting the Meisl gene in mice and cloned them into the pAAV-U6gRNA1-U6gRNA2-TnT-Cre vector individually,which was then packaged into adeno-associated virus(AAV).We infected the cardiomyocyte of Rosa26-LSL-Cas9EGFP P1 mice with PBS or AAV-sgCtrl in vitro and in vivo,and compared the expression levels of enhanced green fluorescent protein(EGFP)in the cardiomyocytes.We injected AAV-sgCtrl and AAV-sgMeis1 into the thoracic cavity of Rosa26-LSL-Cas9EGFP P1 mice.After 3 weeks,the cardiomyocytes were isolated and the sections of heart tissue were prepared.The editing efficiency of Meis1 gene and the relative mRNA level of Meis1 gene and cell cycle-related genes in cardiomyocytes were compared between Meis1 sgRNA group(AAV-sgMeis1)and control group(AAV-sgCtrl).The expression levels of Ki-67 antigen(KI67),a cell cycle-related marker,Phospho-Histone 3(PH3),a mitotic marker,and Aurora B kinase(AURKB),a cytokinesis marker,in heart tissue were compared between the two groups.Results:Part Ⅰ:Real-time PCR results showed that lentivirus in each group could induce cardiomyocyte overexpression of target genes in vitro,and compared with rtTA(Ctrl)group,mRNA relative expression levels of Ccna2,Ccnb1,Ccnb2 and other cell cycle related genes were significantly up-regulated in cardiomyocytes of OSKM group and c-Myc group(P all<0.05),but there was no significant change in OSK group.Immunofluorescence staining showed that,compared with rtTA(Ctrl)group,the proportion of cytoskeletal CTNT depolymerization in cardiomyocytes in OSK and OSKM groups was significantly up-regulated(P all<0.05),but there was no significant change in c-Myc group.Compared with rtTA(Ctrl)group,Edu labeling and KI67,PH3 and AURKB protein expression levels in myocytes of OSKM and c-Myc groups were significantly up-regulated(P all<0.05),but there was no significant change in OSK group.The results of RNA-Seq showed that the cardiomyocytes of rtTA(Ctrl)group were more similar to those of P7 mice,while the cardiomyocytes of OSK group were more similar to those of P1 mice.The up-regulated genes of OSK group were mainly concentrated in cytoskeletal rearrangement,cardiac development and proliferation-related signaling pathways such as PI3K-AKT,mTOR and ErbB.The down-regulated genes were mainly concentrated in the functions of cell cycle,apoptosis and inflammatory signaling pathways such as NF-κB and TNF.Compared with the rtTA(Ctrl)group,the proportion of cytokinesis in the OSK and OSKM groups was significantly up-regulated by the MADM-ML-11TG/GT tool in mice(P all<0.05),but there was no significant change in c-Myc group.Part Ⅱ:Compared with the PBS group,adeno-associated virus can specifically induce the expression of EGFP and Cas9 in cardiomyocytes both in vitro and in vivo.Compared with the control group,the AAV-sgMeis1 group achieved a gene editing efficiency of[(56.67±4.16)%],among which the frame-shifting mutation rate accounted for[(28.67±5.13)%],and the relative mRNA level of Meis1 was significantly downregulated(P<0.05),indicating that administering Meis1 sgRNA AAV could significantly induce Meis1 knockdown in cardiomyocytes.Furthermore,the expression of cell cycle-related genes in cardiomyocytes and heart tissues were detected.Compared with the control group,the relative mRNA levels of Ccna2,Ccnb1,Cdk1,Foxm1,and E2f1 in cardiomyocytes of AAV-sgMeis1 group were significantly upregulated(all P<0.05),and the protein expression levels of cell cycle-related markers K167,PH3,and AURKB in heart tissue were significantly upregulated(all P<0.05).Conclusions:Part Ⅰ:The results showed that the OSK factor promoted the dedifferentiation of cardiomyocytes and caused the remodeling of cardiomyocytes and the regression to a more immatured state.Along with the cytoskeletal rearrangement of cardiomyocytes and the changes of cell proliferation related signaling pathways,the cardiomyocytes in this state were more likely to complete cytokinesis,which confirmed the important role of the reprogramming factor OSK in the process of myocardial proliferation.Part Ⅱ:Cardiomyocyte-specific knockout of Meis1 gene in juvenile mice can be achieved by using CASAAV technique,which promotes the in-situ myocardial proliferation.It confirms the feasibility of CASAAV technique in the rapid construction of gene knockout model and its clinical transformation potential in the field of cardiac regeneration. |
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