| Objective:1.To investigate the effect of specific endothelial cells with a fibrotic-like phenotype(EndoFP)on chronic heart failure and the mechanism of exacerbating chronic heart failure through interaction with cardiomyocytes/fibroblasts using single cell sequencing technology combined with in vivo and in vitro studies.2.To explore the effects of Xinyin Tablet,a representative formula of Yiqi Yangyin Huoxue LiShui method,on the cardiac function of mice with chronic heart failure,to reveal the regulation of EndoFP by Xinyin Tablet and the screening of effective ingredients,providing new research ideas and strategies for the mechanism of Xinyin Tablet in treating chronic heart failure.Methods:Ⅰ.Mechanisms of the interaction between EndoFP and cardiomyocytes/fibroblasts in heart failure1.Using single-cell sequencing(scRNA-Seq)data,cell collection was performed to build a library,t-SNE downscaling and K-mean clustering were applied to downscale clustering of cell populations,the enrichment algorithm of the SignleR package was applied to marker cardiac cells,and heart failure was screened out by manual collection of marker genes combined wi th comprehensive analysis of cell identification using the SingleR package.The specific EndoFP cell population associated with heart failure was screened and validated by animal experiments.2 Based on CellPhoneDB and other relevant biomaterial tools combined with in vitro and in vivo experiments,we explored the interaction pattern between EndoFP and cardiomyocytes/fibroblasts,and screened and validated the interaction key factors by analyzing RNA-seq data from mouse and human-derived cardiac tissue specimens.2.1 By establishing a TAC model,setting up animal experimental subgroups(sham,TAC,Snail_KD),performing cardiac ultrasound,and finally isolating hearts for histological examination(WGA staining,Masson staining,EndoFP cell population marker staining)and exploring the effects of EndoFP on cardiac function and pathology in animal models.2.2 In vitro construction of EndoFP and cardiomyocyte/fibroblast co-culture system to explore the key mechanisms of EndoFP interaction with cardiomyocytes and fibroblasts.Ⅱ.Screening of active ingredients based on Xinyin tablets to modulate specific EndoFP associated with chronic heart failure1.TAC models were established;echocardiography was performed 8 weeks after drug administration to assess cardiac function in mice,and myocardial injury markers(ANP,BNP,β-MHC),inflammatory response(IL-6,Tgfb1,Tnf),and fibrosis-related markers(Sppl,Collal,Fnl,Col3al)mRNA levels were assessed to evaluate the degree of myocardial hypertrophy,inflammation and myocardial fibrosis,and immunofluorescence was used to show the expression of EndoFP-related proteins and to investigate the effect of Xinyin tablets on EndoFP in chronic heart failure.2.Based on the transcriptome data established by the previous group and based on the enrichment analysis and molecular docking,we predicted the effective regulatory components of EndoFP regulation by Xinyin tablets.3.To detect the effect of different concentrations of leonurine on EndoFP activity by CCK-8,as well as the expression of EndoFP-related proteins after leonurine intervention by qPCR and immunofluorescence double-labeling in vitro assay.Results:Ⅰ.Mechanism of EndoFP interaction with cardiomyocytes/fibroblasts in heart failure1.Identification of specific EndoFP associated with chronic heart failure and validation in animal experimentsThe mouse heart in a chronic pressure overload model were analyzed by single cell sequencing to identify a subpopulation of specialized cells delineated as associated with chronic heart failure,and each cell in this specialized subpopulation has a high EndoMT score.In TAC model mice,the EndoMT signature was increased at 2 and 5 weeks after TAC(highest value at 2 weeks).Heart tissue immunofluorescence results showed an increased in CD31+Coll protein co-expression at 2 weeks after TAC compared to baseline,but its decrease at 8 weeks,qPCR data results showed that the change in mRNA level of EndoFPrelated markers(Collal,Col3a1,Vim and Snail)also showed a trend of increase at 2 weeks compared to baseline(P<0.05),but the mRNA levels of Acta2 showed almost no change(P>0.05).2.Prediction of the interaction pattern between EndoFP and cardiomyocytes/fibroblasts based on CellPhoneDB and other related toolsThe results of predicted intercellular interactions based on CellPhoneDB and other related tools for ligand-receptor relationships showed higher interaction scores between EndoFP and cardiomyocytes/fibroblasts compared with normal endothelial cells and the presence of ligand-receptor interactions.3.Inhibition of EndoFP significantly improved cardiac function and reduced myocardial hypertrophy and fibrosis in TAC model mice.Transcriptomic data suggest that EndoFP may influence the process of cardiac hypertrophy and fibrosis.Inhibition of EndoFP significantly improved cardiac function in mice in the TAC group(P<0.01).The area of cardiomyocytes was significantly reduced(P<0.05).Myh7,Nppa and Nppb mRNA levels were significantly reduced(P<0.01 or P<0.05).Collagen fiber deposition was significantly reduced(P<0.05).Collal,Fnl,and Col3a1 mRNA levels were significantly reduced(P<0.001 or P<0.05).4.EndoFP may be involved in the Osteopontin signaling pathwayTranscriptome data analysis yielded 872 differential genes,and further GSEA enrichment analysis showed that the Osteopontin signaling pathway was the most widely involved pathway in the EndoFP gene.RNA-seq data analysis of 366 patients with/without heart failure showed that SPP1 expression was positively associated with EndoFP signature,fibrosis and hypertrophy,and negatively associated with left ventricular ejection fraction(LVEF).Immunofluorescence results of animal tissues showed increased Opn protein expression in cardiomyocytes and fibroblasts in the TAC group compared with the shamoperated group.Opn protein expression was decreased in cardiomyocytes and fibroblasts of mice in the TAC+Snail_KD group compared to the TAC group.5.Construction of co-culture system to clarify the role of EndoFP interacting with cardiomyocytes and fibroblasts5.1 EndoFP affects fibroblast activation,migration and apoptosisImmunofluorescence staining showed that the expression of OPN and Collal proteins increased in fibroblasts after co-culture with EndoFP(P<0.0001).The mRNA levels of profibrotic and pro-inflammatory mediators(Tnf,116,Tgfbl and Spp1)were inreased(P<0.01 or P<0.001 or P<0.0001),promoting fibroblast migration and anti-apoptosis(P<0.01).5.2 EndoFP affects cardiomyocyte hypertrophy,ROS,calcium transients and apoptosisThe area of cardiomyocytes co-cultured with EndoFP was significantly increased(P<0.05).qPCR results showed significantly higher levels of Nppa,Nppb,Myh7 and Spp1 mRNA(P<0.01 or P<0.001 or P<0.0001)and significantly higher levels of ROS(P<0.0001).Fluo-4AM staining of extracted primary cardiomyocytes followed by electrical stimulation using passage of 1 Hz showed that Ca2+ transient amplitude was significantly reduced in cardiomyocytes co-cultured with EndoFP(P<0.01).Opn protein expression was significantly increased(P<0.0001),and apoptosis was significantly decreased in cardiomyocytes(P<0.0001).6.IGFBP5 may be a key factor regulating the interaction of EndoFP with cardiomyocytes/fibroblastsPrediction of key factors of EndoFP interaction with cardiomyocytes/fibroblasts by single-cell sequencing showed that the expression level of IGFBP5 protein was increased in the hearts of patients with hypertrophic cardiomyopathy or dilated cardiomyopathy compared with the normal heart group(P<0.01 or P<0.001),and the mRNA levels of IGFBP5 in the cardiac tissues of patients were positively correlated with the mRNA levels of NPPA,NPPB,SNAI1 and COL1A1.By adding neutralizing antibodies to IGFBP5,a significant decrease in Opn protein expression in cardiomyocytes/fibroblasts after IGFBP5 inhibition compared with co-cultured cardiomyocytes/fibroblasts with EndoFP(P<0.05),and a significant increase in Ca2+ transient amplitude in cardiomyocytes(P<0.05).Fibroblasts showed significantly reductions in Coll protein expression and migration(P<0.05).7.Rab5a may be a new regulatory gene involved in the EndoFP processThe results of gene co-expression network using differential gene linkage analysis indicated that Rab5a may be a new regulatory gene of the EndoFP process.In vitro experiments showed a significant increase in the mRNA level of Rab5a during the EndoFP process(P<0.001).Snail,Postn,Colla1 and Col3a1 mRNA expression levels were significantly reduced after knockdown of Rab5a expression(P<0.01 or P<0.05).Immunofluorescence staining results showed that knockdown of Rab5a significantly reduced EndoFP-related protein CD31 and Coll co-expressing cells in mouse endothelial cells and heart tissues.Ⅱ.Screening of active ingredients based on Xinyin tablets to regulate specific EndoFP associated with chronic heart failure1.Xinyin tablets significantly improved cardiac function in TAC model mice.Compared with the TAC group,the LVEF and LVFS values in the XYP and Peri groups were improved to different degrees and the LVIDd and LVIDs values were reduced to different degrees after the administration of Xinyin Tablets and perindopril,with statistically significant differences(P<0.05).2.Xinyin tablets significantly improved myocardial hypertrophy in TAC model mice.Compared with the TAC group,the mRNA levels of Anp,Bnp and β-Mhc,markers of myocardial hypertrophy in mice,were significantly reduced after treatment with Xinyin tablets and perindopril,which improved cardiomyocyte hypertrophy,and the difference was statistically significant(P<0.05).3.Xinyin tablets significantly improved myocardial fibrosis in TAC mice.Compared with the TAC group,the mRNA levels of Col 1 a 1,Fn1,Spp1 and Vim,markers of myocardial fibrosis,were significantly reduced in mice after treatment with Xinyin tablets and perindopril,which reduced the collagen accumulation in cardiac tissue,and the difference was statistically significant(P<0.0001).4.Xinyin tablets significantly reduced the cardiac inflammation index in TAC mice.The mRNA levels of IL-1β and IL-6 and Tgf-b1 were significantly reduced in mice treated with Xinyin tablets and perindopril compared with the TAC group,and the differences were statistically significant(P<0.01).5.GSEA enrichment analysis showed that the Xinyin tablet negatively regulated Snai 1,and the enrichment fraction of the Xinyin tablet for the up-regulated gene set of Snail knockdown was 0.697(adjusted P<0.01)and for the down-regulated gene set of Snail knockdown was-0.659(adjusted P<0.01).6.Molecular docking simulations were performed between the structural crystals of Snail protein and the nine effective small molecules of the Xinyin sheet,and the highest score was obtained for the docking of leonurine with Snail protein crystals.7.Immunofluorescence results showed that leonurine intervention significantly reduced the co-expression level of EndoFP-related CD31+Col1 protein.8.Real-time qPCR results showed that the levels of EndoFP-related markers Snail,Postn,Collal and Col3a1 mRNA were significantly reduced after leonurine intervention compared with the control group(P<0.05).Conclusions:1.IGFBP5 may be a key molecule regulating the interaction of EndoFP with cardiomyocytes/fibroblasts and accelerating the pathological progression of chronic heart failure.2.Rab5a may be a new key regulatory gene involved in the EndoFP process.3.The small molecule component of Xinyin tablets,Leonurine,may be one of the effective ingredient for the regulation of specific EndoFP associated with chronic heart failure. |
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