| BackgroundHeart failure(HF)is a major cardiovascular disease that threatens human health in the 21st century.Nearly 50%of heart failure patients die from sudden cardiac death caused by ventricular arrhythmias.Effective prevention and treatment of ventricular arrhythmias in heart failure is a problem in the cardiovascular field.Ventricular arrhythmias in heart failure are associated with substrate remodeling and electrical remodeling.They also involve multiple pathological processes,with multicellular populations interacting with each other to form a complex mechanism for these arrhythmias.The increase of calcium/calmodulin-dependent protein kinase II(CaMKII)is one of the key pathological mechanisms of the disease.Clinical studies have pointed out that TCM has unique advantages in improving the quality of survival,improving cardiac function,and reducing the number of occurrences in patients with ventricular arrhythmias in heart failure.Therefore,it is hypothesized that ShenLian FuMai granules may intervene in ventricular arrhythmias in heart failure by improving CaMKIIδC overexpression,and its mechanism of action may be related to the regulation of cell subpopulation composition and gene expression,as well as the interaction between different cell populations.Objectives1 This study explores and summarizes Professor Lin Qian’s medication experience and rules in the treatment of tachyarrhythmia through data mining technology.It is expected to provide a reference for the clinical application of Chinese medicine in the treatment of tachyarrhythmia,and better use of modern technology to inherit the valuable experience of famous Chinese medicine practitioners.2 In this study,a mouse model of ventricular arrhythmia in heart failure caused by conditional overexpression of CaMKIIδC was established.Single-cell nuclear sequencing was used to investigate the possible molecular mechanisms of CaMKIIδC overexpression-induced ventricular arrhythmias in heart failure treated with Shenlian Fumai granules in terms of cell subpopulation composition,differential gene expression,and interactions among different cell populations.Part Ⅰ.Study on the medication pattern of Professor Lin Qian in the treatment of tachyarrhythmia based on the analysis of data mining technologyMethodsThe method was to collect outpatient prescriptions that met the inclusion criteria in the database of the Information Center of Dongzhimen Hospital of Beijing University of Traditional Chinese Medicine.After data cleaning and standardization of the names of Chinese medicines,Microsoft Excel 2021 for Mac was used to analyze the frequency of the use of Chinese medicines,the four natures of drugs,the five flavors,the channel tropism,and the efficacy.An Excel database was established with the above keywords,and the basis of the determination criteria was referred to the Chinese Pharmacology and the Pharmacopoeia of the People’s Republic of China.The Apriori algorithm of SPSS Modeler 18.0 was used for association rule analysis,and the results of the visualized data were imported into Cytoscape v3.7.1 software for complex network view.SPSS Statistics 26.0 software was used to perform cluster analysis on the drugs with the top 30 frequency rankings.Results1 Prescription inclusion results:A total of 117 prescriptions of patients who met the criteria for the first consultation were included in this study,involving 90 herbs of traditional Chinese medicine drugs,with a total of 1647 times.The top 10 herbs in terms of frequency of use were Salvia miltiorrhiza,Codonopsis pilosula,Astragalus membranaceus,Coptis chinensis,Polygala tenuifolia,Radix paeoniae rybra,Ligusticum chuanxiong,Magnetitum,Radix paeoniae alba,Cinnamomum cassia.2 Drug efficacy classification statistics:The 90 drugs involved can be divided into 24 categories according to drug efficacy,of which the top 6 categories of drugs used more frequently are blood invigorating,removing blood stasis,tonifying Qi,calming the mind,clearing heat and cooling blood,clearing heat and drying dampness,and warming the liver,and the cumulative frequency of use of these 6 categories of drugs reached 77.29%.3 Results of the four natures of drugs,five flavors,channel tropism,and efficacy of Chinese medicines:The medicinal properties of the herbs used are mainly warm,cold,slightly cold and mild,the tastes of the herbs are mainly bitter,sweet,and pungent,and the frequency of the drugs used to the liver,spleen,heart and lung meridians is higher.4 Drug association rule analysis:The pairs of drugs with the highest support were Codonopsis pilosula-Salvia miltiorrhiza,Astragalus membranaceus-Salvia miltiorrhiza and Polygala tenuifolia-Salvia miltiorrhiza;the highest corner drugs were Astragalus membranaceus-Codonopsis pilosula and Salvia miltiorrhiza,Polygala tenuifolia-Codonopsis pilosula and Salvia miltiorrhiza,Radix paeoniae rybra-Codonopsis pilosula and Salvia miltiorrhiza,and Ligusticum chuanxiong-Codonopsis pilosula and Salvia miltiorrhiza.5 Drug cluster analysis:By systematic cluster analysis method,the commonly used drugs were divided into 8 categories.Category 1:Semen lepidii,Polyporus umbellatus,Morus alba,Lycopuslucidus,Carthamus tinctorius,Peach kernel,Alisma plantago-aquatica,Poria cocos,Leonurusj aponicus;Category 2:Crataegus pinnatifida;Category 3:Angelica sinensis,Radix curcumae,Astragalus membranaceus;Category 4:Coptis chinensis,Cinnamomum cassia;Category 5:Pinellia ternata,Euonymus alatus,Radix paeoniae alba,Prepared licorice,Radix paeoniae rybra,Ligusticum chuanxiong,Magnetitum,Salvia miltiorrhiza,Codonopsis pilosula,Poly gala tenuifolia;Category 6:Spine date seed;Category 7:Cornus officinalis,Dioscorea opposita;Category 8:Curcuma Longa,Caulis spatholobi,Pueraria lobata.ConclusionBased on the theory of Qi and Blood in Chinese medicine,Prof.Lin Qian proposed that the basic pathogenesis of tachyarrhythmia is Qi deficiency,blood stasis,and phlegm-heat disturbing the heart.The basic treatment is to benefit Qi and activate blood,clear the heart and resolve phlegm.Drugs are mainly "Codonopsis pilosula,Salvia miltiorrhiza,Coptis chinensis,Pinellia ternata,Euonymus alatus,Ligusticum chuanxiong,Radix paeoniae rybra,Radix paeoniae alba,Polygala tenuifolia,Spine date seed,Prepared licorice" and so on.The basic formula is summarized and named "ShenLian FuMai granules" while focusing on both symptoms and causes,and adding or subtracting medicines appropriately according to the condition.Part II Experimental studiesExperiment 1:Effect of ShenLian FuMai granules on cardiac function and arrhythmia in heart failure ventricular arrhythmia mice caused by overexpression of CaMKIIδCMethodsIn this part,CaMKIIδC conditional overexpression mice were constructed by CRISPR/Cas9 technology.The CaMKIIδC overexpression-causing ventricular arrhythmia in heart failure mouse model was established by intraperitoneal injection of tamoxifen,and was divided into the control group(Control),the model group(Model),the traditional Chinese medicine group(SLFM),and the CaMKII inhibitor group(KN93).The cardiac function was detected by echocardiography,the induction of arrhythmia by isoproterenol was detected by electrocardiography,the pathological changes were observed by HE and Masson staining,and the expression and cellular localization of CaMKIIδC tagged protein was detected by immunofluorescence and Western blot.Results1 Genotype identification and sequencing of CaMKIIδC conditional overexpression mice:the genotype identification of F1 and F2 generation mice by PCR and the sequencing results of F1 generation mice were consistent with the theoretical primer size and theoretical sequence.2 Echocardiography:Compared with the control group,the model group showed that LVEF and FS were significantly decreased(P<0.01),and LVIDs,LVIDd,and LVW were significantly increased(P<0.01).Compared with the model group,the SLFM group showed that LVEF and FS were significantly increased(P<0.01),LVIDs and LVW were decreased(P<0.05),and the KN93 group showed that LVEF and FS were increased(P<0.05).3 ECG results in the awake state:Heart rate(HR):In the general state,compared with the control group,the HR of the model group was decreased(P<0.05),compared with the model group,the HR of the SLFM group was not statistically different(P>0.05),and the HR of the KN93 group was increased(P<0.05).There was no statistical difference between the groups compared after isoproterenol induction(P>0.05).QRS interval:In the general state,compared with the control group,the QRS interval of the model group was significantly prolonged(P<0.01).Compared with the model group,the QRS interval of the SLFM group was shortened(P<0.05)and the QRS interval of the KN93 group was significantly shortened(P<0.01);the QRS interval of all groups after isoproterenol evocation was not statistically different(P>0.05).QTc interval:In the general state,compared with the control group,the QTc interval of the model group was significantly prolonged(P<0.01).Compared with the model group,there was no statistical difference in the SLFM and KN93 groups(P>0.05).4 Arrhythmogenesis:After isoproterenol induction,compared with the control group,the arrhythmia scores of the model group were increased(P<0.01).Compared with the model group,the arrhythmia scores of the SLFM and KN93 groups were decreased(P<0.01).After isoproterenol induction,arrhythmia scores were increased in all groups compared with the general state(P<0.05).Compared with the control group,the incidence of premature ventricular contractions was significantly increased in the model group(P<0.01),and compared with the model group,the incidence of premature ventricular contractions was decreased in both the SLFM and KN93 groups(P<0.05).One mouse in the Model group developed ventricular tachycardia after isoproterenol induction,which lasted about 10s.5 HE staining:Compared with the control group,the hearts of mice in the Model group showed obvious enlargement of the ventricular cavity,disorganized and loose myocardial arrangement,extensive swelling of cardiomyocytes,and visible vacuole formation with inflammatory cell infiltration,and the SLFM and KN93 groups showed improvement on the above pathological conditions.6 Masson staining:Compared with the control group,myocardial tissue in the Model group showed mild myocardial fibrosis with a significantly increased CVF(P<0.01).Compared with the Model group,mice in the SLFM and KN93 groups showed reduced myocardial fibrosis and significantly decreased CVF in the heart(P<0.01),and there was no statistical difference in CVF between the SLFM and KN93 groups(P>0.05).7 Immunofluorescence showed that CaMKIIδC-Flag protein expressed in the Model,SLFM,and KN93 groups was mainly localized in the cytoplasm.Western Blot:Compared with the control group,CaMKⅡδC-Flag protein expression level of the model group was significantly increased(P<0.01);compared with the model group,CaMKIIδC-Flag protein expression level of the SLFM group was decreased(P<0.05),and there was no statistically significant difference in the CaMKIIδC-Flag protein expression level in the KN93 group,but there was a trend toward reduction(P=0.055);compared with the KN93 group,the CaMKIIδC-Flag protein expression level of the SLFM group was not statistically different(P>0.05).ConclusionIn this study,CaMKIIδC overexpressing Rosa26LSL/+:Cre mice were successfully constructed.It was demonstrated that ShenLian FuMai granules could inhibit the expression level of CaMKIIδC,improve cardiac function,incidence of ventricular arrhythmia,and shorten the QRS time and QTc interval.Experiment 2:Single-cell sequencing technology to investigate the efficacy mechanism of ShenLian FuMai granules in the treatment of ventricular arrhythmias in heart failure caused by CaMKIIδC overexpressionMethodsIn this part,three mouse heart samples were obtained from each group of Control,Model,and SLFM groups,respectively,and were subjected to snRNA-seq by isolating single-cell nuclei via the 10X Genomics system.Sequencing data were obtained and analyzed by downconverting and clustering,followed by cell-type annotation based on the cell-type signature genes,and differential gene analysis was performed on the cell subpopulations,respectively,Gene Ontology(GO)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis,Pseudotime analysis,and cell communication analysis were performed on the cell subpopulations.Finally,we explored the proportion of cell subpopulations as well as molecular and pathway alterations in disease progression and after treatment with ShenLian FuMai granules,and analyzed the key targets and pathways at four levels:cardiomyocyte(CM)subpopulations,fibroblast(FB)subpopulations,endothelial cell(ECs)subpopulations,and inter-cellular communication,respectively.Results1 High-quality data of 89,546 mouse heart cell nuclei were obtained by quality control of snRNA-seq data,and a total of 40 individual subpopulations(C0-C39)were distinguished.These 40 cell subpopulations were named based on the cell type signature genes reported in the literature,and finally,a total of 13 different cell types were classified.2 A total of 8 cell subpopulations were identified in cardiomyocytes,and ShenLian FuMai granules could inhibit the cell proliferation of subpopulation 4.Through differential gene enrichment analysis,68 Hub genes and 16 pathways were screened,and it was found that ShenLian FuMai granules might be involved in the regulation of calcium channel activity,calcium ion transport and other biological processes,and inhibiting CaMKIIδC overexpression by modulating calcium signaling pathway,and play a role in improving electrical remodeling.In addition,the focused-to pathway was found to be closely related to cardiac hypertrophy,apoptosis,and cell adhesion.The ShenLian FuMai granules may pass through focal adhesion,actin cytoskeleton regulation,MAPK,Rap 1,FoxO,Apelin,AMPK,PI3K/AKT,adrenergic signaling in cardiomyocytes,Ras,HIF-1,cAMP,JAK-STAT,VEGF,ErbB,and other signaling pathways related to cardiac hypertrophic proliferation,apoptosis,and so on play a role in improving myocardial remodeling.The CALM1,CALM2,RYR2,CACNA1C,CAMK2A,PTK2B,and ITPR1 are associated with calcium signaling,ACTN1,ACTN4,TLN1,PXN,ACTB,RAC1,VCL are associated with myocardial contraction and hypertrophy,BCL2L11,CDKN1A,PIK3CA,MAPK1,MAPK9 associated with programmed cell death,cell growth and development may be key targets for the treatment of ventricular arrhythmias in heart failure.3 A total of 8 cell subpopulations were identified in fibroblasts,and ShenLian FuMai granules could inhibit cell proliferation in subpopulation 2 and subpopulation 5.Through differential gene screening,11 Hub genes and 9 pathways were identified,and it was found that ShenLian FuMai granules may improve extracellular matrix deposition,regulate ion transport,regulate ATP metabolism and other biological processes.The targets and pathways focused on are closely related to myocardial fibrosis,so ShenLian FuMai granules may play a role in improving myocardial remodeling through the regulation of actin cytoskeleton regulation,focal adhesion,extracellular matrix receptor interactions,PI3K/AKT,MAPK,HIF-1,FoxO,calcium signaling pathway,VEGF,and other pathways.The ITGA5,COL4A1,COL4A2,COL6A1,COL6A2,COL4A4,and COL5A1 involved in collagen synthesis,cell proliferation adhesionrelated targets may be key targets for the treatment of ventricular arrhythmias in heart failure.4 A total of 10 subpopulations of endothelial cells were identified,and there was no statistically significant difference in the proportion of cell subpopulations between groups(P<0.05).Through differential gene screening,36 Hub genes and 11 pathways were identified,and it was found that ShenLian FuMai granules might be involved in biological processes such as myocardial contraction,ion transport,membrane potential regulation,intercellular junction,ATP metabolism,CM proliferation regulation,matrix adhesion,etc.,and play a therapeutic role by inhibiting the overexpression of CaMKIIδC through the modulation of the calcium signaling pathway.In addition,the targets enriched to have a close relationship with cell adhesion,proliferation migration,and signaling,ShenLian FuMai granules may play a role through the regulation of PI3K/AKT,MAPK,cAMP,Ras,HIF-1,Rap1,cGMP-PKG,ErbB,calcium signaling pathway,focal adhesion pathway,and other pathways.The ITGB3,CDH5,CDH2,ERBB4,VEGFA,KDR,NOS3,PTPN11,ITGA5,TGFB1,and ABL1 are associated with cell adhesion and proliferation and migration,and CALM2,CACNA1C,RYR2,PLCG1,PLCB1,PRKCA,and CALR targets associated with calcium signaling may be key targets for the treatment of ventricular arrhythmias in heart failure.5 Cell communication analysis constructed a complex communication network among various cell subpopulations.It was found that ShenLian FuMai granules may mediate cellular communication among cardiomyocytes,fibroblasts,and endothelial cell subpopulations through the VEGF,PDGF,and FGF pathways.These pathways can trigger downstream signaling cascades and activate the MAPK signaling pathway,PI3K/AKT signaling pathway,etc.,to play a regulatory role.In addition,it was found that VEGFA,PDGFD,and FGF 13 may be potential targets for the treatment of ventricular arrhythmias in heart failure.ConclusionThis part is divided into four levels:cardiomyocyte subpopulation,fibroblast subpopulation,endothelial cell subpopulation,and inter-subpopulation communication,respectively,to explore the key targets and pathways of the action of ginseng replenishment particles.Ultimately,it was found that ShenLian FuMai granules may improve ventricular arrhythmias in heart failure caused by CaMKIIδC overexpression in terms of regulating calcium signaling,improving myocardial remodeling,and cell death. |
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