Study On The Pathological Molecular Mechanism And Risk Prediction Of Arrhythmogenic Cardiomyopath

Part 1:Heart-specific LncRNAs identified by deep RNA sequencing contribute to pathogenesis in Arrhythmogenic cardiomyopathyBackground:Arrhythmogenic cardiomyopathy(ACM)is an inherited disease that is characterized by fibrofatty replacement of myocardium,predominantly in the right ventricle(RV),and ventricular arrhythmias.It is one of main causes of sudden cardiac death in young people with limited understanding of molecular mechanism.Increasing evidence suggests that long noncoding RNAs(lncRNAs)play essential roles in a variety of biological processes despite being poorly defined.LncRNAs are emerging as important regulators of cardiovascular diseases.To date,however,the transcriptional alterations of lncRNAs from ACM heart have never been explored.Objective:This study aims to identify lncRNAs in ACM genome wide and investigate their functions.Methods:This study was approved by the Ethics Committee of Fuwai Hospital,Beijing,China.Extremely deep RNA-seq was performed for ACM right ventricles(RVs,n=9)and left ventricles(LVs,n=6),normal RVs(n=5)and LVs(n=6),and dilated cardiomyopathy(DCM)RVs(n=4).Comprehensive transcriptome of these heart diseases was revealed by the deep RNA sequencing,specially to the lncRNAs that may be involved in pathogenesis.Novel lncRNAs were accurately confirmed by epigenetic remarks,which were annotated using normal human heart epigenetic data from the ENCODE database.We systematically compared the transcriptomes between ACM,DCM,and normal hearts to screen out the ACM-specific lncRNAs.Then,Pathway enrichment analysis of target genes of these ACM-specific lncRNAs were performed by Genomic Regions Enrichment of Annotations Tool(GREAT)and Gene Set Enrichment Analysis(GSEA).To predict the potential functions of these ACM-specific lncRNAs,we performed correlation analysis among the large independent cohort between lncRNAs and pathogenic signatures associated with ACM,involved in adipogenesis,cardiac calcium signaling,fibrosis,and Wnt signaling pathways.An independent cohort containing 24 ACM,18 DCM,and 18 donor hearts were used to validate the gene and lncRNAs expression results.Next,we validated the expression and distribution of LncRNA AC1 by in situ hybridization.And we isolated the primary cells of human myocardium including human cardiomyocytes(hCM)and human cardiac fibroblast(hCF)to detect the LncRNA AC1 expression specifically.Finally,we performed gain-of-function study and adipogenic medium induce assay to study the LncRNA AC 1 function in iPSC-CM and cardiac fibroblast.Results:Each ACM patient was clinically diagnosed according to revised Task Force Criteria,and was confirmed by pathological examination.We obtained 5.41 billion reads,with an average 180 million reads per sample in the deep RNA-seq.Integrating with epigenetic marks,we identified 5,443 novel lncRNAss.Compared with normal and DCM,1,925 lncRNAs transcripts specifically upregulated in ACM RVs were identified.Enrichment analysis revealed that these lncRNAss were associated with ACM phenotypes like RV enlargement and lipid metabolism dysregulation.of which,LncRNA AC1 was significantly positively correlated with adipogenic genes expression,including CEBPA(r=0.851，P<0.0001)，PPARG(r=0.835,P<0.0001),PLIN1(r=0.793,P<0.0001),and ADIPOQ(r2=0.892,P<0.0001)in ACM samples.And overexpression of LncRNA AC 1 promoted adipogenic genes expression and droplet deposition in human derived iPSC-CM and cardiac fibroblast.Conclusion:In summary,this study provides for the first time a fundamental resource for human cardiac lncRNA profiles in ACM including both LV and RV.We identified novel ACMspecifically high lncRNAs related to adipogenesis and validated them in-vitro,which may promote the potential development of basic translation study for ACM.Part 2:Mass spectrometry imaging-based single-cell metabolomics profiles metabolic signatures of arrhythmogenic cardiomyopathyBackground:Arrhythmogenic cardiomyopathy(ACM)has been growing as a serious public health burden,leading as the one of main causes of sudden cardiac death in young people.At least 14 genes have been identified as contributing to this disease,with desmosome genes,including plakophilin-2(PKP2),desmoglein-2(DSG2),desmoplakin(DSP),desmocollin-2(DSC2),and plakoglobin(JUP),accounting for more than 50%of ACM cases.The heart is a high energy-consuming organ that works uninterruptedly,with lipids acting as the main energy sources for cardiomyocytes(CMs).Previous studies have recognized the essential roles of metabolic remodeling and associated impaired signaling in the process of ACM.Metabolite acts as the final downstream product of biological activities,serving as the fundamental bridge between genotype and phenotype.Recent advances in single-cell analysis have greatly enhance the understanding of the cellular heterogeneity.Such heterogeneity is well recognized to reflect the varieties of underlying disease pathogenesis.Therefore,single-cell metabolomics,particularly further lipid metabolism is appreciated as a factor of structural and functional integrity of individual CMs.Mass spectrometry enables the simultaneous detection of hundreds of label-free biomolecules from complex sample.Time-of-flight secondary ion mass spectrometry(ToF-SIMS),as one of the mass spectrometry imaging techniques,has been successfully employed in the single-cell metabolomics analysis(especially for lipidomic)with high resolution.Objective:Constructing a detection system for single-cell metabolomics of mammalian CMs.And further proposing possible metabolic pathways in the mechanisms of ACM.Methods:The cardiac-specific DSP knockout mice were generated as previously described.Echocardiographic experiments were performed in mice at the age of 6 months,using an ultrasound system(Vivid 7;GE Healthcare,USA).ToF-SIMS was applied to study the metabolic features of mouse CMs.ToF-SIMS surface analyses were measured using a 30 keV Bi3+primary ion beam as analytical source,depth profiles were measured using a 10 keV Ar2000+sputter gun,and charge compensation were performed by using a 20eV electron flood gun.Mass spectra and chemical mapping of single cells were recorded by delayed extraction(DE)mode.ToF-SIMS spectra and chemical mapping were analyzed by SurfaceLab 7.1 software.Pathway enrichment analysis of the differential metabolites was conducted using the MetaboAnalyst 5.0.SHapley Additive exPlanation(SHAP)method was applied by executing package ’shap’ to analyze feature importance for prediction of heart failure.Results:Cardiac M-mode echocardiography exhibited ventricular dilatation and systolic dysfunction in ACM mice at 6 months,obtaining phenotypes of ACM.The freshly isolated CMs demonstrated a normal morphological,functional and molecular characteristic,which could reveal the pathophysiologic status in vivo.Single cell metabolomics was acquired through ToF-SIMS with a delayed extraction mode,exhibiting high sensitivity and spatial resolution.The CMs lipids database was set up by assigning the mass peaks to corresponding positive and negative ions.The scores plot from OPLS-DA presented distinct differences between the metabolomes of two groups(ACM VS.control).There was a significant decrease in the membrane expression of metabolites in the cardiomyocytes of ACM group.The major species of these metabolites were found to be phosphatidylinositol(PI),phosphatidylethanolamine(PE)and phosphatidylcholine(PC).Functional enrichment analysis of these differential metabolites was mainly enriched in pathways associated with lipoprotein metabolism and transmembrane transport.However,the intracellular metabolic differences are predominantly lysophosphatidic acid(LPA),phosphatidylinositol(PI)and phosphatidylethanolamine(PE)).MetaboAnalyst enrichment analysis indicated the differential intracellular metabolites were related to lipid metabolism and signal transductionConclusion:A single-cell metabolomics method for mammalian CMs based on ToF-SIMS was developed.This study provides insight into the mechanism of ACM through depth profile of single-cell metabolomics.Part 3:Plasma testosterone and arrhythmic events in male patients with arrhythmogenic cardiomyopathyBackground:Arrhythmogenic Cardiomyopathy(ACM)is associated with life-threatening ventricular arrhythmia and progressive ventricular dysfunction.Nowadays,as implantable cardioverter defibrillators(ICD)proved to be useful for the prevention of SCD in ACM,the remodeling of the cardiac structure and progressive heart failure are becoming other essential threats for disease prognosis.Depending on the trend of disease progression(the abnormalities of electrophysiology or ventricular dysfunction progression),different treatments should be taken to achieve good clinical efficacy.Thus,it is necessary to identify risk predictors that could specifically predict the patients susceptible to adverse HF events or Malignant arrhythmic events.Previous studies suggested that sex hormones play an important role in the onset and prognosis of ACM.Objective:This study aimed to investigate the role of testosterone in predicting major adverse cardiac events in the Chinese ACM cohort.Methods:99 ACM patients(median age,40 years;70.7%male)and 96 healthy controls(median age,41 years;62.5%male)were enrolled.The circulating levels of testosterone were measured by enzyme-linked immunosorbent assays(ELISA).The median follow-up time of all ACM male patients was 17 months(interquartile range/IQR 9-29).Cox proportionalhazards regression was used to analyse the effect of plasma testosterone and other welldescribed risk factors on Malignant arrhythmic events in male ACM patients.Results:The male ACM patients had significantly elevated levels of total testosterone(TT,6.390(4.438～8.768)ng/mL VS.3.617(2.073～4.479)ng/mL,P<0.0001,data shown as the median with IQR),bioavailable testosterone(BT,4.11(1.990～6.545)ng/mL VS.1.32(0.7965～2.0350)ng/mL,P<0.0001,median with IQR),and free testosterone(FT,0.2055(0.1000～0.4073)ng/mL VS.0.0768(0.0405～0.1105)ng/mL,p<0.0001,median with IQR)than healthy male volunteer,whereas no differences of that were observed among female counterparts.There was no significant correlation between the baseline clinical characteristics and testosterone levels in male ACM patients(Spearman’s correlation test,P>0.05).During the follow-up,the levels of testosterone were higher in male patients who experienced Malignant arrhythmic events(N=22)than in those who did not(N=25)(TT,9.034(7.222～15.370)ng/mL VS.4.633(3.363～6.375)ng/mL,P<0.001;BT,7.485(2.070～9.163)ng/mL VS.3.300(1.685～4.690)ng/mL,P<0.05;FT,0.453(0.221～0.758)ng/mL VS.0.161(0.075～0.337)ng/mL P<0.05,data expressed as median(IQR)and adjusted by Dunn’s multiple comparisons test),whereas such distinction was not observed among patients with Significant structural progression events(N=16).Through multivariable adjustments,the Cox regression analysis showed the level of plasma total testosterone(HR=1.325,95%confidence interval=1.171～1.498,P<0.001)was an independent predictor for Malignant arrhythmic events.Conclusion:The levels of plasma testosterone in ACM male patients are higher than those in healthy males.Testosterone level,without relation to the baseline cardiac function and future significant structural progression events,is a strong predictor of future adverse arrhythmic events in male patients with ACM.Therefore,our results suggest that testosterone may be a useful biomarker in arrhythmic risk prediction in the ACM.