Basic Translational Research On Clinicopathological Typing And Metabolic Mechanisms Of Arrhythmogenic Cardiomyopathy

Part 1:A comprehensive clinicopathology study of arrhythmogenic cardiomyopahty:’Fuwai classification’Objectives:Arrhythmogenic cardiomyopathy(ACM)is an inherited cardiomyopathy characterized by the fibro-fatty replacement predominantly in the right ventricle.Recent studies show large heterogeneity in its genetic and pathological presentation of this disease.In addition to desmosome genes,at least 10 more genes have been identified to be responsible for ACM.And the pathology involvement extends from RV dominant phenotype to bi-ventricular involvement and even left-dominant phenotype.This study aims to provide a comprehensive atlas of ACM,establish a novel classification,and illustrate the relationships among clinical characteristics,genotype and pathological profiles of patients with this disease.Methods:We collected 60 explanted ACM hearts and performed standard pathology examinations.Masson staining of six representative sections of each heart were performed.Digital pathology combined with image segmentation was developed to calculate distribution of myocardium,fibrosis and adipose tissue.Unsupervised clustering algorithm was used to establish the classification of ACM patients.The clinical characteristics of patients,their genotype and cardiac magnetic resonance imaging(CMR)findings were assessed along with pathological characteristics,to validate and describe the novel clinic-pathological classification of ACM,which was named as‘Fuwai Classification’by an international committee consisting of ACM experts.Results:An unsupervised clustering based on fibrofatty distribution containing 4 subtypes was constructed.Patients in Fuwai Cluster 1 mainly carried desmosomal mutations(except for desmoplakin)and were subjected to transplantation at early age;this group showed extensive fat replacement in the entire right ventricle and fibrofatty infiltration in posterolateral wall of left ventricle,which was consistent with classical’desmosomal cardiomyopathy’.Fuwai Cluster 2 mostly had non-desmosomal mutations and showed regional fibrofatty replacement in right ventricle;the fibrosis distribution in Cluster 2 was mainly interstitial perimyocyte pattern spreading around the full-thickness section of left ventricle.Patients in Fuwai Cluster 3 showed parallel progression of left and right ventricle,and included patients with desmoplakin mutations.Fuwai Cluster 4 was typical left-dominant ACM,although the genetic background of these patients was not yet clear.Correlation analysis revealed that multiple parameters of echocardiograph and ECG were highly associated with severity of myocardial remodeling.Besides,multivariate regression analysis demonstrated precordial QRS amplitude as an independent indicator of the residual myocardium of right ventricle,which was validated in predicting death and transplant events in the validation cohort(n=92).Conclusions:This study provides a novel classification of ACM with distinct genetic backgrounds indicating different potential pathogenesis.Fuwai Cluster 1 is distinct in genotype and clinicopathology and can be defined as‘desmosomal cardiomyopathy’.Precordial QRS amplitude is an independent indicator reflecting the right ventricular remodeling,which may be able to predict transplant/death events for ACM patients.Part 2:The basic translational study of ketone body metabolism remodeling in arrhythmogenic cardiomyopahtyObjectives:Arrhythmogenic cardiomyopathy(ACM)is an inherited cardiomyopathy,leading to lethal arrhythmia and cardiac dysfunction.It is one of the main causes of sudden cardiac(SCD)death among adult under 35 years old and athletes.SCD could be the first symptom of patients with ACM.Establishing a biomarker for early diagnosis and better risk stratification of patients and their relatives is quintessential for preventing ACM,however clinical indicators predicting adverse progression remain lacking.Recent findings suggest metabolic dysregulation is present in ACM.We performed this study to identify metabolic indicators that predicted major adverse cardiac events(MACE)in ACM patients and their relatives.Methods:Firstly,we enrolled a discovery cohort to reveal the ketone metabolism in ACM including 13 ACM with heart and blood,13 donor heart and healthy blood(normal control),13 dilated cardiomyopathy(DCM,left ventricle failure)blood,and 20 pulmonary artery hypertension(PAH,right ventricle failure)blood.Proteomic analysis combined with qPCR,Western Blot and Northern Blot was used to identify the metabolism related proteins expression.β-hydroxybutyrate(β-OHB)assay was used to detect the plasma concentration among these groups.β-OHB assay of Matched coronary artery and sinus blood and targeted metabolomics profiling of human myocardium were then performed to illuminate the ketone body production and utilization in hearts.Secondly,we developed ACM induced pluripotent stem cell-derived cardiomyocytes(iPSC-CMs)disease model to verify the ketone metabolism from early to advanced phases.A previously reported mouse model(Myh6-Cre:Dspw/f)of ACM was also involved to measure alterations of ketone body enzymes in liver and heart during disease progression.Thirdly,we included validation cohort with 221 individuals,incuding 65 ACM probands with their 94 first/second-degree relatives,and 62 unrelated healthy volunteers.We evaluated the relationship between plasma β-OHB concentrations and MACE occurrence,and early disease progression.Results:Comparing explanted hearts from patients with ACM and healthy donors,we identified deregulated metabolic pathways using quantitative proteomics.Right ventricles from patients with AC displayed elevated ketone metabolic enzymes,OXCT1 and HMGCS2,suggesting higher ketone metabolism in ACM.Plasma β-OHB in ACM patients was higher than in patients with DCM,PAH,and healthy volunteers,suggesting that elevated ketone bodies might be a signature in ACM.Analysis of matched coronary artery and sinus plasma suggested potential ketone body synthesis at early-stage ACM,which was validated using patient-derived iPSC-CMs in vitro.Transgenic mice model validated that ketogenic activity was increased in heart but not liver at early stage,whereas at end stage,the hepatic ketogenesis was increased as a compensatory response to heart failure.Targeted metabolomics analysis in RVs from end-stage ACM revealed a"burned-out" state,with predominant medium-chain fatty acid rather than ketone body utilization.In an independent validation cohort,65 probands with mostly non-heart failure manifestations of ACM had higher plasma β-OHB than 62 healthy volunteers(P<0.001).ACM Probands with MACE had higher β-OHB than those without MACE(P<0.001).Among 94 relatives of probands,higher plasma β-OHB distinguished 25 relatives having suspected AC from non-affected relativesConclusions:This study demonstrates that cardiac ketogenesis occurs in ACM.Plasmaβ-OHB may be used as a potential biomarker to predict not only MACE in ACM probands,but also disease progression in ACM patients and their family members,particularly useful for individuals at the concealed phase.