Fatty Properties And Lipidomics Studies Of Arrhythmogenic Right Ventricular Cardiomyopathy

Background:Arrhythmogenic right ventricular cardiomyopathy(ARVC)is a rare type of cardiomyocyte disease featured by preferentially fibro-fatty infiltration of right ventricle walls.Conventionally,there are two types of adipose,namely white adipose and brown adipose.The functions of white adipose primarily include metabolic energy storage and hormone production,whereas brown adipose functions via thermogenesis in response to cold exposure.The white adipocytes can be differentiated from stromal-vascular cells through entire life cycle,whereas brown adipocytes develop only during fetal period and decrease overtime in adult life.The determinant transcription factors of white adipocytes during differentiation are PPAR-y and RXR,whereas brown adipocytes are regulated by both PRDM-16 and PGC-1.Currently,the epicardial fat are considered to be consisting of white adipocytes,however,other reports discovered that epicardial fat was uncoupling protine-1(UCP-1)positive which is the unique biomarker of brown adipocyte,suggesting the epicardial fat could be brown adipose.The fibro-fatty infiltration usually progresses from epicardium towards endocardium.Due to epicardial adipose tissue could be differentiated from epicardium,the possibility of infiltrating adipose in ARVC caused by over-adipogenesis of epicardial adipose tissue or epicardium can therefore unable to be excluded.The issue of ARVC infiltrating adipose to be whether white or brown adipose remains undetermined.Identification of ARVC infiltrating adipose is indicative in uncovering its cellular origin as well as the exact underlying mechanism of abnormal adipogenesis.Objective:this study sought to identify the nature of infiltrating adipose of ARVC.Methods:the full section tissue of ventricular walls of ARVC patients(n=8),human suprasternal adipose(n=10),human anterior mediastinum adipose(n=10),human epicardial adipose(n=10)and human subepidermal adipose were collected.Both immunohistochemical staining and western immunoblotting were utilized to detect the expression of UCP-1 among above tissues;In addition,Masson trichrome staining and HE staining were both adopted to validate the morphometric characteristics of above tissues.Results:As demonstrated by both the immunohistochemical staining and western immunoblotting results,the infiltrating adipose of ARVC right ventricle walls are UCP-1 negative,whereas both human suprasternal and anterior-mediastinal adipose are UCP-1 positive,and both human epicardial adipose and subepidermal adipose are UCP-1 negative.ARVC infiltrating adipose is morphometrically analogous to the typical white adipocytes:the diameter of adipocytes ranges from 15 to 150 um which are usually filled by a unilocular lipid droplet,cytomembrane could be clearly demarcated at which flatten nucleus is situated.Both human suprasternal and anterior mediastinal adipose contain typical brown adipocytes:the diameter of adipocytes ranges from 10-25um which are usually filled by miniscule multilocular lipid droplets,cytomembrane could not be clearly demarcated with round nucleus eccentrically situated.human epicardial adipose as well as human subepidermal adipose exhibit classical morphological features of white adipocytes.Conclusions:The infiltrating adipose of ARVC right ventricle walls is white adipose,both human suprasternal adipose and human anterior mediastinal adipose contain portion of brown adipose,both human epicardial adipose and subepidermal adipose are typical white adipose.Background:arrhythmogenic right ventricular cardiomyopathy is a type of rare cardiomyocytes disease featured by primarily fibro-fatty infiltration of right ventricle walls.The infiltration eventually exacerbates over time,with unavailable effective therapies to prevent it at present time.Investigation of the lipids composition and changing trend of infiltrating adipose tissue is necessary for comprehending the underlying mechanism of adipogenesis and early diagnosis or even clinical intervening of ARVC.Objective:By utilizing liquid chromatography-mass spectrometer techniques,to delineate the lipidomic characteristics of ARVC right ventricular infiltrating adipose tissue(ARVIAT)that are expected to be distinct from normal right ventricular epicardial adipose tissue(NRVEAT).Methods:Approximate 50 mg of either ARVIAT(n=32)or NRVEAT(n=10)preserved in liquid nitrogen were harvested,total lipids contained in equivalent to 45 mg of tissue were then extracted by HPLC-grade dichloromethane:methanol(volume ratio,2:1)mixture from the raw homogenized lysate produced via violent oscillation.Lipids were then analyzed by both positive and negative mode liquid chromatography-mass spectrometer(LC-MS)(Thermo Q-Exactive).The preliminary data was processed via data-dependent MS/MS.Lipids ranging from 240-2000 in positive mode and from 200-2000 in negative mode were selected after searching through the database of Lipid search or Trace Finder.The raw data of lipids was then normalized within the same lipid class and analyzed through multivariate statistical analysis and hierarchical cluster analysis to identify the potential lipid biomarker able to differentiate ARVIAT from NRVEAT.Results:A total of 599 lipids were detected.The ingredient of lipids are identical between ARVIAT and NRVEAT.According to the principal component analysis,the global characteristics,however,are significantly different between the two groups.Comparing to that of NRVEAT,there are 87 and 69 subtypes of lipids are proved to be relatively elevated or decreased in ARVIAT,respectively.The major differential lipids are phosphatidylethanolamine(PE,24 subtypes elevated,10 decreased),phosphatidylcholine(PC,23 elevated,9 decreased),cardiolipin(CL,7 elevated,22 decreased)and triglyceride(TG,11 elevated,12 decreased).The plasmalogens PC such as PC(16:0p/18:2)and PC(16:0p/20:4)in ARVIAT are significantly decreased as compared to NRVEAT.The TG species containing 16:1 and 18:3 acyl-chains in ARVIAT such as TG(16:0/16:0/16:1)(p<0.01),TG(16:1/16:1/18:1)(p<0.001),TG(16:1/18:1/18:2)(p<0.001)TG(16:1/18:1/18:3)(p<0.001)and TG(20:1/18:1/18:3)(p<0.001)are significantly declined compared to NRVEAT.Palmitoleic acid(16:1)(p<0.01)and linolenic acid(18:3)(p<0.01)in ARVIAT are significantly elevated;Stearic acid(18:0)(p<0.01)in ARVIAT is significantly declined compared to NRVEAT,whereas total lipids containing stearic acid(18:0)had no significant difference between the two groups.Receiver-Operating Characteristic curve analysis identified the potential lipid biomarkers capable of differentiating ARVIAT from NRVEAT which include CL(18:2/18:2/18:2/18:1)(AUC=0.98,95%CI 0.883-1),CL(18:2/20:3/18:2/18:1)(AUC=0.98,95%CI 0.883-1),TG(16:1/16:1/18:1)(AUC=0.98,95%CI 0.888-1)and TG(16:1/18:1/18:3)(AUC=1,95%CI 0.916-1).Conclusions:The composition of lipids in ARVIAT is a distinct entity as compared to NRVEAT:The distribution of lipids in ARVIAT,including phospholipids such as PE,PC and CL,neutral lipids such as TG and diglyceride(DG),and Fatty acids(FA),significantly varies from that of NRVEAT,suggesting that multiple pathways involved in lipids metabolism of ARVIAT are perturbed.The significant decrease of Plasmalogens-PC in ARVIAT may indicate the increased oxidative stress.ARVIAT may have malfunction in synthesizing TG by incorporating such as palmitic acid or palmitoleic acid or in lipolysis of TG containing the corresponding fatty acyls.Stearic acid in ARVIAT is significantly decreased,whereas there is no significant decrease of total lipids containing stearic acid in ARVIAT,indicating the ARVIAT may tend to release stearic acid as the main substrate for energy supply,which may increase cardiac lipotoxicity and consequently underlie the pathology of development of heart failure in ARVC patients.