The Role Of TWEAK/Fn14 In Atrial Fibrillation And Its Mechanism

BackgroundAtrial fibrillation(AF)is a rapid supraventricular tachycardia characterized by atrial uncoordinated activation followed by atrial invalid contraction.AF was first confirmed by electrocardiogram more than 100 years ago,and since then it has become increasingly recognized as a major health burden worldwide.According to the 2010 Global Burden of Disease Study,after eliminating age factors,the global prevalence of AF is 596 per 100,000 men,33 out of every 100,000 women,and about 33 million.In Australia,Europe,and the United States,the prevalence of adult AF is about 1-4%,and in older people over 80 is greater than 13%.About 3 to 5 million Americans have AF,and as the population age,about 8 million Americans will have AF in 2050.By 2060,the number of Europeans suffering from AF will rise from the current 880,000 to 18 million.The incidence of AF increases rapidly with age and is<1%for people younger than 60 years old,12%for people aged 75-84 and>1/3 for people older than 80 years old.The most terrible complication of AF is cerebral embolism with an average annual risk of 4.4%.The quality of life of patients with AF decreased significantly.AF not only increases the risk of heart failure,myocardial infarction,tachycardia,pulmonary infection,dementia,and chronic kidney disease,but also augments mortality.Life expectancy in patients with AF is 8-10 years less than that of non-AF patients.After eliminating the age factor,the mortality rate of people with AF.over 35 years old in the world is 3.8/100,000 for men and 4.2/100,000 for women.However,the pathogenesis of AF remains unclear.At present,the mechanism of AF is considered to include two major categories:electrophysiological and pathophysiological mechanisms.The former contains a trigger mechanism and the latter includes a maintenance mechanism.The onset of AF needs a trigger,while maintenance requires a corresponding substrate.The substrate of AF is closely related to its pathophysiological mechanism.The four important pathophysiological mechanisms leading to AF include structural remodeling,electrical remodeling,autonomic nervous system changes,and Ca2+ handling abnormalities.Each mechanism can be caused by a variety of heart diseases and can promote the development of AF.Conversely,AF can lead to abnormal changes in each mechanism of AF promotion.Atrial remodeling caused by AF increases the susceptibility of the heart to AF and the persistence of AF.This self-enhancing characteristic of AF is M AF begets AF." Atopic ectopic impulses in the atria can maintain AF or trigger AF reentry by maintaining a substrate of reentry,which has conditions for proper reentry and induction and maintenance of AF.AF is an irregular atrial rhythm that can be maintained by regular sources of excitation or drive.Whether it is a rapid ectopic impulse or a single fast-rotating reentry loop,AF can be triggered by the spatial heterogeneity of the atrium "fibrillation conductio|".The balance between persistence and conductivity of this "fibrillation conduction" determines the basis for the induction and maintenance of AF.Atrial structural remodeling refers to the increase of myocardial interstitial tissue proliferation or fibrosis,the decrease of cell membrane stability,and the changes in the structure,morphology and quantity of some organelles.Pathological changes in structural remodeling include atrial enlargement,atrial muscle hypertrophy,atrial myocyte necrosis,atrial myocyte apoptosis,atrial fibrosis,and changes in extracellular matrix composition of atrial tissue.Structural remodeling is characterized by atrial enlargement and tissue fibrosis.Under normal functional conditions,atrial size is an important determinant of the persistence of AF.Fibrosis promotes AF because of disruption of fiber bundle continuity and interference with local conduction.In addition,the interaction of fibroblastic cardiomyocytes can lead to cardiac arrhythmias caused by bioelectrical activity of cardiomyocytes.Atrial fibrosis is a common endpoint for most AF development and can predict recurrence.Moreover,AF can lead to atrial fibrosis,which can result in resistance to treatment in patients with persistent arrhythmias.Recent studies have confirmed that inflammation plays an important role in AF,and some inflammatory cytokines such as IL-6 and TNF-a are closely associated with AF.As a new member of the tumor necrosis factor superfamily,tumor necrosis factor-like weak inducer of apoptosis(TWEAK)is a cytokine that has been clearly characterized by multiple functions and can regulate a variety of biological processes such as proliferation,migration,apoptosis,differentiation,angiogenesis,and inflammation.In 1997,Chicheportiche et al.found that the TWEAK gene is located on chromosome 17p13.I and composed of 249 amino acids.It is a type ? membrane protein with a hydrophobic bond at its N-terminus,allowing its N-terminus to be inserted into the cell membrane,and the C-terminal extracellular region contains its receptor binding site.Because it has a TNF homology domain consisting of a?-sandwich structure,it belongs to the TNF superfamily.TWEAK is a membrane-bound protein at the beginning of cell endoplasmic reticulum synthesis and is rapidly hydrolyzed by furin into a soluble fragment of 156 amino acids with biological properties.In 2001,fibroblast growth factor-inducible molecule 14(Fn14)was confirmed to be a receptor for TWEAK and is the smallest member of the TNF receptor superfamily to date.The human Fn14 gene is located on chromosome 16p13.3 and is a type I membrane protein encoding 129 amino acids.It is then hydrolyzed by the signal peptide into a cell surface receptor consisting of 102 amino acids.TWEAK is widely expressed in various tissues and organs such as heart,blood vessels,pancreas,small intestine,brain,lung,ovary and skeletal muscle,and is also detected at low levels in the liver and kidney.High expression of Fn14 mRNA was observed in heart,kidney,lung and placenta.In the cardiovascular system,TWEAK and Fn14 mRNA or protein can be expressed in ventricular myocytes,cardiac fibroblasts,human endothelial cells,and smooth muscle cells.TWEAK works by binding to its receptor Fn14.This interaction activates a variety of intracellular signaling pathways including the classical and non-canonical NF-?B pathways and the MAPK pathway.Studies have suggested that the TWEAK/Fn14 axis is closely associated with diverse cardiovascular diseases such as coronary heart disease,heart failure,hypertension,pulmonary hypertension,abdominal aortic aneurysm,and peripheral arterial disease.However,the role of TWEAK/Fn14 in the pathogenesis of AF remains unclear.In this study,the concentration of soluble TWEAK(sTWEAK)in serum of patients with AF was measured,the expression of TWEAK in peripheral blood mononuclear cell(PBMCs)was observed,and the expression of Fnl4 in right atrial appendage of patients with AF was analyzed,which is to explore whether TWEAK/Fnl4 is closely related to the onset of atrial fibrillation and whether sTWEAK is a serum marker of AF.Objectives1.To study the role of TWEAK/Fn14 in the pathogenesis of AF;2.To investigate whether sTWEAK is a serologic biomarker of AF.Methods1.Study subjectThis study selected 62 patients admitted to the Department of Cardiology,Qilu Hospital of Shandong University from March 2014 to June 2015.They were divided into two groups:AF group(n=42)and control group(n=20).Among them,there were 25 males and 17 females with AF,with an average age of 57.76±10.14 years.In the AF subgroup,there were 9 males and 11 females with an average age of 58.55±9.17 years in the paroxysmal AF group.There were 16 males and 6 females with a mean age of 5 7.05±11.10 years in the persistent AF group.Twenty patients in the control group were from those who underwent radiofrequency ablation in the Department of Cardiology for paroxysmal supraventricular tachycardia,including 10 males and 10 females with an average age of 55.25±8.42 years.2.Clinical data collectionClinical data were collected such as smoking history,drinking history,hypertension,diabetes,history of coronary heart disease,and medication history(ACEI or ARB,beta blockers,and calcium antagonists).Blood pressure,height and weight were measured on the day of admission,and body mass index(BMI)was calculated from body weight(Kg)/height(m)2.3.ELISA assaySerum TWEAK and CRP concentrations were determined by ELISA.4.Western BlottingTWEAK expression in the peripheral blood mononuclear cells and Fn14 level in the right atrial appendage were detected by western blotting.5.Immunohistochemical stainingThe expression of Fn14 protein in right atrial appendage was observed by immunohistochemical staining.6.Statistical analysisData processing was performed using SPSS 17.0 software.The measurement data was first subjected to a normal distribution by Kolmogorov-Smirnov test,and expressed as mean±standard deviation if obeying a normal distribution.The mean comparison between the two groups was performed using an independent sample t test,and the mean between groups was compared using one-way ANOVA.The count data is tested by ?2.When the theoretical frequency is less than 1,Fisher's exact probability method is used.When both variables are subject to a normal distribution,correlation analysis is performed using pearson correlation.Univariate and multivariate logistic stepwise regression analysis were used to determined whether sTWEAK is a risk factor for the onset of AF.P<0.05 was considered to be statistically significant.Results1.Baseline characteristicsThe subjects in the AF group had some similarity to those in the control group in age,gender,body mass index,systolic blood pressure,diastolic blood pressure,past history such as hypertension,diabetes,coronary heart disease,smoking history and drinking history,combined medication including angiotensin-converting enzyme inhibitor(ACEI),angiotensin receptor blocker(ARB),beta receptor blockers,calcium antagonists.In terms of other parameters,there were no significant differences in alanine aminotransferase,total cholesterol,triglycerides,high-density lipoprotein cholesterol and low-density lipoprotein cholesterol,urea nitrogen,creatinine,fasting blood glucose,and left ventricular ejection fraction(LVEF).However,the peripheral blood leukocyte count,left atrial diameter and serum C-reactive protein(CRP)concentration in the AF group were significantly higher than those in the control group(P<0.05).while serum soluble TWEAK was dramaticlly lower than the control group(P<0.05).2.Baseline characteristics in patients with AFThe patients in the paroxysmal AF group had some similarity to those in the persistent AF group in age,gender,body mass index,systolic blood pressure,diastolic blood pressure,past history such as hypertension,diabetes,coronary heart disease and drinking history,and combined medications including ACEI,ARB,and ?-receptor blockers and calcium ion antagonists.In the history of smoking,patients with paroxysmal AF had a significantly more smoking history than those with persistent AF.In terms of biochemical parameters,there were no significant differences in peripheral blood leukocyte counts,alanine aminotransferase,total cholesterol,triglycerides,high-density lipoprotein cholesterol and low-density lipoprotein cholesterol,urea nitrogen,creatinine,and fasting blood glucose.Although LVEF was normal in both groups,LVEF in patients with persistent AF were observably lower than those with paroxysmal AF.The left atrial diameter,serum levels of CRP and sTWEAK in patients with persistent AF were significantly higher than those with paroxysmal AF(P<0.05).3.Correlation between serum sTWEAK level and CRP concentration and left atrial diameter in patients with AFSerum soluble TWEAK levels were positively correlated with CRP concentration in patients with AF(R2=0.626,P<0.0001),but negatively correlated with left atrial diameter(R2=0.224,P=0.002).4.Analysis of risk factors for AFWith AF as the dependent variable,age,body mass index,history of hypertension,white blood cell count,left atrial diameter,CRP and TWEAK as independent variables,univariate regression analysis was performed,and white blood cell count,left atrial diameter and TWEAK were found to significant correlations(P<0.05).With AF as the dependent variable,white blood cell count,left atrial diameter and TWEAK as independent variables,multivariate regression analysis showed that TWEAK was a protection factor for AF(OR=0.269,95%CI=0.088-0.826,P=0.022).5.Expression of TWEAK in peripheral blood mononuclear cells(PBMCs)in the study populationWestern blot analysis showed that the expression of TWEAK in PBMCs of patients with AF was significantly higher than that of the control group(P<0.05).6.High expression of Fn14 in right atrial appendage of patients with AF(1)Baseline characteristics in patients undergoing cardiac surgeryThe patients in the AF group had some similarity to those in the control group in age,gender,body mass index,systolic blood pressure,diastolic blood pressure,past history such as hypertension,diabetes,coronary heart disease,smoking history and drinking history,combined medication including ACEI or ARB,?-blockers,and calcium ion antagonists.In terms of other parameters,there were no significant differences in white blood cell count,total cholesterol,triglycerides,high-density lipoprotein cholesterol and low-density lipoprotein cholesterol,urea nitrogen,creatinine,fasting blood glucose,and LVEF.However,the left atrial diameter of the AF group was slightly larger than that of the sinus rhythm group(41.38± 10.94 mm vs.58.71 ± 19.40 mm,P = 0.049).(2)Right atrial appendage tissue from patients with AF showed hypertrophy of atrial myocytesMyocardial fibers in the right atrial appendage of the sinus rhythm group were arranged regularly,with small cells and mild interstitial fibrosis.The muscle fibers of the AF group had different sizes,loose and disordered cells,large cells,different cell nucleus,obvious nuclear atypia,broken and dissolved myofilament,and a large amount of collagen hyperplasia in the interstitium.The right atrial appendage from patients with AF showed that the area of atrial myocytes was significantly higher than that of the control group,indicating that the atrial myocytes were markedly hypertrophied(P<0.05).(3)High expression of Fn14 in right atrial appendage of patients with AFWestern blotting and immunohistochemistry confirmed that Fn14 protein was highly expressed in right atrial appendage of patients with AF(P<0.05).The positive signal for immunohistochemical staining was brown particles.In the sinus rhythm group,the right atrial appendage myocytes displayed uniform and sparse light brown particles in the cytoplasm,while the AF group showed dense dark brown particles in the cardiomyocytes.Conclusions.1.TWEAK/Fn14 is closely related to AF;2.sTWEAK may be a serologic biomarker of AF.IntroductionAtrial fibrillation(AF)is the most common sustained cardiac arrhythmia in the general population,serving as a primary risk factor of stroke,which is currently the 5th leading cause of death in the U.S.and 2nd in the world.AF prevalence continues to increase,affecting 6-12 million U.S.citizens by 2050 and nearly 18 million Europeans by 2060,which creates great concerns as it propels toward epidemic status.In the UK,the direct cost of AF accounts for about 1%of the total cost of health care,while in the US,it was $6-26 billion in 2008,mainly due to complications of AF such as stroke and its treatment costs.With the advent of social aging,AF will become a serious public health and economic burden.AF can lead to stroke,thromboembolism,heart failure,myocardial infarction,renal impairment,cognitive decline and dementia.Currently,although we have made substantial progress in understanding the pathophysiological mechanism of AF,the cause and maintenance mechanism of atrial fibrillation have not been fully clarified.At present,available treatment options all have moderate effectiveness,likely because of our limited understanding of the mechanisms promoting initiation,progression,and maintenance of AF.A better understanding of the molecular mechanisms of AF will help to identify and implement safer and more effective therapies for AF.Diverse factors can increase the risk of AF,such as gene mutation,smoking,alcohol consumption,aging,obesity,inflammation and so on.Accumulating evidence suggests that inflammatory markers such as IL-6,IL-1?,myeloperoxidase(MPO)and tumor necrosis factor-alpha(TNF-a)are positively correlated with the progression of AF from paroxysm to persistence,and can predict the outcome of ablation of AF.Atrial remodeling constitutes the pathophysiological substrate on which AF occurs and maintains.Atrial remodeling occurs at both the macro and micro levels.Macroscopic changes are marked by atrial dilatation,while significant microscopic changes include atrial myocyte hypertrophy,myolysis,dedifferentiation,fibrosis,apoptosis,mitochondrial and sarcoplasmic reticulum disintegration.Basic and clinical studies have confirmed that TNF-? can promote atrial hypertrophy and fibrosis,and then participate in atrial structural remodeling,which is closely related to the occurrence of AF.As a new member of the tumor necrosis factor superfamily,fibroblast growth factor-inducible molecule 14(Fn14)was proved to be the receptor of tumor necrosis factor-like weak inducer of apoptosis(TWEAK)in 2001 and is the smallest superfamily of TNF receptors so far.Human Fn14 gene is located on chromosome 16p13.3 and is a type I membrane protein encoding 129 amino acids.It is then hydrolyzed by signal peptide to a cellular surface receptor composed of 102 amino acids.At the organ level,Fn14 is highly expressed in the heart,kidney,lung and placenta.At the cellular level,Fn14 gene or protein can be expressed in ventricular myocytes,atrial myocytes,cardiac fibroblasts,smooth muscle cells,human endothelial cells,monocytes/macrophages.Combination of TWEAK and Fn14 regulates proliferation,migration,apoptosis,differentiation,inflammation and angiogenesis of various cells and tissues by activating various intracellular signaling pathways,including classical as well as non-classical NF-?B pathways and mitogen-activated protein kinase(MAPK)pathways.It is suggested that Fn14 may be involved in left ventricular fibrosis in SHR rats and TWEAK can induce hypertrophy of ventricular myocytes in mice,partly by binding to Fn14 and then activating the NF-kappa B pathway to promote ventricular hypertrophy.TWEAK can also activate the proliferation and collagen synthesis of rat fibroblasts through binding to Fn14 as well as activating NF-?B,ERK1/2 and p38 MAPK pathways.In addition,our recent research has confirmed that TWEAK/Fn14 can promote HL-1 atrial myocyte hypertrophy by activating JAK2/STAT3 signaling pathway.However,the role and mechanism of Fn14 in the cell model of AF induced by rapid pacing remain unclear.The purpose of this study is to explore:(1)whether rapid pacing can facilitate hypertrophy in HL-1 atrial myocyte;(2)the role of Fn14 in this process and its possible mechanism.Materials and methodsHL-1 cell culture and tachypacingHL-1 atrial myocytes derived from the AT-1 mouse atria were kindly provided by Dr.William Claycomb(Louisiana State University Health Science Center,New Orleans,LA,USA).The cells were cultured and maintained in supplemented Claycomb Medium(Sigma-Aldrich,St.Louis,Mo)as previously described.The cells were serum-starved for 24 h on the third day after passaging and then used for rapid pacing.The spontaneous rate of confluent HL-1 cells is 0.5-1 Hz.After serum starvation for 24 h,the HL-1 atrial myocytes were subjected to field stimulation(paced group)or cultured parallelly in the absence of stimulation(control group).Stimulated atrial myocytes were paced as previously described with a C-Pac100TM-culture pacer and C-Dish100TM culture dishes(IonOptix Corporation,Netherlands)with a 10-ms duration and 4 Hz square-wave pulses(30V pulse-voltage)for 24 h.Western blottingTotal protein was extracted from HL-1 cells using standard protocols in a RIPA buffer with 1 mM PMSF.Equal amounts of protein from different experimental groups were separated by 8-12%SDS-PAGE and transferred to PVDF membranes(Millipore,Eschborn,Germany),which were blocked for one hour with 5%non-fat milk in TBST at room temperature,then incubated overnight at 4? with primary antibodies against ANP(Millipore,Temecula,CA,USA),Troponin T(Novus,Littleton,CO,USA),a-tubulin(Millipore,Temecula,CA,USA),Fn14(Abcam,Cambridge,UK),phospho-STAT3(at Tyr705),and STAT3(all Cell Signaling Technology,Danvers,MA),and then incubated with appropriate secondary HRP-conjugated secondary antibody for one hour at room temperature.The protein signals were detected using a chemiluminescence kit(Millipore,MA,USA).Statistical analysisAll statistical analyses were performed using SPSS 17.0(SPSS,Chicago,IL,USA).Statistical data of three independent experimental results,i.e.measurement data were tested by Kolmogorov-Smirnov test whether they obeyed normal distribution or not.If data obeyed normal distribution,they were expressed as mean±standard deviation.Independent sample t test was used to compare the mean between the two groups.All p-values were two-sided,a p-value of<0.05 was considered statistically significant.ResultsHL-1 cell culture and inverted microscope observationHL-1 cells were cultured in Claycomb complete medium(Claycomb medium 87ml,10%fetal bovine serum 10ml,100 U/ml penicillin,10Og/ml streptomycin 1 ml,2 mM L-glutamine 1 ml and 0.1 mM norepinephrine).The culture flask T25 was pre-coated with gelatin and fibronectin and placed in a cell incubator with 5%CO2 and 37? constant temperature.The cells grew well,when their cell density is basically integrated,morphological observations were made under an inverted microscope.HL-1 cells exhibited various forms,mainly triangular,irregular,rod-shaped and fusiform.After 72 hours,the cells were observed to grow and fuse into pieces,and most cells showed regular pulsation.Rapid pacing can promote HL-1 cell hypertrophyAfter 24 hours of rapid pacing of HL-1 cells,Western Blotting showed a significant increase in ANP and Troponin T protein expression in the paced group compared with the control group,and the results were statistically significant(P<0.05).The findings showed that electrical stimulation with high frequency can up-regulate the expression levels of the cell hypertrophic markers ANP and Troponin T,promote the atrial myocyte hypertrophy,and further induce the structural remodeling of atrial myocytes.Rapid pacing can facilitate high expression of Fn14 in HL-1 cellsAfter 24 hours of electrical stimulation of HL-1 cells,the expression of Fn14 protein in the paced group was significantlv increased compared with the control group(P<0.05).the result indicated that high-frequency electrical stimulation can upregulate the expression level of TWEAK receptor Fn14,suggesting that Fn14 may be involved in the process of promoting atrial myocyte hypertrophy,and then participate in the structural remodeling of atrial myocytes.Rapid pacing can activate STAT3 signaling pathway in HL-1 cellsWestern Blotting results demonstrated that after 24 hours of electrical stimulation of HL-1 cells,there was no significant increase in total STAT3 expression in the paced group,but the expression of p-STAT3 was remarkably increased(P<0.05).The findings indicate that high-frequency electrical stimulation can activate the expressio n of p-STAT3 protein in the signaling pathway.The Fn14/STAT3 signaling pathway may be involved in the process of electrical stimulation to promote atrial myocyte hypertrophy and then participate in the structural remodeling of atrial myocytes.ConculusionFn14 may involved in rapid pacing-induced HL-1 atrial myocytes hypertrophy by activating STAT3 pathway.