Inflammation As A Predictor Of Atrial Fibrillation, And It's Relationship To Prothrombic State And Myocardiac Remodelling

BackgroudAtrial fibrillation(AF),the most commonly encountered arrhythmia in clinical practice,is associated with a 2-fold increase in total and cardiovascular mortality,as well as the potential for substantial morbidity,including stroke,congestive heart failure,and cardiomyopathy.Its incidence and prevalence are increasing,and it represents a growing clinical and economic burden.Owing to relative inefficacy and side effects of current pharmacological and Non-pharmacological therapy for AF,it remains a great challenge to improve primary and secondary AF prevention strategies to reduce this potentially enormous health burden.However the pathogeneses,especially risk factors underlying this arrhythmia should be fully understood at first step.Recent findings have demonstrated a mechanistic link between inflammatory processes and the development of AF,a link that may be a target for more effective treatment and prevention strategies.Evidence for an inflammatory contribution to at least some forms of AF was initially suggested by high incidences(25%to 40%) of AF after cardiac surgery.Till now,more and more study have showed that atrial fibrillation associated closely with inflammation,which didn't depend on the underlying cardiovascular disease.Some of these study found that significantly more inflammatory cells infiltrated or more inflammatory factors expressed within the atrial tissue of atrial fibrillation individuals, some others discovered the levels of circulatory inflammatory markers elevated, when compared to the respective indexes of non-af individuals.Moreover,some pleiotropic agents such as ACEIs,ARB,fish oil,etc.,and corticosteroids had been found to reduce the morbidity of AF in many clinical trials.However,the studies that showed correlation between AF and fibrillation are mostly cross-sectional,and could only imply the"coexistence"but not causal relation of AF and inflammation.Futhermore,there are still some studies showed different results,which didn't support the relationship between AF and inflammation at all.As a result,unlike atheroclerosis,AF was not accepted as a kind of inflammatory disease by the majority.Tumor necrosis factor(TNF)-αis essentially produced by monocytes and macrophages,and,in turn,it is the strongest known paracrine activator of monocytes and macrophages.Upon stimulation,these cells secrete a variety of products including interleukin(IL)-6,stimulating the liver to produce the acute phase reactant C-reactive protein(CRP).CRP is an acknowledged indicator of increased systemic inflammation across a wide range of diseases.TNF-αand CRP both are found in considerable quantities in atherosclerotic lesions,and they have also been associated with increased cardiovascular risk in numerous large population-based studies. Hitherto,a lot of studies has investigated the role of GRP in AF,but seldom ones have concentrated on the role of TNF-αin either the pathogenesis or prothrombotic state in AF.Some SNPs in TNF-ahave been found to associated with the level of circulatory TNF-αand some kinds of inflammatory disease,such as COPD and CHD,but to our knowledge,no one has investigate the relationship between SNPs in the TNF-αpromoter and AF.Both thrombophlebitis and pyemia are typical examples of hypercoagulatory states caused by inflammation.In fact,inflammation had been showed as an important reason that lead to thrombotic events in a lot of studies of atherosclerosis and heart failure.Further investigations have showed a prothrombotic state with blood hypercoagulation,platelet activation,damaged atrial endothelium and atrial blood stasis in AF,while these changes are due to the AF itself,or coexistent cardiovascular risk factors remains a matter of debate.Moreover,the relationship between inflammation and this prothrombotic state,especially blood hypercoagulation and platelet activation,has been seldom investigated.Nevertheless,this could be very important for our decision to chose an anti-thrombotic therapy,when the association of AF with inflammation get much clearer.Untill now,no study has specially focused on the changes of coagulation,platelet and endothelial fuction in idiopathic AF and lone AF,which should help to answer whether AF per se or the possibly coexisting cardiovascular disease cause the prothrombotic state in AF patient.In addition,it is somewhat vague the recommendation of antithrombotic therapies for these two specified AF populations in 2006 ACC/AHA/ESC AF guideline.The objectives of this study include:To investigate whether the levels of several inflammatory markers elevate in AF.To examine the proportion of genotype at the SNPs sites of TNF-αpromoter in AF and sex,age and risk factors matched controls, and make clear whether some of them imply great risk to the pathogeneses of AF.To explore the independent risk factors for AF.To investigate whether AF patients have hypercoagulatory state and platelet activation and if so,the influencing factors promoting prothrombotic statte and the role of inflammation in prothrombotic state. To study the association of AF with the incidence of bradicardia and the changes of cardiac morphology.To evaluate the differences of inflammatory markers and ultorsonic indexes of atrium between different types of AF.To observe the effect of duration of AF history on inflammation markers and ultrasonic indexes of atrium.Study population and methods102 AF patents were recruited into the study group,117 sex and age matched individual were recruited into the control group.Cases of AF were identified by ECG or charge diagnosis.Exclusion criteria were as follows:＞75 years old,overt atherosclerosis,congenital heart disease,rheumatic heart disease,myocardiopathy, LV thickening,heart dysfunction,diabete,chronic inflammatory disease,surgery and severe disease,tumor and function failure of important organ.Detailed medical history,physical examination,routine biochemical testing (including Fib and CRP testing)were performed in addition to a 12-lead electrocardiograms(ECG).By transthorasic echocardiography,valvular functions, left ventricular functions(ejection fraction),interventricular septum(IVSd),left atrial diameter(LAd)and right atrial diameter(RAd)were evaluated.LAd was measured in parasternal long axis view.Patients with AF who would be submitted to radiofrequecy ablation still underwent tranesophageal echocardiography in 72 hours before procedure.Each blood sample was obtained from a peripheral vein in drying tube and potassium citrate containing tube in the morning situation of fasting.Serum was immediately extracted,aliquoted,and stored at-80℃until analysis by a batch.Whole blood in the former used to extract genomic DNA with QIAamp^?DNA Blood Mini Kits within 3 days.The procedure included mainly cells lysis,column adsorption, washing and elution.DNA samples were then comfirmed by electrophoresis in agarose gel and their concentration were determined by the method of optical density before being stored at-20℃.The primers for PCR amplification of target fragment of TNF-αpromoter were designed using software Prime Premier 5 and synthesized by Shanghai Shenggong Co.Ltd.PCR amplification for each DNA sample was carried out using Promega PCR kit(GoTaq^?PCR Core SystemsⅠ).The main procedure include denaturation,annealing and extension,for about 30-35 circles.After comfirmed by electrophoresis in agarose gel,PCR products were send to Guangzhou Jingrui company for purifying and sequenceing.The sequencing results were read using Chroma software and blasted at Pubmed to determine genotype at the SNPs sites for every subjectSerum TNF-α,sP-Selectin and vWF were determined using commercially available enzyme-linked immunosorbentassay(ELISA)kits according to manufacturer's instructions.The interassay coefficient of variation is within 5%and intraassay coefficient of variation is within 10%according to manufacturer's instructions.The Curve Expert 1.3 was used to make standard curve and transfer optical density into concentration for each indices.Continuous data are expressed as mean±SD if normally distributed and as median (interquartile range)if nonparametrically distributed.Differences in clinical features, blood indices between two groups were evaluated with 2-sample t tests for normally distributed continuous variables,Mann-Whitney U tests for nonparametrically distributed continuous variables,and chi-square tests for categorical variables.Because the distribution of TNF-α,CRP,Fib,vWF,GPT,LAd,RAd,neutrophil and monocyte was skewed,logarithms transformation were applied to remove skewness of these variables before they were included into multivariate linear regression analysis and analysis of covariance.To account for covariate imbalance,analysis of covariance was introduced with the potential confounding factors as covariate.Correlation was evaluated with the Pearson coefficient when both variables are normally numberic ones,and with the Spearman coefficient when one or above of them aren't normally numberic variables.Partial correlation was introduced to control for the factors that potentially influence the correlation between two variable.A stepwise multiple logistic regression analysis was performed for identifying independent predictors for the presence of AF with sex,age and those varialbles showing difference with significance less than 0.20 between subjects with AF and those without AF in univariate analysis as explanatory variables.The odd ratio of the presence of-308A to the absence of-308A for AF was estimated by the function of"risk estimate"in the crosstab model of SPSS 13.0.Multiple variates linear regression analyses were performed respectively for identifying factors that determine important indices such as TNF-α,CRP,Fib,and sP-Selectin,including age,sex,grouping(AF or not),and variables associated with each of these indices with significance of less than 0.20 in univariate analysis.A p value of＜0.05 was accepted as significant in all analyses.All statistical analyses were made by using SPSS 13.0 program(SPSS Inc.;Chicago,Illinois)Results219 individuals were enrolled totally,with 102 AF patients in the AF group and 109 non-AF individuals in the control group.The proportion of male/female are 62/40 in AF group and 48/31 in control group(only 71 individuals has a sex record), and the proportions showed no significant differentce between two groups(P＞0.05). The AF group and the control group had a mean age of 56.7±10.9(29.0 to 75.0)years old and 56.7±10.9(38.5 to 75.0)years old respectively,with no significant difference found between two groups.The AF group include a sample of 87 paroxysmal AF,6 persistent AF and 9 long-persistent AF,most of whom were submitted to radiofrequency therapy.the control group include a sample of 30 individuals with AVNRT,49 healthy individuals and 38 DNA specimens of healthy persons.41 patients in the AF group and 45 individuals in the control group complicated with hypertension.Two groups had a similar proportion of hypertension(P＞0.05).Less frequent alleles weren't found at the SNPs site of-851,-575,-419 and-244 in TNF-αpromoter.The overall proportion of less frequent genotypes at the SNPs site of-863,-857,-806,-782,-646,-572,-570 and-238 of TNF-αpromoter is 24.8%, 24.3%,7.1%,1.0%,1.9%,2.4%,1.0%and 1.9%respectively.The ratioes of less frequent genotypes to ancestral genotype at these sites between the AF group and the control group all showed no significant difference(P＞0.05).The proportions of less frequent genotypes-308(GA or AA)and ancestral genotype-308GG were 21%and 79%,respectively,in the AF group,with 1 case being homozygote of-308A,and those in the control group were 10.1%and 89.9% respectively,with no-308AA.The distribution of the-308 genotypes was significantly different between the AF group and the control group(P=0.035)with the former having a higher frequency of less frequent genotypes.The presence of the -308A allele(including both homozygous and heterozygous subjects)was associated with an increased risk for AF(odds ratio 2.368,95%CI:1.078-5.205),compared with -308G homozygous subjects,which implied a higher incidence of AF in less frequent -308 genotypes subjects.In univariate analysis,when compared with the control group,the AF group has a significantly higher levels of circulatory TNF-α(3.84pg/ml(3.07,4.49)versus 2.85 pg/ml(2.28,3.60),P＜0.001),CRP(1.70mg/L(1,3.12)versus l.00mg/L(0.95,2.10), P=0.015),Fib(3.03g/L(2.62,3.65)versus 2.58g/L(2.28,3.02),P=0.002), sP-Selectin(26.5±5.8 ng/ml versus 21.3±3.8ng/ml,P＜0.001)and GPT(22.50 U/L(17.00,29.50)versus 18.00 U/L(13.50,23.50),P=0.002).The level of blood vWF show no significant difference between the two groups.These implied subjiects with AF had more severe inflammation and prothrombotic state than those without AF.No statistically significant differences in indices of blood cells,blood glucose, blood lipid,blood electrolytes,hepatic function,renal function and thyroid function, were found when comparing the AF group and the control group.These implied the above indices didn't influence the differences of the other indices between the two groups.Both fibrinogen and sP-selectin levels were significantly higher in individual with paroxysmal AF than those in the control group(Fib:3.20±0.84 and 2.77±0.60 g/L, respectively,P=0.005;sP-selectin:26.2±5.9 and 21.5±3.9 ng/mL,P＜0.001).Both differences were still significant after adjusted for age,sex and risk factors in analysis of covariance.The incidences of bradicardia in the AF group and the control group were 29.7% and 6.8%,respectively,with a significant difference between the two groups(P＜0.001).The subjects with AF had a higher incidence of mitral regurgitation then those without AF(52.3%and 14.9%,P＜0.001).In univariate analysis,some of the echocardiographic indices were significantly higher in the patients of the AF group than those in the control group.The indices include LAD(33.0mm(35.0,38.0)and 28.0mm(30.0,33.0),P＜0.001),RAD(47.0mm(43.0,51.0)and 41.0mm(37.0,43.0), P＜0.001)and IVSD(10.6(10.3,10.8)and 9.6(9.2,9.9),P＜0.001).These imply subjuects with AF have higher incidence of bradycardia and more severe myocardium remodeling,especially atrium remodelling.No statistically significant difference of TNF-αwere found when comparing the groups defined by the presence and absence of the-308A allele(3.37(2.72,4.44)and 3.36(2.83,4.16),P=0.600).In multivariate linear regression model,the presence of AF and monocyte number were showed to be independent factor affecting serum TNF-α,with regression coefficient being 0.331(P=0.002)and 0.199(P=0.046),respectively.The presence of-308A wasn't a independent factor influencing TNF-α.A multivariate linear analysis using serum CRP level as dependent variable showed 1gFib,1gvWF,1gNeu and lgIVSD were independent predictors,with regression coefficient being 0.343(P =0.002),0.288(P=0.005),0.239(P=0.023),0.347(P=0.001)respectively.TNF-α,sP-Selectin and LAd were showed to be independent predictors for the presence of AF in a multivariate logistic regression model with OR being(TNF-α: 1.375,95%CI(1.024-1.846),P=0.035;sP-Selectin:1.248,95%CI(1.121-1.389);P＜0.001 and LAd:1.321,95%CI(1.125-1.552),P=0.001).There were statistically positive correlations between inflammatory indices (TNF-αor CRP)and prothrombotic indices(Fib or sP-Selectin),among which the efficient for the correlation between CRP and Fib was 0.560(P＜0.001).After controlling for sex,age and grouping(AF or not),the correlations between TNF-αand Fib,TNF-αand sP-Selectin,and between CRP and sP-Selectin became statistically insignificant,while the statistical significance for the correlation between CRP and Fib was retained(r=0.421,P＜0.001).Grouping(that is,AF or not),sex,age and lgCRP were independent determinors of Fib.Their standard coefficients were 0.302(P=0.004),-0.396(P＜0.001),0.221 (P=0.032)and 0.265(P=0.013),which implied the presence of AF,woman,elder and inflammation all associated with higher level of Fib.The presence of AF was the only independent factor influencing the level of sP-Selectin in a multivariate linear regression.Its standard coefficients was 0.446(P＜0.001).Subgroup analysis showed raised Fib and sP-selectin levels in the subjects with idiopathic AF,compared to those in the subjects without risk factors,which implied a hypercoagulable state and platelet activation in idiopathic AF.Compared with age- and risk factors-matched control,the subjects with lone AF had a higher levels of sP-selectin but not Fib,which implied lone AF associated with platelet activation rather than hypercoagulation.The association of LAD with RAD was statistically significant in univariate analysis.This should imply the simultaneous morphous changes in left and right atrium.The levels of inflammatory markers and prothrombotic factors were not independent predictors for both atrial remodelling.LAd were 30.6±3.8,35.1±4.0 and 43.4±7.1mm for subjuects without AF,with paroxysmal AF and with persistent AF respectively.Analysis of variance showed significant difference in LAd between these three groups,with persistent AF patients having a higher LAd than paroxysmal AF patients and both groups having a higher LAd than subjects without AF.RAd were 42.8±4.8,46.2±6.2 and 55.8±7.0mm for subjuects without AF,with paroxysmal AF and with persistent AF respectively.Analysis of variance showed significant difference in RAd between these three groups,with persistent AF patients having a higher RAd than paroxysmal AF patients and both groups having a higher RAd than subjects without AF. Conclusion1.We didn't found less frequent alleles at the site of-851,-575,-419 and-244 in TNF-αpromoter.2.The subjects with AF had a higher rate of less frequent genotypes at-308 than those without AF.The presence of less frequent allele-308 A represent an underlying diathesis predisposing to this common arrhythmia.3.TNF-α,sP-selectin and LAd were independent predictors for AF.This should imply that inflammation plays a role in the pathogenesis of AF.4.The subject with AF had statistically higher level of Fib.Sex,age and inflammation were the independent factors affecting plasma fibrinogen level.Female,old age and high level of CRP implied high level of fibrinogen.5.The level of sP-selectin was higher in the subjects with AF.Inflammation wasn't a significant influential factor.6.Patients with paroxysmal AF had higher levels of fibrinogen and sP-selectin during sinus rhythm compared to those in control groups.7.The levels of plasma fibrinogen and serum sP-selectin were both higher in the subjects with idiopathic AF got higer,which implied that idiopathic AF associated with hypercoagulatory state and platelet activation.The level of serum sP-selectin but not that of plasma fibrinogen was higher in the subjects with lone AF,which implied lone AF associated with platelet activation.8.Both LAD and RAD were significantly larger in subjects with AF,which didn't rely on inflammation.9.Subjects with persistent and long-persistent AF had higher level of inflammatory markers and larger atrium than subjects with paroxysmal AF.