The Mechanism Of TWEAK/FN14 In Atrial Remodeling And Atrial Fibrillation Anticoagulant Therapy

BackgroudAtrial fibrillation(AF)is the most common sustained arrhythmia in clinical practice,and its underlying substrates are the focus of research on novel therapeutic targets.Atrial myocyte hypertrophy is one of the most important structural remodeling features in AF,and its main substrate.Atrial myocyte hypertrophy can lead to conduction heterogeneity and is one of the most important substrates in AF.However,the molecular mechanisms underlying atrial myocyte hypertrophy are largely unknown.AF is clearly associated with increased levels of known inflammatory markers and inflammation plays a role in the genesis and perpetuation of AF.TWEAK is a member of the tumor necrosis factor(TNF)pro-inflammatory cytokine superfamily.Consistent with leukocytes as a main cellular source of TWEAK,TWEAK is expressed broadly by human peripheral blood-derived innate immune cells.TWEAK is expressed as a type Ⅱ transmembrane protein that can be processed to generate a soluble cytokine.Elevated circulating levels of TWEAK are sufficient to cause cardiac dilation and progression to heart failure in adult TWEAK transgenic overexpression mice.However,it has been reported that decreased concentrations of sTWEAK have been observed in diseases related to chronic low-grade inflammation,and TWEAK may have evolved to guard against the development of a potentially excessive inflammatory response.Previous studies have demonstrated that subjects with carotid stenosis showed a reduced plasma sTWEAK level compared with healthy subjects,and sTWEAK concentrations negatively correlated with the carotid intima-media thickness.Decreased sTWEAK concentration is significantly and independently associated with long-term cardiovascular mortality in patients with peripheral arterial disease.TWEAK blood plasma levels are reduced in monocrotaline-treated rats while Fn14 is upregulated in the heart.TWEAK is a multifunctional cytokine that functions mainly through the fibroblast growth factor-inducible molecule 14(Fn14)receptor.Fn14 is a tightly regulated receptor that has been associated with various downstream signaling cascades.Although Fn14 usually is expressed at very low levels under physiological conditions,it can be induced in cardiovascular disease,and the TWEAK/Fn14 axis is involved in the pathogenesis of various cardiovascular diseases.Adult TWEAK transgenic mice exhibit cardiomyocyte hypertrophy and cardiac dilation,while Fn14-/-mice are protected against TWEAK-induced cardiac dilation.Activation of TWEAK/Fn14 signaling in adult rat primary cardiac myocytes causes cardiomyocyte hypertrophy.However,the expression of Fn14 in atrial structural remodeling remain undefined.Janus kinase(JAK)/signal transducer and activator of transcription(STAT)pathway was initially discovered as a major cytokine signal transduction pathway.JAK-STAT signaling pathway plays an important role in cardiac pathophysiology and has been implicated in pressure overload-induced cardiac hypertrophy and remodeling.The activation of JAK/STAT pathway by IL-6 may contribute to the pathogenesis of cardiac hypertrophy,and phospho-STAT3 levels are elevated in atrial tissues from AF patients.TWEAK/Fn 14 upregulates pro-inflammatory cytokine secretion via STAT3 pathways in hepatic stellate cells.We hypothesized that upregulated Fn14 expression may facilitate TWEAK-induced atrial myocyte hypertrophy and that,conversely,Fn14 inhibition may have a protective role on atrial structural remodeling.The present study therefore explored the potential role and underlying mechanism of Fn 14 in HL-1 atrial myocyte hypertrophy under conditions of TWEAK stimulation.Objectives1.To study the effect of TWEAK on the atrial myocyte hypertrophy.2.To study the effect of TWEAK on the Fn14 expression in HL-1 atrial myocyte.3.To study the mechanism of TWEAK affecting the atrial remodeling.Methods1.Cell cultureHL-1 atrial myocytes derived from adult mouse atria were obtained from Dr.William Claycomb.We chose a series of TWEAK concentrations to explore the effect of TWEAK on hypertrophy of HL-1 atrial myocytes.2.Transfection of small interfering RNA(siRNA)HL-1 myocytes were cultured in antibiotic-free growth medium until they had reached 60%confluency.The cells were incubated with a mixture of specific siRNA to negative control(NC),Fnl4,JAK2 or STAT3 and Lipo-fectamine 2000 in antibiotic-free and serum-free medium for 12h.The HL-1 cells were divided into the following groups according to different interventions:control group,TWEAK group,TWEAK+siRNA-NC group,TWEAK+ siRNA-Fn14 group/TWEAK+ siRNA-JAK2 group/TWEAK+ siRNA-STAT3 group.3.Cell immunofluorescenceHL-1 atrial myocytes were cultured on Millicell EZ slide 8-well.HL-1 cells were treated with TWEAK for 24 h after transfection of siRNA Fn14.After treatment,cells were washed with PBS,then fixed with 4%paraformaldehyde for 20 min at room temperature.Cells were washed thrice with PBS,blocked in 5%bovine serum albumin buffer for 30 min,and then were incubated overnight at 4℃ with the primary antibodies against Fn14(1:100 dilution).Cells were gently washed thrice with PBS before incubation with FITC-conjugated secondary antibody(1:200 dilution)for 30 min at 37℃.Nuclei were stained with DAPI.4.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 SDS-PAGE and transferred to PVDF membranes,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 TWEAK,Fn14,ANP,Troponin T,β-actin,phospho-JAK1(at Tyr1022/1023),JAK1,phospho-JAK2(at Tyr1007/1008),JAK2,phospho-TYK2(at Tyr1054/1055),TYK2,phospho-STAT1(at Tyr701),STAT1,phospho-STAT3(at Tyr705),and STAT3,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.5.Statistical analysisAll statistical analyses were performed using SPSS 17.0.Continuous variables are represented as means±SD,and were compared using unpaired t test and one-way ANOVA.Categorical variables were presented as counts and percentages then analyzed using either a Chi-square test or Fisher’s exact test.A difference with a p value of less than 0.05(2-sided)was considered statistically significant.Results1.TWEAK increased Fn14 expression in HL-1 atrial myocytesBased on previous studies,we chose a series of TWEAK concentrations to explore the effect of TWEAK on Fn14 expression of HL-1 atrial myocytes.The Fn14 protein level increased at 100,200 and 400 ng/mL with TWEAK(p<0.05).Thus,Fnl4 expression was positively regulated by TWEAK in HL-1 cells in vitro,and the concentration of 100 ng/ml as used as TWEAK stimulation in the following vitro experiments.2.TWEAK induced hypertrophy of HL-1 atrial myocytesMeanwhile,we evaluated the expression of hypertrophy marker genes in TWEAK-treated HL-1 atrial myocytes at different concentration.Relative to NC treatment,TWEAK at 50,100,200 and 400 ng/ml increased ANP expression(p<0.05),and at the latter 3 concentration also Troponin T expression increased(p<0.05).3.Fn14 inhibition attenuated the increased hypertrophy induced by TWEAK stimulation in HL-1 atrial myocytesTo determine the effect of Fn14 on atrial myocytes hypertrophy,we used Fn14-specific siRNA to knock down Fn14 expression in HL-1s.Fn14 expression significantly decreased after transfection with Fn14-specific siRNA as compared with siNC treatment(p<0.05).Immunofluorescence assay also revealed greater Fn 14 protein level with TWEAK stimulation than control conditions,and reduced Fn14 protein expression following Fn14 inhibition for 24 hours(p<0.05).TWEAK stimulation significantly increased HL-1 atrial myocytes hypertrophy marker genes expression,while inhibition of Fn14 attenuated the increased ANP and Troponin T protein expression induced by TWEAK(p<0.05).Therefore,TWEAK-induced HL-1 atrial myocytes hypertrophy was reduced following the inhibition of Fn14,rendering Fn14 responsible for TWEAK-induced HL-1s hypertrophy.4.Activation of JAK2 pathway by TWEAK/Fn 14 in HL-1 atrial myocytesTo assess whether JAKs pathway was associated with TWEAK/Fn14 activation in HL-1 atrial myocytes,we explored JAKs expression with TWEAK treatment.p-JAK2 expression significantly increased after TWEAK treatment for 24 hours(p<0.05).p-JAK1,p-TYK2 protein expression were also evaluated and no obvious changes were observed after TWEAK stimulation.5.Activation of STAT3 pathway by TWEAK/Fn14 in HL-1 atrial myocytesTo assess whether STATs pathway was associated with TWEAK/Fn14 activation in HL-1 atrial myocytes,we explored STATs expression with TWEAK treatment.p-STAT3 expression significantly increased after TWEAK treatment for 24 hours(p<0.05).p-STAT1 protein expression was unchanged after TWEAK stimulation.6.Fnl4 inhibition attenuated activation of JAK2/STAT3 pathway induced by TWEAK stimulation in HL-1 atrial myocytesIn addition,western blot analysis showed that inhibition of Fn14 effectively decreased TWEAK-induced p-JAK2 and p-STAT3 expression(p<0.05).7.Fn14 mediated TWEAK-induced hypertrophy via JAK2/STAT3 pathway in HL-1 atrial myocytesBecause JAK2/STAT3 pathway can be modulated by TWEAK/Fnl4 axis,we explored the potential role of the JAK2/STAT3 pathway in TWEAK-modulated hypertrophy.Following the blockade of JAK2 expression by siRNA(p<0.05),p-STAT3,ANP and Troponin T protein levels decreased significantly(p<0.05).Similarly,siRNA knockdown of STAT3(p<0.05)decreased ANP and Troponin T expression(p<0.05).Therefore,TWEAK upregulated ANP and Troponin T expression by activating the JAK2/STAT3 pathway.Conclusion1.Fn14 expression was upregulated in response to TWEAK treatment in HL-1 atrial myocytes.2.TWEAK increased the expression of hypertrophy marker genes ANP and Troponin T,and Fn14 knockdown counteracted the effect.3.Inhibition of JAK2,STAT3 by specific siRNA attenuated TWEAK-induced HL-1 atrial myocytes hypertrophy.BackgroundAtrial fibrillation(AF)is the most common cardiac arrhythmia in clinical practice and increases the risk of thrombus in the left atrial appendage(LAA)which is strongly associated with an increased risk of ischemic stroke.Embolic strokes caused by AF are typically severe,more commonly disabling and fatal compared with strokes not associated with AF.Transesophageal echocardiography(TEE)is a sensitive method for intracardiac thrombus identification.When AF lasts for 2 days,atrial thrombi may take place in up to 14%patients on TEE examination.The prevalence of thrombus in AF patients varies from 10%to 18%,with about 90%of thrombi found in the LAA.The administration of vitamin K antagonists(VKA)has been the mainstay in the treatment of intracardiac thrombus with a target international normalized ratio(INR)level of 2.0 to 3.0.Maintenance of INR values in the therapeutic range is associated with decreased thrombus size by inducing a relative predominance in plasma fibrinolytic activity over anticoagulation-inhibited thrombin activity.The use of VKA,such as warfarin,exerts excellent protective effects against thromboembolic complication but requires restrictions on food and drugs;therapeutic monitoring and dose adjustment are always needed due to its narrow therapeutic window,and high intra-and inter-patient variability.Dabigatran etexilate,a novel oral direct thrombin inhibitor,has been approved as an alternative to warfarin for the prevention of stroke and systemic thromboembolism in patients with non-valvular AF.Recent studies indicated that dabigatran can be applied for the treatment and prevention of venous thromboembolism(VTE),with similar effects on VTE recurrence and a lower risk of hemorrhage compared with warfarin.Some case reports also showed that intracardiac thrombus resolved after anticoagulation therapy with dabigatran.In the trial,we compared the efficacy and safety of dabigatran,administered at a fixed dose of 150 mg bid with dose-adjusted warfarin for the treatment of atrial thrombus in patients with non-valvular AF.Objectives1.To study the effect of dabigatran in the treatment of atrial thrombus in patients with non-valvular AF.2.To compare the the efficacy and safety of fixed-dose dabigatran with dose-adjusted warfarin in anticoagulation therapy for atrial thrombus,and investigate whether dabigatran can effectively replace warfarin.3.To explore the mechanism of anticoagulant therapy for atrial thrombus in non-valvular AF.Methods1.Inclusion criteria(1)Age ≥18 years;(2)Presence of non-valvular AF as evidenced by 12-lead electrocardiogram(ECG)or 24h Holter monitoring;(3)Thrombus in atrium/atrial appendage detected by TEE.2.Exclusion criteria(1)Hypersensitivity to the active ingredient or any excipients;(2)Patients with severe renal impairment(CrCL<30 mL/min);(3)Clinically active bleeding;(4)Significant risk factors for major bleeding,including current or recent gastrointestinal ulceration,malignant neoplasms at high risk of bleeding,recent brain or spinal injury or surgery,recent intracranial haemorrhage,known or suspected esophageal varices,arteriovenous malformations,vascular aneurysms or major intraspinal or intracerebral vascular abnormalities;(5)Concomitant treatment with any other anticoagulants except switching therapy to or from dabigatran or when unfractionated heparin is given to maintain an open central venous or arterial catheter;(6)Severe hepatic impairment or liver disease;(7)Concomitant treatment with systemic ketoconazole,cyclosporine,itraconazole,tacrolimus and dronedarone.3.TEE examinationTEE examination was performed using a 5-MHz multiplane probe before and after 3-month anticoaguation treatment.The probe was inserted into the esophagus after topical anesthesia.Images of the LAA were acquired in different tomographic planes to explore the lobes entirely.The RAA was usually examined in a mid-esophageal section.Thrombus was diagnosed by the presence of an echo-dense mass in the atrium or the atrial appendage.4.GroupingThe patients were divided into two groups according to the anticoagulant selected by the patients:Group D:dabigatran with a fixed dose of 150 mg;Group W:dose-adjusted warfarin with a target INR of 2.0 to 3.0.5.Follow-upThe patients ware assessed after 1 month and then 3 months in the clinic;meanwhile they were requested to contact the investigator immediately if symptoms developed that were suggestive of stroke,thromboembolism or major bleeding.Other adverse events,laboratory examinations,and adherence were assessed routinely.The trial was approved by the ethics committee of Qilu Hospital of Shandong University and informed consents were obtained from each patients.6.Statistical analysisAll statistical analyses were conducted with SPSS Statistics version 17.0 software.Continuous variables were expressed as mean ± SD and compared using Mann-Whitney U and Wilcoxon W test.Categorical variables were presented as frequency and percentage(%)and between-group comparisons were performed using the Chi-square test and Fisher’s exact test.The thrombus therapeutic effect,represented by the ratio of decreased thrombus area to original area,was compared by Wilcoxon W test between the 2 groups.Difference with p value<0.05(2-sided)was considered statistically significant.Results1.Thrombus characteristics detected by first TEEThe thrombus area ranges from 0.1 to 4.48 cm2 and the locations of thrombi were mainly in LAA.The thrombus in warfarin group were larger than in dabigatran group(p<0.05).2.Patient characteristics41 patients who had atrial thrombus detected by TEE were enrolled.Among them,19 patients received dabigatran 150 mg bid and the remaining 22 patients received dose-adjusted warfarin based on the patients’ individual choice.There was no difference in baseline data between the two groups.3.Monitoring and follow-upThe mean number of INR examination values obtained in the warfarin group was 11 during the therapy.Time in therapeutic range of INR(TTR)was 72%with less than 5%time above the therapeutic range.During the 3 months,the dabigatran was discontinued about two weeks in group D because of gastrointestinal discomfort.One patient in the group W experienced ischemic stroke.No major bleeding occurred in both two groups.4.Thrombus characteristics detected by repeated TEENo thrombus was detected by repeated TEE in 17 patients in group D(17 of 19,89.5%)and 17 patients in group W(17 of 22,77.3%).The rest patients were still found thrombus by secondary TEE.In the group D and group W respectively,thrombus appeared to have decreased in 5 patients(1 vs.4),while in 2 patients(1 vs.1)the thrombi appeared to have increased in size instead.5.Comparison of thrombus therapeutic effect between the two groupsThe thrombus therapeutic effect,represented by the ratio of decreased thrombus area to original area,was compared by Wilcoxon W test.There was no statistical difference in the therapeutic effect of the two groups(p>0.05).Conclusion1.Dabigatran has similar effect compared with warfarin for the treatment of atrial thrombus in patients with non-valvular AF.2.Fixed-does dabigatran(150 mg bid)is a far more convenient medication than warfarin because it need no routine laboratory monitoring and dose adjustment.3.Uninterrupted dabigatran is particularly essential and crucial and drug discontinuance would affect thrombus therapeutic effect.