Long-term Effects And Mechanism Of Right-sided Ganglionated Plexi Ablation On Structure And Function Of Target Atrial Tissues In Canine Models

BackgroundAtrial fibrillation(AF)is a common sustained arrhythmia characterized by atrial rthytm disorder in clinical,and the cardiac autonomic nervous system plays an important role in its initiation and maintenance.Cardiac nervous system involves both the intrinsic and extrinsic cardiac autonomic nervous system.Ganglionated plexi(GP)which located on the pericardium adjacent to big vessels and axons of GP composed the former.The latter collectively includes the ganglia in the brain and along the spinal cord and their axons.GPs are particularly well supplied by both adrenergic and vagal nerve endings and acted as "integration centers",which are usually embedded in the fat pads.There are four major GPs:the anterior right GP,the inferior right GP,the superior left GP,the inferior left GP.These GPs modulates and controls the release of neurotransmitters within the myocardium.Both sympathetic and vagal activation induce shortening of the atrial effective refractory period(AERP)and action potential duration,these changes lead to the occurrence of AF.Radiofrequency ablation including rcumferential pulmonary vein ablation,pulmonary vein isolation,complex fractionated atrial electrogram ablation and GP ablation.Nevertheless,no matter what kind of procures the long-term success rate is unsatisfactory.The long-term recurrence of single ablation on AF reached 11%-50%,and more than 20-40%patients needed the second,even third times catheter ablation.But the success rate of multiple ablations was still 7%-24%,the recurrence rate increased with time after ablation.AF recurrence is divided into these types:electrical pulmonary vein(PV)reconnection,generation of non-PV triggers,reentry activities formation,local or systemic inflammatory response and the atrial substrates change caused by catheter ablation.The normal cardiac function greatly depended on the structure,electrophysiology and cardiac innervation.The cardiac intrinsic nerve system consists of ganglionate plexi which located on the root of pulmonary vein and the nerve fibers which connect the nerve plexuses.Ablation the ganglionated plexi may destroy the normal electrophysiology and structure of cardiac,which could change the atrial substrate and further cause the structure and electrophysiology remodeling that induce the recurrence of AF.The progression of atrial fibrosis induced by ablation and structural remodeling are easily identified as atrial enlargement,which is the surrogate marker for AF substrates.Moreover,no matter what kind of ablation strategies would destroy the intrinsic cardiac autonomic nervous system at different degrees.The modulation in autonomic neural active,proliferation and heterogeneity of nerve that induce autonomic neural remodeling lead to the late recurrence of AF.In this study,we ablated the anterior and inferior right GP in a canine model to explore long-term effects on atrial size,electrophysiological characteristics,neuron remodeling and ultrastructural of target atrial tissues.We also explored the role of cardiac automatic neuron remodeling and the change of atrial size induced AF.Objectives1.To observe the changes of atrial size and electrophysiology.2.To observe the change of ultrastructural in target tissue and neuron remodeling after right-side GP ablation in different follow-up.3.To investigate the effect and potential mechanism of atrial sturcter,ultrastructural and neuron remodeling on the inducibility of AF after GP ablation..Materials and Methods1.Animals model and grouping(1)Thirty-two adult mongrel dogs of either sex were divided into four groups(n=8).Group 1(control group):perform right lateral thoracotomy at the fourth intercostal space,and exposed the posterior side of heart without GP ablation.Groups 2 to 4(ablation group)underwent right thoracotomy and exposed anterior right GP and inferior right GP,followed by GP ablation.Group 1 and 2 were observed for 1 month,group 3 for 6 month,group 4 for 12 months.(2)Canine models of right-side GP ablation:All canaines were anesthetized with pentobarbital 3%?,and assisted breatging withventilator.Supplemental doses were administered to maintain stable anesthesia.Standard heart electricity was monitored.Perform right lateral thoracotomy at the fourth intercostal space,and anterior right GP and inferior right GP were exposed by lifting up the pericardium without GP ablation.The anterior right GP situated in the fat pad near the base of right pulmonary veins and adjacent to the caudal end of the sinoatrial node;the inferior right GP located in the fat pad close to the junction of the inferior vena cava and both atria.The GP was localized by applying high-frequency stimulation(HFS;20 Hz;square wave pulse;0.1 ms duration;2-5 V)with a variable output stimulator.In this voltage range,progressive slowing of the sinus rate or AV conduction was observed and was directly related to the voltage applied.Radiofr-equency energy was delivered(30 W for 30 s to 60 s per application)under direct visual.Flush the catheter tip with small amounts of saline during radiofrequency current delivery to prevent the tissue charring.The end point of ablation was defined as the disappearance of vagal response induced by highfrequency stimulation in the GP area.2.EchocardiographyAll animals performed transthoracic echocardiography at baseline(before ablation)and the end of experiment(on months 1,6,and 12).Hair was clipped chest and the neck,continuous ECG monitor was performed.Measurements of the left atrial diameter(LAD)were performed with a 2-D-guided M-mode using a long-axis right-sided parasternal.Right atrial diameters(RAD1 and RAD2)were measured during apical 4-chamber with M-mode.Atrial volumes were measured at two points:(1)mid-diastole volume,just prior to onset of atrial contraction(MDV),(2)end-diastole volume,at the point of minimal left atrial volume(EDV).For both the left atrial and right atrial,atrial ejection fraction(AEF)was calculated using the Equation:(MDV-EDV)/MDV.All the parameters were measured under the sinus rhythm,and three times averaged.2.Electrophysiology Study Three bipolar electrodes were placed at the right atrial free wall,high right atrium,and right atrial appendage.Before and immediately after ablation,an electrophysiology study was performed on the dogs in epicardial GP ablation group 2 with 4.Atrial effective refractory period(AERP)was determined as the longest S1-S2 interval that failed to produce a propagated response.Data before ablation,immediately after ablation,and follow-up after ablation were collected form each animals.AF was defined as the fast atrial rate that persisted for at least 30 s after the end of burst stimulation.3.Tissue HarvestingThe animals were killed under anesthesia after the last electrophysiological studies,and the target tissues were obtained from all animals.4.Immunohistochemistry StudiesImmunohistochemical staining was performed to detect the distribution of nerve.The nerve markers,including tyrosine hydroxylase(TH)and choline acetyltransferase(ChAT)were stained.Nerve densities,were determined in control group and ablation groups.5.Western blot Western blot was used to detect the expression of CHAT,TH and growth-associated protein 43(GAP43).6.Ultrastructural AnalysisAfter echocardiography was measured,the target atrial tissues were processed for electron microscopy.Target atrial tissue,a strip of tissue,with approximately1.0mm3,was selected and cut into small blocks,which were then fixed in cacodylate-buffered 2.5%glutaraldehyde at 4? for 2 hours,and postfixed in cacodylate-buffered 1%osmium tetroxide/0.5%potassium ferrocyanide at 4? for 2 hours.Every tissue block was dehydrated in a graded series of ethanols,and embedded in epoxy resin.Ultrathin sections were stained with uranyl acetate and lead citrate and viewed under a transmission electron microscope.7.Statistical analysisAll data were presented as medians with first and third quartiles.Comparisons were assessed by unpaired t-test with SPSS Software(version 17.0,SPSS China).AERPs between different times were compared using a generalized estimated equation method.ANOVA was used for multiple comparison of nerves densities and protein expression between the control group and ablation groups.A value of P<0.05 was considered statistically significant.ResultsAt the end of the study,twenty-nine dogs were evaluated during the study.7 dogs completed the experimental protocol in group 1,6 in group 4,and 8 dogs in group2 and 3.1.Echocardiography measurementsIn left atrial,no significant change of all the parameters were observed at 1,6 and 12 months after ablation compared with baseline.In right atrial,1 month after ablation,RAD1,RAD2,RA-MDV and RAEF showed reduction after 1 month but it showed no change in 6 and 12 months after ablation compared with baseline.RA-EDV remained stable after ablation in all groups compared with baseline.2.Effect of GP Ablation on AERPAERP was significantly longer immediately after GP ablation than that at preablation at all sites(P<0.001).These AERP variations persisted at all sites within 1 month after GP ablation.However,these changes reverted to preablation levels after 6 and 12 months(P>0.05).3.Effect of GP Ablation on AF InducibilityIn control group,AF was not induced after right thoracotomy and after 1 month.In particular,AF was induced in 5 of 8 dogs after 1 month in group 2,and in all animals after 6 months in group 3,and after 12 months in group 4.4.Nerve DensityAt 1 month after GP ablation,TH-and CHAT-positive nerves in the right atrium were significantly reduced compared with the control group(P<0.01).However,these nerve density changes reverted to preablation levels after 6 and 12 months.5.Protein Expression of GAP43.TH,and CHAT The protein expression level of GAP43 was upregulated after ablation compared with that in the control group.Compared with the control group,the expression of TH and CHAT in the ablation groups were downregulated 1 month after GP ablation but reverted to control level until 6-12 months.6.Ultrastructural FindingsA visible intercalated disc is present with normal mitochondria,plasma membrane and sarcoplasmic reticulum in control group.1 month after ablation significant ultrastructural abnormalities were observed in target tissue.6 months and 12 months after ablation were also abnormal,but the extent of injury was slightly less than 1 month.However,it appeared severe interstitial collagen deposition with relatively few damaged organelles.Conclusion1.RAD1.RAD2,RA-MDV and RAEF showed reduction and AERP were significantly prolonged.The nerve densities decreased and ultrastructural showed abnormalities 1 month after ablation.2.RAD1,RAD2,RA-MDV,RAEF and AERP reverted to preablation levels after 6 and 12 months and the AF was easily to induce.The nerve density and protein expression were reverted to preablation.The extent of injury was slightly less than 1 month.Targeted atrial neuron remodeling and atrial function change may be an important mechanism underlying the observed electrophysiological changes.3.The changs of atrial structer,electricsiology,neuron remodeling after GP ablation may contribute to the inducibility of AF.BackgroundThe mechanism of cardiac autonomic nerve remodeling after catheter ablation is not clear so far.Such as,the incomplete denervation,the differences between different ablation techniques on the injure of nerves,the regeneration induced by high frequency stimulation during catheter ablation and the increased expression of nerve growth factors after ablation,but the above mechanisms can't explain the cardiac autonomic nerve remodeling after catheter ablation.Recently,the rapid development of molecular biological techniques have penetrate into all fields,the appearance of biochip technology and high throughput sequencing technique provide a powerful methods for the development of modern medicine and translational medicine.These techniques promote medical science to transit from the "system,blood,tissue and cell levle" to the "DNA,RNA,protein,and their interactions level",provide new means for revealing the mechanisms of disease from molecular and gene levels.Long non-coding RNAs(lncRNAs)which are longer than 200 nucleotides,but have no protein-coding function.Previously,they were regarded as the 'noise' of gene transcription,but growing evidence revealed the important roles of IncRNAs in gene expression regulatory network.It was considered that the quantity of noncoding RNAs is in direct proportion to the species' complexity,which explains the phenotypes of human are more complex than other species even they have similar quantity of protein-coding genes.The characteristic of conservation of IncRNAs may be lower than mRNA,however,their promoter region and secondary structures have highly conserved elements.LncRNAs can modulate gene expression at the epigenetic,transcriptional and post-transcriptional levels.LncRNAs mediate X-in activation,gene imprinting.Chromatin modifications,transcriptional activation,transcription interference and some other activities of life.It also plays an important regulatory role on development of organism,growth,age and die.LncRNAs are involved in the development of nervous system and cardiac,about neuronal differential,brain development,synaptic plasticity,peripheral nervous damage and repair and the occurrence of neurodegenerative disease.Besides,lncRNAs can also participate in heart failure,myocardial infarction,myocardiopathy and as blood markers of cardiac diseases.Our previous study have found that lncRNA regulate the cardiac autonomic neural remodeling of AF.If IncRNAs participate in the cardiac autonomic neural remodeling after GP ablation are not clear.ObjectivesThe aim of the study is to establish GP ablation canine model with short-term and long-term;characterize the lncRNAs expression in AF inducibility canine after GP ablation by high throughput sequencing technique;identify deferential expression lncRNAs in cardiac autonomic nerve remodeling after GP ablation.Materials and Methods1.Animals model and groupGroup:twelve adult mongrel dogs of either sex were divided into four groups(n=3).Group 1(control group):observation for 1 month.Perform right lateral thoracotomy at the fourth intercostal space,and anterior right GP and inferior right GP were exposed by lifting up the pericardium without GP ablation.Groups 2(ablation group):observation for 1 month,underwent right thoracotomy and exposed anterior right GP and inferior right GP,followed by GP ablation.Group3 was control group for 6 months,group 4 was ablation group for 6 months.2.Validation the cardiac autonomic nerve remodeling after GP ablationAs mentioned in the first part.3.High-throughput sequencing and verificationEvery group canine right atrial tissues were obtained,and isolate the total RNA from each tissue by using TRIzol reagent.Examine the expression of lncRNAs and mRNAs through high-throughput sequencing.Analyze the expression of random selected differential lncRNAs by real-time quantitative PCR to determine the accuracy of the results.4.Statistical analysisAll data were presented as mean ± SD.Comparisons were assessed by unpaired t-test with SPSS Software(version 17.0,SPSS China).Analysis of variance(ANOVA)was used to compare data between four experimental groups.A value of P<0.05 was considered statistically significant.Results1.High-throughput sequencing results analysisIn total,32705 genes were acquired and the novel genes were 2590,the differential expression genes between group 1 and group2 were 160,group 1 and 3 were 1615 and group3 and 4 were 558.Based on the results,there are 7596 lncRNAs were detected,and the novel were 3356.The differential expression lncRNAs between group 1 and 2 were 11,group1 and 3 were 33 and group3 and 4 were 36.2.Experimental resultsThe sequencing results were reliable confirmed by detecting the randomly selected 6 lncRNAs by qRT-PCR.ConclusionWe identify series of differential expression lncRNAs in GP ablation canine models,and many of the differential expression IncRNAs were involved in cardiac autonomic neuron remodeling after GP ablation.