Effects And Mechanisms Of α7 Nicotinic Acetylcholine Receptor On β-amyloid-induced Atrial Remodeling Of Atrial Fibrillation

Background:Atrial fibrillation(AF)is the most common arrhythmia in clinical settings,and Alzheimer’s disease(AD)is a common kind of Senile dementia.Studies have shown that AF and AD are age-dependent diseases with similar genetic and biochemical characteristics and common trigger factors,but the specific mechanism is still unclear.The formation ofβ-Amyloid(Aβ)deposition is the mainstream pathological hypothesis of AD,and Aβmay be found in the heart and brain of AD patients.Amyloid deposits like those in AD were found in the pathological tissues of patients with idiopathic dilated cardiomyopathy(IDCM),atherosclerosis and other cardiovascular diseases.Therefore,Aβdeposition may be a systemic syndrome that accumulates multiple organs including brain,heart,etc.The role of Aβin atrial fibrillation is also worthy of further study.αNicotinic acetylcholine receptor(α7n Ach R)is a calcium ion highly permeable ion channel,which is expressed on the surface of hippocampal neurons,autonomic synapses,macrophages and myocardium,which plays a role in signal transduction.Aβcan bindα7n Ach R with high affinity and can be activated directlyα7n Ach R to regulate cell electrophysiological activities by significantly increasing calcium influx.Calcium/calmodulin dependent protein kinase II(CaMKⅡ)is an important calcium related regulatory molecule,which can increase the risk of AD and AF by mediating oxidative stress response.However,the effect of Aβonα7n Ach R in cardiomyocytes has not been reported yet,while the effect of Aβ-α7nAChR on atrial fibrillation is also unclear.In this study,clinical human atrial muscle tissue,APPPS1 transgenic animal model and HL-1 cardiomyocyte line were used as research carriers.Through single-cell RNA sequencing technology,morphological technology,electrophysiological technology and molecular biology technology,this study progressed layer by layer from four aspects:clinical apparent difference,animal atrial remodeling,cell phenotype and molecular mechanism.We have systematically analyzed the correlation between AF and ad in mechanism and explored the effect of Aβdeposition on atrial remodeling in atrial fibrillation,clarified whether there is a regulatory effect ofα7nAChR to Aβon atrial fibrillation,and studied the changes and related molecular mechanisms of oxidative stress and mitochondrial dysfunction.This study aims to provide theoretical support for the cross research between AF and AD,and improve new targets for the treatment based on their common mechanism.Methods:1.Expression and correlation ofα7nAChR and Aβin human atrial tissue with atrial fibrillationSix elderly patients with degenerative mitral valve disease treated in the Department of cardiovascular surgery of the General Hospital of Northern Theater Command from September 2019 to January 2020 were selected.The myocardial tissue at the base of the patient’s auricle was obtained during the operation,with a size of about(0.5×0.5cm~2)as the research object.According to the preoperative diagnosis of atrial fibrillation,the patients who obtained myocardial tissue were divided into atrial fibrillation group(AF)and no atrial fibrillation group(no-AF).Cardiomyocytes were isolated,differential genes were obtained by single cell RNA sequencing,GO analysis and enrichment analysis were carried out,the differential protein expression level was verified by Western blot,the level of fibrosis was detected by Masson staining,and the level of oxidative stress was detected by Mito Sox staining.2.Effect and preliminary mechanism of specific inhibition of myocardialα7nAChR on atrial structure and electrical remodeling in APP/PS1 transgenic mice with atrial fibrillationSix male APP/PS1 and WT mice aged 3 months,6 months and 12 months were used to determine the relationship between age growth and Aβaccumulation andα7nAChR expression level.12-month-old male APP/PS1 mice and 12 same month-old WT mice were used for follow-up study.According to the injection of AAV virus,mice were divided into four groups:WT+AAV-CON group,WT+AAV-Chrna7 group),APP/PS1+AAV-CON group and APP/PS1+AAV-Chrna7 group.Mouse genotypes were identified by mouse tail PCR;AAV virus was injected through caudal vein,and the virus transfection was verified by immunofluorescence experiment;Morris water maze was used to detect the learning and memory ability of mice;Basic physiological signs such as blood pressure and heart rate were detected;Atrial dilation was assessed by echocardiography;Assessment of susceptibility to atrial fibrillation by trans-jugular atrial electrical stimulation;The conduction ability of atrial muscle was evaluated by cardiac surface field potential mapping under isolated cardiac perfusion of Langendorff mice;The levels of atrial fibrosis,oxidative stress and apoptosis were quantitatively evaluated by Masson staining,Mito Sox staining and TUNEL staining,respectively;The level of inflammatory factors was detected by ELISA;Western blot was used to detectα7nAChR,CaMKⅡ and MAPK key molecular levels.3.Effect and mechanism ofα7nAChR mediated CaMKⅡ/MAPK pathway on oxidative stress injury and mitochondrial dysfunction induced by Aβin HL-1 cell lineThe cardio-myoblast cell line HL-1 was taken as the main research object.In order to explore the role ofα7nAChR in Aβ-induced cardiomyocyte injury,HL-1 cells were divided into four groups according to different drug treatments:Control group,α-BTX(α7nAChR inhibitor)group,Aβgroup and Aβ+α-BTX group.In order to clarify the mediating role and downstream mechanism pathway of CaMKⅡ inα7nAChR affecting Aβ-induced cardiomyocyte injury,this study was divided into five groups according to different drug treatments given to HL-1 cells:Aβgroup、Aβ+α-BTX group,Aβ+α-BTX+o CaMKⅡδgroup,Aβ+α-BTX+AngⅡ,Aβ+α-BTX+si CaMKⅡδgroup.The viability of CCK-8 cells was measured;Fluo-3 Am or 7-AAD/Annexin V were detected by flow cytometry to evaluate the level of calcium ion or apoptosis,respectively;ROS fluorescent probe DHE staining and GSH/GSSG was used to evaluate the level of oxidative stress;The mitochondrial function was evaluated by JC-1 staining,Mito-Tracker Red staining,m PTP staining and NAD~+/NADH detection;Lipofectamine 3000 was used to transfect interference virus or overexpression virus;Western blot was used to detectα7nAChR,CaMKⅡ and MAPK key molecular levels.Results:1.Differential genes APP,CHRNA7 and CAMK2D were obtained by single cell RNA sequencing.Go analysis showed that these differential genes were enriched in the regulation of amyloid fiber formation,calcium ion transmembrane transport and MAPK signal pathway.WB verified these gene related expression proteins of APP,α7nAChR,CaMKⅡ,p38 and ERK.Compared with no-AF,atrial tissue in atrial fibrillation also has increased levels of fibrosis and oxidative stress.2.The expression level ofα7nAChR and Aβin the atrium of APP/PS1 mice was age-dependent,and 12-month-old APP/PS1 mice could be used as the follow-up research object of this part of the experiment.APP/PS1 mice showed significant atrial dilation and myocardial fibrosis.AAV-Chrna7 had a protective effect on atrial structural remodeling induced by APP/PS1 genotype mutation.APP/PS1 mice had strong susceptibility and persistence of atrial fibrillation,which could be attenuated by inhibiting myocardiumα7nAChR.Direct promotion of atriumα7nAChR activity could lead to ectopic electrical activity in the atrium of APP/PS1 mice;Conversely,the atriumα7nAChR inhibition had a protective effect on atrial electrical activity in APP/PS1 mice.APP/PS1 genotype mutation could induce oxidative stress injury,apoptosis and pro-inflammatory factor in mouse atrial muscle,which could be enhanced by inhibiting myocardiumα7nAChR expression decreased.The increase of oxi-CaMKⅡ,the phosphorylation of MAPK pathway molecules(p38,ERK)and the transcription factors AP-1(c-Fos,c-Jun)in the atrium of APP/PS1 mice could be controlled by down-regulation ofα7nAChR.3.Through time and dose-dependent experiments,50μM Aβand 50nmα-BTX was administered for 72 hours as a treatment for HL-1 cell lines.Aβtreatment could promote the intracellular calcium level of HL-1 cells,andα-BTX inhibited this promotion.Aβtreatment induced the increase of oxidative stress level of HL-1 cells,which could be suppressed byα-BTX.For mitochondrial function,Aβtreatment led to the decrease of mitochondrial membrane potential,destroy the integrity of mitochondria,and then induced apoptosis.This damage effect could be reversed byα-BTX.Overexpression of CaMKⅡ(o CaMKⅡ)and AngiotensinⅡ(AngⅡ)could significantly activate MAPK pathway,in which AngⅡplayed a stronger role.However,knockout of CaMKⅡ(si CaMKⅡ)could not further down regulate the phosphorylation of MAPK key proteins(p38 and ERK)on the basis ofα-BTX.Conclusion:1.AD and AF may have mechanism commonality,and the role of Aβ-α7nAChR may be the key.2.Aβpromotes oxidative stress injury,mitochondrial dysfunction and inflammatory level throughα7nAChR,which leads to atrial remodeling in atrial fibrillation,and specifically down regulates cardiomyocytesα7nAChR can ameliorate the above effects.3.CaMKⅡ/MAPK pathway activation in Aβ-α7nAChR plays an important mediating role in promoting atrial fibrillation induced by atrial fibrillation.