| Objective: To establish an active immune rabbit model with enhanced β1 adrenergic receptor autoantibody(β1ARAb)expression,and to study the effects of increased expression of β1ARAb on atrial electrophysiological characteristics and atrial fibrillation(AF)induction rate.TMT quantitative proteomics technique was used to identify proteins with statistically significant differences in expression levels between the left atrial tissue of the immune group and the control rabbits after the modeling was completed.And these identified differentially expressed proteins were subjected to subsequent protein functional annotation,biological analysis,and construction of protein-protein interaction network,to explore the possible mechanism of β1ARAb overexpression at the molecular level to induce AF,and to further verify the mechanism of action detected by proteomics in animal models and patients with paroxysmal AF.Methods: 1)The extracellular second loop functional epitope peptide(ECL2)of β1 adrenergic receptor(β1AR)was designed and synthesized.Sixteen adult male New Zealand white rabbits were randomly divided into control group and immunized group.The immunized group was injected with synthetic ECL2 peptide of β1AR mixed with Freund’s adjuvant(2 mg every time,once every two weeks for 4 times)to establish an active immune rabbit model of overexpression of β1ARAb,while the control group was injected with Freund’s adjuvant without synthetic ECL2 peptide of β1AR(once every two weeks for 4 times).Every two weeks,the serum β1ARAb expression level of the two groups was measured by enzyme linked immunosorbent assay(ELISA),the serum cAMP expression level of the two groups before and after modeling was compared by ELISA method,and the expression level of β1AR protein in the left atrium of the two groups was measured by Western blotting to verify whether the model was successful or not.After the establishment of the animal model,atrial electrophysiological examination was performed in all experimental rabbits,including measurement of resting heart rate and spontaneous arrhythmia within 5minutes,measurement of atrial effective refractory period(AERP),and recording the induction rate and duration of atrial arrhythmia after Burst stimulation,and measurement of left atrial appendage conduction velocity and conduction heterogeneity;2)Three rabbits in the immune group and three rabbits in the control group were selected from the experimental rabbits after the first stage of modeling,and the proteins in the left atrium were extracted by quantitative proteomics TMT technique.According to the steps of trypsin hydrolysis,TMT labeling,high performance liquid chromatography(HPLC)classification,liquid chromatography-mass spectrometry(LC-MS)analysis and database comparison,the differential expression was compared.The differentially expressed proteins in the left atrium of the immunized group and the control group were identified and screened when the differential expression was more than 1.3 as the threshold of significantly up-regulated and less than 1/1.3 as the threshold of significantly down-regulated,and P value was less than 0.05.The biological functions of differentially expressed proteins in left atrium of immune group and control group were preliminarily discussed by means of bioinformatics analysis,such as protein functional annotation,pathway analysis,functional enrichment,cluster analysis,protein interaction network analysis;3)According to the results of TMT quantitative proteomics and bioinformatics analysis in the second stage,it was verified that the increased expression of β1ARAb promoted the occurrence of AF in active immune rabbit model induced by atrial fibrosis.The diameters of four-chamber heart were recorded by transthoracic echocardiography before and 8 weeks after immunization,and the left ventricular ejection fraction(LVEF)was calculated.After the model was made and the experimental animals were killed,the differences of tissue structure and fibrosis degree of left atrium between the two groups were observed by hematoxylin-eosin staining,Masson staining,Sirius red staining and transmission electron microscope examination.Western blotting and real-time reverse transcription-polymerase chain reaction(RT-qPCR)were used to detect the difference of protein and mRNA expression of type collagen I,type collagen III,transforming growth factor β1(TGF-β1),Smad3 and Smad7 in left atrium between the two groups;4)A total of 70 patients newly diagnosed as paroxysmal AF in the Heart Center of Xinjiang Medical University,Affiliated Hospital One from July 2017 to March 2018 were continuously selected,and the general clinical data and echocardiographic results were recorded.Fasting blood was collected in the early morning after admission.The levels of serum β1ARAb and circulating fiber markers(type III procollagen N-terminal peptide,type I collagen pre-C terminal peptide,galactose lectin 3)were measured by ELISA method.Correlation analysis was used to study the correlation between serum β1ARAb level and general clinical data,circulating fiber markers and left atrial diameter.Results: 1)There was no significant difference in serum β1ARAb titer between the immunization group and the control group at baseline(0.19±0.01 vs.0.19±0.02,P=0.878).From the second week,the serum β1ARAb titer of the immunization group increased gradually,which was significantly higher than that of the baseline,while there was no significant difference between the serum β1ARAb titer of the control group at each time point and the baseline.There was no significant difference in the baseline serum cAMP level between the immunization group and the control group(23.89±1.93 vs.24.44±2.49 ng/mL,P=0.624),but the serum cAMP level in the immunization group was higher than that in the control group after modeling making(56.63±13.38 vs.24.70±2.64 ng/mL,P<0.001).Compared with the control group,the expression of β1AR protein in the left atrium of the immunized group decreased significantly after eight weeks of modeling(0.40±0.05 vs.0.29±0.02,P < 0.001).Electrophysiological examination showed that the resting heart rate of rabbits in the immune group was higher than that in the control group(207.13±8.63 vs.177.13±6.17 bpm,P< 0.001),the AERP was lower than that in the control group(70.00±5.49 vs.96.46±3.27 ms,P<0.001),the incidence of total induced arrhythmias in the immune group was higher than that in the control group(38/40 vs.3/40,P<0.001),and the induction rate of AF in the immune group was higher than that in the control group(55% vs.0%,P<0.001).There was no significant difference in the incidence of spontaneous AF between the two groups.In the immunization group,the electric conduction velocity of left atrial appendage decreased significantly(34.38±8.48 vs.61.50±13.40 cm,P<0.001),and the conduction heterogeneity increased significantly(2.63±0.40 vs.1.52±0.25,P<0.001);2)According to the results of quality control,such as the length distribution of peptide,the relationship between protein molecular weight and coverage,the distribution of quality error,as well as the results of principal component analysis and relative standard deviation,these results showed that the differentially expressed proteins identified by proteomics method were reliable and reproducible.A total of 4043 proteins were identified,of which 3429 were quantifiable.According to the set differential threshold,a total of 213 differentially expressed proteins were identified between the two groups,including 88 up-regulated proteins and 125 down-regulated proteins.According to the results of bioinformatics analysis such as gene ontology enrichment,Kyoto encyclopedia of genes and genomes,cluster analysis,etc.,differentially expressed proteins in the left atrium of the immune group and the control group play an important role in cellular and molecular biological behavior,such as cell metabolism,collagen and fiber junction,lipid metabolism,extracellular matrix and so on.The two groups of differentially expressed proteins are involved in the autoimmune pathway and the cell fiber synthesis pathway;3)The left atrial diameter after modeling in the immune group was significantly higher than that in the baseline and the control group(10.93±0.99 vs.9.13±0.64 mm,10.93±0.99 vs.9.77±0.82 mm,P<0.05).There was no significant change in the left atrial diameter in the control group before and after modeling.The LVEF of the immune group after modeling was significantly lower than that of the baseline state and the control group(54.47±9.68 vs.67.43±5.77%,54.47±9.68 vs.63.71±6.82%,P<0.05).There was no significant change in LVEF before and after modeling in the control group.HE staining showed that the cardiomyocytes in the control group arranged regularly and tightly,the stroma was less,and the fibers were regular and neatly arranged.In the immune group,the cardiomyocytes were scattered,the size of myocardial nucleus was irregular and poor clarity,and the extracellular matrix was significantly increased.Masson staining and Sirius red staining showed that the amount of collagen fibers between cardiomyocytes in the control group was less,while a large number of collagen tissues were distributed among cardiomyocytes in the immune group,and even connected with each other in a network,the overall distribution was uneven.The quantitative results showed that the area ratio of left atrial fibrosis in the immune group was higher than that in the control group(Sirius red staining: 16.76±6.40% vs.4.85±0.40%,Masson staining: 15.17±3.46% vs.4.92±1.72%,P<0.001).The results of correlation analysis showed that there was a significant positive correlation between the level of serum β1ARAb and the ratio of left atrial fiber area after eight weeks(Masson staining: r=0.895,Sirius red staining: r=0.786,P < 0.001).Transmission electron microscope showed that the nuclear membrane of cardiomyocytes in the control group was intact,the cytoplasm was abundant and arranged neatly,and the pathological changes such as autophagy,fibroproliferation,fibrinolysis,breakage,mitochondrial swelling and vacuolization could be seen in the immune group.The results of Western blotting showed that the protein expression of collagen I,collagen III,TGF-β1 and Smad3 in the immunized group increased(0.397±0.093 vs.0.221±0.072,0.379±0.072 vs.0.177±0.056,0.486±0.108 vs.0.194±0.093,0.257±0.092 vs.0.163±0.041,P < 0.05),while the expression of Smad7 decreased(0.123±0.036 vs.0.181±0.052,P<0.05).The results of RT-qPCR showed that the mRNA expression of collagen I,collagen III,TGF-β1 and Smad3 in the immunized group increased(2.368±0.471 vs.1.093±0.153,2.880±0.688 vs.1.351±0.323,2.880±0.643 vs.1.230±0.233,2.373±0.428 vs.1.172±0.195,P<0.05),while the expression of Smad7 decreased(0.739±0.090 vs.1.110±0.123,P<0.05).4)A total of 70 patients with paroxysmal AF were included,with an average age of 60.57±13.25 years old,accounting for 55.7% of males.The level of β1ARAb in patients with enlarged left atrium was higher than that in patients with normal left atrial diameter(8.87±3.16 vs.6.75±1.34 ng/mL,P=0.005),and the level of LVEF in patients with enlarged left atrium was lower than that in patients with normal left atrial diameter(60.21±4.87% vs.62.95±5.47%,P=0.045).Correlation analysis showed that the level of serum β1ARAb at 8 weeks was positively correlated with circulating fiber markers(r=0.694,0.316,0.545,respectively)and left atrial diameter(r=0.272,all P<0.05).Conclusion: Multiple injections of ECL2 peptide of β1AR could successfully establish an active immune model with increased expression of β1ARAb.This model showed electrophysiological manifestations of increased induction rate of AF,shortening of AERP,decrease of atrial conduction velocity and increase of conduction heterogeneity.Proteomics and bioinformatics analysis have found that the fibrotic pathway is involved in the molecular biological process of atrial remodeling mediated by the enhanced expression of β1ARAb.The increased expression of β1ARAb can lead to atrial enlargement,decrease of cardiac function,increase of interstitial fibrosis area and up-regulation of fibrosisrelated protein molecules,and the serum level of β1ARAb is positively correlated with fibrosis area.The level of serum β1ARAb in patients with paroxysmal AF was positively correlated with biochemical markers of atrial fibrosis and left atrial diameter. |
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