Expression Of PTPN1 In Patients With Atrial Fibrillation And Its Effect On Cardiac Fibroblasts Fibrosis

Objective To investigate the expression of protein tyrosine phosphatase non-receptor type 1(PTPN1)in the right atrial appendage of patients with atrial fibrillation(AF)and sinus rhythm(SR),and its serum level on the prediction of early recurrence after AF radiofrequency catheter ablation(RFCA),and the effect of silencing PTPN1 on angiotensin Ⅱ(ANG Ⅱ)induced fibrosis of primary rat cardiac fibroblasts(CFS).Methods The right atrial appendage tissues of patients with valvular heart disease who underwent thoracotomy in our hospital since July 2020 were collected and divided into AF group and SR group.The fibrosis level of atrial tissue was assessed by the expression of collagen Ⅲ and α-SMA detected by Western blot and Masson staining of histopathological sections.The expression of PTPN1 was evaluated by Western blot and immunohistochemistry(IHC).The patients with persistent AF admitted to hospital for RFCA from January to September 2021 were collected.The preoperative serum PTPN1 level was detected by enzyme linked immunosorbent assay(ELISA).The patients were followed up for 3 months.The factors affecting early recurrence were evaluated by univariate and multivariate logistic regression analysis.CFS fibrosis model was established by Ang II stimulation,and the expression of PTPN1 protein during fibrosis was detected by Western blot.PTPN1 siRNA was transfected by lipofectamine,and the transfection efficiency was evaluated by quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot.After transfection,CFS fibrosis was induced by Ang Ⅱ,and the expression of collagen Ⅲ protein was detected.Finally,the single gene bioinformatics analysis of PTPN1 was carried out.Results A total of 36 patients with valvular heart disease were included,18 in each of the AF and SR groups.The left atrial diameter(LAD)in the AF group was significantly higher than that in the SR group(P<0.05).The protein expressions of collagen Ⅲ and α-SMA in the right atrial appendage tissue of the AF group and the collagen deposition showed by Masson staining were significantly higher than those of the SR group(P<0.05).Compared with SR group,PTPN1 protein expression was significantly increased in AF group,and IHC of tissue sections also showed that PTPN1 expression was increased in AF group.A total of 67 patients with persistent AF who underwent RFCA were included and were followed up for 3 months.Among them,28 patients had early recurrence(recurrence group),the recurrence rate was 41.8%,and 39 patients had no recurrence(non-recurrence group).The duration of AF,serum PTPN1 level and LAD in the recurrence group were significantly higher than those in the non-recurrence group.Multivariate Logistic regression analysis showed that the duration of AF was the independent risk factor for early postoperative recurrence.The expressions of Collagen Ⅲ and Ptpnl in CFs stimulated by Ang Ⅱ were higher than those in the control group.After transfection of Ptpnl siRNA,the mRNA and protein expressions of Ptpnl were significantly decreased.After transfection of Ptpnl siRNA,CFs were stimulated with Ang Ⅱ,and Western Blot showed that silencing of Ptpnl attenuated Ang Ⅱ-induced elevation of Collagen Ⅲ.Bioinformatics analysis shows that PTPN1 is located in the cytoplasm and other parts,involving biological processes such as protein dephosphorylation.Its molecular functions include phosphoprotein phosphatase pctivity and participate in the platelet derived growth factor receptor beta(PDGFR-β)signal pathway,and interact with proteins such as caveolin 1(CAV 1).Conclusion(1)The expression level of PTPN1 in right atrial appendage in AF group is higher than that in SR group;(2)The early recurrence rate of patients with persistent AF after RFCA was 41.8%;(3)The duration of AF,serum PTPN1 level and LAD in early recurrence group were significantly higher than those in non-recurrence group;(3)The duration of AF is the independent risk factor for early recurrence after RFCA;(4)Silencing PTPN1 can reduce Ang Ⅱ induced CFS fibrosis;(5)PTPN1 may be involved in fibrosis through the interaction of fibrosis-related proteins and signaling pathways.Part Ⅰ Expression of PTPN1 in Right Atrial Appendage of Patients with Atrial FibrillationBackground and Objective The occurrence and development of AF involves many factors,such as atrial electrical remodeling and structural remodeling.Among them,structural remodeling with atrial fibrosis as the main manifestation can lead to left atrial dysfunction and delay of electromechanical conduction.The expression of PTPN1 was increased in animal models of liver fibrosis and colorectal cancer,but decreased in esophageal cancer.At present,the expression of PTPN1 in AF patients is not clear.In the first part of the study,the expression of PTPN1 was compared between the two groups by collecting the right atrial appendage tissue samples of AF and SR patients.Methods The right atrial appendage tissue of patients with valvular heart disease who underwent thoracotomy in our hospital from July 2020 was collected,and divided into AF group and SR group according to the rhythm of the heart,and the clinical baseline data of the two groups of patients were recorded.The expressions of collagen fiber-related proteins Collagen Ⅲ andα-SMA were detected by Western Blot,and the collagen volume fraction(CVF)measured by Masson staining of histopathological sections was used to evaluate the expression of collagen fibers in the right atrial appendage of the two groups of patients.The protein expression of PTPN1 in the right atrial appendage tissue of the two groups of patients was detected by Western Blot,and the expression of PTPN1 was evaluated by IHC staining of histopathological sections.Results A total of 36 patients with valvular heart disease who underwent thoracotomy were included.Among them,there were 18 patients in the AF group and 18 patients in the SR group.The LAD in the AF group was significantly higher than that in the SR group(52.61±8.07 mm vs 45.61±7.42 mm,P<0.05).There was no significant difference between the two groups in age,gender,combined basic diseases,types of valvular disease,left ventricular ejection fraction and other clinical baseline data.In terms of collagen fiber expression in the right atrial appendage tissue of the two groups of patients,the protein expressions of collagen Ⅲ and α-SMA in the right atrial appendage tissue of the AF group were significantly higher than those of the SR group(P<0.05).Masson staining also showed that the CVF%of right atrial appendage in AF group was significantly higher than that in SR group(P<0.05).In terms of PTPN1 expression,compared with patients in SR group,the expression of PTPN1 protein in right atrial appendage tissue of patients in AF group was significantly higher(P<0.05),and IHC of pathological tissue section also showed that the expression of PTPN1 was significantly higher in patients in AF group(P<0.05).Conclusion The fibrosis level of right atrial appendage and the expression level of PTPN1 in patients with AF were significantly increased.PTPN1 may be involved in atrial fibrosis in patients with AF.Part Ⅱ Predictive Effect of Serum PTPN1 Level on Early Recurrence after RadioFrequency Catheter Ablation in Patients with Persistent Atrial FibrillationBackground and Objective Atrial fibrosis is an important factor for recurrence after catheter ablation of AF.Serological markers reflecting fibrosis were associated with the severity of atrial fibrosis in patients with AF,and these markers also appeared to be predictive of recurrence after catheter ablation in patients with AF.The first part of this study showed that the levels of PTPN1 and fibrosis in the right atrial appendage of patients with AF increased.In the second part of the study,the expression level of PTPN1 in the preoperative serum of patients with persistent AF undergoing RFCA was detected to understand its predictive effect on the early recurrence after catheter ablation.Methods Adult patients with persistent AF who received RFCA in our hospital from January 2021 to September 2021 were collected.The expression level of serum PTPN 1 before operation was detected by ELISA.The baseline data,serological indexes,transthoracic and transesophageal echocardiographic parameters and oral drugs after catheter ablation were recorded.The patients were followed up 3 months after the operation by clinical symptoms,ECG and Holter.Univariate analysis and multivariate logistic regression analysis were used to evaluate the factors affecting early postoperative recurrence.Results A total of 67 patients with persistent AF who underwent RFCA were included in this study and were followed up for 3 months.Among them,28 patients had early recurrence(recurrence group),the recurrence rate was 41.8%,and 39 patients did not recur(non-recurrence group).In the baseline data,the duration of AF in the recurrence group was significantly longer than that in the non-recurrence group(P<0.05).Serum PTPN1 level detected by ELISA was significantly higher in the recurrence group[4.13(3.57,5.38)ng/ml vs 3.72(3.17,4.55)ng/ml,P<0.05].There were no significant differences in preoperative renal function,blood lipids,blood glucose and D-dimer.The LAD in the recurrence group was significantly greater than that in the non-recurrence group(P<0.05),and there was no statistical difference in left ventricular ejection fraction,left ventricular end-diastolic volume and spontaneous echo contrast.Postoperative oral medications(statins,anticoagulants,angiotensin converting enzyme inhibitors/angiotensin receptor blockers,beta blockers,and antiarrhythmic drugs amiodarone/dronedarone)was not statistically different.Multivariate Logistic regression analysis showed that the duration of AF was an independent risk factor for early recurrence after catheter ablation.Conclusion(1)The early recurrence rate of patients with persistent AF after RFCA is 41.8%;(2)The duration of AF,serum PTPN1 level and LAD were significantly increased in patients with early recurrence;(3)Multivariate logistic regression analysis showed that the duration of AF was an independent risk factor for early recurrence after RFCA.Part Ⅲ Effect of Ptpn1 on Ang Ⅱ Induced Cardiac Fibroblasts Fibrosis and its Bioinformatics AnalysisBackground and Objective The mechanism of AF fibrosis involves many factors.Abnormal activation of CFs and subsequent excessive synthesis and irregular deposition of extracellular matrix proteins are the main features of atrial fibrosis.Based on the research in the previous two parts,this part intends to investigate whether silencing of Ptpnl can alleviate Ang Ⅱ-induced fibrosis in rat primary CFs at the cellular level,and conduct a Gene Ontology(GO)function analysis,signal pathway analysis and search for proteins interacting with PTPN1 through bioinformatics methods to establish a theoretical basis for subsequent research.Methods The primary CFs of SD suckling mice were extracted and the cells were identified by immunofluorescence staining.CFs fibrosis model was established by Ang Ⅱ stimulation,and the protein expression of Ptpnl during cell fibrosis was detected by Western blot.Ptpnl siRNA was transfected by lipofectamine,and the transfection efficiency was evaluated by qRT-PCR and Western blot.After transfection,the cells were stimulated with Ang Ⅱ to induce CFs fibrosis,and the protein expression of collagen Ⅲ was detected.In the single-gene bioinformatics analysis,the human species PTPN1 gene was retrieved through the Ensembl platform to query the biological process,molecular function and cellular localization involved in the GO function..The signal pathways involved in PTPN1 gene were retrieved through GeneCards platform.Finally,the protein network interacting with PTPN1 was searched by STRING platform.Results The extracted rat primary CFs was identified by vimentin immunofluorescence staining and accorded with the characteristics of CFs.The expressions of collagen Ⅲ and Ptpn1 protein in Ang Ⅱ stimulated cells were higher than those in the control group.After the cells were transfected with Ptpn1 siRNA,the mRNA expression level of Ptpn1 was significantly reduced by qRT-PCR,and the protein expression level of Ptpn1 was also significantly reduced by Western blot.After transfection of Ptpn1 siRNA,CFs was stimulated with Ang Ⅱ.The results of Western blot showed that silencing Ptpn1 could reduce the increase of collagen Ⅲ induced by Ang Ⅱ.Bioinformatics analysis showed that PTPN1 is located in the cytoplasm,mitochondrial matrix,endoplasmic reticulum and other parts.The biological processes involved include protein dephosphorylation,regulation of signal transduction,positive regulation of Jun kinase activity,etc.The molecular functions include phosphoprotein phosphatase activity,insulin receptor binding,cadherin binding,etc.In terms of signal pathway,PTPN1 participates in immune response IFN-α/β Signal pathway,PDGFR-β signal pathway,insulin signal pathway,etc.PTPN1 interacts with many proteins,such as epidermal growth factor receptor,insulin-like growth factor 1 receptor and CAV1,etc.Conclusion(1)Silencing of Ptpn1 can alleviate Ang Ⅱ-induced fibrosis in rat primary CFs;(2)PTPN1 may be involved in fibrosis through the interaction of fibrosis-related proteins and signaling pathways.