Mechanism Of PLB Involved In Inducing Atrial Myocyte Apoptosis Promoting Atrial Fibrosis

Backgroud and Objective:Atrial fibrillation(AF),one of the common arrhythmias,affects 3% of adults.The presence of AF and its complications affects the quality of many patients' life.Although the treatment of AF has made some progress,there are still problems with high recurrence rate.The reason is that the specific mechanism is not completely clear.In recent years,numerous studies have pointed out that the occurrence and maintenance of AF is closely related to atrial remodeling.The early stage of atrial remodeling is mainly electrophysiological remodeling,and the main performance in the middle and late stages is structural remodeling.Structural remodeling of the atria is an important cause of AF maintenance and recurrence.In the presence of AF,atrial myocytes undergo apoptosis,mostly results from different stimuli,including stretching,hypoxia,inflammation,and oxidative stress.Decrease in atrial myocytes after apoptosis causes changes in the composition of myocardial tissue.After cell loss,fibroblasts activate and repair myocardial tissue,improve extracellular matrix protein secretion,promotes interstitial fibrosis,and consequently causes atrium remodeling.Normal systolic and diastolic function of cardiomyocytes depends on the circulation of intracellular calcium ions between sarcoplasmic reticulum and cytoplasm.In cytoplasm,calcium ions are pumped back into sarcoplasmic reticulum by the sarcoplasmic reticulum Ca2+-ATPase(SERCA2a),which is released into cytoplasm during the systolic phase by the type 2 ryanodine receptors(RYR2).Phospholamban(PLB)is a 52 amino acid peptide chain with both phosphorylated pentamers and dephosphorylated monomers.Monomer PLB binds to SERCA2 a to reduce its activity and acts as negative regulator of SERCA2 a.Phosphorylated pentamer PLB loses its inhibitory effect on SERCA2 a.Changes in expression and phosphorylation level of PLB affect the normal activity of intracellular calcium ions by altering the activity of SERCA2 a.Calcium overload caused by abnormal calcium ion activity may cause atrial myocyte apoptosis which participates in atrial remodeling.Thus expression and phosphorylation level of PLB may play an important role in the occurrence and maintenance of AF.In the first part of the study,we reviewed the clinical data of two groups of patients with rheumatic valvular disease,summarized the characteristics of AF patients,detected and compared the fibrotic level,atrial myocyte apoptosis and apoptosis gene expression in the atrial tissue of the two groups,to investigate the significance of atrial myocyte apoptosis in atrial remodeling of AF.In the second part,we detected the gene expression levels of CaMKII and PLB and the protein levels of PLB,PLBp17 and CaMKIIp286 in the right atrial tissue of the two groups.After PLB mRNA was down-regulated by siRNA silencing in HL-1 cell line,we examined the apoptosis rate,in order to explore the mechanism of expression and phosphorylation level of PLB in atrial remodeling in AF.Methods:Part 1 Significance of apoptosis and apoptosis-related genes in atrial myocytes examined in atrial remodeling of atrial fibrillation1.Clinical data: 695 patients with rheumatic heart disease undergoing mitral valve replacement surgery from September 2016 to October 2017 were divided into two groups according to dynamic electrocardiogram and medical history data.Two hundred and twelve cases were assigned to sinus rhythm(SR)group and 483 to AF group.2.Tissue specimen collection: After the establishment of extracorporeal circulation,the right atrial tissue was obtained before the cardiac arrest,followed by removement of adipose tissue and blood by physiological saline.The tissue was divided into three parts.The first part was made into fast frozen sections,and the second part was made into paraffin sections,and the third part was used for tissue total RNA extraction.Tissue were grouped according to the dynamic electrocardiogram and medical history data,and the right atrial tissue specimens from the SR group(10 cases)and the AF group(15 cases)were obtained and preserved according to the methods mentioned above.3.Masson staining and Sirius red staining were used to detect and compare the fibrotic levels in the two groups.4.Immunofluorescence was used to detect and compare the apoptosis rate of atrial myocytes.5.RT-qPCR was used to detect and compare the mRNA levels of apoptosis-related genes in atrial tissue of the two groups.Part 2 Changes in expression and phosphorylation level of PLB are involved in inducing apoptosis of atrial myocytes.1.Tissue specimen collection: After the establishment of extracorporeal circulation,the right atrial tissue was obtained before the cardiac arrest,followed by removement of adipose tissue and blood by physiological saline.The first part was used for tissue total RNA extraction,and the second part was used for total tissue protein extraction.Tissue were grouped according to the dynamic electrocardiogram and medical history data,and the right atrial tissue specimens from the SR group(11 cases)and the AF group(19 cases)were obtained and preserved according to the methods mentioned above2.RT-qPCR was used to detect and compare the levels of CaMKII and PLB mRNA in the atrial tissue of the two groups..3.Western Blot was used to detect the protein expression levels of PLB,PLBp17 and CaMKIIp286.4.After PLB mRNA was down-regulated by siRNA silencing in HL-1 cell line,the apoptosis rate was detected by flow cytometry after 48 hours of interference.Results:Part 1 the atrial myocyte apoptosis and apoptosis-related genes in the atrial remodeling of atrial fibrillation.1.Clinical data showed that compared with the SR group,the prevalence of advanced age(P<0.01),long hospital stay(P<0.05),pulmonary hypertension was significantly higher in the AF group(P <0.01).The incidence of complicated left atrial thrombus and cerebral infarction markedly rose(P<0.01,P<0.05).While NYHA class II was dramatically reduced,class III and IV both predominantly surged(both P< 0.01).LADd,RADd and RVDd were larger(all P<0.01),EF became lessened noteworthily(P<0.01),and tricuspid regurgitation augmented(P<0.01).2.Baseline data of tissue specimens: Compared with the SR group,LADd and RADd remarkably increased in patients with AF(P<0.01),and EF was significantly lower(P<0.01).3.The results of Masson staining and Sirius red staining showed that the degree of fibrosis in the AF group was significantly severer than that in the SR group(P<0.01),and the collagen fibers were mainly type I and type III when fibrosis occurred.4.After TUNEL staining,the rate of apoptotic cells was calculated under fluorescence microscope.The statistical results showed that the apoptotic rate of AF group was significantly higher than that of SR group(P<0.01).5.The expressions of TP53,BAX and SNAI2 mRNA in AF group were significantly higher than those in SR group,BCL2 and BMP4 were lower than in SR group(P<0.05).Part 2 Changes in expression and phosphorylation level of PLB are involved in inducing apoptosis of atrial myocytes.1.Baseline data of tissue specimens: Compared with the SR group,LADd(P<0.05)and RADd P<0.01)overtly increased in patients with AF,and EF was distinctly decreased(P<0.05).2.RT-qPCR results showed that compared with the SR group,the expression levels of CaMKII(P<0.05)and PLB genes(P<0.05)in atrial myocytes of AF group were higher.3.Western Blot results showed that compared with the SR group,the protein levels of CaMKII p286(P<0.05)and PLB p17(P<0.05)increased in the AF group,and the PLB protein level in the AF group was lower(P<0.05).4.Apoptosis detection by flow cytometry after 48 hours of siRNA silencing of PLB gene showed that the apoptosis rate of the experimental group was obviously higher than that in the negative control group and the blank group(P<0.05).Conclusions:1.Atrial remodeling with myocardial fibrosis occurs during AF:The right atrial tissue fibrosis was severer in AF group,indicating that the structural remodeling of AF was more serious.The AF group mainly exhibited expansion of each heart chamber and increased tricuspid regurgitation?2.Atrial remodeling and fibrosis in AF are associated with atrial myocyte apoptosis:In the atrial tissue of AF group,the apoptotic ratio of atrial myocytes increased and the expression of apoptosis-related genes were disordered accordingly.the expression of TP53,BAX and SNAI2 increased,while BCL2 and BMP4 decreased,3.PLB participates in atrial remodeling by regulating atrial myocyte apoptosis:CaMKII and PLB mRNA were up-regulated in AF group,protein expression levels of CaMKIIp286 and PLB p17 were increased,while the monomer PLB level was decreased.The HL-1 cell line was cultured,and the expression of PLB gene was interferenced by siRNA.Forty-eight hours after interference,the apoptosis level of cells was detected by flow cytometry,and the apoptosis rate of siRNA group was found to be increased.that PLB It is suggested that the PLB plays an important role in atrial remodeling by regulating atrial myocyte apoptosis.