The Relationship Between Sirtuin 3 Expression And Oxidative Stress In Atrial Tissue In Atrial Fibrillation

Background:Atrial fibrillation(AF)is the most common sustained arrhythmia which affect about 0.77%of the population ages 18years and above in our country.The lifetime risk to develop AF at the age of 40 years was 25%in Europe.AF carried a significantly elevated risk for death mainly attribute to the complications of stroke and heart failure.There are still lack of very effective methods for the treatment of AF,especially for the treatment of persistent and permanent AF.Therefore,better understanding of mechanisms underlying AF is essential for the treatment of AF.A lot of studies have shown that atrial remodeling,including electrical remodeling and structure remodeling,plays an extremely important role in the onset and maintain of AF.Oxidative stress has been demonstrated to play a role in the remodeling of AF,But the signaling pathway and regulatory mechanism of oxidative stress in AF are not fully understood.The silence information regulator 2(Sirtuin)is an important class III deacetylase and is considered a pivotal mediator of longevity and DNA repair.Seven mammalian homologues of Sirtuin have been identified and are designated as Sirtuinl(SIRTl)through Sirtuin7(SIRT7).Sirtuin3(SIRT3)is the most robust mitochondrial deacetylase and has been shown to particularly regulate the production of ROS as well as the detoxification of ROS through activation of antioxidant enzymes.Klishadi et al found that decreased SIRT3 protein level subsequent to ischemia reperfusion(IR)might be a novel signaling mechanism involved in IR injury and Losartan exerts anti-ischemic and antioxidant effects partly by normalizing the SIRT3 protein level in ischemic tissue of the heart.Sultana et al observed reduced activity of SIRT3 and increased acetylation of MnSOD in diabetic rat heart.They also found garlic could reduce level of ROS and attenuate tissue damage through activating SIRT3-MnSOD axis.We hypothesize that SIRT3 may play a role in oxidative stress in AF.The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors(statins)are potent inhibitors of cholesterol biosynthesis.Apart from lipid-lowering effect,other important effects of statins such as stabling plaque,anti-inflammatory and antioxidant stress were also found in recent years.Shida et al demonstrated that the beneficial effects of fluvastatin on diabetic cardiomyopathy might result,at least in part,from improving coronary microvasculature through reduction in myocardial oxidative stress.Antoniades et al found that atorvastatin could inhibit atrial oxidative stress in patients receiving coronary artery bypass graft(CABG)operation after administrated for a short time before operation.We suspect that statins may reduce oxidative stress in AF through acting on SIRT3.Objective:1.To evaluate the feasibility of developing a rabbit model of atrial fibrillation by rapid atrial pacing under different duration.2.To evaluate the role of SIRT3 in oxidative stress occurred in atrial tissue of rabbits after long time rapid atrial pacing.3.To determine whether atorvastatin can relieve oxidative stress induced by rapid pacing through effect on SIRT3.Methods:1.Establishment of AF model by rapid atrial pacing:1%sodium pentobarbital was intravenously injected bolus at 2ml/kg into rabbit ear vein,and then was injected continuously under the speed of lml/kg/h by use of syringe pump for anesthesia.The rabbit was fixed on the operation table,intubated,and ventilated with a ventilator(50 cycles/min,10 ml/kg).Heart rhythm was monitored on electrocardiogram.The right internal jugular vein was separated after neck incision.The proximal segment of vein was ligated and a 5Fr decapolar Webster electrophysiology(EP)catheter was inserted into the right atrium through a 5Fr sheath slowly until a large biphasic A wave was appeared on intracardiac distal bipolar ECG.The distal bipolar was connected to a programmed stimulator.Atrial pacing was performed at 600 beats/min with a 2-ms rectangular pulse width and two times the diastolic threshold.2.Group distribution:40 New Zealand white rabbits were randomly divided into four groups in average by use of randomized block design:Sham operation group(Sham group),short time pacing group(SP group,persistent rapid atrial pacing at 600 beats/min for 6 hours),long time pacing group(LP group,persistent rapid atrial pacing at 600 beats/min for 12 hours),atorvastatin intervention group(AI group,atorvastatin was administered at the dose of 2.5mg/kg for 7 days before rapid atrial pacing at 600 beats/min for 12 hours).3..Main outcomes measures:The atrial structural histopathology changes were observed using light microscope and transmission electron microscope.Fibrosis in left atrial tissue was semiquantitatively assessed by morphometric determination of collagen volume fraction using Image-ProPlus6.0 software after Masson staining.The level of malondialdehyde(MDA),total reactive oxygen species(ROS)and the manganese superoxide superoxide dismutase(MnSOD)activity in left atrial tissue were measured by colorimetric determination.The protein expression of SIRT3,MnSOD as well as forkhead transcription factor 3a(FOXO3a)in nucleus and in cytoplasm were determined by western-blot.The mRNA levels of SIRT3,FOXO3a and MnSOD were detected by Real-time quantitative reverse transcription polymerase chain reaction.Result:1.The EP catheter was inserted into the right atrium of every rabbit successfully.The stimulation could capture atrium of all rabbits.All of 40 rabbits survived to the end of experiment except for 4 rabbits died during the procedure,including one rabbit in Sham group,two rabbits in LP group and one rabbit in AI group.2.The changes of atrial histological structure:Observed under HE staining:Compared with Sham group,the significant interstitial fibrosis,interstitial tissue edema,irregular size of individual cells,disorganization of cell arrangement were observed in LP and AI group while these changes were more severe in LP group than those in AI group;Slight disorganization of cell arrangement other than interstitial fibrosis were observed in SP group.Observed under Masson staining:Compared with Sham group,a larger amount of interstitial fibrosis and collagen distribution throughout the tissue were found in LP group while which were attenuated significantly in AI group.The collagen volume fraction(CVF)in AI group was lower than that in LP group.There were only slight interstitial fibrosis in SP group and the CVF was similar in SP group and Sham group.Histological changes under electron microscope:All degrees of histological ultrastructure damages including disorganization of sarcomere,abnormality of gap junction in mitochondria nuclear membrane,disarrangement of mitochondria were found in all rapid pacing rabbits,while those damages were more severe in LP group and slight in SP group.3.The levels of MDA and ROS were higher in LP group than those in SP and Sham group while the activity of MnSOD was lower in LP group than that in SP and Sham group(P<0.05);The levels of MDA and ROS were lower in AI group than those in LP group while the activity of MnSOD was higher in AI group than that in LP(P<0.05);Similar differences were not found between SP and Sham group(P>0.05).4.The protein expression levels of MnSOD and SIRT3 were lower in LP group than those in Sham group(P<0.05).Compared with LP group,increase expressions of SIRT3 and nuclear FOXO3a were observed in AI group(P<0.05).There was no difference for protein expression of MnSOD between LP group and AI group(P>0.05).The expression differences of MnSOD,SIRT3 and FOXO3a protein were not found between Sham group and SP group(P>0.05).There was negative correlation between the expression of SIRT3 and the content of ROS in atrial tissue of rabbits receiving rapid atrial pacing(P<0.01).5.The mRNA expression levels of SIRT3 gene and MnSOD gene were lower in LP group than those in Sham group and SP group(P<0.05).There were no difference for mRNA expressions of SIRT 3 and MnSOD between SP group and Sham group(P>0.05).The mRNA expression levels of SIRT3 gene and MnSOD gene were higher in AI group than those in LP group(P<0.05),while the mRNA expression differencesof FOXO3a gene were not found among four groups.Conclusion:1.Oxidative stress and histologic structure damages are found in rabbit atrial tissue after persistent atrial rapid pacing at 600beats/min for 12 hours,which is proved to be an efficient method for the development of atrial fibrillation animal model.2.The down-regulated expression of SIRT3 is related to the oxidative stress and structure remodeling in atrial tissue of rabbit AF model induced by rapid atrial pacing.3.Atorvastatin can up-regulate the expression of SIRT3 and attenuate oxidative stress and histologic structure damages in atrial tissue of AF rabbit induced by rapid atrial pacing.Background:Atrial fibrillation(AF)is the most common sustained arrhythmia which affecting about 0.77%of the population ages 18 years and above in our country.The lifetime risk to develop AF at the age of 40 years was 25%in Europe.AF carried a significantly elevated risk for death mainly attribute to the complications of stroke and heart failure.There are still lack of very effective methods for the treatment of AF,especially for the treatment of persistent and permanent AF.Therefore,better understanding of mechanisms underlying AF is essential for the treatment of AF.A lot of studies have shown that atrial remodeling,including electrical remodeling and structure remodeling,plays a extremely important role in the onset and maintain of AF.Oxidative stress has been demonstrated to play a role in the remodeling of AF,But the signaling pathway and regulatory mechanism of oxidative stress in AF are not fully understood.The silence information regulator 2(SIR2,sirtuins)is an important class III deacetylase and is considered a pivotal mediator of longevity and DNA repair.Seven mammalian homologues of sirtuins have been identified and are designated as sirtuinl(SIRT1)through sirtuin7(SIRT7).Sirtuin3(SIRT3)is the most robust mitochondrial deacetylase and has been shown to particularly regulate the production of reactive oxygen species(ROS)as well as the detoxification of ROS through activation of antioxidant enzymes.Klishadi et al found decreased SIRT3 protein level subsequent to ischemia reperfusion(IR)might be a novel signaling mechanism involved in IR injury and Losartan exerts anti-ischemic and antioxidant effects partly by normalizing the SIRT3 protein level in ischemic tissue of the heart.Sultana et al observed reduced activity of SIRT3 and increased acetylation of MnSOD in diabetic rat heart.They also found garlic could reduce level of ROS and attenuate tissue damage through activating SIRT3-MnSOD axis.We hypothesize that SIRT3 may play a role in oxidative stress in AF.Objective:1.To evaluate the role of SIRT3 in oxidative stress in atrial tissue of patients with atrial fibrillation by determining the level of SIRT3 and oxidative stress state in right atrial appendage tissue from AF patients with rheumatic valvular heart disease.2.To determine whether the malondialdehyde(MDA)level in peripheral blood can be used to evaluate oxidative stress state in atrial tissue accurately by analyzing the correlation between MDA level in peripheral blood and MDA level in atrial tissue.Methods:40 patients with mitral stenosis resulting from rheumatic heart disease scheduled to underwent cardiac surgery in XuZhou Medical College Affiliated Hospital were divided into 2 groups according to the concomitant of AF:AF group(n=28),and sinus rhythm group(SR group,n=12).Theleft atrial diameter(LAD)and left ventricular ejection fraction(LVEF)were measured by transthoracic echocardiogram before operation.The peripheral blood sample was acquired before operation and right atrial appendage(RAA)tissue were acquired during operation.The level of MDA in peripheral blood was tested by thiobarbituric acid method.The atrial structural histopathology changes were observed under light microscope by use of HE staining.The atrial fibrosis as well as collagen volume fraction(CVF)was evaluated under Masson staining.The level of MDA and ROS in RAA tissue were measured by colorimetric determination.The protein expression of SIRT3,MnSOD were determined by western-blot.Real-time quantitative reverse transcription polymerase chain reaction was used to detect the mRNA level of SIRT3,FOXO3a and MnSOD in RAA tissue.Results:1.The LAD in AF group was larger than that in SR group(P<0.01).Compared with SR group,a larger amount of interstitial fibrosis and collagen distribution throughout the RAA tissue were found in AF group.CVF in AF group was much higher than that in SR group(P<0.01).2.The level of MDA in peripheral blood was higher in AF group than that in SR group(P<0.01).3.The le-vels of MDA and ROS in atrial tissue were higher in AF group than those in SR group(P<0.01).4.There was moderate liner correlationship between MDA level in RAA tissue and MDA level in peripheral blood(r=0.65,R2=0.42,P<0.01).5.The protein expression of SIRT3 and MnSOD in atrial tissue was lower in AF group than that in SR group(P<0.05).6.The mRNA expressions of SIRT3 and MnSOD in atrial tissue were lower in AF group than those in SR group(P<0.05)while there was no difference for the mRNA expression of FOXO3a between two groups(P>0.05).7.The protein expression SIRT3 in AF group correlates negatively to the level of ROS and LAD(P<0.01),while correlates positively to the level of MnSOD protein(P<0.01).Conclusions:1.Oxidative stress exist in atrial tissue of AF patients complicated with rheumatic valvular disease.2.The level of SIRT3 expression in atrial tissue is reduced in patients with AF3.There are correlation between SIRT3 expression and oxidative stress in atrial tissue of AF patients with rheumatic heart disease.