The Role Of Electrophysiological Changes Of Kv1.5 And Oxidative Stress In Atrial Remodeling During Atrial Fibrillation

ObjectiveAtrial fibrillation is the kind of most often clinically observed arrhythmia,which is the independent risk factor of the mortality of population.But there is not any effective methods that could solve atrial fibrillation,and the biggest barrier is the unclear mechanism of atrial fibrillation.Since the concept of atrial remodeling proposed by Wijffels in 1995,a large number of basic and clinical researches were developed.And now there is the consistent opion that atrial remodeling is the molecular basis of the genesis and development of atrial fibrillation,essentially including the shortage of myocardial action potential duration(APD) and absolute refractory period(ARP).So many factors affecting the atrial remodeling determined the complexity of atrial remodeling.Recently,some studies found kv1.5 was only expressed on the atrium and never or little on the ventricle without physiological function,kv1.5 is one of the main elements of atrial repolarization,which is the molecular basis of atrial ultra rapid delayed rectifier potassium currents(Ikur) playing the important role in repolarization and affecting APD.Some researches showed there were disorders of energy metabolism by different extent resulting in oxidative stress.Oxygen free radicals could directly affect the character of kv1.5 channel proved by cell culture study in vitro,which showed oxygen free radicals increased the currents amplitude during repolar phase and enhanced the opening of the channel with the shortage of APD.How does the oxygen free radicals affect human atrial kv1.5 channel is not reported and unclear.Though it could not be concluded according to forthcoming researches,the oxidative stress of the atrium would be the new target of study and treatment for atrial fibrillation.This study pay attention to human artial myocytes,detect the expression of kv1.5,oxidatases and reductases and influence of oxygen free radicals on kv1.5 channel with the purpose of studying the contribution of this mechanism to the atrial remodeling.Materimals and Methods1.Sample collectionThe right atrial appendages were from the patients who received the valvular replacement with rheumatic heart disease and divided into two groups,one is sinus rhythm group and the other is atrial fibrillation group according to the heart rhythm.2.Methods(1) The mRNA expression of the gene was analyzed by RT-PCR.The total RNA was abstracted from the tissue by the means of Trizol method,and then was reverse transcripted into cDNA by the two-step RT-PCR kit.The amplification of the genes of kv1.5,flavin containing monooxygenase-1,monoamine oxidase B,glutathione peroxidase-1 and heme oxygenase-2 were attained through the kits and the inside comparative gene was GAPDH.The amplificated products were dissociated by agarose gel electrophoresis and calculated the expression.(2) The protein expression of kv1.5 was analyzed by Western Blot.The total protein was abstracted from the tissue by the means of RIPA method and then determined the concentration of the protein.Electrophoresis,transferring, hybridization of antigen and antibody and ECL kit were under use to calculate the protein expression of kv1.5.(3) The effects of oxidative radicals on channel kv1.5 were detected by the patch clamp technique.Myocardium sample was cut into small block of 1mm³ with fine scissors.Single myocytewas isolated with enzymatic dissociation techniquesand the cells with best quality were collected and stored in KB solution.A few drops of isolated myocytes were placed in bath of 1cm³ on microscope.5 min later,column-shaped myocytes with strong membranous reflection and clear were prepared for patch clamp.4-AP and H₂O₂ were added in the bathing solution, respectively.Natium currentwas deactivated with -40mv holding potential.When whole cell recording finished,holding potential was set to -40mv,commanding potential was depolarized from -60mv to +60mv,with step pulse at 10mv,holding duration of 200ms and stimulating interval of 3s.Recorded current was analyed with program of Pclamp 5.5.1,Clampan,Clampex and Clampfit.(4) Statistical analysisStatistical analysis was performed with SPSS13.0.Data were expressed as means±SD((?)+s).Inter groups comparison was performed with student t test.When P is less than 0.05,the difference was considered statically significant.Results1.The mRNA expression of the gene was analyzed by RT-PCRThe expression of kv1.5 was significantly lower in AF group as compared with SR group(P＜0.05).The expression of FMO-1 and MAO-B were significantly higher and the expression of HMOX-2 and GPX-1 significantly lower in AF group as compared with SR group(P＜0.05).2.The protein expression of kv1.5 was analyzed by Western Blot.The protein expression of kv1.5 was significantly lower in AF group as compared with SR group(P＜0.05).3.The effects of oxidative radicals on channel kv1.5 were detected by the patch clamp technique.The pA/pF(4.12±1.37) of Ikur about +50mV in AF group was significantly less than that(8.91±1.86) in SR group,P＜0.01;The pA/pF(2.75±0.84) of Ikur in 4-AP+AF group was significantly less than that(4.12±1.37) in AF group,P＜0.01;The pA/pF (3.86±1.18) of Ikur in H₂O₂+4-AP+AF group was significantly larger than that(2.75±0.84) in 4-AP+AF group,P＜0.01.The APD(310±21ms) in AF group was significantly shorter than that(425±17ms) in SR group,P＜0.01;The APD(387±19ms) in 4-AP+AF group was significantly longer than that(310±21ms) in AF group,P＜0.01;The APD(297±22ms) in H₂O₂+4-AP+AF group was significantly shorter than that(387±19ms) in 4-AP+AF group,P＜0.01.DiscussionRecent clinical researches found that the treatment for atrial remodeling was very effective in preventing AF,which makes us to think that studying the mechanism of atrial remodeling in depth and finding how to reverse the remodeling should be most important for clinical treatment on AF.Many studies have showed that there are the dysfunction of energy metabolism in atrial myocytes during AF,which resulting in oxidative stress.Carnes found that oxidative redicals were increased during atrial pacing and Vitamine C could reduced them and delayed the happening of electrical remodeling.The expressions of the oxidases were increased and the expressions of reductases were decresed,so the physiological balance between the production and clearance of oxido-reductase was disturbed.It showed that oxidative stress might play a very vital role in the happening and the pathologic progression of AF and offers novel insight into potential treatment with antioxidants.Just similar to the results of Van wagoner's research,that the pA/pF of Ikur in AF group was significantly less than thatin SR group was related to that the expression of kv1.5 was significantly lower in AF group.Recently,some studies found kv1.5 was only expressed on the atrium and never or little on the ventricle without physiological function,kv1.5 is one of the main elements of atrial repolarization,which is the molecular basis of atrial Ikur playing the important role in repolarization and affecting APD.The mathematics model found the down regulation of Ikur may recover the APD to the normal,which might be a compensative way.Our study found that the pA/pF of Ikur in AF group was significantly less than thatin SR group;in 4-AP+AF group significantly less than in AF group;in H₂O₂+4-AP+AF group significantly larger than in 4-AP+AF group and the APD in AF group was significantly shorter than thatin SR group;in 4-AP+AF group significantly longer than in AF group;in H₂O₂+4-AP+AF group significantly shorter than in 4-AP+AF group.All of the results suggested that oxidative redicals might change the opening characters of kv1.5 and more oxidative redicals could enhance the open of kv1.5,increase the pA/pF of kv1.5 and reduce the APD,which providing conditions for the happening and persistence of AF.Our research found oxygen free radicals change the character of kv1.5,increase pA/pF and reduce APD,which explaining why simple use of specific blockade of Ikurcan not control the AF totally in some extent.Conclusions(1)The mRNA and protein expresstion of kv1.5 decreased during atrial fibrillation;(2)There was imbalance between the production and clearance of oxygen free radicals in fibrillating atrial myocytes,which implied that oxidative stress played a important role in the happening and perpetuation of atiral fibrillation;(3)Oxygen free radicals could affect the opening character of channel kv1.5,which revealed that the disorders of energy metabolism and oxidative stress is a kind of mechanism of the genesis and development of atrial fibrillation.