| Background and objectiveAtrial fibrillation(AF), the most common clinical arrhythmia, is seen in patients with structural heart disease, most commonly rheumatic heart disease (RHD) in China, but some patients suffer from AF with no obvious cause. AF can lead further damage to heart function, increased incidence of stroke and multiplied mortality.By now, Prevention and treatment of AF are all not ideal, mainly because the mechanisms of incidence and maintenance of AF are not fully clear, but in recent years, mechanisms of electrical remodeling of AF have undoubtedly become a major hot spot. After AF, a series of atrial structural and functional changes called atrial remodeling occur, which show electrophysiologically shorten action potential duration and effective refractory period, reduced adaptation for frequency. The electrophysiological changes after AF are also called electrical remodeling. Electrical remodeling makes multiwavelet reentry easy to maintain and difficult to terminate and is involved with a series of changes of various ion channels. Ion channels more expressed in atrium than in ventricle may be potential ideal target for the prevention and treatment of AF.Calcium-activated potassium channels are widely distributed in many kinds of cells, activated by intracellular calcium(<1.0μM) and divided into three types--large conductance (BK), intermediate conductance (IK) and small conductance (SK) by electrical conductivity. SK2 is a subtype of SK, whoseαsubunit is the functional subunit of the channel coded by KCNN2 gene. SK2 can specifically blocked be very low concentration(100 pM 10 nM) of apamin, a kind of peptide from bee venom. Studies have shown that SK2 expression was more significantly in atrium than in ventricle. Short-term rapid pacing in pulmonary vein can result in increased SK2 expression, more SK2 protein transferring to membrane and increased current density in the whole-cell mode, which are in relationships with significant shortening of action potential duration, and SK2 is considered a possible ideal target for treatment of AF. Another recent animal experiments have indeed proved that application of SK2 blockers can prevent the onset of AF. However, above conclusions were all from animal studes and changes SK2 in patients suffering from persistent AF are unknown.This study involving differences of SK2 gene and protein expression between left and right atrium and between atrium and ventricle between in patients suffering from persistent AF, changes of gene and protein expression of SK2α-subunit and functional SK2 in people with persistent AF, as well as changes of the SK2 accessory subunit of calmodulin (CaM) in persistent AF patients. The purpose is our study to explore SK2 change in the role of electrical remodeling in people suffering from persistent AF and to find if SK2 is ideal target for prevention and treatment of persistent AF.Methods and results1 SK2-specific distribution of various parts of heart1.1 RHD patients undertaken mitral valve replacement were selected. parts of right atrial appendage and left atrial appendage were gotten at the same time before cardiopulmonary bypass (CPB), and each specimen was divided into two parts for immunohistochemistry (IHC) and reverse transcription polymerase chain reaction (RT-PCR), to observe if there were different SK2 gene and protein expression between left and right atrium.1.2 Patients with congenital heart disease(CHD) and hypertrophy right ventricular outflow who undertaken deformity correction were selected. Parts of right atrial appendage and right ventricular outflow were gotten during the operations and each specimen was divided into two parts for IHC and RT-PCR to observe if there were different SK2 gene and protein expression between atrium and ventricle. 1.3 As it's difficult to isolate human atrial and ventricular myocytes, we isolated atrial and ventricular myocytes from SD rats by Langendoff perfusion apparatus and recoded Ik,ca current (or Apamin-sensitive potassium current) in the whole cell mode in order to observe whether there were differences in SK2 channel functionally between atrium and ventricle.2 SK2 changes after suffering from persistent AF1.1 According to the existence of persistent AF( >7days), RHD patients who undertaken mitral valve replacement surgery were divided into two groups, AF group and normal sinus rhythm (NSR) group. Parts of right atrial appendage of these patients were obtained before CPB to discover a series of changes in atrial structural remodeling by routine HE staining and Masson staining and to observe gene and protein expression changes of SK2αsubunit and CaM after suffering from persistent AF by IHC and RT-PCR1.2 Also according to the existence of persistent AF, RHD patients who undertaken mitral valve replacement surgery were divided into two groups, AF group and NSR group. Parts of right atrial appendage of these patients were obtained before CPB to isolate atrial myocytes enzymatically, which to be used to record action potentials and apamin sensitive K+ current in whole cell mode in order to record Ik,ca. About 50% of calcium-resistant cardiacites with undamaged structure could be gotten by application of Langendoff perfusion system. Myocardial cells with undamaged structure were shown membrane integrity, strong refraction and clear cross striations, which could be used for patch clamp experiments.Results1 SK2 in various parts of heart1.1 left atrium vs right atrium8 cases of RHD patients with persistent AF were selected, and there was no difference in SK2 gene and protein expression between left atrium and right atrium.1.2 atrium vs ventricle6 cases of CHD patients, including 3 cases of Tetralogy of Fallot(TOF), 2 Ventricular septal defect (VSD), 1 Pulmonary stenosis(PS), all of them were NSR. no difference in relative expression of KCNN2 gene expression was found between human atrium and ventrile, but expression of SK2 protein in atrium increased more significantly than in ventricle.1.3 Comparison of Ik,ca between atrial myocytes and ventricular myocytes of SD ratIk,ca, also called apamin sensitive K+ current, of rat atrial and ventricular muscle cells was obtained by digital subtraction. Ik,ca is an apamin ensitive K+ current with inward rectifying properties, whose reversal potential was about -60mV, close to the Nernst equation. Ik,ca could be recorded in atrial and ventricular myocytes . When pulse voltage was during -50 40mV, Ik,ca current density was significantly more greater than in atrial muscle myocytes than in ventricular ones. measured values of membrane capacitance were 21.36±3.23pF and 48.25±4.15pF in atrial and ventricular muscle cells respectively, and the difference was statistically significant.2 SK2 changes after suffering from persistent AF2.1 Baseline clinical data of the two groups18 patients with NSR and 23 patients with persistent AF were selected in our study. Comparing with NSR group, patients in AF group sustained a greater proportion of mitral stenosis, had larger left atrial diameters and more increased heart-chest rate significantly (P<0.05). Patients in AF group were older with a higher pulmonary artery systolic pressure than patients in NSR group, but the difference did not reach statistical significance. In the respect of male-female ratio, left ventricular diastolic diameter, left ventricular systolic diameter and NYHA classification in cardiac function, there were also no difference between NSR and AF group statistically.2.2HE staining and Masson stainingAtrial myocytes showed rod-like shape at longitudinal section and round-like shape at horizontal section, there were filaments shown in cytoplasm and a nucleus In most cases located in the central of cell. Multiple cells were arranged in dense bundles, with branches connected. Atrial myocytes of patients in AF group, whose arrangement were in disorder, were larger in the size and also were surrounded by more collagen tissue than in NSR group .2.3 SK2αsubunit gene and protein expression changes after suffering from persistent AFRelative KCNN2 gene expression in atrium of patients in AF group and NSR group groups of were 0.70±0.30 and 1.25±0.52, the difference was statistically significant (P <0.01). SK2 protein expression in atrium of patients in AF group also decreased, down by 13% compared with NSR group, and IOD were 2525900±772807.4 in NSR group and 2206917±1170312 in AF group respectively, but the difference in SK2 protein expression between the two group did not reach statistical signific level(P>0.05). The intracellular distribution of SK2 protein did not change significantly between the two group.2.4 Changes in CaM (SK2 channel auxiliary subunit) of patients with persistent AFAfter persistent AF CaM mRNA levels of atrial tissue did not change. CaM protein expression in IHC was brown, mainly in the atrial muscle cell membrane, looked like relatively strong staining rings; CaM protein also expressed cytoplasm in a small amount, but did not expressed in the nucleus. Distribution of CaM overlaped with the SK2 protein. Difference in CaM protein expression from atrium between the two groups did not reach statistical significance, and CaM intracellular distribution did not change after suffering from persistent AF.2.5 Ik,ca current changes in patients with persistent AF atrial myocytes Compared with the NSR group, action potential duration of atrial myocytes in AF group were was significantly shortened. Apamin sensitive K+ current or Ik,ca could be obtained by digital subtraction in atrial myocytes from patients with NSR and persistent AF. In general, compared with the NSR group, Ik,ca current density of atrial myocytes from in AF group upregrated , and when pulse voltage at -10 +40 mV, the difference of Ik,ca current density of atrial myocytes between the two groups were significative statistically. Membrane capacitance of atrial muscular cells from patients with persistent AF patients increased significantly.DiscussionA variety of research methods are used to study ion channels in general from gene, protein and function, but RT-PCR, IHC and patch clamp are the 3 most classical experimental methods. In Our study, RT-PCR, IHC and patch clamp, the three kinds of classical experimental methods, were used to study SK2. To investigate the distribution of SK2 in different parts of heart found around the heart, we found no differences of SK2 gene and protein expression existed between left and right atrium, and SK2 expression level of right atrial appendage could represent the level of overall atrium. Ventricular tissue was also found a compared KCNN2 gene expression level in atrial tissue, but SK2 protein expression in ventricular tissue was significantly reduced, this result was inconsistent to previous results of animal experiments, indicating that SK2 expression in human ventricular tissue may exist an unclear protranslation mechanism. Ik,ca current density of atrial myocytes from SD rat was found was significantly greater than ventricular myocytes, and the results suggest that specific blocker SK2 channel mainly affect electrophysiological properties of atrial muscle cells, so SK2 channels may be used as target of new ideal antiarrhythmic drugs for prevention and treatment of atrial arrhythmias in the future.SK2 changes after persistent AF in atrium from people suffering from RHD patients mainly involving mitral valve were to explore in various levels including gene, protein, function by classical experimental methods for the first time. Because paroxysmal AF is relatively rare when RHD patients accept MVR , so there were only patients with persistent AF compared with those with NSR. Compared with NSR group, Patients in AF group were suffering more from MS and displayed more obvious left atrial enlargement. characteristics of atrial remodeling in MS patients were obvious left atrial enlargement, providing more space for reentry and benefiting maintenance of AF. In addition to left atrial enlargement, HE staining and Masson staining of atrium specimens from patients also showed that more collagen tissue proliferated in atrium in AF group and cardiac muscle cells arranged in disorder. Interstitial fibrosis causing by collagen tissue proliferation is an important part of substrate leading to AF. Interstitial fibrosis can disrupt the order of myocardial cells, so that signal transmission between cells slow down, which is conducive to the maintenance of reentry.KCNN2 mRNA levels testing of right atrial appendage from patients with NSR and persistent AF were found KCNN2 expression in all atrium in the two groups, but unlike previous animal studies ,this research shown that KCNN2 mRNA in right atrial appendage from patients with persistent AF was at the lower level. In addition, right atrial myocytes from patients with either NSR or persistent AF showed a positive protein expression of SK2 protein, and SK2 protein expressed mainly on membrane of atrial myocytes. SK2 distribution is consistent in atrial myocytes of two groups of patients. Persistent AF patients had a low protein expression level of SK2 channel, which was not the same results with previous animal experiments.Right atrial appendage tissue of patients with NSR and persistent AF were also used to test CaM gene expression, but after the occurrence of persistent AF, CaM mRNA levels of right atrium did not change. Right atrial myocytes in NSR and AF group showed positive CaM protein expression, CaM protein distributionin the two groups had not difference. These results suggest that persistent AF do not affect on gene and protein expression level of CaM, and upregrated apamin sensitive K+ current density has nothing to do with CaM.Apamin sensitive current could be recorded by digital subtraction, and apamin sensitive current density of atrial myocytes in NSR group was greater. Current density may be affected by many factors and one important factor is gene expression level of the channel. Decreased KCNN2 gene expression in atrium of patients with persistent AF can result in reduced density of apamin sensitivity K+ current. However, intracellular Ca2+ overload significantly after AF and increased intracellular Ca2+ can upregrate SK2 channel functionally, so we can say increased Ca2+ in atrial mycardiocyte more impacted functional level of SK2 than downregulated SK2 gene and protein expression.ConclusionIn summary, SK2 gene and protein expression were the same in right and let atrium, so SK2 gene and protein expression of right atrium could reflect SK2 gene and protein expression of left atrium. SK2 gene expression in ventricule shown no difference with that in atrium, but SK2 protein expression and apamin sensitive K+ current in atrium increased significantly compared with ventricule. Different from previous animal experiments, after suffering from persistent AF, gene expression and protein expression of SK2αsubunit downregrated, gene expression and protein expression of CaM (auxiliary subunit of SK2) unchanged, and functional level of SK2 upregrated, suggesting that apamin sensitive current density were not only effected by SK2 subunits gene and protein expression level, but also by Ca2+ concentration in atrial myocytes. SK2 channel can be looked as an ideal target treatment for patients persistent AF, and it is promising for SK2 channel blockers as a new generation of antiarrhythmic drugs to treat atrial fibrillation.
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