Effects Of Huangyangning On Transient Outward Potassium Channels And Inward Rectifier Potassium Channels In Rat Atrial Myocytes Pretreated With Hydrogen Peroxide

Objective:Atrial fibrillation（AF）is one of the most common arrhythmias in clinic,which seriously affects the quality of life of patients,increases the risk of heart failure and stroke,and its pathogenesis is complex and diverse.Studies have shown that the increased level of oxidative stress in patients with atrial fibrillation accelerates cardiomyocyte apoptosis and fibrosis,and promotes structural remodeling;clinical observation of Huangyangning in the treatment of arrhythmia has significant effect and little side effects,but whether it can inhibit the occurrence and development of atrial fibrillation by changing electrical remodeling is not clear.The aim of this study was to explore the mechanism of Huangyangning in treating atrial fibrillation by influencing inward rectifier potassium current and instantaneous outward potassium current.Method:1.Isolation of cardiomyocytes:Atrial myocytes were isolated from clean male SD rats（330-360g）by Langendorff device.The atrial muscle cells were injured by adding hydrogen peroxide（100 micromol）into the myocardial culture medium,and then randomly divided into three groups.Huangyangning,amiodarone,Mitotempo（antioxidant）and Huangyangning+Mitotempo were added to observe K⁺channel under whole-cell patch clamp.Change.2.Experimental grouping:The experiment was divided into six groups:Control group,H₂O₂ group,H₂O₂+Huangyangning group,H₂O₂+amiodarone group,H₂O₂+Mitotempo group,H₂O₂+Huangyangning+Mitotempo group.3.Whole-cell patch clamp method was used to measure the effects of hydrogen peroxide on IK1 and Ito channels in atrial myocytes.4.The changes of IK1 and Ito channels in atrial myocytes were observed by injecting Huangyangning,amiodarone,Mitotempo and Huangyangning+Mitotempo after H₂O₂cell injury.5.Immunofluorescence method was used to observe the expression of cell membrane-associated proteins（kir2.1,Kv4.3）in each group.Result:1.The results of inward rectifier potassium current I-V curve showed that there was no significant change in the effect of H₂O₂ on IK1 current density compared with the blank group（p>0.05）,H₂O₂+Huangyangning group,H₂O₂+amiodarone group,H₂O₂+Mitotempo group,H₂O₂+Mitotempo+The current density of the Huangyangning group decreased（p<0.05）.Compared with the H₂O₂ group,the current density decreased in the H₂O₂+Huangyangning group,H₂O₂+amiodarone group,H₂O₂+Mitotempo group,H₂O₂+Mitotempo+Huangyangning group.（p<0.05）;Compared with H₂O₂+Huangyangning group,the current density of H₂O₂+amiodarone group,H₂O₂+Mitotempo group,H₂O₂+Mitotempo+Huangyangning group did not change much（p>0.05）.2.The results of transient outward potassium current I-V curve showed that the current current density of H₂O₂ group was significantly reduced compared with the blank group（p<0.05）,while H₂O₂+Huangyangning group,H₂O₂+amiodarone group,H₂O₂+Mitotempo group,H₂O₂+Mitotempo+The current density of the Huangyangning group increased（p<0.05）.Compared with the H₂O₂ group,the current density increased in the H₂O₂+Huangyanging group,H₂O₂+amiodarone group,H₂O₂+Mitotempo group,H₂O₂+Mitotempo+Huangyangning group.（p<0.05）;Compared with H₂O₂+Huangyangning group,the current density of H₂O₂+amiodarone group,H₂O₂+Mitotempo group,H₂O₂+Mitotempo+Huangyangning group did not change much（p>0.05）.3.Ito steady-state activation curve results show that there is no significant change between the groups,using the Boltzmann equation to fit the semi-activated voltage（V1/2）and the slope factor k;compared with the blank group,the V1/2 value of the H₂O₂ group There was no significant difference（p>0.05）.There was no significant difference in H₂O₂+Huangyangninggroup,H₂O₂+amiodaronegroup,H₂O₂+Mitotempo+Huangyangning group（p>0.05）,but H₂O₂+Mitotempo group the V1/2value increased（p<0.05）;compared with the H₂O₂ group,the V1/2 value of the H₂O₂+Huangyangning group decreased（p<0.05）;compared with the H₂O₂+Huangyangning group,the V1/2 of the H₂O₂+Mitotempo group was increased（p<0.05）.Compared with the blank group,the k value of H₂O₂ group and H₂O₂+amiodarone group increased（p<0.05）,but there was no significant change in H₂O₂+Huangyanging group,H₂O₂+Mitotempo group,H₂O₂+Mitotempo+Huangyangning group（p>0.05）;compared with H₂O₂ group,there was no significant difference in K value between H₂O₂+Huangyangning group,H₂O₂+amiodarone group,H₂O₂+Mitotempo group,H₂O₂+Mitotempo+Huangyangning group（p>0.05）.Compared with the H₂O₂+Huangyangning group,there was no significant difference in K values between the other groups（p>0.05）.In addition,there was no significant change in the steady-state inactivation curve of Ito.There was no significant difference in V1/2 and k value between the other groups compared with the blank group（p>0.05）.4.The recovery curve of Ito after inactivation showed that the time constant tau value of the recovery after inactivation was obtained by exponential equation fitting.Compared with the blank group,the recovery time constant of H₂O₂+Huangyangning group and H₂O₂+Mitotempo group was significantly shorter（p<0.05）.;There was no significant difference in H₂O₂ group,H₂O₂+amiodarone group,H₂O₂+Mitotempo+Huangyangning group（p>0.05）;Compared with H₂O₂ group,there was no significant difference in recovery time constant between all other groups（p>0.05）;Compared with the H₂O₂+Huangyangning group,there was no significant difference in the recovery time constants of all other groups（p>0.05）.5.Immunofluorescence staining,co-staining of I_K1 channelαsubunit Kir2.1 andα-SMA.After laser confocal observation,it was found that there was no significant change in green fluorescence intensity on the cell membrane in H₂O₂ group compared with the blank group;Compared with H₂O₂ group,the green fluorescence intensity of cell membrane in H₂O₂+amiodarone group,H₂O₂+Huangyangning group,H₂O₂+Mitotempo,H₂O₂+Mitotempo+huangyangning group was weakened to different degrees,among which H₂O₂+Huangyangning group>H₂O₂+amiodarone group>H₂O₂In the+Mitotempo group>H₂O₂+Mitotempo+Huangyangning group,the fluorescence expression on the membrane of H₂O₂+Mitotempo+Huangyangning group was the weakest.Co-staining of the Ito channelαsubunit Kv4.3 andα-SMA was observed.After laser confocal observation,it was found that the red fluorescence intensity of the cell membrane was weakened in the H₂O₂ group compared with the blank group,while the H₂O₂+boxwood was compared with the H₂O₂ group.The red fluorescence intensity on the cell membrane of the Ning group and the H₂O₂+Mitotempo group was similar,while the red fluorescence intensity on the cell membrane of the H₂O₂+amiodarone group was slightly lower than that of the H₂O₂+Huangyangning group and the H₂O₂+Mitotempo group,and the H₂O₂+Mitotempo+Huangyangning group was red on the cell membrane.The increase in fluorescence intensity is most pronounced.Conclusion:1.H₂O₂ had no effect on IK1 but decreased Ito significantly.Huangyangning inhibited IK1 and increased Ito,which may play a role in the treatment of atrial fibrillation by affecting the electrophysiological characteristics of Ito.2.H₂O₂ had no significant effect on the expression of Kir2.1,but decreased the expression of Kv4.3.Huangyangning increased Ito by increasing the expression of Kv4.3.