Effect Of Huangyangning On L-type Calcium Channel In Rat Atrial Myocytes Pretreated With Hydrogen Peroxide

ObjectiveThe pathogenesis of atrial fibrillation is related to atrial electrical remodeling,L-type calcium channel is involved in the process of atrial electrical remodeling.Clinical studies have shown that Cyclovirobuxine D is effective in the treatment of atrial fibrillation.The aim of this study was to investigate the effects of Cyclovirobuxine D on L-type calcium current,L-type calcium channel electrophysiological characteristics and the expression of L-type calcium channelαsubunit in adult SD rat atrial myocytes pretreated with hydrogen peroxide.To explain that Cyclovirobuxine D may play a role in the treatment of atrial fibrillation by affecting the electrophysiological characteristics of L-type calcium channel.MethodsPreparation of atrial myocytes from SD rats:atrial myocytes of SD rats were isolated by acute enzymatic hydrolysis using Langendorff perfusion device system,retrograde perfusion of isolated heart,gradient recalcification,Calcium-tolerant cardiomyocytes suitable for electrophysiological recording were obtained so that the whole-cell patch clamp technique could be carried out normally.Experimental groups:the obtained atrial myocytes were randomly divided into six groups:Control group,H₂O₂group,H₂O₂+Amiodarone group,H₂O₂+Cyclovirobuxine D group,H₂O₂+Mito TEMPO group,H₂O₂+Cyclovirobuxine D+Mito-tempo group.Whole-cell patch clamp technique was used to record L-type calcium current(I_{Ca L}),L-type calcium channel activation curve,inactivation curve and recovery curve.The expression of L-type calcium channelαsubunit Cav1.2 was detected by immuneofluore-scence.Results1.Effects of Cyclovirobuxine D on L-type calcium current density and I-V curve of atrial myocytes in rats:compared with Control group,L-type calcium current density in H₂O₂ group was higher than that in Control group（p<0.05）,and compared with H₂O₂ group,L-type calcium current density in Amiodarone group,Cyclovirobuxine D group,Mito TEMPO group and Cyclovirobuxine D+Mito TEMPO group were decreased（p<0.05）,and compared with Amiodarone group,L-type calcium current density in Cyclovirobuxine D group,Mito TEMPO group and Cyclovirobuxine D+Mito TEMPO group all increased（p<0.05）,but there was no significant difference among Cyclovirobuxine D group,Mito TEMPO group and Cycloviro-buxine D+Mito TEMPO group（p>0.05）.2.Effect of Cyclovirobuxine D on I_Ca-L I-V curve of rat atrial myocytes.Compared with Control group,the I_Ca-L I-V curve of rat atrial myocytes was shifted downward in H₂O₂ group,upward in Amiodarone group,Cyclovirobuxine D group,Mito TEMPO group and Cycloviro-buxine D+Mito TEMPO group,and the I-V curve of Amiodarone group was shifted most obviously.3.Effect of Cyclovirobuxine D on steady-state activation,inactivation and recovery curve of I_Ca-L in rat atrial myocytes.Activation curve:compared with Control group,H₂O₂ group moved to the left;Compared with H₂O₂ group,Amiodarone group,Cyclovirobuxine D group,Mito TEMPO group,Cyclovirobuxine D+Mito TEMPO group were moved to the left.Inactivation curve:compared with Control group,H₂O₂ group and Cyclovirobuxine D group,Amiodarone group,Mito TEMPO group,Cyclovirobuxine D+Mito TEMPO group all moved to the left;Recovery curve:compared with Control group,H₂O₂ group and Cyclovirobuxine D group,Amiodarone group,Mito TEMPO group and Cyclovirobuxine D+Mito TEMPO group all moved to the right.4.Effect of Cyclovirobuxine D on the expression of L-type calcium channelαsubunitCav1.2.Compared with Control group,the expression of H₂O₂ group was increased,and compared with H₂O₂ group,the expression of Amiodarone group,Cyclovirobuxine D group,Mito TEMPO group and Cyclovirobuxine D+Mito TEMPO group was decreased,and the expression of Amiodarone group was the weakest.There was no significant difference among Cyclovirobuxine D group,Mito TEMPO group and Cyclovirobuxine D+Mito TEMPO group.Conclusion1.H2O2 can increase L-type calcium current;and Cyclovirobuxine D can inhibit L-type calcium current and may play a role in the treatment of atrial fibrillation by affecting the electrophysiological characteristics of L-type calcium channels.2.H₂O₂ may increase I_{Ca L} increasing the expression of I_Ca-LCav1.2 protein.Cycloviro-buxine D may reduce I_{Ca L} by reducing the expression of I_Ca-LCav1.2 protein.