Investigation Of Connexin 43 In The Canine Atrial Myocytes Model By Isoproterenol And Rapid Electric Pacing

Objective This subject uses RNA interference technology to silence canine connexin 43(Cx43)gene and establishes Cx43 low expression canine atrial myocytes model.Establishing sympathetic nerve atrial myocytes(AF)cell model by isoproterenol and rapid electric pacing canine atrial myocytes,to investigate the role of Cx43 low expression on canine atrial myocytes gap junction channel conduction.Methods 1.Transfection and determination of the Cx43 low expression canine atrial myocytes model.The canine atrial myocytes were cultured and divided into three groups:Cx43 normal expression(NE)Group,without any intervention;negative control(NC)group,transfected for 48 hours with miRNA-NC;Cx43 low expression(LE)Group,transfected for 48 hours with Cx43siRNA.Realtime fluorescence quantitative PCR(RT-PCR)was used to detect the expression level of Cx43 mRNA and western blotting(WB)was used to detect the expression level of Cx43 protein in atrial myocytes.2.Investigation of connexin 43 in the canine atrial myocytes model by isoproterenol and rapid electric pacing.①The canine atrial myocytes were cultured and divided into four groups:control group,without any intervention;isoproterenol group,perfused for 30 minutes with isoprenaline;high frequency electric stimulation group,high frequency stimulated for 24 hours under the electric field;isoprenaline and high frequency electric stimulation group,perfused for 30 minutes with isoprenaline and high frequency stimulated for 24 hours under the electric field.RT-PCR was used to detect the expression level of Cx43 mRNA and WB was used to detect the expression level of Cx43 protein in atrial myocytes.② The canine atrial myocytes were cultured and divided into eight groups:negative control(NC)group,transfected for 48 hours with miRNA-NC;negative control isoproterenol group,transfected for 48 hours with miRNA-NC,perfused for 30 minutes with isoprenaline;negative control high frequency electric stimulation group,transfected for 48 hours with miRNA-NC,high frequency stimulated for 24 hours under the electric field;negative control isoprenaline and high frequency electric stimulation group,transfected for 48 hours with miRNA-NC,perfused for 30 minutes with isoprenaline and high frequency stimulated for 24 hours under the electric field;Cx43 low expression(LE)Group,transfected for 48 hours with Cx43siRNA;Cx43 low expression isoproterenol group,transfected for 48 hours with Cx43siRNA,perfused for 30 minutes with isoprenaline;Cx43 low expression high frequency electric stimulation group,transfected for 48 hours with Cx43siRNA,high frequency stimulated for 24 hours under the electric field;Cx43 low expression isoprenaline and high frequency electric stimulation group,transfected for 48 hours with Cx43siRNA,perfused for 30 minutes with isoprenaline and high frequency stimulated for 24 hours under the electric field.RT-PCR was used to detect the expression level of Cx43 mRNA,immunofluorescence(IF)was used to detect the expression level of Cx43 protein in atrial myocytes and scrape-loading and dye transfer(SLDT)was used to detect the cell communication functions of gap junction channels.Results 1.The expression level of Cx43 mRNA and Cx43 protein in atrial myocytes were decreased significantly in LE group than in NE group and in NC group(P<0.05).2.①No significant chance was found in the expression level of Cx43 mRNA and Cx43 protein in atrial myocytes between isoprenaline group and control group(P>0.05).The expression level of Cx43 mRNA and Cx43 protein in atrial myocytes were decreased significantly in high frequency electric stimulation group and isoproterenol joint high frequency electric stimulation group than in control group(P<0.05).② The expression level of Cx43 mRNA and Cx43 protein in atrial myocytes were decreased significantly in LE groups than in NC groups(P<0.05).LY fluorescence transmission distance in the vicinity of atrial myocytes scratch were shorter significantly in LE groups than in NC groups(P<0.05).Conclusion 1.Isoproterenol transient stimulation can not inhibit the expression level of Cx43,but high frequency electric stimulation and isoproterenol joint high frequency electric stimulation can inhibit the expression level of Cx43 in canine atrial myocytes.2.LV-Cx43siRNA can inhibit significantly the expression level of Cx43 in canine atrial myocytes and establish Cx43 low expression canine atrial myocytes model.3.Reduce the number of Cx43 in the atrial myocytes can lead to the signaling dysfunction of intercellular gap junction channel,it may play an important role in occurrence and maintenance of sympathetic nerve AF.