Involvement Of Src And PKC? In The Down-regulation Of Ikur Induced By TNF-? In Cardiomyocytes

Atrial fibrillation?AF?is one of the most common arrhythmias,which can lead to thromboembolism and heart failure,causing disability and death.The pathophysiological changes related with AF include electrical remodeling,structural remodeling,autonomic remodeling and Ca²⁺handling abnormalities.Ultra-rapid delayed rectifier K⁺current?Ikur?is a specific current of cardiomyocytes,which participates in the repolarization of action potentials.Ikur can participate in the electrical remodeling of cardiomyocytes by affecting action potential duration?APD?,thereby playing an important role in the occurrence and development of AF.Inflammation is closely related to the occurrence of atrial fibrillation.Tumor necrosis factor-??TNF-??,as an important inflammatory factor,plays a role in the regulation of variety of ion currents,thereby affecting electrical remodeling of AF.Based on the above research background,we investigated the role of TNF-?in the regulation of Ikur during atrial fibrillation.Objective:To elucidate whether TNF-?is involved in the regulation of Ikur in atrial fibrillation and the possible molecular mechanism.Methods:1.Whole-cell patch-clamp was used to study the current density of Ikur in cardiomyocytes from left atrial appendages?LAAs?obtained from patients with sinus rhythm?SR?or AF;2.Western blot was used to detect the expression of Kv1.5,TNF-?,Src,PKC?Protein kinase C?and PKC?/p-PKC?protein in LAAs from patients with SR or AF;3.H9c2cells and HL-1 cells were treated with different concentration of TNF-?,TNF-?and Src inhibitor-PP1 or PCK?inhibitor G?6976.Whole-cell patch-clamp was used to study the current density of Ikur in cells from each group;4.H9c2 cells and HL-1 cells were treated with different concentration of TNF-?,TNF-?and Src inhibitor-PP1 or PCK?inhibitor G?6976.Western blot was used to detect the expression of Kv1.5,TNF-?,Src,p-Src,PKC,PKC?and p-PKC?protein in cells from each group.Results:1.Compared with SR controls,the current density of Ikur was significantly decreased in atrial myocytes isolated from LAAs obtained from patients with AF;2.Reduction of Kv1.5 protein levels was accompanied by increased expression of TNF-?in AF patients;meanwhile,expression of Src and PKC-?activation were both increased in LAAs from AF patients;3.TNF-?treatment in H9c2 cells and HL-1 cells can significantly inhibit the current density of Ikur in a concentration-dependent manner;4.In H9c2 cells and HL-1 cells,the expression of Kv1.5 rather than Kv3.1b protein was markedly reduced in a concentration-dependent manner after treating with TNF-?;meanwhile,expression of Src,p-Src,PKC,PKC?,and p-PKC?protein were significantly increased,which suggests that TNF-?can inhibit expression of Kv1.5 and activate Src kinase and PKC?in H9c2 cells and HL-1 cells;5.In H9c2cells and HL-1 cells,both of Src inhibitor PP1 and PCK?inhibitor G?6976 could reverse the down-regulation of Ikur induced by TNF-?;6.In H9c2 cells and HL-1 cells,PP1 could reverse the decrease of Kv1.5 protein expression induced by TNF-?,whereas G?6976 had no effect.Conclusions:In cardiomyocytes,TNF-?is involved in atrial electrical remodeling by depressing Ikur via activation of Src kinase and PKC-?,eventually leading to the occurrence and development of AF.