C-JNK信号通路对糖尿病大鼠心肌Ito钾通道重构的影响

目的研究c-JNK信号通路在糖尿病(DM)大鼠左室心肌细胞电压门控钾通道(Kv)重构中的作用和内在调控机制。方法将50只健康SD大鼠随机分为DM组n=25,采用链尿佐菌素(STZ)诱导成模]和对照组(sham)(n=25)。应用全细胞膜片钳方法记录DM组与sham组大鼠心室肌瞬时外向钾电流(Ito);使用非放射性JNK激酶分析kit进行c-Jun活性测定。应用JNK抑制剂SP600125(10M)对DM大鼠心肌细胞进行体外孵育,观察孵育前后心肌细胞Ito的变化。用硫氧还蛋白还原酶(TrxR)抑制剂金诺芬(AF)对经JNK抑制剂SP600125孵育的大鼠心肌细胞进行处理,观察处理前后心肌细胞Ito的变化。应用抗Kv4.2抗体对Kv4.2的含量进行检测,检测结果使用UVP生物成像系统进行分析。结果DM组心肌JNK活性显著升高超过1倍,而Ito显著降低Sham组:(31.2±3.4)pA/pF,n=16;DM组:(15.4±3.1)pA/pF,n=17;P<0.05]。DM大鼠心室肌细胞经JNK抑制剂SP600125(10 M)处理4 h后,Ito电流密度可恢复至Sham组水平DM+SP600125组:(31.9±3.8)pA/pF,n=18;Sham组:(31.2±3.4)pA/pF,n=16;P<0.05];且sham组经SP600125处理后的最大Ito电流强度(29.8±3.4)pA/pF,n=9]和未经处理的sham组无统计学差异。DM心肌经膜渗透性蛋白抑制剂JNKI-1(10 M)处理后,Ito密度也有显著增加,而sham组经相同处理后无改变。TrxR抑制剂AF显著抑制了SP600125对DM大鼠心肌Ito电流的增大作用DM+AF+SP600125:(15.5±3.2)pA/pF,n=17],而AF对sham组Ito无明显影响。JNK抑制剂SP600125治疗后DM大鼠心肌的Kv4.2蛋白表达量显著增大,尽管未完全恢复到sham组心肌水平,但与先前在DM大鼠心肌所观察到的Ito电流改变一致。而JNK抑制并没有明显改变sham组心肌的Kv4.2蛋白表达量。结论DM大鼠心肌钾通道重构是氧化还原敏感的,可能通过持续性激活c-JNK信号通路促进Ito重构。在DM心肌中,JNK活性显著增高,Kv通道的电流密度降低;抑制JNK信号通路后可显著改善Kv通道重构,这一过程可能被硫氧还原蛋白系统所调控。

Influence of c-JNK signaling pathway on myocardial Ito potassium channel remodeling in rats with diabetes mellitus

Objective To study the effect and internal regulative mechanism of signaling pathway of c-Jun N-terminal kinase(c-JNK)in the remodeling of voltage-gated potassium channel(Kv)of left ventricular cardiomyocytes in rats with diabetes mellitus(DM).Methods Healthy SD rats(n=50)were randomly divided into DM group(n=25,modeled by using STZ)and sham group(n=25).The changes of myocardial transient outward potassium current(Ito)were recorded by using whole-cell patch-clamp technique,and c-Jun activity was determined by using non-radioactive JNK kinase assay kit.The cardiomyocytes from DM rats were incubated in vitro by using JNK inhibitor-SP600125(10 M)for observing the changes of cardiomyocyte Ito before and after the incubation.The cardiomyocytes from DM rats incubated with JNK inhibitor-SP600125 were treated with thioredoxin reductase(TrxR)inhibitors-auranofin(AF)for observing the changes of cardiomyocyte Ito before and after the treatment.The content of Kv4.2 was detected by using anti-Kv4.2 antibody,and the results were analyzed by applying UVP bioimaging system.Results The activity of myocardial JNK increased significantly in DM group,and Ito decreased significantly(31.2±3.4)pA/pF,n=16 in sham group and(15.4±3.1)pA/pF,n=17 in DM group,P<0.05].After the treatment with SP600125 for 4 h in DM group,Ito density was recovered to the level of sham group(31.9±3.8)pA/pF,n=18 in DM group and(31.2±3.4)pA/pF,n=16 in sham group,P<0.05].After the treatment with SP600125 in sham group,the maximum Ito intensity(29.8±3.4)pA/pF,n=9]had no statistical difference compared with that in sham group before treatment.After the treatment with JNKI-1(10 M),Ito density increased significantly in DM group,while there was no the same change in sham group.AF inhibited significantly Ito increase due to SP600125 in DM group(15.5±3.2)pA/pF,n=17],while AF had no significant influence in sham group.After the treatment with SP600125,the expression of Kv4.2 protein increased significantly in DM group but did not recover to the same level as that in sham group,which was similar to the change of Ito in DM group.The expression of Kv4.2 protein was not significantly influenced by AF in sham group.Conclusion The remodeling of myocardial Kv is redox sensitivity,which may be improved through activating continuously c-JNK signaling pathway.The activity of JNK increases significantly and Kv current density decreases in DM rats.The inhibitions of c-JNK signaling pathway can significantly improve Kv remodeling,and this process may be regulated by thioredoxin system.