c-JNK信号通路对糖尿病大鼠心肌钾通道重构的氧化还原调控机制
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摘要
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)重构中的作用及机制。方法将45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(n=20,采用普通饮食饲养)。应用全细胞膜片钳方法记录DM组与对照组大鼠心室肌瞬时外向钾电流(I_(to))密度;使用非放射性JNK激酶分析元件进行c-Jun活性测定。应用JNK抑制剂SP600125(10μmol/L)对DM大鼠心肌细胞进行体外孵育,观察孵育前后心肌细胞I_(to)密度的变化。采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I_(to)密度的变化。使用抗Kv4.2抗体对Kv4.2的含量进行检测,检测结果采用UVP生物成像系统进行分析。结果与对照组比较,DM组心肌JNK活性明显升高超过1倍,而I_(to)密度(对照组:30.2±3.3pA/pF,n=16;DM组:15.3±2.1pA/pF,n=17)则明显降低(P<0.05)。DM大鼠心室肌细胞经JNK抑制剂SP600125处理4h后,I_(to)密度可恢复至对照组水平(DM+SP600125组:32.3±3.7pA/pF,n=18;对照组:30.2±3.3pA/pF,n=16;P<0.05);且对照组经SP600125处理后的最大I_(to)密度(对照+SP600125组:31.6±3.4pA/pF,n=18)和未经处理的对照组比较差异无统计学意义。DM心肌经膜渗透性蛋白抑制剂JNKI-1(10μmol/L)处理后,I_(to)密度也明显增加,而对照组经相同处理后无改变。TrxR抑制剂金诺芬明显抑制了SP600125对DM大鼠心肌I_(to)的增大作用(DM+SP600125+AF:15.7±3.3pA/pF,n=15),而对对照组I_(to)无明显影响。JNK抑制剂SP600125处理后DM大鼠心肌的Kv4.2蛋白表达量明显增加,尽管未完全恢复到对照组心肌水平,但与先前在DM大鼠心肌中观察到的I_(to)改变一致。而JNK抑制并未明显改变对照组心肌的Kv4.2蛋白表达量。结论 DM大鼠心肌钾通道重构对氧化还原敏感,可能通过持续性激活c-JNK信号通路促进I_(to)重构。在DM心肌中,JNK活性明显增高,Kv通道的电流密度降低;抑制JNK信号通路后可明显改善Kv通道重构,这一过程可能被硫氧还原蛋白系统所调控。
Objective To investigate the role of c-Jun NH_2-terminal kinase(c-JNK) signaling pathway on voltage-gated potassium channel(Kv) remodeling in left ventricular myocytes of diabetic rats, and explore the intrinsic regulatory mechanism. Methods Forty-five SD rats were randomly divided into DM group(n=25, modeling with streptozotocin induction) and control group(n=20, fed with normal diet). Transient outward potassium current(I_(to)) of rats' ventricular myocytes in DM group and control group was recorded by whole-cell patch-clamp method. The c-Jun activity was detected using a non-radioactive JNK kinase assay kit(Cell Signaling Technology). JNK inhibitor SP600125 was used to incubate the cardiomyocytes of diabetes rats in vitro, and then the changes of I_(to) in cardiomyocytes were observed. Thioredoxin reductase(TrxR) inhibitor--auranofin(AF) was used to treat the rats' cardiomyocytes incubated with SP600125, and then the changes of I_(to) in cardiomyocytes were observed. The content of Kv4.2 was tested using anti-Kv4.2 antibody, and the results were analyzed using a UVP bioimaging system. Results The JNK activity in DM group rose more than 1 times compared with control group, while the density of I_(to) decreased significantly(Control: 30.2±3.3 pA/pF, n=16; DM: 15.3±2.1 pA/pF, n=17; P<0.05). The ventricular myocytes of DM rats were treated with SP600125(10μmol/Lol/L) for 4 hours, then the I_(to) density increased to control group level(DM+SP600125: 32.3±3.7 pA/pF, n=18; Control: 30.2±3.3 pA/pF, n=16; P<0.05). There was no significant difference in the maximum I_(to) density between the treated with SP600125(Control+SP600125: 31.6±3.4 pA/pF, n=18) and untreated control groups. The I_(to) density in DM myocardial cells significantly increased after treatment with the membrane permeable protein inhibitor JNKI-1(10μmol/L), and no changes were found in control group after the same treatment. The augmentation effect of SP600125 on I_(to) current in DM myocytes was significantly inhibited by Trx R inhibitor auranofin(1μmol/L)(DM+SP600125+AF: 15.7±3.3 pA/pF, n=15), while AF did not change the I_(to)density in control group. The expression of Kv4.2 protein was significantly increased in DM rats after administration of SP600125, which was consistent with the changes of I_(to) current observed in the myocardium of DM rats, although not fully restored to the level of control group myocardium. JNK inhibitor did not markedly alter the expression of Kv4.2 protein in control group myocardium. Conclusions Kv channel remodeling in DM rat's myocardium is redox-regulated, and the I_(to) remodeling might be assisted with the persistent activation of c-JNK signaling pathway. It has showed that c-JNK activity is significantly increased in DM rat heart and the current density of Kv channels is reduced. The inhibition of JNK signaling pathway can markedly improve Kv channel reconstruction and the process may be regulated by thioredoxin system.
Objective To investigate the role of c-Jun NH_2-terminal kinase(c-JNK) signaling pathway on voltage-gated potassium channel(Kv) remodeling in left ventricular myocytes of diabetic rats, and explore the intrinsic regulatory mechanism. Methods Forty-five SD rats were randomly divided into DM group(n=25, modeling with streptozotocin induction) and control group(n=20, fed with normal diet). Transient outward potassium current(I_(to)) of rats' ventricular myocytes in DM group and control group was recorded by whole-cell patch-clamp method. The c-Jun activity was detected using a non-radioactive JNK kinase assay kit(Cell Signaling Technology). JNK inhibitor SP600125 was used to incubate the cardiomyocytes of diabetes rats in vitro, and then the changes of I_(to) in cardiomyocytes were observed. Thioredoxin reductase(TrxR) inhibitor--auranofin(AF) was used to treat the rats' cardiomyocytes incubated with SP600125, and then the changes of I_(to) in cardiomyocytes were observed. The content of Kv4.2 was tested using anti-Kv4.2 antibody, and the results were analyzed using a UVP bioimaging system. Results The JNK activity in DM group rose more than 1 times compared with control group, while the density of I_(to) decreased significantly(Control: 30.2±3.3 pA/pF, n=16; DM: 15.3±2.1 pA/pF, n=17; P<0.05). The ventricular myocytes of DM rats were treated with SP600125(10μmol/Lol/L) for 4 hours, then the I_(to) density increased to control group level(DM+SP600125: 32.3±3.7 pA/pF, n=18; Control: 30.2±3.3 pA/pF, n=16; P<0.05). There was no significant difference in the maximum I_(to) density between the treated with SP600125(Control+SP600125: 31.6±3.4 pA/pF, n=18) and untreated control groups. The I_(to) density in DM myocardial cells significantly increased after treatment with the membrane permeable protein inhibitor JNKI-1(10μmol/L), and no changes were found in control group after the same treatment. The augmentation effect of SP600125 on I_(to) current in DM myocytes was significantly inhibited by Trx R inhibitor auranofin(1μmol/L)(DM+SP600125+AF: 15.7±3.3 pA/pF, n=15), while AF did not change the I_(to)density in control group. The expression of Kv4.2 protein was significantly increased in DM rats after administration of SP600125, which was consistent with the changes of I_(to) current observed in the myocardium of DM rats, although not fully restored to the level of control group myocardium. JNK inhibitor did not markedly alter the expression of Kv4.2 protein in control group myocardium. Conclusions Kv channel remodeling in DM rat's myocardium is redox-regulated, and the I_(to) remodeling might be assisted with the persistent activation of c-JNK signaling pathway. It has showed that c-JNK activity is significantly increased in DM rat heart and the current density of Kv channels is reduced. The inhibition of JNK signaling pathway can markedly improve Kv channel reconstruction and the process may be regulated by thioredoxin system.
引文
[1]Li XY,Liu G,Guo JH.Research progress of changes of potassium channels in diabetic myocardium[J].Chin J Cardiac Pacing Electrophysiol,2012,26(3):261-264.[李学永,刘刚,郭继鸿.糖尿病心肌钾离子通道改变研究进展[J].中国心脏起搏与心电生理杂志,2012,26(3):261-264.]
[2]Veit F,Pak O,Brandes RP.Hypoxia-dependent reactive oxygen species signaling in the pulmonary circulation:focus on ion channels[J].Antioxid Redox Signal,2015,22(6):537-552.
[3]Zheng MQ,Li XY,Liu G,et al.Role of thioredoxin system to remodeling in diabetes rat hearts[J].J Clin Cardiol,2013,29(8):573-576.[郑明奇,李学永,刘刚,等.硫氧还蛋白系统对糖尿病大鼠心肌Ito电流的影响[J].临床心血管病杂志,2013,29(8):573-576.]
[4]Li X,Tang K,Xie B,et al.Regulation of Kv4 channel expression in failing rat heart by the thioredoxin system[J].Am J Physiol Heart Circ Physiol,2008,295(1):H416-H424.
[5]Nicholson CK,Lambert JP,Molkentin JD,et al.Thioredoxin 1is essential for sodium sulfide-mediated cardioprotection in the setting of heart failure[J].Arterioscler Thromb Vasc Biol,2013,33(4):744-751.
[6]Zschauer TC,Matsushima S,Altschmied J,et al.Interacting with thioredoxin-1-disease or no disease?[J].Antioxid Redox Signal,2013,18(9):1053-1062.
[7]Liu YN,Xu QL,Guo XH,et al.Effects of MAPKs signaling on heat stress-induced apoptosis of pulmonary microvascular endothelial cells and its mechanism[J].Med J Chin PLA,2017,42(4):279-284.[刘亚楠,徐秋林,郭晓华,等.MAPKs家族在中暑小鼠肺微血管内皮细胞凋亡中的作用及机制研究[J].解放军医学杂志,2017,42(4):279-284.]
[8]Karczewski KJ,Snyder M,Altman RB,et al.Coherent functional modules improve transcription factor target identification,cooperativity prediction,and disease association[J].PLoSGenet,2014,10(2):e1004122.
[9]Ouedraogo R,Wu X,Xu SQ,et al.Adiponectin suppression of high-glucose-induced reactive oxygen species in vascular endothelial cells evidence for involvement of a cAMP signaling pathway[J].Diabetes,2006,55(6):1840-1846.
[10]Adams V,Linke A,Krakel N,et al.Impact of regular physical activity on the NAD(P)H oxidase and angiotensin receptor system in patients with coronary artery disease[J].Circulation,2005,111(5):555-562.
[11]Li S,Zheng MQ,Rozanski GJ.Glutathione homeostasis in ventricular myocytes from rat hearts with chronic myocardial infarction[J].Exp Physiol,2009,94(7):815-824.
[12]Zheng MQ,Tang K,Zimmerman MC,et al.Role of gammaglutamyl transpeptidase in redox regulation of K+channel remodeling in postmyocardial infarction rat hearts[J].Am JPhysiol Cell Physiol,2009,297(2):C253-C262.
[13]Goolsby TV,Lombardo FA.Extravasation of chemotherapeutic agents:prevention and treatment[J].Semin Oncol,2006,33(1):139-143.
[14]Prakasam A,Ghose S,Oleinik NV,et al.JNK1/2 regulate Bid by direct phosphorylation at Thr59 in response to ALDH1L1[J].Cell Death Dis,2014,5:e1358.
[15]Chen XM,Liu XL,Wu W.Relationship between c-junN terminal kinase 1 and early injury in the myocardium of diabetic rats[J].Chin Gen Pract,2016,19(27):3281-3285.[陈昕明,刘晓亮,吴伟.c-junN端蛋白激酶1磷酸化与糖尿病大鼠早期心肌损伤关系研究[J].中国全科医学,2016,19(27):3281-3285.]
[16]Liang H,Li X,Li S,et al.Oxidoreductase regulation of Kv currents in rat ventricle[J].J Mol Cell Cardiol,2008,44(6):1062-1071.
[17]Kaprielian R,Wickenden AD,Kassiri Z,et al.Relationship between K+channel down-regulation and Ca2+in rat ventricular myocytes following myocardial infarction[J].J Physiol,1999,517(1):229-245.
[18]Nishida K,Otsu K.The role of apoptosis signal-regulating kinase 1in cardiomyocyte apoptosis[J].Antioxid Redox Signal,2006,8(9-10):1729-1736.
[19]Izumiya Y,Kim S,Izumi Y,et al.Apoptosis signal-regulating kinase 1 plays a pivotal role in angiotensinⅡ-induced cardiac hypertrophy and remodeling[J].Circ Res,2003,93(9):874-883.
[20]Liu Y,Min W.Thioredoxin promotes ASK1 ubiquitination and degradation to inhibit ASK1-mediated apoptosis in a redox activityindependent manner[J].Circ Res,2002,90(12):1259-1266.
[2]Veit F,Pak O,Brandes RP.Hypoxia-dependent reactive oxygen species signaling in the pulmonary circulation:focus on ion channels[J].Antioxid Redox Signal,2015,22(6):537-552.
[3]Zheng MQ,Li XY,Liu G,et al.Role of thioredoxin system to remodeling in diabetes rat hearts[J].J Clin Cardiol,2013,29(8):573-576.[郑明奇,李学永,刘刚,等.硫氧还蛋白系统对糖尿病大鼠心肌Ito电流的影响[J].临床心血管病杂志,2013,29(8):573-576.]
[4]Li X,Tang K,Xie B,et al.Regulation of Kv4 channel expression in failing rat heart by the thioredoxin system[J].Am J Physiol Heart Circ Physiol,2008,295(1):H416-H424.
[5]Nicholson CK,Lambert JP,Molkentin JD,et al.Thioredoxin 1is essential for sodium sulfide-mediated cardioprotection in the setting of heart failure[J].Arterioscler Thromb Vasc Biol,2013,33(4):744-751.
[6]Zschauer TC,Matsushima S,Altschmied J,et al.Interacting with thioredoxin-1-disease or no disease?[J].Antioxid Redox Signal,2013,18(9):1053-1062.
[7]Liu YN,Xu QL,Guo XH,et al.Effects of MAPKs signaling on heat stress-induced apoptosis of pulmonary microvascular endothelial cells and its mechanism[J].Med J Chin PLA,2017,42(4):279-284.[刘亚楠,徐秋林,郭晓华,等.MAPKs家族在中暑小鼠肺微血管内皮细胞凋亡中的作用及机制研究[J].解放军医学杂志,2017,42(4):279-284.]
[8]Karczewski KJ,Snyder M,Altman RB,et al.Coherent functional modules improve transcription factor target identification,cooperativity prediction,and disease association[J].PLoSGenet,2014,10(2):e1004122.
[9]Ouedraogo R,Wu X,Xu SQ,et al.Adiponectin suppression of high-glucose-induced reactive oxygen species in vascular endothelial cells evidence for involvement of a cAMP signaling pathway[J].Diabetes,2006,55(6):1840-1846.
[10]Adams V,Linke A,Krakel N,et al.Impact of regular physical activity on the NAD(P)H oxidase and angiotensin receptor system in patients with coronary artery disease[J].Circulation,2005,111(5):555-562.
[11]Li S,Zheng MQ,Rozanski GJ.Glutathione homeostasis in ventricular myocytes from rat hearts with chronic myocardial infarction[J].Exp Physiol,2009,94(7):815-824.
[12]Zheng MQ,Tang K,Zimmerman MC,et al.Role of gammaglutamyl transpeptidase in redox regulation of K+channel remodeling in postmyocardial infarction rat hearts[J].Am JPhysiol Cell Physiol,2009,297(2):C253-C262.
[13]Goolsby TV,Lombardo FA.Extravasation of chemotherapeutic agents:prevention and treatment[J].Semin Oncol,2006,33(1):139-143.
[14]Prakasam A,Ghose S,Oleinik NV,et al.JNK1/2 regulate Bid by direct phosphorylation at Thr59 in response to ALDH1L1[J].Cell Death Dis,2014,5:e1358.
[15]Chen XM,Liu XL,Wu W.Relationship between c-junN terminal kinase 1 and early injury in the myocardium of diabetic rats[J].Chin Gen Pract,2016,19(27):3281-3285.[陈昕明,刘晓亮,吴伟.c-junN端蛋白激酶1磷酸化与糖尿病大鼠早期心肌损伤关系研究[J].中国全科医学,2016,19(27):3281-3285.]
[16]Liang H,Li X,Li S,et al.Oxidoreductase regulation of Kv currents in rat ventricle[J].J Mol Cell Cardiol,2008,44(6):1062-1071.
[17]Kaprielian R,Wickenden AD,Kassiri Z,et al.Relationship between K+channel down-regulation and Ca2+in rat ventricular myocytes following myocardial infarction[J].J Physiol,1999,517(1):229-245.
[18]Nishida K,Otsu K.The role of apoptosis signal-regulating kinase 1in cardiomyocyte apoptosis[J].Antioxid Redox Signal,2006,8(9-10):1729-1736.
[19]Izumiya Y,Kim S,Izumi Y,et al.Apoptosis signal-regulating kinase 1 plays a pivotal role in angiotensinⅡ-induced cardiac hypertrophy and remodeling[J].Circ Res,2003,93(9):874-883.
[20]Liu Y,Min W.Thioredoxin promotes ASK1 ubiquitination and degradation to inhibit ASK1-mediated apoptosis in a redox activityindependent manner[J].Circ Res,2002,90(12):1259-1266.