缝隙连接在长QT综合征室性心律失常发生中的作用及其机制研究
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摘要
长QT综合征(Long QT Syndrome, LQTS)是一种先天遗传性,或者后天获得的心脏复极延迟的疾病。长QT综合征表现为心电图上QT间期延长,它可以引起室性早搏及尖端扭转性室速(Torsade de Pointes, TdP)等致命性的心律失常,心源性晕厥和猝死为长QT综合征常见并发症。长QT综合征患者心电图上QT间期异常延长,临床上长QT综合征患者反复发生心源性晕厥、并常导致猝死的发生。
近年来许多研究表明,除了心脏复极时程的延长以外,跨室壁复极离散度(transmural dispersion of repolarization,TDR)的增加在长QT综合征早期后除极(early afterdepolarization, EAD)的产生,以及心律失常TdP的诱发和维持中也起着重要作用。长QT综合征中增大的TDR可使得EAD能够跨室壁传导,EAD跨室壁传导导致心脏的触发活动(比如R-on-T期前收缩)和致命性的心律失常TdP的产生。长QT综合征TDR的增加同时也可以通过二相折返的机制来维持TdP的发生。心脏各层心肌细胞有着显著的电生理特性和功能上的差异,近年来一些研究表明心室壁各层心肌细胞表面离子通道表达和功能的差异,使得跨室壁复极离散度异常增加。
心脏各层心肌细胞有着显著的电生理特性和功能上的差异导致了跨室壁复极离散度的形成,而心肌细胞间之间通过缝隙连接耦连。心肌细胞的缝隙连接位于闰盘处,动作电位通过缝隙连接从一个细胞扩布到相邻的细胞。跨膜离子及小分子物质可通过心肌细胞的缝隙连接直接交换,心肌细胞的缝隙连接在维持正常的心脏节律和细胞间的代谢物交换方面扮演着重要角色。由于整体心脏中心肌细胞间存在缝隙连接,小分子物质通过缝隙连接顺着电化学梯度在心肌细胞间传递,从而减少了心肌细胞间的电生理异质性。在整体心脏中,由于心肌细胞间良好的缝隙连接耦连,使得跨室壁复极的差异小于单个心肌细胞间动作电位时程的差异。近来有研究结果显示,在正常和心衰的心脏中,缝隙连接蛋白Cx43的表达、分布和磷酸化均影响了跨室壁复极的异质性。缝隙连接蛋白磷酸化状态的改变可影响缝隙连接通道的开放和代谢,造成缝隙连接的解耦连和功能下降。Carbenoxolone(缝隙连接解耦连剂)可增加复极离散度,而增加各层心肌细胞间缝隙连接的耦连,可减少跨室壁复极时程的差异。缝隙连接耦连增强剂抗心律失常肽AAP10和HP-5能够增加缝隙连接功能,进而减少动作电位时程的离散度。增加心肌细胞间缝隙连接的耦连,可以防止病理状态跨室壁复极离散度的异常增加。
心肌肥厚和心衰等多种心脏疾病的跨室壁复极离散度增加,同时其伴有心肌细胞间缝隙连接失耦连。这些研究结果表明改变心肌细胞间缝隙连接耦连,可能影响心脏各层心肌细胞间复极特性的差异的大小。因而我们推测增加心脏心肌细胞间缝隙连接的耦连,可以减少跨室壁复极离散度,进而预防长QT综合征室性心律失常的发生。本研究检测了缝隙连接激动剂抗心律失常肽(AAP10)对兔LQT3和犬LQT2模型跨室壁复极离散度和室性心律失常的影响并探讨其作用机制。
第一部分抗心律失常肽对兔LQT3模型缝隙连接耦连和室性心律失常的影响
目的：跨室壁复极离散度(transmural dispersion of repolarization, TDR)的增加可导致长QT综合征心律失常的发生。心肌细胞的缝隙连接也参与了跨室壁复极离散度的形成和维持。本实验研究了缝隙连接激动剂抗心律失常肽(AAP10),对兔LQT3模型缝隙连接磷酸化,以及室性心律失常的影响并探讨其作用机制。
方法：心肌细胞钠通道开放剂海葵毒素ATX-Ⅱ(20 nM, Calbiochem Corp., La Jolla,CA,USA)和兔左室楔型心肌块用于建立LQT3模型。抗心律失常肽AAP10(缝隙连接激动剂,Chinese Peptide Co., Hangzhou, Zhejiang, China)用于增加心肌细胞间缝隙连接耦连。采用浮置玻璃微电极法,同步记录心外膜、心内膜心肌细胞跨膜动作电位,同时记录跨室壁心电图。免疫印迹法(Western blot)检测心肌细胞缝隙连接蛋白43(Connexin43, Cx43)去磷酸化水平的变化。
基础步长1000 ms起搏1小时(电生理稳定),药物均溶解于台氏液,通过左室楔型心肌组织块冠状动脉插管灌流。左室楔型心肌组织块随机分为正常组、LQT3组、AAP-100nM和AAP-500nM干预组：正常组灌流正常台式液(n=10)、LQT3组灌流含20 nM ATX-Ⅱ的台式液(n=10)、AAP-100nM干预组灌流含20 nM ATX-Ⅱ和100 nMAAP10的台式液(n=10)、AAP-500nM干预组灌流含20 nM ATX-Ⅱ和500 nM AAP10的台式液(n=10)。
在基础状态下、AAP10预处理时和灌流ATX-Ⅱ的LQT3阶段,观察并记录左室楔型心肌组织块自发性心律失常,以及程序刺激诱发的左室楔型心肌组织块心律失常。QT间期为起搏信号到T波下降支与基线交叉点间的距离；动作电位复极90%时程(APD90)为动作电位0期到复极至90%振幅的时程；跨室壁复极离散度(TDR)为同一心搏心内膜和心外膜复极时间差值。
结果：与正常组相比较,LQT3组QT间期和跨室壁复极离散度(TDR)显著增大。LQT3组早期后除极(EAD),R-on-T期前收缩,尖端扭转性室速(TdP)的发生率也显著高于正常组,并伴有去磷酸化Cx43水平增高(均P<0.001)。正常组未发生早期后除极(EAD),R-on-T期前收缩,尖端扭转性室速(TdP)。与正常组相比较,LQT3组中10／10产生EAD,10／10产生R-on-T期前收缩(P<0.001)。LQT3组有5／10出现自发性TdP(P=0.033),9／10诱发出TdP(P<0.001)。与LQT3组相比较,AAP-500nM干预组的QT间期以及TDR显著缩短(均P<0.001)。AAP-500nM干预组的早期后除极(EAD),R-on-T期前收缩,尖端扭转性室速(TdP)发生率显著小于LQT3组(P=0.003,P=0.001,P=0.02),AAP-500nM干预组伴随Cx43去磷酸化水平降低(P<0.001)。
结论：缝隙连接激动剂AAP10防止Cx43去磷酸化,减少了兔LQT3模型的跨室壁复极离散度和心律失常发生率,说明兔LQT3模型的缝隙连接可能参与了跨室壁复极离散度的形成。
第二部分抗心律失常肽对犬LQT2模型缝隙连接重构和室性心律失常的影响
目的：跨室壁复极离散度(transmural dispersion of repolarization, TDR)的增加可导致长QT综合征心律失常的发生。心肌细胞的缝隙连接也参与了跨室壁复极离散度的形成和维持。本实验研究了缝隙连接激动剂抗心律失常肽(AAP10),对犬LQT2模型缝隙连接磷酸化,以及室性心律失常的影响并探讨其作用机制。
方法：应用心肌细胞IKr通道阻断剂d-sotalol (100μM, Sigma, St. Louis, MO, USA)在犬左室楔型心肌块建立LQT2模型。抗心律失常肽AAP10(缝隙连接激动剂,ChinesePeptide Co., Hangzhou, Zhejiang, China)用于增加心肌细胞间缝隙连接耦连。采用浮置玻璃微电极法同步记录心内膜、中层(M细胞)和心外膜心肌细胞跨膜动作电位及跨室壁心电图。免疫印迹法(Western blot)检测心肌细胞缝隙连接蛋白43 (Connexin43,Cx43)总的Cx43和去磷酸化Cx43水平的变化。应用荧光免疫组化技术检测上述各组心肌细胞缝隙连接蛋白Cx43表达、空间分布和磷酸化水平的改变。
基础步长1000 ms起搏1小时(电生理稳定),药物均溶解于台氏液,通过左室楔型心肌组织块冠状动脉插管灌流。随机分为正常组、LQT2组和AAP10干预组：正常组(n=10)灌流正常台式液、LQT2组(n=10)灌流含100μM d-sotalol的台式液、AAP10干预组(n=10)灌流含100μM d-sotalol和500 nM AAP10的台式液。
在基础状态、AAP10预处理阶段和灌流d-sotalol阶段,观察并记录左室楔型心肌组织块自发性心律失常,以及程序刺激诱发的左室楔型心肌组织块心律失常。QT间期为起搏信号到T波下降支与基线交叉点间的距离；动作电位复极90%时程(APD90)为动作电位0期到复极至90%振幅的时程；TDR为同一心搏M细胞和心外膜复极时间差值。
结果：正常组没有发生尖端扭转性室速(TdP)。与正常组相比较,LQT2组QT间期和跨室壁复极离散度(TDR)显著增大,LQT2组尖端扭转性室速(TdP)发生率也显著高于正常组。与正常组相比较,LQT2组有4／10出现自发性TdP(P=0.087),7／10诱发出TdP(P<0.05)。LQT2组同时伴有去磷酸化Cx43水平增高(均P<0.001)。与LQT2组相比较,AAP-500nM干预组QT间期和TDR显著缩短(均P<0.001)。AAP-500nM干预组TdP的发生率显著小于LQT2组(P<0.05),并伴随去磷酸化Cx43水平降低(P<0.001)。
结论：缝隙连接激动剂AAP10防止Cx43去磷酸化,减少了犬LQT2模型的跨室壁复极离散度和心律失常发生率,说明犬LQT2模型的缝隙连接可能参与了跨室壁复极离散度的形成。
Long QT syndrome (LQTS) is either an inherited or acquired disorder of delayed ventricular repolarization characterized by an excessively prolonged QT interval on the ECG. It often manifests clinically as recurrent syncope or sudden cardiac death as the consequence of polymorphic ventricular tachycardia known as torsades de pointes (TdP). LQTS is characterized by abnormally prolonged ventricular repolarization that predispose the affected individuals to arrhythmia, typically polymorphic ventricular tachycardia in the form of torsade de pointes (TdP) and an increased risk of sudden cardiac death.
In addition to prolonged repolarization time, an increasing number of studies have indicated that amplified transmural dispersion of repolarization (TDR) is essential for the development of TdP in both congenital and acquired LQTS. The increase in TDR facilitates transmural propagation of EAD, produces trigger beat (i.e., R-on-T extrasystole) capable of initiating TdP, and also contributes to the maintenance of TdP by serving as a functional reentrant substrate. In vitro studies have revealed that TDR is a result of significant heterogeneity in the expression of ion channels among different cell types across the ventricular wall. Important electrical and functional heterogeneity exists between the myocytes from different regions of the heart.
TDR is mainly attributed by significant heterogeneities of ion channel expression among different cell types, of which adjacent myocytes are coupled via gap junction. Gap junctions are composed of intercellular channels that allow the transfer of electrical current and small molecules between adjacent cells. The constituent proteins of gap junction channels, connexins, play a critical role in impulse propagation and electrical synchronization between myocytes. In an intact heart, such intrinsic electrophysiological heterogeneity diminishes due to the existence of gap junctions that permits movement of small molecules along the electrochemical gradient and thus helps in electrical synchronization of adjacent myocytes. In the intact heart where cells are well-coupled, transmural repolarization heterogeneity is significantly smaller than the intrinsic difference in action potential duration (APD) among these cells. Recent studies have found that expression and distribution of connexin43 (Cx43), the principle constitutional protein of gap junction in the ventricle, influence transmural electrical heterogeneities in both normal and failing hearts. Changes in phosphorylation of connexin can result in gap junction uncoupling by its effects on the gating of gap junction channels and connexin turnover dynamics. A gap junction uncoupler, carbenoxolone, enhances dispersion of repolarization, whereas high-degree coupling between myocardial layers diminishes or eradicates transmural dispersion in repolarization time and masks M cells. Agents such as antiarrhythmic peptide 10 (AAP10) and antiarrhythmic peptide HP-5, which facilitate the function of gap junctions, reduce dispersion of action potential duration (APD) and may have antiarrhythmic action. Also, augmentation of gap junction coupling can prevent abnormal amplification of TDR.
Increased TDR and uncoupling of the gap junctions are observed in many heart diseases such as ventricular hypertrophy and heart failure. All of these findings indicate that changes in intercellular coupling of gap junctions can modify the intrinsic difference in repolarization properties of cells spanning the ventricular wall. Therefore, we hypothesized that increasing gap junction coupling may be capable of reducing transmural heterogeneities of repolarization and hence, preventing ventricular arrhythmias under conditions of LQTS. To test this hypothesis, we investigated the effects of AAP10, a gap junction enhancer, on TDR and induction of TdP in a rabbit LQT3 and canine LQT2 model.
Objective Increased transmural dispersion of repolarization (TDR) contributes importantly to the development of torsades de pointes (TdP) in long QT syndrome (LQTS). Intercellular electrical coupling via gap junctions plays an important role in maintaining TDR in both normal and diseased hearts. This study examined the effects of antiarrhythmic peptide AAP10, a gap junction enhancer, on TDR and induction of TdP in a rabbit LQT3 model.
Methods An arterially perfused rabbit left ventricular preparation and sea anemone toxinⅡ(ATX-Ⅱ,20 nM) were used to establish a LQT3 model. Transmural ECG as well as action potentials from both endocardium and epicardium were simultaneously recorded. Changes in nonphosphorylated connexin43 (Cx43) were measured by immunoblotting.
The ventricular wedge preparations were allowed to equilibrate in the tissue bath for 1 hour prior to electrical recordings. Unless noted otherwise, all drugs used in this study were dissolved in Tyrode's solution and infused into the wedge preparation via the cannulated artery. ATX-II (20 nM, Calbiochem Corp., La Jolla, CA, USA) was used to augment the late INa and create a model of LQT3. AAP10 (Chinese Peptide Co., Hangzhou, Zhejiang, China), a gap junction enhancer, was used to enhance gap junction coupling. After baseline data acquisition, preparations were divided into four groups:
Control group (n=10):wedge preparations were kept perfusion with Tyrode's solution; LQT3 group (n=10):wedge preparations were perfused with ATX-II (20 nM) to mimic a LQT3 situation; AAP-100nM group (n=10) and AAP-500nM group (n=10):15 minutes prior to ATX-II administration, wedge preparations were pretreated with 100 nM and 500 nM AAP10, respectively.
The development of spontaneous and programmed electrical stimulation (PES)-induced TdP was assessed at baseline conditions, pretreatment of AAP10, and in the presence of ATX-II. PES-induced arrhythmias were evaluated by use of single extrastimulus (S2) applied to the endocardial surface of the wedge. The QT interval was defined as the time from the onset of the QRS to the point at which the final downslope of the T wave crossed the isoelectric line. APD was measured at 90% repolarization (APD90). TDR was defined as the difference between the endocardial and the epicardial epolarization times across the left ventricular wall.
Results Compared with the control group, the QT interval, TDR, early afterdepolariztion (EAD), R-on-T extrasystole, and TdP increased sharply with augmented nonphosphorylated Cx43 in the LQT3 group (P<0.001 for both). EAD and R-on-T extrasystole were induced by ATX-II (20 nM) in 10 of 10 preparations (P<0.001 vs. control) at a BCL of 2000 ms, among which five (P=0.033 vs. control) spontaneously degenerated into TdP in the LQT3 group. A single extrastimulus reproducibly induced TdP in nine of 10 preparations in the LQT3 group (P<0.001 vs. control). Interestingly, compared with the LQT3 group,500 nM AAP10 reduced QT interval, TDR (P<0.001 for both), and prevented EAD, R-on-T extrasystole, and TdP (P=0.003, P=0.001, P=0.02) with a parallel decrease in nonphosphorylated Cx43 in the presence of ATX-Ⅱ(P<0.001).
Conclusions Gap junction enhancer AAP10 is capable of abbreviating the QT interval, reducing TDR, and suppressing TdP in a rabbit LQT3 model probably via its effect by preventing dephosphorylation of Cx43. These data suggest that increasing intercellular coupling may reduce TDR and, therefore, prevent TdP in LQTS.
Objective Gap junctions contribute to the transmural heterogeneity of repolarization in the normal heart and under conditions of prolonged QT interval in the diseased heart. This study examined whether enhancing of gap junction coupling can reduce transmural dispersion of repolarization (TDR) and prevent torsade de pointes (TdP) in a canine LQT2 model.
Methods Canine left ventricular wedge preparations were perfused with delayed rectifier potassium current (IKr) blocker d-sotalol to mimic LQT2 and the antiarrhythmic peptide 10 (AAP10) was used as a gap junction coupling enhancer. Western blot and immunofluorescence were used for examining the expression and distribution of Cx43.
The ventricular wedge preparations were allowed to equilibrate in the tissue bath for 1 h prior to electrical recordings. Unless noted otherwise, all drugs used in this study were dissolved in Tyrode's solution and delivered to the wedge preparation via the cannulated artery. d-Sotalol (Sigma, St. Louis, MO, USA), a blocker of the rapidly activating delayed rectifier potassium current (IKr), was used to create the LQT2 model. Antiarrhythmic peptide 10 (AAP10), a peptide that enhances gap junction coupling, was obtained from Chinese Peptide (Hangzhou, Zhejiang, China).
Three groups of experiments were carried out. Control group (n=10):wedge preparations were kept perfusion with Tyrode's solution; LQT2 group (n=10):wedge preparations were perfused with d-sotalol (100μM) to mimic a LQT2 situation;
AAP10 group (n=10):wedge preparations were pretreated with 500 nM AAP10 for 15 min, then perfused with both d-sotalol (100μM) and AAP10 (500 nM). Development of spontaneous and programmed electrical stimulation (PES)-induced TdP was assessed at baseline conditions, after pretreatment of AAP10, and in the presence of d-sotalol. The QT interval was defined as the time from the onset of the QRS to the point atwhich the final downslope of the T wave crossed the isoelectric line. APD was measured at 90% repolarization (APD90). TDR was defined as the difference between the longest and shortest repolarization times of intracellular action potentials recorded across the ventricular wall.
Results As compared with the control group, the LQT2 group had significantly augmented TDR and higher incidence of TdP associated with increased nonphosphorylated connexin 43 (Cx43). AAP10 prevented augmentation of TDR and induction of TdP while rescuing Cx43 phosphorylation. There was no significant change in the quantity and spatial distribution of Cx43.
Pretreatment with AAP10 significantly attenuated effects of d-sotalol on APD90 in the M cells (P<0.001), but not in the endocardial or epicardial cells. As a net effect, the QT interval and TDR were significantly lower in the AAP 10 group as compared with the LQT2 group (P<0.001). AAP10 pretreatment completely suppressed spontaneous TdP (from 4/10 to 0/10; P=0.087) and significantly reduced PES-induced TdP episodes (7/10 in d-sotalol group versus 1/10 in AAP10+d-sotalol group; P=0.02). Pretreatment with AAP 10 did not alter QT interval, APD90, TDR, or QRS duration (P=NS). No arrhythmia was observed in the samples receiving AAP 10 alone.
Conclusions The present study showed that gap junction enhancer AAP 10 reduces TDR and incidence of TdP by preventing dephosphorylation of Cx43 in a canine ventricular model of LQT2. These results imply that dephosphorylation of Cx43 may be involved in the genesis of transmural heterogeneity of repolarization, and ultimately ventricular arrhythmias in LQT2.
近年来许多研究表明,除了心脏复极时程的延长以外,跨室壁复极离散度(transmural dispersion of repolarization,TDR)的增加在长QT综合征早期后除极(early afterdepolarization, EAD)的产生,以及心律失常TdP的诱发和维持中也起着重要作用。长QT综合征中增大的TDR可使得EAD能够跨室壁传导,EAD跨室壁传导导致心脏的触发活动(比如R-on-T期前收缩)和致命性的心律失常TdP的产生。长QT综合征TDR的增加同时也可以通过二相折返的机制来维持TdP的发生。心脏各层心肌细胞有着显著的电生理特性和功能上的差异,近年来一些研究表明心室壁各层心肌细胞表面离子通道表达和功能的差异,使得跨室壁复极离散度异常增加。
心脏各层心肌细胞有着显著的电生理特性和功能上的差异导致了跨室壁复极离散度的形成,而心肌细胞间之间通过缝隙连接耦连。心肌细胞的缝隙连接位于闰盘处,动作电位通过缝隙连接从一个细胞扩布到相邻的细胞。跨膜离子及小分子物质可通过心肌细胞的缝隙连接直接交换,心肌细胞的缝隙连接在维持正常的心脏节律和细胞间的代谢物交换方面扮演着重要角色。由于整体心脏中心肌细胞间存在缝隙连接,小分子物质通过缝隙连接顺着电化学梯度在心肌细胞间传递,从而减少了心肌细胞间的电生理异质性。在整体心脏中,由于心肌细胞间良好的缝隙连接耦连,使得跨室壁复极的差异小于单个心肌细胞间动作电位时程的差异。近来有研究结果显示,在正常和心衰的心脏中,缝隙连接蛋白Cx43的表达、分布和磷酸化均影响了跨室壁复极的异质性。缝隙连接蛋白磷酸化状态的改变可影响缝隙连接通道的开放和代谢,造成缝隙连接的解耦连和功能下降。Carbenoxolone(缝隙连接解耦连剂)可增加复极离散度,而增加各层心肌细胞间缝隙连接的耦连,可减少跨室壁复极时程的差异。缝隙连接耦连增强剂抗心律失常肽AAP10和HP-5能够增加缝隙连接功能,进而减少动作电位时程的离散度。增加心肌细胞间缝隙连接的耦连,可以防止病理状态跨室壁复极离散度的异常增加。
心肌肥厚和心衰等多种心脏疾病的跨室壁复极离散度增加,同时其伴有心肌细胞间缝隙连接失耦连。这些研究结果表明改变心肌细胞间缝隙连接耦连,可能影响心脏各层心肌细胞间复极特性的差异的大小。因而我们推测增加心脏心肌细胞间缝隙连接的耦连,可以减少跨室壁复极离散度,进而预防长QT综合征室性心律失常的发生。本研究检测了缝隙连接激动剂抗心律失常肽(AAP10)对兔LQT3和犬LQT2模型跨室壁复极离散度和室性心律失常的影响并探讨其作用机制。
第一部分抗心律失常肽对兔LQT3模型缝隙连接耦连和室性心律失常的影响
目的：跨室壁复极离散度(transmural dispersion of repolarization, TDR)的增加可导致长QT综合征心律失常的发生。心肌细胞的缝隙连接也参与了跨室壁复极离散度的形成和维持。本实验研究了缝隙连接激动剂抗心律失常肽(AAP10),对兔LQT3模型缝隙连接磷酸化,以及室性心律失常的影响并探讨其作用机制。
方法：心肌细胞钠通道开放剂海葵毒素ATX-Ⅱ(20 nM, Calbiochem Corp., La Jolla,CA,USA)和兔左室楔型心肌块用于建立LQT3模型。抗心律失常肽AAP10(缝隙连接激动剂,Chinese Peptide Co., Hangzhou, Zhejiang, China)用于增加心肌细胞间缝隙连接耦连。采用浮置玻璃微电极法,同步记录心外膜、心内膜心肌细胞跨膜动作电位,同时记录跨室壁心电图。免疫印迹法(Western blot)检测心肌细胞缝隙连接蛋白43(Connexin43, Cx43)去磷酸化水平的变化。
基础步长1000 ms起搏1小时(电生理稳定),药物均溶解于台氏液,通过左室楔型心肌组织块冠状动脉插管灌流。左室楔型心肌组织块随机分为正常组、LQT3组、AAP-100nM和AAP-500nM干预组：正常组灌流正常台式液(n=10)、LQT3组灌流含20 nM ATX-Ⅱ的台式液(n=10)、AAP-100nM干预组灌流含20 nM ATX-Ⅱ和100 nMAAP10的台式液(n=10)、AAP-500nM干预组灌流含20 nM ATX-Ⅱ和500 nM AAP10的台式液(n=10)。
在基础状态下、AAP10预处理时和灌流ATX-Ⅱ的LQT3阶段,观察并记录左室楔型心肌组织块自发性心律失常,以及程序刺激诱发的左室楔型心肌组织块心律失常。QT间期为起搏信号到T波下降支与基线交叉点间的距离；动作电位复极90%时程(APD90)为动作电位0期到复极至90%振幅的时程；跨室壁复极离散度(TDR)为同一心搏心内膜和心外膜复极时间差值。
结果：与正常组相比较,LQT3组QT间期和跨室壁复极离散度(TDR)显著增大。LQT3组早期后除极(EAD),R-on-T期前收缩,尖端扭转性室速(TdP)的发生率也显著高于正常组,并伴有去磷酸化Cx43水平增高(均P<0.001)。正常组未发生早期后除极(EAD),R-on-T期前收缩,尖端扭转性室速(TdP)。与正常组相比较,LQT3组中10／10产生EAD,10／10产生R-on-T期前收缩(P<0.001)。LQT3组有5／10出现自发性TdP(P=0.033),9／10诱发出TdP(P<0.001)。与LQT3组相比较,AAP-500nM干预组的QT间期以及TDR显著缩短(均P<0.001)。AAP-500nM干预组的早期后除极(EAD),R-on-T期前收缩,尖端扭转性室速(TdP)发生率显著小于LQT3组(P=0.003,P=0.001,P=0.02),AAP-500nM干预组伴随Cx43去磷酸化水平降低(P<0.001)。
结论：缝隙连接激动剂AAP10防止Cx43去磷酸化,减少了兔LQT3模型的跨室壁复极离散度和心律失常发生率,说明兔LQT3模型的缝隙连接可能参与了跨室壁复极离散度的形成。
第二部分抗心律失常肽对犬LQT2模型缝隙连接重构和室性心律失常的影响
目的：跨室壁复极离散度(transmural dispersion of repolarization, TDR)的增加可导致长QT综合征心律失常的发生。心肌细胞的缝隙连接也参与了跨室壁复极离散度的形成和维持。本实验研究了缝隙连接激动剂抗心律失常肽(AAP10),对犬LQT2模型缝隙连接磷酸化,以及室性心律失常的影响并探讨其作用机制。
方法：应用心肌细胞IKr通道阻断剂d-sotalol (100μM, Sigma, St. Louis, MO, USA)在犬左室楔型心肌块建立LQT2模型。抗心律失常肽AAP10(缝隙连接激动剂,ChinesePeptide Co., Hangzhou, Zhejiang, China)用于增加心肌细胞间缝隙连接耦连。采用浮置玻璃微电极法同步记录心内膜、中层(M细胞)和心外膜心肌细胞跨膜动作电位及跨室壁心电图。免疫印迹法(Western blot)检测心肌细胞缝隙连接蛋白43 (Connexin43,Cx43)总的Cx43和去磷酸化Cx43水平的变化。应用荧光免疫组化技术检测上述各组心肌细胞缝隙连接蛋白Cx43表达、空间分布和磷酸化水平的改变。
基础步长1000 ms起搏1小时(电生理稳定),药物均溶解于台氏液,通过左室楔型心肌组织块冠状动脉插管灌流。随机分为正常组、LQT2组和AAP10干预组：正常组(n=10)灌流正常台式液、LQT2组(n=10)灌流含100μM d-sotalol的台式液、AAP10干预组(n=10)灌流含100μM d-sotalol和500 nM AAP10的台式液。
在基础状态、AAP10预处理阶段和灌流d-sotalol阶段,观察并记录左室楔型心肌组织块自发性心律失常,以及程序刺激诱发的左室楔型心肌组织块心律失常。QT间期为起搏信号到T波下降支与基线交叉点间的距离；动作电位复极90%时程(APD90)为动作电位0期到复极至90%振幅的时程；TDR为同一心搏M细胞和心外膜复极时间差值。
结果：正常组没有发生尖端扭转性室速(TdP)。与正常组相比较,LQT2组QT间期和跨室壁复极离散度(TDR)显著增大,LQT2组尖端扭转性室速(TdP)发生率也显著高于正常组。与正常组相比较,LQT2组有4／10出现自发性TdP(P=0.087),7／10诱发出TdP(P<0.05)。LQT2组同时伴有去磷酸化Cx43水平增高(均P<0.001)。与LQT2组相比较,AAP-500nM干预组QT间期和TDR显著缩短(均P<0.001)。AAP-500nM干预组TdP的发生率显著小于LQT2组(P<0.05),并伴随去磷酸化Cx43水平降低(P<0.001)。
结论：缝隙连接激动剂AAP10防止Cx43去磷酸化,减少了犬LQT2模型的跨室壁复极离散度和心律失常发生率,说明犬LQT2模型的缝隙连接可能参与了跨室壁复极离散度的形成。
Long QT syndrome (LQTS) is either an inherited or acquired disorder of delayed ventricular repolarization characterized by an excessively prolonged QT interval on the ECG. It often manifests clinically as recurrent syncope or sudden cardiac death as the consequence of polymorphic ventricular tachycardia known as torsades de pointes (TdP). LQTS is characterized by abnormally prolonged ventricular repolarization that predispose the affected individuals to arrhythmia, typically polymorphic ventricular tachycardia in the form of torsade de pointes (TdP) and an increased risk of sudden cardiac death.
In addition to prolonged repolarization time, an increasing number of studies have indicated that amplified transmural dispersion of repolarization (TDR) is essential for the development of TdP in both congenital and acquired LQTS. The increase in TDR facilitates transmural propagation of EAD, produces trigger beat (i.e., R-on-T extrasystole) capable of initiating TdP, and also contributes to the maintenance of TdP by serving as a functional reentrant substrate. In vitro studies have revealed that TDR is a result of significant heterogeneity in the expression of ion channels among different cell types across the ventricular wall. Important electrical and functional heterogeneity exists between the myocytes from different regions of the heart.
TDR is mainly attributed by significant heterogeneities of ion channel expression among different cell types, of which adjacent myocytes are coupled via gap junction. Gap junctions are composed of intercellular channels that allow the transfer of electrical current and small molecules between adjacent cells. The constituent proteins of gap junction channels, connexins, play a critical role in impulse propagation and electrical synchronization between myocytes. In an intact heart, such intrinsic electrophysiological heterogeneity diminishes due to the existence of gap junctions that permits movement of small molecules along the electrochemical gradient and thus helps in electrical synchronization of adjacent myocytes. In the intact heart where cells are well-coupled, transmural repolarization heterogeneity is significantly smaller than the intrinsic difference in action potential duration (APD) among these cells. Recent studies have found that expression and distribution of connexin43 (Cx43), the principle constitutional protein of gap junction in the ventricle, influence transmural electrical heterogeneities in both normal and failing hearts. Changes in phosphorylation of connexin can result in gap junction uncoupling by its effects on the gating of gap junction channels and connexin turnover dynamics. A gap junction uncoupler, carbenoxolone, enhances dispersion of repolarization, whereas high-degree coupling between myocardial layers diminishes or eradicates transmural dispersion in repolarization time and masks M cells. Agents such as antiarrhythmic peptide 10 (AAP10) and antiarrhythmic peptide HP-5, which facilitate the function of gap junctions, reduce dispersion of action potential duration (APD) and may have antiarrhythmic action. Also, augmentation of gap junction coupling can prevent abnormal amplification of TDR.
Increased TDR and uncoupling of the gap junctions are observed in many heart diseases such as ventricular hypertrophy and heart failure. All of these findings indicate that changes in intercellular coupling of gap junctions can modify the intrinsic difference in repolarization properties of cells spanning the ventricular wall. Therefore, we hypothesized that increasing gap junction coupling may be capable of reducing transmural heterogeneities of repolarization and hence, preventing ventricular arrhythmias under conditions of LQTS. To test this hypothesis, we investigated the effects of AAP10, a gap junction enhancer, on TDR and induction of TdP in a rabbit LQT3 and canine LQT2 model.
Objective Increased transmural dispersion of repolarization (TDR) contributes importantly to the development of torsades de pointes (TdP) in long QT syndrome (LQTS). Intercellular electrical coupling via gap junctions plays an important role in maintaining TDR in both normal and diseased hearts. This study examined the effects of antiarrhythmic peptide AAP10, a gap junction enhancer, on TDR and induction of TdP in a rabbit LQT3 model.
Methods An arterially perfused rabbit left ventricular preparation and sea anemone toxinⅡ(ATX-Ⅱ,20 nM) were used to establish a LQT3 model. Transmural ECG as well as action potentials from both endocardium and epicardium were simultaneously recorded. Changes in nonphosphorylated connexin43 (Cx43) were measured by immunoblotting.
The ventricular wedge preparations were allowed to equilibrate in the tissue bath for 1 hour prior to electrical recordings. Unless noted otherwise, all drugs used in this study were dissolved in Tyrode's solution and infused into the wedge preparation via the cannulated artery. ATX-II (20 nM, Calbiochem Corp., La Jolla, CA, USA) was used to augment the late INa and create a model of LQT3. AAP10 (Chinese Peptide Co., Hangzhou, Zhejiang, China), a gap junction enhancer, was used to enhance gap junction coupling. After baseline data acquisition, preparations were divided into four groups:
Control group (n=10):wedge preparations were kept perfusion with Tyrode's solution; LQT3 group (n=10):wedge preparations were perfused with ATX-II (20 nM) to mimic a LQT3 situation; AAP-100nM group (n=10) and AAP-500nM group (n=10):15 minutes prior to ATX-II administration, wedge preparations were pretreated with 100 nM and 500 nM AAP10, respectively.
The development of spontaneous and programmed electrical stimulation (PES)-induced TdP was assessed at baseline conditions, pretreatment of AAP10, and in the presence of ATX-II. PES-induced arrhythmias were evaluated by use of single extrastimulus (S2) applied to the endocardial surface of the wedge. The QT interval was defined as the time from the onset of the QRS to the point at which the final downslope of the T wave crossed the isoelectric line. APD was measured at 90% repolarization (APD90). TDR was defined as the difference between the endocardial and the epicardial epolarization times across the left ventricular wall.
Results Compared with the control group, the QT interval, TDR, early afterdepolariztion (EAD), R-on-T extrasystole, and TdP increased sharply with augmented nonphosphorylated Cx43 in the LQT3 group (P<0.001 for both). EAD and R-on-T extrasystole were induced by ATX-II (20 nM) in 10 of 10 preparations (P<0.001 vs. control) at a BCL of 2000 ms, among which five (P=0.033 vs. control) spontaneously degenerated into TdP in the LQT3 group. A single extrastimulus reproducibly induced TdP in nine of 10 preparations in the LQT3 group (P<0.001 vs. control). Interestingly, compared with the LQT3 group,500 nM AAP10 reduced QT interval, TDR (P<0.001 for both), and prevented EAD, R-on-T extrasystole, and TdP (P=0.003, P=0.001, P=0.02) with a parallel decrease in nonphosphorylated Cx43 in the presence of ATX-Ⅱ(P<0.001).
Conclusions Gap junction enhancer AAP10 is capable of abbreviating the QT interval, reducing TDR, and suppressing TdP in a rabbit LQT3 model probably via its effect by preventing dephosphorylation of Cx43. These data suggest that increasing intercellular coupling may reduce TDR and, therefore, prevent TdP in LQTS.
Objective Gap junctions contribute to the transmural heterogeneity of repolarization in the normal heart and under conditions of prolonged QT interval in the diseased heart. This study examined whether enhancing of gap junction coupling can reduce transmural dispersion of repolarization (TDR) and prevent torsade de pointes (TdP) in a canine LQT2 model.
Methods Canine left ventricular wedge preparations were perfused with delayed rectifier potassium current (IKr) blocker d-sotalol to mimic LQT2 and the antiarrhythmic peptide 10 (AAP10) was used as a gap junction coupling enhancer. Western blot and immunofluorescence were used for examining the expression and distribution of Cx43.
The ventricular wedge preparations were allowed to equilibrate in the tissue bath for 1 h prior to electrical recordings. Unless noted otherwise, all drugs used in this study were dissolved in Tyrode's solution and delivered to the wedge preparation via the cannulated artery. d-Sotalol (Sigma, St. Louis, MO, USA), a blocker of the rapidly activating delayed rectifier potassium current (IKr), was used to create the LQT2 model. Antiarrhythmic peptide 10 (AAP10), a peptide that enhances gap junction coupling, was obtained from Chinese Peptide (Hangzhou, Zhejiang, China).
Three groups of experiments were carried out. Control group (n=10):wedge preparations were kept perfusion with Tyrode's solution; LQT2 group (n=10):wedge preparations were perfused with d-sotalol (100μM) to mimic a LQT2 situation;
AAP10 group (n=10):wedge preparations were pretreated with 500 nM AAP10 for 15 min, then perfused with both d-sotalol (100μM) and AAP10 (500 nM). Development of spontaneous and programmed electrical stimulation (PES)-induced TdP was assessed at baseline conditions, after pretreatment of AAP10, and in the presence of d-sotalol. The QT interval was defined as the time from the onset of the QRS to the point atwhich the final downslope of the T wave crossed the isoelectric line. APD was measured at 90% repolarization (APD90). TDR was defined as the difference between the longest and shortest repolarization times of intracellular action potentials recorded across the ventricular wall.
Results As compared with the control group, the LQT2 group had significantly augmented TDR and higher incidence of TdP associated with increased nonphosphorylated connexin 43 (Cx43). AAP10 prevented augmentation of TDR and induction of TdP while rescuing Cx43 phosphorylation. There was no significant change in the quantity and spatial distribution of Cx43.
Pretreatment with AAP10 significantly attenuated effects of d-sotalol on APD90 in the M cells (P<0.001), but not in the endocardial or epicardial cells. As a net effect, the QT interval and TDR were significantly lower in the AAP 10 group as compared with the LQT2 group (P<0.001). AAP10 pretreatment completely suppressed spontaneous TdP (from 4/10 to 0/10; P=0.087) and significantly reduced PES-induced TdP episodes (7/10 in d-sotalol group versus 1/10 in AAP10+d-sotalol group; P=0.02). Pretreatment with AAP 10 did not alter QT interval, APD90, TDR, or QRS duration (P=NS). No arrhythmia was observed in the samples receiving AAP 10 alone.
Conclusions The present study showed that gap junction enhancer AAP 10 reduces TDR and incidence of TdP by preventing dephosphorylation of Cx43 in a canine ventricular model of LQT2. These results imply that dephosphorylation of Cx43 may be involved in the genesis of transmural heterogeneity of repolarization, and ultimately ventricular arrhythmias in LQT2.
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19. Carlsson L, Drews L, Duker G. Rhythm anomalies related to delayed repolarization in vivo:influence of sarcolemmal Ca++ entry and intracellular Ca++ overload. The Journal of pharmacology and experimental therapeutics.1996;279(1):231-239.
20. Hirano Y, Moscucci A, January CT. Direct measurement of L-type Ca2+ window current in heart cells. Circ Res.1992;70(3):445-455.
21. Burt JM. Block of intercellular communication:interaction of intracellular H+ and Ca2+. The American journal of physiology.1987;253(4 Pt 1):C607-612.
22. White RL, Doeller JE, Verselis VK, Wittenberg BA. Gap junctional conductance between pairs of ventricular myocytes is modulated synergistically by H+ and Ca++. The Journal of general physiology.1990;95(6):1061-1075.
23. Noma A, Tsuboi N. Dependence of junctional conductance on proton, calcium and magnesium ions in cardiac paired cells of guinea-pig. The Journal of physiology. 1987;382:193-211.
24. Laird DW. Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation. Biochim Biophys Acta.2005; 1711 (2):172-182.
25. Solan JL, Lampe PD. Key connexin 43 phosphorylation events regulate the gap junction life cycle. JMembr Biol.2007;217(1-3):35-41.
26. Solan JL, Lampe PD. Connexin phosphorylation as a regulatory event linked to gap junction channel assembly. Biochim Biophys Acta.2005;1711(2):154-163.
27. Cooper CD, Solan JL, Dolejsi MK, Lampe PD. Analysis of connexin phosphorylation sites. Methods.2000;20(2):196-204.
28. Shimizu W, Antzelevitch C. Differential effects of beta-adrenergic agonists and antagonists in LQT1, LQT2 and LQT3 models of the long QT syndrome. J Am Coll Cardiol.2000;35(3):778-786.
29. Dhein S. Peptides acting at gap junctions. Peptides.2002;23(9):1701-1709.
30. Conrath CE, Wilders R, Coronel R, de Bakker JM, Taggart P, de Groot JR, Opthof T. Intercellular coupling through gap junctions masks M cells in the human heart. Cardiovasc Res.2004;62(2):407-414.
31. Roden DM. Drug-induced prolongation of the QT interval. The New England journal of medicine.2004;350(10):1013-1022.
32. Wit AL, Duffy HS. Drug Development for Treatment of Cardiac Arrhythmias. Targeting the Gap Junctions. American journal of physiology.2007.
33. Alexander DB, Goldberg GS. Transfer of biologically important molecules between cells through gap junction channels. Curr Med Chem.2003;10(19):2045-2058.
34. Muller A, Gottwald M, Tudyka T, Linke W, Klaus W, Dhein S. Increase in gap junction conductance by an antiarrhythmic peptide. Eur J Pharmacol. 1997;327(1):65-72.
35. Jozwiak J, Dhein S. Local effects and mechanisms of antiarrhythmic peptide AAP10 in acute regional myocardial ischemia:electrophysiological and molecular findings. Naunyn Schmiedebergs Arch Pharmacol.2008.
36. Dhein S, Weng S, Grover R, Tudyka T, Gottwald M, Schaefer T, Polontchouk L. Protein kinase Calpha mediates the effect of antiarrhythmic peptide on gap junction conductance. Cell communication & adhesion.2001;8(4-6):257-264.
37. Zhou Y, Yang W, Lurtz MM, Ye Y, Huang Y, Lee HW, Chen Y, Louis CF, Yang JJ. Identification of the calmodulin binding domain of connexin 43. J Biol Chem. 2007;282(48):35005-35017.
38. Lurtz MM, Louis CF. Intracellular calcium regulation of connexin43. Am J Physiol Cell Physiol.2007;293(6):C1806-1813.
1. Evans WH, Martin PE. Gap junctions:structure and function (Review). Mol Membr Biol.2002;19(2):121-136.
2. Mese G, Richard G, White TW. Gap junctions:basic structure and function. J Invest Dermatol.2007;127(11):2516-2524.
3. Salameh A. Life cycle of connexins:regulation of connexin synthesis and degradation. Adv Cardiol.2006;42:57-70.
4. Laird DW. Life cycle of connexins in health and disease. Biochem J.2006;394(Pt 3):527-543.
5. Teunissen BE, Bierhuizen MF. Transcriptional control of myocardial connexins. Cardiovasc Res.2004;62(2):246-255.
6. Saffitz JE, Laing JG, Yamada KA. Connexin expression and turnover:implications for cardiac excitability. Circ Res.2000;86(7):723-728.
7. Desplantez T, Dupont E, Severs NJ, Weingart R. Gap junction channels and cardiac impulse propagation. J Membr Biol.2007;218(1-3):13-28.
8. Jansen JA, van Veen TA, de Bakker JM, van Rijen HV. Cardiac connexins and impulse propagation. J Mol Cell Cardiol.48(1):76-82.
9. Maass K, Shibayama J, Chase SE, Willecke K, Delmar M. C-terminal truncation of connexin43 changes number, size, and localization of cardiac gap junction plaques. Circ Res.2007;101(12):1283-1291.
10. Chaldoupi SM, Loh P, Hauer RN, de Bakker JM, van Rijen HV. The role of connexin40 in atrial fibrillation. Cardiovasc Res.2009;84(1):15-23.
11. Severs NJ, Bruce AF, Dupont E, Rothery S. Remodelling of gap junctions and connexin expression in diseased myocardium. Cardiovasc Res.2008;80(1):9-19.
12. Singh B. Atrial fibrillation:from ion channels to bedside treatment options. J Electrocardiol.2009;42(6):660-670.
13. Burashnikov A, Antzelevitch C. New pharmacological strategies for the treatment of atrial fibrillation. Ann Noninvasive Electrocardiol.2009;14(3):290-300.
14. Naccarelli GV, Gonzalez MD. Atrial fibrillation and the expanding role of catheter ablation:do antiarrhythmic drugs have a future? J Cardiovasc Pharmacol. 2008;52(3):203-209.
15. Harris AL. Emerging issues of connexin channels:biophysics fills the gap. Q Rev Biophys.2001;34(3):325-472.
16. Peters NS, Coromilas J, Severs NJ, Wit AL. Disturbed connexin43 gap junction distribution correlates with the location of reentrant circuits in the epicardial border zone of healing canine infarcts that cause ventricular tachycardia. Circulation. 1997;95(4):988-996.
17. Zhou SH, He XZ, Liu QM, Du WH, Li XP, Zhou T, Tang JQ, Li RS. Study on the spatial distribution pattern of Cx40 gap junctions in the atria of patients with coronary heart disease. Cardiol J.2008; 15(1):50-56.
18. Sun Q, Tang M, Pu J, Zhang S. Pulmonary venous structural remodeling in a canine model of chronic atrial dilation due to mitral regurgitation. Can J Cardiol. 2008;24(4):305-308.
19. Boengler K, Dodoni G, Rodriguez-Sinovas A, Cabestrero A, Ruiz-Meana M, Gres P, Konietzka I, Lopez-Iglesias C, Garcia-Dorado D, Di Lisa F, Heusch G, Schulz R. Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning. Cardiovasc Res.2005;67(2):234-244.
20. Jain SK, Schuessler RB, Saffitz JE. Mechanisms of delayed electrical uncoupling induced by ischemic preconditioning. Circ Res.2003;92(10):1138-1144.
21. Beardslee MA, Lerner DL, Tadros PN, Laing JG, Beyer EC, Yamada KA, Kleber AG, Schuessler RB, Saffitz JE. Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia. Circ Res. 2000;87(8):656-662.
22. Rakotovao A, Tanguy S, Toufektsian MC, Berthonneche C, Ducros V, Tosaki A, de Leiris J, Boucher F. Selenium status as determinant of connexin-43 dephosphorylation in ex vivo ischemic/reperfused rat myocardium. J Trace Elem Med Biol.2005;19(1):43-47.
23. Akar FG, Spragg DD, Tunin RS, Kass DA, Tomaselli GF. Mechanisms underlying conduction slowing and arrhythmogenesis in nonischemic dilated cardiomyopathy. Circ Res.2004;95(7):717-725.
24. Chen HH, Baty CJ, Maeda T, Brooks S, Baker LC, Ueyama T, Gursoy E, Saba S, Salama G, London B, Stewart AF. Transcription enhancer factor-1-related factor-transgenic mice develop cardiac conduction defects associated with altered connexin phosphorylation. Circulation.2004;110(19):2980-2987.
25. Ai X, Pogwizd SM. Connexin 43 downregulation and dephosphorylation in nonischemic heart failure is associated with enhanced colocalized protein phosphatase type 2A. Circ Res.2005;96(1):54-63.
26. Simon AM, Goodenough DA, Li E, Paul DL. Female infertility in mice lacking connexin 37. Nature.1997;385(6616):525-529.
27. Simon AM, McWhorter AR. Vascular abnormalities in mice lacking the endothelial gap junction proteins connexin37 and connexin40. Dev Biol.2002;251(2):206-220.
28. Cai WJ, Kocsis E, Scholz D, Luo X, Schaper W, Schaper J. Presence of Cx37 and lack of desmin in smooth muscle cells are early markers for arteriogenesis. Mol Cell Biochem.2004;262(1-2):17-23.
29. Kwak BR, Mulhaupt F, Veillard N, Gros DB, Mach F. Altered pattern of vascular connexin expression in atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2002;22(2):225-230.
30. Kruger O, Beny JL, Chabaud F, Traub O, Theis M, Brix K, Kirchhoff S, Willecke K. Altered dye diffusion and upregulation of connexin37 in mouse aortic endothelium deficient in connexin40. J Vasc Res.2002;39(2):160-172.
31. Mather S, Dora KA, Sandow SL, Winter P, Garland CJ. Rapid endothelial cell-selective loading of connexin 40 antibody blocks endothelium-derived hyperpolarizing factor dilation in rat small mesenteric arteries. Circ Res. 2005;97(4):399-407.
32. Sandow SL, Tare M, Coleman HA, Hill CE, Parkington HC. Involvement of myoendothelial gap junctions in the actions of endothelium-derived hyperpolarizing factor. Circ Res.2002;90(10):1108-1113.
33. Sendao Oliveira AP, Bendhack LM. Relaxation induced by acetylcholine involves endothelium-derived hyperpolarizing factor in 2-kidney 1-clip hypertensive rat carotid arteries. Pharmacology.2004;72(4):231-239.
34. Sofola OA, Knill A, Hainsworth R, Drinkhill M. Change in endothelial function in mesenteric arteries of Sprague-Dawley rats fed a high salt diet. J Physiol. 2002;543(Pt 1):255-260.
35. Onaka U, Fujii K, Abe I, Fujishima M. Antihypertensive treatment improves endothelium-dependent hyperpolarization in the mesenteric artery of spontaneously hypertensive rats. Circulation.1998;98(2):175-182.
2. Chiang CE, Roden DM. The long QT syndromes:genetic basis and clinical implications. J Am Coll Cardiol. 2000;36(1):1-12.
3. Shimizu W, McMahon B, Antzelevitch C. Sodium pentobarbital reduces transmural dispersion of repolarization and prevents torsades de Pointes in models of acquired and congenital long QT syndrome. Journal of cardiovascular electrophysiology. 1999;10(2):154-164.
4. Shimizu W, Antzelevitch C. Effects of a K(+) channel opener to reduce transmural dispersion of repolarization and prevent torsade de pointes in LQT1, LQT2, and LQT3 models of the long-QT syndrome. Circulation.2000;102(6):706-712.
5. Viitasalo M, Oikarinen L, Swan H, Vaananen H, Glatter K, Laitinen PJ, Kontula K, Barron HV, Toivonen L, Scheinman MM. Ambulatory electrocardiographic evidence of transmural dispersion of repolarization in patients with long-QT syndrome type 1 and 2. Circulation.2002; 106(19):2473-2478.
6. Liu J, Laurita KR. The mechanism of pause-induced torsade de pointes in long QT syndrome. Journal of cardiovascular electrophysiology.2005; 16(9):981-987.
7. Kumar NM, Gilula NB. The gap junction communication channel. Cell. 1996;84(3):381-388.
8. Saffitz JE, Davis LM, Darrow BJ, Kanter HL, Laing JG, Beyer EC. The molecular basis of anisotropy:role of gap junctions. Journal of cardiovascular electrophysiology.1995;6(6):498-510.
9. Akar FG, Spragg DD, Tunin RS, Kass DA, Tomaselli GF. Mechanisms underlying conduction slowing and arrhythmogenesis in nonischemic dilated cardiomyopathy. Circ Res.2004;95(7):717-725.
10. Poelzing S, Akar FG, Baron E, Rosenbaum DS. Heterogeneous connexin43 expression produces electrophysiological heterogeneities across ventricular wall. American journal of physiology.2004;286(5):H2001-2009.
11. Poelzing S, Rosenbaum DS. Altered connexin43 expression produces arrhythmia substrate in heart failure. American journal of physiology.2004; 287(4): H1762-1770.
12. Yan GX, Rials SJ, Wu Y, Liu T, Xu X, Marinchak RA, Kowey PR. Ventricular hypertrophy amplifies transmural repolarization dispersion and induces early afterdepolarization. American journal of physiology.2001;281 (5):H1968-1975.
13. Yan GX, Wu Y, Liu T, Wang J, Marinchak RA, Kowey PR. Phase 2 early afterdepolarization as a trigger of polymorphic ventricular tachycardia in acquired long-QT syndrome:direct evidence from intracellular recordings in the intact left ventricular wall. Circulation.2001;103(23):2851-2856.
14. Lankipalli RS, Zhu T, Guo D, Yan GX. Mechanisms underlying arrhythmogenesis in long QT syndrome. Journal of electrocardiology.2005;38(4 Suppl):69-73.
15. Dhein S, Larsen BD, Petersen JS, Mohr FW. Effects of the new antiarrhythmic peptide ZP123 on epicardial activation and repolarization pattern. Cell communication & adhesion.2003;10(4-6):371-378.
16. Weng S, Lauven M, Schaefer T, Polontchouk L, Grover R, Dhein S. Pharmacological modification of gap junction coupling by an antiarrhythmic peptide via protein kinase C activation. Faseb J.2002;16(9):1114-1116.
17. Dhein S, Manicone N, Muller A, Gerwin R, Ziskoven U, Irankhahi A, Minke C, Klaus W. A new synthetic antiarrhythmic peptide reduces dispersion of epicardial activation recovery interval and diminishes alterations of epicardial activation patterns induced by regional ischemia. A mapping study. Naunyn Schmiedebergs Arch Pharmacol.1994;350(2):174-184.
18. Muller A, Schaefer T, Linke W, Tudyka T, Gottwald M, Klaus W, Dhein S. Actions of the antiarrhythmic peptide AAP10 on intercellular coupling. Naunyn Schmiedebergs Arch Pharmacol.1997;356(1):76-82.
19. Carlsson L, Drews L, Duker G. Rhythm anomalies related to delayed repolarization in vivo:influence of sarcolemmal Ca++ entry and intracellular Ca++ overload. The Journal of pharmacology and experimental therapeutics.1996;279(1):231-239.
20. Hirano Y, Moscucci A, January CT. Direct measurement of L-type Ca2+ window current in heart cells. Circ Res.1992;70(3):445-455.
21. Burt JM. Block of intercellular communication:interaction of intracellular H+ and Ca2+. The American journal of physiology.1987;253(4 Pt 1):C607-612.
22. White RL, Doeller JE, Verselis VK, Wittenberg BA. Gap junctional conductance between pairs of ventricular myocytes is modulated synergistically by H+ and Ca++. The Journal of general physiology.1990;95(6):1061-1075.
23. Noma A, Tsuboi N. Dependence of junctional conductance on proton, calcium and magnesium ions in cardiac paired cells of guinea-pig. The Journal of physiology. 1987;382:193-211.
24. Laird DW. Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation. Biochim Biophys Acta.2005; 1711 (2):172-182.
25. Solan JL, Lampe PD. Key connexin 43 phosphorylation events regulate the gap junction life cycle. JMembr Biol.2007;217(1-3):35-41.
26. Solan JL, Lampe PD. Connexin phosphorylation as a regulatory event linked to gap junction channel assembly. Biochim Biophys Acta.2005;1711(2):154-163.
27. Cooper CD, Solan JL, Dolejsi MK, Lampe PD. Analysis of connexin phosphorylation sites. Methods.2000;20(2):196-204.
28. Shimizu W, Antzelevitch C. Differential effects of beta-adrenergic agonists and antagonists in LQT1, LQT2 and LQT3 models of the long QT syndrome. J Am Coll Cardiol.2000;35(3):778-786.
29. Dhein S. Peptides acting at gap junctions. Peptides.2002;23(9):1701-1709.
30. Conrath CE, Wilders R, Coronel R, de Bakker JM, Taggart P, de Groot JR, Opthof T. Intercellular coupling through gap junctions masks M cells in the human heart. Cardiovasc Res.2004;62(2):407-414.
31. Roden DM. Drug-induced prolongation of the QT interval. The New England journal of medicine.2004;350(10):1013-1022.
32. Wit AL, Duffy HS. Drug Development for Treatment of Cardiac Arrhythmias. Targeting the Gap Junctions. American journal of physiology.2007.
33. Alexander DB, Goldberg GS. Transfer of biologically important molecules between cells through gap junction channels. Curr Med Chem.2003;10(19):2045-2058.
34. Muller A, Gottwald M, Tudyka T, Linke W, Klaus W, Dhein S. Increase in gap junction conductance by an antiarrhythmic peptide. Eur J Pharmacol. 1997;327(1):65-72.
35. Jozwiak J, Dhein S. Local effects and mechanisms of antiarrhythmic peptide AAP10 in acute regional myocardial ischemia:electrophysiological and molecular findings. Naunyn Schmiedebergs Arch Pharmacol.2008.
36. Dhein S, Weng S, Grover R, Tudyka T, Gottwald M, Schaefer T, Polontchouk L. Protein kinase Calpha mediates the effect of antiarrhythmic peptide on gap junction conductance. Cell communication & adhesion.2001;8(4-6):257-264.
37. Zhou Y, Yang W, Lurtz MM, Ye Y, Huang Y, Lee HW, Chen Y, Louis CF, Yang JJ. Identification of the calmodulin binding domain of connexin 43. J Biol Chem. 2007;282(48):35005-35017.
38. Lurtz MM, Louis CF. Intracellular calcium regulation of connexin43. Am J Physiol Cell Physiol.2007;293(6):C1806-1813.
1. Evans WH, Martin PE. Gap junctions:structure and function (Review). Mol Membr Biol.2002;19(2):121-136.
2. Mese G, Richard G, White TW. Gap junctions:basic structure and function. J Invest Dermatol.2007;127(11):2516-2524.
3. Salameh A. Life cycle of connexins:regulation of connexin synthesis and degradation. Adv Cardiol.2006;42:57-70.
4. Laird DW. Life cycle of connexins in health and disease. Biochem J.2006;394(Pt 3):527-543.
5. Teunissen BE, Bierhuizen MF. Transcriptional control of myocardial connexins. Cardiovasc Res.2004;62(2):246-255.
6. Saffitz JE, Laing JG, Yamada KA. Connexin expression and turnover:implications for cardiac excitability. Circ Res.2000;86(7):723-728.
7. Desplantez T, Dupont E, Severs NJ, Weingart R. Gap junction channels and cardiac impulse propagation. J Membr Biol.2007;218(1-3):13-28.
8. Jansen JA, van Veen TA, de Bakker JM, van Rijen HV. Cardiac connexins and impulse propagation. J Mol Cell Cardiol.48(1):76-82.
9. Maass K, Shibayama J, Chase SE, Willecke K, Delmar M. C-terminal truncation of connexin43 changes number, size, and localization of cardiac gap junction plaques. Circ Res.2007;101(12):1283-1291.
10. Chaldoupi SM, Loh P, Hauer RN, de Bakker JM, van Rijen HV. The role of connexin40 in atrial fibrillation. Cardiovasc Res.2009;84(1):15-23.
11. Severs NJ, Bruce AF, Dupont E, Rothery S. Remodelling of gap junctions and connexin expression in diseased myocardium. Cardiovasc Res.2008;80(1):9-19.
12. Singh B. Atrial fibrillation:from ion channels to bedside treatment options. J Electrocardiol.2009;42(6):660-670.
13. Burashnikov A, Antzelevitch C. New pharmacological strategies for the treatment of atrial fibrillation. Ann Noninvasive Electrocardiol.2009;14(3):290-300.
14. Naccarelli GV, Gonzalez MD. Atrial fibrillation and the expanding role of catheter ablation:do antiarrhythmic drugs have a future? J Cardiovasc Pharmacol. 2008;52(3):203-209.
15. Harris AL. Emerging issues of connexin channels:biophysics fills the gap. Q Rev Biophys.2001;34(3):325-472.
16. Peters NS, Coromilas J, Severs NJ, Wit AL. Disturbed connexin43 gap junction distribution correlates with the location of reentrant circuits in the epicardial border zone of healing canine infarcts that cause ventricular tachycardia. Circulation. 1997;95(4):988-996.
17. Zhou SH, He XZ, Liu QM, Du WH, Li XP, Zhou T, Tang JQ, Li RS. Study on the spatial distribution pattern of Cx40 gap junctions in the atria of patients with coronary heart disease. Cardiol J.2008; 15(1):50-56.
18. Sun Q, Tang M, Pu J, Zhang S. Pulmonary venous structural remodeling in a canine model of chronic atrial dilation due to mitral regurgitation. Can J Cardiol. 2008;24(4):305-308.
19. Boengler K, Dodoni G, Rodriguez-Sinovas A, Cabestrero A, Ruiz-Meana M, Gres P, Konietzka I, Lopez-Iglesias C, Garcia-Dorado D, Di Lisa F, Heusch G, Schulz R. Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning. Cardiovasc Res.2005;67(2):234-244.
20. Jain SK, Schuessler RB, Saffitz JE. Mechanisms of delayed electrical uncoupling induced by ischemic preconditioning. Circ Res.2003;92(10):1138-1144.
21. Beardslee MA, Lerner DL, Tadros PN, Laing JG, Beyer EC, Yamada KA, Kleber AG, Schuessler RB, Saffitz JE. Dephosphorylation and intracellular redistribution of ventricular connexin43 during electrical uncoupling induced by ischemia. Circ Res. 2000;87(8):656-662.
22. Rakotovao A, Tanguy S, Toufektsian MC, Berthonneche C, Ducros V, Tosaki A, de Leiris J, Boucher F. Selenium status as determinant of connexin-43 dephosphorylation in ex vivo ischemic/reperfused rat myocardium. J Trace Elem Med Biol.2005;19(1):43-47.
23. Akar FG, Spragg DD, Tunin RS, Kass DA, Tomaselli GF. Mechanisms underlying conduction slowing and arrhythmogenesis in nonischemic dilated cardiomyopathy. Circ Res.2004;95(7):717-725.
24. Chen HH, Baty CJ, Maeda T, Brooks S, Baker LC, Ueyama T, Gursoy E, Saba S, Salama G, London B, Stewart AF. Transcription enhancer factor-1-related factor-transgenic mice develop cardiac conduction defects associated with altered connexin phosphorylation. Circulation.2004;110(19):2980-2987.
25. Ai X, Pogwizd SM. Connexin 43 downregulation and dephosphorylation in nonischemic heart failure is associated with enhanced colocalized protein phosphatase type 2A. Circ Res.2005;96(1):54-63.
26. Simon AM, Goodenough DA, Li E, Paul DL. Female infertility in mice lacking connexin 37. Nature.1997;385(6616):525-529.
27. Simon AM, McWhorter AR. Vascular abnormalities in mice lacking the endothelial gap junction proteins connexin37 and connexin40. Dev Biol.2002;251(2):206-220.
28. Cai WJ, Kocsis E, Scholz D, Luo X, Schaper W, Schaper J. Presence of Cx37 and lack of desmin in smooth muscle cells are early markers for arteriogenesis. Mol Cell Biochem.2004;262(1-2):17-23.
29. Kwak BR, Mulhaupt F, Veillard N, Gros DB, Mach F. Altered pattern of vascular connexin expression in atherosclerotic plaques. Arterioscler Thromb Vasc Biol. 2002;22(2):225-230.
30. Kruger O, Beny JL, Chabaud F, Traub O, Theis M, Brix K, Kirchhoff S, Willecke K. Altered dye diffusion and upregulation of connexin37 in mouse aortic endothelium deficient in connexin40. J Vasc Res.2002;39(2):160-172.
31. Mather S, Dora KA, Sandow SL, Winter P, Garland CJ. Rapid endothelial cell-selective loading of connexin 40 antibody blocks endothelium-derived hyperpolarizing factor dilation in rat small mesenteric arteries. Circ Res. 2005;97(4):399-407.
32. Sandow SL, Tare M, Coleman HA, Hill CE, Parkington HC. Involvement of myoendothelial gap junctions in the actions of endothelium-derived hyperpolarizing factor. Circ Res.2002;90(10):1108-1113.
33. Sendao Oliveira AP, Bendhack LM. Relaxation induced by acetylcholine involves endothelium-derived hyperpolarizing factor in 2-kidney 1-clip hypertensive rat carotid arteries. Pharmacology.2004;72(4):231-239.
34. Sofola OA, Knill A, Hainsworth R, Drinkhill M. Change in endothelial function in mesenteric arteries of Sprague-Dawley rats fed a high salt diet. J Physiol. 2002;543(Pt 1):255-260.
35. Onaka U, Fujii K, Abe I, Fujishima M. Antihypertensive treatment improves endothelium-dependent hyperpolarization in the mesenteric artery of spontaneously hypertensive rats. Circulation.1998;98(2):175-182.