心房颤动与细胞缝隙连接关系及其治疗新进展

心房颤动（AF）是临床上最常见的快速性房性心律失常之一,有极高的致残率和致死率。AF的发生和维持的机制是困扰心血管医生的难题。缝隙连接是存在于相邻细胞间的一种由特殊膜蛋白通道组成的,在相邻细胞间中构成一个通道,它对多细胞生命体的协同行为非常重要。心脏组织的缝隙连接与心律失常有着密切的关系。近年研究发现,肺静脉肌袖及心房组织细胞缝隙连接蛋白的变化与AF的发生及维持机制密切相关。本文主要综述心房组织的细胞缝隙连接与AF的关系及相关治疗的新进展。     

缝隙连接、连接蛋白43及其与心律失常的关系

缝隙连接(GJ)通道是介导心肌细胞间电化学信息交流,保证心脏整体活动的协调性和同步性的特殊通道。在致心律失常发生上,GJ通道介导的细胞间电耦联障碍甚至比膜离子通道功能紊乱起了更重要的作用。连接蛋白43(Cx43)是心室GJ通道的主要构成成份,其表达和分布的异常将导致心室肌细胞的整体性异常,从而传导速度和传导各向异性发生改变,产生折返和传导阻滞。以GJ通道为作用靶点的新一代抗心律失常药的出现将为心律失常的治疗带来新的希望。     

异型缝隙连接通道和磷酸化对心脏缝隙连接的调变

目的 检测由缝隙连接蛋白(connexin，Cx)43和Cx45组成的多种异型缝隙连接通道(her—eromultimeric gap junction channels，HGJC)和磷酸化对缝隙连接(gap junction，GJ)的调变作用。方法 将转染了编码为Cx43或Cx45的DNA后的Hela细胞放置在一起共同培养组成双侧和单侧异型GJ通道。显微注射若丹明123(rhodamine123，Rh)检测经200nmol／L十四(烷)酰佛波醇乙酸酯(12-0-tetrade—canoylphorbol-13-acetae，TPA)处理前后，在紫外光显示下由Cx43和Cx45所组成的不同GJ通道对荧光染料的偶联率(coupling ratio)。结果 在不同的GJ中，同型GJ通道Cx43(homotypie Cx43，HoCx43)偶联率最高。从Cx45侧注入荧光染料的单侧异型GJ通道45(mono-heteromeric Cx45-Cx43／45，MH45)偶联率较之从Cx43／45侧注入荧光染料的MH45、双侧异型GJ通道Cx43／45(bi-heteromeric Cx43／45，BH43／45)及同型GJ通道Cx45(homotypic Cx45，HoCx45)等的偶联率是最低的。根据HoCx43或HoCx45通道的偶联率对各型通道偶联率进行标准化处理。BH43／45和MH43通道的偶联率均较HoCx43降低。对MH45通道来说，从Cx43／45侧注射的通道偶联率大于从Cx45侧注射的偶联率。TPA处理后HoCx43的偶联率降低，而当Cx43和Cx45组合成BH43／45和MH43通道后其偶联率下降更显著。结论 Cx43和Cx45共同表达可构成BH43／45、MH43和MH45等异型通道，而这些通道可降低细胞间的通讯并对磷酸化的作用不敏感。单侧异型GJ通道的偶联率取决于染料注射的方向。     

细胞间隙连接通讯及其通路蛋白与大肠癌发生关系的研究进展

细胞间隙连接通讯(gap junctional intercellular communication,GJIC)是多细胞生物体内普遍存在的一种通讯方式,他参与离子和其他小分子信号物质的转运.GJIC对细胞的生长、增殖和分化起重要的调控作用,他的改变与肿瘤的发生密切相关.大肠癌是人类常见的恶性肿瘤之一,目前研究表明,大肠癌的发生是一个多步骤、多基因、多阶段的过程,包括癌基因激活、抑癌基因失活、DNA转录表达失控、DNA损伤等.不论何种原因的细胞转化,其最终表现为细胞周期失控、细胞无限增殖.本文就GJIC及其通路蛋白、细胞因子与大肠癌的发生的研究进展作一综述.     

病毒性心肌炎连接蛋白的变化

目的观察小鼠急性病毒性心肌炎时连接蛋白的变化，了解连接蛋白与病毒性心肌炎的关系。方法Balb／c4周龄雄性小鼠26只，随机分为模型组（20只）和正常对照组（6只），模型组予腹腔接种柯萨奇病毒B组3型（CVB3）建立小鼠急性病毒性心肌炎模型。在接种病毒后第14天处死所有存活小鼠，观察心肌形态学改变，并采用聚合酶链式反应（PCR）及免疫组织化学技术，检测各组小鼠心肌组织中connexin43（Cx43）、connexin40（Cx40）和connexin45（Cx45）基因水平及蛋白水平的变化。结果模型组小鼠心肌HE染色可见明显的炎性细胞浸润和心肌坏死等炎症改变；与正常对照组相比，Cx43和Cx45mRNA表达明显减少（分别为0．11±0．02vs0．34±0．05，0．21±0．02vs0．41±0．06）。Cx40mRNA表达较对照组无统计学差异，三者蛋白水平的变化与基因水平变化相一致。结论急性病毒性心肌炎小鼠心肌中connexin43和connexin45表达明显减少，上述连接蛋白的变化与病毒性心肌炎致心律失常的潜在作用有关。     

Age-related changes in gap junctional protein of the rat heart

OBJECTIVE:

Connexin 43 (Cx43), a membrane protein involved in the control of cell-to-cell communication, is thought to play a role in physiological processes such as tissue homeostasis, growth regulation and development. The aim of the present study was to investigate the change of Cx43 expression in aged myocardium.

METHODS AND RESULTS:

Sixteen male Sprague-Dawley rats (adult: 10 weeks old, n=8; aged: two years old, n=8) were used in the present study. In an isolated rat heart Langendorff model, hearts were perfused for 10 min with a modified Krebs-Henseleit bicarbonate buffer. Contractile functions were measured and all hearts were stained with anti-Cx43 antibody for fluorescence microscopic examinations. There were no significant differences observed in heart rate (234±8.2 beats/min versus 231±15.6 beats/min), left ventricular developed pressure (112.5±6.3 mmHg versus 107.2±2.5 mmHg), first derivative of the left ventricular pressure (1450.4±165.1 mmHg/s versus 1384.6±95.4 mmHg/s) and coronary flow (17.4±0.7 mL/min versus 21.3±1.8 mL/min) between adult and aged rats, respectively. However, significant differences were observed in left ventricular weight (adult versus aged; 0.639±0.108 g versus 1.124±0.257 g, P=0.04) and in fluorescence examinations where there was reduced distribution of Cx43 in aged myocardium compared with adult myocardium.

CONCLUSIONS:

These results demonstrated that the role of Cx43 may be more important than previously reported, and that this protein is partially responsible for the maintenance of cellular structure in myocardial development.     

Remodeling of gap junctions in mouse hearts hypertrophied by forced retinoic acid signaling

BACKGROUND: Beta-MHC-hRARalpha transgenic mice express a constitutively active (truncated) form of the human retinoic acid receptor which triggers development of dilated cardiomyopathy. In those hearts, we studied expression of gap junction proteins in relation to electrical impulse propagation. METHODS AND RESULTS: As compared to wildtype mice, hearts of 4-6 month old mice with 7-12 inserted hRARalpha copies are marked by an increased heart weight/body weight- and heart weight/tibia length ratio. 3-extremity lead ECGs revealed prolongation of the Q-j interval suggesting delayed ventricular activation. Mapping of electrical activity of epi- and endocardial left ventricular free wall revealed activation delay, increased heterogeneity in conduction and regional conduction block. Ventricular tachycardias did not occur spontaneously nor could be induced by ventricular pacing. Immunohistochemical analysis showed profound and heterogeneous redistribution and down-regulation of the gap junction protein connexin43 (Cx43) in the left ventricular free wall. Here, hRARalpha expression induced re-expression of the hypertrophic markers alpha-skeletal actin and beta-MHC, and in 3 out of 10 severely affected mice, re-expression of Cx40. Concomitant with changes in expression/distribution of Cx43, changes in expression and distribution of beta-catenin and N-cadherin (two other intercalated disk associated proteins) were observed. CONCLUSIONS: Beta-MHC-hRARalpha transgenic hearts show heterogeneous re-expression of (early) sarcomeric genes while expression of connexin43, N-cadherin and beta-catenin is down-regulated. We postulate that the resulting aberrant ventricular activation does not trigger development of lethal arrhythmias due to the small size of remaining healthy ventricular tissue where the transgene is not expressed.     

增加缝隙连接耦联对兔长QT综合征心律失常的影响

目的:观察增加缝隙连接耦联对兔长QT综合征(LQTS)模型室性心律失常的影响。方法:应用心肌细胞快速延迟整流钾通道(IKr)阻滞剂E-4031(0.5μmol/L)在兔左室楔型心肌块建立LQT2模型,随机分为正常组、LQT2组、100 nmol/L缝隙连接激动剂(AAP-10)干预组(AAP-100组)、500 nmol/L AAP-10干预组(AAP-500组),每组10只。采用浮置玻璃微电极法同步记录心内膜、心外膜心肌细胞跨膜动作电位及跨室壁心电图。结果:LQT2组QT间期,跨室壁复极离散度(TDR)、早期后除极(EAD)、R-on-T期前收缩和尖端扭转性室性心动过速(TdP)发生率均显著高于正常组(P<0.05)。在LQT2模拟状态下,500 nmol/L AAP显著缩短了QT间期和TDR(P<0.05),降低了EAD、R-on-T期前收缩和TdP的发生率(P<0.05)。结论:增加缝隙连接耦联能减少兔LQT2模型TDR和室性心律失常发生率。     

血管紧张素-(1-7)在拮抗血管紧张素Ⅱ诱导心肌细胞Cx43间隙连接上调中的作用

目的探讨血管紧张素-(1-7)[Ang-(1-7)]在血管紧张素Ⅱ(AngⅡ)诱导心肌细胞Cx43间隙连接中作用。方法AngⅡ处理培养心肌细胞24h。PD98059和Ang-(1-7)在AngⅡ刺激细胞前1h加到培养基中,对照组加等体积药物溶剂DMSO。用Western blot分析和电镜观察心肌细胞Cx43表达和间隙连接。结果Western blot分析显示用10-9~10-6mol/L AngⅡ刺激细胞24h,Cx43的表达与对照组相比呈浓度依赖性增加;用AngⅡ0.1μmol/L刺激心肌细胞24h,与对照组相比Cx43表达上调、磷酸化ERK1/2活性增加(P<0.01),ERK1/2激酶特异性抑制剂1μmol/LPD98059和0.1μmol/L Ang-(1-7)能阻断AngⅡ上调Cx43表达和磷酸化ERK1/2活性增加。电镜观察证明用AngⅡ0.1μmol/L刺激心肌细胞24h,AngⅡ处理组细胞间隙连接数目和大小较对照组增加(P<0.05),0.1μmol/L Ang-(1-7)能阻断AngⅡ增加心肌细胞间隙连接数目和大小。结论Ang-(1-7)通过抑制磷酸化ERK1/2活性增加,从而拮抗AngⅡ上调培养新生鼠心肌细胞Cx43间隙连接。     

Modulation of cardiac gap junction expression and arrhythmic susceptibility

Connexin43 (Cx43), the predominant ventricular gap junction protein, is critical for maintaining normal cardiac electrical conduction, and its absence in the mouse heart results in sudden arrhythmic death. The mechanisms linking reduced Cx43 abundance in the heart and inducibility of malignant ventricular arrhythmias have yet to be established. In this report, we investigate arrhythmic susceptibility in a murine model genetically engineered to express progressively decreasing levels of Cx43. Progressively older cardiac-restricted Cx43 conditional knockout (CKO) mice were selectively bred to produce a heart-specific Cx43-deficient subline ("O-CKO" mice) in which the loss of Cx43 in the heart occurs more gradually. O-CKO mice lived significantly longer than the initial series of CKO mice but still died suddenly and prematurely. At 25 days of age, cardiac Cx43 protein levels decreased to 59% of control values (P<0.01), but conduction velocity was not significantly decreased and no O-CKO mice were inducible into sustained ventricular tachyarrhythmias. By 45 days of age, cardiac Cx43 abundance had decreased in a heterogeneous fashion to 18% of control levels, conduction velocity had slowed to half of that observed in control hearts, and 80% of O-CKO mice were inducible into lethal tachyarrhythmias. Enhanced susceptibility to induced arrhythmias was not associated with altered invasive hemodynamic measurements or changes in ventricular effective refractory period. Thus, moderately severe reductions in Cx43 abundance are associated with slowing of impulse propagation and a dramatic increase in the susceptibility to inducible ventricular arrhythmias.     

心脏连接蛋白43羧基末端相互作用蛋白的酵母双杂交筛选

目的研究心脏连接蛋白43（Cx43）羧基末端与哪些心肌细胞内蛋白质存在相互作用。方法①通过PCR方法得到编码心脏Cx43羧基末端（AA235-382）的cDNA片段,并在其两端分别加上EcoRI和BamHI酶切位点,应用EcoRI/BamHI酶切PCR产物及pGBKT7空载体,凝胶分离后应用T4连接酶进行连接;②通过化学转化法将pG-BKT7-Cx43-CT转化酵母菌AH109;③通过“尿素/SDS”法从被转化的酵母菌中提取蛋白质;④应用抗C-myc抗体,通过Western blot方法检测”诱饵”蛋白的表达（即Gal4-BD-C-myc-Cx43-CT融合蛋白）;⑤检测”诱饵”蛋白自我激活报告基因与否后,将已被“诱饵“质粒转化的AH109与人心脏cDNA文库进行杂交,筛选阳性克隆分离阳性克隆中的cDNA,并测序。结果①诱饵载体测序结果证明pGBKT7中的“Gal4 DNA binding domain-C-myc”与Cx43的羧基末端在同一读框中;②“诱饵”质粒转化AH109酵母菌成功率100%;③从被转化的AH109中能提取到浓度满意的总蛋白;④Western blot能检测到特异性条带,其位置与Gal4-BD-C-myc-Cx43-CT的分子量相当;⑤“诱饵”蛋白不能自激活报告基因Ade2和Mel1,但可自激活His3,5mmol/L的3-AT可有效抑制“诱饵”蛋白本身自激活报告基因His3,以利于下一步的杂交筛选,筛选得到10个准阳性克隆。结论心脏连接蛋白43羧基末端能与心肌细胞中的多种蛋白质存在相互作用,这些蛋白质可能参与对间隙连接通道的功能调控。     

Is there a role for remodeled connexins in AF? No simple answers

Gap junctions provide direct cytoplasmic continuity between cells forming a low resistivity barrier to electrical propagation. As such, aberrant regulation of these low resistive conduits has been blamed for electrical conduction disorders in diseased myocardium. While there is a plethora of evidence that abnormalities in gap junctional communication underlie many forms of ventricular arrhythmias, the role of gap junctions in atrial conduction disorders has been less well studied. The atria are the most heterogeneous cardiac structures in terms of the gap junction proteins, connexins (Cx), which are present. Cx40 is the primary, or most abundant, gap junction protein in atria although Cx43 is also abundantly expressed. Cx45 is also expressed in atria, although at low levels. This heterogeneity in connexins leads to a complexity that makes understanding the role of cell coupling in conduction disorders and arrhythmogenesis difficult. In this review we focus on what is known about atrial connexins and their role in atrial fibrillation but also on the challenges presented in understanding the complex interplay between the individual connexin isoforms.     

不同β受体阻滞剂对大鼠心肌间隙连接结构作用的对比研究

目的比较三代β受体阻滞剂的代表药物卡维地洛、美托洛尔及普萘洛尔对大鼠心肌缺血再灌注损伤心肌间隙连接（GJ）结构的不同作用。方法将大鼠随机分为假手术组、缺血再灌注组、卡维地洛组、美托洛尔组及普萘洛尔组。除假手术组只穿线不结扎外,其余各组均结扎左冠状动脉前降支30min,然后松开结扎线复灌4h,建立心肌缺血再灌注损伤模型。于再灌4h末用免疫荧光和激光扫描共聚焦显微镜技术观察心肌间隙连接蛋白43（CX43）的分布及组成变化,用激光扫描共聚焦显微镜对CX43进行定量。结果与假手术组相比,缺血再灌注组CX43-GJ结构明显异常。与缺血再灌注组比较,卡维地洛组、美托洛尔组和普萘洛尔组CX43-GJ损伤减轻。各药物治疗组间比较,卡维地洛组CX43-GJ结构损伤最轻。结论各种β受体阻滞剂均具有保护心肌GJ结构的作用,以卡维地洛的作用最明显。     

Effect of resistive barrier location on the relationship between T-wave alternans and cellular repolarization alternans: a 1-D modeling study

Structural inhomogeneities in cardiac tissue have been associated with increased cellular repolarization alternans in animal experiments and increased T-wave alternans (TWA) in clinical studies. However, the effect of structural inhomogeneities on the relationship between cellular alternans and TWA has not been thoroughly investigated. We created 1-dimensional multicellular fiber models with and without a resistive barrier in various fiber regions and paced each model to induce cellular alternans. The models demonstrate that a resistive barrier in one fiber region substantially alters cellular repolarization alternans throughout the fiber. A midmyocardial or subepicardial barrier increase both TWA amplitude and maximum cellular alternans magnitude, relative to a fiber without a barrier. In addition, a direct relationship exists between TWA amplitude and maximum cellular alternans magnitude, which was highly dependent on barrier location. These results suggest that the position of a structural inhomogeneity within the myocardium may have substantial effects on dynamic repolarization instability and arrhythmogenicity.     

间隙连接蛋白32,43的表达对胃癌侵袭转移潜能的影响

目的:研究间隙连接蛋Cx32,Cx43表达水平对胃癌侵袭转移潜能的影响．方法:采用免疫组化SP法和间接免疫荧光方法分别检测Cx32,Cx43在正常胃组织及不同病理分型的胃癌组织和不同分化程度胃癌细胞株中的表达,并分析该蛋白表达对胃癌侵袭转移潜能的影响．结果:免疫组化SP法结果显示:Cx32,Cx43 阳性棕黄色颗粒位于细胞膜和细胞质内,在正常胃组织中的阳性表达率显著高于胃癌组织(Cx32:X2=23．1,P<0．05,Cx43:X2=11．6, P<0．05),Cx32,Cx43在高中分化胃癌的阳性表达率显著高于低分化胃癌(Cx32:X2= 32．1,P<0．05;Cx43:X2=22．6,P<:0．05)．间接免疫荧光法结果显示:在转化的人胃细胞系 (GES-1)、胃高分化腺癌细胞系(N87)中Cx32, Cx43的表达均为阳性,但表达率和表达部位有显著差异．在GES-1中Cx32,Cx43绿色荧光沿其细胞膜呈颗粒状或细线状分布,其阳性表达率均为100％;在N87中,Cx32绿色荧光沿其细胞膜呈颗粒状或细线状分布,其阳性表达率为49%,而Cx43荧光分布在其细胞质中,其阳性表达率为55％;在BGC-823中Cx32,Cx43的表达均为阴性．结论:Cx32,Cx43是影响胃癌转移潜能的一个重要因素．     

Mechanism of oleic acid-induced gap junctional disassembly in rat cardiomyocytes

This study investigated the mechanism of oleic acid (OA) on gap junctions and identified the protein kinase C (PKC) isoforms involved in OA-mediated gap junction disassembly in cardiomyocytes. Control cardiomyocytes showed continuous staining of the plasma membrane at cell-cell contact areas using antibodies reacting with connexin 43 (Cx43). The spontaneous contraction rate of cultured cardiomyocytes was reduced in a time-dependent manner by OA. In addition, Cx43 expression at cell-cell junction decreased, suggesting the disassembly of gap junction. Staining for PKC and PKCalpha, which were shown to colocalize with Cx43, also decreased with increased duration of OA treatment. The effects of OA on these distributional changes at cell junctions were reversed by 24 h incubation in fresh culture medium devoid of OA. Immunoprecipitation assays confirmed the biochemical binding between Cx43 and PKC/PKCalpha, and this protein interaction was not affected by OA. This may provide the basis for simultaneous detachment of Cx and PKC/PKCalpha from the cell-cell junction to the cytosol upon OA stimulation. Western blot analysis showed that OA-induced Cx43 Ser368 phosphorylation, and that this effect could be blocked by cotreatment with the general PKC inhibitor, calphostin C, the PKC inhibitor, eV1-2, or the Src kinase inhibitor, PP1, but not by the PKCalpha inhibitor, G?6976. eV1-2 also prevented the OA-induced disassembly of gap junctions. Taken together, these data suggest that OA-induced Cx43 Ser368 phosphorylation is mediated by activation of PKC and Src kinase and might be responsible for OA-induced gap junctional disassembly.     

卡维地洛对大鼠心肌间隙连接通讯的影响及作用机制研究

目的研究卡维地洛对大鼠心肌缺血再灌注损伤、心肌间隙连接通讯（GJIC）及连接蛋白43（CX43）的影响,验证卡维地洛可通过改变CX43磷酸化状态抑制GJIC起到防止心肌再灌注损伤的假设.方法将大鼠随机分为假手术组、缺血再灌注组和卡维地洛组.结扎左冠状动脉前降支致缺血30 min后复灌4 h,建立心肌缺血再灌注损伤模型,于再灌注4 h末测定心肌酶及心肌梗死范围变化;制作Langendorf心脏灌流模型,随机分为假手术组、缺血再灌注组、卡维地洛组和庚醇组,于整体缺血30 min末期用改良的划痕标记染料示踪技术测定GJIC;用Western blot技术检测缺血30 min CX43磷酸化状态的改变.结果与假手术组相比,缺血再灌注组心肌酶及心肌梗死范围明显增加,GJIC无明显变化,非磷酸化CX43升高.与缺血再灌注组比较,卡维地洛组心肌酶及心肌梗死范围显著减少,同间隙连接抑制剂庚醇作用相似,GJIC也明显受到抑制,伴非磷酸化CX43水平明显上升.结论卡维地洛具有使CX43去磷酸化进而抑制GJIC防止心肌再灌注损伤的作用.     

血吸虫可溶性虫卵抗原诱发脑血管内皮细胞缝隙连接蛋白Cx37mRNA及蛋白的表达

目的 通过观察血吸虫可溶性虫卵抗原(SEA)孵育培养的脑血管内皮细胞缝隙连接(GJ)蛋白的表达和分布,探讨脑动脉GJ在脑血吸虫病病理发生中的作用。方法 使用血吸虫SEA孵育培养幼兔脑基底动脉血管内皮细胞,实验分对照组、SEA 1～5组：加入SEA（质量浓度分别为10.0％、5.0％、3.3％、2.5％、2.0％）,应用逆转录—聚合酶链式反应(RT-PCR)技术测定兔脑血管内皮细胞GJ蛋白Cx37 mRNA的表达;应用Western 印迹技术测定兔脑血管内皮细胞GJ蛋白Cx37蛋白的表达。结果 对照组和SEA 1 ～5组的GJ蛋白Cx37 mRNA水平分别为0.239±0.037、0.260±0.043、0.218±0.310、0.647±0.040、0.419±0.036、0.513±0.038;其中SEA 3～5组的GJ蛋白Cx37 mRNA水平高于对照组（P均＜0.05）。对照组和SEA 1～5组GJ 蛋白Cx37蛋白表达分别为0.401±0.045、0.485±0.048、0.749±0.052、1.119±0.063、1.015±0.057、0.605±0.047,其中SEA 2 ～5组Cx37蛋白表达高于对照组（P均＜0.05）。结论 血吸虫SEA孵育培养的兔脑血管内皮细胞GJ蛋白Cx37 mRNA及其蛋白水平高于对照组,提示GJ蛋白在SEA及其分泌物浸润脑动脉沉积于脑组织,从而在诱发脑血吸虫病的病理发生机制中可能起重要的作用。