多西环素对缺氧大鼠心肌细胞缝隙连接蛋白43表达的影响

目的探讨多西环素对缺氧大鼠H9c2心肌细胞缝隙连接蛋白43(connexin 43,Cx 43)表达的影响及其可能的机制。方法培养大鼠H9c2心肌细胞,用Western Blotting法检测缺氧6 h、12 h、24 h后Cx43总蛋白的表达量及基质金属酶抑制剂(多西环素)、PI3K抑制剂(LY294002)、ERK1/2抑制剂(U0126)干预缺氧H9c2心肌细胞后Cx43总蛋白的表达量改变。结果缺氧组较空白对照组Cx43总蛋白表达量明显降低,差异有统计学意义(P<0.01)。缺氧6 h时,多西环素和U0126干预组较缺氧组Cx43总蛋白表达量明显增高,差异有统计学意义(P<0.05);缺氧12 h时,U0126干预组Cx43总蛋白表达量仍较缺氧组增高,差异有统计学意义(P<0.05);缺氧24 h时,多西环素和U0126干预后Cx43总蛋白表达量与缺氧组比较,差异无统计学意义(P>0.0.05)。LY294002干预组Cx43总蛋白表达量在观察时间范围内均较缺氧组显著降低,差异有统计学意义(P<0.01)。结论在培养的大鼠H9c2心肌细胞中,多西环素通过抑制基质金属酶增加缺氧心肌细胞Cx43总蛋白表达量,该作用至少部分抑制由ERK1/2信号介导的传导通路。     

自发性高血压大鼠肥厚左室心肌组织微小RNA-1与缝隙连接蛋白43的改变

目的观察自发性高血压大鼠(SHR)肥厚左室心肌组织微小RNA-1(miRNA-1)、缝隙连接蛋白43(Cx43)表达的变化及其关系,以探讨高血压性心肌肥厚发生室性心律失常(VA)的分子机制。方法 10只17周龄雄性SHR大鼠做为左室肥厚组(LVH组),10只8周龄雄性SHR大鼠做为对照组,通过病理学、心肌细胞横径的测量、实时荧光定量聚合酶链反应、免疫组织化学法及western blotting检测等方法 ,比较两组大鼠左室心肌组织病理学改变、miRNA-1及Cx43蛋白表达。结果①与对照组比较,LVH组的收缩压、舒张压升高,左室质量指数及心肌细胞横径均明显增大(P均0.05);miRNA-1表达水平明显升高,以及Cx43蛋白表达水平降低(0.27±0.10vs0.60±0.13,P0.05);②LVH组大鼠左室心肌组织miRNA-1与Cx43蛋白的表达水平呈显著负相关(r=-0.661,P0.05)。结论 miRNA-1可能通过抑制Cx43表达而参与高血压LVH发生VA。     

血管紧张素Ⅱ下调肥厚心肌细胞缝隙连接蛋白Cx43的表达

目的 研究血管紧张素Ⅱ诱导心肌细胞肥大后连接蛋白43(Cx43)表达的变化及与细胞周期分布的关系.方法 分离培养大鼠心肌细胞,用血管紧张素Ⅱ诱导心肌细胞肥大,72 h后用RT-PCR,Western-blot和免疫荧光方法 观察心肌细胞Cx43基因和蛋白表达,用流式细胞仪测定法观察心肌细胞周期分布变化以及与Cx43表达量的关系.结果 血管紧张素Ⅱ处理后的心肌细胞表现细胞肥大且细胞活力增强,S期、G2-M期细胞百分比增加,细胞内G2-M(二倍体)DNA含量降低,Cx43蛋白表达明显低于正常对照组,呈浓度依赖性下调,Cx43 mRNA表达水平显著下调,Cx43蛋白表达下调与细胞周期分布的改变相关.结论 血管紧张素Ⅱ诱导心肌细胞肥大后Cx43基因及Cx43蛋白表达出现浓度依赖性下调,这一改变与心肌肥大过程中的细胞周期变化有关,提示血管紧张素Ⅱ可能通过调控Cx43基因的表达而参与缝隙连接重构过程,而Cx43表达的变化可能与心肌肥大的机制有关.     

丹参预防自发性高血压大鼠左心室肥厚

目的　观察丹参对自发性高血压大鼠左室肥厚的作用。方法　 18只 8周龄的自发性高血压大鼠随机分成 3组 :一组于 8周处死 ,另两组分别经腹腔注射丹参和蒸馏水 (1g/kg·d) ,共 10周。测量大鼠尾动脉收缩压 (SBP)及左心室重量指数 (LVMI)。应用HE、VG染色 ,结合计算机图像分析技术 ,检测心肌细胞的直径 (TDM)、面积 (CA)、心肌组织胶原体积比例 (CVF)、血管周围胶原面积和管腔面积比例 (PVCA)。结果　与 8周龄的自发性高血压大鼠相比 ,18周龄大鼠的SBP、LVMI、心肌细胞的直径、面积、CVF、PVCA显著增加 (P <0 0 1) ,丹参治疗组大鼠反映左室肥厚的各项指标上升不明显 (P >0 0 5 ) ,但收缩压仍显著升高 (P <0 0 1)。结论　长期应用丹参治疗可预防自发性高血压大鼠左室肥厚的形成。     

稳心颗粒对去甲肾上腺素诱导的心肌细胞H9C2增殖及其Cx43 mRNA表达的影响

目的观察稳心颗粒(WXKL)对去甲肾上腺素(NE)诱导的心肌细胞H9C2增殖及其缝隙连接蛋白Cx43mRNA表达的影响。方法将H9C2细胞分为四组:对照组用常规培养基培养,NE组用加入NE的常规培养基培养,WXKL组用加入WXKL的常规培养基培养,NE+WXKL组用加入含有NE和WXKL的培养基培养。培养24 h后用MTT法测定心肌细胞活力;在培养72 h后测定心肌细胞直径和细胞蛋白,提取RNA,对Cx43 mRNA进行RT-PCR扩增,琼脂糖凝胶电泳观察结果。结果与对照组相比,NE组H9C2细胞增殖率下降(P<0.05)、直径增大且蛋白含量增加(P均<0.05),其Cx43 mRNA表达减少(P<0.05);与NE组相比,NE+WXKL组H9C2细胞增殖率上升(P<0.05)、直径减小且蛋白含量下降(P均<0.05),其Cx43 mRNA表达上升(P<0.05)。结论 WXKL可改善NE诱导的H9C2细胞的肥大以及Cx43的表达下调,可能是其抗心律失常的机制之一。     

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。     

自发性高血压大鼠心脏肥大过程中心肌细胞凋亡与增殖

目的　观察不同周龄自发性高血压大鼠 (SHR)左心室心肌细胞凋亡与增殖的变化 ,探讨心肌细胞凋亡与增殖在SHR心脏肥大过程中的作用。方法　心脏肥厚指数 (Cardiachypertrophicindex ,CHI)按心脏重量 (mg)与体重 (g)的比值计算 ;末端脱氧核苷酸转移酶介导的dUTP末端标记法 (TUNEL)和透射电镜技术检测SHR心肌细胞凋亡 ;免疫组化技术检测心肌细胞增殖核抗原 (proliferatingcellnuclearantigen ,PCNA)。结果　 (1)与同周龄组WKY相比 ,12w、2 4wSHR心脏肥厚指数显著升高 (P <0 0 1) ,SHR心脏肥厚指数随周龄增加表现出增大的趋势 (SHR16vsSHR12 ,P>0 0 5 ;SHR2 0 vsSHR12 ,P <0 0 1;SHR2 0 vsSHR16,0 0 5

0 1)。 (2 ) 12周龄SHR与同周龄WKY相比 ,左心室心肌细胞凋亡指数 (APOI)无显著增加 (P >0 0 5 ) ,2 4周龄SHR左心室心肌细胞凋亡指数显著低于同周龄WKY(P <0 0 5 ) ;从 12周龄到 2 0周龄 ,SHR左心室心肌细胞APOI逐渐增加 (SHR16vsSHR12 ,0 0 5

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血管紧张素Ⅱ对心肌缝隙连接蛋白43重构的影响及贝那普利的干预作用

目的采用大鼠肾上腹主动脉部分结扎的模型,观察心肌缝隙连接蛋白（Connexin,Cx）43含量和分布的变化以及贝那普利干预的防治效果。方法将30只雄性Wistar大鼠随机等分为对照组（A组）、腹主动脉结扎组（B组）、结扎+贝那普利组（C组）。喂养8周后处死,用放射免疫法测定血浆血管紧张素Ⅱ（AngⅡ）浓度和心肌组织AngⅡ浓度,用免疫组化方法显示大鼠心肌Cx43的分布特征,半定量统计分析。结果与A组相比,B组大鼠血浆、心肌AngⅡ浓度显著升高（均P<0.01）,但B组大鼠心房肌及心室肌Cx43含量均较A组显著减少（分别为P<0.05和P<0.01）,且分布紊乱;而C组Cx43含量较B组显著增多（P<0.01）,且分布不规律程度减轻。结论AngⅡ升高可能导致心肌Cx43重新分布及表达减少;贝那普利能有效减轻Cx43的重构。     

脑利钠肽对新生大鼠心室肌细胞缝隙连接蛋白Cx43含量的影响

观察脑利钠肽(BNP)对缝隙连接蛋白Cx43含量的影响,探讨BNP对心室肌细胞电生理特性的直接效应。以培养新生大鼠心室肌细胞为实验模型,分为实验组和对照组,实验组以不同浓度(1×10-8,1×10-7,1×10-6mol/L)的BNP处理心肌细胞24h后,通过免疫印迹法(Westernblot)反映心肌细胞Cx43总蛋白含量变化;通过免疫组化法在激光共聚焦显微镜下观察GJ通道的密度变化。结果:Westernblot显示BNP处理组和对照组比较Cx43条带显色密度减弱并有统计学意义(P<0.01),且随着BNP的剂量越大,Cx43条带密度减弱越明显。在共聚焦显微镜下观察到的Cx43的特异性荧光经过强度定量分析后得出实验组GJ通道Cx43密度比对照组减弱(P<0.01)。结论:一定浓度的BNP可使GJ蛋白Cx43的含量下调。BNP有可能参予心室肌细胞GJ的重构,从而在“致心律失常基质”的形成中起作用。     

丹参预防自发性高血压大鼠左心室肥厚

目的观察丹参对自发性高血压大鼠左室肥厚的作用.方法 18只8周龄的自发性高血压大鼠随机分成3组一组于8周处死,另两组分别经腹腔注射丹参和蒸馏水(1 g/kg*d),共10周.测量大鼠尾动脉收缩压(SBP)及左心室重量指数(LVMI).应用HE、VG染色,结合计算机图像分析技术,检测心肌细胞的直径(TDM)、面积(CA)、心肌组织胶原体积比例(CVF)、血管周围胶原面积和管腔面积比例(PVCA).结果与8周龄的自发性高血压大鼠相比,18周龄大鼠的SBP、LVMI、心肌细胞的直径、面积、CVF、PVCA显著增加(P<0.01),丹参治疗组大鼠反映左室肥厚的各项指标上升不明显(P>0.05),但收缩压仍显著升高(P<0.01).结论长期应用丹参治疗可预防自发性高血压大鼠左室肥厚的形成.     

Remodelling of cardiac gap junction connexin 43 and arrhythmogenesis

BACKGROUND: In cardiac muscle, the gap junction plays a pivotal role in electrical cell-to-cell coupling and impulse propagation between cells. The function of the gap junction depends on the regulation of connexin in the gap junction channel. A dysfunction of the gap junction is possibly caused by the downregulation of connexin or one of arrhythmogenic factors. The mechanisms of ventricular fibrillation, a lethal tachyarrhythmia, have been studied in relation to the remodelling of connexin. OBJECTIVES: To determine what type of connexin 43 (Cx43) remodelling contributes to the generation of ventricular fibrillation and what factors induce the modelling of Cx43. METHODS: Aconitine-induced ventricular fibrillation was induced in hearts isolated from adult rats. Alterations in the electrical activity, the phosphorylation of Cx43 and the expression of Cx43 were evaluated by both intracellular and extracellular recording of the action potentials, Western blotting and immunohistochemistry, respectively. Flutter activity after the application of aconitine shifted spontaneously to fibrillation, showing an electrical interaction between neighbouring cells in close proximity to one another. The facility of the shift from flutter to fibrillation was evaluated as a susceptibility of the heart to fibrillation in relation to gap junction function. The effects of phorbol 12-myristate 13-acetate, angiotensin II (AII) analogues, AII antagonists, the diabetic state, protein kinase A (PKA) activator, cyclic AMP analogues, d-sotalol (class III antiarrhythmic drug) and PKA inhibitors on the susceptibility of the heart to fibrillation were examined. RESULTS: Pathological hearts with heterogeneous expression of Cx43 at the gap junction, such as phorbol 12-myristate 13-acetate-and AII analogue-treated hearts, as well as diabetic hearts, showed a significantly higher susceptibility to fibrillation. On the other hand, hearts with augmentative expression of Cx43 at the gap junction, such as hearts pretreated with a PKA activator, a cyclic AMP analogue (8-bromo-cyclic AMP) or d-sotalol, showed a significantly lower susceptibility to fibrillation. At the beginning of fibrillation, an increase in the cardiac tissue AII level, an augmentation of the protein kinase C (PKC)-epsilon activity, the presence of PKC-mediated hyperphosphorylation, a suppression of the PKA-mediated phosphorylation of Cx43 and a reduction in the expression of Cx43 at the gap junction were observed. These alterations in Cx43 expression were also observed to increase as the fibrillation advanced. CONCLUSIONS: Augmentation of PKC-mediated phosphorylation and suppression of PKA-mediated phosphorylation induces the downward remodelling of Cx43. Such remodelling of Cx43 induces asynchronous electrical activities and makes the ventricular tissue susceptible to fibrillation. PKC is activated by AII. The fibrillation itself remodels Cx43, thereby causing a vicious cycle. As a result, PKC inhibitors, AII antagonists and PKA activators are considered to possibly have a protective effect against the initiation or advancement of ventricular fibrillation.     

Relating specific connexin co-expression ratio to connexon composition and gap junction function

Cardiac connexin 43 (Cx43), Cx40 and Cx45 are co-expressed at distinct ratios in myocytes. This pattern is considered a key factor in regulating the gap junction channels composition, properties and function and remains poorly understood.This work aims to correlate gap junction function with the connexin composition of the channels at accurate ratios Cx43:Cx40 and Cx43:Cx45.Rat liver epithelial cells that endogenously express Cx43 were stably transfected to induce expression of accurate levels of Cx40 or Cx45 that may be present in various areas of the heart (e.g. atria and ventricular conduction system). Induction of Cx40 does not increase the amounts of junctional connexins (Cx43 and Cx40), whereas induction of Cx45 increases the amounts of junctional connexins (Cx43 and Cx45). Interestingly, the non-junctional fraction of Cx43 remains unaffected upon induction of Cx40 and Cx45. Co-immunoprecipitation studies show low level of Cx40/Cx43 heteromerisation and undetectable Cx45/Cx43 heteromerisation. Functional characterisation shows that induction of Cx40 and Cx45 decreases Lucifer Yellow transfer. Electrical coupling is decreased by Cx45 induction, whereas it is decreased at low induction of Cx40 and increased at high induction.These data indicate a fine regulation of the gap junction channel make-up in function of the type and the ratio of co-expressed Cxs that specifically regulates chemical and electrical coupling. This reflects specific gap junction function in regulating impulse propagation in the healthy heart, and a pro-arrhythmic potential of connexin remodelling in the diseased heart.     

心力衰竭大鼠心脏缝隙连接蛋白43的变化及厄贝沙坦的干预作用

目的观察压力超负荷所致心力衰竭大鼠心室肌中缝隙连接蛋白43表达的变化以及厄贝沙坦对心力衰竭时缝隙连接重构的干预作用。方法采用腹主动脉缩窄法建立大鼠心力衰竭模型,随机分为厄贝沙坦组和心力衰竭组,分别用厄贝沙坦(50 mg/kg)和安慰剂治疗,另设假手术对照组。术后16周用颈总动脉插管法测定心功能,用免疫印迹法检测心肌细胞缝隙连接蛋白43蛋白表达的变化,并以透射电镜观察缝隙连接空间重构的变化。结果心力衰竭大鼠心肌中缝隙连接蛋白43表达明显下调并出现空间重构,厄贝沙坦可明显上调缝隙连接蛋白43蛋白的表达并改善缝隙连接的空间重构。结论压力超负荷所致心力衰竭大鼠心室肌存在明显缝隙连接重构,这可能是心力衰竭时心肌电生理重构的机制之一,而此过程与血管紧张素Ⅱ的作用有关,血管紧张素受体拮抗剂厄贝沙坦可以明显改善心力衰竭时出现的缝隙连接重构。     

Gap junction remodeling in hypertrophied left ventricles of aortic-banded rats: prevention by angiotensin II type 1 receptor blockade

Remodeling of gap-junctional organization in hypertrophied left ventricle (LV) in response to pressure overload in rats induced by abdominal aorta banding was investigated by immunoconfocal and electron microscopy. Eight to 12 weeks after banding, rats developed significant LV hypertrophy. In contrast to control LV myocytes, which showed connexin43 (Cx43) labeling largely confined to the intercalated disks, LV myocytes from aortic-banded rats showed dispersion of punctate Cx43 labeling over the entire cell surface. In LV tissues sectioned longitudinally, the proportion of Cx43 label at the intercalated disk decreased significantly (control, 0.87 v aortic-banded, 0.62). En-face views of intercalated disks of hypertrophied myocardium revealed a reduction of Cx43 gap junctions in the disk center, giving rise to a significant decrease in the proportion of the disk occupied by gap-junctional membrane (control, 0.32 v aortic-banded, 0.24). Electron microscopy of hypertrophied LV tissue revealed that Cx43-containing gap junctions were frequently displaced from their usual locations to form side-to-side contacts distant from the disk, and also appeared as annular profiles. In aortic-banded rats treated with the angiotensin II (AII) type 1 receptor (AT1) antagonist, losartan (10 mg/kg/day, 11 weeks) not only LV hypertrophy, but also the gap junction disorganization was markedly reduced. These results suggest that LV hypertrophy induced by pressure overload is associated with Cx43 gap junction disorganization and that AII may play an important role either directly or indirectly in gap-junctional remodeling.     

大鼠心室肌缝隙连接蛋白增龄性变化对心律失常发生率的影响

目的 研究大鼠心室肌缝隙连接蛋白(Cx43)增龄性改变及增龄导致室性心律失常发生率增高的原因.方法 随机取3～6月龄(幼年组)、9～12月龄(青年组)、18～21月龄(中年组)及24～26月龄(老年组)F344健康雄性大鼠,通过监测其肢体导联心电图,记录其心律失常发生情况.各组心室肌分别用苏木素-伊红、马森三色复合染色...     

Statins reduce connexin40 and connexin43 expression in atherosclerotic aorta of rabbits

BACKGROUND: Gap junction protein connexin43 (Cx43) expression was enhanced in proliferating smooth muscle cells (SMCs) in the neointima of atherosclerotic lesions. HMG-CoA Reductase Inhibitors (statins) can reduce Cx43 expression in vivo and in vitro. Connexin40 (Cx40) is also a very important connexin in SMCs of arterial wall. METHODS: We observed the expression of Cx40 and Cx43 in a rabbit model of a high-cholesterol diet and investigated the effect of lovastatin (10 mg.kg-1.d-1, 2 weeks) or fluvastatin (10 mg.kg-1.d-1, 2 weeks) on these changes by the methods of western blotting, RT-PCR, immunohistochemistry, and transmission electron microscope. RESULTS: There was abundant expression of Cx40 mRNA and protein in SMCs of rabbit aorta. Besides Cx43, Cx40 expression was also obviously upregulated in atherosclerotic plaques. Treatment with statins reduced the over-expression of Cx43 and Cx40 in atherosclerotic lesion. Cx40 and Cx43 gap junction quantity from each of the arteries obtained at the different drug treatment levels revealed no significant difference. Neointimal SMCs had abundant, large gap junctions, whereas normal SMCs had smaller, less frequent junctions. Statins also normalized the enlarged gap junctions. CONCLUSIONS: These results provide novel in vivo evidence for the key role of gap junctions in atherogenesis and the possible mechanism in antiatherogenic effect of statins.     

增龄对犬肺静脉缝隙连接蛋白的影响

目的探讨增龄对犬肺静脉缝隙连接蛋白数量及分布形式的影响。方法6只成年（1～2岁）及5只老年杂种犬（〉6岁），麻醉后行心内电生理检查以评价房颤的诱发率。然后取左上肺静脉的肌袖组织，用免疫荧光共聚焦显微镜观察Cx40和Cx43的表达和分布。结果所有的成年犬均未诱发出持续性房颤，而5只老年犬中有4只诱发出了持续性房颤（χ2，P〈0，05）。Cx40和Cx43的表达和分布在两组间无显著性差异。结论增龄并不影响犬肺静脉肌袖细胞间缝隙连接蛋白的表达水平和分布形式。     

Decreased endothelial size and connexin expression in rat caudal arteries during hypertension

OBJECTIVES : Hypertension is accompanied by endothelial dysfunction. The present study has investigated endothelial cell morphology and connexin expression in the caudal artery of the rat during the development of hypertension. METHODS : A significant increase in systolic blood pressure was detected from 9 weeks of age in spontaneously hypertensive male rats (SHR) compared to normotensive Wistar-Kyoto (WKY) rats, reaching a maximum by 11-12 weeks of age. Immunohistochemistry was used to quantify cell size and expression of connexins (Cxs) 37, 40 and 43 in the endothelium of prehypertensive (3-week-old) and hypertensive (12-week-old) rats. RESULTS : At 12 weeks, the size of endothelial cells and the expression of all three Cxs per endothelial cell were significantly less in SHR than WKY rats. At 3 weeks, there was no significant difference in cell size nor in the expression of Cxs 37 or 43; however, expression of Cx40 was significantly lower in SHR than in WKY rats. Between 3 and 12 weeks in WKY rats, there was no change in endothelial cell size, nor in the expression of Cxs 37, 40 and 43. In SHR, both cell size and Cx expression per endothelial cell were significantly decreased during the same developmental period, with a significant decrease in the density of Cx40 plaques. CONCLUSION : The development of hypertension in the SHR is accompanied by significant decreases in endothelial cell size and expression of Cx40, which may contribute to the endothelial dysfunction present in hypertension.     

Gap junction remodelling in human heart failure is associated with increased interaction of connexin43 with ZO-1

AIMS: Remodelling of gap junctions, involving reduction of total gap junction quantity and down-regulation of connexin43 (Cx43), contributes to the arrhythmic substrate in congestive heart failure. However, little is known of the underlying mechanisms. Recent studies from in vitro systems suggest that the connexin-interacting protein zonula occludens-1 (ZO-1) is a potential mediator of gap junction remodelling. We therefore examined the hypothesis that ZO-1 contributes to reduced expression of Cx43 gap junctions in congestive heart failure. METHODS AND RESULTS: Left ventricular myocardium from healthy control human hearts (n = 5) was compared with that of explanted hearts from transplant patients with end-stage congestive heart failure due to idiopathic dilated cardiomyopathy (DCM; n = 5) or ischaemic cardiomyopathy (ICM; n = 5). Immunoconfocal and immunoelectron microscopy showed that ZO-1 is specifically localized to the intercalated disc of cardiomyocytes in control and failing ventricles. ZO-1 protein levels were significantly increased in both DCM and ICM (P = 0.0025), showing a significant, negative correlation to Cx43 levels (P = 0.0029). There was, however, no significant alteration of ZO-1 mRNA (P = 0.537). Double immunolabelling demonstrated that a proportion of ZO-1 label is co-localized with Cx43, and that co-localization of Cx43 with ZO-1 is significantly increased in the failing ventricle (P = 0.003). Interaction between the two proteins was confirmed by co-immunoprecipitation. The proportion of Cx43 that co-immunoprecipitates with ZO-1 was significantly increased in the failing heart. CONCLUSION: Our findings suggest that ZO-1, by interacting with Cx43, plays a role in the down-regulation and decreased size of Cx43 gap junctions in congestive heart failure.     

Connexin expression in cultured neonatal rat myocytes reflects the pattern of the intact ventricle

OBJECTIVE: Primary cultures of neonatal rat ventricular myocytes have become a widely used model to examine a variety of functional, physiological and biochemical cardiac properties. In the adult rat, connexin43 (Cx43) is the major gap junction protein present in the working myocardium. In situ hybridization studies on developing rats, however, showed that Cx40 mRNA displays a dynamic and heterogeneous pattern of expression in the ventricular myocardium around birth. The present studies were performed to examine the expression pattern of the Cx40 protein in neonatal rat heart, and to examine the connexins present in cultures of ventricular myocytes obtained from those hearts. METHODS: Cryosections were made of hearts of 1-day-old Wistar rats. Cultures of ventricular myocytes obtained from these hearts by enzymatic dissociation were seeded at various densities (to obtain > 75, approximately 50%, and < 25% confluency) and cultured for 24, 48 or 96 h. Cx40 and Cx43 were detected by immunofluorescence and immunoblotting. RESULTS: Immunohistochemical stainings confirmed that gap junctions in the atrium and His-Purkinje system were composed of at least Cx43 and Cx40. From the subendocardium towards the subepicardium Cx40 expression gradually decreased, resulting in the sole expression of Cx43 in the subepicardial part of the ventricular wall. In ventricular myocytes cultured at high density (> 75% confluency) Cx43 and Cx40 immunoreactivity could be detected. In contrast to Cx43 immunolabeling which showed a homogeneous distribution pattern, Cx40 staining was heterogeneous, i.e. in some clusters of cells abundant labeling was present whereas in others no Cx40 staining could be detected. The pattern of Cx43 immunoreactivity was not altered by the culture density. In contrast, in isolated ventricular myocytes cultured at low density (< 25% confluency) the relative number of cell-cell interfaces that were Cx40-immunopositive decreased as compared to high density cultures (35 vs. 70%). Western blots did not reveal significant differences in the level of Cx40 and Cx43 expression at different culture densities. CONCLUSIONS: These results show that cultured ventricular myocytes retained typical features of the native neonatal rat ventricular myocardium with regard to their composition of gap junctions. This implicates that these cultures may serve as a good model for studying short-term and long-term regulation of cardiac gap junction channel expression and function.