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.     

益气活血药对心肌梗死大鼠心脏结构和Cx43表达的影响

目的探讨益气活血药对心肌梗死(myocardial infarction,MI)大鼠心脏结构及心肌电偶联的关键结构缝隙连接蛋白43(connexin 43,Cx43)表达的影响。方法结扎雄性SD大鼠冠状动脉前降支建立MI模型,将30只MI大鼠随机分为模型组、益气活血组(参芍片0.25 g/kg联合复方丹参片0.75 g/kg)和倍他乐克组(12 mg/kg),每组10只,于造模成功后24 h开始灌胃,1次/日,连续4周。另设假手术组10只,给予等量去离子水灌胃。4周后取材进行苏木素-伊红染色观察心肌组织病理,图像分析测量左室内径和心肌梗死百分比,免疫组织化学法检测心肌Cx43的表达。结果与假手术组比较,术后4周模型组大鼠左室内径明显增大,心肌Cx43表达的平均光密度值(average optical density,AOD)、阳性表达面积(Area)和积分光密度(integral optical density,IOD)明显降低(P<0.01)。与模型组比较,益气活血组和倍他乐克组的左室内径和心肌梗死百分比均明显减少(P<0.05或P<0.01),在心肌Cx43的表达上,仅益气活血组心肌Cx43表达的AOD和IOD明显增加(P<0.05或P<0.01)。结论益气活血治疗能减少MI大鼠左室内径和心肌梗死百分比,增加心肌Cx43表达强度和部分好转Cx43弥散表达状态,对MI后缺血心肌有保护作用,可能是稳定MI后心肌细胞间电偶联的机制之一。     

缺氧对肥大心肌细胞凋亡和缝隙连接蛋白43表达的影响

目的探讨缺氧对正常心肌细胞和肥大心肌细胞缝隙连接蛋白43(Cx43)表达的影响。方法将培养的乳鼠心肌细胞或诱导肥大后的心肌细胞分别缺氧24 h,并以Hoechst33258染色法检测缺氧对心肌细胞凋亡的影响,Western blot和免疫荧光法检测Cx43的表达。结果培养的心肌细胞经血管紧张素Ⅱ诱导48 h后出现肥大,肥大心肌细胞缺氧24 h比正常心肌细胞出现更明显的凋亡,缺氧明显下调心肌细胞Cx43的表达,而肥大心肌细胞Cx43的表达下调更为明显。结论缺氧导致肥大心肌细胞Cx43的表达显著下调,细胞凋亡更明显,可能与缺氧时肥大心肌的电生理重构和恶性心律失常的产生机制有关。     

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，72h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，Cx43mRNA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。     

冷刺激对乳鼠心肌细胞缝隙连接蛋白43的影响及药物干预的研究

目的：在急性冷刺激乳鼠心肌细胞模型下,评价缝隙连接蛋白43（Connexin43,Cx43）的表达及研究急性冷刺激时乳鼠心肌细胞间传导的变化及其机制。
　　方法：乳鼠心肌细胞原代培养,随机分为正常对照组,实验4℃组;实验0℃组;并在实验4℃组和实验0℃组分别加入100 nmol/L抗心律失常肽（APP）10,每组乳鼠8只。采用流式细胞术（FCM）分析各组细胞的凋亡率,应用聚合酶链反应(PCR)和蛋白免疫印迹法（Western blot）检测Cx43及其磷酸化水平在基因和蛋白上的表达。
　　结果：FCM结果显示：实验4℃组和实验0℃组随着冷刺激程度加强和低温时间的延长,心肌细胞的凋亡率增加;同时PCR结果显示：实验4℃组和实验0℃组Cx43 mRNA呈现不同程度的下降;Western blot结果显示：实验4℃组和实验0℃组随着刺激强度和低温时间的延长,Cx43蛋白表达亦出现不同程度的下降;磷酸化的Cx43(P-Cx43)蛋白表达亦下降,而AAP10干预后可提高两组Cx43和P-Cx43蛋白的表达。
　　结论：心肌细胞在急性冷刺激时Cx43在基因和蛋白水平上表达均有不同程度的下降,P-Cx43表达亦下降,而AAP10能提高Cx43和P-Cx43蛋白的表达从而改善细胞间的传导。     

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。     

肺动脉高压大鼠肺小动脉内皮细胞间和右心室心肌中缝隙连接蛋白43表达变化的初步研究

目的:研究肺动脉高压大鼠及波生坦(bosentan)干预后肺小动脉内皮细胞间及右心室心肌中缝隙连接蛋白(Cx)43表达的变化,并探究其意义.方法:10只SD大鼠为正常对照组(n=10),不做任何处理;其余57只通过腹腔内注射野百合碱(60 mg/kg)两周后,随机将大鼠分成2组,肺动脉高压SD大鼠+安慰剂组(n＝35),肺动脉高压SD大鼠+波生坦组(n＝22),予以安慰剂或波生坦300 mg/(kg·d)治疗,每天一次,共计3周.通过免疫荧光的方法测定Cx43在肺小动脉内皮细胞间、右心室心肌组织中表达的变化.结果:在正常对照组的肺小动脉内皮细胞间未检测到Cx43,但在肺动脉高压SD大鼠+安慰剂组中肺小动脉内皮细胞间出现了Cx43的异常表达,肺动脉高压SD大鼠+波生坦组Cx43的表达水平显著下降,三组之间两两比较差异均有统计学意义(P<0.01).在右心室心肌纵切面,正常对照组的Cx43多数集中于与心肌细胞长轴垂直的端端连接处,少数位于与长轴平行的细胞侧侧连接处;而在肺动脉高压SD大鼠+安慰剂组Cx43的表达在端端连接处明显减少,在侧侧连接处明显增加;肺动脉高压SD大鼠+波生坦组Cx43的表达与正常对照组相似.三组单位面积的右心室心肌组织Cx43的表达面积的半定量分析差异无统计学意义(P>0.05).结论:肺小动脉内皮细胞间Cx43的异常表达在肺动脉高压的发病机制中可能起着非常重要的作用.右心室心肌Cx43的分布紊乱可能是导致右心室重塑的机制之一.     

Ghrelin对大鼠心肌梗死后电重构的影响

目的 观察Ghrelin对大鼠心肌梗死后电重构的作用.方法 将65只SD大鼠随机分成假手术组(n=15)和心肌梗死组(n=50).假手术组开胸后剪开心包腔,不结扎冠状动脉.心肌梗死组大鼠结扎冠状动脉前降支制作心肌梗死模型.存活7d的大鼠随机分成Ghrelin干预组(n=17)和梗死对照组(n=17).Ghrelin干预组按Ghrelin 100 μg/kg的剂量皮下注射,2次/d.梗死对照组和假手术组皮下注射等量生理盐水.28 d后采用程控刺激进行在体电生理测定,采用免疫组化方法检测缝隙连接蛋白43(Cx43)的分布,免疫印迹法检测Cx43蛋白表达,实时定量PCR法检测Cx43 mRNA表达.结果 与梗死对照组相比,Ghrelin能显著降低室性心动过速(室速)的诱发率(P＜0.05),心律失常评分也显著降低(P＜0.05).与假手术组相比,梗死心肌内Cx43表达显著降低(P＜0.05),Ghrelin干预使梗死周边心肌Cx43及其mRNA水平增加(P＜0.05).结论 Ghrelin能改善心肌梗死后大鼠心脏的电生理学特性,增加心肌Cx43的表达,防止室性心律失常的发生,可能对心血管系统具有保护作用.     

微小RNA-1调控自发性高血压大鼠肥厚左室心肌组织连接蛋白43的表达

目的观察微小RNA-1(miRNA-1)对自发性高血压大鼠(SHRs)肥厚左室心肌组织中连接蛋白43(Cx43)表达的调控及其意义。方法 18只17周龄雄性SHRs随机分为干预组(n=6)、阴性对照组(n=6)和空白对照组(n=6),通过脂质体瞬时转染技术,干预组由尾静脉注射miRNA-1抑制剂,两对照组分别注入miRNA-1抑制剂阴性对照和无血清培养基混合物,并通过实时荧光定量PCR、免疫组织化学法及western blot等技术,检测大鼠左室心肌组织miRNA-1及Cx43蛋白表达水平的改变。结果与阴性对照组和空白对照组比较,干预组miRNA-1表达水平明显降低,伴随着Cx43蛋白表达水平增高(0.45±0.15 vs 0.27±0.14,0.23±0.10,P均<0.05)。结论抑制miRNA-1表达能使SHR肥厚左室心肌组织Cx43蛋白水平升高。     

心脏缝隙连接蛋白Cx43磷酸化水平与室性心律失常的关系

心室肌的主要构成蛋白——缝隙连接蛋白43(Connexin43,Cx43)的面积、数量、空间分布及其功能状态的改变将会影响到心肌细胞间的电传导。其中,Cx43蛋白磷酸化水平改变通过改变其蛋白的构像而影响到缝隙连接通道的开放与关闭,由此导致心肌电的传导异常而发生室性心律失常。     

Transmural distribution of connexins in rodent hearts

INTRODUCTION: Electrophysiologic heterogeneity across the ventricular wall is a result of differential transmural expression of various ion channel proteins that underlie the different action potential waveforms observed in epicardial, midmyocardial, and endocardial regions. Cardiac connexins mediate cell-to-cell communication, are critical for normal impulse propagation, and play a role in electrophysiologic remodeling in disease states. However, little is known about the transmural distribution of cardiac gap junction proteins. METHODS AND RESULTS: Connexin expression in epicardium, midmyocardium, and endocardium was assessed immunohistochemically in mouse and rat hearts. The total connexin protein content within different ventricular regions was measured by immunoblotting. Connexin43 is twice as abundant in midmyocardium and endocardium compared with epicardium in the mouse but not in the rat. Connexin45 is expressed equally across the left ventricular wall. CONCLUSION: Epicardial myocytes express significantly less Cx43 and therefore may be less well coupled than midmyocardial and endocardial myocytes. A transmural gradient of connexin43 expression across the left ventricular free wall likely results in differences in the stoichiometry of connexins expressed in different regions of the heart.     

Cardiac Connexin 43 and Ischemic Cardioprotection

The connexin 43 （ Cx43 ） proteins, which is the predominant protein that can form gap junctions and non- junctional hemichannels in ventricular myocardium, are central to the cardioprotection afforded by ischemic preconditioning （IP） and maybe ischemic postconditioning （ PC ） too. Recent studies showed that recruitment of Cx43 to the mitochondria in IP might play a role in the production of reactive oxygen species （ROS） that mediates IP. The localization of Cx43 at mitochondria appears to be important for the achieved cardioprotection and opens a new door for us to reveal the exact mechanisms of ischemia/reperfusion （I/R） injury and cardioprotection, and it might be new targets of pharmacological modulator to achieved cardioprotection.     

Voltage-Gated Na+ Channel Activity and Connexin Expression in C×43-Deficient Cardiac Myocytes

INTRODUCTION: Dynamic interplay between active and passive electrical properties of cardiac myocytes is based on interrelationships between various channels responsible for depolarizing and repolarizing ionic currents and intercellular conductances. Mice with targeted disruption of the connexin43 (Cx43) gene have hearts completely devoid of Cx43, the principal gap junctional protein expressed in mammalian hearts. METHODS AND RESULTS: To determine whether cardiac myocytes that develop in an abnormal environment of reduced intercellular coupling have altered active membrane properties, we studied whole cell action potentials, Na+ channel currents, and Na+ channel expression and distribution via immunoblotting and confocal immunofluorescence in neonatal ventricular myocytes isolated from Cx43 wild-type, heterozygous, and homozygous null hearts. Action potential morphology, peak Na+ current, activation and inactivation kinetics, and Na+ channel protein expression and distribution were not different among myocytes isolated from wild-type, heterozygous, or null hearts. Active membrane properties and Na+ channel activity were completely normal in Cx43-deficient myocytes isolated from hearts that have been shown to exhibit markedly reduced Cx43 expression, gap junction number, and epicardial conduction delay. CONCLUSION: Despite a genetic inability to produce Cx43 and a developmental history that culminates in marked gross cardiac morphologic abnormalities, premature death, and myocardial inexcitability ex vivo, cardiac Na+ channel distribution and function appear to be normal in Cx43 null hearts. Although intimate structural and functional interrelationships have been described between ion channels and gap junction channels, expression and function of Na+ channels is not affected by the absence of Cx43.     

犬交感神经末梢、Cx43的分布与阵发性房颤的相关性研究

目的探讨房颤的发生率与交感神经末梢和缝隙连接蛋白分布的关系。方法测定犬右心耳(RAA)、左心耳(LAA)、左房(LA)、右房(RA)、左上肺静脉(LSPV)、左下肺静脉(LIPV)、右上肺静脉(RSPV)和右下肺静脉(RIPV)的不应期(AERP),并给予诱发房颤。随后取上述组织做成石蜡标本,HE染色确定基本结构后,通过免疫组织化学显示交感神经末梢、缝隙连接蛋白(Cx43)的分布。结果犬的房颤诱发率与缝隙连接蛋白分布数量有关。交感神经与缝隙连接蛋白43(Cx43)之间有相关性。结论逢隙连接蛋白的空间分布不均性及其表达,排列方式的变化,是房颤发生的基质,交感神经可能通过影响缝隙连接蛋白43来促进房颤的发生和维持。     

Upregulation of Angiotensin II Receptor and Connexin 43 in Increased Suburothelial Myofibroblasts in the Rat Inflammatory Bladder

Objectives: Signaling pathways in suburothelial layer are involved in the bladder sensory response. The expression of angiotensin II type 1 (AT1) receptors and connexin 43 (Cx43) in suburothelial myofibroblasts was investigated in an acute bladder inflammation model. Methods: Adult female Wistar rats underwent urethral catheterization and received 0.2 mL intravesical infusion of 0.4 M HCl to establish acute bladder inflammation model or 0.2 mL of sterile saline as control (n = 10 rats/group). Eight days after treatment, cystometry was performed. Suburothelial myofibroblasts were also collected and subjected to immunohistochemical staining to examine AT1 receptor and Cx43 expression. Results: Eight days after treatment with HCl to induce acute bladder inflammation, the frequency and basal pressure of the bladder was significantly increased compared with those in control rats. The number of suburothelial myofibroblasts was significantly increased in acute bladder inflammation rats, as was the expression of AT1 receptor and Cx43. Conclusion: These results suggest that the increased number of suburothelial myofibroblasts, upregulation of AT1 receptor and Cx43 expression may be associated with the pathogenesis of hyperactivation of bladder sensory signaling pathways in acute inflammatory bladder.     

扶正化瘀胶囊对心肌梗死大鼠心肌缝隙连接蛋白43表达的影响

目的研究扶正化瘀胶囊对心肌梗死大鼠心肌缝隙连接蛋白43(connexin43,CX43)表达的影响。方法SD雄性大鼠随机分为假手术组、模型组以及扶正化瘀胶囊组和卡托普利组,每组各6只,采用结扎左冠状动脉前降支造成心肌梗死。术后10d开始灌胃给药,持续8周。用免疫组织化学方法观察心肌CX43表达的变化。结果在心肌梗死区域内,模型组与假手术组比较,CX43排列紊乱,阳性表达面积、平均光密度值和积分光密度均显著低于假手术组(P<0.01)。在梗死边缘区,扶正化瘀胶囊组CX43平均光密度值高于模型组(P<0.01),积分光密度高于模型组(P<0.05),CX43排列紊乱有所减轻,阳性表达面积与模型组无统计学意义。卡托普利组CX43平均光密度值高于模型组(P<0.01),积分光密度和阳性表达面积均高于模型组(P<0.05),CX43排列紊乱也有减轻。结论扶正化瘀胶囊能够部分改善心肌梗死大鼠心肌的CX43重构。     

压力超负荷心力衰竭大鼠心室肌电生理失稳态及缝隙连接蛋白Cx43 的变化

目的观察压力超负荷所致心力衰竭（HF）大鼠心室肌电生理失稳态和缝隙连接蛋白Cx43表达的变化。方法采用腹主动脉缩窄法建立压力超负荷大鼠HF模型,取左室舒张末压≥15mmHg的存活大鼠入HF组,另设假结扎对照组。术后32周两组大鼠以颈总动脉插管法和心脏B超测定心功能,并检测电生理指标,以免疫印迹方法检测心肌细胞Cx43蛋白表达的变化,通过透射电镜观察心室肌缝隙连接分布的变化。结果HF大鼠出现明显电生理失稳态和心功能不全,左室舒张末压明显增高而心室有效不应期明显延长,左室射血分数明显下降,同时大鼠心室肌中缝隙连接蛋白Cx43表达明显下调（0．929±0．095VS1．250±0．083,P〈0．05）并出现空间重构。结论压力超负荷所致HF大鼠心室肌存在明显缝隙连接重构,这可能是导致HF时心肌电生理重构的重要机制。     

Reduced intercellular coupling leads to paradoxical propagation across the Purkinje-ventricular junction and aberrant myocardial activation

Ventricular tachycardia is a common heart rhythm disorder and a frequent cause of sudden cardiac death. Aberrant cell-cell coupling through gap junction channels, a process termed gap junction remodeling, is observed in many of the major forms of human heart disease and is associated with increased arrhythmic risk in both humans and in animal models. Genetically engineered mice with cardiac-restricted knockout of Connexin43, the major cardiac gap junctional protein, uniformly develop sudden cardiac death, although a detailed electrophysiological understanding of their profound arrhythmic propensity is unclear. Using voltage-sensitive dyes and high resolution optical mapping techniques, we found that uncoupling of the ventricular myocardium results in ectopic sites of ventricular activation. Our data indicate that this behavior reflects alterations in source-sink relationships and paradoxical conduction across normally quiescent Purkinje-ventricular muscle junctions. The aberrant activation profiles are associated with wavefront collisions, which in the setting of slow conduction may account for the highly arrhythmogenic behavior of Connexin43-deficient hearts. Thus, the extent of gap junction remodeling in diseased myocardium is a critical determinant of cardiac excitation patterns and arrhythmia susceptibility.     

阿托伐他汀对血管紧张素Ⅱ引起的大鼠心肌连接蛋白43重构的防治作用

目的 观察阿托伐他汀对血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)升高致大鼠心肌缝隙连接蛋白(connexin,Cx)43重构的模型的防治作用.方法 将30只雄性Wistar大鼠随机按随机数字表法分为对照组(A组)、盐酸异丙基肾上腺素(B组)、盐酸异丙基肾上腺素加阿托伐他汀组(C组),每组IO只.喂养8周后处...     

Connexin 43 gene-modified BMSCs for treating myocardial infarction

Background Stem cells have multilineage differentiation capacity,which makes bone marrow mesenchymal stem cells(BMSCs) derived relatively easy and provides a promising alternative treatment for myocardial infarction(MI).The in vivo cardiac differentiation and functional effects of unmodified BMSCs after MI are controversial.Poor moderate survival benefits of BMSCs-implanted rats were caused by incomplete electromechanical integration induced by tissue heterogeneity between myocytes and engrafted BMSCs in th...