迷走神经对快速起搏犬上腔静脉肌袖缝隙连接蛋白40,43重构的影响

目的研究迷走神经对犬上腔静脉(SVC)肌袖缝隙连接蛋白40(Cx40)、43(Cx43)重构的影响。方法24只杂种犬,随机等分为3组:假手术组(Sham组)、切除上腔静脉-主动脉根部(SVC-AO)脂肪垫组(RM组)和保留SVC-AO脂肪垫组(RS组)。RM组和RS组植入起搏器,500～650次/分起搏6W;Sham组保留SVC-AO脂肪垫植入起搏器但不起搏。以程序刺激或猝发刺激诱发心房颤动(AF),取SVC肌袖组织行Cx40、Cx43免疫组化染色并半定量分析,透射电镜观察缝隙连接(GJ)的超微结构。结果RS组有5只犬诱发出持续性AF(5/7),而Sham组和RM组无1只犬能诱发持续性AF。RM组和RS组的Cx40、Cx43蛋白表达均显著高于Sham组(P<0.05);RS组的Cx40、Cx43蛋白表达显著高于RM组(P<0.05)。Sham组和RM组的Cx40、Cx43端/侧比值均显著高于RS组(P<0.05)。与RM组比较,RS组GJ通道变短且宽,出现肌溶解及线粒体空泡状改变。结论迷走神经可能通过介导SVC肌袖Cx40、Cx43重构而使AF易于发生和维持;切除犬SVC-AO脂肪垫可减弱此效应。     

氧化应激对心房颤动心房间质纤维化影响的实验研究

目的 观察氧化应激对慢性心房快速起搏诱发心房颤动(房颤)犬心房肌间质纤维化的影响.方法 20只犬分为假手术组、对照组和普罗布考组.无菌条件下开胸手术,植入高频起搏器(400次/min)心房快速起搏6周,建立房颤犬模型.普罗布考组于起搏前1周服用普罗布考(100 mg/kg),至起搏6周结束.采用Masson染色观察各组犬心房肌胶原容积分数(CVF);采用Western印迹法检测各组犬心房肌MMP-9、TMP-1表达;分别于起搏前、起搏6周后测量各组犬左心房最大容积(LAVmax)、最小容积(LAVmin)和射血分数(LAEF);测定左心耳最大容积(LAAVmax)、最小容积(LAAVmin)和射血分数(LAAEF);记录左心耳最大正向血流速度(V-LAA+)和负向血流速度(V-LAA-);比色法检测各组犬心房肌氧化应激相关指标.结果 与假手术组相比,对照组犬心房肌CVF值显著增加,普罗布考能够显著降低房颤犬心房肌CVF值.与假手术组比较,对照组犬心房肌MMP-9表达显著上调(P＜0.01),TMP-1表达显著下调(P＜0.01);与对照组犬比较,普罗布考组犬心房肌MMP-9表达显著降低(P＜0.01),TMP-1表达显著升高(P＜0.01).心房快速起搏6周后,对照组犬LAVmax、LAVmin、LAAVmax和LAAVmin明显增大,LAEF、LAAEF、V-LAA+和V-LAA-显著减小.与假手术组犬比较,对照组犬心房肌MDA-水平显著增加(P＜0.01),与对照组犬比较,普罗布考组犬心房肌MDA水平显著降低(P＜0.01),心房肌氧化应激指标(MDA)与CVF值呈正相关(r=0.976,P＜0.001).结论 长期心房快速起搏诱发房颤犬心房肌存在氧化应激,心房肌MMP-9表达显著上调,TIM-1表达显著下调,心房肌纤维化程度显著增加.普罗布考能够通过抑制氧化应激,抑制房颤犬心房肌MMP-9表达上调,促进TIM-1表达上调,防止房颤犬心房结构重构,改善心房功能,对房颤防治有益.     

高位胸段硬膜外阻滞对心房颤动自主神经重构的影响

目的:观察高位胸段硬膜外阻滞(HTEB)对长期右心耳快速起搏诱发心房颤动(房颤)犬心房自主神经重构的影响,并探讨心房肌神经生长因子(NGF)在心房自主神经重构中的作用。方法:18只犬随机分为假手术组、对照组和HTEB组。对照组和HTEB组犬给予持续快速心房起搏建立房颤模型,HTEB组给予硬膜外腔注射0.5%利多卡因行高位胸段硬膜外阻滞。通过Masson染色检测犬心房肌胶原容积分数(CVF)改变,免疫组化法测定犬心房肌神经生长相关蛋白43(GAP43)及酪氨酸羟化酶(TH)表达,蛋白免疫印迹法检测犬心房肌NGF、GAP43及TH蛋白表达情况。结果:与对照组相比,右心耳快速起搏6周后,HTEB组犬心房肌CVF值显著降低;GAP43及TH神经萌出量显著减少(P0.05);NGF、GAP43及TH蛋白表达量显著减少(P0.05~0.01)。与假手术组比较,HTEB组犬心房肌CVF值显著增加;GAP43及TH神经萌出量显著增加(P0.05~0.01);NGF、GAP43及TH蛋白表达量显著增加(P0.05)。结论:长期右心耳快速起搏诱发犬房颤致心房肌神经不均一萌出,诱发自主神经重构,而NGF在其中发挥重要作用。HTEB可有效阻止房颤犬心房肌NGF表达上调,减轻自主神经重构。     

风湿性心脏病持续性心房颤动患者右心耳缝隙连接蛋白40和43mRNA水平的研究

研究风湿性心脏病持续性心房颤动(AF)患者心房缝隙连接蛋白(Cx)40和43mRNA表达水平的改变与AF之间的关系。选择我院35例风湿性心脏病行换瓣患者的右心耳,依心脏节律分为窦性心律组(SR)14例,AF组21例。应用逆转录聚合酶链反应技术,检测AF、SR患者右心耳组织Cx40、Cx43mRNA的表达量。结果:风湿性心脏病AF患者其右心耳Cx40mRNA的表达量明显降低,而Cx43mRNA的表达量无明显改变。结论Cx40表达水平下调可能参与了AF的发生维持。     

慢性快速心房起搏心房颤动犬内皮型一氧化氮合酶mRNA表达变化的研究

为研究慢性快速心房起搏心房颤动(简称房颤)犬模型中心内膜内皮型一氧化氮合酶(eNOS)mRNA表达的变化,探讨其与心房结构重构、血栓形成的关系。13只健康犬随机分为假手术组和起搏组,应用埋藏式高频率心脏起搏器快速起搏心房(400次 /分) 6周,取左、右心房,左、右心耳及主动脉内膜。通过逆转录 聚合酶链反应 (RT PCR),以β actin为内参照,测定犬心内膜eNOSmRNA表达的变化,同时检测血浆NO代谢产物硝酸盐 (NOx)的含量。结果:正常犬心脏eNOSmRNA表达存在差异,左房、左心耳明显高于右房、右心耳;起搏 6周后左房、左心耳eNOSmRNA表达起搏组明显低于假手术组,而右房、右心耳、主动脉无明显差别,血浆NOx起搏组亦明显低于假手术组。结论:正常犬心脏eNOS基因表达是不平衡的,左房明显高于右房。房颤犬eNOSmRNA表达降低可能是心房结构重构,血栓形成的重要因素之一。     

心房颤动犬心房肌肾素-血管紧张素系统改变

目的探讨慢性心房快速起搏诱发心房颤动(房颤)犬心房肌肾素血管紧张素系统(RAS)的改变。方法13只犬随机分为假手术组(n=6)和起搏组(n=7)。起搏组犬无菌开胸后在右心房缝植5对心外膜记录电极,电极尾端经皮下由犬背部穿出;在右心耳缝植螺旋型起搏电极,连接实验用AOO高频起搏器(400次/min),心房快速起搏6周,建立房颤犬模型;假手术组犬仅缝植心外膜记录电极和起搏电极而不起搏。经心外膜电极记录各组犬房颤诱发情况;采用放射免疫方法检测两组犬左心房及右心房组织血管紧张素Ⅰ(AngⅠ)、血管紧张素Ⅱ(AngⅡ)含量及肾素活性;采用逆转录聚合酶链反应(RTPCR)检测两组犬心房肌肾素、血管紧张素原和血管紧张素转换酶mRNA表达水平改变。结果(1)起搏组7只犬均经短阵快速刺激(burstpacing)诱发出房颤,房颤诱发率和平均持续时间较假手术组显著增加(P<0.01);(2)起搏组犬心房肌AngⅠ、AngⅡ含量较假手术组犬心房肌明显升高(P<0.01),肾素活性亦显著增加(P<0.01);同一组犬左心房与右心房组织AngⅠ、AngⅡ含量及肾素活性差异无统计学意义(P>0.05);(3)起搏组犬心房肌肾素、血管紧张素原和血管紧张素转换酶mRNA表达较假手术组犬心房肌显著上调(P<0.01)。结论慢性心房快速起搏诱发房颤犬心房肌AngⅠ、AngⅡ含量及肾素活性显著增加,可能是局部RAS基因表达上调的结果,提示房颤伴随心房肌RAS激活。     

心房颤动患者心房组织连接蛋白40和连接蛋白43基因转录表达的研究

目的：探讨心 房颤动（房颤）患者心房组织连接蛋白40（connexin40,Cx40)和连接蛋白43（Cx43)基因转录的变化，方法：39例风湿性心瓣膜病患者，心脏外科手术时取右心耳组织，通过逆转录－聚合酶链反应，以GAPDH为内参照，测量Cx40和Cx43的mRNA表达量。结果：阵发性房颤组和慢性房颤组心房组织Cx40的mRNA表达水平均显著高于窦性心律组（分别为P＜0．05，P＜0．01），而慢性房颤组心房组织Cx40的mRNA水平又明显高于阵发性房颤组（P＜0．01），窦性心律组，阵发性房颤组和慢性房颤组之间心房组织Cx43的mRNA水平无明显差异（均为P＞0．05），结论：心房Cx40基因转录上调是促进房颤发生和持续的重要因素。     

环肺静脉隔离对阵发性心房颤动患者心房有效不应期的影响

目的比较环肺静脉隔离术(CPVI)前后心房各部位有效不应期(ERP)的变化。方法入选2015年4月至2015年12月入住天津医科大学总医院行首次射频消融术(RFCA)的阵发性心房颤动(房颤)患者30例。CARTO 3系统以FAM模式完成右心房(RA)、左心房(LA)电解剖模型构建。并于上腔静脉(SVC)、中位右心房(MRA)、右心耳(RAA)、左心房前壁(LA-A)、左心房顶部(LA-R)、左心房后壁(LA-P)、左心耳(LAA)、左上肺静脉(LSPV)、左下肺静脉(LIPV)、右上肺静脉(RSPV)、右下肺静脉(RIPV)、冠状静脉窦近端(CSp)、冠状静脉窦远端(CSd)定位取点。所有患者均完成双侧CPVI,并达到肺静脉(PV)-LA双向传导阻滞。于CPVI术前、术后应用心房S1S2程序刺激分别测定心房各部位ERP。并计算右心房(RA,包括SVC、MRA、RAA、CSp)、左心房(LA,包括LA-A、LA-R、LA-P、LAA、CSd)、肺静脉(PV,包括LSPV、LIPV、RSPV、RIPV)的ERP。观察CPVI术前、术后心房电生理特性的改变。结果 (1)CPVI术前心房各部位ERP比较:RAA(197.4±28.6)ms最小(P0.01);其次为PV,分别为LSPV(213.0±47.5)ms、LIPV(208.9±45.9)ms、RSPV(209.3±43.6)ms、RIPV(213.5±48.1)ms和LAA(218.1±27.7)ms。LA、RA及PV比较:PV ERP(211.2±35.2)ms最短,其次为RA ERP(227.0±23.7)ms及LA ERP(241.0±21.5)ms(P0.05)。(2)CPVI术前、术后各部位比较:RAA[(197.4±28.6)ms比(208.6±32.2)ms,P=0.003],CSp[(234.7±29.1)ms比(246.9±29.7)ms,P=0.007],LA-R[(242.9±28.9)ms比(258.3±26.9)ms,P=0.003],LA-P[(252.2±28.5)ms比(261.1±30.2)ms,P=0.039],CSd[(238.6±28.3)ms比(250.3±23.6)ms,P=0.009]。LA、RA及PV比较:RA[(227.0±23.7)ms比(235.9±21.7)ms,P=0.002),LA[(241.0±21.5)ms比(249.7±19.9)ms,P=0.001),术后ERP均较术前延长。(3)术前共诱发房性心律失常90例次,以RAA(17)、LAA(12)及PV(36)诱发次数多,上述部位ERP均较短。术后共诱发房性心律失常8例次,位于RAA(4)、LAA(3)及SVC(1)。结论 (1)阵发性房颤患者心房各部位ERP比较,以PV、LAA与RAA最短;该部位程序刺激易诱发房性心律失常;(2)阵发性房颤患者ERP比较:PV的ERP最短,其次为RA和LA;PV与LA之间存在较大的ERP梯度变化;(3)CPVI使RA、LA的ERP延长;(4)CPVI使心房内程序刺激诱发房性心律失常的诱发率明显降低。     

心房肌缝隙连接蛋白40、43表达改变与心房颤动的关系

目的研究心房颤动患者心房肌缝隙连接蛋白(Cx)40、43表达的改变,探讨Cx在房颤发生与维持中的作用.方法39例接受开胸手术者分为房颤组和窦性心律组,手术时取右心耳及左心房各约100mg,采用Western blot与免疫组织化学技术,检测心房肌Cx40、Cx43表达量与分布的改变.结果房颤组Cx40表达量在左心房、右心耳较窦性心律组高,Cx43表达量在左心房较窦性心律组高,在右心耳无差异.免疫组化显示房颤组Cx40、Cx43均分布紊乱,聚集于胞浆或核周.结论房颤患者心房肌Cx40、Cx43表达增高,且分布异常,提示心房肌Cx40、Cx43表达改变与心房颤动的发生与维持有关.     

心房肌缝隙连接蛋白40、43表达改变与心房颤动的关系

目的：研究心房颤动患者心房肌缝隙连接蛋白(Cx)40、43表达的改变，探讨Cx在房颤发生与维持中的作用。方法：39例接受开胸手术者分为房颤组和窦性心律组，手术时取右心耳及左心房各约100mg，采用Western blot与免疫组织化学技术，检测心房肌Cx40、Cx43表达量与分布的改变。结果：房颤组Cx40表达量在左心房、右心耳较窦性心律组高，Cx43表达量在左心房较窦性心律组高，在右心耳无差异。免疫组化显示房颤组Cx40、Cx43均分布紊乱，聚集于胞浆或核周。结论：房颤患者心房肌Cx40、Cx43表达增高，且分布异常，提示心房肌Cx40、Cx43表达改变与心房颤动的发生与维持有关。     

起搏治疗对心脏自主神经基质改变的实验研究

目的观察窦房结和房室结功能障碍对心脏神经基质的影响,以及右心耳（RAA）和右心室心尖部（RVA）起搏对神经重构的作用。方法28只犬随机分为窦房结损伤组、RAA起搏组、房室结损伤组和RVA起搏组。7只健康犬作为对照组。14只犬用20％甲醛滤纸片外敷窦房结区损伤窦房结,其中7只犬将起搏电极导线缝合固定于RAA上,以90~．／min行心房起搏。另外14只犬于房室交界区注入无水乙醇损伤房室结,其中7只犬将起搏电极导线缝合固定于RVA,以907~／min起搏心室。起搏60d后二次麻醉,取出心脏。所有犬均于RAA、房间隔（As）、左心耳（LAA）、RVA、室问隔（Vs）、左心室心尖部（LVA）取材。运用免疫组化技术测定心肌中的新生神经（GAP43标测）和交感神经（TH标测）密度。结果（1）窦房结损伤组RAA的新生神经和交感神经密度低于正常对照组（P〈0．01）,但AS、LAA及心室的新生神经和交感神经密度与对照组相似（P〉0．05）;（2）RAA起搏组RAA的新生神经与交感性神经密度高于窦房结损伤组（P〈0．01）,与对照组接近（P〉0．05）;（3）房室结损伤组心房和心室各部位的新生神经和交感神经密度高于对照组（P〈0．01）;（4）RVA起搏组心房和心室的新生神经和交感神经密度与房室结损伤组差异无统计学意义（P〉0．05）。结论窦房结和房室结功能障碍可造成心脏神经基质的改变。RAA起搏可以逆转窦房结功能低下造成的神经重构,而RVA起搏不能逆转。     

Differential densities of cholinergic nerves in canine supraventricular regions of hearts

PurposeCholinergic nerve plays an important role in the induction and maintenance of atrial fibrillation (AF). Cholinergic innervation at supraventricular tissues is considered to be the histological basis and intervention-associated target site for the arrhythmia; however, the distribution of cholinergic nerve in supraventricular tissues has not been clearly studied. In this study, we investigated the cholinergic nerve innervation in canine supraventricular regions of hearts.MethodsWe performed histological and immunohistochemical staining on canine tissues of left atrial appendage (LAA), right atrial appendage (RAA), left atrium (LA), right atrium (RA), atrial septum (AS), crista terminalis (CT), pulmonary vein (PV), and super vena cava (SVC) using hematoxylin and eosin (H&E) and antibodies to choline acetyltransferase.ResultsNormal canine cardiovascular histological structures were shown from H&E staining. Cholinergic nerve densities at LAA and RAA were significantly higher than LA, which was higher than RA, but no significant difference was observed between LAA and RAA. Furthermore, RA was significantly higher than AS, CT, PV, and SVC and there were no significant differences among the latter four.ConclusionThe heterogeneity of different densities of cholinergic nerve innervation of canine supraventricular regions establishes the histological basis of cholinergic nerve-mediated pathological conditions.     

Gap junctional remodeling in relation to stabilization of atrial fibrillation in the goat

OBJECTIVE: It has been postulated that high atrial rate induced changes at the level of the gap junctions ('gap junctional remodeling', i.e. changes in distribution, intercellular orientation and expression of gap junction proteins), could be part of the vicious circle of electrophysiologic and structural changes leading to sustained atrial fibrillation (AF). To obtain experimental evidence in favour of such a postulate the timing of this remodeling process was studied in relation to the development of sustained AF in a goat model. METHODS AND RESULTS: Thin sections from the left (LAA) and right atrial appendage (RAA) from goats in sinus rhythm (SR) or AF, induced through programmed endocardial burst pacing for time periods between 0 and 16 weeks, were immunolabeled with antibodies against connexin(Cx)40 and Cx43 and analysed by immunofluorescence and confocal laser scanning microscopy. During SR the distribution pattern for Cx43 was completely homogeneous (LAA and RAA) and for Cx40 mostly homogeneous (LAA: all five goats, RAA: three out of five goats). The distribution pattern for Cx43 remained stable during AF, while the Cx40 distribution pattern became increasingly heterogeneous, both in the LAA and RAA, with increasing duration of pacing. This increase in heterogeneity in Cx40 distribution correlated (Spearman rank order) with an increase in stability of AF and the occurrence of structural changes (myolysis) in atrial myocytes. The Cx40/Cx43 immunofluorescence signal ratio in both the LAA and RAA appeared to be significantly lower in AF (1-16 weeks) as compared to SR (0 weeks); going from 0 to 16 weeks average ratios decreased 54.5% (n=5; P=0.026) in the LAA and 35.8 (n=5; P=0.034) in the RAA. Western blot analyses revealed similar decreases in the total Cx40/Cx43 protein ratio, on average 50.0% (n=5; P=0.008) and 47.8% (n=5; P=0.02) in the LAA and RAA, respectively. No changes were measured in the levels of Cx40 or Cx43 mRNA, as was assessed through RT-PCR. CONCLUSION: The time course of changes in the distribution and content of Cx40 gap junctions as observed during endocardial burst pacing of the goat atrium suggests that Cx40 gap junctional remodeling might be involved in the pathogenesis of sustained atrial fibrillation.     

Differential densities of muscarinic acetylcholine receptor and I(K,ACh) in canine supraventricular tissues and the effect of amiodarone on cholinergic atrial fibrillation and I(K,ACh)

BACKGROUND: Vagal nerve plays an important role in the induction and maintenance of atrial fibrillation (AF). This study investigated the differential densities of M2 receptor and acetylcholine-induced inward rectifier K+ current (I(K,ACh)) in atrial appendage, atrium, pulmonary vein (PV) and super vena cava (SVC) to discuss the role of atrial appendage and PV in cholinergic AF. METHODS AND RESULTS: In 10 dogs, action potential duration was determined at 24 sites during bilateral cervical vagal stimulation and amiodarone administration. AF could be induced at first in right atrial appendage (RAA) and right atrium (RA) without left atrial appendage (LAA) and left atrium (LA). Amiodarone decreased the initiation of AF in vivo. Western blot and patch clamp were used to determine M2 receptor and I(K,ACh) in RAA, LAA, RA, LA, PV and SVC. The densities of M2 receptor and I(K,ACh) in LAA, RAA and LA were higher than that in RA, PV and SVC (21.34 +/- 0.92 vs. 8.24 +/- 0.45 pA/pF, p < 0.05). Furthermore, the densities of the M2 receptor and I(K,ACh) in LAA and RAA were higher than that in LA (21.34 +/- 0.92 vs. 14.17 +/- 0.65 pA/pF, p < 0.05). After amiodarone administration, densities of I(K,ACh) in LA and RA were not different, but densities of I(K,ACh )were also less in atrium than in atrial appendage. CONCLUSIONS: Densities of the M2 receptor and I(K,ACh) are higher in atrial appendage than other sites. Atrial appendage perhaps plays an important role in initiation of cholinergic AF. However, PV and SVC less often play an important role in vagotonic paroxysmal AF. Reduced dispersion of I(K,ACh) is the mechanism for amiodarone to therapy AF.     

持续快速心房起搏对犬肺静脉及心房组织连接蛋白43和Ⅲ型胶原的影响

目的　探讨持续快速心房起搏对犬肺静脉和心房组织连接蛋白 43(Cx43)和Ⅲ型胶原的影响。方法　16只杂种犬,随机分为持续快速心房起搏组(8只)和正常对照组 (8只 ),前者以 400次 /min的频率持续起搏 10周,建立心房颤动(房颤)动物模型。分别取两组犬的左上肺静脉、左房游离壁和右心耳等部位的心肌组织进行Cx43的免疫荧光半定量分析和Ⅲ型胶原纤维定量分析。结果10周后快速心房起搏组所有犬均可诱发出持续性房颤。快速心房起搏组犬肺静脉、左房游离壁和右心耳部位的Cx43水平显著高于正常对照组犬各相应的部位 (肺静脉: 3370 .91±275. 11与1405 .82±90. 38, P<0. 05;左房游离壁: 2448. 68±272 .10与 1467. 12±147 .93,P<0. 05;右心耳: 2331 .96±199 .61与 1288. 27±216 .22, P<0 .05)。快速心房起搏组犬肺静脉Cx43的水平显著高于左心房游离壁和右心耳(P<0. 05),而左心房和右心耳部位的Cx43水平差异无统计学意义 (P>0. 05)。持续快速心房起搏组犬肺静脉、左房游离壁和右心耳等部位的Ⅲ型胶原含量显著高于正常对照组犬各相应部位(肺静脉: 3301 97±309 70与 1404 56±178 02, P<0 05;左房游离壁: 2477 86±190. 43与1479. 20±187 .17, P<0 .05;右心耳: 2045 .92±139 .43与 1417. 07±139. 43,P<0 .05 )     

风湿性心脏瓣膜病心房颤动患者心房结构重构及胶原的表达

目的检测风湿性心脏瓣膜病心房颤动患者心房结构重构及胶原的表达,探讨胶原在心房结构重构中的意义。方法选择行开胸心脏手术的风湿性心脏瓣膜病患者39例,将持续性心房颤动患者24例为房颤组,窦性心律患者15例为窦律组。取患者心房组织标本,应用HE染色及Masson染色进行组织学检查;免疫组织化学法检测心房组织中Ⅰ型、Ⅲ型胶原蛋白的表达。结果房颤组患者较窦律组左心房内径明显增大。房颤组患者心房有肌溶解、心肌肥厚及广泛间质纤维化的改变。与窦律组比较,房颤组患者的左、右心房组织胶原容积分数均明显增大,左、右心房Ⅰ型胶原蛋白的表达明显增加(P0.05),而Ⅲ型胶原蛋白表达无明显差异(P0.05)。结论心房颤动患者心房结构发生明显的病理改变,其中心房间质纤维化为主要特征,左心房改变最为明显。     

Immunohistochemical localization and semi-quantification of atrial natriuretic polypeptide (ANP) in formalin-fixed-paraffin-embedded normal human hearts--comparative study with radioimmunoassay

The presence of atrial natriuretic polypeptide (ANP) was immunohistochemically demonstrated in the formalin-fixed and paraffin-embedded tissues of 25 autopsied normal human hearts using monoclonal antibody. The ANP amounts were immunohistochemically semiquantified and compared with amounts measured by radioimmunoassay (RIA). The 25 autopsied hearts were divided into 5 groups according to the interval of formalin fixation or the length of time between death and fixation. Formalin-fixation intervals were one week in group 1A and 1B; 1 year in group 2; 4 to 5 years in group 3 and 10 to 12 years in group 4. The hearts of group 1A, 2, 3 and 4 were fixed within 5 hours after death. Those of group 1B were fixed 14 to 18 hours at 4 degrees C. After fixation, the left and right atrial appendages (LAA and RAA), the left and right atrial free walls (LA and RA), the left and right ventricular free walls (LV and RV) and the ventricular septum (VS) were transmurally dissected from each heart. They were embedded in paraffin, cut into 4 microns sections and immunohistochemically stained by the avidin-biotin-peroxidase complex (ABC) method using monoclonal antibody against alpha-human ANP. Under a light microscope, they were evaluated semiquantitatively according to the incidence of ANP-positive cells and the intensity of immunostaining. For every heart in group 1A, the tissue concentrations of ANP in the different parts were also measured separately by RIA before fixation. ANP-positive myocytes were noted in the atria of all hearts of all groups, but no in any ventricular myocytes. Both their incidence and grade in the atria were similar among groups 1A, 1B and 2. However, they were less in group 3, and least in group 4 among all groups. For all groups, they decreased in the following order: LAA greater than RAA not equal to LA greater than RA; the inner 1/3 greater than the middle 1/3 greater than the outer 1/3 of the atrial walls. The order in LAA, RAA, LA and RA.(ABSTRACT TRUNCATED AT 250 WORDS)     

犬心房颤动模型缝隙连接蛋白40、45与心肌纤维化的相关性

目的 心房颤动(房颤)的发生及维持机制与心房结构重构和电重构有关.缝隙连接蛋白(connexin,Cx)是心肌闰盘的重要组成结构.一旦Cx出现重构,可影响心肌细胞电传导的极性,出现传导阻滞或折返,引发心律失常.实验通过快速心房起搏建立房颤模型,观察其对心房Cx40和Cx45及心房心肌纤维化的影响,并对两者相关性进行研究.方法 16只健康杂种犬随机分为模型组和对照组,2组犬均在X线下置入心房"J"型电极于右心耳,模型组予以400次/min快速起搏,而对照组维持窦性心律.连续起搏10周,分别在2、4、6、8周检测肢导联心电图.对于10周后未出现房颤的犬予以房颤诱发.实验结束后,取左心房组织制备心肌组织切片.Masson染色观察心房心肌胶原改变,电镜观察心房心肌超微结构及闰盘改变,放射免疫法测定血清中Ⅲ型前胶原氨基端肽和Ⅳ型胶原,免疫组化法检测Cx40及Cx45的表达.结果 模型组在快速起搏10周后均未出现自发性房颤,但其中有2只犬分别出现心房扑动和房性早搏,模型组和对照组予以Burst刺激后,模型组房颤诱发率可达66.7%,而对照组均正常.与对照组相比,模型组心房心肌胶原容积分数(collagen volume fraction,CVF)增加(P＜0.05),尤以心内膜和心房肌细胞间质纤维化明显.电镜下,模型组心房肌细胞超微结构可见肌纤维紊乱、断裂,胶原纤维增生,闰盘结构扭曲、扩张,部分闰盘缝隙消失.模型组血清中Ⅲ型前胶原氨基端肽和Ⅳ型胶原水平较对照组显著增高(P＜0.05);模型组心房心肌Cx40表达较对照组增加(P＜0.05),而Cx45蛋白改变二组差异无统计学意义(P＞0.05).将心房心肌组织CVF与Cx40行相关性分析,结果显示CVF与Cx40呈正相关(r=0.671).结论 犬心房快速起搏能诱导左心房组织心肌纤维化和Cx40表达增加,但对Cx45无影响,同时发现Cx40的改变程度受心房心肌纤维化程度的影响.

Abstract：

Objective Electrical and structural remodeling are of importance for the occurrence and maintenance of atrial fibrillation. We observed association between atrial connexin protein expression and fibrosis in a canine model of prolonged rapid atrial pacing. Methods "J"-type electrodes were placed in the right atrial appendage under the guidance of X-ray in 16 dogs, Animals in model group ( n = 8) received fast pacing (400 beats/min ) for 10 weeks while animals in control group (n =8) maintained at sinus rhythm.Limb-lead ECGs were recorded at 2,4,6,8 weeks respectively. Burst stimulation was applied to induce atrial fibrillation in all animals after 10 weeks, animals were sacrificed thereafter and the left atrial tissues were taken for myocardial collagen measurement ( Masson staining) and myocardial ultrastructure examination and detection of protein expression of connexin ( Cx ) 40 and 45 ( immune staining). Procollagen type Ⅲ N-terminal peptide and type Ⅳ collagen in serum were also detected by radioimmunoassay. Results Two dogs died in model group due to atrial rupture induced cardiac tamponade or lung emboli. Spontaneously atrial fibrillation was not observed in all animals, but two dogs developed atrial flutter and atrial premature beats. Atrial fibrillation was induced by burst stimulation in 4 out of 6 dogs in model group and in none of the dogs in control group. Atrial myocardial collagen volume fraction was significantly increased in model group compared with the control group (P ＜ 0. 05). Ultrastructure examination in atrial tissue evidenced disorder,fracture,collagen fiber proliferation, mitochondrial swelling, blurred cristae, and intercalated disc distortion,expansion, part of gap junction disappears in model group. The serum levels of procollagen type Ⅲ N-terminal peptide and type Ⅳ collagen in model group were significantly higher than in the control group ( P ＜ 0. 05 ). The protein expression of Cx40 in atrial myocardium in model group was significantly higher than in control group (P ＜ 0. 05 ), while Cx45 protein expression was similar between two groups (P ＞0. 05). The left atrial CVF was positively correlated with Cx40 ( r = 0. 671, P ＜ 0. 01 ). Conclusion Increased myocardial fibrosis is positively correlated with upregulation of myocardial Cx40 protein expression in left atrium in rapid atrial paced canine.