Connexin43基因与先天性心脏病发病机制的研究
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摘要
先天性心脏病(简称先心病)是人类最常见的出生缺陷之一，其发病机理的研究已经成为热点课题，研究者试图找出其发病的原因并加以预防，减少发病率，以提高出生人口的质量。遗传因素日渐受到重视，某些控制心脏发育基因的突变或功能缺陷可能是先心病的分子基础。其中，连接蛋白43(Connexin43，Cx43)基因作为先心病的重要候选基因之一正被日益关注。Cx43是哺乳动物心脏中最主要的连接蛋白，其在细胞膜上聚合而成的缝隙连接通道是介导相邻细胞间离子、小分子代谢物和信号分子跨膜交流的重要结构基础。近年来一系列动物模型的研究提示Cx43在心脏形态发育中具有重要作用，Cx43基因缺陷或过量表达均可导致以右室流出道肥厚梗阻为主的心脏畸形。但目前关于人类先心病与Cx43基因异常之间关系的研究还非常有限，Cx43基因在先心病发病中所起的作用迄今还未有明确的结论。本研究通过Cx43基因敲除(Cx43KO)小鼠，观察其胚胎期的心脏发育及其可能的病理机制。同时，对汉族先心病患者进行一个大样本的Cx43基因突变检测的研究，较全面地观察Cx43基因突变是否与先心病发病相关，并检测先心病患者心肌Cx43基因的表达，为阐述先心病的病理机制提供有价值的资料。
第一部分Cx43基因敲除小鼠心脏表型及相关基因的表达
研究目的
观察Cx43基因敲除(Cx43KO)小鼠心脏异常发育的表型及其胚胎期心脏中Cx40和Cx45的时空表达规律，探讨其发生心脏畸形的可能机制。
材料和方法
2月龄Cx43KO杂合小鼠交配，选用其后代胚胎期(embryonic day，ED)13.5天至出生后1天小鼠作为研究对象，采用PCR方法鉴定基因型。根据基因型分为Cx43KO纯合子(Cx43-／-)、杂合子(Cx43+／-)及野生型(Cx43+／+)；C57BL6小鼠作为对照组。小鼠进行显微解剖观察心脏大体结构，心脏组织切片，HE染色观察心脏形态学结构。同时选取ED10.5～15.5的Cx43KO纯合子及野生型小鼠胚胎，免疫组化ABC法检测Cx40、Cx45在心脏的时空表达，SCIM显微图像分析系统对染色强度进行定量分析。
结果
1．Cx43KO新生小鼠在解剖显微镜下观察发现：纯合型Cx43KO小鼠心脏主要表现为肺动脉近右室流出道端明显膨隆突出；而野生型、杂合型和C57BL6小鼠心脏大体结构未见异常。
2．心脏切片HE染色显示：胚胎期及新生纯合型Cx43KO小鼠心脏主要表现为右室流出道明显梗阻；野生型、杂合型和C57BL6小鼠心脏形态学结构未见异常。
3．野生型小鼠胚胎心脏Cx40从ED11.5开始在右室表达，随后逐渐分布于各房室，ED14.5后在心室的表达开始减弱，出生后主要分布于心房、希氏束及浦氏纤维；Cx45在ED10.5表达于心脏流入道、流出道心肌及房室垫，但随后表达开始下调，仅在流出道继续表达，成年后主要在室间隔部分表达。Cx43KO小鼠Cx40和Cx45的时空表达在ED10.5～15.5这一心脏分隔、瓣膜发育的关键时期却较野生型明显减弱(P＜0.05)。
结论
1．Cx43KO小鼠存在明显的心脏发育异常，主要表现为右室流出道梗阻。
2．Cx43KO小鼠心脏发育过程中Cx40和Cx45时空表达出现异常，这可能是其发生心脏畸形的相关的潜在机制。
第二部分Cx43基因在先天性心脏病中的突变检测
研究目的
检测先心病患者中Cx43基因的突变，观察Cx43基因突变与人类先心病的关系，为阐述先心病的病因提供有价值的资料。
材料和方法
研究对象包括试验组250例不同类型的先心病患者，40例健康儿童为对照组。试验组分成复杂先心病和简单先心病两个亚组：复杂先心病182例，主要以心脏圆锥动脉干畸形为主；68例简单先心病包括室间隔缺损(VSD)、房间隔缺损(ASD)、动脉导管未闭(PDA)和肺动脉狭窄(PS)。基因组DNA全部从外周血提取，同时亦从心脏手术中收集部分先心病患者的右室流出道心肌中提取DNA，与外周血相对照。在Cx43编码序列两侧设计一对引物(F1和R3)，扩增片段长度为1348bp。其中上游引物设计在内含子中，以避免基因组中Cx43假基因的非特异扩增。同时在编码区内设计4条引物(R1、F2、R2和F3)，并与2条外侧引物一起配成3对作为测序引物。PCR产物纯化后进行DNA直接测序。
结果
1．所有标本均成功由PCR扩增出Cx43基因的全长编码序列，PCR产物的长度为1348bp，与所设计的片段大小预期值符合，特异性理想。
2．所有研究对象的Cx43基因测序后同GenBank人类Cx43编码序列进行比较，仅有1例患者(法洛四联症)存在1个单核苷酸的多态性(single nucleotide polymorphism,SNP)，为924位核苷酸G→A。其余研究对象的测序结果均未发现Cx43基因突变。
结论
1．人类先心病中Cx43基因编码区可能不存在规律性的基因位点突变。
2．Cx43基因外显子的突变在先心病中可能很少见，进一步说明人类心脏的发育是一个涉及多基因、多环节、多因子的精细复杂的过程，Cx43基因参与人类先心病的发病需要更深入的研究。
第三部分Cx43基因在法洛四联症心肌中表达的研究
研究目的
检测法洛四联症(tetralogy of Fallot，TOF)患者心肌Cx43基因的表达。为解释Cx43基因与某些先心病的关系提供证据。
材料和方法
试验组为16例TOF的右室流出道心肌，4例单纯VSD和1例原发性肺动脉高压患者的右室流出道心肌为对照，通过RT-PCR的方法检测Cx43 mRNA的表达差异，SCIM显微图像分析系统对灰度值进行半定量分析。进一步对心肌切片行免疫组化ABC法，观察心肌细胞中Cx43蛋白的表达。
结果
1．与对照相比，TOF患者Cx43基因表达水平明显增高。从Cx43／β-actin的比值可见，TOF患者Cx43基因mRNA水平显著高于对照(P＜0.01)。
2．免疫组化提示TOF右室心肌细胞具有较高的Cx43表达，而对照心肌则未见明显的Cx43表达。
结论
1．TOF右室流出道心肌Cx43的高表达提示胚胎期心脏发育过程中可能存在Cx43时空表达紊乱。
2．Cx43基因转录表达的异常可能影响相关心脏发育因子的调控作用，从而导致参与心脏发育基因表达紊乱，这可能是TOF发病的一种潜在机制。
3．TOF作为人类最常见的先天性圆锥动脉干畸形，其心肌Cx43基因的异常表达为探讨Cx43基因功能缺陷导致先心病提供了新的线索。
总结
Cx43KO小鼠存在心脏畸形和Cx40、Cx45基因表达异常，表明Cx43基因与心脏发育密切相关，提示其在心脏发育过程中存在精细的调节机制。就人类而言，大多数先心病患者中可能不存在规律性的Cx43基因突变，提示Cx43基因突变引起先心病并不多见，Cx43基因可能是心脏发育中的重要环节之一；TOF心肌Cx43基因的异常表达提示其功能缺陷对先心病发病所起的作用。人类心脏的发育是一个多基因、多信号和多环节参与的复杂过程，Cx43基因异常可能导致这一过程出现紊乱，同时引起相关心脏发育因子的异常，从而导致先心病的发生。因此揭示Cx43与其它相关基因在心脏发育中相互作用及调控机制需要更深入的研究。
Congenital heart disease (CHD) is one of the common congenital defects in human and the pathogenesis of CHD has already been more highlighted. Scientists have been working hard to find out the causes of CHD in order to prevent the occurrence of CHD beforehand and improve the quality of population. Hereditary factors have been recognized in process of research and mutations of some genes controlling human heart development maybe the molecular basis of CHD. Connexin43 (Cx43), the major gap junction protein in the mammalian heart, makes up gap junction channels that mediate the cell-to-cell diffusion of ions, small metabolites, and small cell signaling molecules. Recently, a series of animal models suggest that Cx43 plays an important role in the morphogenesis of heart, especially the pulmonary conotruncal region. However, the role of Cx43 gene in the development of CHD in human being is still unclear. Hence, in this study Cx43 gene knockout mice was used to observe the heart morphology and further reveal the pathogenesis mechanism of the heart malformations in Cx43 gene defect mouse model. Cx43 gene coding sequence was also detected in a large number of Chinese Han patients with CHD to identify the association of Cx43 gene mutation with CHD. Furthermore, Cx43 expression in the myocardium was detected in some patients with CHD to illustrate the potential mechanism of CHD.
Part I Cardiac Morphology and Expression of Associated Genes in Cx43 Gene Knockout Mice
Objective
To observe cardiac morphology of Cx43 knockout (Cx43KO) mice and investigate the spatio-temporal expression of Cx40 and Cx45 in Cx43KO mice. Materials and methods
The objects were offsprings of mice of ED13.5 to 1 day after birth by the mating of 2-month-old heterozygous Cx43KO mice, which included Cx43KO homozygotes
(Cx43-/-), heterozygotes (Cx43+/-) and wild types (Cx43+/+) genotyped by PCR method. C57BL6 mice were used as control. Micro-dissection and HE staining were used to examine the structures of the mouse hearts. Immuno-histochemistry was used to detect Cx40 and Cx45 expression in the myocardium of Cx43KO mice from ED10.5 tol5.5. Results
1. Cx43-/- mice died within 24hr after birth with a swelling and blockage of the conotruncal region, which led to the obstruction of outlet of right ventricle and enlargement of right ventricle. In control C57BL6 mice did not exhibit any heart malformations.
2. HE staining showed that the homozygous Cx43KO mice had severe right ventricular outflow tract obstruction (RVOTO). In contrast, C57BL6 mice did not exhibit any heart malformations.
3. With the wide type mouse fetal heart development, Cx40 expression was detected in right ventricle in ED11.5. Subsequently it was distributed in atria and ventricles. Peak expression of Cx40 was in ED14.5, then it faded. After birth it was only detected in atria and His-Purkinje conductive system. Cx45 expression was detected in inflow tract, outflow tract and atrio-ventricular cushion in ED10.5. Then it faded and distributed in outflow tract. After birth it was detected only in interventricular septum. Cx40 and Cx45 expression in Cx43KO mouse fetal hearts were obviously weakened as compared with wild type (P<0.05).
Conclusions
1. Cx43 gene defect is obviously associated with abnormal heart morphogenesis, especially in the conotruncal outflow region.
2. Lower expression of Cx40 and Cx45 in Cx43KO mouse myocardium maybe the potential mechanism of heart malformations.
Part II Mutations of Cx43 in Patients with Congenital Heart
Disease
Objective
To detect the mutations of Cx43 gene in patients with congenital heart disease (CHD), and illustrate the relationship between Cx43 gene mutations and CHD in the human being. Materials and methods
Two hundred and fifty patients with CHD were recruited in this study, among which 182 patients had a variety of complex heart defects and 68 patients had simple heart defects, such as atrial septal defect (ASD), ventricular septal defect (VSD), pulmonary stenosis (PS) and patent ductus arteriosus (PDA). Forty children without CHD were selected as control. Samples were obtained from peripheral blood in all subjects, and from myocardial tissue in some patients. The entire coding region of Cx43 was amplified by PCR with the primers F1 and R3. A small portion of an intron in the 5' untranslated region of the gene (the coding region contains no introns) was designed to exclude any contribution from a processed pseudogene. Then the PCR products were purified and directly sequenced by using sequencing primers F1, F2, F3, R1, R2 and R3. Sequencing products were run on ABI 3700 DNA sequencer and both coding and the antisense strand of the Cx43 gene were sequenced. Results
The entire Cx43 coding sequences of all subjects were analyzed and compared with human Cx43 coding sequence. No Cx43 mutation was found except for a single nucleotide polymorphism (SNP) in a patient with TOF. Conclusions
1. Our results indicate that Cx43 gene mutations may be very rare in patients with CHD.
2. The morphogenesis of the human heart is a very complex process, in which a lot of genes are involved. Cx43 may take an important role in the signal pathway for the heart development.
Part III Expression of Cx43 in Patients with Tetralogy of Fallot
Objective
To detect expression Cx43 in patients with tetralogy of Fallot (TOF), which
would give some clues to pathogenesis of the disease. Materials and methods
Sixteen patients with TOF were recruited in this study and 4 patients with VSD and 1 patient with primary pulmonary hypertension were selected as control. The myocardial samples were obtained from right ventricle outlet of hearts. They were analyzed by RT-PCR. Moreover, immuno-histochemistry was used to observe Cx43 expression in the myocardium. Results
1. More Cx43 in mRNA level was observed as compared with controls.
2. Plenty of Cx43 protein was expressed in the myocardium in patients with TOF, while no expression of Cx43 was observed in the control.
Conclusions
1. The higher expression of Cx43 in the myocardium indicates that the turbulence of Cx43 spatio-temporal expression in heart morphogenesis in patients with TOF.
2. The abnormal expression of Cx43 in the myocardium gives a clue to the potential pathogenesis of TOF.
Summary
Cx43 gene is involved in heart morphogenesis with a complicated delicate mechanism. However, Cx43 gene mutations rarely exist in patients with CHD. Cx43 gene defect may cause turbulence of some related genes in the process of cardiomorphogenesis and lead to heart malformations.
第一部分Cx43基因敲除小鼠心脏表型及相关基因的表达
研究目的
观察Cx43基因敲除(Cx43KO)小鼠心脏异常发育的表型及其胚胎期心脏中Cx40和Cx45的时空表达规律，探讨其发生心脏畸形的可能机制。
材料和方法
2月龄Cx43KO杂合小鼠交配，选用其后代胚胎期(embryonic day，ED)13.5天至出生后1天小鼠作为研究对象，采用PCR方法鉴定基因型。根据基因型分为Cx43KO纯合子(Cx43-／-)、杂合子(Cx43+／-)及野生型(Cx43+／+)；C57BL6小鼠作为对照组。小鼠进行显微解剖观察心脏大体结构，心脏组织切片，HE染色观察心脏形态学结构。同时选取ED10.5～15.5的Cx43KO纯合子及野生型小鼠胚胎，免疫组化ABC法检测Cx40、Cx45在心脏的时空表达，SCIM显微图像分析系统对染色强度进行定量分析。
结果
1．Cx43KO新生小鼠在解剖显微镜下观察发现：纯合型Cx43KO小鼠心脏主要表现为肺动脉近右室流出道端明显膨隆突出；而野生型、杂合型和C57BL6小鼠心脏大体结构未见异常。
2．心脏切片HE染色显示：胚胎期及新生纯合型Cx43KO小鼠心脏主要表现为右室流出道明显梗阻；野生型、杂合型和C57BL6小鼠心脏形态学结构未见异常。
3．野生型小鼠胚胎心脏Cx40从ED11.5开始在右室表达，随后逐渐分布于各房室，ED14.5后在心室的表达开始减弱，出生后主要分布于心房、希氏束及浦氏纤维；Cx45在ED10.5表达于心脏流入道、流出道心肌及房室垫，但随后表达开始下调，仅在流出道继续表达，成年后主要在室间隔部分表达。Cx43KO小鼠Cx40和Cx45的时空表达在ED10.5～15.5这一心脏分隔、瓣膜发育的关键时期却较野生型明显减弱(P＜0.05)。
结论
1．Cx43KO小鼠存在明显的心脏发育异常，主要表现为右室流出道梗阻。
2．Cx43KO小鼠心脏发育过程中Cx40和Cx45时空表达出现异常，这可能是其发生心脏畸形的相关的潜在机制。
第二部分Cx43基因在先天性心脏病中的突变检测
研究目的
检测先心病患者中Cx43基因的突变，观察Cx43基因突变与人类先心病的关系，为阐述先心病的病因提供有价值的资料。
材料和方法
研究对象包括试验组250例不同类型的先心病患者，40例健康儿童为对照组。试验组分成复杂先心病和简单先心病两个亚组：复杂先心病182例，主要以心脏圆锥动脉干畸形为主；68例简单先心病包括室间隔缺损(VSD)、房间隔缺损(ASD)、动脉导管未闭(PDA)和肺动脉狭窄(PS)。基因组DNA全部从外周血提取，同时亦从心脏手术中收集部分先心病患者的右室流出道心肌中提取DNA，与外周血相对照。在Cx43编码序列两侧设计一对引物(F1和R3)，扩增片段长度为1348bp。其中上游引物设计在内含子中，以避免基因组中Cx43假基因的非特异扩增。同时在编码区内设计4条引物(R1、F2、R2和F3)，并与2条外侧引物一起配成3对作为测序引物。PCR产物纯化后进行DNA直接测序。
结果
1．所有标本均成功由PCR扩增出Cx43基因的全长编码序列，PCR产物的长度为1348bp，与所设计的片段大小预期值符合，特异性理想。
2．所有研究对象的Cx43基因测序后同GenBank人类Cx43编码序列进行比较，仅有1例患者(法洛四联症)存在1个单核苷酸的多态性(single nucleotide polymorphism,SNP)，为924位核苷酸G→A。其余研究对象的测序结果均未发现Cx43基因突变。
结论
1．人类先心病中Cx43基因编码区可能不存在规律性的基因位点突变。
2．Cx43基因外显子的突变在先心病中可能很少见，进一步说明人类心脏的发育是一个涉及多基因、多环节、多因子的精细复杂的过程，Cx43基因参与人类先心病的发病需要更深入的研究。
第三部分Cx43基因在法洛四联症心肌中表达的研究
研究目的
检测法洛四联症(tetralogy of Fallot，TOF)患者心肌Cx43基因的表达。为解释Cx43基因与某些先心病的关系提供证据。
材料和方法
试验组为16例TOF的右室流出道心肌，4例单纯VSD和1例原发性肺动脉高压患者的右室流出道心肌为对照，通过RT-PCR的方法检测Cx43 mRNA的表达差异，SCIM显微图像分析系统对灰度值进行半定量分析。进一步对心肌切片行免疫组化ABC法，观察心肌细胞中Cx43蛋白的表达。
结果
1．与对照相比，TOF患者Cx43基因表达水平明显增高。从Cx43／β-actin的比值可见，TOF患者Cx43基因mRNA水平显著高于对照(P＜0.01)。
2．免疫组化提示TOF右室心肌细胞具有较高的Cx43表达，而对照心肌则未见明显的Cx43表达。
结论
1．TOF右室流出道心肌Cx43的高表达提示胚胎期心脏发育过程中可能存在Cx43时空表达紊乱。
2．Cx43基因转录表达的异常可能影响相关心脏发育因子的调控作用，从而导致参与心脏发育基因表达紊乱，这可能是TOF发病的一种潜在机制。
3．TOF作为人类最常见的先天性圆锥动脉干畸形，其心肌Cx43基因的异常表达为探讨Cx43基因功能缺陷导致先心病提供了新的线索。
总结
Cx43KO小鼠存在心脏畸形和Cx40、Cx45基因表达异常，表明Cx43基因与心脏发育密切相关，提示其在心脏发育过程中存在精细的调节机制。就人类而言，大多数先心病患者中可能不存在规律性的Cx43基因突变，提示Cx43基因突变引起先心病并不多见，Cx43基因可能是心脏发育中的重要环节之一；TOF心肌Cx43基因的异常表达提示其功能缺陷对先心病发病所起的作用。人类心脏的发育是一个多基因、多信号和多环节参与的复杂过程，Cx43基因异常可能导致这一过程出现紊乱，同时引起相关心脏发育因子的异常，从而导致先心病的发生。因此揭示Cx43与其它相关基因在心脏发育中相互作用及调控机制需要更深入的研究。
Congenital heart disease (CHD) is one of the common congenital defects in human and the pathogenesis of CHD has already been more highlighted. Scientists have been working hard to find out the causes of CHD in order to prevent the occurrence of CHD beforehand and improve the quality of population. Hereditary factors have been recognized in process of research and mutations of some genes controlling human heart development maybe the molecular basis of CHD. Connexin43 (Cx43), the major gap junction protein in the mammalian heart, makes up gap junction channels that mediate the cell-to-cell diffusion of ions, small metabolites, and small cell signaling molecules. Recently, a series of animal models suggest that Cx43 plays an important role in the morphogenesis of heart, especially the pulmonary conotruncal region. However, the role of Cx43 gene in the development of CHD in human being is still unclear. Hence, in this study Cx43 gene knockout mice was used to observe the heart morphology and further reveal the pathogenesis mechanism of the heart malformations in Cx43 gene defect mouse model. Cx43 gene coding sequence was also detected in a large number of Chinese Han patients with CHD to identify the association of Cx43 gene mutation with CHD. Furthermore, Cx43 expression in the myocardium was detected in some patients with CHD to illustrate the potential mechanism of CHD.
Part I Cardiac Morphology and Expression of Associated Genes in Cx43 Gene Knockout Mice
Objective
To observe cardiac morphology of Cx43 knockout (Cx43KO) mice and investigate the spatio-temporal expression of Cx40 and Cx45 in Cx43KO mice. Materials and methods
The objects were offsprings of mice of ED13.5 to 1 day after birth by the mating of 2-month-old heterozygous Cx43KO mice, which included Cx43KO homozygotes
(Cx43-/-), heterozygotes (Cx43+/-) and wild types (Cx43+/+) genotyped by PCR method. C57BL6 mice were used as control. Micro-dissection and HE staining were used to examine the structures of the mouse hearts. Immuno-histochemistry was used to detect Cx40 and Cx45 expression in the myocardium of Cx43KO mice from ED10.5 tol5.5. Results
1. Cx43-/- mice died within 24hr after birth with a swelling and blockage of the conotruncal region, which led to the obstruction of outlet of right ventricle and enlargement of right ventricle. In control C57BL6 mice did not exhibit any heart malformations.
2. HE staining showed that the homozygous Cx43KO mice had severe right ventricular outflow tract obstruction (RVOTO). In contrast, C57BL6 mice did not exhibit any heart malformations.
3. With the wide type mouse fetal heart development, Cx40 expression was detected in right ventricle in ED11.5. Subsequently it was distributed in atria and ventricles. Peak expression of Cx40 was in ED14.5, then it faded. After birth it was only detected in atria and His-Purkinje conductive system. Cx45 expression was detected in inflow tract, outflow tract and atrio-ventricular cushion in ED10.5. Then it faded and distributed in outflow tract. After birth it was detected only in interventricular septum. Cx40 and Cx45 expression in Cx43KO mouse fetal hearts were obviously weakened as compared with wild type (P<0.05).
Conclusions
1. Cx43 gene defect is obviously associated with abnormal heart morphogenesis, especially in the conotruncal outflow region.
2. Lower expression of Cx40 and Cx45 in Cx43KO mouse myocardium maybe the potential mechanism of heart malformations.
Part II Mutations of Cx43 in Patients with Congenital Heart
Disease
Objective
To detect the mutations of Cx43 gene in patients with congenital heart disease (CHD), and illustrate the relationship between Cx43 gene mutations and CHD in the human being. Materials and methods
Two hundred and fifty patients with CHD were recruited in this study, among which 182 patients had a variety of complex heart defects and 68 patients had simple heart defects, such as atrial septal defect (ASD), ventricular septal defect (VSD), pulmonary stenosis (PS) and patent ductus arteriosus (PDA). Forty children without CHD were selected as control. Samples were obtained from peripheral blood in all subjects, and from myocardial tissue in some patients. The entire coding region of Cx43 was amplified by PCR with the primers F1 and R3. A small portion of an intron in the 5' untranslated region of the gene (the coding region contains no introns) was designed to exclude any contribution from a processed pseudogene. Then the PCR products were purified and directly sequenced by using sequencing primers F1, F2, F3, R1, R2 and R3. Sequencing products were run on ABI 3700 DNA sequencer and both coding and the antisense strand of the Cx43 gene were sequenced. Results
The entire Cx43 coding sequences of all subjects were analyzed and compared with human Cx43 coding sequence. No Cx43 mutation was found except for a single nucleotide polymorphism (SNP) in a patient with TOF. Conclusions
1. Our results indicate that Cx43 gene mutations may be very rare in patients with CHD.
2. The morphogenesis of the human heart is a very complex process, in which a lot of genes are involved. Cx43 may take an important role in the signal pathway for the heart development.
Part III Expression of Cx43 in Patients with Tetralogy of Fallot
Objective
To detect expression Cx43 in patients with tetralogy of Fallot (TOF), which
would give some clues to pathogenesis of the disease. Materials and methods
Sixteen patients with TOF were recruited in this study and 4 patients with VSD and 1 patient with primary pulmonary hypertension were selected as control. The myocardial samples were obtained from right ventricle outlet of hearts. They were analyzed by RT-PCR. Moreover, immuno-histochemistry was used to observe Cx43 expression in the myocardium. Results
1. More Cx43 in mRNA level was observed as compared with controls.
2. Plenty of Cx43 protein was expressed in the myocardium in patients with TOF, while no expression of Cx43 was observed in the control.
Conclusions
1. The higher expression of Cx43 in the myocardium indicates that the turbulence of Cx43 spatio-temporal expression in heart morphogenesis in patients with TOF.
2. The abnormal expression of Cx43 in the myocardium gives a clue to the potential pathogenesis of TOF.
Summary
Cx43 gene is involved in heart morphogenesis with a complicated delicate mechanism. However, Cx43 gene mutations rarely exist in patients with CHD. Cx43 gene defect may cause turbulence of some related genes in the process of cardiomorphogenesis and lead to heart malformations.
引文
1 刘薇廷，宁寿葆，华邦杰等．上海市杨浦、徐汇区小儿先天性心脏病发病率及其特点。中华儿科杂志，1995，33(6)：347-349．
2 Hoffman JI, Kaplan S. The incidence of congenital heart disease. J Am Coil Cardiol, 2002, 39 (12): 1890-1900.
3 Bruzzone R, White TW, Paul DL. Connections with connexins: the molecular basis of direct intercellular signaling. Eur J Biochem, 1996, 238(1): 1-27.
4 Goodenough DA, Goliger JA, Paul DL. Connexins, connexons and intercellular communication. Annu Rev Biochem, 1996, 65: 475-502.
5 Reaume AG, de Sousa PA, KulkamiS, et al. Cardiac malformation in neonatal mice lacking connexin43. Science, 1995, 267(5205): 1831-1834.
6 Huang GY, Wessels A, Smith BR, et al. Alteration in connexin 43 gap junction gene dosage impairs conotruncal heart developmentDev Biol, 1998, 198(1): 32-44.
7 Ya J, Erdtsieck-Ernste EBHW, Boer PAJ, et al. Heart defects in cormexin43-deficient mice. Circ Res, 1998, 82(3): 360-366.
8 Sullivan R, Huang GY, Meyer RA, et al. Heart malformations in transgenic mice exhibiting dominant negative inhibition of gap junctional communication in neural crest cells. Dev Biol, 1998, 204(1): 224-234.
9 Ewart JL, Cohen MF, Meyer RA, et al. Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene. Development, 1997, 124(7): 1281-1292.
10 Huang GY, Cooper ES, Waldo K, et al. Gap junction-mediated cell-cell communication modulates mouse neural crest migration. J Cell Biol, 1998, 143(6): 1725-1734.
11 Britz-Cunningham SH, Shah MM, Zuppan CW, et al. Mutations of the connexin43 gap-junction gene in patients with heart malformations and defects of laterality. N Engl J Med, 1995, 332(20): 1323-1329.
12 Splitt MP, Burn J, Goodship J, et al. Connexin43 mutation in sporadic and familial defects of laterality. N Engl J Meal, 1995, 333: 941-942.
13 Gebbia M, Towbin JA, Casey B. Failure to detect connexin43 mutations in 38 cases of sporadic and familial heterotaxy. Circulation, 1996, 94(8): 1909-1912.
14 Splitt MP, Tsai MY, Burn J, Goodship JA. Absence of mutations in the regulatory domain of the gap junction protein connexin 43 in patients with visccroatrial heterotaxy. Heart, 1997, 77: 369-370.
15 Debrus S, Tuffery S, Matsuoka R, et al. Lack of evidence for connexin 43 gene mutations in human autosomal recessive lateralization defects. J Molec Cell Cardiol, 1997, 29(5): 1423-1431.
16 Dasgupta C, Martinez AM, Zuppan CW, et al. Identification of cormexin43 (α1) gap junction gene mutations in patients with hypoplastic left heart syndrome by denaturing gradient gel electrophoresis (DGGE). Mutat Res, 2001, 479(1-2): 173-186.
17 Frank C, Hong Gu, Steven M, et al. High Incidence of Cardiac Malformations in Connexin40-Deficient Mice. Circ Res, 2003, 93(3): 201-206.
18 Madoka K, Kiyomasa N, Kei-ichiro Net al. Loss of connexin45 causes a cushion defect in early cardiogenesis. Development, 2000, 127(16): 3501-3512.
19 Nishii K, Kumai M, Egashira K, et al. Mice lacking connexin45 conditionally in cardiac myocytes display embryonic lethality similar to that of germline knockout mice without endocardial cushion defect. Cell Communication & Adhesion, 2003, 10(4-6): 365-9.
20 庞玉生，沈媛，黄国英等。应用PCR测定Cx43转基因小鼠的基因型。广西医科大学学报，2003，20(5)：657-658．
21 Lo CW, Cohen MF, Huang GY, et al. Cx43 gap junction gene expression and gap junction communication in mouse neural crest cells. Dev G-enet, 1997, 20(2): 119-132.
22 谢利剑，黄国英，赵晓晴等。Connexin43基因缺陷新生小鼠心脏组织病理学研究。中华医学杂志，2005，85(18)：1249-1251．
23 Yu Q, Shen Y, Chatterjee B, et al. ENU induced mutations causing congenital cardiovascular anomalies. Dev, 2004, 131 (24): 6211-6223.
24 Gros D, Jongsma HJ. Connexins in mammalian heart function. BioEssays, 1996, 18: 719-730.
25 Delorme B, Dahl E, Jarry-Guichard T, et al. Expression pattern of connexin gene products at the early developmental stages of the mouse cardiovascular system. Circ Res, 1997, 81(3): 423-437.
26 Delorme B, Dahl E, Jarry-Guichard T, et al. Developmental regulation of connexin 40 gene expression in mouse heart correlates with the differentiation of the conduction system. Dev Dyn, 1995, 204(4): 358-371.
27 Sebastien A, Magali T, Therese J, et al. Down regulation of Connexin 45 Gene Products During Mouse Heart Development. Circ Res, 1999, 84: 1365-1379.
28 Chen P, Xie LJ, Huang GY, et al. Mutations of connexin43 in fetuses with congenital heart malformations. Chin Med J, 2005, 118(12): 971-976.
29 Forsthoefel KF, Papp AC, Snyder PJ, et al. Optimization of DNA extraction from formalin-fixed tissue and its clinical application in Duchenne muscular dystrophy. Am J Clin Pathol, 1992, 98(1): 98-104.
30 Fishman GI, Eddy RL, Shows TB, et al. The human connexin gene family of gap junction proteins: distinct chromosomal locations but similar structures. Genomics, 1991, 10(1): 250-256.
31 Devor EJ. Use of molecular beacons to verify that the serine hydroxymethyltransferase pseudogene SHMT-psl is unique to the order Primates. Genome Biol. 2001, 2(2): 101.
32 Zhang J Dyer KD. Rosenberg HF, et al. Evolution of the rodent eosinophil-associated RNase gene family by rapid gene sorting and positive selection. Proc Natl Acad Sci USA, 2000, 97(9): 4701-4706.
33 王秀敏，尚世强。脊椎动物假基因。国外医学分子生物学分册，2003，25(6)：327-330．
34 王秀敏，韩蓓，顾学范。CYP21基因启动子结构与功能突变研究。国外医学分子生物学分册，2001，23(1)：52-54．
35 Toyofuku T, Yabuki M, Otsu K, et al. Direct association of the gap junction protein cormexin43 with ZO-1 in cardiac myocytes. J Biol Chem, 1998, 273 (21 ): 12725-12731.
36 Levin M, Mercola M. Gap junction-mediated transfer of left-right patterning signals in the early chick blastoderm is upstream of shh asymmetry in the node. Development, 1999, 126(21): 4703-4714.
37 Thomas MA, Zosso N, Scerri I, et al. A tyrosine-based sorting signal is involved in connexin43 stability and gap junction turnover. J Cell Sci, 2003, 116(11), 2213-2222.
38 Pizzuti A, Flex E, Mingarelli RA, et al. A homozygous GJA1 gene mutation causes a Hallermarm-Streiff/ODDD spectrum phenotype. Human Mutation, 2004, 23(3): 286.
39 Paznekas WA, Boyadjiev SA, Shapiro RE, et al. Connexin 43 (GJA1) Mutations Cause the Pleiotropic Phenotype of Oculodentodigital Dysplasia. Am. J. Hum. Genet, 2003, 72(2): 408-418.
40 李广镰，张开滋，Cheng TO主编．心血管遗传病学．北京：北京医科大学中国协和医科大学联合出版社．1994：29-58．
41 钟秋安，仇小强。先天性心脏病的病因研究进展。中国儿童保健杂志，2003，11(4)：259-261．
42 GELB BD. Recent advances in the understanding of genetic causes of congenital heart defects. Front Biosci, 2000, 5: 321-333
43 Basson CT, Bachinshy DR, Lin RC, et al. Mutations in human TBX5 cause limb and cardiac malformations in Holt-Oram syndrome. Nat Genet, 1997, 15(1): 30-35.
44 Schott JJ, Benson DW, Basson CT, et al. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science, 1998, 281 (5373): 108-111.
45 GOLDMUNTZ E, GEIGER E, BENSON DW, et al. Nkx 2.5 mutations in patients with tetralogy of fallot. Circulation, 2001, 104(21): 2565-2568.
46 Kurihara Y, Kurihara H, Oda H, et al. Aortic arch malformations and ventricular septal defect in mice deficient in endothelin-1. J Clin Invest. 1995 96(1): 293-300.
47 Meyer D, Birchmeier C. Multiple essential functions of neuregulin in development Nature 1995 378(6555): 386-390.
48 Molkentin JD, Olson EN. GATA4: a novel transcriptional regulator of cardiac hypertrophy? Circulation, 1997, 96(11): 3833-3835.
49 van Kempen MJ, Fromaget C, Gros D, et al. Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart. Circ Res, 1991, 68(6): 1638-51.
50 Fishman GI, Hertzberg EL, Spray DC, et al. Expression of cormexin43 in the developing rat heart. Circ Res, 1991, 68(3): 782-787.
51 陈萍，黄国英，谢利剑等。Cx43基因在人类及小鼠胎心发育中的时空表达规律。中国实验动物学报，2005，13(1)：16-19．
52 Kolcz J, Rajwa B, Drukala J, et al. Three-dimensional visualization of connexin 43 on the human cardiomyocytes. Appl lmmunohistochem Mol Morphol., 2002, 10(3): 247-252.
53 Egger G, Liang G, Aparicio A, et al. Epigenetics in human disease and prospects for epigenetic therapy. Nature, 2004, 429: 457-63.
54 Morgan, HD, Santos F, Green K, et al. Epigenetic reprogrammirtg in mammals. Hum Mol Genet, 2005, 14(Suppl 1): R47-58.
2 Hoffman JI, Kaplan S. The incidence of congenital heart disease. J Am Coil Cardiol, 2002, 39 (12): 1890-1900.
3 Bruzzone R, White TW, Paul DL. Connections with connexins: the molecular basis of direct intercellular signaling. Eur J Biochem, 1996, 238(1): 1-27.
4 Goodenough DA, Goliger JA, Paul DL. Connexins, connexons and intercellular communication. Annu Rev Biochem, 1996, 65: 475-502.
5 Reaume AG, de Sousa PA, KulkamiS, et al. Cardiac malformation in neonatal mice lacking connexin43. Science, 1995, 267(5205): 1831-1834.
6 Huang GY, Wessels A, Smith BR, et al. Alteration in connexin 43 gap junction gene dosage impairs conotruncal heart developmentDev Biol, 1998, 198(1): 32-44.
7 Ya J, Erdtsieck-Ernste EBHW, Boer PAJ, et al. Heart defects in cormexin43-deficient mice. Circ Res, 1998, 82(3): 360-366.
8 Sullivan R, Huang GY, Meyer RA, et al. Heart malformations in transgenic mice exhibiting dominant negative inhibition of gap junctional communication in neural crest cells. Dev Biol, 1998, 204(1): 224-234.
9 Ewart JL, Cohen MF, Meyer RA, et al. Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene. Development, 1997, 124(7): 1281-1292.
10 Huang GY, Cooper ES, Waldo K, et al. Gap junction-mediated cell-cell communication modulates mouse neural crest migration. J Cell Biol, 1998, 143(6): 1725-1734.
11 Britz-Cunningham SH, Shah MM, Zuppan CW, et al. Mutations of the connexin43 gap-junction gene in patients with heart malformations and defects of laterality. N Engl J Med, 1995, 332(20): 1323-1329.
12 Splitt MP, Burn J, Goodship J, et al. Connexin43 mutation in sporadic and familial defects of laterality. N Engl J Meal, 1995, 333: 941-942.
13 Gebbia M, Towbin JA, Casey B. Failure to detect connexin43 mutations in 38 cases of sporadic and familial heterotaxy. Circulation, 1996, 94(8): 1909-1912.
14 Splitt MP, Tsai MY, Burn J, Goodship JA. Absence of mutations in the regulatory domain of the gap junction protein connexin 43 in patients with visccroatrial heterotaxy. Heart, 1997, 77: 369-370.
15 Debrus S, Tuffery S, Matsuoka R, et al. Lack of evidence for connexin 43 gene mutations in human autosomal recessive lateralization defects. J Molec Cell Cardiol, 1997, 29(5): 1423-1431.
16 Dasgupta C, Martinez AM, Zuppan CW, et al. Identification of cormexin43 (α1) gap junction gene mutations in patients with hypoplastic left heart syndrome by denaturing gradient gel electrophoresis (DGGE). Mutat Res, 2001, 479(1-2): 173-186.
17 Frank C, Hong Gu, Steven M, et al. High Incidence of Cardiac Malformations in Connexin40-Deficient Mice. Circ Res, 2003, 93(3): 201-206.
18 Madoka K, Kiyomasa N, Kei-ichiro Net al. Loss of connexin45 causes a cushion defect in early cardiogenesis. Development, 2000, 127(16): 3501-3512.
19 Nishii K, Kumai M, Egashira K, et al. Mice lacking connexin45 conditionally in cardiac myocytes display embryonic lethality similar to that of germline knockout mice without endocardial cushion defect. Cell Communication & Adhesion, 2003, 10(4-6): 365-9.
20 庞玉生，沈媛，黄国英等。应用PCR测定Cx43转基因小鼠的基因型。广西医科大学学报，2003，20(5)：657-658．
21 Lo CW, Cohen MF, Huang GY, et al. Cx43 gap junction gene expression and gap junction communication in mouse neural crest cells. Dev G-enet, 1997, 20(2): 119-132.
22 谢利剑，黄国英，赵晓晴等。Connexin43基因缺陷新生小鼠心脏组织病理学研究。中华医学杂志，2005，85(18)：1249-1251．
23 Yu Q, Shen Y, Chatterjee B, et al. ENU induced mutations causing congenital cardiovascular anomalies. Dev, 2004, 131 (24): 6211-6223.
24 Gros D, Jongsma HJ. Connexins in mammalian heart function. BioEssays, 1996, 18: 719-730.
25 Delorme B, Dahl E, Jarry-Guichard T, et al. Expression pattern of connexin gene products at the early developmental stages of the mouse cardiovascular system. Circ Res, 1997, 81(3): 423-437.
26 Delorme B, Dahl E, Jarry-Guichard T, et al. Developmental regulation of connexin 40 gene expression in mouse heart correlates with the differentiation of the conduction system. Dev Dyn, 1995, 204(4): 358-371.
27 Sebastien A, Magali T, Therese J, et al. Down regulation of Connexin 45 Gene Products During Mouse Heart Development. Circ Res, 1999, 84: 1365-1379.
28 Chen P, Xie LJ, Huang GY, et al. Mutations of connexin43 in fetuses with congenital heart malformations. Chin Med J, 2005, 118(12): 971-976.
29 Forsthoefel KF, Papp AC, Snyder PJ, et al. Optimization of DNA extraction from formalin-fixed tissue and its clinical application in Duchenne muscular dystrophy. Am J Clin Pathol, 1992, 98(1): 98-104.
30 Fishman GI, Eddy RL, Shows TB, et al. The human connexin gene family of gap junction proteins: distinct chromosomal locations but similar structures. Genomics, 1991, 10(1): 250-256.
31 Devor EJ. Use of molecular beacons to verify that the serine hydroxymethyltransferase pseudogene SHMT-psl is unique to the order Primates. Genome Biol. 2001, 2(2): 101.
32 Zhang J Dyer KD. Rosenberg HF, et al. Evolution of the rodent eosinophil-associated RNase gene family by rapid gene sorting and positive selection. Proc Natl Acad Sci USA, 2000, 97(9): 4701-4706.
33 王秀敏，尚世强。脊椎动物假基因。国外医学分子生物学分册，2003，25(6)：327-330．
34 王秀敏，韩蓓，顾学范。CYP21基因启动子结构与功能突变研究。国外医学分子生物学分册，2001，23(1)：52-54．
35 Toyofuku T, Yabuki M, Otsu K, et al. Direct association of the gap junction protein cormexin43 with ZO-1 in cardiac myocytes. J Biol Chem, 1998, 273 (21 ): 12725-12731.
36 Levin M, Mercola M. Gap junction-mediated transfer of left-right patterning signals in the early chick blastoderm is upstream of shh asymmetry in the node. Development, 1999, 126(21): 4703-4714.
37 Thomas MA, Zosso N, Scerri I, et al. A tyrosine-based sorting signal is involved in connexin43 stability and gap junction turnover. J Cell Sci, 2003, 116(11), 2213-2222.
38 Pizzuti A, Flex E, Mingarelli RA, et al. A homozygous GJA1 gene mutation causes a Hallermarm-Streiff/ODDD spectrum phenotype. Human Mutation, 2004, 23(3): 286.
39 Paznekas WA, Boyadjiev SA, Shapiro RE, et al. Connexin 43 (GJA1) Mutations Cause the Pleiotropic Phenotype of Oculodentodigital Dysplasia. Am. J. Hum. Genet, 2003, 72(2): 408-418.
40 李广镰，张开滋，Cheng TO主编．心血管遗传病学．北京：北京医科大学中国协和医科大学联合出版社．1994：29-58．
41 钟秋安，仇小强。先天性心脏病的病因研究进展。中国儿童保健杂志，2003，11(4)：259-261．
42 GELB BD. Recent advances in the understanding of genetic causes of congenital heart defects. Front Biosci, 2000, 5: 321-333
43 Basson CT, Bachinshy DR, Lin RC, et al. Mutations in human TBX5 cause limb and cardiac malformations in Holt-Oram syndrome. Nat Genet, 1997, 15(1): 30-35.
44 Schott JJ, Benson DW, Basson CT, et al. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science, 1998, 281 (5373): 108-111.
45 GOLDMUNTZ E, GEIGER E, BENSON DW, et al. Nkx 2.5 mutations in patients with tetralogy of fallot. Circulation, 2001, 104(21): 2565-2568.
46 Kurihara Y, Kurihara H, Oda H, et al. Aortic arch malformations and ventricular septal defect in mice deficient in endothelin-1. J Clin Invest. 1995 96(1): 293-300.
47 Meyer D, Birchmeier C. Multiple essential functions of neuregulin in development Nature 1995 378(6555): 386-390.
48 Molkentin JD, Olson EN. GATA4: a novel transcriptional regulator of cardiac hypertrophy? Circulation, 1997, 96(11): 3833-3835.
49 van Kempen MJ, Fromaget C, Gros D, et al. Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart. Circ Res, 1991, 68(6): 1638-51.
50 Fishman GI, Hertzberg EL, Spray DC, et al. Expression of cormexin43 in the developing rat heart. Circ Res, 1991, 68(3): 782-787.
51 陈萍，黄国英，谢利剑等。Cx43基因在人类及小鼠胎心发育中的时空表达规律。中国实验动物学报，2005，13(1)：16-19．
52 Kolcz J, Rajwa B, Drukala J, et al. Three-dimensional visualization of connexin 43 on the human cardiomyocytes. Appl lmmunohistochem Mol Morphol., 2002, 10(3): 247-252.
53 Egger G, Liang G, Aparicio A, et al. Epigenetics in human disease and prospects for epigenetic therapy. Nature, 2004, 429: 457-63.
54 Morgan, HD, Santos F, Green K, et al. Epigenetic reprogrammirtg in mammals. Hum Mol Genet, 2005, 14(Suppl 1): R47-58.