[Show abstract][Hide abstract] ABSTRACT: GJA1 gene encodes a gap junction protein known as connexin 43 (Cx43). Cx43 is abundantly expressed in the ventricular myocardium and in cardiac neural crest cells. Cx43 is proposed to play an important role in human congenital heart disease, as GJA1 knock-out mice die neonatally from outflow tract obstruction. In addition, patients with visceroatrial heterotaxia or hypoplastic left heart syndrome were reported to have point mutations in GJA1 at residues that affect protein kinase phosphorylation and gating of the gap junction channel. However, as these clinical findings were not replicated in subsequent studies, the question remains about the contribution of GJA1 mutations in human congenital heart disease (CHD).
We analyzed the GJA1 coding sequence in 300 patients with CHD from two clinical centers, focusing on outflow tract anomalies. This included 152 with Tetralogy of Fallot from over 200 patients exhibiting outflow tract anomalies, as well as other structural heart defects including atrioventricular septal defects and other valvar anomalies. Our sequencing analysis revealed only two silent nucleotide substitutions in 8 patients. To further assess the possible role of Cx43 in CHD, we also generated two knock-in mouse models with point mutations at serine residues subject to protein kinase C or casein kinase phosphorylation, sites that are known to regulate gating and trafficking of Cx43, respectively.
Both heterozygous and homozygous knock-in mice were long term viable and did not exhibit overt CHD.
The combined clinical and knock-in mouse mutant studies indicate GJA1 mutation is not likely a major contributor to CHD, especially those involving outflow tract anomalies.
Full-text · Article · Oct 2011 · Journal of cardiovascular disease research
[Show abstract][Hide abstract] ABSTRACT: The connexin43 knockout (Cx43 KO) mouse dies at birth with an enlarged conotruncal region, which leads to the obstruction of the right outflow tract (OFT). Since myocardialization of the proximal OFT septum is one of the key events during heart development, we investigated the process in the Cx43 KO embryo hearts. Rho associated coiled-coil forming protein kinase 1 (ROCK1), is a recently found key molecule to regulate the myocardialization of OFT, but its spatiotemporal expression pattern during myocardialization remains unknown. The objective of this study was to investigate the differentially expressed pattern of ROCK1 between Cx43 KO and wild type embryo hearts, and its relationship with the delayed myocardialization in Cx43 KO embryo hearts.
Using immunohistochemistry, the processes of myocardiolization were investigated both in Cx43 KO and wild type embryo hearts. The differentially expressed pattern of ROCK1 between Cx43 KO and wildtype embryo hearts was evaluated both at the mRNA and protein level by real-time RT-PCR and immunohistochemistry.
The expression of α-sarcomeric actin (α-SCA) in the proximal OFT septum of Cx43 KO embryos was delayed. Meanwhile, it was shown that the downregulation of ROCK1 coincided with delayed myocardialization. The expression of ROCK1 protein was mainly limited to the proximal outflow tract septum from embryo day (E) E11.5 to E15.5. Its expression pattern was similar with that of α-SCA. Real-time RT-PCR found that the expression level of Rock-1 mRNA began at a low level on E11.5 and reached peak at E13.5 and E14.5.
ROCK1 may have an important role in the process of myocardialization of the proximal OFT septum. Downregulation of ROCK1 is likely to contribute to the aberrant myocardialization in Cx43 KO embryo hearts.
Preview · Article · Jul 2011 · Chinese medical journal
[Show abstract][Hide abstract] ABSTRACT: Connexin 43 knockout (Cx43 KO) mice exhibit conotruncal malformations and coronary artery defects. We observed epicardial blisters in the Cx43 KO hearts that suggest defects in epicardial epithelial-mesenchymal transformation (EMT), a process that generates coronary vascular progenitors. Analysis using a three-dimensional collagen gel invasion assay showed that Cx43 KO epicardial cells are less invasive and that, unlike wild-type epicardial cells, they fail to organize into thin vessel-like projections. Examination of Cx43 KO hearts using Wt1 as an epicardial marker revealed a disorganized pattern of epicardial cell infiltration. Time-lapse imaging and motion analysis using epicardial explants showed a defect in directional cell migration. This was associated with changes in the actin/tubulin cytoskeleton. A defect in cell polarity was indicated by a failure of the microtubule-organizing center to align with the direction of cell migration. Forced expression of Cx43 constructs in epicardial explants showed the Cx43 tubulin-binding domain is required for Cx43 modulation of cell polarity and cell motility. Pecam staining revealed early defects in remodeling of the primitive coronary vascular plexuses in the Cx43 KO heart. Together, these findings suggest an early defect in coronary vascular development arising from a global perturbation of the cytoarchitecture of the cell. Consistent with this, we found aberrant myocardialization of the outflow tract, a process also known to be EMT dependent. Together, these findings suggest cardiac defects in the Cx43 KO mice arise from the disruption of cell polarity, a process that may be dependent on Cx43-tubulin interactions.
[Show abstract][Hide abstract] ABSTRACT: To investigate the spatio-temporal expression of connexin (Cx) 40 and Cx45 genes in Cx43 knockout embryonic mouse hearts.
Cx43 knockout heterozygous mice were raised. PCR was performed to identify genotypes of their offsprings. The homozygote (Cx43-/-) was used for study and the wild type (Cx43+/+) was used as control. Immuno-histochemistry was used to detect the Cx40 and Cx45 expression in different parts of the fetal hearts at the embryonic days (EDs) 10.5, 11.5, 12.5, 13.5, 14.5, and 15.5, respectively. SCIM microscopic image analytic system was used for quantitative analysis of staining intensity.
(1) Cx40 expression was detected in ventricles of Cx43+/+ fetal heart as early as ED10.5 with the intensity represented by A value of 8.6. Subsequently it was distributed in the atria and ventricles with the peak expression observed at ED14.5 (A value = 94.8), and faded afterwards. Less Cx40 expression was observed in the Cx43-/- fetal hearts as compared with Cx43+/+ although its expression pattern was similar in both groups. (2) Cx45 expression was detected in ventricles at ED 10.5 (A value = 20.0). It was subsequently distributed in the atria and ventricles with the peak expression observed at ED12.5 (A value = 49.6), and then faded. Less Cx45 expression was observed in the Cx43-/- fetal hearts as compared with Cx43+/+ although its expression pattern was similar as well in both groups.
Down-regulated expression of the genes Cx40 and Cx45 may be associated with the abnormal heart development in Cx43 knockout animals.
No preview · Article · Apr 2009 · Zhonghua yi xue za zhi
[Show abstract][Hide abstract] ABSTRACT: Heterotaxy arises from a failure of the embryo to establish normal left-right asymmetry and is known to affect 3% of infants with congenital heart disease. A recessive mutation causing heterotaxy was recovered in a mouse mutagenesis screen focused on congenital heart defects. Homozygote mutants exhibit abnormal situs in the thoracic and abdominal cavities. Dextrocardia, levocardia, or mesocardia was seen together with right pulmonary isomerism and complex structural heart defects in the single ventricle spectrum. A dominant chamber of left ventricular morphology positioned on the left or right is seen together with transposition of the great arteries. Right atrial isomerism with or without total anomalous pulmonary venous connection was observed in half of the mutants. Because ciliary motion at the embryonic node is required for the specification of laterality, we examined the tracheal epithelia of newborn mice as a proxy for the nodal cilia. However, videomicroscopy showed no defect in ciliary motion. Genome scanning using polymorphic microsatellite markers mapped the mutation to a 3.3 Mb interval on mouse chromosome 7. None of the genes previously described for familial heterotaxy were found in this interval, indicating a novel mutation in this mouse model of heterotaxy.
Full-text · Article · Feb 2008 · Pediatric Research
[Show abstract][Hide abstract] ABSTRACT: Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder associated with ciliary defects and situs inversus totalis, the complete mirror image reversal of internal organ situs (positioning). A variable incidence of heterotaxy, or irregular organ situs, also has been reported in PCD patients, but it is not known whether this is elicited by the PCD-causing genetic lesion. We studied a mouse model of PCD with a recessive mutation in Dnahc5, a dynein gene commonly mutated in PCD. Analysis of homozygous mutant embryos from 18 litters yielded 25% with normal organ situs, 35% with situs inversus totalis, and 40% with heterotaxy. Embryos with heterotaxy had complex structural heart defects that included discordant atrioventricular and ventricular outflow situs and atrial/pulmonary isomerisms. Variable combinations of a distinct set of cardiovascular anomalies were observed, including superior-inferior ventricles, great artery alignment defects, and interrupted inferior vena cava with azygos continuation. The surprisingly high incidence of heterotaxy led us to evaluate the diagnosis of PCD. PCD was confirmed by EM, which revealed missing outer dynein arms in the respiratory cilia. Ciliary dyskinesia was observed by videomicroscopy. These findings show that Dnahc5 is required for the specification of left-right asymmetry and suggest that the PCD-causing Dnahc5 mutation may also be associated with heterotaxy.
Full-text · Article · Jan 2008 · Journal of Clinical Investigation
[Show abstract][Hide abstract] ABSTRACT: To explore the etiology of the conotruncal malformations in Cx43 knockout mice.
The objects were C57/BL6 mice of E11.5 to 1 day after birth by the mating of 2 month old heterozygous mice which included Cx43 (knockout, KO) homozygotes (Cx43-/-), heterozygotes (Cx43+/-) and wild-types (Cx43+/+) genotyped by PCR method. Microdissection and HE staining were used to examine the structures of hearts. The expression of the alpha-SCA, alpha-SMA, AP-2alpha were detected by immunohistochemistry. AP-2alpha mRNA was detected by in situ hybridization.
Cx43-/- mice died within 24 h after birth with a swelling and blockage of the conotruncal region, which led to the obstruction of OFT and enlargement of right ventricle. HE staining showed plenty of abnormal tissues in this region forming many pouches. No apparent malformations were observed in Cx43+/- and Cx43+/+ mice. The expression of alpha-SCA in the proximal OFT septum was delayed obviously in Cx43-/- predominantly at E13.5 and E14.5. The expression of alpha-SMA in the OFT in Cx43+/- and Cx43-/- was stronger than that of Cx43+/+ mice, and mostly located in the hyperplastic conotruncal region especially at E13.5-E15.5 in Cx43-/- mice. The expression could still be observed at the birth day in Cx43-/- mice, which was not observed in Cx43+/+ mice. The expression of AP-2alpha and AP-2alpha mRNA at E13.5 increased in Cx43-/- and abnormally located in the proximal OFT septum.
Cx43 KO mice are characterized by hyperplasia in conotruncal region. Cx43 KO mice exhibited a delayed myocardialization and the developmental immaturity of cardiomyocytes. The abnormal distribution of cardiac neural crest cells is likely to contribute to the conotruncal malformations in Cx43-deficient mice.
No preview · Article · Nov 2005 · Zhonghua yi xue za zhi
[Show abstract][Hide abstract] ABSTRACT: Gap junction channels formed by connexin43 (Cx43) protein are important in cardiac morphogenesis, and Cx43 gene is thought to be associated with congenital heart malformation (CHM). This study was undertaken to detect the mutations of Cx43 in fetuses with CHM.
Cx43 extron DNA was amplified by PCR from 16 fetuses with a variety of CHM. The PCR products were analyzed by SSCP and DNA sequencing. Thirty children who had no CHM were selected as controls.
Eight homozygous mutations of Cx43 were observed in a fetus with double outlet right ventricule (DORV), five of the 8 mutations were missense mutations including Arg239Trp, Ser251Thr, Ala253Pro, Pro283Leu and Thr290Asn, and the remaining 3 were silent polymorphisms including Gly252Gly, Pro256Pro and Thr275Thr. No mutations were found in other fetuses and the control group.
Mutations of Cx43 may be associated with congenital conotruncal anomalies. PCR-SSCP is an effective method for screening the mutations of Cx43.
No preview · Article · Jul 2005 · Chinese medical journal
[Show abstract][Hide abstract] ABSTRACT: To investigate the characteristics of heart morphology in neonatal mice with connexin43 gene defects.
Two mouse lines were used in this study, which included connexin43 knockout (Cx43KO) mice and CMV43(CT) transgenic mice. PCR analysis was carried out to identify the genotypes of two transgenic mice. Sections with HE staining were analyzed to display the morphologic structures of the hearts in neonatal mice. C57BL6/SJ mice were used as control.
All 11 homozygous Cx43KO mice died within 24 hr after birth showing severe right ventricular outflow tract obstruction (RVOTO). Out of 20 homozygous CMV43(CT) transgenic mice, 12 mice died within 48 hr after birth showing not only RVOTO, but also atrial septal defect (ASD), ventricular septal defect (VSD) and other cardiac defects, while the remaining 8 mice were alive without heart defects. Both heterozygous Cx43KO and CMV43(CT) transgenic mice had normal hearts.
Connexin43 gene defect is obviously associated with abnormal heart morphogenesis. The different types and different dosage of the gene defects may lead to different phenotypes of hearts.
No preview · Article · Jun 2005 · Zhonghua yi xue za zhi