Cx40 Gene Research Articles

Loss of the Tbx5 gene in the ventricular conduction system induces spontaneous ventricular arrhythmias

The ventricular conduction system (VCS) and the working myocardium (WM) derive from the same myogenic progenitors during the embryonic development. The VCS development is regulated by numerous genes including the T-box transcription factor 5 (Tbx5). In human, Tbx5 mutations cause Holt-Oram syndrome, an autosomal dominant disease associated with heart abnormalities such as arrhythmias. In adult mice, the deletion of the Tbx5 gene provoked a slowing of ventricular conduction due to a loss of the gap junction Connexin 40 and dysfunction of cardiac channels. However, it is still unknown how Tbx5 deletion affects the development and the function of the VCS. The aim is to study the consequences of the Tbx5 deletion in embryonic progenitors of the ventricular conduction system on cardiac function in adult mice. Conditional deletion of Tbx5 alleles in mice was induced by expressing the Cre recombinase under the control of the Connexin 40 (Cx40) gene. We studied two groups of mutants: Tbx5 deletion at embryonic days E13-14 targeting both the ventricular conduction system and the working myocardium (“VCS + WM” group; n = 40) and mice deleted at E18-P2 targeting only the ventricular conduction system (“VCS” group; n = 17). Surface ECGs were achieved monthly until 3 months of age, when immunostaining was performed on the left VCS. Immunostaining showed a reduced expression of the two major connexines in the ventricle (Cx40 and Cx43) and a reduced number of conductive cells in both mutants, suggesting a defective maturation of the VCS (Fig. 1). Furthermore, the loss of Cx43 was correlated with that of Tbx5, reflecting a direct transcriptional effect. Surface ECG revealed a significant increase of the ventricular activation duration in both mutants (P < 0.01). Moreover, premature ventricular contractions (PVCs) and a left QRS axis deviation were observed in both mutants. Finally, these PVCs disappeared with age in both mutants under resting conditions, and are unmasked when the heart is challenged with a β-1 adrenergic agonist. Our results provide new insights into Tbx5 targets in the VCS. Tbx5 deletion impacts the maturation and the anatomy of the VCS as proved by the deviation of the ventricular electrical axis, which even occurs when the VCS is already formed. Tbx5 appears to have a direct impact on the Gja1 gene, encoding the gap junction Cx43, which is reduced at the junction between the VCS and the WM in both mutants and may be responsible for PVCs.

Single and fractionated ionizing radiation induce alterations in endothelial connexin expression and channel function

Radiotherapy is an effective treatment for most tumor types. However, emerging evidence indicates an increased risk for atherosclerosis after ionizing radiation exposure, initiated by endothelial cell dysfunction. Interestingly, endothelial cells express connexin (Cx) proteins that are reported to exert proatherogenic as well as atheroprotective effects. Furthermore, Cxs form channels, gap junctions and hemichannels, that are involved in bystander signaling that leads to indirect radiation effects in non-exposed cells. We here aimed to investigate the consequences of endothelial cell irradiation on Cx expression and channel function. Telomerase immortalized human Coronary Artery/Microvascular Endothelial cells were exposed to single and fractionated X-rays. Several biological endpoints were investigated at different time points after exposure: Cx gene and protein expression, gap junctional dye coupling and hemichannel function. We demonstrate that single and fractionated irradiation induce upregulation of proatherogenic Cx43 and downregulation of atheroprotective Cx40 gene and protein levels in a dose-dependent manner. Single and fractionated irradiation furthermore increased gap junctional communication and induced hemichannel opening. Our findings indicate alterations in Cx expression that are typically observed in endothelial cells covering atherosclerotic plaques. The observed radiation-induced increase in Cx channel function may promote bystander signaling thereby exacerbating endothelial cell damage and atherogenesis.

ФЕНОТИПОВІ ОЗНАКИ І ПОЛІМОРФІЗМ RS10465885 ГЕНА КОНЕКСИНУ-40 ЯК ПРЕДИКТОРИ ВИНИКНЕННЯ РЕЦИДИВУ АРИТМІЇ У ПАЦІЄНТІВ З ФІБРИЛЯЦІЄЮ ПЕРЕДСЕРДЬ НЕКЛАПАННОГО ҐЕНЕЗУ ПІСЛЯ ВІДНОВЛЕННЯ СИНУСОВОГО РИТМУ ПРИ 1-РІЧНОМУ СПОСТЕРЕЖЕННІ: РЕЗУЛЬТАТИ НЕЙРОМЕРЕЖЕВОГО АНАЛІЗУ

Atrial fibrillation (AF) is a violation of the heart rhythm associated with 1 % (or even more) of the health budget expenditure in Europe. Despite the fact that there is a constant increase in the socio-economic burden of AF, and there has been significant progress in understanding the pathophysiology of this arrhythmia, the effectiveness of its treatment is still far from satisfactory. One of the reasons for the insufficient effectiveness of modern strategies for the prevention and treatment of AF is the existence of limitations in understanding the complex pathophysiology of this disturbance of the heart rhythm.The aim of the study – to leran the phenotypic and genotypic (rs10465885 polymorphism in connexin-40 [Cx40] gene) predictors of arrhythmia recurrence in patients with non-valvular atrial fibrillation (AF) after sinus rhythm restoring (SRR ) at 1-year follow-up (AF360), in particular by the use of artificial neural networks (ANN ) analysis.Materials and Methods. We enrolled 104 patients (pts) with non-sustained non-valvular AF (average age (53±10) years, 80 (76.9 %) men). The distribution of rs10465885 polymorphic variants in Cx40 gene (n=73) was as follows: TT – 17 (23.3 %) pts, CT – 33 (45.2 %), СС – 23 (31.5 %). We analyzed 122 cases of SR restoring: 32 (26.2 %) – pharmacological cardioversion (29 pts); 63 (51.6 %) – electrical cardioversion (53 pts); 27 (22.2 %) – radiofrequency catheter ablation (22 pts). AF360 occurred in 76 (65.5 %) of 116 cases. In order to identify AF360 predictors, we used logistic regression analysis, as well as ANN analysis (building the linear [LIN] and nonlinear [multilayer perceptron (MLP )] ANN`s).Results and Discussion. Genetic algorithm Input Selection revealed 16 parameters, associated with AF360, including SRR type, rs10465885 polymorphism, and certain clinical (in particular, CHA 2DS 2-VAS c score), lab and echo parameters. CHA 2DS 2-VAS c score was the only independent AF360 predictor, according to the results of multivariable logistic regression analysis. The area under curve (AU C) for MLP , included all 16 variables, revealed by the Genetic algorithm, was significantly higher than in linear LIN: 0.874 (95 % confidence interval [CI] 0.798–0.929) vs. 0.678 (CI 0.583–0.763), respectively (p&lt;0.001). In order to obtain the maximal reduction of predictors, we built the MLP , included the set of 5 variables (MLP 5): SRR type; rs10465885 polymorphism; the heart failure presence and its stage; AF type (recurrent or first diagnosed); and antero-posterior left atrial dimension. MLP 5 AU C (0.808 (95 % CI 0.723–0.876)) was significantly higher than those for LIN (р = 0.027).Conclusion. AF360 was non-linearly associated with SRR type, rs10465885 polymorphism in Cx40 gene, as well as certain phenotypic parameters. The further search of the most significant genetic and epigenetic predictors of AF recurrence at different terms after SRR is of crucial importance.

Characterizing the role of atrial natriuretic peptide signaling in the development of embryonic ventricular conduction system

Patients born with congenital heart defects frequently encounter arrhythmias due to defects in the ventricular conduction system (VCS) development. Although recent studies identified transcriptional networks essential for the heart development, there is scant information on the mechanisms regulating VCS development. Based on the association of atrial natriuretic peptide (ANP) expression with VCS forming regions, it was reasoned that ANP could play a critical role in differentiation of cardiac progenitor cells (CPCs) and cardiomyocytes (CMs) toward a VCS cell lineage. The present study showed that treatment of embryonic ventricular cells with ANP or cell permeable 8-Br-cGMP can induce gene expression of important VCS markers such as hyperpolarization-activated cyclic nucleotide-gated channel-4 (HCN4) and connexin 40 (Cx40). Inhibition of protein kinase G (PKG) via Rp-8-pCPT-cGMPS further confirmed the role of ANP/NPRA/cGMP/PKG pathway in the regulation of HCN4 and Cx40 gene expression. Additional experiments indicated that ANP may regulate VCS marker gene expression by modulating levels of miRNAs that are known to control the stability of transcripts encoding HCN4 and Cx40. Genetic ablation of NPRA revealed significant decreases in VCS marker gene expression and defects in Purkinje fiber arborisation. These results provide mechanistic insights into the role of ANP/NPRA signaling in VCS formation.

Circadian expression of connexins in the mouse heart

Significant circadian variations exist in the frequency of cardiac arrhythmia; however, the underlying mechanism is largely unknown. Connexins are essential in the propagation of electrical activity throughout the heart and are an important determinant of conduction velocity. Their dysfunction is related to the genesis of cardiac arrhythmia. In this study, we investigated if cx40 and cx43 expressed circadianly in the mouse heart using suprachiasmatic nuclei (SCN) lesion and pharmacological autonomic nervous system block mouse. Significant 24-h variations were observed in the expression of cx40 and cx43 in the sham-operated mice. In the SCNX mouse, all genes examined lost circadian rhythm. In the ANSB mouse, the expressions of Bmal1 was dampened significantly but still had circadian rhythm, whereas the two connexin gene expressions lost rhythm. These results suggest that cx40 and cx43 gene expressions have clear circadian rhythm and might be regulated by the central clock in the SCN through the ANS.

PP.36.15

Objective: To study the allelic polymorphism of connexin-40 (Cx40) gene (rs10465885) in patients with non-valvular atrial fibrillation (AF) in Ukrainian population according to the presence of left ventricular (LV) hypertrophy (H). Design and method: We enrolled 112 patients (pts) before the age of 65 years (mean age (50 ± 10) years; males – 86 (76,8%), females – 26 (23,2%)) with non-valvular AF, associated with essential hypertension (n = 76, 67,9%), coronary heart disease (n = 67, 59,8%), myocardial fibrosis (n = 40, 35,7%) and metabolic cardiomyopathy (n = 5, 4,5%). Among them 47 (42,0%) pts had paroxysmal AF, 45 (40,2 %) – persistent and 20 (17,8%) – stable. We performed 2D-Echo with LV myocardial mass index assessment. LV H was diagnosed according to ASE guidelines (2006) and was presented in 68 (60,7%) pts. The single nucleotide polymorphism rs10465885 in promoter B Cx40 gene was genotyped by means of real time polymerase chain reaction (T – reference (“wild” type) allele, C – minor allele). Results: Genotype and allelic distribution of rs10465885 in pts with AF but without LV H was as follows: T/ T – 23% (n = 10); C/T – 41% (n = 18); C/C – 36% (n = 16); allele T – 43% (n = 38); C – 57% (n = 50). The distribution in pts with LV H was as follows: T / T – 28% (n = 19); C / T – 54% (n = 37); C / C – 18% (n = 12); allele T – 55,1% (n = 75), C – 44,9% (n = 61). The difference of rs10465885 genotype and allelic distribution was not statistically significant in pts with or without LV H (p = 0,08 for both). Recessive model was the best one to describe the studied association with the lowest value of Akaike information criterion (pts without LV H: C/C – 36%, T/T + C/T – 64%; with LV H: C/C – 18%, T/T + C/T – 82%, p = 0,03; odds ratio 0,38 (95% confidence interval 0,15–0,89)). Conclusions: The absence of LV H in pts before the age of 65 years with non-valvular AF is associated with higher frequency of rs10465885 minor allele homozygotes.

The loss of cellular junctions in epithelial lung cells induced by cigarette smoke is attenuated by corilagin.

Cigarette smoke (CS) contains over 4700 compounds, many of which can affect cellular redox balance through free radicals production or through the modulation of antioxidant enzymes. The respiratory tract is one of the organs directly exposed to CS and it is known that CS can damage the integrity of lung epithelium by affecting cell junctions and increasing epithelium permeability. In this study, we have used a human lung epithelial cell line, Calu-3, to evaluate the effect of CS on lung epithelial cell junctions levels, with special focus on the expression of two proteins involved in intercellular communication: connexins (Cx) 40 and 43. CS exposure increased Cx40 gene expression but not of Cx43. CS also induced NFκB activation and the formation of 4HNE-Cxs adducts. Since corilagin, a natural polyphenol, is able to inhibit NFκB activation, we have determined whether corilagin could counteract the effect of CS on Cxs expression. Corilagin was able to diminish CS induced Cx40 gene expression, 4HNE-Cx40 adducts formation, and NFκB activation. The results of this study demonstrated that CS induced the loss of cellular junctions in lung epithelium, possibly as a consequence of Cx-4HNE adducts formation, and corilagin seems to be able to abolish these CS induced alterations.

Mutations of the Connexin 37 and 40 Gap-Junction Genes in Patients with Acute Myocardial Infarction

There is evidence showing the association between connexin 37 (Cx37) C1019T polymorphism and acute myocardial infarction (AMI). Since there has been no study that takes the polymorphisms of Cx37 and connexin 40 (Cx40) into consideration at the same time, we investigated the association between AMI and the polymorphism gene of two gap junction proteins Cx 37 and Cx40 which are important in the electrical coupling between arterial myocytes. 200 patients with acute myocardial infarction (AMI) and 185 healthy controls were included in this study. Cx37 and Cx40 genotypes were determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). We identified that single nucleotide polymorphism (SNP) of Cx37 was significantly associated with AMI disease. Significant differences between cases and controls were observed for Cx37 genotype frequencies (p < 0.001, Chi2 = 16.528). The T allele of Cx37 had more frequency in the AMI group compared to the control group. (53.75% vs. 40%; p < 0.05). Subsequent analysis identified that, in contrast to the previous studies, there is a significant difference in women (p < 0.01) but not in men. We also found that the SNP of Cx40 was not significantly associated with AMI disease (p > 0.05). Our study showed that the -44A allele and -44AA genotype were not significantly different in the AMI and control groups (p > 0.05). It is suggested that the polymorphism in the Cx37 gene (but not Cx40 gene) potentially plays a significant role in the manifestation of AMI disease in Iranian population.

Novel GermlineGJA5/Connexin40 Mutations Associated with Lone Atrial Fibrillation Impair Gap Junctional Intercellular Communication

Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia worldwide. Here, we investigate the molecular and cellular mechanisms of lone AF-linked germline mutations in the connexin40 (Cx40) gene, GJA5. The entire coding region of GJA5 was sequenced in 68 unrelated patients with lone AF. A novel germline heterozygous missense mutation in Cx40 (p.I75F) was identified in one index patient. The mutation was also present in the proband's father with lone AF but was not found in the unaffected family members who were examined and 200 unrelated healthy control individuals. Electrophysiological studies revealed no electrical coupling of the cell pairs expressing the mutant alone and a significant reduction in gap junction coupling conductance when the mutant was coexpressed with wild-type (wt) Cx40 or Cx43. Interestingly, another lone AF-linked Cx40 mutant p.L229M did not show any apparent coupling defect when expressed alone or together with wt Cx40 but specifically reduced the gap junction coupling when coexpressed with wt Cx43. This study is the first to demonstrate that the germline familial mutations in Cx40 impair the gap junctions through different mechanisms, which may predispose the mutant carriers to AF.

Low prevalence of connexin-40 gene variants in atrial tissues and blood from atrial fibrillation subjects.

BackgroundThe atrial gap junction protein connexin-40 (Cx40) has been implicated to play an important role in atrial conduction and development of atrial fibrillation (AF). However, the frequency of Cx40 mutations in AF populations and their impact on Cx40 expression remains unclear. In this study, we sought to identify polymorphisms in the Cx40 gene GJA5, investigate the potential functional role of these polymorphisms, and determine their allelic frequencies. The prevalence of nonsynonymous Cx40 mutations in blood and atrial tissue was also compared to mutation frequencies reported in prior studies.MethodsWe conducted direct sequencing of the GJA5 coding and 3′ UTR regions in blood samples from 91 lone AF subjects and 67 atrial tissue-derived samples from a lone cohort, a mixed AF cohort, and several transplant donors. Reporter gene transfection and tissue allelic expression imbalance assays were used to assess the effects of a common insertion/deletion polymorphism on Cx40 mRNA stability and expression.ResultsWe identified one novel synonymous SNP in blood-derived DNA from a lone AF subject. In atrial tissue-derived DNA from lone and mixed AF subjects, we observed one novel nonsynonymous SNP, one rare previously reported synonymous SNP, and one novel 3′ UTR SNP. A previously noted 25 bp insertion/deletion polymorphism in the 3′ UTR was found to be common (minor allele frequency = 0.45) but had no effect on Cx40 mRNA stability and expression. The observed prevalence of nonsynonymous Cx40 mutations in atrial tissues derived from lone AF subjects differed significantly (p = 0.03) from a prior atrial tissue study reporting a high mutation frequency in a group of highly selected young lone AF subjects.ConclusionsOur results suggest that Cx40 coding SNPs are uncommon in AF populations, although rare mutations in this gene may certainly lead to AF pathogenesis. Furthermore, a common insertion/deletion polymorphism in the Cx40 3′ UTR does not appear to play a role in modulating Cx40 mRNA levels.

Role of connexin40 in the autoregulatory response of the afferent arteriole

Connexins in renal arterioles affect autoregulation of arteriolar tonus and renal blood flow and are believed to be involved in the transmission of the tubuloglomerular feedback (TGF) response across the cells of the juxtaglomerular apparatus. Connexin40 (Cx40) also plays a significant role in the regulation of renin secretion. We investigated the effect of deleting the Cx40 gene on autoregulation of afferent arteriolar diameter in response to acute changes in renal perfusion pressure. The experiments were performed using the isolated blood perfused juxtamedullary nephron preparation in kidneys obtained from wild-type or Cx40 knockout mice. Renal perfusion pressure was increased in steps from 75 to 155 mmHg, and the response in afferent arteriolar diameter was measured. Hereafter, a papillectomy was performed to inhibit TGF, and the pressure steps were repeated. Conduction of intercellular Ca(2+) changes in response to local electrical stimulation was examined in isolated interlobular arteries and afferent arterioles from wild-type or Cx40 knockout mice. Cx40 knockout mice had an impaired autoregulatory response to acute changes in renal perfusion pressure compared with wild-type mice. Inhibition of TGF by papillectomy significantly reduced autoregulation of afferent arteriolar diameter in wild-type mice. In Cx40 knockout mice, papillectomy did not affect the autoregulatory response, indicating that these mice have no functional TGF. Also, Cx40 knockout mice showed no conduction of intercellular Ca(2+) changes in response to local electrical stimulation of interlobular arteries, whereas the Ca(2+) response to norepinephrine was unaffected. These results suggest that Cx40 plays a significant role in the renal autoregulatory response of preglomerular resistance vessels.

EP News: Basic and Translational

Igarashi et al (Circulation 2012;125:216, PMID 22158756) randomized 30 Yorkshire swines into 2 groups (sinus rhythm and atrial fibrillation [AF]) and each group into 3 subgroups: sham-operated control, gene therapy with adenovirus expressing connexin 40 (Cx40), and gene therapy with adenovirus expressing Cx43. Sinus rhythm animals had strong transgene expression, but no atrial conduction changes. In animals with AF, controls had reduced and lateralized Cx43 expression, and Cx43 gene transfer restored expression and cellular location to sinus rhythm control levels. In the AF group, both Cx40 and Cx43 gene transfer improved conduction and reduced AF relative to controls. The authors conclude that connexin gene therapy preserved atrial conduction and prevented AF.

Lack of association between connexin40 polymorphisms and coronary artery disease

ObjectiveCx40 is a gap junction protein important for cell-cell communication in the endothelium. Polymorphisms in the promoter region of the human Cx40 gene, −44G>A and +71A>G, were shown to reduce Cx40 transcription by half. As mice with an endothelial-specific deletion of Cx40 are more susceptible to atherosclerosis, this study was designed to discover a correlation between these polymorphisms and atherosclerosis in European populations. Methods and results803 patients referred to the Geneva University Hospitals for elective coronary angiography were divided according to the number of significantly stenosed vessels (from 0 to 3) and were genotyped for the Cx40 polymorphisms. Genotype distribution in the control group was −44GG/+71AA=59.8%, −44AG/+71AG=35.1% and −44AA/+71GG=5.2%. Surprisingly, this distribution was similar in the CAD group, with −44GG/+71AA=58.5%, −44AG/+71AG=37.6% and −44AA/+71GG=3.8% (p=0.67). Moreover, no significant association between histological carotid plaque composition of culprit lesions and Cx40 polymorphisms could be detected in 583 Dutch patients of the Athero-Express study. ConclusionsDespite a clear antiatherogenic role of Cx40 in mice, our study could not detect an association of Cx40 promoter polymorphisms and CAD in human. Moreover, a correlation with atherosclerotic plaque stability or hypertension could not be demonstrated either. Connexin polymorphisms affecting channel function may be of greater importance for cardiovascular disease than polymorphisms affecting the expression level of the protein.

Connexin Gene Transfer Preserves Conduction Velocity and Prevents Atrial Fibrillation

Several lines of evidence have suggested that maintenance of atrial fibrillation (AF) depends on reentrant mechanisms. Maintenance of reentry necessitates a sufficiently short refractory period and/or delayed conduction, and AF has been associated with both alterations. Fibrosis, cellular dysfunction, and gap junction protein alterations occur in AF and cause conduction delay. We performed this study to test the hypothesis that gap junction protein overexpression would improve conduction and prevent AF. Thirty Yorkshire swine were randomized into 2 groups (sinus rhythm and AF), and each group into 3 subgroups: sham-operated control, gene therapy with adenovirus expressing connexin (Cx) 40, and gene therapy with adenovirus expressing Cx43 (n=5 per subgroup). All animals had epicardial gene painting; the AF group had burst atrial pacing. All animals underwent terminal study 7 days after gene transfer. Sinus rhythm animals had strong transgene expression but no atrial conduction changes. In AF animals, controls had reduced and lateralized Cx43 expression, and Cx43 gene transfer restored expression and cellular location to sinus rhythm control levels. In the AF group, both Cx40 and Cx43 gene transfer improved conduction and reduced AF relative to controls. Connexin gene therapy preserved atrial conduction and prevented AF.

Connexins and Atrial Fibrillation

Atrial fibrillation (AF) is an extremely common arrhythmia with important consequences and presently suboptimal therapeutic options.1 A great deal of research has been performed to understand the detailed mechanisms of AF, with the hope that a better appreciation of the fundamental determinants of arrhythmogenesis will lead to the development of novel, more successful treatment possibilities.2 Article see p 216 One aspect of AF pathophysiology that has elicited great interest has been changes in gap junction/connexin physiology. An important role for altered gap junction function and the potential importance of gap junctions as a therapeutic target for AF were first emphasized by Spach and Starmer over 15 years ago.3 Since that time, our understanding of gap junction physiology, biophysics, and molecular biology has increased enormously,4,5 as have studies on their role in AF pathogenesis and management.2 Gap junctions contain transmembrane ion-channel proteins called connexins (Figure,panel A). Connexons (containing 6 connexin molecules each) in the gap junctions of adjacent cardiomyocytes line up and attach, transferring ions or molecules <1 kDa freely between cells, coupling them electrically (Figure, panel B). Cardiac tissues express a variety of connexins, including connexin (Cx)40, Cx43, Cx45, Cx30.2/31.9, and Cx37,5 with all but Cx37 present in cardiomyocytes and the most important connexins in atrial tissue being Cx40 and Cx43. There is evidence that posttranslational modification, particularly connexin phosphorylation, is important in governing connexin localization and function.6 Figure. A , Connexons contain 6 connexins each, arranged in a circle. Connexins …

Connexin Gene Transfer Preserves Conduction Velocity and Prevents Atrial Fibrillation

Introduction: Fibrosis, cellular dysfunction and gap junction protein alterations occur in AF and cause conduction delay. We performed this study to test the hypothesis that gap junction protein overexpression would improve conduction and prevent AF. Methods and Results: Thirty Yorkshire swine were randomized into sinus rhythm (SR) and AF groups (n=15 per group). Each group included 3 subgroups: sham-operated control, gene therapy with adenovirus expressing connexin (Cx) 40 and Cx43, with 5 animals in each subgroup. All animals had epicardial gene painting; the AF group had burst atrial pacing. All animals underwent terminal study 7 days after gene transfer. SR animals had strong transgene expression but no atrial conduction changes. In AF animals, controls had reduced and lateralized Cx43 expression, and Cx43 gene transfer restored the expression of total and phosphorylated Cx43 to SR control levels. Immunohistochemical analysis revealed connexin localization to the intercalated disk after gene transfer. In the AF group, both Cx40 and Cx43 gene transfer improved conduction and reduced AF relative to controls. Conclusions: Atrial-specific gene therapy with gap junction protein preserved atrial conduction velocity and prevented AF.

Inducible Cx40‐Cre expression in the cardiac conduction system and arterial endothelial cells

The Connexin-40 (Cx40) gene encodes a gap junction protein that plays an important role in cell-cell communication in cardiomyocytes of the atria and cardiac conduction system and endothelial cells of large arteries. During embryonic development, Cx40 expression is tightly regulated and correlates with progressive ventricular conduction system (VCS) differentiation and vessel function. We have generated Cx40(Cre) mice carrying a CreERT2-IRESmRFP cassette by targeted recombination. In Cx40(Cre) mice, the pattern of expression of RFP is identical to that of the endogenous Cx40 gene and a Cx40(GFP) allele. Using a LacZ-based Cre reporter mouse line, tamoxifen dependent Cre recombination was observed throughout the spatio-temporal profile of Cx40 expression in the VCS and arterial endothelial cells. Cx40(Cre) mice can therefore be used to direct inducible genetic modification in Cx40 expressing cells.

E0678 Protein and mRNA expression of Cx40 in crista terminalis of patients suffered from rheumatic heart disease with chronic atrial fibrillation

ObjectiveTo investigate the protein expression and mRNA expression of Cx40 in crista terminalis of patients suffered from rheumatic heart disease with chronic atrial fibrillation. And evaluate the function of the...

Distinct phenotypes after cell‐specific deletion of Cx40: Loss in endothelial cells impairs conduction in arterioles and loss in renin‐producing cells elevates blood pressure

The gap junction protein connexin40 (Cx40) is important in cardiovascular and renal physiology as concluded from mice globally deficient for Cx40. They exhibit hypertension, renin excess, and impaired conduction of dilations along the endothelium. However, the exact contribution of endothelial Cx40 vs. Cx40 expressed in renin‐producing cells is unknown. In mice carrying a cell‐specific deletion of Cx40 in endothelial (EC) or renin‐producing cells (RPC) arteriolar responses and blood pressure were measured in vivo.Local stimulation of arterioles using ACh or bradykinin (Bk) elicited dilations that conducted rapidly without attenuation of the amplitude to upstream, remote sites (1200 μm) in mice carrying a floxed Cx40 gene (control). In contrast, remote dilations were significantly attenuated in EC‐Cx40ko (ACh by 28%, Bk by 32%) but not in RPC‐Cx40ko. However, local and remote dilations initiated by adenosine were comparable in all 3 groups of mice. Interestingly, awake EC‐Cx40ko were normotensive (EC‐Cx40ko: 115, control: 113 mmHg) whereas RPC‐Cx40ko exhibited a significantly elevated mean arterial pressure (138 mmHg) and heart weight.These data demonstrate that EC‐specific deletion of Cx40 impairs conduction of endothelium‐dependent dilations, however, per se does not increase blood pressure. Conversely, elevated pressure in RPC‐Cx40ko does not affect such signal conduction along arterioles.

Absence of Connexin 40 gene polymorphism, as a marker of undetected atrial fibrillation in patients with unexplained cerebral ischemic events

Atrial fibrillation (AF) is a major cause of cerebral infarction. Idiopathic AF is strongly associated with the human minor connexin 40 (Cx40) promotor polymorphism. We examined the prevalence of the minor Cx40 allele in patients with cerebral ischemia and no other cardiovascular disease (CVD), as an indication of underlying idiopathic AF. In patients with cerebral ischemia without prior CVD (n = 225), DNA analysis of the Cx40 minor allele (-44 G-->A) was performed. Patients were divided into those with a normal electrocardiographic (ECG) findings (group A, n = 164), with ECG abnormalities (group B, n = 51) and those with normal ECG and documented episodes of AF (group C, n = 10). On the basis of echocardiography (ECHO) data availability, further subgroups were defined: normal ECG and ECHO (group D, n = 45); ECG or ECHO abnormalities (group E, n = 22); and normal ECG and ECHO and documented AF episodes (group F, n = 8). The prevalence of Cx40 promotor polymorphism was compared among all the subgroups. The average age was 58.7 years (+/-11.5) and 64.4% were men. Patients with episodes of AF and those with abnormal ECG or ECHO results (B+C or E+F) did not show a higher prevalence of the minor allele genotype (AA vs. GG) compared with the normal ECG/ECHO groups (A or D) (group A, odds ratio = 1.04, 95% confidence interval: 0.26-4.11 and group D, odds ratio = 0.38, 95% confidence interval: 0.04-3.63). In patients with cerebral ischemic events, without prior CVD, a higher prevalence of the Cx40 gene polymorphism, as a marker of underlying idiopathic AF appeared to be absent.