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Cardiovascular Research 2004 62(2):299-308; doi:10.1016/j.cardiores.2004.02.010
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Copyright © 2004, European Society of Cardiology

Genetically modified mice: tools to decode the functions of connexins in the heart—new models for cardiovascular research

Daniel Gros*, Laurent Dupays1, Sébastien Alcoléa2, Sonia Meysen, Lucile Miquerol and Magali Théveniau-Ruissy

Laboratoire de Génétique et Physiologie du Développement (UMR CNRS 6545), Campus de Luminy, Case 907, Institut de Biologie du Développement de Marseille, Université de la Méditerranée, Marseilles cedex 13288, France

* Corresponding author. Tel.: +33-491-26-97-32; fax: +33-491-26-97-26. Email address: gros{at}ibdm.univ-mrs.fr

Received 3 November 2003; revised 23 January 2004; accepted 18 February 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Cx43, a major...
 3. Cx40, a major...
 4. Cx45 is essential...
 5. Conclusions
 References
 
It has long been known that gap junctions are required for the propagation of electrical impulse in the heart. A good deal later, the connexins (Cxs), which are probably exclusive components of the junctional channels that constitute the gap junctions, were identified. More recently, the in vivo functions of cardiac Cxs have been investigated by the analysis of genetically modified mice. These studies have confirmed that Cxs are involved in cardiac impulse conduction, and, unexpectedly, in heart morphogenesis. In addition, cardiac abnormalities described in mice genetically modified for Cx genes, and those observed in certain human cardiac diseases, have been proven to be similar.

KEYWORDS Connexins; Arrhythmia (mechanisms); Conduction (block); Sudden death; Developmental biology


   1. Introduction
Top
 Abstract
 1. Introduction
2. Cx43, a major...
 3. Cx40, a major...
 4. Cx45 is essential...
 5. Conclusions
 References
 
Of the 20 connexin (Cx) genes identified in the mouse genome [1] only three, Cx45, -43, and -40 have so far been shown to be expressed in the cardiomyocytes [2]. Their expression is not, however, restricted to the myocardium. Each of these genes has a unique expression pattern in the adult and the developing mouse heart [2]. Cx37 is also a cardiac Cx. It is synthesized in the endocardial cells, but no heart defects have to date been detected in Cx37-deficient mice. Other Cxs, which remain to be identified, are probably expressed in the mouse heart.

The biophysical properties of the junctional channels formed by these Cxs have been extensively investigated in vitro in experimentally controlled situations [3,4]. The in vivo situation is more complex because these Cxs are probably involved in various types of channels (homo- and heteromeric, homo- and heterotypic channels) whose distribution in the cardiac tissues is unknown. An in vitro reductionistic approach is required to elucidate the functioning of these channels, but only detailed analyses of genetically modified mice will make it possible to demonstrate the in vivo functions of Cxs. The investigation of human cardiovascular diseases induced by mutations of cardiac Cx genes, or of genes modifying the expression of cardiac Cxs, could also provide clues as to the roles of these proteins.

The aim of this review is to analyze the results from studies of genetically modified mice for the Cx genes expressed in the cardiomyocytes. These genes are involved in both cardiac electrical impulse propagation and cardiogenesis.


   2. Cx43, a major determinant of electrical impulse propagation in the ventricles
Top
 Abstract
 1. Introduction
 2. Cx43, a major...
3. Cx40, a major...
 4. Cx45 is essential...
 5. Conclusions
 References
 
Cx43 is widely distributed throughout vertebrate tissues, and it is the major Cx of the mammalian heart. In the mouse it is abundantly expressed in all the cardiac compartments with the exception of the sino-atrial (SA) and atrio-ventricular (AV) nodes, the His bundle, and the proximal parts of the bundle branches [2] (Fig. 1). Mouse embryos (129SvXC57BL/6 genetic background) in which both alleles of the Cx43 gene had been disrupted survived until birth but died of asphyxiation shortly after delivery [5,6]. Death is the consequence of an obstruction of the right ventricular outflow tract, which prevents the blood flow from reaching the lungs. Heterozygous mutants are viable and able to breed.


Figure 1
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  		Fig. 1 Expression pattern of Cxs associated with cardiomyocytes in the adult mouse heart. (A) Diagram of the heart. The conduction system includes the atrio-ventricular (AV) node, the His bundle (HB), the bundles branches (BB), and the Purkinje fibers (PF). SA node: sino-atrial node, IVS: interventricular septum. (B, C, and D) Expression (dark grey) of Cx43, -40 and -45, respectively. The same expression patterns, with a few minor variations, are also found in the heart of other mammals [2,51,65]. The patterns of Cx43, -40 and -45 in the adult heart result from the spatio-temporal regulation of their expression during cardiogenesis. The expression patterns of these Cxs at various stages of embryonic heart are illustrated in Ref. [29].

 
2.1. Abnormalities of heart morphogenesis and the coronary arteries in Cx43 knockout mice
The first abnormalities detected during the cardiac development of the Cx43–/– knockout mouse embryos are a delay in the normal looping of the heart tube (looping which normally occurs at the embryonic stage E9, i.e., 9 days post-conception, equivalent to 20–22 days post-conception in human), and, subsequently, the progressive development of intertrabecular pouches at the junction of the right ventricle (RV) and the outflow tract [7]. This phenotype shows some variability, with some mice exhibiting double pouches while others exhibit only a single bulge on one side of the outflow tract [5,8]. Further investigations indicated that the walls of the pouches were sometimes continuous with distal coronary artery branches [9]. The tissue lining the interior of the pouches was shown to consist of a proliferating cardiac myosin-free tissue, expressing markers of vascular smooth muscle cells [9]. Heavy extracellular matrix deposits were also detected in the wall of these pouches [9]. These observations suggested that the formation of the pouches involved the abnormal distribution of vascular smooth muscle cells and fibroblasts, both of which are derived from the proepicardial organ that, in wild-type mice, expresses Cx43. This organ is the source of cells that migrate over the tubular heart to form the epicardium. Subsequently, the epicardial cells infiltrate the myocardium to give rise to the cardiac fibroblasts, and the vascular smooth muscle cells of the coronary arteries.

Modifications of the conotruncal region of the developing heart due to the lack of Cx43 are attributed to changes in the migratory behavior of a subpopulation of neural crest cells, the cardiac crest cells, that express Cx43, and are involved in the development of the cardiac outflow tract [10,11]. Studies of transgenic mice exhibiting a gain [12,13] or loss [13,14] of Cx43, confined to cardiac crest cells, and in vitro experiments [13,15], have provided evidence for a role played by Cx43 junctional channels in the ability of these cells to participate in right ventricular outflow tract morphogenesis. The migration rate of cardiac neural crest cells was shown to increase with overexpression of Cx43, and decrease with deficiency of Cx43. In addition, in the Cx43–/– mice, cardiac crest cells migrate into the outflow tract, but in reduced numbers. These observations, and others, suggest that some aspects of the cardiac crest cell role are quantitatively modulated by Cx43-mediated cell–cell communication and that these probably affect events modulating tissue remodeling in the outflow tract [16].

Cardiac malformations in the Cx43–/– mice also include the coronary arteries. They give rise to discontinuities and decrease of smooth muscle myosin expression in the wall of these arteries, reduction of their diameter, and modifications of their major patterning [9]. The same set of patterning abnormalities were observed in both homo- and heterozygous mutants [9] but the influence of the genetic background on the modifications of the patterning cannot be excluded [7]. The abnormalities of the coronary arteries were shown to be associated with an increase in the rate of migration and proliferation of the proepicardial cells whereas the ability of these cells to differentiate into vascular smooth muscle cells is not affected by the lack of Cx43 [9]. Transgenic mice with a loss of Cx43 function restricted to cardiac crest cells [13,14] exhibit an outflow tract obstruction similar to that of the Cx43 knockout mice, but never have intertrabecular pouches. In addition, the behaviour of their proepicardial cells is not altered [9]. These results, associated with the presence of smooth muscle cell and fibroblast markers in the tissue lining the wall of the pouches, indicate that the proepicardial cells also play an essential role in pouch formation.

It is known that the formation of the coronary arteries is dependent on the neural crest cells, although, unlike proepicardial cells, they do not contribute structurally to these vessels [11]. Their role is presumably only a regulatory one, but various coronary artery patterning defects, such as anomalous origin, absence of the left or the right coronary artery, and the development of accessory vessels, similar to those observed in Cx43–/– mice, have been previously described in neural crest-ablated chick embryos [17]. Given that the neural crest and proepicardial cells are both found at the base of the heart where the coronary arteries form from the aorta, signaling through interactions between these two cell types certainly plays a role in modulating the initial patterning of the coronary arteries. Whatever the mechanism of these interactions, which has not yet been elucidated, it is likely that the normal development of the coronary arteries requires a precise balance between these two cell types whose migration from their sources is modulated by Cx43. Multiple coronary anomalies observed in the Cx43–/– mice have also been described in human, and, consequently, this Cx is a candidate gene for mutations that may play a significant role in human coronary artery anomalies [9].

2.2. Severe Cx43 deficiency in the ventricular cardiomyocytes results in arrhythmias and sudden death
Arrhythmias have been commonly observed in Cx43–/– embryos at E17.5, and as a rule after birth [18]. In perfused hearts, isolated from Cx43+/– adult mice, and exposed to acute ischemia, spontaneous ventricular tachyarrhythmias occur twice as frequently as in wild-type mouse hearts, and last longer [19]. Thus, reduction of Cx43 expression, and consequently, of the electrical coupling, may play a critical role in ventricular arrhythmogenesis. This has been elegantly confirmed by investigating Cx43-conditional knockout mice.

To circumvent the postnatal lethality that occurs in Cx43–/– mice, Gutstein et al. [20] have generated mice with a cardiac-restricted inactivation of Cx43 by the use of the Cre/loxP system. Cx43 expression was reduced by 95% and 86% in the ventricles of adult {alpha}-MHC CKO ({alpha}-myosin heavy chain conditional knockout) and MLC2v CKO (myosin light chain 2v conditional knockout) mice (resulting from the crossing of {alpha}-MHC/Cre or MLC2v/Cre mice with Cx43flox/flox mice), respectively. Only 10% of the ventricular myocytes of these mice immunostained positively for Cx43 [21]. In cardiomyocytes with Cx43 deficiency, the organization of adherens junctions and desmosomes was not altered [22]. The heart of these mice has a normal structure, and its contractile performance was similar to that of the control hearts. In addition, no compensatory increase in the expression of other Cxs known to be synthesized in cardiomyocytes was observed. All these mice succumb to sudden cardiac arrest resulting from spontaneous ventricular arrhythmias by 2 months of age.

Another way to overcome the postnatal lethality of Cx43–/– mice is to induce the deletion of the Cx43 gene at the adult stage, in all cells that express it. This has been performed with Cx43Cre-ER(T)/flox mice (resulting from the crossing of Cx43Cre-ER(T)/+ mice with Cx43flox/flox mice) in which the efficient deletion of the Cx43 gene is induced with 4-hydroxytamoxifen [23,24]. During the 3-week treatment, adult control mice survived, unaffected, whereas induced Cx43Cre-ER(T)/flox mice died. Telemetric ECG recordings indicated that sudden death was initiated by severe ventricular tachyarrhythmias. Analysis of the hearts of the induced transgenic mice revealed that expression of Cx43 in the ventricles does not reach 10% of its normal value. Further investigations have shown that sustained tachycardias in induced Cx43Cre-ER(T)/flox mice were based on a stable reentry circuit in the right ventricle, and fibrillatory conduction in the left ventricle [24].

Severe reduction of Cx43 in heart may also be induced by activation of the c-Jun N-terminal kinase (c-JNK). In the transgenic mice expressing MKK7D (a specific activator of c-JNK) under the control of the murine {alpha}-MHC promoter, the ventricular expression level of Cx43 was only 13% of its control value [25]. These transgenic mice died prematurely between 6 and 8 weeks of age. The cause of their death has not been identified, but one may assume, given the above data, that these mice demonstrate fatal ventricular arrhythmias.

The above results indicate that: (i) there is no intrinsic cardiomyocyte-autonomous requirement for Cx43 during heart development, in agreement with studies which have shown that the cardiac developmental defects resulting from germline inactivation of Cx43 come from alterations of non-myocyte lineages; and (ii) severe deficiency of Cx43 expression, alone, may serve as a critical event in the formation of an arrhythymogenic substrate. Therefore, the conditional knockout mouse lines described above represent new genetic models to investigate ventricular arrhythmias and sudden death resulting from low electrical coupling.

In the chimeric mice, generated from Cx43–/– embryonic stem cells and wild-type blastocystes, regions of cardiac muscle with normal Cx43 expression are adjacent to regions devoid of Cx43 [26]. These mice develop normally, but the presence of discrete areas of conduction delay of varying dimension were detected in the ventricular walls. In addition, this loss of synchronous activation was found to diminish the contractile performance of the ventricles. Spontaneous ventricular arrhythmias were also observed but this phenomenon affected only a small minority of the investigated hearts. Heterogeneous expression of Cx43 has also been observed in the heart of the transgenic mice that express a constitutively active form of the human retinoic acid receptor (hRAR{alpha}) under the control of the β-MHC promoter [27]. These mice develop a dilated cardiomyopathy, and Cx43 is both downregulated (more than 50% decrease) and heterogeneously redistributed in the left ventricular free wall. The size of the regions almost devoid of Cx43 ranges from small clusters of 5–10 cells to about 40% of the wall. Mapping of the cardiac electrical activity revealed a delayed ventricular activation with increased heterogeneity of propagation. However, ventricular arrhythmias did not occur spontaneously in these transgenic mice, nor could they be induced by ventricular pacing [27]. Analysis of the last two mouse lines clearly indicates that even moderate and heterogeneous expression of Cx43 in the ventricular tissue is sufficient to prevent arrhythmias, in most of the cases.

Electrical uncoupling induced by acute ischemia enhances arrhythmogenesis [19], but it may also protect the heart by limiting intercellular spread of chemical mediators of injury. This hypothesis was checked by comparing ventricular remodeling and infarct size in wild-type and Cx43+/– mice following coronary ligation [28]. Infarct size measured histologically eight days after infarction was 29% smaller in Cx43+/– mouse heart (17±14% of total left ventricular area) than in wild-type mouse heart (24±15%). Ten weeks after coronary occlusion, the infarct size was still smaller (6±5% vs. 12±7%). Thus, Cx43-deficient mice develop much smaller infarcts than wild-type mice, and this results in a paradoxical situation in which new therapies, designed to decrease the risk of arrhythmias by enhancing intercellular communication, could lead to larger infarcts caused by persistent coronary occlusion.

2.3. Cx43 plays a major role in electrical impulse propagation in the ventricles during late embryonic life
The propagation of electrical impulse was investigated in the ventricles of Cx43-deficient mice at various stages of embryonic development. No reduction in the conduction velocity (CV) was detected in Cx43–/– embryos at E12.5 [18]. The compensating presence of other Cxs expressed in the myocytes (Cx40 and -45) [29,30], at this stage, may explain these results. In contrast a marked reduction in the CV was observed in the right ventricle at E15.5, and in both ventricles at E17.5 [18]. At the latter stage, for example, the CV in the RV was reduced from 8.4–8.7 cm/s in wild-type and Cx43+/– embryos to 1.1 cm/s in Cx43–/– embryos. Analysis of action potentials excluded a genotype-dependent reduction of excitatory current density, suggesting that reduction of the upstroke velocity of the action potentials could not be the primary cause of the slowing of propagation [18]. Thus, Cx43 is largely responsible for the conduction of electrical impulse in the ventricles during late embryonic life. Low amounts of Cx40 and -45 still expressed in the working ventricular myocytes, or unidentified Cxs, would account for the residual conduction.

2.4. How much reduction of Cx43 protein is required to result in a significant slowing of electrical impulse propagation in the adult ventricles?
The disruption of one allele of the Cx43 gene results in about 50% reduction in Cx43 protein expression in the atrial and ventricular tissues, and unchanged expression of the Cx40 protein [31–34]. The number of Cx43 gap junctions is concomitantly reduced without any change in the size of the remaining junctions [35]. Action potentials recorded from ventricular myocytes isolated from neonatal wild-type, Cx43+/– or Cx43–/– mice are similar, and the sodium channel activity in these very same myocytes is not dependent on the genotype [36]. In addition, heterozygous Cx43 deficiency does not significantly affect cardiac defects such as concentric remodeling, mild systolic dysfunction and fibrosis, associated with ageing in mice [37].

The epicardial CV, measured using a linear electrode array, was found to be similar in the atria of adult Cx43+/– and wild-type mice (0.35–0.4 m/s at 31 °C) [32], suggesting that the abundant expression of Cx40 in this tissue may prevent the development of a conduction phenotype when Cx43 is reduced by half. In contrast, a very significant slowing (38–44%) of the CV was observed in the ventricles of the Cx43+/– mice as compared with wild type mice [31,32] (0.18–0.25 vs. 0.32–0.38 m/s; 31 °C). This slowing of conduction was confirmed by Eloff et al. [33] using an optical mapping technique (CVmax: 0.42 and 0.58 m/s in Cx43+/– and wild-type mice, respectively; CVmin: 0.28 vs. 0.38 m/s; 37 °C). These results were challenged by Morley et al. [38], who, using a similar optical mapping method, did not observe any difference between the ventricular CVs measured in wild-type and Cx43+/– mice (CVmax: 0.63 and 0.628 m/s in Cx43+/– and wild-type mice, respectively; CVmin: 0.395 vs. 0.402 m/s; 37–39 °C). The discrepancies between these results obtained with similar techniques are difficult to explain, all the more so since a possible effect due to the genetic background of the Cx43+/– mice has been eliminated [33]. They may relate to some of the methodological and instrumental differences that have been discussed [33]. Gustein et al. [20], using the same technique as Morley et al. [38], found a very significant slowing of propagation ({approx}50%) in the Cx43 CKO mice (CVmax: 0.62 and 0.36 m/s in control and {alpha}-MHC CKO mice, respectively; CVmin: 0.38 vs. 0.17 m/s) in which expression of Cx43 in the ventricles is only 5–16% of this normal value (Section 2.2.). Van Rijen et al. [24], using an electrode mapping technique, have also reported that a 70–95% decrease of Cx43 was required to result in a significant reduction of the CV in the ventricles, a 50% reduction resulting in no change. In summary, and somewhat schematically, a 50% reduction of the CV in the ventricles is reached with a 50 [31–33] or 90% reduction [20,24] of Cx43, according to different authors. The difference is considerable.

By getting around the problem of organ complexity, can in vitro experimental data help to elucidate the precise effect of a reduction in Cx43 expression on the propagation velocity of electrical impulse? Measurements of electrical properties by microelectrodes and optical mapping were carried out on synthetic strands of cultured neonatal ventricular myocytes isolated from wild-type and Cx43+/– mice [39]. The CV was found to be independent of the genotype, but maximum upstroke velocity of the action potentials (dV/dt max) was increased, and action potential duration was reduced in the Cx43+/– strands. Computer simulation of propagation and dV/dtmax revealed a relatively low dependence of the CV on Cx43 gap junction coupling (a conclusion also previously reported by Jongsma and Wilders [40]), and suggested that the difference in dV/dtmax was due to an upregulation of INa in the myocytes of Cx43+/– strands. Thus, in this model, the absence of a slowing of the CV associated with about 50% decrease of Cx43 may be explained by the dominating role of myoplasmic resistance and the compensatory increase of dV/dtmax [39]. The requirement for additional mechanisms to maintain the propagation of influx is also suggested by the investigations of Yao et al. [21]. These authors have evaluated the electrical coupling in ventricular cardiomyocyte pairs isolated from the heart of adult control and Cx43 CKO mice generated by Gutstein et al. [20] (Section 2.2). In cell pairs isolated from conditional mice the junctional conductance was found to be reduced to a few nS, including 21% of cell pairs with no detectable electrical coupling, compared with about 600 nS in control cell pairs. Theoretical models indicate that this very low degree of coupling is not expected to support levels of conduction measured in vivo in these mice (0.36 and 0.17 m/s for CVmax and CVmin, respectively) [20], and suggest that additional mechanisms (upregulation of INa for example) are required to maintain the propagation of influx when gap junctional conductance is severely reduced [21].

2.5. Cx40 or Cx32 can only partially fulfil the functions of Cx43 in the heart
The knockout techniques are used to evaluate the in vivo functions of the deleted gene. A complementary approach is to replace the gene of interest by another gene (knockin or KI technique). This technique has been proven to be suitable to differentiate the functions of genes encoding closely related proteins. Cx43KICx32 and Cx43KICx40 mice have been generated by Plum et al. [41]. Homozygous mutants (Cx43KICx32/KICx32 and Cx43KICx40/KICx40 mice) of both lines survived until adulthood though a significant number of them (40%) die during the first three weeks after birth. The hearts of Cx43KICx32/KICx32 neonates showed morphological defects similar to those of Cx43–/– mice, but to a much lesser extent. In contrast, the hearts of Cx43KICx40/KICx40 neonates were comparatively normal. The parameters of ECGs recorded from adult hetero- (Cx43+/KICx32, Cx43+/KICx40) and homozygous Cx43KICx32 and Cx43KICx40 mice were no different from those of control wild-type mice. However, Cx43KICx40 mice, both hetero- and homozygotes, were more prone to spontaneous arrhythmias than control mice. Interestingly, the frequency of arrhythmias was low in Cx43KICx32 mice as compared with Cx43KICx40 mice. These results indicate that Cx43–/– mice can be rescued to some extent by either Cx40 or Cx32 expression, but in no case are both normal structure and function of the heart restored. Thus, Cx43 has unique properties that are indispensable for normal morphogenesis and functioning of the heart.


   3. Cx40, a major determinant of electrical impulse propagation in the cardiac conduction system (CCS)
Top
 Abstract
 1. Introduction
 2. Cx43, a major...
 3. Cx40, a major...
4. Cx45 is essential...
 5. Conclusions
 References
 
At E12.5 Cx40 is strongly expressed in both the atria and the ventricles. From this stage, its expression is downregulated in the ventricular walls [30], resulting at the adult stage in a pattern restricted to the atria and the cardiac conduction system. In the latter, Cx40 is detected in the AV node, the His bundle and its two branches, and the Purkinje fibers [2] (Fig. 1). Cx40 has never been detected in the adult ventricular working myocytes. Cx40 knockout mice have been generated by two independent groups [42,43]. Lethality was reported to be either extremely low [42,44], or high [6] suggesting a strong influence of the genetic background. Surviving mice are viable and fertile. Expression levels of Cx37, -43, and -45 genes in the heart of these mice are similar to those measured in wild-type mice [43].

3.1. Cx40 is involved in the propagation of electrical impulse from the atria to the ventricles
Numerous studies, summarized in Table 1, have focused on electrical impulse propagation in the heart of the Cx40–/– mice [42,43,45–50]. There are obvious discrepancies between these results, which can be attributed to differences in the genetic background of the mice analyzed, the recording techniques and the stimulation protocols used, the size of the samples and the methods of sedation. However, these investigations agree in identifying two types of defects: anomalies of the cardiac influx propagation at various levels of the CCS, and an increased incidence of inducible atrial arrhythmias.


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  		Table 1 Main characteristics of the electrophysiological phenotype of the Cx40–/– mice heart

 
Alteration of impulse conduction has been detected in the AV node [45,46,48], the His bundle [48–50], and the bundle branches [42,45,47,50], in agreement with the distribution of Cx40 in the CCS. In addition, the right bundle branch was shown to be more vulnerable that the left branch [47,50], a characteristic also frequently observed in patients with cardiovascular diseases. In the absence of Cx40, the impulse propagation in the basal part of the CCS (i.e., the AV node, the His bundle, and the proximal parts of the branches) depends upon Cx45 only. In the apex region, encompassing the distal part of the branches and the Purkinje fibers, both Cx45 and Cx43 participate in the impulse conduction. Given the high level of expression of Cx40 in the atria, one might expect that all studies would report slow conduction in this compartment. This is, however, not the case, and several authors have indicated that no change was detected in the atria [47–49], suggesting that Cx43, alone, in this tissue, was able to ensure normal influx propagation. Similarly, spontaneous arrhythmias have been detected in some cases, and not in others [42,46,49]. In contrast, the Cx40–/– mice unambiguously exhibit a proneness to atrial fibrillation upon stimulation [45,46,49]. Finally, disruption of both alleles of the Cx40 gene results in sino-atrial conduction disturbances [45,46,49], indicating that, even though Cx40 is not expressed in the SA node, Cx40 deficiency in the working perinodal atrial myocytes may impair SA function.

ECGs have been recorded in the Cx40+/–/Cx43+/– double heterozygotes [6]. Analysis of ECG parameters indicated that haplodeficiency of both Cx40 and Cx43 has additive effects in the ventricles (increase of QRS complex duration), but not in the atria.

3.2. Cardiac malformations in Cx40-deficient mice
The occurrence of cardiac malformations in surviving Cx40-deficient mice was first reported by Kirchhoff et al. [6] who have identified atrial and ventricular septation defects, at low incidence, and in some cases, hypertrophy. More systematic studies were carried out by Gu et al. [44] on fetuses and newborns. Their analyses indicate that the cardiac malformations in the C40+/– mice include: bifid atrial appendages, ventricular septal defects, tetralogy of Fallot, and aortic arch abnormalities. In Cx40–/– mice resulting from the crossing of Cx40+/– mice, the most common abnormalities were double-outlet right ventricle, tetralogy of Fallot, and partial endocardial cushion defects. The overall incidence of cardiac malformations was 18% and 33% in Cx40+/– and Cx40–/– mice, respectively. In the heart of Cx40–/– mice resulting from the crossing of Cx40–/– mice the incidence of malformations reached 44%, and the conotruncal malformations (double-outlet right ventricle or tetralogy of Fallot) were predominant. These results indicate that Cx40 may be involved in cardiogenesis, and more especially in the septation process. Given that malformations were present only in a fraction of the animals, and that the investigated mice were of mixed genetic background the above data also suggest the existence of genetic modifiers that influence cardiogenesis, and which either compensate for the absence of Cx40, or are modulated by the Cx40 protein.

Cx40–/–/Cx43+/– and Cx40+/–/Cx43–/– double mutant mice die around birth [6]. All embryos with these genotypes have cardiac abnormalities. In the Cx40–/–/Cx43+/– embryos, the most frequent defects were abnormal AV connections, which were more severe than those described in the Cx40–/–/Cx43+/+ embryos. Defects of the ventricular septum, premature closure of the ductus arteriosus, and subcutaneous edema were also observed. The heart of the Cx40+/–/Cx43–/– embryos was characterized by an obstruction of the right ventricular outflow tract similar to that described for Cx40+/+/Cx43–/– animals. These results demonstrate that Cx43 haplodeficiency aggravates the morphological phenotype resulting from Cx40 deficiency, whereas Cx40 haplodeficiency does not worsen the Cx43–/– phenotype.

The expression pattern of Cx40 in the human heart is similar to that of the mouse heart [51]. Consequently, one might expect that Cx40 deficiency in human recapitulates the cardiac anomalies described in the mouse. This is indeed the case. Dominant mutations of Tbx5 and Nkx2–5 result in both humans and mice in a cardiac electrophysiological and morphological phenotype that is strikingly reminiscent of that of Cx40-deficient mice [52]. Furthermore it has been shown that Cx40 was a downstream target of both Tbx5 and Nkx2–5 [52]. Thus, the human phenotype is probably due, at least in part, to the low expression of Cx40 in the heart.

3.3. Cx45 can only partially fulfil the functions of Cx40 in the heart
Cx45 is expressed in the CCS along with Cx40, but at very low abundance compared with that of Cx40 [2]. Is Cx45 able to functionally replace Cx40? The Cx40KICx45/KICx45 transgenic mice have a normal heart structure, but mapping of cardiac electrical activation indicated that the replacement of Cx40 by Cx45 resulted in a significant reduction of the CV in the left atrium, and a normal CV in the right atrium [53]. The conduction delay in the AV node was unaffected, whereas a partial loss of function became apparent in the right bundle branch, but not in the left branch [53]. Thus, Cx45 only partially fulfils the conduction function of Cx40 in the heart, perhaps in part because of the low unitary conductance of the Cx45 channels compared with that of Cx40 channels [3,4].


   4. Cx45 is essential for embryonic heart development
Top
 Abstract
 1. Introduction
 2. Cx43, a major...
 3. Cx40, a major...
 4. Cx45 is essential...
5. Conclusions
 References
 
At the early stages of cardiogenesis, Cx45 is expressed in all mouse heart compartments, including the AV canal, then it is downregulated [29]. In the adult myocardium, Cx45 is synthesized in small amounts in the SA node, the various parts of the CCS and the most peripheral regions of the interventricular septum [54] (Fig. 1). Its expression in the working cardiomyocytes is controversial. Most investigators have not detected it in these cells where its expression could be species-dependent.

4.1. Germline disruption of both alleles of the Cx45 gene is lethal
Germline disruption of both alleles of the Cx45 gene is lethal, and all embryos die in utero around E10 [55,56]. Cardiac contractions are initiated at E8.5 in the knockout embryos, as in the wild-types, but AV conduction blocks appear at E9.5, associated with the absence of coordination between the contractions of the primitive ventricle and those of the outflow tract [56]. So far, only Cx45 has been shown to be expressed in the AV canal at the early stage of cardiogenesis [29]. Its absence in the knockout embryos may account for the AV conduction blocks. Other cardiac abnormalities were observed which include a looping defect, dilatation of the chambers, reduced trabeculation and endocardial cushion defects. The latter defects may result from impairment of the epithelial–mesenchymal transformation of the cardiac endothelium which may be due to the disruption of the Ca2+/calcineurin/NF-ATc1 pathway [57]. Besides the cardiac defects, several other abnormalities that may contribute to the lethal phenotype have also been observed. They include the interruption of vascular development (which may be due to defective TGFβ signaling in the vasculature of the yolk sac), an impaired placental function and massive apoptosis [56].

4.2. Cardiomyocyte-specific inactivation of both alleles of the Cx45 gene does not prevent lethality
Cx45 conditional knockout mice (resulting from the crossing of Cx45flox/flox mice with cardiac {alpha}-actin/Cre mice) were generated with the aim of circumventing the embryonic lethality of Cx45–/– animals [58]. At E9.5, activity of the Cre recombinase in the conditional knockout embryos was restricted to the cardiomyocytes. Surprisingly, the embryos die at around E10, indicating that the heart defects were the cause of death. These defects were similar to those previously described in the non-conditional knockouts [55], except for the endocardial cushions that were well-developed, suggesting that the formation of these structures is not dependent on Cx45 expression in the cardiomyocytes. The vasculature development also was impaired, but to varying degrees between embryos. In both types of knockouts, contractions of the outflow tract were weak or absent, probably resulting in reduction of intracardiac blood flow-induced forces. These observations must be considered in the light of recent experiments carried out on zebrafish heart that indicate that reduction of intracardiac haemodynamics resulted in cardiac defects including the lack of heart looping and impaired valve formation [59]. The phenotype of Mlc2a–/– mouse embryos characterized by severely diminished atrial contractions also suggests that fluid forces play a crucial role at the earliest stages of cardiac morphogenesis [60]. Thus, the abnormal haemodynamics which likely occur in the Cx45 knockout embryos may explain some aspects of the cardiac and vasculature morphological phenotypes. Whatever the mechanisms involved, these results indicate the requirement of Cx45 for the normal progress of cardiogenesis.

4.3. Cx45 and Cx40 have additive roles
Cx40 and Cx45 being both expressed in the CCS, it is reasonable to wonder whether these two Cxs have additive or compensatory functions. Only a few double mutant mice for both Cx40 and -45 (Cx40–/–/Cx45+/– mice) survived until adulthood [61]. About 60% of them die during embryogenesis or just after birth. A high incidence of septal defects and a conduction delay in the atria were observed. These preliminary results suggest that these Cxs have additive roles in cardiac morphology and function.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Cx43, a major...
 3. Cx40, a major...
 4. Cx45 is essential...
 5. Conclusions
References
 
Analysis of genetically modified mice for Cx genes expressed in the cardiomyocytes raises four essential points.

(i) Cx43, -40 and -45 are involved in cardiogenesis. This role may be either direct (Cx45) or indirect (Cx43), though for a given Cx synergic action of the two modes cannot be ruled out.
(ii) Cx43, -40 and -45 are involved in electrical impulse propagation in embryonic and adult heart. The junctional channels they form are required for the propagation to occur, but impulse conduction velocity is modulated by a variety of other factors.
(iii) Cx43, -40 and -45 have a unique function in the heart, and one given Cx can only partially replace another one.
(iv) Genetically modified mice for Cx43 and Cx40 genes develop cardiac anomalies that mimic those of known human diseases (sudden death syndrome, primary and secondary conduction blocks, etc.). These mouse models should prove highly useful to decipher the mechanisms that come into play in these diseases, and for the development of future therapies. Reciprocally, are there human cardiac genetic diseases which are unambiguously induced by Cx gene mutations? Mutations in the coding region of the human Cx43 gene have been shown to be associated with deafness [62] and oculodentodigital dysplasia [63]. In the first case, no cardiac defect was reported; in the second one, cardiac abnormalities were detected but very rarely, and when detected, they occurred along with numerous other defects. Atrial standstill is a rare arrhythmia characterized by the absence of electrical and mechanical activity in the atria. A familial form of this syndrome has been attributed to the coinheritance of a cardiac sodium channel mutation and rare polymorphisms in the basal promoter of the human Cx40 gene [64]. Thus, human cardiac diseases specifically caused by mutations on Cx genes have not often been described, either because they are lethal, or because they have not yet been identified. The example of the genetically modified mice suggests there are probably many other human cardiovascular diseases resulting from mutations on Cx genes that have yet to be discovered.


   Notes
 
1 Present address: National Institute for Medical Research, Mill Hill, London, UK. Back

2 Present address: Laboratoire de Physiopathologie et Pharmacologie Cellulaire et Moléculaire (U INSERM 533), Nantes, France. Back

Time for primary review 20 days


   References
Top
 Abstract
 1. Introduction
 2. Cx43, a major...
 3. Cx40, a major...
 4. Cx45 is essential...
 5. Conclusions
 References
 

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