12
Original article Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants Laurent Dupays a,1 , Thérèse Jarry-guichard a , David Mazurais b,2 , Thierry Calmels b , Seigo Izumo c,3 , Daniel Gros a , Magali Théveniau-Ruissy a, * a Laboratoire de Génétique et Physiologie du Développement (UMR CNRS 6545), Institut de Biologie du Développement de Marseille, Université de la Méditerranée, Campus de Luminy, case 907, 13288 Marseille cedex 9, France b Bioprojet-Biotech, 35762 Saint-Grégoire, France c Cardiovascular Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Received 17 December 2004; received in revised form 8 February 2005; accepted 16 February 2005 Abstract In humans, mutations of the gene encoding the transcription factor Nkx2-5 result in the heart in electrical conduction defects and morpho- logical abnormalities. In this organ Nkx2-5 is expressed in both the myocardium and the endocardium. Connexins (Cxs) are gap junction channel proteins that have been shown to be involved in both heart development and cardiac electrical conduction, suggesting a possible correlation between expression of Cxs and Nkx2-5. To evaluate this correlation, the expression of Cxs has been investigated in the cardiovas- cular system of wild-type and Nkx2-5–/– 9.2 days post-conception (dpc) mouse embryos. The disruption of the Nkx2-5 gene results in the loss of Cx43 in the heart, due in part to the poor development of the ventricular trabecular network, as well as specific downregulation of Cx45 gene expression. In addition, the nuclear translocation of NFATc1 in the endocardial endothelial cells is inhibited in the Nkx2-5–/– embryos. These results indicate for the first time that Nkx2-5 is involved in the transcriptional regulation of the Cx45 gene expression. In the mutant embryos the aorta is collapsed, and the vascular endothelial Cxs, Cx40 and Cx37, are no longer expressed in its posterior region. Poor development of the trabeculae and downregulation of Cx45 may contribute both to failure of the myocardial function and to hemo- dynamic insufficiency. The latter, in turn, may result in the dysregulation of Cx40 and -37 expressions along the whole length of the aorta. Direct or indirect effects of Nkx2-5 inactivation on the Cx45 gene expression could explain the absence of the endocardial cushions in the heart of Nkx2-5–/– embryos. © 2005 Elsevier Ltd. All rights reserved. Keywords: Nkx2-5/Csx; Heart development; Connexins; Connexin45; Connexin43; Connexin40; NFATc1 1. Introduction Connexins (Cxs) are transmembrane proteins, which form intercellular channels aggregated into gap junctions. In the heart these channels provide low electrical resistance path- ways that are essential for the coordinated spread of cardiac impulse and the synchronization of the contraction of the car- diomyocytes [1]. Twenty Cx genes have been identified in the mouse genome [2], and the Cx43, -40 and -45 proteins have been shown to be expressed in the cardiomyocytes of the mammal heart. Furthermore, each of these Cxs has a unique pattern of expression in the adult myocardium (see [3]). Hence, in the mouse heart, Cx43 is abundantly synthe- sized in the atrial and ventricular walls, and the distal part of the conduction system; Cx40 is strongly expressed in the atria and the conduction system (except for the sinoatrial node); Cx45 is present in the conduction system (including the sinoatrial node). Residual expression of Cx45 in the mature ventricular working cardiomyocytes has also been reported. * Corresponding author. Tel.: +33 4 91 26 97 34; fax: +33 4 91 26 97 26. E-mail address: [email protected] (M. Théveniau-Ruissy). 1 Present address: National Institute for Medical Research, Mill Hill, Lon- don NW7 1AA, UK 2 Present address: Station SCRIBE-Inra. Laboratoire de Physiologie des Poissons, Campus de Beaulieu, 35042 Rennes cedex, France 3 Present address: Novartis Institutes for Biomedical Research, 100 Tech- nology Square, Cambridge, MA, USA Journal of Molecular and Cellular Cardiology 38 (2005) 787–798 www.elsevier.com/locate/yjmcc 0022-2828/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2005.02.021

Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants

Embed Size (px)

Citation preview

Page 1: Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants

Original article

Dysregulation of connexins and inactivation of NFATc1in the cardiovascular system of Nkx2–5 null mutants

Laurent Dupays a,1, Thérèse Jarry-guichard a, David Mazurais b,2, Thierry Calmels b,Seigo Izumo c,3, Daniel Gros a, Magali Théveniau-Ruissy a,*

a Laboratoire de Génétique et Physiologie du Développement (UMR CNRS 6545), Institut de Biologie du Développement de Marseille, Université de laMéditerranée, Campus de Luminy, case 907, 13288 Marseille cedex 9, France

b Bioprojet-Biotech, 35762 Saint-Grégoire, Francec Cardiovascular Division, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

Received 17 December 2004; received in revised form 8 February 2005; accepted 16 February 2005

Abstract

In humans, mutations of the gene encoding the transcription factor Nkx2-5 result in the heart in electrical conduction defects and morpho-logical abnormalities. In this organ Nkx2-5 is expressed in both the myocardium and the endocardium. Connexins (Cxs) are gap junctionchannel proteins that have been shown to be involved in both heart development and cardiac electrical conduction, suggesting a possiblecorrelation between expression of Cxs and Nkx2-5. To evaluate this correlation, the expression of Cxs has been investigated in the cardiovas-cular system of wild-type and Nkx2-5–/– 9.2 days post-conception (dpc) mouse embryos.

The disruption of the Nkx2-5 gene results in the loss of Cx43 in the heart, due in part to the poor development of the ventricular trabecularnetwork, as well as specific downregulation of Cx45 gene expression. In addition, the nuclear translocation of NFATc1 in the endocardialendothelial cells is inhibited in the Nkx2-5–/– embryos. These results indicate for the first time that Nkx2-5 is involved in the transcriptionalregulation of the Cx45 gene expression. In the mutant embryos the aorta is collapsed, and the vascular endothelial Cxs, Cx40 and Cx37, are nolonger expressed in its posterior region.

Poor development of the trabeculae and downregulation of Cx45 may contribute both to failure of the myocardial function and to hemo-dynamic insufficiency. The latter, in turn, may result in the dysregulation of Cx40 and -37 expressions along the whole length of the aorta.Direct or indirect effects of Nkx2-5 inactivation on the Cx45 gene expression could explain the absence of the endocardial cushions in theheart of Nkx2-5–/– embryos.© 2005 Elsevier Ltd. All rights reserved.

Keywords: Nkx2-5/Csx; Heart development; Connexins; Connexin45; Connexin43; Connexin40; NFATc1

1. Introduction

Connexins (Cxs) are transmembrane proteins, which formintercellular channels aggregated into gap junctions. In theheart these channels provide low electrical resistance path-ways that are essential for the coordinated spread of cardiac

impulse and the synchronization of the contraction of the car-diomyocytes [1]. Twenty Cx genes have been identified inthe mouse genome [2], and the Cx43, -40 and -45 proteinshave been shown to be expressed in the cardiomyocytes ofthe mammal heart. Furthermore, each of these Cxs has aunique pattern of expression in the adult myocardium (see[3]). Hence, in the mouse heart, Cx43 is abundantly synthe-sized in the atrial and ventricular walls, and the distal part ofthe conduction system; Cx40 is strongly expressed in the atriaand the conduction system (except for the sinoatrial node);Cx45 is present in the conduction system (including thesinoatrial node). Residual expression of Cx45 in the matureventricular working cardiomyocytes has also been reported.

* Corresponding author. Tel.: +33 4 91 26 97 34; fax: +33 4 91 26 97 26.E-mail address: [email protected] (M. Théveniau-Ruissy).

1 Present address: National Institute for Medical Research, Mill Hill, Lon-don NW7 1AA, UK

2 Present address: Station SCRIBE-Inra. Laboratoire de Physiologie desPoissons, Campus de Beaulieu, 35042 Rennes cedex, France

3 Present address: Novartis Institutes for Biomedical Research, 100 Tech-nology Square, Cambridge, MA, USA

Journal of Molecular and Cellular Cardiology 38 (2005) 787–798

www.elsevier.com/locate/yjmcc

0022-2828/$ - see front matter © 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.yjmcc.2005.02.021

Page 2: Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants

Besides the cardiomyocytes, Cx40 is also expressed in vas-cular endothelial cells, along with Cx37 [4]. Furthermore, bothCx37 and Cx45 [5] are synthesized in the endocardial endot-helial cells.

The role of myocardial Cxs in the functioning of the hearthas been in part deciphered by studies of Cx-deficient mice(reviewed by [3]). Deletion of the Cx40 gene results in anoma-lies of the impulse propagation in the atria and the conduc-tion system of adult mice. Specific inactivation of the Cx43gene expression in the cardiomyocytes has generated trans-genic mice which all undergo sudden cardiac death inducedby spontaneous ventricular arrhythmias. Thus, Cx43 and-40 are clearly involved in impulse propagation in the adultheart. Unexpectedly, Cx45 is the only identified Cx generequired for normal heart development in the early stages ofcardiogenesis. Specific inactivation of the Cx45 gene in thecardiomyocytes results in conduction blocks that are detectedsoon after the first contraction has occurred at 8.5 days post-conception (dpc), and all the conditional mutant embryos diearound 10 dpc [6].

At 8.5 dpc, the stage of the first contractions of the mouseheart, Cx45, -43 and -40 genes have been shown to be acti-vated in the heart as demonstrated by the presence of theirmRNAs [5,7,8]. From this stage, there is a dynamic modula-tion of the pattern of expression of Cxs which certainly reflectsthe adaptation necessary for optimum propagation of the car-

diac impulse at the various stages of heart development ([8],Fig. 1). This requires the fine tuning of the regulation of theexpression of Cxs in conjunction with the morphogeneticevents of cardiogenesis.

Little is known about the in vivo transcriptional regulationof the Cx genes in the heart [9]. However, a few data provideclues suggesting that transcription factors such asCxs/Nkx2-5, Tbx5, and HF-1b/Sp4 might be involved in thisregulation. Nkx2-5, one of the earliest known markers of thedeveloping heart, plays a pivotal role in cardiac morphogen-esis [10,11]. In humans, mutations of NKX2-5 have been cor-related with the occurrence at high penetrance of severe andprogressive AV conduction blocks and atrial septal defects[12,13]. In transgenic mice the expression of a mutant DNAnon-binding Nkx2-5 protein (Nkx2-5(I183P)), similar to thatfound in patients, results in profound cardiac conductiondefects and heart failure [14]. Three weeks after birth, thelevel of expression of both Cx40 and -43 proteins is dramati-cally decreased in the heart of the transgenic mice, suggest-ing that Nkx2-5 could play a specific role in the regulation ofboth genes. Haplo-insufficiency of Nkx2-5 in the mouse resultsin cardiac defects similar to those seen in humans with NKX2-5 mutations. These anomalies concern atrial septal defectsand arrhythmias (AV conduction block, atrial fibrillation, ven-tricular tachychardia) [15–17]. In these mice the expressionof Cx40 is downregulated in the atria, whereas the expres-

Fig. 1. Whole-mount in situ hybridization of specific probes for the Cx40, -43, -45, and -37 genes in wild-type (+/+) (a–d) and homozygous mutant (–/–) (e–h)mouse embryos of 9.2 dpc. Note the wide AV canal typical of the mutant embryos (arrows in e–h), the bulbous aspect of the ventricle (Insert in e), and thesomewhat narrow outflow tract (arrowhead). Panel a: Cx40 transcript (panel a) was detected in the atrium (at), the ventricle (v), the pharyngeal arches (*), thesomites (arrowheads), dorsal aorta (da) and umbilical vessels (uv). Panel e: no Cx40 transcript was seen in the heart or in the caudal vessels. Panel b: Cx43transcript was observed as a faint labeling in the ventricle (arrowhead, and insert) and in various regions of the embryos. Panel f: no cx43 transcript was detectedin the heart. Panel c: Cx45 transcript was widely distributed in the embryos. Panel g: no Cx45 signal was detected in the heart. Panel d: Cx37 transcript wasobserved in the heart and the vessels of the embryos. Panel h: Cx37 transcript was restricted to the heart and the anterior parts of the aorta. No signal wasdetected in the caudal vessels.

788 L. Dupays et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 787–798

Page 3: Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants

sion of Cx43 is unchanged in both the atria and the ventricles[16,17].

To investigate the regulation of cardiac Cxs by Nkx2-5 wehave analyzed Nkx2-5 null mutant mouse embryos and com-pared them with heterozygous (Nkx2-5+/–) and wild-type(Nkx2-5+/+) embryos. Our data indicate that inactivation ofthe Nkx2-5 gene results in Cx45 deficiency in the embryonicmyocardium, and in the inactivation of NFATc1 in theendocardium. These results may explain some aspects of thephenotype of Nkx2-5 null mutants.

2. Materials and methods

2.1. Generation of Nkx2-5 deficient mice

Homozygous mutant mice completely null for Nkx2-5(Nkx2-5–/– mice) were generated by inbreeding heterozy-gous animals [11]. Mice were mated overnight, and the fol-lowing morning was considered as 0.5 dpc when the femaleswere pregnant. Genotyping was carried out as described pre-viously [11].

2.2. In situ hybridization

Whole-mount in situ hybridizations and control experi-ments were carried out with wild-type embryos of 9.2 dpc(22–25 somites), and heterozygous and homozygous mutantembryos at the same stage, as described previously [7,8].

2.3. RT-PCR experiments

Hearts were dissected from 9.2 dpc embryos, and freed ofsurrounding tissues. Each heart was individually investi-gated. RNA was extracted and reverse transcribed using theoligo(dT) primer in the Supercript II amplification system(GIBCO-BRL). The reactions of amplification were per-formed using primers specific for the murine sequences ofthe glyceraldehyde-3-phosphate dehydrogenase (GAPDH),Cx40, Cx43 and Cx45 genes as described previously [7,8,18].vascular endothelial growth factor (VEGF) transcript wasamplified using nested PCR. The first primer pair used wasidentical to that described by Miquerol et al. [19]; the secondpair was: 5′-GCCCTGGAGTGCGTGC-CCACGTCAG-AGAGCA-3′ for the sense primer), and 5′-TCTGGCT-TTGTTCTGTCTTTC-3′ for the antisense primer. All theRNA samples were tested for DNA contamination in theabsence of reverse transcription. 10 RNA samples, extractedfrom 10 hearts, were probed for each type of embryo col-lected (wild-type embryos, heterozygous and homozygousmutant embryos).

2.4. Immunofluorescence experiments

The preparation of the samples, and the analysis by immu-nofluorescence of sections from 9.2 dpc embryos were car-

ried out as described previously [8] using a Zeiss Ax-ioskop2 MOT microscope. The specificity of the anti-Cx45,anti-Cx43 and anti-Cx40 rabbit antibodies has been previ-ously demonstrated [8]. Desmin and PECAM (platelet endot-helial cell-adhesion molecule) were used as markers of myo-cardium and endothelial cells, respectively. Anti-PECAM(clone CD31, PharMingen) antibody was a rat monoclonal;anti-desmin (clone DE-U-10, Sigma Aldrich Chimie), andnuclear factor of activated T-cells (anti-NFATc1) (clone 7A6,PharMingen) antibodies were mouse monoclonals. Second-ary antibodies used were TRITC (or FITC)-conjugated don-key anti-rabbit (or anti-mouse) IgGs, CyTM3-conjugated don-key anti-rabbit IgGs (all from Jackson ImmunoResearch Lab),and Alexa 488 (or 594)-conjugated horse (or donkey) anti-mouse (or anti-rat) IgGs (Molecular Probes).

2.5. Videomicroscopy

9.2 dpc embryos were collected from pregnant mice andkept in DMEM medium maintained at 37 °C during record-ing. Video-images of 10 hearts from wild-type embryos andof 17 hearts from homozygous mutant embryos were takenwith a camera (Sony SSC-DC 198P) connected to a LeicaMZ6 stereomicroscope. Recordings were processed usingAdobe Premiere 6.0. All embryos were genotyped as de-scribed above after recording.

3. Results

3.1. Cx40, -43 and -45 gene products were undetectable inthe heart of Nkx2-5 homozygous null embryos

The phenotype of Nkx2-5 heterozygous and homozygousnull embryos has been described previously by Tanaka et al.[11]. This phenotype is similar to that of mutant mice gener-ated by Lyons et al. [10] in which the Nkx2-5 gene was inter-rupted in the homeodomain. The homozygous mutants diebetween 9.5 and 11.5 dpc [11]. In this study, the embryoswere collected at stage 9.2 dpc, a stage at which the inter-atrial and inter-ventricular septa are not yet formed. Only thehomozygous mutant embryos with a beating heart were inves-tigated. The expression of the Tbx5 gene transcript, which isexpressed in the mouse heart as early as 8 dpc [20], was shownto be maintained in the homozygous mutants (not shown) aspreviously described by Yamagishi et al. [21], indicating thatthe embryos were collected before lethality. Similar controlswere previously performed by Tanaka et al. [11] on mutantembryos at 9.5 dpc by performing in situ hybridization andsemiquantitive RT-PCR showing that the expression ofa-cardiac actin was not significantly disturbed in Csx/Nkx2.5null mutant embryos. Besides, the expression patterns oftwenty different genes were investigated for the expressionat 9.5 dpc. Ten of these genes, in addition to the structuralgenes a-MHC and b-MHC, were shown not to be affected bythe absence of Nkx2-5 at the investigated stage [11].

789L. Dupays et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 787–798

Page 4: Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants

3.1.1. In situ hybridizationFig. 1 illustrates the expression of the transcripts of the

Cx40, -43, -45 and Cx37 genes in wild-type (Fig. 1a–d), andhomozygous mutant embryos (Fig. 1e–h). Pattern and inten-sity of hybridization signals were similar in both wild-typeand heterozygous mutant embryos (not shown). The patternof expression of these transcripts in wild-type embryos hasbeen described in detail previously [5,7,8,22]. In wild-typeembryos Cx40 transcript was detected in the primitive atriumand ventricle, the anterior somites, and blood vessels (Fig. 1a).No labeling was observed in the heart of homozygous mutantembryos (Fig. 1e) nor in the caudal vessels. Faint labelingwas detected in the outermost curvature of the ventricle ofwild-type embryos treated with Cx43-specific probe (Fig. 1b).This labeling was absent in homozygous mutants whereasintensity of signals in the other sites of expression wasunchanged (Fig. 1f). Cx45 transcript was widely distributedin the tissues and organs of wild-type embryos, including themyocardium (Fig. 1c). It was also present in the mesenchy-mal and endocardial cells of the AV canal [5,8]. Consistently,no signal was detected in the heart of homozygous mutants(Fig. 1g). Cx37 gene transcript, which is synthesized in thevascular and endocardial endothelial cells, but not in cardi-omyocytes [7], was detected in the heart of both wild-typeand homozygous mutants (Fig. 1d, h).

3.1.2. RT-PCR

RT-PCR experiments were carried out on RNA extractsfrom isolated heart (Fig. 2). RNA extracts from wild-type andheterozygous mutant embryos were both shown to expressthe Cx45, -43 and -40 genes (Fig. 2, lanes 2 and 4). In con-trast, no signal corresponding to these transcripts was detectedfrom RNA extracted from hearts of Nkx2-5–/– embryos(Fig. 2, lane 6).

3.1.3. Immunofluorescence

In the heart, Cx40 was detected in the roof of the atriumand the outermost curvature of the ventricular wall, includ-ing the trabeculae (Fig. 3a–b); Cx43 was seen in the outer-most myocytes of the ventricular wall and the developing tra-beculae (Fig. 4a–c), expression in the latter structures beingthe most prevalent, whereas the outermost myocytes of allthe cardiac compartments, including theAV canal, were Cx45-positive (Fig. 5a–c). These results were similar to thosedescribed previously [5,7,8]. In contrast, in the myocardiumof Nkx2-5 homozygous null mutants Cx40, -43 and -45 werenot detected (Cx40, Fig. 3c, d; Cx43, Fig. 4d–f; Cx45,Fig. 5d–f) though their expression was unchanged in variousother tissues and organs (Cx40, endothelial cells of the ante-rior part of the aorta, for example, not shown; Cx43, Fig. 4b,c, e, f; Cx45, Fig. 5g–i). These data indicate that the Cx geneproducts known to be expressed in the cardiomyocytes ofwild-type embryos were no longer detectable in these cellsafter disruption of the Nkx2-5 gene.

3.2. Cx37 and -40 gene products were expressedaccording a rostro-caudal gradient in the aortaof Nkx2-5–/– embryos

Examination of homozygous mutant embryos indicatedthat inactivation of the Nkx2-5 gene resulted in a rostro-caudal gradient of expression of Cx40 and Cx37 transcriptsin the vessels (Fig. 1e, h). These transcripts were detected inthe branchial arteries and the anterior parts of the aorta(paired), and its derivatives, but were undetectable in the pos-terior part of the aorta and the umbilical vessels. Observationof sections of embryos treated with anti-PECAM antibodyrevealed that the aorta was collapsed along its whole lengthin the null mutants as compared with wild-type embryos(Fig. 3e, g; Fig. 6a, c, e, g). The narrowing of this vesselresulted in an abnormally small lumen, and was associatedwith an irregular morphology of the endothelial cell layer. Inaddition double immunofluorescence experiments have dem-onstrated that both Cx40 (Fig. 3g, h) and Cx37 (Fig. 6g, h)proteins were undetectable in the endothelial cells of the bloodvessels in the posterior region of the Nkx2-5–/– embryos.These results were in agreement with the patterns of hybrid-ization signals described above.

Fig. 2. Representative data of expression of the transcripts of the Cx40, -43,and -45 genes in the heart of wild-type mouse embryos (+/+), and hetero-zygous (+/–) and homozygous mutant (–/–) embryos of 9.2 dpc. PCR expe-riments were carried out after (lanes 2, 4 and 6) and without (lanes 3, 5 and7) reverse transcription. Lane 1: 100 bp ladder. The transcripts of the threecardiac Cxs are detected in RNA extracted from the heart of wild-type andheterozygous mutant embryos (lanes 2 and 4), but were undetectable in RNAfrom homozygous mutants (lane 6).

790 L. Dupays et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 787–798

Page 5: Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants

3.3. Abnormal cardiac contractions in Nkx2-5–/– embryos

Videomicroscopy was used to capture and analyze cardiaccontractions in mutant embryos (n = 17) and compare themwith those of wild-type embryos (n = 10) (Fig. 7). In 75% ofthe Nkx2-5–/– mutant embryos (12/17) the sequential activa-tion of the cardiac compartments was normal (i.e. atrium, ven-tricle, then outflow tract), without AV block, but the contrac-tions were slow and peristaltic (Fig. 7b) resembling thecontractions which occur in earlier wild-type embryos at8.5 dpc. The frequency of cardiac beats was 32 ± 2 per min

in these embryos, compared with 60 ± 3 for wild-typeembryos. In the other mutant embryos (5/17) the ventriclewas first activated, then the contractions slowly propagatedbi-directionally to both the atrium and the outflow tract(Fig. 7c) indicating a shift of the pacemaker area. The fre-quency of beats in these embryos was 20 ± 2 per min. Bothmutant phenotypes were observed in embryos from the samelitter. In addition, observation of the blood mass in the car-diac chambers of the mutant embryos indicated that it wassubjected to back and forth movements and that it was inef-ficiently, or not at all, ejected from the heart. Video-images ofthe cardiac contractions of wild-type and mutant embryos areavailable in the supplementary material.

3.4. Cytoplasmic localization of NFATc1 inthe endocardium of Nkx2-5–/– embryos

Examination of sections of wild-type embryos treated withanti-NFATc1 antibodies has revealed that this transcriptionfactor was expressed in the endocardium, including its AVregion, and localized in the nuclei of most of the endocardialcells (Fig. 8a–g), as described previously [5,23,24]. In con-trast, in the endocardium of the Nkx2-5 homozygous nullembryos NFATc1 was localized in the cytoplasm of most ofthe cells (Fig. 8 h–n) and therefore inactivated. A statisticalanalysis has indicated that NFATc1 was nuclear in91.5 ± 2.9% and 9.2 ± 2.1% of the endocardial cells of wild-type embryos and homozygous null mutants, respectively(Fig. 9).

VEGF has been shown to be involved in the regulation ofNFATc1 (Johnson et al., 2003). The level of expression ofVEGF transcript was assessed by RT-PCR experiments inRNA extracted from hearts of both wild-type and homozy-gous mutants embryos. No significant difference was detectedbetween the two genotypes (not shown).

4. Discussion

Nkx2-5 is initially expressed in the precardiac mesoderm,and the adjacent pharyngeal mesoderm in 7.5 dpc mouseembryo. By 8.5 dpc it is homogenously expressed in the atrialand ventricular myocardium, and continues to have robustexpression throughout the embryonic and neonatal stages, andinto adulthood [10,25]. In addition, experiments by Stanleyet al. [26] have suggested that Nkx2-5 is expressed in earlyendocardial cells, or in their precursors.

4.1. Loss of Cx43 expression in the myocardium of mutantembryos

The Cx40, -43 and -45 genes are expressed in the heart of9.2 dpc wild-type embryos. Our data indicate that at the samestage the products of these genes are undetectable by in situhybridization, RT-PCR and immunofluorescence in the heartof Nkx2-5–/– embryos. Downregulation of Cx40 in atrial and

Fig. 3. Double-labeling immunofluorescence experiments on frozen sec-tions of wild-type (+/+) (panels a, b, e and f) and homozygous mutant (–/–)(panels c, d, g and h) mouse embryos of 9.2 dpc. Immunoreactivity to anti-desmin (des), anti-PECAM (PECAM), and anti-Cx40 (Cx40) antibodies isshown in panels a and c, e and g, and b, d, f, h, respectively. Panels a–d:cross-sections at the level of the heart (at: atrium, v: ventricle; oft: outflowtract); panels e–h: cross-sections in the posterior part of the dorsal aorta.Note that the aorta has collapsed in the homozygous mutant embryos (panelg). In sections of wild-type embryos Cx40 was detected in the roof of theatrium and the outermost curvature of the ventricular wall, including thetrabeculae (arrows in b), and between the vascular endothelial cells (panelf). In sections of homozygous mutant embryos Cx40 was detected neither inthe myocardium (panel d), nor in the posterior region of the aorta (panel h).Note the absence of trabeculae in the ventricle of mutant embryos (panel c).Bar in a: 150 µm for panels a–d; bar in e: 50 µm for panels e–h.

791L. Dupays et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 787–798

Page 6: Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants

ventricular myocardium is in agreement with the resultsreported by Tanaka et al. [16] and Jay et al. [17] in Nkx2-5 +/-adult mice, and more recently by Linhares et al. [27] in Nkx2-5–/– mouse embryos. In addition, in in vitro experiments, thelatter authors have identified a functional Nkx2-5 binding sitein the proximal promoter region of the mouse Cx40 gene.

Expression in transgenic mice of a Nkx2-5 protein whichdoes not bind DNA [14], or overexpression of Nkx2-5 in ratneonatal ventricular myocytes [28] have also suggested thatNkx2-5 may regulate the Cx43 gene. The transcriptional regu-lation of this gene, which has been shown to consist of sixexons with three promoters, generating up to nine tran-scripts, all being expressed in the mouse heart [29], is farfrom being elucidated, and no Nkx2-5 binding sites have yetbeen identified in the promoters of this gene. In addition, theexpression levels of Cx43 in the heart of Nkx2-5 +/- miceand wild-type mice are similar [16,17]. In 9.2 dpc wild-type

embryo heart, Cx43 which is regarded as a molecular markerof the developing cardiac chambers [30] is expressed in out-ermost myocytes of the ventricular wall and in the trabecularnetwork, expression in the latter structure being predomi-nant. Deletion of the Nkx2-5 gene results in poor develop-ment or even the absence of trabeculae ([10,11] and Figs. 2and 3). Consequently, the loss of Cx43 expression in theNkx2-5 mutant embryo heart is in large part due to the poordevelopment of the ventricular trabeculae. This abnormalityalso contributes to the loss of Cx40 expression.

4.2. The Cx45 gene is an in vivo target of Nkx2-5

In the heart of the 9.2 dpc wild-type embryos Cx45 is onlyexpressed in the outermost cardiomyocytes of the walls(Fig. 5, and see [5,8]). Consequently, the Cx45 deficiency inthe heart of Nkx2-5–/– embryos is not due to the poor devel-

Fig. 4. Double-labeling immunofluorescence experiments on frozen sections of wild-type (+/+) (panels a–c) and homozygous mutant (–/–) (panels d–f) mouseembryos of 9.2 dpc. Immunoreactivity to anti-desmin (des) and anti-Cx43 antibodies (Cx43) is shown in panels a, d, and b, e, respectively. Panels c and f aremerged pictures of a and b, and d and e, respectively. In sections of wild-type embryos Cx43 was seen as small dots distributed both in the outermost myocar-dium layer, and in the trabeculae (arrows in panels b and c, and insert corresponding to the white rectangle). In addition, this Cx was strongly expressed in thebody wall (arrowheads in panels b and c). In sections of homozygous mutant embryos Cx43 was not detected in the myocardium but its expression wasunchanged in the body wall (arrowheads in panels e and f). Note the absence of trabeculae in the ventricle of mutant embryos (panel f). at: atrium; v: ventricle.Bar in a: 100 µm for panels a–f.

792 L. Dupays et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 787–798

Page 7: Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants

opment or the absence of trabeculae, and regulation of theCx45 gene by Nkx2-5 could be therefore considered. Therough structure of the Cx45 gene is known: it consists of threeexons with the transcription start site located upstream of thefirst exon [31], but to our knowledge the basal promoter ofthis gene has not yet been identified, and the mechanismsinvolved in the regulation of its transcriptional activity arenot known. Thus, though our results indicate, for the first time,that the Cx45 gene is an in vivo downstream target of Nkx2-5,the absence of more precise data on the potential interactionsof Nkx2-5 with the Cx45 gene makes it impossible to deter-mine how Nkx2-5 can modulate, directly or indirectly, theCx45 gene expression.

The cardiovascular phenotype of the Nkx2-5 mutantembryos is certainly due in part to the downregulation ofCx45. The conditional Cx45 mutant embryos (resulting fromthe crossing of a-actin-Cre mice with Cx45flox/flox mice) inwhich the Cx45 gene was specifically inactivated in the car-diomyocytes die around 10 dpc [6], indicating the require-ment of high Cx45 expression level for normal heart devel-

opment. Thus, Cx45 deficiency in the myocardium of theNkx2-5 mutant embryos could be enough to result in the deathof the embryos. However, other cardiac genes including forexample Anf(Nppa), Carp, Chisel, eHand/Hand1, SM-22,Cited1, Mef2c, Irx4, Nmyc, Msx2, Csm, Hop [11,32–35] arealso downregulated in the Nkx2-5–/– embryos. Their low lev-els of expression or the absence of expression may also con-tribute to the cardiac phenotype observed.

4.3. Ineffıciency of cardiac contractions may contribute tothe vascular phenotype of the Nkx2-5–/– embryos

The cardiac impulse propagation and contractions areabnormal in the Nkx2-5–/– embryos. In 75% of the embryosthe heart rate was low (32 beats per min versus 60 in wild-type embryos), suggesting an immaturity or a dysfunction ofthe sinoatrial (SA) node (type 1 embryos). In the wild-typemice electrical coupling between the myocytes of the SA nodeis mediated by Cx45 gap junction channels [36]. Conse-quently, downregulation of Cx45 in the Nkx2-5–/– embryos

Fig. 5. Double-labeling immunofluorescence experiments on frozen sections of wild-type (+/+) (panels a–c) and homozygous mutant (–/–) (panels d–i) mouseembryos of 9.2 dpc. Panels a, d, and g: phase contrast micrographs. Immunoreactivity to anti-desmin (des) and anti-Cx45 (Cx45) antibodies is shown in panelsb, e, h, and c, f, i, respectively. Panels a–f illustrate sections in the ventricular wall of the heart; panels g–i, in the body wall. In sections of wild-type embryosCx45 was detected in various tissues and organs including the myocardium (panels a–c). However, note the very low expression of Cx45 in the myocytes of thetrabeculae (tr) compared to the robust expression of Cx45 in the outermost myocytes of the ventricular wall (panels b and c). In sections of homozygous mutantembryos no immunoreactivity to anti-Cx45 antibodies was seen in the myocardium (panels d–f) whereas the expression of Cx45 was unchanged in other tissuesand organs, as for example in the first branchial arch (panels g–i).Bar in a: 20 µm for panels a–i.

793L. Dupays et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 787–798

Page 8: Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants

certainly contributes to the alterations of the SA node prop-erties. Interestingly, it has been shown that cardiomyocytesdifferentiated in vitro from Cx45–/– ES cells present arrhyth-mias associated with high and irregular contraction rates com-pared with cardiomyocytes derived from wild-type ES cells[37]. In a significant number of cases (25%) the contractionsof the heart of Nkx2-5–/– mutant embryos occurred first inthe ventricle (type 2 embryos), indicating that the SA nodewas not functional and was replaced by another pacemaker,which was probably the AV node. It has been experimentallydemonstrated that in the absence of a functional SA node, theAV node imposes a rhythm, which is slower than the sinusrhythm [38]. The comparison of beat frequencies betweentype 1 (32 beats per min) and type 2 (20 beats per min) Nkx2-5–/– embryos supports this hypothesis. However, recentresults indicate that the AV node may be absent in the Nkx2-5neo/neo embryos of 9.5 dpc, which do not express Nkx2-5

[39]. The discrepancy between these data and our observa-tions could result from the difference of modifications of theNkx2-5 gene alleles present in the two types of embryos inves-tigated (Nkx2-5–/–, [11]; Nkx2-5neo/neo, [39]). Alternatively,the reported absence of Nkx2-5 may result in the cardiacdownregulation of the molecular marker used to visualize theAV node (see [40]).

Besides abnormal contractions, and probably as a conse-quence of these defects, the heart of the Nx2-5–/– embryosfails to eject blood efficiently. It is known that blood pres-sure, blood flow and shear stress modulate the diameter ofvessels [41]. In agreement with these observations the hemo-dynamic insufficiency of the Nkx2-5–/– embryos is reflectedin the vascular phenotype which is characterized by the nar-rowness of the aorta. Other mouse lines with mutations affect-ing early cardiogenesis also show vascular defects. This isthe case for example for mice knock-out for N-cadherin,Mefc2, and Cx45 [22,42–44]. In addition, the vascular phe-notype of N-cadherin null embryos was shown to be second-ary to the cardiac defects [45].

An intriguing finding is the expression gradient of Cx37and -40 gene products in the aorta of the Nkx2-5–/– embryos.The heterogeneous expression of these Cxs in this vessel can-not be simply explained by the absence of Nkx2-5 since thistranscription factor has never been detected in the endothe-lial cells which line it. In the vascular wall Cx45 and -43 areexpressed predominantly in the smooth muscle cells of maturevessels [22,46] whereas Cx37 and -40 are synthesized in theendothelial cells as early as 8–8.5 dpc. Mice knock-out forboth Cx37 and -40 die perinatally with a highly penetrantphenotype of vascular dysmorphogenesis, congestion, andhaemorrhage in different tissues but the major systemic ves-sels, including the aorta, appear normal [47]. Thus, the gra-dient of Cx gene products in the aorta of the Nkx2-5 nullmutant embryos may not account for the dysmorphology ofthis vessel. These two Cx genes have been shown to beinvolved in the conduction of vasomotor signals in the vesselwall (reviewed by [4]), and the expression of vascular Cxsappears to be affected by shear stress, blood pressure and envi-ronmental conditions [4]. Thus, an epigenetic factor such asthe hemodynamic insufficiency may contribute to creatingthe expression gradient of Cx37 and -40 in the aorta, and con-sequently generate an antero-posterior loss of the functionsof the endothelial cells in this vessel.

4.4. Inactivation of the Nkx2-5 gene prevents nucleartranslocation of NFATc1 in the endocardium

The endocardial cushions are embryonic structures, derivedfrom endocardium, which contribute to the formation of thecardiac valves [48]. The absence of endocardial cushions is acharacteristic feature of Nkx2-5–/– embryos [5,10,11].NAFTc1, a transcription factor which is expressed in theendocardium [26,49], can switch from an inactivated state inthe cytoplasm to an activated state in the nucleus. Our resultsindicate that inactivation of the Nkx2-5 gene prevents the

Fig. 6. Double-labeling immunofluorescence experiments on frozen sec-tions of wild-type (+/+) (panels a, b, e and f) and homozygous mutant (–/–)(panels c, d, g and h) mouse embryos of 9.2 dpc. Cross-sections were treatedwith both anti-PECAM (panels a, c, e and g) and anti-Cx37 (panels b, d, fand h) antibodies. Panels a–d illustrate sections in the anterior regions of theaorta; panels e–h, in the posterior regions. Note that the aorta is collapsed inthe homozygous mutant embryos. In the wild-type embryos Cx37 proteinwas expressed in the endothelial cells along the whole length of the aorta (band f). In the homozygous mutants this Cx was detected in the anterior regionsof the aorta (panel d) but was undetectable in the posterior regions (h).Bar in a: 50 µm for panels a–h.

794 L. Dupays et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 787–798

Page 9: Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants

nuclear localization of NFATc1 in the endothelial endocar-dial cells at 9.2 dpc and consequently inactivates its transcrip-tional functions.

Mistranslocation of NFATc1 in the endocardial cells hasbeen previously described in the Cx45 knock-out mouseembryos, which also display cardiac cushion defects [5]. Thisphenomenon has been correlated with inactivation of theCa2+/calcineurin/NFATc1 pathway in the endocardial cells,which are normally connected through Cx45 gap junctionchannels in wild-type embryos [5,8]. This hypothesis has beenstrengthened by the results of Nishii et al. [6] who have dem-onstrated that mouse embryos, in which the Cx45 gene hasbeen inactivated only in the cardiomyocytes, have no cardiaccushion defects. These results have suggested the existenceof a network of events in which Cx45 deficiency results ininactivation of NFATc1, and consequently that of theCa2+/calcineurin/NFATc1 pathway, leading to the absence ofendocardial cushions. Such a network could also be opera-tive in the endocardium of the Nkx2-5–/– embryos. How-ever, the mechanism by which Nkx2-5 may regulate theCa2+/calcineurin/NFATc1 pathway (through the downregula-

tion of Cx45 or modulation of another factor) in the endocar-dium is unclear. Nkx2-5 is not expressed in the endocardiumat E8.5, E9.5 or later [10,11] but the observations of Stanleyet al. [26] have suggested that it is expressed very early in theprogenitor cells of the endocardium. It may be therefore sug-gested that inactivation of Nkx2-5 in the precursors of theendocardial cells may affect, at 9.2 dpc, the cells of theendocardial line, which then may interpret this situation bypreventing NFATc1 from translocating in the nucleus. Alter-natively, Nkx2-5 deficiency in the myocardium may inacti-vate, through an unknown cross-talk pathway, NFATc1 in theendocardium. In conclusion, although a direct effect of theNkx2-5 inactivation on the mistranslocation of NFATc1 inthe endocardium cannot be excluded, the above hypothesessuggest that indirect effects are prevalent.

In addition, epigenesis could also regulate the formationof endocardial cushions. Recent data do indeed indicate thatlack of endocardial cushion formation in the zebrafish heartoccurs secondarily to poor myocardial function or lack ofblood flow [50]. Accordingly, the abnormal cardiac contrac-

Fig. 7. Representative video-images of spontaneous contraction patterns of wild-type (Nkx2-5+/+) and homozygous mutant (Nkx2-5–/–) mouse embryos of9.2 dpc. The profile of the hearts is drawn under each frame (at: atrium; v: ventricle; oft: outfow tract). Cardiac compartments which contract are in red. Inwild-type embryos (10 hearts) strong atrial contractions preceding the ventricular and outflow tract contractions were observed (panels a). In Nkx2-5 mutantscontractions were initiated from either the atrium (12 hearts out of 17) (panels b), or the ventricle (5 hearts out of 17) (panels c). In the first case, the hearts hada slow peristaltic beat with a normal sequence; in the second case, even slower contractions propagated bidirectionally to both the atrium and the outflow tract.Time is indicated in second in the first and last frames of each recording.

795L. Dupays et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 787–798

Page 10: Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants

Fig. 8. Subcellular localization of NFATc1 in the endocardium of wild-type (+/+) (panels a–g) and homozygous mutant (–/–) (panels h–n) mouse embryos of9.2 dpc. Panels a and h show transverse sections of the embryos at the level of the cardiac region (nt: neural tube, at: atrium, avc, atrio-ventricular canal, lv, leftventricle, oft, outflow tract). Regions 1, 2, 3 and 4 are shown at higher magnifications in panels b–d, e–g, i–k and l–n, respectively. Sections were treated withanti-NFATc1 antibodies (panels a, b, e, h, i, l) and the nuclei were counterstained with Hoescht dye (panels a, c, f, h, j, m). Panels d, g, k and n are mergedpictures of panels b and c, e and f, i and j, and l and m, respectively. Panel a: NFATc1 is localized in the nucleus of the endocardial cells. Arrowheads in panelsb–d and e–g indicate the endocardial cells of the atrium and the avc, respectively. Panel h: NFATc1 is localized in the cytoplasm of the endocardial cells. Seepanels i–k for the endocardial cells of the atrium (upper arrowheads) and the avc (lower arrowheads), and panels l–n for the endocardial cells of the ventricle(arrowheads). The localization of NFATc1 is also illustrated in the inserts of panels d and n.Bars in a and h: 50 µm; bars in b: 10 µm for panels b–g and i–n.

796 L. Dupays et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 787–798

Page 11: Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants

tions of the Nkx2-5 mutant heart may also indirectly contrib-ute to the absence of endocardial cushions in these embryos.

In summary, the disruption of the Nkx2-5 gene results inthe mouse heart in the loss of Cx43 (due in part to the poordevelopment of the ventricular trabecular network), and down-regulation of Cx45 gene expression. Both phenomena, poordevelopment of the trabecular network and downregulationof the Cx45 gene expression, contribute to failure of myocar-dial function and hemodynamic insufficiency. The latter, inturn, may result in dysregulation of the expression of vascu-lar endothelial Cxs, Cx40 and Cx37, along the whole lengthof the aorta. Downregulation of Cx45 (in conjunction—ornot—with hemodynamic insufficiency) may prevent the for-mation of the endocardial cushions through inhibition of thenuclear translocation of NFATc1 in the endocardium.

Acknowledgements

This work was supported by the European Community(contract QLG1-1999-00516), the French Ministry of Re-search (ACI “Biologie du Développement et PhysiologieIntégrative”), the Fondation de France, the AssociationFrançaise contre les Myopathies and the Fondation pour laRecherche Médicale (fellowship to L.D.).

References

[1] Rohr S. Role of gap junctions in the propagation of the cardiac actionpotential. Cardiovasc Res 2004;62:309–22.

[2] Sohl G, Willecke K. Gap junctions and the connexin protein family.Cardiovasc Res 2004;62:228–32.

[3] Gros D, Dupays L, Alcolea S, Meysen S, Miquerol L, Theveniau-Ruissy M. Genetically modified mice: tools to decode the functions ofconnexins in the heart-new models for cardiovascular research. Car-diovasc Res 2004;62:299–308.

[4] Haefliger JA, Nicod P, Meda P. Contribution of connexins to thefunction of the vascular wall. Cardiovasc Res 2004;62:345–56.

[5] Kumai M, Nishii K, Nakamura K, Takeda N, Suzuki M, Shibata Y.Loss of connexin45 causes a cushion defect in early cardiogenesis.Development 2000;127:3501–12.

[6] Nishii K, Kumai M, Egashira K, Miwa T, Hashizume K, Miyano Y,et al. Mice lacking connexin45 conditionally in cardiac myocytesdisplay embryonic lethality similar to that of germline knockout micewithout endocardial cushion defect. Cell Commun Adhes 2003;10:365–9.

[7] Delorme B, Dahl E, Jarry-Guichard T, Briand JP, Willecke K, Gros D,et al. Expression pattern of connexin gene products at the earlydevelopmental stages of the mouse cardiovascular system. Circ Res1997;81:423–37.

[8] Alcolea S, Theveniau-Ruissy M, Jarry-Guichard T, Marics I, Tzoua-nacou E, Chauvin JP, et al. Downregulation of connexin45 geneproducts during mouse heart development. Circ Res 1999;84:1365–79.

[9] Teunissen BE, Bierhuizen MF. Transcriptional control of myocardialconnexins. Cardiovasc Res 2004;62:246–55.

[10] Lyons I, Parsons LM, Hartley L, Li R, Andrews JE, Robb L, et al.Myogenic and morphogenetic defects in the heart tubes of murineembryos lacking the homeo box gene Nkx2-5. Genes Dev 1995;9:1654–66.

[11] Tanaka M, Chen Z, Bartunkova S, Yamasaki N, Izumo S. The cardiachomeobox gene Csx/Nkx2.5 lies genetically upstream of multiplegenes essential for heart development. Development 1999;126:1269–80.

[12] Benson DW, Silberbach GM, Kavanaugh-McHugh A, Cottrill C,Zhang Y, Riggs S, et al. Mutations in the cardiac transcription factorNKX2.5 affect diverse cardiac developmental pathways. J Clin Invest1999;104:1567–73.

[13] Schott JJ, Benson DW, Basson CT, Pease W, Silberbach GM,Moak JP, et al. Congenital heart disease caused by mutations in thetranscription factor NKX2-5. Science 1998;281:108–11.

[14] Kasahara H, Wakimoto H, Liu M, Maguire CT, Converso KL, Shioi T,et al. Progressive atrioventricular conduction defects and heart failurein mice expressing a mutant Csx/Nkx2.5 homeoprotein. J Clin Invest2001;108:189–201.

[15] Biben C, Weber R, Kesteven S, Stanley E, McDonald L, Elliott DA,et al. Cardiac septal and valvular dysmorphogenesis in mice heterozy-gous for mutations in the homeobox gene Nkx2-5. Circ Res 2000;87:888–95.

[16] Tanaka M, Berul CI, Ishii M, Jay PY, Wakimoto H, Douglas P, et al. Amouse model of congenital heart disease: cardiac arrhythmias andatrial septal defect caused by haploinsufficiency of the cardiac tran-scription factor Csx/Nkx2.5. Cold Spring Harb Symp Quant Biol2002;67:317–25.

[17] Jay PY, Berul CI, Tanaka M, Ishii M, Kurachi Y, Izumo S. Cardiacconduction and arrhythmia: insights from Nkx2.5 mutations in mouseand humans. Novartis Found Symp 2003;250:227–38 discussion238–41, 276–9].

[18] Dupays L, Mazurais D, Rucker-Martin C, Calmels T, Bernot D,Cronier L, et al. Genomic organization and alternative transcripts ofthe human connexin40 gene. Gene 2003;305:79–90.

[19] Miquerol L, Langille BL, Nagy A. Embryonic development is dis-rupted by modest increases in vascular endothelial growth factor geneexpression. Development 2000;127:3941–6.

[20] Bruneau BG, Logan M, Davis N, Levi T, Tabin CJ, Seidman JG, et al.Chamber-specific cardiac expression of Tbx5 and heart defects inHolt-Oram syndrome. Dev Biol 1999;211:100–8.

Fig. 9. Percentage of endocardial cells from wild-type (Nkx2-5+/+) andhomozygous mutant (Nx2.5–/–) embryos in which NFATc1 is localized inthe nucleus. N = 4 embryos for each genotype; n = 754 and 626 endocardialcells for Nkx2-5+/+ and Nkx2-5–/– embryos, respectively. Student’s t-testindicates that the difference of distribution of NFATc1 between the two geno-types was highly significant (P < 0.0001). Data are mean ± S.D. [48].

797L. Dupays et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 787–798

Page 12: Dysregulation of connexins and inactivation of NFATc1 in the cardiovascular system of Nkx2–5 null mutants

[21] Yamagishi H, Yamagishi C, Nakagawa O, Harvey RP, Olson EN,Srivastava D. The combinatorial activities of Nkx2.5 and dHAND areessential for cardiac ventricle formation. Dev Biol 2001;239:190–203.

[22] Kruger O, Plum A, Kim JS, Winterhager E, Maxeiner S, Hallas G,et al. Defective vascular development in connexin45-deficient mice.Development 2000;127:4179–93.

[23] de la Pompa JL, Timmerman LA, Takimoto H, Yoshida H, Elia AJ,Samper E, et al. Role of the NF-ATc transcription factor in morpho-genesis of cardiac valves and septum. Nature 1998;392:182–6.

[24] Ranger AM, Grusby MJ, Hodge MR, Gravallese EM, De laBrousse FC, Hoey T, et al. The transcription factor NF-ATc is essen-tial for cardiac valve formation. Nature 1998;392:186–90.

[25] Kasahara H, Bartunkova S, Schinke M, Tanaka M, Izumo S. Cardiacand extracardiac expression of Csx/Nkx2.5 homeodomain protein.Circ Res 1998;82:936–46.

[26] Stanley EG, Biben C, Elefanty A, Barnett L, Koentgen F, Robb L,et al. Efficient Cre-mediated deletion in cardiac progenitor cells con-ferred by a 3’UTR-ires-Cre allele of the homeobox gene Nkx2-5. Int JDev Biol 2002;46:431–9.

[27] Linhares VL, Almeida NA, Menezes DC, Elliott DA, Lai D,Beyer EC, et al. Transcriptional regulation of the murine con-nexin40 promoter by cardiac factors Nkx2-5, GATA4 and Tbx5.Cardiovasc Res 2004;64:402–11.

[28] Teunissen BE, Jansen AT, Van Amersfoorth SC, O’Brien TX,Jongsma HJ, Bierhuizen MF. Analysis of the rat connexin43 proximalpromoter in neonatal cardiomyocytes. Gene 2003;322:123–36.

[29] Pfeifer I, Anderson C, Werner R, Oltra E. Redefining the structure ofthe mouse connexin43 gene: selective promoter usage and alternativesplicing mechanisms yield transcripts with different translational effi-ciencies. Nucleic Acids Res 2004;32:4550–62.

[30] Christoffels VM, Habets PE, Franco D, Campione M, De Jong F,Lamers WH, et al. Chamber formation and morphogenesis in thedeveloping mammalian heart. Dev Biol 2000;223:266–78.

[31] Baldridge D, Lecanda F, Shin CS, Stains J, Civitelli R. Sequence andstructure of the mouse connexin45 gene. Biosci Rep 2001;21:683–9.

[32] Bruneau BG, Bao ZZ, Tanaka M, Schott JJ, Izumo S, Cepko CL, et al.Cardiac expression of the ventricle-specific homeobox gene Irx4 ismodulated by Nkx2-5 and dHand. Dev Biol 2000;217:266–77.

[33] Palmer S, Groves N, Schindeler A, Yeoh T, Biben C, Wang CC, et al.The small muscle-specific protein Csl modifies cell shape and pro-motes myocyte fusion in an insulin-like growth factor 1-dependentmanner. J Cell Biol 2001;153:985–98.

[34] Harvey RP. Patterning the vertebrate heart. Nat Rev Genet 2002;3:544–56.

[35] Ueyama T, Kasahara H, Ishiwata T, Yamasaki N, Izumo S. Csm, acardiac-specific isoform of the RNA helicase Mov10l1, is regulatedby Nkx2.5 in embryonic heart. J Biol Chem 2003;278:28750–7.

[36] Verheijck EE, Van Kempen MJ, Veereschild M, Lurvink J,Jongsma HJ, Bouman LN. Electrophysiological features of the mousesinoatrial node in relation to connexin distribution. Cardiovasc Res2001;52:40–50.

[37] Egashira K, Nishii K, Nakamura K, Kumai M, Morimoto S, ShibataY.Conduction abnormality in gap junction protein connexin45-deficientembryonic stem cell-derived cardiac myocytes. Anat Rec 2004;280A:973–9.

[38] Eyster JAEMA. The origin and conduction of the heart beat. PhysiolRev 1921;1:1–43.

[39] Jay PY, Harris BS, Maguire CT, Buerger A, Wakimoto H, Tanaka M,et al. Nkx2-5 mutation causes anatomic hypoplasia of the cardiacconduction system. J Clin Invest 2004;113:1130–7.

[40] Pashmforoush M, Lu JT, Chen H, Amand TS, Kondo R, Pradervand S,et al. Nkx2-5 pathways and congenital heart disease; loss of ventricu-lar myocyte lineage specification leads to progressive cardiomyopa-thy and complete heart block. Cell 2004;117:373–86.

[41] Beny JL. Information Networks in the Arterial Wall. News Physiol Sci1999;14:68–73.

[42] Radice GL, Rayburn H, Matsunami H, Knudsen KA, Takeichi M,Hynes RO. Developmental defects in mouse embryos lackingN-cadherin. Dev Biol 1997;181:64–78.

[43] Lin Q, Lu J, Yanagisawa H, Webb R, Lyons GE, Richardson JA, et al.Requirement of the MADS-box transcription factor MEF2C for vas-cular development. Development 1998;125:4565–74.

[44] Bi W, Drake CJ, Schwarz JJ. The transcription factor MEF2C-nullmouse exhibits complex vascular malformations and reduced cardiacexpression of angiopoietin 1 and VEGF. Dev Biol 1999;211:255–67.

[45] Luo Y, Ferreira-Cornwell M, Baldwin H, Kostetskii I, Lenox J, Lie-berman M, et al. Rescuing the N-cadherin knockout by cardiac-specific expression of N- or E-cadherin. Development 2001;128:459–69.

[46] van Kempen MJ, Jongsma HJ. Distribution of connexin37, con-nexin40 and connexin43 in the aorta and coronary artery of severalmammals. Histochem Cell Biol 1999;112:479–86.

[47] Simon AM, McWhorter AR. Vascular abnormalities in mice lackingthe endothelial gap junction proteins connexin37 and connexin40.Dev Biol 2002;251:206–20.

[48] Armstrong EJ, Bischoff J. Heart valve development: endothelial cellsignaling and differentiation. Circ Res 2004;95:459–70.

[49] Schulz RA, Yutzey KE. Calcineurin signaling and NFAT activation incardiovascular and skeletal muscle development. Dev Biol 2004;266:1–16.

[50] Bartman T, Walsh EC, Wen KK, McKane M, Ren J, Alexander J, et al.Early myocardial function affects endocardial cushion developmentin zebrafish. PLoS Biol 2004;2:E129.

798 L. Dupays et al. / Journal of Molecular and Cellular Cardiology 38 (2005) 787–798