RESUMO
With the advance in chromatin immunoprecipitation followed by high-throughput sequencing, there has been a dramatic increase in our understanding of distal enhancer function. In the developing heart, the identification and characterisation of such enhancers have deepened our knowledge of the mechanisms of transcriptional regulation that drives cardiac differentiation. With next-generation sequencing techniques becoming widely accessible, the quantity of data describing the genome-wide distribution of cardiac-specific transcription factor and chromatin modifiers has rapidly increased and it is now becoming clear that the usage of enhancers is highly dynamic and complex, both during the development and in the adult. The identification of those enhancers has revealed new insights into the transcriptional mechanisms of how tissue-specific gene expression patterns are established, maintained, and change dynamically during development and upon physiological stress.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Organogênese/genética , Animais , Humanos , Modelos Biológicos , Estatística como AssuntoRESUMO
Desmosomes are anchoring junctions that exist in cells that endure physical stress such as cardiac myocytes. The importance of desmosomes in maintaining the homeostasis of the myocardium is underscored by frequent mutations of desmosome components found in human patients and animal models. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a phenotype caused by mutations in desmosomal components in â¼ 50% of patients, however, the causes in the remaining 50% of patients still remain unknown. A deficiency of inhibitor of apoptosis-stimulating protein of p53 (iASPP), an evolutionarily conserved inhibitor of p53, caused by spontaneous mutation recently has been associated with a lethal autosomal recessive cardiomyopathy in Poll Hereford calves and Wa3 mice. However, the molecular mechanisms that mediate this putative function of iASPP are completely unknown. Here, we show that iASPP is expressed at intercalated discs in human and mouse postmitotic cardiomyocytes. iASPP interacts with desmoplakin and desmin in cardiomyocytes to maintain the integrity of desmosomes and intermediate filament networks in vitro and in vivo. iASPP deficiency specifically induces right ventricular dilatation in mouse embryos at embryonic day 16.5. iASPP-deficient mice with exon 8 deletion (Ppp1r13l(Δ8/Δ8)) die of sudden cardiac death, displaying features of ARVC. Intercalated discs in cardiomyocytes from four of six human ARVC cases show reduced or loss of iASPP. ARVC-derived desmoplakin mutants DSP-1-V30M and DSP-1-S299R exhibit weaker binding to iASPP. These data demonstrate that by interacting with desmoplakin and desmin, iASPP is an important regulator of desmosomal function both in vitro and in vivo. This newly identified property of iASPP may provide new molecular insight into the pathogenesis of ARVC.
Assuntos
Arritmias Cardíacas , Cardiomiopatia Hipertrófica Familiar , Morte Súbita , Desmossomos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Repressoras , Substituição de Aminoácidos , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Sequência de Bases , Cardiomiopatia Hipertrófica Familiar/genética , Cardiomiopatia Hipertrófica Familiar/metabolismo , Cardiomiopatia Hipertrófica Familiar/patologia , Bovinos , Linhagem Celular Transformada , Desmina/genética , Desmina/metabolismo , Desmoplaquinas/genética , Desmoplaquinas/metabolismo , Desmossomos/genética , Desmossomos/metabolismo , Desmossomos/patologia , Modelos Animais de Doenças , Feminino , Humanos , Filamentos Intermediários , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Mutação de Sentido Incorreto , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Deleção de SequênciaRESUMO
RATIONALE: The ventricular conduction system controls the propagation of electric activity through the heart to coordinate cardiac contraction. This system is composed of specialized cardiomyocytes organized in defined structures including central components and a peripheral Purkinje fiber network. How the mammalian ventricular conduction system is established during development remains controversial. OBJECTIVE: To define the lineage relationship between cells of the murine ventricular conduction system and surrounding working myocytes. METHODS AND RESULTS: A retrospective clonal analysis using the alpha-cardiac actin(nlaacZ/+) mouse line was carried out in three week old hearts. Clusters of clonally related myocytes were screened for conductive cells using connexin40-driven enhanced green fluorescent protein expression. Two classes of clusters containing conductive cells were obtained. Mixed clusters, composed of conductive and working myocytes, reveal that both cell types develop from common progenitor cells, whereas smaller unmixed clusters, composed exclusively of conductive cells, show that proliferation continues after lineage restriction to the conduction system lineage. Differences in the working component of mixed clusters between the right and left ventricles reveal distinct progenitor cell histories in these cardiac compartments. These results are supported by genetic fate mapping using Cre recombinase revealing progressive restriction of connexin40-positive myocytes to a conductive fate. CONCLUSIONS: A biphasic mode of development, lineage restriction followed by limited outgrowth, underlies establishment of the mammalian ventricular conduction system.
Assuntos
Sistema de Condução Cardíaco/crescimento & desenvolvimento , Ventrículos do Coração/crescimento & desenvolvimento , Fatores Etários , Animais , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Transgênicos , Gravidez , Estudos RetrospectivosRESUMO
Development of the mammalian heart is mediated by complex interactions between myocardial, endocardial, and neural crest-derived cells. Studies in Drosophila have shown that the Slit-Robo signaling pathway controls cardiac cell shape changes and lumen formation of the heart tube. Here, we demonstrate by in situ hybridization that multiple Slit ligands and Robo receptors are expressed in the developing mouse heart. Slit3 is the predominant ligand transcribed in the early mouse heart and is expressed in the ventral wall of the linear heart tube and subsequently in chamber but not in atrioventricular canal myocardium. Furthermore, we identify that the homeobox gene Nkx2-5 is required for early ventral restriction of Slit3 and that the T-box transcription factor Tbx2 mediates repression of Slit3 in nonchamber myocardium. Our results suggest that patterned Slit-Robo signaling may contribute to the control of oriented cell growth during chamber morphogenesis of the mammalian heart.
Assuntos
Coração/embriologia , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Gravidez , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas RoundaboutRESUMO
Tbx2 is a member of the T-box family of transcription factors that play important roles during heart development. In the embryonic heart tube, Tbx2 is expressed in non-chamber myocardium (outflow tract and interventricular canal) and has been shown to block chamber formation. We have developed a genetic system to conditionally misexpress Tbx2 in the embryonic mouse heart at early stages of development. We show that Tbx2 expression throughout the myocardium of the heart tube both represses proliferation and impairs secondary heart field (SHF) progenitor cell deployment into the outflow tract (OFT). Repression of proliferation is accompanied by the upregulation of Ndrg2 and downregulation of Ndrg4 expression, both genes believed to be involved in cell growth and proliferation. Impaired deployment of SHF cells from the pharyngeal mesoderm is accompanied by downregulation of the cell adhesion molecules Alcam and N-cadherin in the anterior part of the embryonic heart. Tbx2 misexpression also results in downregulation of Tbx20 within the OFT, indicating complex and region-specific transcriptional cross-regulation between the two T-box genes.
Assuntos
Diferenciação Celular/fisiologia , Coração/embriologia , Miocárdio/citologia , Células-Tronco/citologia , Proteínas com Domínio T/biossíntese , Proteínas Adaptadoras de Transdução de Sinal , Animais , Padronização Corporal , Caderinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Transgênicos , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas/metabolismo , Células-Tronco/fisiologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
Elevated levels of the cardiac transcription factor Hand1 have been reported in several adult cardiac diseases but it is unclear whether this change is itself maladaptive with respect to heart function. To test this possibility, we have developed a novel, inducible transgenic system, and used it to overexpress Hand1 in adult mouse hearts. Overexpression of Hand1 in the adult mouse heart leads to mild cardiac hypertrophy and a reduction in life expectancy. Treated mice show no significant fibrosis, myocyte disarray or congestive heart failure, but have a greatly reduced threshold for induced ventricular tachycardia, indicating a predisposition to cardiac arrhythmia. Within 48 h, they show a significant loss of connexin43 protein from cardiac intercalated discs, with increased intercalated disc beta-catenin expression at protein and RNA levels. These changes are sustained during prolonged Hand1 overexpression. We propose that cardiac overexpression of Hand1 offers a useful mouse model of arrhythmogenesis and elevated HAND1 may provide one of the molecular links between the failing heart and arrhythmia.
Assuntos
Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cardiomegalia/genética , Cardiomegalia/metabolismo , Eletrofisiologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The homeodomain transcription factor NKX2-5 is known to be essential for both normal heart development and for heart function. But little is yet known about the identities of its downstream effectors or their function during differentiation of cardiac progenitor cells (CPCs). We have used transgenic analysis and CRISPR-mediated ablation to identify a cardiac enhancer of the Furin gene. The Furin gene, encoding a proprotein convertase, is directly repressed by NKX2-5. Deletion of Furin in CPCs is embryonic lethal, with mutant hearts showing a range of abnormalities in the outflow tract. Those defects are associated with a reduction in proliferation and premature differentiation of the CPCs. Deletion of Furin in differentiated cardiomyocytes results in viable adult mutant mice showing an elongation of the PR interval, a phenotype that is consistent with the phenotype of mice and human mutant for Nkx2-5. Our results show that Furin mediate some aspects of Nkx2-5 function in the heart.
Assuntos
Furina/genética , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Proteína Homeobox Nkx-2.5/metabolismo , Organogênese/genética , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Sistemas CRISPR-Cas , Diferenciação Celular/genética , Proliferação de Células/genética , Embrião de Mamíferos , Furina/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Animais , Mutagênese , Mutação , Miocárdio/metabolismo , Miócitos Cardíacos/fisiologia , Células-Tronco/fisiologiaRESUMO
Chromatin remodelling is essential for cardiac development. Interestingly, the role of histone chaperones has not been investigated in this regard. HIRA is a member of the HUCA (HIRA/UBN1/CABIN1/ASF1a) complex that deposits the variant histone H3.3 on chromatin independently of replication. Lack of HIRA has general effects on chromatin and gene expression dynamics in embryonic stem cells and mouse oocytes. Here we describe the conditional ablation of Hira in the cardiogenic mesoderm of mice. We observed surface oedema, ventricular and atrial septal defects and embryonic lethality. We identified dysregulation of a subset of cardiac genes, notably upregulation of troponins Tnni2 and Tnnt3, involved in cardiac contractility and decreased expression of Epha3, a gene necessary for the fusion of the muscular ventricular septum and the atrioventricular cushions. We found that HIRA binds GAGA rich DNA loci in the embryonic heart, and in particular a previously described enhancer of Tnni2/Tnnt3 (TTe) bound by the transcription factor NKX2.5. HIRA-dependent H3.3 enrichment was observed at the TTe in embryonic stem cells (ESC) differentiated toward cardiomyocytes in vitro. Thus, we show here that HIRA has locus-specific effects on gene expression and that histone chaperone activity is vital for normal heart development, impinging on pathways regulated by an established cardiac transcription factor.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Regulação da Expressão Gênica , Coração/embriologia , Chaperonas de Histonas/fisiologia , Miócitos Cardíacos/citologia , Fatores de Transcrição/fisiologia , Troponina I/metabolismo , Troponina/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteína Homeobox Nkx-2.5/genética , Proteína Homeobox Nkx-2.5/metabolismo , Camundongos , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Troponina/genética , Troponina I/genéticaRESUMO
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.
Assuntos
Conexinas/fisiologia , Bloqueio Cardíaco/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Arritmias Cardíacas/metabolismo , Conexinas/genética , Morte Súbita Cardíaca/etiologia , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Camundongos , Camundongos Transgênicos , Modelos AnimaisRESUMO
The homeobox transcription factors NKX2-5 and MEIS1 are essential for vertebrate heart development and normal physiology of the adult heart. We show that, during cardiac differentiation, the two transcription factors have partially overlapping expression patterns, with the result that as cardiac progenitors from the anterior heart field differentiate and migrate into the cardiac outflow tract, they sequentially experience high levels of MEIS1 and then increasing levels of NKX2-5. Using the Popdc2 gene as an example, we also show that a significant proportion of target genes for NKX2-5 contain a binding motif recognized by NKX2-5, which overlaps with a binding site for MEIS1. Binding of the two factors to such overlapping sites is mutually exclusive, and this provides a simple regulatory mechanism for spatial and temporal synchronization of a common pool of targets between NKX2-5 and MEIS1.
Assuntos
Moléculas de Adesão Celular/metabolismo , Elementos Facilitadores Genéticos , Proteínas de Homeodomínio/metabolismo , Proteínas Musculares/metabolismo , Miocárdio/metabolismo , Proteínas de Neoplasias/metabolismo , Organogênese/genética , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Moléculas de Adesão Celular/genética , Embrião de Mamíferos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteína Homeobox Nkx-2.5 , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Musculares/genética , Proteína Meis1 , Proteínas de Neoplasias/genética , Motivos de Nucleotídeos , Ligação Proteica , Transdução de Sinais , Fatores de Transcrição/genética , Troponina/genética , Troponina/metabolismo , Troponina I/genética , Troponina I/metabolismoRESUMO
The human Cx40 gene (NT_004434.5) was sorted out from the GenBank database and as a result of a BLAST homology search, two ESTs (BE784549 from a human lung database, and BE732411 from a human placenta database) overlapping with the coding exon 2 sequence and upstream regions of the gene were identified. These ESTs correspond to two transcripts 1A and 1B, which diverge from each other in their 5' regions. The transcript 1A corresponds to the only transcript previously identified for the mouse and rat Cx40 genes; whereas the transcript 1B is a new transcript. The human Cx40 gene therefore comprises three exons: exon 1A (100 bp), exon 1B (132 bp) and coding exon 2, with the exons 1A and 1B at 14 and 1.3 kb of the exon 2, respectively. The expression of these transcripts is cell-type specific. Transcript 1A is expressed in endothelial cells. Its expression was demonstrated in human umbilical vein endothelial cells (HUVEC). Transcript 1B is expressed in placental cytotrophoblasts. Its expression was demonstrated in malignant trophoblastic cells, BeWo, JAR and JEG-3, and purified cytotrophoblasts from human first trimester placental tissues. Interestingly, both transcripts 1A and 1B are expressed in the right atrial appendages (RAA), although the cell-type expression of the two transcripts in this particular tissue has not yet been determined. Both transcripts were found to be expressed in the various heart regions investigated, where transcript 1B was found to always occur rarely in comparison with transcript 1A. Transcripts 1A and 1B are both more abundant in the atria than in the ventricles. Luciferase reporter gene assays demonstrated that two genomic regions containing the exons 1A and 1B induced a cell-type specific expression. The 1.2 kb sequence, containing the exon 1A, induced an increase of the luciferase activity in HUVEC; whereas the 1.9 kb sequence, containing the exon 1B, induces an increase of expression of the luciferase activity in BeWo cells. The DNA sequence upstream of the exon 1A contains SP1 binding sites, but no TATA- or CAAT-box; whereas the region upstream of the exon 1B is preceded by three CAAT-boxes. Thus, in contrast to the mouse and rat Cx40 genes, the human Cx40 gene organized in three exons and generates two transcripts, which are cell-type specific.
Assuntos
Processamento Alternativo , Conexinas/genética , RNA/metabolismo , Região 5'-Flanqueadora/genética , Regiões 5' não Traduzidas/genética , Regiões 5' não Traduzidas/metabolismo , Sequência de Bases , Células Cultivadas , DNA/química , DNA/genética , Etiquetas de Sequências Expressas , Feminino , Genes/genética , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Dados de Sequência Molecular , Gravidez , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/genética , RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , Transcrição Gênica/genética , Células Tumorais Cultivadas , Proteína alfa-5 de Junções ComunicantesRESUMO
AIMS: The aim of this study was to characterize ventricular activation patterns in normal and connexin40-deficient mice in order to dissect the role of connexin40 in developing the conduction system. METHODS AND RESULTS: We performed optical mapping of epicardial activation between ED9.5-18.5 and analysed ventricular activation patterns and times of left ventricular activation. Mouse embryos deficient for connexin40 were compared with normal and heterozygous littermates. Morphology of the primary interventricular ring (PIR) was delineated with the help of T3-LacZ transgene. Four major types of ventricular activation patterns characterized by primary breakthrough in different parts of the heart were detected during development: PIR, left ventricular apex, right ventricular apex, and dual right and left ventricular apices. Activation through PIR was frequently present at the early stages until ED12.5. From ED14.5, the majority of hearts showed dual left and right apical breakthrough, suggesting functionality of both bundle branches. Connexin40-deficient embryos showed initially a delay in left bundle branch function, but the right bundle branch block, previously described in the adults, was not detected in ED14.5 embryos and appeared only gradually with 80% penetrance at ED18.5. CONCLUSION: The switch of function from the early PIR conduction pathway to the mature apex to base activation is dependent upon upregulation of connexin40 expression in the ventricular trabeculae. The early function of right bundle branch does not depend on connexin40. Quantitative analysis of normal mouse embryonic ventricular conduction patterns will be useful for interpretation of effects of mutations affecting the function of the cardiac conduction system.
Assuntos
Conexinas/deficiência , Sistema de Condução Cardíaco/metabolismo , Ventrículos do Coração/metabolismo , Potenciais de Ação , Animais , Fascículo Atrioventricular/embriologia , Fascículo Atrioventricular/metabolismo , Bloqueio de Ramo/genética , Bloqueio de Ramo/metabolismo , Conexinas/genética , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Sistema de Condução Cardíaco/embriologia , Ventrículos do Coração/embriologia , Óperon Lac , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Morfogênese , Penetrância , Imagens com Corantes Sensíveis à Voltagem , Proteína alfa-5 de Junções ComunicantesRESUMO
The precise origins of myocardial progenitors and their subsequent contribution to the developing heart has been an area of considerable activity within the field of cardiovascular biology. How these progenitors are regulated and what signals are responsible for their development are, however, much less well understood. Clearly, not only is there a need to identify factors that regulate the transition from proliferation of cardioblasts to differentiation of cardiac muscle, but it is also necessary to identify factors that maintain an adequate pool of undifferentiated myocyte precursors as a prerequisite to preventing organ hypoplasia and congenital heart disease. Here, we report how upregulation of the basic helix-loop-helix (bHLH) transcription factor Hand1, restricted exclusively to Hand1-expressing cells, brings about a significant extension of the heart tube and extraneous looping caused by the elevated proliferation of cardioblasts in the distal outflow tract. This activity is independent of the further recruitment of extracardiac cells from the secondary heart field and permissive for the continued differentiation of adjacent myocardium. Culture studies using embryonic stem (ES) cell-derived cardiomyocytes revealed that, in a Hand1-null background, there is significantly elevated cardiomyocyte differentiation, with an apparent default mesoderm pathway to a cardiomyocyte fate. However, Hand1 gain of function maintains proliferating precursors resulting in delayed and significantly reduced cardiomyocyte differentiation that is mediated by the prevention of cell-cycle exit, by G1 progression and by increased cell division. Thus, this work identifies Hand1 as a crucial cardiac regulatory protein that controls the balance between proliferation and differentiation in the developing heart, and fills a significant gap in our understanding of how the myocardium of the embryonic heart is established.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Miócitos Cardíacos/fisiologia , Transdução de Sinais/fisiologia , Animais , Western Blotting , Primers do DNA , Imunofluorescência , Hibridização In Situ , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tomografia ÓpticaRESUMO
In humans, mutations of the gene encoding the transcription factor Nkx2-5 result in the heart in electrical conduction defects and morphological 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 cardiovascular 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 hemodynamic 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.