Magali Théveniau-Ruissy

Expression Pattern of Connexin Gene Products at the Early Developmental Stages of the Mouse Cardiovascular System

The synchronized contraction of myocytes in cardiac muscle requires the structural and functional integrity of the gap junctions present between these cells. Gap junctions are clusters of intercellular channels formed by transmembrane proteins of the connexin (Cx) family. Products of several Cx genes have been identified in the mammalian heart (eg, Cx45, Cx43, Cx40, and Cx37), and their expression was shown to be regulated during the development of the myocardium. Cx43, Cx40, and Cx45 are components of myocyte gap junctions, and it has also been demonstrated that Cx40 was expressed in the endothelial cells of the blood vessels. The aim of the present work was to investigate the expression and regulation of Cx40, Cx43, and Cx37 during the early stages of mouse heart maturation, between 8.5 days post coitum (dpc), when the first rhythmic contractions appear, and 14.5 dpc, when the four-chambered heart is almost completed. At 8.5 dpc, only the reverse-transcriptase polymerase chain reaction technique has allowed identification of Cx43, Cx40, and Cx37 gene transcripts in mouse heart, suggesting a very low activity level of these genes. From 9.5 dpc, all three transcripts became detectable in whole-mount in situ-hybridized embryos, and the most obvious result was the labeling of the vascular system with Cx40 and Cx37 anti-sense riboprobes. Cx40 and Cx37 gene products (transcript and/or protein) were demonstrated to be expressed in the vascular endothelial cells at all stages examined. By contrast, only Cx37 gene products were found in the endothelial cells of the endocardium. In heart, Cx37 was expressed exclusively in these cells, which rules out any direct involvement of this Cx in the propagation of electrical activity between myocytes and the synchronization of contractions. Between 9.5 and 11.5 dpc, Cx40 gene activation in myocytes was demonstrated to proceed according to a caudorostral gradient involving first the primitive atrium and the common ventricular chamber (9.5 dpc) and then the right ventricle (11.5 dpc). During this period of heart morphogenesis, there is clearly a temporary and asymmetrical regionalization of the Cx40 gene expression that is superimposed on the functional regionalization. In addition, comparison of Cx40 and Cx43 distribution at the above developmental stages has shown that these Cxs have overlapping (left ventricle) or complementary (atrial tissue and right ventricle) expression patterns.

Downregulation of Connexin 45 Gene Products During Mouse Heart Development

The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The gap junction channels that couple the myocytes are responsible for this propagation process. The gap junction channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Three members of this family have been demonstrated to be synthesized in the cardiomyocytes: Cx40, Cx43, and Cx45. In addition, each of them has been shown to form channels with unique and specific electrophysiological properties. Understanding the conduction phenomenon requires detailed knowledge of the spatiotemporal expression pattern of these Cxs in heart. The expression patterns of Cx40 and Cx43 have been previously described in the adult heart and during its development. Here we report the expression of Cx45 gene products in mouse heart from the stage of the first contractions (8.5 days postcoitum [dpc]) to the adult stage. The Cx45 gene transcript was demonstrated by reverse transcriptase-polymerase chain reaction experiments to be present in heart at all stages investigated. Between 8.5 and 10.5 dpc it was shown by in situ hybridization to be expressed in low amounts in all cardiac compartments (including the inflow and outflow tracts and the atrioventricular canal) and then to be downregulated from 11 to 12 dpc onward. At subsequent fetal stages, the transcript was weakly detected in the ventricles, with the most distinct expression in the outflow tract. Cx45 protein was demonstrated by immunofluorescence microscopy to be expressed in the myocytes of young embryonic hearts (8.5 to 9.5 dpc). However, beyond 10.5 dpc the protein was no longer detected with this technique in the embryonic, fetal, or neonatal working myocardium, although it could be shown by immunoblotting that the protein was still synthesized in neonatal heart. In the major part of adult heart, Cx45 was undetectable. It was, however, clearly seen in the anterior regions of the interventricular septum and in trace amounts in some small foci dispersed in the ventricular free walls. Cx45 gene is the first Cx gene so far demonstrated to be activated in heart at the stage of the first contractions. The coordination of myocytes during the slow peristaltic contractions that occur at this stage would thus appear to be controlled by the Cx45 channels.

The del22q11.2 Candidate Gene Tbx1 Controls Regional Outflow Tract Identity and Coronary Artery Patterning

TBX1, encoding a T-box containing transcription factor, is the major candidate gene for del22q11.2 or DiGeorge syndrome, characterized by craniofacial and cardiovascular defects including tetralogy of Fallot and common arterial trunk. Mice lacking Tbx1 have severe defects in the development of pharyngeal derivatives including cardiac progenitor cells of the second heart field that contribute to the arterial pole of the heart. The outflow tract of Tbx1 mutant embryos is short and narrow resulting in common arterial trunk. Here we show by a series of genetic crosses using transgene markers of second heart field derived myocardium and coronary endothelial cells that a subdomain of myocardium normally observed at the base of the pulmonary trunk is reduced and malpositioned in Tbx1 mutant hearts. This defect is associated with anomalous coronary artery patterning. Both right and left coronary ostia form predominantly at the right/ventral sinus in mutant hearts, proximal coronary arteries coursing across the normally coronary free ventral region of the heart. We have identified Semaphorin3c as a Tbx1-dependent gene expressed in subpulmonary myocardium. Our results implicate second heart field development in coronary artery patterning and provide new insights into the association between conotruncal defects and coronary artery anomalies.

Tbx3 Is Required for Outflow Tract Development

Conotruncal and ventricular septal congenital heart anomalies result from defects in formation and division of the embryonic outflow tract. Cardiac remodeling during outflow tract and ventricular septation converts the tubular embryonic heart into a parallel circulatory system with an independent left ventricular outlet and right ventricular inlet. Tbx3 encodes a T-box–containing transcription factor expressed in the developing conduction system of the heart. Mutations in TBX3 cause ulnar–mammary syndrome. Here we show that mice lacking Tbx3 develop severe outflow tract defects, including connection of both the aorta and pulmonary trunk with the right ventricle, in addition to aortic arch artery anomalies and abnormal communication between the right atrium and left ventricle. Alignment defects are preceded by a delay in caudal displacement of the arterial pole of the heart during aortic arch artery formation. Embryonic anterior–posterior patterning and cardiac chamber development are unaffected in Tbx3 mutant embryos. However, the contribution of second heart field derived progenitor cells to the arterial pole of the heart is impaired. Tbx3 is expressed in pharyngeal epithelia and neural crest cells in the pharyngeal region, suggesting an indirect role in second heart field deployment. Loss of Tbx3 affects multiple signaling pathways regulating second heart field proliferation and outflow tract morphogenesis, including fibroblast growth factor signaling, leading to a failure of normal heart tube extension and consequent atrioventricular and ventriculoarterial alignment defects.

Clonal analysis reveals a common origin between nonsomite-derived neck muscles and heart myocardium

Significance Head muscles, derived from the first and second pharyngeal arches, share common progenitors with myocardial cells of the heart. This is in contrast to somite-derived skeletal muscles of the trunk and limbs. Neck muscles, located in the transition zone between head and trunk, have both a somitic and nonsomitic origin. We now demonstrate a clonal relationship between nonsomitic neck muscles and myocardial cells located in the atria, inflow and outflow regions of the heart. This is distinct from that of the two head muscle lineages. Formation of these neck muscles, like those in the head, depends on a gene regulatory network shared with myocardial progenitors. We thus identify a third clonal group within cardiopharyngeal mesoderm, with implications for human malformations. Neck muscles constitute a transition zone between somite-derived skeletal muscles of the trunk and limbs, and muscles of the head, which derive from cranial mesoderm. The trapezius and sternocleidomastoid neck muscles are formed from progenitor cells that have expressed markers of cranial pharyngeal mesoderm, whereas other muscles in the neck arise from Pax3-expressing cells in the somites. Mef2c-AHF-Cre genetic tracing experiments and Tbx1 mutant analysis show that nonsomitic neck muscles share a gene regulatory network with cardiac progenitor cells in pharyngeal mesoderm of the second heart field (SHF) and branchial arch-derived head muscles. Retrospective clonal analysis shows that this group of neck muscles includes laryngeal muscles and a component of the splenius muscle, of mixed somitic and nonsomitic origin. We demonstrate that the trapezius muscle group is clonally related to myocardium at the venous pole of the heart, which derives from the posterior SHF. The left clonal sublineage includes myocardium of the pulmonary trunk at the arterial pole of the heart. Although muscles derived from the first and second branchial arches also share a clonal relationship with different SHF-derived parts of the heart, neck muscles are clonally distinct from these muscles and define a third clonal population of common skeletal and cardiac muscle progenitor cells within cardiopharyngeal mesoderm. By linking neck muscle and heart development, our findings highlight the importance of cardiopharyngeal mesoderm in the evolution of the vertebrate heart and neck and in the pathophysiology of human congenital disease.

Connexin 30 is expressed in the mouse sino-atrial node and modulates heart rate

AIMS This study aimed at characterizing expression and the functional role of the Gjb6 gene, encoding for connexin 30 (Cx30) protein, in the adult mouse heart. METHODS AND RESULTS The expression of the Gjb6 gene in the mouse heart was investigated by RT-PCR and sequencing of amplified cDNA fragments. The sites of Gjb6 expression were identified in the adult heart using transgenic mice with reporter genes (Cx30(LacZ/LacZ) and Cx30(LacZ/LacZ)/Cx40(EGFP/EGFP) mice), as well as anti-HCN4 (hyperpolarization activated cyclic nucleotide-gated potassium channel 4) or anti-connexin antibodies. Cine-magnetic resonance imaging and telemetric ECG recordings were used to evaluate the impact of Cx30 deficiency on cardiac physiology. Gjb6 was shown to be expressed in the sinoatrial (SA) node of the adult mouse heart. Eighty from 100 nuclei on average were LacZ-positive in the SA node of Cx30(LacZ/LacZ) mice. No significant LacZ expression was seen in other cardiac tissues. Cx30 protein was identified in low abundance in the SA node of wild-type mice, as indicated by immunofluorescence experiments. Telemetric ECG recordings indicated that Cx30-deficient mice displayed a mean daily heart rate (HR) that was 9% faster than that measured in control mice (572 +/- 38 b.p.m. vs. 524 +/- 23, P < 0.05). This moderate tachycardia was still observed after inhibition of the autonomic nervous system, demonstrating that Cx30 deficiency resulted in changes in the intrinsic electrical properties of the SA node. Consistent with this hypothesis, Cx30(LacZ/LacZ) displayed a significant reduction of SDNN (standard deviation of the interbeat interval) compared with control mice. Increase of both the cardiac index (20%) and the end-diastolic volume to body weight ratio (16%) with no deficiency in ejection fraction or stroke volume were observed in mutant mice. An increase in cardiac index was interpreted as being a direct consequence of high HR, whereas large end-diastolic volume may be an indirect consequence of prolonged high HR. CONCLUSION Cx30 is functionally expressed, in low abundance, in the SA node of the adult mouse heart where it participates in HR regulation.

Connexin expression in cultured neonatal rat myocytes reflects the pattern of the intact ventricle.

OBJECTIVE Primary cultures of neonatal rat ventricular myocytes have become a widely used model to examine a variety of functional, physiological and biochemical cardiac properties. In the adult rat, connexin43 (Cx43) is the major gap junction protein present in the working myocardium. In situ hybridization studies on developing rats, however, showed that Cx40 mRNA displays a dynamic and heterogeneous pattern of expression in the ventricular myocardium around birth. The present studies were performed to examine the expression pattern of the Cx40 protein in neonatal rat heart, and to examine the connexins present in cultures of ventricular myocytes obtained from those hearts. METHODS Cryosections were made of hearts of 1-day-old Wistar rats. Cultures of ventricular myocytes obtained from these hearts by enzymatic dissociation were seeded at various densities (to obtain > 75, approximately 50%, and < 25% confluency) and cultured for 24, 48 or 96 h. Cx40 and Cx43 were detected by immunofluorescence and immunoblotting. RESULTS Immunohistochemical stainings confirmed that gap junctions in the atrium and His-Purkinje system were composed of at least Cx43 and Cx40. From the subendocardium towards the subepicardium Cx40 expression gradually decreased, resulting in the sole expression of Cx43 in the subepicardial part of the ventricular wall. In ventricular myocytes cultured at high density (> 75% confluency) Cx43 and Cx40 immunoreactivity could be detected. In contrast to Cx43 immunolabeling which showed a homogeneous distribution pattern, Cx40 staining was heterogeneous, i.e. in some clusters of cells abundant labeling was present whereas in others no Cx40 staining could be detected. The pattern of Cx43 immunoreactivity was not altered by the culture density. In contrast, in isolated ventricular myocytes cultured at low density (< 25% confluency) the relative number of cell-cell interfaces that were Cx40-immunopositive decreased as compared to high density cultures (35 vs. 70%). Western blots did not reveal significant differences in the level of Cx40 and Cx43 expression at different culture densities. CONCLUSIONS These results show that cultured ventricular myocytes retained typical features of the native neonatal rat ventricular myocardium with regard to their composition of gap junctions. This implicates that these cultures may serve as a good model for studying short-term and long-term regulation of cardiac gap junction channel expression and function.

0245 : Dissecting progenitor cell contributions to the developing heart

Cardiac progenitor cells of the second heart field (SHF) contribute to the poles of the elongating embryonic heart. Perturbation of SHF development leads to a spectrum of congenital heart defects. Recent evidence suggests that distinct regions of the heart are pre-patterned in the SHF. For example the dell22q11.2 or DiGeorge syndrome gene Tbx1 is required in the SHF for development of the inferior wall of the embryonic outflow tract, giving rise to subpulmonary myocardium. Characterization of the expression of an enhancer trap transgene at the Hes1 locus, encoding a transcriptional repressor, has identified a complementary Notch-dependent Hes1 + TBX1 – subpopulation of SHF cells giving rise to future subaortic myocardium. Using transcriptomic analysis we have characterized the genetic signatures of future subaortic and subpulmonary myocardium and identified Pparg among the genes enriched in future subpulmonary myocardium. Genetic and explant analyses have shown that Hes1 controls the molecular signature of future subaortic myocardium through direct transcriptional repression of Ppar g. Our results reveal that distinct genetic regulatory networks control different progenitor cell contributions to the developing heart. We also investigated the potential role of Hes1 in the maintenance of residual SHF progenitors in the fetal heart. Our initial results have identified Hes1 + cells in the fetal heart and suggest that Hes1 deletion impacts negatively on residual progenitor cell numbers. Together, our study identifies a role for Hes1 in the regulation of cardiac progenitor cell fate and maintenance in the definitive heart of clinical importance for heart repair.

344 Molecular patterning of the cardiac outflow tract and coronary arteries of the mouse heart

Background: The prevalence of obesity in children is increasing worldwide. We used 2D speckle strain imaging to investigate whether severely overweight children without hypertension, dyslipidemia, diabetes or sleep apnea, show early cardiac abnormalities. We also investigated the relation between these myocardial features and severity of obesity, fat mass percentage, inflammation and insuline resistance index.

305 Conotruncal and coronary artery development in two mouse models of congenital heart defects

Conotruncal heart defects are among the most frequent congenital heart diseases. Coronary artery anomalies are commonly associated with outflow tract malformations. The molecular and cellular mechanisms underlying their development have yet to be unravelled. TBX1, encoding a T-box transcription factor, is the major DiGeorge syndrome (del22q11.2) candidate gene and is required for pharyngeal and cardiovascular development. Tbx1-/- embryos have severe cardiac anomalies including a common arterial trunk. DiGeorge syndrome patients have a high incidence of conotruncal defects including persistant truncus arteriosus and tetralogy of Fallot. We have shown that the common arterial trunk in Tbx1-/- embryos has an aorta-like phenotype associated with severe reduction of a subpopulation of second heart field progenitor cells normally contributing to myocardium at the base of pulmonary trunk. Underdevelopment of subpulmonary myocardium is thought to be the primary defect in human conotruncal defects like tetralogy of Fallot. Anomalous coronary artery patterning occurs in Tbx1-/- hearts. Semaphorin3c, encoding a neurovascular guidance molecule is expressed in a Tbx1-dependent domain in the subpulmonary myocardium. Disruption of the semaphorin signaling pathway during heart morphogenesis results in outflow tract defects and anomalies of the aortic arch arteries. Sema3c-/- embryos also display common arterial trunk with interruption of the aortic arch but coronary artery patterning appears normal. Here we present a comparative analysis of the evolution of common trunk in these two models and investigate potential genetic interaction between these genes. Future subaortic and subpulmonary regions are prefigured in the E10.5 outflow tract. Using a candidate gene approach and microarray analysis at E10.5 we aim to identify additional genes expressed in subpulmonary myocardium that may contribute to conotruncal and coronary artery development.