Atrioventricular Endocardial Cushions Research Articles

Crk and Crkl Are Required in the Endocardial Lineage for Heart Valve Development.

Background Endocardial cells are a major progenitor population that gives rise to heart valves through endocardial cushion formation by endocardial to mesenchymal transformation and the subsequent endocardial cushion remodeling. Genetic variants that affect these developmental processes can lead to congenital heart valve defects. Crk and Crkl are ubiquitously expressed genes encoding cytoplasmic adaptors essential for cell signaling. This study aims to explore the specific role of Crk and Crkl in the endocardial lineage during heart valve development. Methods and Results We deleted Crk and Crkl specifically in the endocardial lineage. The resultant heart valve morphology was evaluated by histological analysis, and the underlying cellular and molecular mechanisms were investigated by immunostaining and quantitative reverse transcription polymerase chain reaction. We found that the targeted deletion of Crk and Crkl impeded the remodeling of endocardial cushions at the atrioventricular canal into the atrioventricular valves. We showed that apoptosis was temporally increased in the remodeling atrioventricular endocardial cushions, and this developmentally upregulated apoptosis was repressed by deletion of Crk and Crkl. Loss of Crk and Crkl also resulted in altered extracellular matrix production and organization in the remodeling atrioventricular endocardial cushions. These morphogenic defects were associated with altered expression of genes in BMP (bone morphogenetic protein), connective tissue growth factor, and WNT signaling pathways, and reduced extracellular signal-regulated kinase signaling activities. Conclusions Our findings support that Crk and Crkl have shared functions in the endocardial lineage that critically regulate atrioventricular valve development; together, they likely coordinate the morphogenic signals involved in the remodeling of the atrioventricular endocardial cushions.

Loss of myocardial Hey2/Hrt2 function disrupts rightward shift of atrioventricular cushion tissue and causes tricuspid atresia.

Endocardial cushion tissue is primordia of the valves and septa of the adult heart, and its malformation causes various congenital heart diseases (CHDs). Tricuspid atresia (TA) is defined as congenital absence or agenesis of the tricuspid valve caused by endocardial cushion defects. However, little is known about what type of endocardial cushion defect causes TA. Using three-dimensional volume rendering image analysis, we demonstrated morphological changes of endocardial cushion tissue in developing Hey2/Hrt2 KO mouse embryos that showed malformation of the tricuspid valve, which resembled human TA at neonatal period. In control embryos, atrioventricular (AV) endocardial cushion tissues showed rightward shift to form a tricuspid valve. However, the rightward shift of endocardial cushion tissue was disrupted in Hey2/Hrt2 KO embryos, leading to the misalignment of AV cushions. We also found that muscular tissue filled up the space between the right atrium and ventricle, resulting in the absence of the tricuspid valve. Moreover, analysis using tissue-specific conditional KO mice showed that HEY2/HRT2-expressing myocardium may physically regulate the AV shift. Disruption of rightward cushion movement is an initial cue of TA phenotype, and myocardial HEY2/HRT2 is necessary for the regulation of proper alignment of AV endocardial cushion tissue.

Natural History of a Transitional Atrioventricular Septal Defect in an Adult Patient with Down Syndrome

Background: Atrioventricular septal defects (AVSD) are caused by a lack of development in atrioventricular endocardial cushions. Its spectrum varies from partial to transitional, or intermediate forms with a common AV valve and 2 orifices, or even the complete form. Aim: The aim of this study is to present a case of a woman in the fifth decade of life with Down syndrome and a transitional AVSD, diagnosed with echocardiography. Case presentation: This is a patient in the fifth decade of life with Down syndrome, transitional AVSD, and severe pulmonary hypertension. Her vital signs were normal, oxygen saturation of 89% on room air. Auscultation revealed pronounced pulmonary component of the second heart sound, diastolic murmur in second left intercostal space, and moderate holosystolic heart murmur that radiated to axilla. The diagnosis was made with a transthoracic echocardiogram. The patient is receiving only medical treatment and since her last consult she has New York Heart Association (NYHA) functional class II. Conclusion: This case demonstrates the natural history of a patient in her fifth decade of life with down syndrome and complex congenital heart disease, despite having received only medical treatment, she is in functional class II. Echocardiography is the noninvasive technique of choice in the diagnosis and follow-up of patients with congenital heart diseases, as seen in this case.

RERE deficiency leads to decreased expression of GATA4 and the development of ventricular septal defects.

ABSTRACTDeletions of chromosome 1p36 are associated with a high incidence of congenital heart defects (CHDs). The arginine-glutamic acid dipeptide repeats gene (RERE) is located in a critical region for CHD on chromosome 1p36 and encodes a cardiac-expressed nuclear receptor co-regulator. Mutations affecting RERE cause atrial and ventricular septal defects (VSDs) in humans, and RERE-deficient mice also develop VSDs. During cardiac development, mesenchymal cells destined to form part of the atrioventricular (AV) septum are generated when endocardial cells in the AV canal undergo epithelial-to-mesenchymal transition (EMT) and migrate into the space between the endocardium and the myocardium. These newly generated mesenchymal cells then proliferate to fill the developing AV endocardial cushions. Here, we demonstrate that RERE-deficient mouse embryos have reduced numbers of mesenchymal cells in their AV endocardial cushions owing to decreased levels of EMT and mesenchymal cell proliferation. In the endocardium, RERE colocalizes with GATA4, a transcription factor required for normal levels of EMT and mesenchymal cell proliferation. Using a combination of in vivo and in vitro studies, we show that Rere and Gata4 interact genetically in the development of CHDs, RERE positively regulates transcription from the Gata4 promoter and GATA4 levels are reduced in the AV canals of RERE-deficient embryos. Tissue-specific ablation of Rere in the endocardium leads to hypocellularity of the AV endocardial cushions, defective EMT and VSDs, but does not result in decreased GATA4 expression. We conclude that RERE functions in the AV canal to positively regulate the expression of GATA4, and that deficiency of RERE leads to the development of VSDs through its effects on EMT and mesenchymal cell proliferation. However, the cell-autonomous role of RERE in promoting EMT in the endocardium must be mediated by its effects on the expression of proteins other than GATA4.This article has an associated First Person interview with the first author of the paper.

BMP2 expression in the endocardial lineage is required for AV endocardial cushion maturation and remodeling

Distal outgrowth, maturation and remodeling of the endocardial cushion mesenchyme in the atrioventricular (AV) canal are the essential morphogenetic events during four-chambered heart formation. Mesenchymalized AV endocardial cushions give rise to the AV valves and the membranous ventricular septum (VS). Failure of these processes results in several human congenital heart defects. Despite this clinical relevance, the mechanisms governing how mesenchymalized AV endocardial cushions mature and remodel into the membranous VS and AV valves have only begun to be elucidated. The role of BMP signaling in the myocardial and secondary heart forming lineage has been well studied; however, little is known about the role of BMP2 expression in the endocardial lineage. To fill this knowledge gap, we generated Bmp2 endocardial lineage-specific conditional knockouts (referred to as Bmp2 cKOEndo) by crossing conditionally-targeted Bmp2flox/flox mice with a Cre-driver line, Nfatc1Cre, wherein Cre-mediated recombination was restricted to the endocardial cells and their mesenchymal progeny. Bmp2 cKOEndo mouse embryos did not exhibit failure or delay in the initial AV endocardial cushion formation at embryonic day (ED) 9.5–11.5; however, significant reductions in AV cushion size were detected in Bmp2 cKOEndo mouse embryos when compared to control embryos at ED13.5 and ED16.5. Moreover, deletion of Bmp2 from the endocardial lineage consistently resulted in membranous ventricular septal defects (VSDs), and mitral valve deficiencies, as evidenced by the absence of stratification of mitral valves at birth. Muscular VSDs were not found in Bmp2 cKOEndo mouse hearts. To understand the underlying morphogenetic mechanisms leading to a decrease in cushion size, cell proliferation and cell death were examined for AV endocardial cushions. Phospho-histone H3 analyses for cell proliferation and TUNEL assays for apoptotic cell death did not reveal significant differences between control and Bmp2 cKOEndo in AV endocardial cushions. However, mRNA expression of the extracellular matrix components, versican, Has2, collagen 9a1, and periostin was significantly reduced in Bmp2 cKOEndo AV cushions. Expression of transcription factors implicated in the cardiac valvulogenesis, Snail2, Twist1 and Sox9, was also significantly reduced in Bmp2 cKOEndo AV cushions. These data provide evidence that BMP2 expression in the endocardial lineage is essential for the distal outgrowth, maturation and remodeling of AV endocardial cushions into the normal membranous VS and the stratified AV valves.

MiR-23b and miR-199a impair epithelial-to-mesenchymal transition during atrioventricular endocardial cushion formation.

Valve development is a multistep process involving the activation of the cardiac endothelium, epithelial-mesenchymal transition (EMT) and the progressive alignment and differentiation of distinct mesenchymal cell types. Several pathways such as Notch/delta, Tgf-beta and/or Vegf signaling have been implicated in crucial steps of valvulogenesis. We have previously demonstrated discrete changes in microRNAs expression during cardiogenesis, which are predicted to target Bmp- and Tgf-beta signaling. We now analyzed the expression profile of 20 candidate microRNAs in atrial, ventricular, and atrioventricular canal regions at four different developmental stages. qRT-PCR analyses of microRNAs demonstrated a highly dynamic and distinct expression profiles within the atrial, ventricular, and atrioventricular canal regions of the developing chick heart. miR-23b, miR-199a, and miR-15a displayed increased expression during early AVC development whereas others such as miR-130a and miR-200a display decreased expression levels. Functional analyses of miR-23b, miR-199a, and miR-15a overexpression led to in vitro EMT blockage. Molecular analyses demonstrate that distinct EMT signaling pathways are impaired after microRNA expression, including a large subset of EMT-related genes that are predicted to be targeted by these microRNAs. Our data demonstrate that miR-23b and miR-199a over-expression can impair atrioventricular EMT.

The development of septation in the four-chambered heart.

The past decades have seen immense progress in the understanding of cardiac development. Appreciation of precise details of cardiac anatomy, however, has yet to be fully translated into the more general understanding of the changing structure of the developing heart, particularly with regard to formation of the septal structures. In this review, using images obtained with episcopic microscopy together with scanning electron microscopy, we show that the newly acquired information concerning the anatomic changes occurring during separation of the cardiac chambers in the mouse is able to provide a basis for understanding the morphogenesis of septal defects in the human heart. It is now established that as part of the changes seen when the heart tube changes from a short linear structure to the looped arrangement presaging formation of the ventricles, new material is added at both its venous and arterial poles. The details of these early changes, however, are beyond the scope of our current review. It is during E10.5 in the mouse that the first anatomic features of septation are seen, with formation of the primary atrial septum. This muscular structure grows toward the cushions formed within the atrioventricular canal, carrying on its leading edge a mesenchymal cap. Its cranial attachment breaks down to form the secondary foramen by the time the mesenchymal cap has used with the atrioventricular endocardial cushions, the latter fusion obliterating the primary foramen. Then the cap, along with a mesenchymal protrusion that grows from the mediastinal mesenchyme, muscularizes to form the base of the definitive atrial septum, the primary septum itself forming the floor of the oval foramen. The cranial margin of the foramen is a fold between the attachments of the pulmonary veins to the left atrium and the roof of the right atrium. The apical muscular ventricular septum develops concomitant with the ballooning of the apical components from the inlet and outlet of the ventricular loop. Its apical part is initially trabeculated. The membranous part of the septum is derived from the rightward margins of the atrioventricular cushions, with the muscularizing proximal outflow cushions fusing with the muscular septum and becoming the subpulmonary infundibulum as the aorta is committed to the left ventricle. Perturbations of these processes explain well the phenotypic variants of deficient atrial and ventricular septation.

Expression of the BMP Receptor Alk3 in the Second Heart Field Is Essential for Development of the Dorsal Mesenchymal Protrusion and Atrioventricular Septation

The dorsal mesenchymal protrusion (DMP) is a prong of mesenchyme derived from the second heart field (SHF) located at the venous pole of the developing heart. Recent studies have shown that perturbation of its development is associated with the pathogenesis of atrioventricular (AV) septal defect. Although the importance of the DMP to AV septation is now established, the molecular and cellular mechanisms underlying its development are far from fully understood. Prior studies have demonstrated that bone morphogenetic protein (BMP) signaling is essential for proper formation of the AV endocardial cushions and the cardiac outflow tract. A role for BMP signaling in regulation of DMP development remained to be elucidated. To determine the role of BMP signaling in DMP development. Conditional deletion of the BMP receptor Alk3 from venous pole SHF cells leads to impaired formation of the DMP and a completely penetrant phenotype of ostium primum defect, a hallmark feature of AV septal defects. Analysis of mutants revealed decreased proliferative index of SHF cells and, consequently, reduced number of SHF cells at the cardiac venous pole. In contrast, volume and expression of markers associated with proliferation and active BMP/transforming growth factor β signaling were not significantly altered in the AV cushions of SHF-Alk3 mutants. BMP signaling is required for expansion of the SHF-derived DMP progenitor population at the cardiac venous pole. Perturbation of Alk3-mediated BMP signaling from the SHF results in impaired development of the DMP and ostium primum defects.

Jun Is Required in Isl1-Expressing Progenitor Cells for Cardiovascular Development

Jun is a highly conserved member of the multimeric activator protein 1 transcription factor complex and plays an important role in human cancer where it is known to be critical for proliferation, cell cycle regulation, differentiation, and cell death. All of these biological functions are also crucial for embryonic development. Although all Jun null mouse embryos die at mid-gestation with persistent truncus arteriosus, a severe cardiac outflow tract defect also seen in human congenital heart disease, the developmental mechanisms are poorly understood. Here we show that murine Jun is expressed in a restricted pattern in several cell populations important for cardiovascular development, including the second heart field, pharyngeal endoderm, outflow tract and atrioventricular endocardial cushions and post-migratory neural crest derivatives. Several genes, including Isl1, molecularly mark the second heart field. Isl1 lineages include myocardium, smooth muscle, neural crest, endocardium, and endothelium. We demonstrate that conditional knockout mouse embryos lacking Jun in Isl1-expressing progenitors display ventricular septal defects, double outlet right ventricle, semilunar valve hyperplasia and aortic arch artery patterning defects. In contrast, we show that conditional deletion of Jun in Tie2-expressing endothelial and endocardial precursors does not result in aortic arch artery patterning defects or embryonic death, but does result in ventricular septal defects and a low incidence of semilunar valve defects, atrioventricular valve defects and double outlet right ventricle. Our results demonstrate that Jun is required in Isl1-expressing progenitors and, to a lesser extent, in endothelial cells and endothelial-derived endocardium for cardiovascular development but is dispensable in both cell types for embryonic survival. These data provide a cellular framework for understanding the role of Jun in the pathogenesis of congenital heart disease.

An isolated cleft of the anterior leaflet of mitral valve in an infant: a case report

Background : Isolated cleft of the anterior leaflet of the mitral valve is the occurrence of mitral cleft without ostium primum or ostium secundum defect and it is rare. It results from various degrees of failure of fusion of early embroyonic atrioventricular endocardial cushions and occurs more in children with trisomy 21. It commonly presents with mitral incompetence which worsens as the child grows. Aim : To document a case of congenital isolated cleft of the anterior leaflet of the mitrial valve in an infant. Findings : A nine month old boy presented in our clinic with a two month history of fast breathing, cough, and loss of weight. He was found not to be cyanosed but pale, tachypnoeic, dyspnoeic with basal crepitations. Cadiovascular system examination revealed he had a precordial bulge with tachycardia, cardiomegaly and a holosystolic murmur maximum at the apex. There was tender hepatomegaly. He was diagnosed as having congestive cardiac failure with broncho- pneumonia secondary to an acyanotic congenital heart disease. Plain chest X-ray confirmed the cardiomegaly and bronchopneumonia while an electrocardiography showed bilateral atrial enlargement with right ventricular hypertrophy. The cardiac failure was treated but he represented with recurrent heart failure upto three times within six months. An echocardiogram done on the third admission showed an isolated cleft of the anterior leaflet of the mitral valve and he has been referred for surgery. Conclusion : Isolated cleft of the mitral valve commonly presents with mitral incompetence and eventual heart failure as in our patient. Early surgical intervention is adviced as the width of the cleft tend to increase as the child grows leading to worsening of the mitral insufficiency. The definitive treatment is direct closure of the cleft. The presentation also highlights the challenges of medical practice in resource-poor countries. Keywords : Congenital , heart, isolated cleft, mitral valve, ostium primum, ostium secundum

Multiple essential roles for primary cilia in heart development

BackgroundThe primary cilium is a microtubule-based, plasma membrane-ensheathed protrusion projecting from the basal bodies of almost all cell types in the mammalian body. In the past several years a plethora of papers has indicated a crucial role for primary cilia in the development of a wide variety of organs. We have investigated heart development in cobblestone, a hypomorphic allele of the gene encoding the intraflagellar transport protein Ift88, and uncovered a number of the most common congenital heart defects seen in newborn humans.MethodsWe generated serial sections of mutant cobblestone and wild type embryos in the region encompassing the heart and the cardiac outflow tract. The sections were further processed to generate three-dimensional reconstructions of these structures, and immunofluorescence confocal microscopy, transmission electron microscopy, and in situ hybridization were used to examine signal transduction pathways in the relevant areas. Whole mount in situ hybridization was also employed for certain developmental markers.ResultsIn addition to an enlarged pericardium and failure of both ventricular and atrial septum formation, the cobblestone mutants displayed manifold defects in outflow tract formation, including persistent truncus arteriosus, an overriding aorta, and abnormal transformation of the aortic arches. To discern the basis of these anomalies we examined both the maintenance of primary cilia as well as endogenous and migratory embryonic cell populations that contribute to the outflow tract and atrioventricular septa. The colonization of the embryonic heart by cardiac neural crest occurred normally in the cobblestone mutant, as did the expression of Sonic hedgehog. However, with the loss of primary cilia in the mutant hearts, there was a loss of both downstream Sonic hedgehog signaling and of Islet 1 expression in the second heart field, a derivative of the pharyngeal mesoderm. In addition, defects were recorded in development of atrial laterality and ventricular myocardiogenesis. Finally, we observed a reduction in expression of Bmp4 in the outflow tract, and complete loss of expression of both Bmp2 and Bmp4 in the atrioventricular endocardial cushions. Loss of BMP2/4 signaling may result in the observed proliferative defect in the endocardial cushions, which give rise to both the atrioventricular septa as well as to the septation of the outflow tract.ConclusionsTaken together, our results potentially identify a novel link between Sonic hedgehog signaling at the primary cilium and BMP-dependent effects upon cardiogenesis. Our data further point to a potential linkage of atrioventricular septal defects, the most common congenital heart defects, to genes of the transport machinery or basal body of the cilia.

Endocardial cell epithelial-mesenchymal transformation requires Type III TGFβ receptor interaction with GIPC

An early event in heart valve formation is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endothelial cells in specific regions of the heart tube, the endocardial cushions. The Type III TGFβ receptor (TGFβR3) is required for TGFβ2- or BMP-2-stimulated EMT in atrioventricular endocardial cushion (AVC) explants in vitro but the mediators downstream of TGFβR3 are not well described. Using AVC and ventricular explants as an in vitro assay, we found an absolute requirement for specific TGFβR3 cytoplasmic residues, GAIP-interacting protein, C terminus (GIPC), and specific Activin Receptor-Like Kinases (ALK)s for TGFβR3-mediated EMT when stimulated by TGFβ2 or BMP-2. The introduction of TGFβR3 into nontransforming ventricular endocardial cells, followed by the addition of either TGFβ2 or BMP-2, results in EMT. TGFβR3 lacking the entire cytoplasmic domain, or only the 3C-terminal amino acids that are required to bind GIPC, fails to support EMT in response to TGFβ2 or BMP-2. Overexpression of GIPC in AVC endocardial cells enhanced EMT while siRNA-mediated silencing of GIPC in ventricular cells overexpressing TGFβR3 significantly inhibited EMT. Targeting of specific ALKs by siRNA revealed that TGFβR3-mediated EMT requires ALK2 and ALK3, in addition to ALK5, but not ALK4 or ALK6. Taken together, these data identify GIPC, ALK2, ALK3, and ALK5 as signaling components required for TGFβR3-mediated endothelial cell EMT.

Expression of cellular retinoic acid-binding protein I and II (CRABP I and II) in embryonic mouse hearts treated with retinoic acid.

Cellular retinoic acid binding proteins are considered to be involved in retinoic acid (RA) signaling pathways. Our aim was to compare the expression and localization of cellular retinoic acid binding proteins I and II (CRABP I and II) in embryonic mouse hearts during normal development and after a single teratogenic dose of RA. Techniques such as real-time PCR, RT-PCR, Western blots and immunostaining were employed to examine hearts from embryos at 9-17 dpc. RA treatment at 8.5dpc affects production of CRABP I and II in the heart in the 48-h period. Changes in expression of mRNA for retinaldehyde dehydrogenase II (Raldh2), Crabp1 and Crabp2 genes also occur within the same time window (i.e. 10-11dpc) after RA treatment. In the embryonic control heart these proteins are localized in groups of cells within the outflow tract (OT), and the atrioventricular endocardial cushions. A gradient of labeling is observed with CRABP II but not for CRABP I along the myocardium of the looped heart at 11 dpc; this gradient is abolished in hearts treated with RA, whereas an increase of RALDH2 staining has been observed at 10 dpc in RA-treated hearts. Some populations of endocardial endothelial cells were intensively stained with anti-CRABP II whereas CRABP I was negative in these structures. These results suggest that CRABP I and II are independently regulated during heart development, playing different roles in RA signaling, essential for early remodeling of the heart tube and alignment of the great arteries to their respective ventricles.

The Many Faces of Hypertrophic Cardiomyopathy: From Developmental Biology to Clinical Practice

Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease, characterized by complex pathophysiology, heterogeneous morphology, and variable clinical manifestations over time. Besides cardiac hypertrophy, the HCM phenotype is characterized by a host of manifestations, including mitral valve and subvalvar abnormalities, subaortic and mid-ventricular left ventricular (LV) obstruction, microvascular dysfunction, myocardial fibrosis, disarray, atrial remodeling, myocardial bridging of epicardial coronary arteries, LV apical aneurysms, and autonomic nervous system abnormalities. Such heterogeneous phenotype still lacks a comprehensive explanation, which cannot be accounted solely by genetic heterogeneity, despite the large number of genes and mutations involved. It is likely that pre-natal and acquired features deriving from the primary genetic defect interact with the environment to produce the final result evident in each patient. Based on novel insights provided by cardiac developmental biology, a common lineage ancestry of several HCM manifestations might be traced back to the pluripotent epicardium-derived cells, which early during heart development differentiate into interstitial fibroblasts, coronary smooth muscle cells, and atrio-ventricular endocardial cushions as mesenchymal cells. To date, the different faces of HCM have not been sufficiently liked or explained. We here attempt to address these issues by describing the various components of the disease, their origin, interaction, and clinical significance.

Generation of Smad7(-Cre) recombinase mice: A useful tool for the study of epithelial-mesenchymal transformation within the embryonic heart.

Smad7 can be induced by various transforming growth factor-beta superfamily ligands and negatively modulates their signaling, thus acting in a negative, autocrine feedback manner. Previous analyses have demonstrated that although Smad7 is widely expressed, it is predominantly found in the vascular endothelium. Because of the restricted spatiotemporal reporter expression driven via a novel 4.3 kb Smad7 promoter in endocardial cells overlying the hearts atrioventricular (AV) cushions; we hypothesized that a transgenic Cre line would prove useful for the analysis of endocardial cushion and valve formation. Here we describe a mouse line, Smad7(Cre), where Cre is robustly expressed within both cardiac outflow and AV endocardial cushions. Additionally, as endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme, we crossed the Smad7(Cre) mice to the ROSA26(eGFP-DTA) diphtheria toxin A-expressing mice in order to genetically ablate Smad7(Cre) expressing cells. Ablation of Smad7(Cre) cells resulted in embryonic lethality by E11.5 and largely acellular endocardial cushions.

Developmental origins of hypertrophic cardiomyopathy phenotypes: a unifying hypothesis

The majority of genetic mutations associated with hypertrophic cardiomyopathy (HCM) occur in genes encoding sarcomeric proteins, which are expressed only in cardiomyocytes. However, some manifestations of the HCM phenotype, such as myocardial disarray, interstitial fibrosis, mitral valve abnormalities, and microvascular remodeling, indicate the involvement of other cell lineages. The link between sarcomeric gene defects and these 'extended' HCM phenotypes remains elusive. Based on novel insights provided by cardiac developmental biology, we propose that a common lineage ancestry of the diverse HCM phenotypes not involving the cardiomyocyte can be traced to the pluripotent epicardium-derived cells (EPDCs). During cardiac colonization, EPDCs differentiate into interstitial fibroblasts, coronary smooth-muscle cells, and atrioventricular endocardial cushions as mesenchymal cells. We propose that the cross-talk between healthy EPDCs and abnormally contracting cardiomyocytes might account for the diverse manifestations of HCM, by a putative mechanism of mechanotransduction leading to abnormal gene expression and differentiation.

Timeline and distribution of melanocyte precursors in the mouse heart

Apart from the well-studied melanocytes of the skin, eye and inner ear, another population has recently been described in the heart. In this study, we tracked cardiac melanoblasts using in situ hybridization with a dopachrome tautomerase (Dct) probe and Dct-LacZ transgenic mice. Large numbers of melanoblasts were found in the atrioventricular (AV) endocardial cushions at embryonic day (E) 14.5 and persisted in the AV valves into adulthood. The earliest time Dct-LacZ-positive cells were observed in the AV endocardial cushions was E12.5. Prior to that, between E10.5 and E11.5, small numbers of melanoblasts traveled between the post-otic area and third somite along the anterior and common cardinal veins and branchial arch arteries with other neural crest cells expressing CRABPI. Cardiac melanocytes were not found in the spotting mutants Ednrb s-l/s-l and Kit w-v/w-v, while large numbers were observed in transgenic mice that overexpress endothelin 3. These results indicate that cardiac melanocytes depend on the same signaling molecules known to be required for proper skin melanocyte development and may originate from the same precursor population. Cardiac melanocytes were not found in zebrafish or frog but were present in quail suggesting an association between cardiac melanocytes and four-chambered hearts.

Shared gene expression profiles in developing heart valves and osteoblast progenitor cells

The atrioventricular (AV) valves of the heart develop from undifferentiated mesenchymal endocardial cushions, which later mature into stratified valves with diversified extracellular matrix (ECM). Because the mature valves express genes associated with osteogenesis and exhibit disease-associated calcification, we hypothesized the existence of shared regulatory pathways active in developing AV valves and in bone progenitor cells. To define gene regulatory programs of valvulogenesis relative to osteoblast progenitors, we undertook Affymetrix gene expression profiling analysis of murine embryonic day (E)12.5 AV endocardial cushions compared with E17.5 AV valves (mitral and tricuspid) and with preosteoblast MC3T3-E1 (subclone4) cells. Overall, MC3T3 cells were significantly more similar to E17.5 valves than to E12.5 cushions, supporting the hypothesis that valve maturation involves the expression of many genes also expressed in osteoblasts. Several transcription factors characteristic of mesenchymal and osteoblast precursor cells, including Twist1, are predominant in E12.5 cushion. Valve maturation is characterized by differential regulation of matrix metalloproteinases and their inhibitors as well as complex collagen gene expression. Among the most highly enriched genes during valvulogenesis were members of the small leucine-rich proteoglycan (SLRP) family including Asporin, a known negative regulator of osteoblast differentiation and mineralization. Together, these data support shared gene expression profiles of the developing valves and osteoblast bone precursor cells in normal valve development and homeostasis with potential functions in calcific valve disease.

Atrial Septal Defects in the Adult

Patients with isolated atrial septal defects (ASDs) have benefited from important recent advances in the diagnosis, evaluation, and management of their conditions. This review will focus on adolescent and adult patients with sizeable ASDs who do not have other major associated cardiac defects. There are 3 major types of ASDs or interatrial communications: ostium secundum, ostium primum, and sinus venosus (Figure 1A) defects. The ostium secundum is a true defect of the atrial septum and involves the region of the fossa ovalis. The ostium primum defect is within the spectrum of the atrioventricular (AV) septal defects (Figure 1B; also known as AV canal defects or endocardial cushion defects), the complete form of which also includes a large ventricular septal defect and a common AV valve. The sinus venosus defect is usually located at the junction of the right atrium and superior vena cava and is almost always associated with partial anomalous pulmonary venous return. Two very uncommon types of ASDs may be mentioned briefly: the inferior vena cava form of the sinus venosus defect and the coronary sinus septal defect (in which a defect between the coronary sinus and the left atrium allows a left-to-right shunt to occur through an “unroofed” coronary sinus). Figure 1. A, A indicates superior sinus venosus ASD; B, secundum ASD; C, inferior sinus venosus ASD; D, ostium primum ASD or partial AV septal defect; E, secundum ASD without posterior septal rim; F, coronary sinus ASD; and IVC, inferior vena cava. B, AV junction viewed from the atrial aspect. Normal indicates normal heart; complete AVSD, complete AV septal defect with a common AV junction and a common, single AV valve, guarded by 5 leaflets (counterclockwise: superior bridging, left mural, inferior bridging, right inferior, and right anterosuperior); partial AVSD, partial AVSD or primum ASD with …

A critical role for the EphA3 receptor tyrosine kinase in heart development

Eph proteins are receptor tyrosine kinases that control changes in cell shape and migration during development. We now describe a critical role for EphA3 receptor signaling in heart development as revealed by the phenotype of EphA3 null mice. During heart development mesenchymal outgrowths, the atrioventricular endocardial cushions, form in the atrioventricular canal. This morphogenetic event requires endocardial cushion cells to undergo an epithelial to mesenchymal transformation (EMT), and results in the formation of the atrioventricular valves and membranous portions of the atrial and ventricular septa. We show that EphA3 knockouts have significant defects in the development of their atrial septa and atrioventricular endocardial cushions, and that these cardiac abnormalities lead to the death of approximately 75% of homozygous EphA3−/− mutants. We demonstrate that EphA3 and its ligand, ephrin-A1, are expressed in adjacent cells in the developing endocardial cushions. We further demonstrate that EphA3−/− atrioventricular endocardial cushions are hypoplastic compared to wildtype and that EphA3−/− endocardial cushion explants give rise to fewer migrating mesenchymal cells than wildtype explants. Thus our results indicate that EphA3 plays a crucial role in the development and morphogenesis of the cells that give rise to the atrioventricular valves and septa.