RESUMEN
The creation of a functional 3D bioprinted human heart remains challenging, largely due to the lack of some crucial cardiac cell types, including the atrioventricular canal (AVC) cardiomyocytes, which are essential to slow down the electrical impulse between the atrium and ventricle. By utilizing single-cell RNA sequencing analysis and a 3D bioprinting technology, we discover that stage-specific activation of canonical Wnt signaling creates functional AVC cardiomyocytes derived from human pluripotent stem cells. These cardiomyocytes display morphological characteristics and express molecular markers of AVC cardiomyocytes, including transcription factors TBX2 and MSX2. When bioprinted in prefabricated cardiac tissues, these cardiomyocytes successfully delay the electrical impulse, demonstrating their capability of functioning as the AVC cardiomyocytes in vitro. Thus, these findings not only identify canonical Wnt signaling as a key regulator of the AVC cardiomyocyte differentiation in vitro, but, more importantly, provide a critical cellular source for the biofabrication of a functional human heart.
Asunto(s)
Defectos de los Tabiques Cardíacos , Miocitos Cardíacos , Vía de Señalización Wnt , Humanos , Miocitos Cardíacos/metabolismo , Cojinetes Endocárdicos , Ventrículos Cardíacos , Diferenciación CelularRESUMEN
BACKGROUND: Essential patterning processes transform the heart tube into a compartmentalized organ with distinct chambers separated by an atrioventricular canal (AVC). This transition involves the refinement of expression of genes that are first found broadly throughout the heart tube and then become restricted to the AVC. Despite the importance of cardiac patterning, we do not fully understand the mechanisms that limit gene expression to the AVC. RESULTS: We show that the zebrafish gene smarcc1a, encoding a BAF chromatin remodeling complex subunit homologous to mammalian BAF155, is critical for cardiac patterning. In smarcc1a mutants, myocardial differentiation and heart tube assembly appear to proceed normally. Subsequently, the smarcc1a mutant heart fails to exhibit refinement of gene expression patterns to the AVC, and the persistence of broad gene expression is accompanied by failure of chamber expansion. In addition to their cardiac defects, smarcc1a mutants lack pectoral fins, indicating similarity to tbx5a mutants. However, comparison of smarcc1a and tbx5a mutants suggests that perturbation of tbx5a function is not sufficient to cause the smarcc1a mutant phenotype. CONCLUSIONS: Our data indicate an important role for Smarcc1a-containing chromatin remodeling complexes in regulating the changes in gene expression and morphology that distinguish the AVC from the cardiac chambers.
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Cojinetes Endocárdicos , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas de Pez Cebra/metabolismo , Corazón , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulación del Desarrollo de la Expresión Génica , Mamíferos/metabolismoRESUMEN
Endocardial cushion formation is essential for heart valve development and heart chamber separation. Abnormal endocardial cushion formation often causes congenital heart defects. ß-Catenin is known to be essential for endocardial cushion formation; however, the underlying cellular and molecular mechanisms remain incompletely understood. Here, we show that endothelial-specific deletion of ß-catenin in mice resulted in formation of hypoplastic endocardial cushions due to reduced cell proliferation and impaired cell migration. By using a ß-catenin DM allele in which the transcriptional function of ß-catenin is selectively disrupted, we further reveal that ß-catenin regulated cell proliferation and migration through its transcriptional and non-transcriptional function, respectively. At the molecular level, loss of ß-catenin resulted in increased expression of cell cycle inhibitor p21 in cushion endocardial and mesenchymal cells in vivo. In vitro rescue experiments with HUVECs and pig aortic valve interstitial cells confirmed that ß-catenin promoted cell proliferation by suppressing p21. In addition, one savvy negative observation is that ß-catenin was dispensable for endocardial-to-mesenchymal fate change. Taken together, our findings demonstrate that ß-catenin is essential for cell proliferation and migration but dispensable for endocardial cells to gain mesenchymal fate during endocardial cushion formation. Mechanistically, ß-catenin promotes cell proliferation by suppressing p21. These findings inform the potential role of ß-catenin in the etiology of congenital heart defects.
Asunto(s)
Cojinetes Endocárdicos , beta Catenina , Animales , Ratones , Porcinos , beta Catenina/genética , Proliferación Celular/genética , Alelos , Ciclo CelularRESUMEN
The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.
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Cojinetes Endocárdicos , Endocardio , Animales , Cojinetes Endocárdicos/metabolismo , Endocardio/metabolismo , Válvulas Cardíacas , Humanos , Mamíferos , Ratones , Morfogénesis , Miocardio/metabolismoRESUMEN
Background The pathogenesis of congenital heart disease (CHD) remains largely unknown, with only a small percentage explained solely by genetic causes. Modifiable environmental risk factors, such as alcohol, are suggested to play an important role in CHD pathogenesis. We sought to evaluate the association between prenatal alcohol exposure and CHD to gain insight into which components of cardiac development may be most vulnerable to the teratogenic effects of alcohol. Methods and Results This was a retrospective analysis of hospital discharge records from the California Office of Statewide Health Planning and Development and linked birth certificate records restricted to singleton, live-born infants from 2005 to 2017. Of the 5 820 961 births included, 16 953 had an alcohol-related International Classification of Diseases, Ninth and Tenth Revisions (ICD-9; ICD-10) code during pregnancy. Log linear regression was used to calculate risk ratios (RR) for CHD among individuals with an alcohol-related ICD-9 and ICD10 code during pregnancy versus those without. Three models were created: (1) unadjusted, (2) adjusted for maternal demographic factors, and (3) adjusted for maternal demographic factors and comorbidities. Maternal alcohol-related code was associated with an increased risk for CHD in all models (RR, 1.33 to 1.84); conotruncal (RR, 1.62 to 2.11) and endocardial cushion (RR, 2.71 to 3.59) defects were individually associated with elevated risk in all models. Conclusions Alcohol-related diagnostic codes in pregnancy were associated with an increased risk of an offspring with a CHD, with a particular risk for endocardial cushion and conotruncal defects. The mechanistic basis for this phenotypic enrichment requires further investigation.
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Cardiopatías Congénitas , Efectos Tardíos de la Exposición Prenatal , Cojinetes Endocárdicos , Femenino , Cardiopatías Congénitas/diagnóstico , Cardiopatías Congénitas/epidemiología , Cardiopatías Congénitas/etiología , Humanos , Lactante , Nacimiento Vivo , Embarazo , Efectos Tardíos de la Exposición Prenatal/epidemiología , Estudios Retrospectivos , Factores de RiesgoRESUMEN
OBJECTIVES: Endocardial cushions are precursors of the valve septum complex that separates the four heart chambers. Several genes have been implicated in the development of endocardial cushions. Specifically, ERp44 has been found to play a role in the early secretory pathway, but its function in heart development has not been well studied. MATERIALS AND METHODS: In this study, we established conditional and tissue-specific knockout mouse models. The morphology, survival rate, the development of heart and endocardial cushion were under evaluation. The relationship between ERp44 and VEGFA was investigated by transcriptome, qPCR, WB, immunofluorescence and immunohistochemistry. RESULTS: ERp44 knockout (KO) mice were smaller in size, and most mice died during early postnatal life. KO hearts exhibited the typical phenotypes of congenital heart diseases, such as abnormal heart shapes and severe septal and valvular defects. Similar phenotypes were found in cTNT-Cre+/- ; ERp44fl / fl mice, which indicated that myocardial ERp44 principally controls endocardial cushion formation. Further studies demonstrated that the deletion of ERp44 significantly decreased the proliferation of cushion cells and impaired the endocardial-mesenchymal transition (EndMT), which was followed by endocardial cushion dysplasia. Finally, we found that ERp44 was directly bound to VEGFA and controlled its release, further regulating EndMT. CONCLUSION: We demonstrated that ERp44 plays a specific role in heart development. ERp44 contributes to the development of the endocardial cushion by affecting VEGFA-mediated EndMT.
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Cojinetes Endocárdicos/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas de la Membrana/genética , Chaperonas Moleculares/genética , Miocardio/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Proliferación Celular/genética , Proliferación Celular/fisiología , Cardiopatías Congénitas/genética , Proteínas de la Membrana/metabolismo , Mesodermo/metabolismo , Ratones Noqueados , Chaperonas Moleculares/metabolismo , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
ERBB family members and their ligands play an essential role in embryonic heart development and adult heart physiology. Among them, ERBB3 is a binding partner of ERBB2; the ERBB2/3 complex mediates downstream signaling for cell proliferation. ERBB3 has seven consensus binding sites to the p85 regulatory subunit of PI3K, which activates the downstream AKT pathway, leading to the proliferation of various cells. This study generated a human ERBB3 knock-in mouse expressing a mutant ERBB3 whose seven YXXM p85 binding sites were replaced with YXXA. Erbb3 knock-in embryos exhibited lethality between E12.5 to E13.5, and showed a decrease in mesenchymal cell numbers and density in AV cushions. We determined that the proliferation of mesenchymal cells in the atrioventricular (AV) cushion in Erbb3 knock-in mutant embryos was temporarily reduced due to the decrease of AKT and ERK1/2 phosphorylation. Overall, our results suggest that AKT/ERK activation by the ERBB3-dependent PI3K signaling is crucial for AV cushion morphogenesis during embryonic heart development.
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Defectos de la Almohadilla Endocárdica/genética , Cojinetes Endocárdicos/metabolismo , Receptor ErbB-3/metabolismo , Animales , Sitios de Unión , Células CHO , Cricetinae , Cricetulus , Defectos de la Almohadilla Endocárdica/metabolismo , Cojinetes Endocárdicos/embriología , Humanos , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor ErbB-3/química , Receptor ErbB-3/genética , Transducción de SeñalRESUMEN
The etiology of ethanol-related congenital heart defects has been the focus of much study, but most research has concentrated on cellular and molecular mechanisms. We have shown with optical coherence tomography (OCT) that ethanol exposure led to increased retrograde flow and smaller atrioventricular (AV) cushions compared with controls. Since AV cushions play a role in patterning the conduction delay at the atrioventricular junction (AVJ), this study aims to investigate whether ethanol exposure alters the AVJ conduction in early looping hearts and whether this alteration is related to the decreased cushion size. Quail embryos were exposed to a single dose of ethanol at gastrulation, and Hamburger-Hamilton stage 19-20 hearts were dissected for imaging. Cardiac conduction was measured using an optical mapping microscope and we imaged the endocardial cushions using OCT. Our results showed that, compared with controls, ethanol-exposed embryos exhibited abnormally fast AVJ conduction and reduced cushion size. However, this increased conduction velocity (CV) did not strictly correlate with decreased cushion volume and thickness. By matching the CV map to the cushion-size map along the inflow heart tube, we found that the slowest conduction location was consistently at the atrial side of the AVJ, which had the thinner cushions, not at the thickest cushion location at the ventricular side as expected. Our findings reveal regional differences in the AVJ myocardium even at this early stage in heart development. These findings reveal the early steps leading to the heterogeneity and complexity of conduction at the mature AVJ, a site where arrhythmias can be initiated.NEW & NOTEWORTHY To the best of our knowledge, this is the first study investigating the impact of ethanol exposure on the early cardiac conduction system. Our results showed that ethanol-exposed embryos exhibited abnormally fast atrioventricular conduction. In addition, our findings, in CV measurements and endocardial cushion thickness, reveal regional differences in the AVJ myocardium even at this early stage in heart development, suggesting that the differentiation and maturation at this site are complex and warrant further studies.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Cojinetes Endocárdicos/efectos de los fármacos , Etanol/farmacología , Sistema de Conducción Cardíaco/efectos de los fármacos , Animales , Embrión no Mamífero , Cojinetes Endocárdicos/diagnóstico por imagen , Cojinetes Endocárdicos/embriología , Gastrulación , Corazón/diagnóstico por imagen , Corazón/efectos de los fármacos , Corazón/embriología , Sistema de Conducción Cardíaco/diagnóstico por imagen , Sistema de Conducción Cardíaco/embriología , Codorniz , Tomografía de Coherencia Óptica , Imagen de Colorante Sensible al VoltajeRESUMEN
Smoothened is a key receptor of the hedgehog pathway, but the roles of Smoothened in cardiac development remain incompletely understood. In this study, we found that the conditional knockout of Smoothened from the mesoderm impaired the development of the venous pole of the heart and resulted in hypoplasia of the atrium/inflow tract (IFT) and a low heart rate. The blockage of Smoothened led to reduced expression of genes critical for sinoatrial node (SAN) development in the IFT. In a cardiac cell culture model, we identified a Gli2-Tbx5-Hcn4 pathway that controls SAN development. In the mutant embryos, the endocardial-to-mesenchymal transition (EndMT) in the atrioventricular cushion failed, and Bmp signalling was downregulated. The addition of Bmp2 rescued the EndMT in mutant explant cultures. Furthermore, we analysed Gli2+ scRNAseq and Tbx5-/- RNAseq data and explored the potential genes downstream of hedgehog signalling in posterior second heart field derivatives. In conclusion, our study reveals that Smoothened-mediated hedgehog signalling controls posterior cardiac progenitor commitment, which suggests that the mutation of Smoothened might be involved in the aetiology of congenital heart diseases related to the cardiac conduction system and heart valves.
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Cojinetes Endocárdicos/embriología , Cojinetes Endocárdicos/metabolismo , Proteínas Hedgehog/metabolismo , Organogénesis , Transducción de Señal , Nodo Sinoatrial/embriología , Nodo Sinoatrial/metabolismo , Animales , Proteína Morfogenética Ósea 2/genética , Proteína Morfogenética Ósea 2/metabolismo , Biología Computacional/métodos , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ontología de Genes , Inmunohistoquímica , Ratones , Ratones Noqueados , Ratones Transgénicos , Receptor Smoothened/genética , Receptor Smoothened/metabolismoRESUMEN
Cushion tissues, the primordia of valves and septa of the adult heart, are formed in the atrioventricular (AV) and outflow tract (OFT) regions of the embryonic heart. The cushion tissues are generated by the endothelial-mesenchymal transition (EMT), involving many soluble factors, extracellular matrix, and transcription factors. Moreover, neural crest-derived mesenchymal cells also migrate into the OFT cushion. The transcription factor Msx1 is known to be expressed in the endothelial and mesenchymal cells during cushion tissue formation. However, its exact role in EMT during cushion tissue formation is still unknown. In this study, we investigated the expression patterns of Msx1 mRNA and protein during chick heart development. Msx1 mRNA was localized in endothelial cells of the AV region at Stage 14, and its protein was first detected at Stage 15. Thereafter, Msx1 mRNA and protein were observed in the endothelial and mesenchymal cells of the OFT and AV regions. in vitro assays showed that ectopic Msx1 expression in endothelial cells induced p27, a cell-cycle inhibitor, expression and inhibited fibroblast growth factor 4 (FGF4)-induced cell proliferation. Although the FGF signal reduced the EMT-inducing activities of transforming growth factor ß (TGFß), ectopic Msx1 expression in endothelial cells enhanced TGFß signaling-induced αSMA, an EMT marker, expression. These results suggest that Msx1 may support the transformation of endothelial cells due to a TGFß signal in EMT during cushion tissue formation.
Asunto(s)
Proliferación Celular/fisiología , Cojinetes Endocárdicos/embriología , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Factor de Transcripción MSX1/metabolismo , Miocardio/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Animales , Embrión de Pollo , Cojinetes Endocárdicos/metabolismo , Factor de Transcripción MSX1/genética , Antígeno Nuclear de Célula en Proliferación/genética , Transducción de Señal/fisiologíaRESUMEN
NADPH oxidases (NOX) are a major source of reactive oxygen species (ROS) production in the heart. ROS signaling regulates gene expression, cell proliferation, apoptosis, and migration. However, the role of NOX2 in embryonic heart development remains elusive. We hypothesized that deficiency of Nox2 disrupts endocardial to mesenchymal transition (EndMT) and results in congenital septal and valvular defects. Our data show that 34% of Nox2-/- neonatal mice had various congenital heart defects (CHDs) including atrial septal defects (ASD), ventricular septal defects (VSD), atrioventricular canal defects (AVCD), and malformation of atrioventricular and aortic valves. Notably, Nox2-/- embryonic hearts show abnormal development of the endocardial cushion as evidenced by decreased cell proliferation and an increased rate of apoptosis. Additionally, Nox2 deficiency disrupted EndMT of atrioventricular cushion explants ex vivo. Furthermore, treatment with N-acetylcysteine (NAC) to reduce ROS levels in the wild-type endocardial cushion explants decreased the number of cells undergoing EndMT. Importantly, deficiency of Nox2 was associated with reduced expression of Gata4, Tgfß2, Bmp2, Bmp4, and Snail1, which are critical to endocardial cushion and valvoseptal development. We conclude that NOX2 is critical to EndMT, endocardial cushion cell proliferation, and normal embryonic heart development.
Asunto(s)
Transición Epitelial-Mesenquimal/fisiología , Cardiopatías Congénitas/patología , Corazón/embriología , NADPH Oxidasa 2/metabolismo , Animales , Apoptosis , Proliferación Celular , Cojinetes Endocárdicos/embriología , Cojinetes Endocárdicos/metabolismo , Cojinetes Endocárdicos/patología , Transición Epitelial-Mesenquimal/genética , Regulación del Desarrollo de la Expresión Génica , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Ratones , NADPH Oxidasa 2/deficiencia , NADPH Oxidasa 2/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Endocardial cells are specialized endothelial cells that, during embryogenesis, form a lining on the inside of the developing heart, which is maintained throughout life. Endocardial cells are an essential source for several lineages of the cardiovascular system including coronary endothelium, endocardial cushion mesenchyme, cardiomyocytes, mural cells, fibroblasts, liver vasculature, adipocytes, and hematopoietic cells. Alterations in the differentiation programs that give rise to these lineages has detrimental effects, including premature lethality or significant structural malformations present at birth. Here, we will review the literature pertaining to the contribution of endocardial cells to valvular, and nonvalvular lineages and highlight critical pathways required for these processes. The lineage differentiation potential of embryonic, and possibly adult, endocardial cells has therapeutic potential in the regeneration of damaged cardiac tissue or treatment of cardiovascular diseases.
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Endocardio/embriología , Válvulas Cardíacas/embriología , Miocardio/citología , Animales , Desarrollo Embrionario , Cojinetes Endocárdicos/embriología , Válvulas Cardíacas/metabolismo , Humanos , Transducción de SeñalRESUMEN
Atrioventricular valve development requires endothelial-to-mesenchymal transition (EndMT) that induces cushion endocardial cells to give rise to mesenchymal cells crucial to valve formation. In the adult endothelium, deletion of the docking protein FRS2α induces EndMT by activating TGFß signaling in a miRNA let-7-dependent manner. To study the role of endothelial FRS2α during embryonic development, we generated mice with an inducible endothelial-specific deletion of Frs2α (FRS2αiECKO). Analysis of the FRS2αiECKO embryos uncovered a combination of impaired EndMT in AV cushions and defective maturation of AV valves leading to development of thickened, abnormal valves when Frs2α was deleted early (E7.5) in development. At the same time, no AV valve developmental abnormalities were observed after late (E10.5) deletion. These observations identify FRS2α as a pivotal controller of cell fate transition during both EndMT and post-EndMT valvulogenesis.
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Cojinetes Endocárdicos/embriología , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/fisiología , Animales , Recuento de Células , Linaje de la Célula , Defectos de la Almohadilla Endocárdica/embriología , Defectos de la Almohadilla Endocárdica/genética , Cojinetes Endocárdicos/citología , Cojinetes Endocárdicos/patología , Células Endoteliales/citología , Eliminación de Gen , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Mesodermo/citología , Mesodermo/embriología , Ratones , Ratones Endogámicos C57BL , MicroARNs/fisiología , Válvula Mitral/anomalías , Válvula Mitral/embriología , Morfogénesis/genética , Fenotipo , Válvula Tricúspide/anomalías , Válvula Tricúspide/embriologíaRESUMEN
Congenital heart defects can decrease the quality of life and life expectancy in affected individuals, and constitute a major burden for the health care systems. Endocardial cushion defects are among the most prevalent heart malformations in the general population, and are extremely frequent (approximately a 100-fold higher prevalence) in children with Down syndrome. Several genes have been proposed to be involved in the pathogenesis of these malformations, but no common pathogenic DNA variants have been identified so far. Here, we focussed on constitutive, epigenetic alterations of function of selected genes, potentially important for endocardial cushion development. We used two types of microarrays, dedicated for assessment of gene promoter methylation and whole genome expression. First, we compared the gene promoter methylation profiles between two groups of Down syndrome patients, with and without heart defects of endocardial cushion-type. Then, to determine the functional role of the detected methylation alterations, we assessed the expression of the genes of interest. We detected significant hypermethylation of the NRG1 gene promoter region in children with heart defects. NRG1 is a key factor in maturation of endocardial cushions. Supplementary gene expression assessment revealed significantly decreased activity of the ERBB3, SHC3 and SHC4 genes in children with heart defects. The above three genes are closely related to the NRG1 gene and are crucial elements of the NRG/ErbB pathway. The results of this pilot study show that hypermethylation of the NRG1 gene promoter can reflect the functional genome alteration contributing to development of congenital heart defects of endocardial cushion-type.
Asunto(s)
Metilación de ADN , Síndrome de Down/genética , Predisposición Genética a la Enfermedad/genética , Cardiopatías Congénitas/genética , Neurregulina-1/genética , Adolescente , Niño , Cojinetes Endocárdicos/crecimiento & desarrollo , Epigenómica , Receptores ErbB/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Neurregulinas/metabolismo , Proyectos Piloto , Receptor ErbB-3 , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 3 que Contiene Dominios de Homología 2 de SrcRESUMEN
Heart formation involves a complex series of tissue rearrangements, during which regions of the developing organ expand, bend, converge, and protrude in order to create the specific shapes of important cardiac components. Much of this morphogenesis takes place while cardiac function is underway, with blood flowing through the rapidly contracting chambers. Fluid forces are therefore likely to influence the regulation of cardiac morphogenesis, but it is not yet clear how these biomechanical cues direct specific cellular behaviors. In recent years, the optical accessibility and genetic amenability of zebrafish embryos have facilitated unique opportunities to integrate the analysis of flow parameters with the molecular and cellular dynamics underlying cardiogenesis. Consequently, we are making progress toward a comprehensive view of the biomechanical regulation of cardiac chamber emergence, atrioventricular canal differentiation, and ventricular trabeculation. In this review, we highlight a series of studies in zebrafish that have provided new insight into how cardiac function can shape cardiac morphology, with a particular focus on how hemodynamics can impact cardiac cell behavior. Over the long-term, this knowledge will undoubtedly guide our consideration of the potential causes of congenital heart disease.
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Líquidos Corporales/fisiología , Corazón/embriología , Corazón/fisiología , Morfogénesis , Pez Cebra/embriología , Animales , Fenómenos Biomecánicos , Diferenciación Celular/genética , Cojinetes Endocárdicos/citología , Cojinetes Endocárdicos/embriología , Cojinetes Endocárdicos/metabolismo , Endocardio/citología , Endocardio/embriología , Endocardio/metabolismo , Regulación del Desarrollo de la Expresión Génica , Corazón/anatomía & histología , Pez Cebra/genéticaRESUMEN
The bicuspid aortic valve (BAV), a valve with two instead of three aortic leaflets, belongs to the most prevalent congenital heart diseases in the world, occurring in 0.5-2% of the general population. We aimed to understand how changes in early cellular contributions result in BAV formation and impact cardiovascular outflow tract development. Detailed 3D reconstructions, immunohistochemistry and morphometrics determined that, during valvulogenesis, the non-coronary leaflet separates from the parietal outflow tract cushion instead of originating from an intercalated cushion. Nos3-/- mice develop a BAV without a raphe as a result of incomplete separation of the parietal outflow tract cushion into the right and non-coronary leaflet. Genetic lineage tracing of endothelial, second heart field and neural crest cells revealed altered deposition of neural crest cells and second heart field cells within the parietal outflow tract cushion of Nos3-/- embryos. The abnormal cell lineage distributions also affected the positioning of the aortic and pulmonary valves at the orifice level. The results demonstrate that the development of the right and non-coronary leaflets are closely related. A small deviation in the distribution of neural crest and second heart field populations affects normal valve formation and results in the predominant right-non-type BAV in Nos3-/- mice.
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Válvula Aórtica/anomalías , Linaje de la Célula , Enfermedades de las Válvulas Cardíacas/embriología , Mutación/genética , Cresta Neural/patología , Óxido Nítrico Sintasa de Tipo III/genética , Animales , Aorta/metabolismo , Válvula Aórtica/embriología , Enfermedad de la Válvula Aórtica Bicúspide , Embrión de Mamíferos/metabolismo , Cojinetes Endocárdicos/metabolismo , Ratones Endogámicos C57BL , Miocardio/metabolismo , Cresta Neural/metabolismo , Óxido Nítrico Sintasa de Tipo III/deficienciaRESUMEN
Abnormal endocardial cushion formation is a major cause of congenital heart valve disease, which is a common birth defect with significant morbidity and mortality. Although ß-catenin and BMP2 are two well-known regulators of endocardial cushion formation, their interaction in this process is largely unknown. Here, we report that deletion of ß-catenin in myocardium results in formation of hypoplastic endocardial cushions accompanying a decrease of mesenchymal cell proliferation. Loss of ß-catenin reduced Bmp2 expression in myocardium and SMAD signaling in cushion mesenchyme. Exogenous BMP2 recombinant proteins fully rescued the proliferation defect of mesenchymal cells in cultured heart explants from myocardial ß-catenin knockout embryos. Using a canonical WNT signaling reporter mouse line, we showed that cushion myocardium exhibited high WNT/ß-catenin activities during endocardial cushion growth. Selective disruption of the signaling function of ß-catenin resulted in a cushion growth defect similar to that caused by the complete loss of ß-catenin. Together, these observations demonstrate that myocardial ß-catenin signaling function promotes mesenchymal cell proliferation and endocardial cushion expansion through inducing BMP signaling.
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Proteína Morfogenética Ósea 2/metabolismo , Cojinetes Endocárdicos/metabolismo , Miocardio/metabolismo , Organogénesis , Transducción de Señal , beta Catenina/metabolismo , Animales , Proliferación Celular , Cojinetes Endocárdicos/embriología , Endocardio/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Ratones , Modelos Biológicos , Comunicación Paracrina , Ratas , Vía de Señalización WntRESUMEN
Deletions 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.
Asunto(s)
Proteínas Portadoras/metabolismo , Factor de Transcripción GATA4/genética , Regulación del Desarrollo de la Expresión Génica , Defectos del Tabique Interventricular/embriología , Defectos del Tabique Interventricular/genética , Proteínas del Tejido Nervioso/deficiencia , Proteínas Represoras/deficiencia , Alelos , Animales , Proliferación Celular , Embrión de Mamíferos/metabolismo , Cojinetes Endocárdicos/embriología , Cojinetes Endocárdicos/metabolismo , Cojinetes Endocárdicos/patología , Endocardio/embriología , Endocardio/metabolismo , Endocardio/patología , Transición Epitelial-Mesenquimal/genética , Factor de Transcripción GATA4/metabolismo , Mesodermo/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células 3T3 NIH , Proteínas del Tejido Nervioso/genética , Proteínas Represoras/genéticaRESUMEN
BACKGROUND: The origin of the intercalated cushions that develop into the anterior cusp of the pulmonary valve (PV) and the noncoronary cusp of the aortic valve (AV) is not well understood. RESULTS: Cre transgenes in combination with the Rosa TdTomato-EGFP reporter were used to generate three-dimensional lineage mapping of AV and PV cusps during intercalated cushion development. Tie2-Cre;EGFP was used to mark endothelial-derived mesenchymal cells, Wnt1-Cre;EGFP for cardiac neural crest and cardiac Troponin T (Tnnt2)Cre;EGFP, for myocardial lineage. The highest percentage of intercalated cushion cells at embryonic day (E) 12.5 was Tnnt2-Cre; EGFP positive; 68.0% for the PV and 50.0% AV. Neither Tnnt2 mRNA nor Tnnt2-Cre protein was expressed in the intercalated cushions; and the Tnnt2-Cre lineage intercalated cushion cells were also positive for the mesenchymal markers Sox9 and versican. Tnnt2-Cre lineage was present within the forming intercalated cushions from E11.5 and was present in the intercalated cushion derived PV and AV cusps and localized to the fibrosa layer at postnatal day 0. CONCLUSIONS: Intercalated cushions of the developing outflow tract are populated with Tnnt2-Cre derived cells, a Cre reporter previously used for tracing and excision of myocardial cells and not previously associated with mesenchymal cells. Developmental Dynamics 247:1005-1017, 2018. © 2018 Wiley Periodicals, Inc.