Normal development of the heart: a review of new findings / Desarrollo normal del corazón: revisión de nuevos hallazgos

Abstract Development and formation of the heart, the central organ of the circulatory system in vertebrates, starts early during embryonic development (second week), reaching maturity during the first few postnatal months. Cardiogenesis is a highly complex process that requires the active and orderly participation of different cardiac and non-cardiac cell populations. Thus, this process is sensitive to errors that may trigger a variety of heart-development defects, called congenital heart defects, which have a worldwide incidence of 8-10/1000 live births. A good understanding of normal cardiogenesis is required for better diagnosis and treatment of congenital heart diseases. This article reviews normal cardiogenesis by comparing information from classic studies with more recent findings. Information from descriptive anatomical studies of histological sections and selective in vivo marking of chicken embryos were emphasized. In addition, the discovery of heart fields has fueled the investigation of cardiogenic events that were believed to be understood and has contributed to proposals for new models of heart development.

Resumen El corazón, órgano central del aparato circulatorio de los vertebrados, comienza a formarse muy temprano en el desarrollo embrionario (segunda semana de gestación) y alcanza su forma madura durante los primeros meses posteriores al nacimiento. La cardiogénesis se caracteriza por ser un proceso altamente complejo, dependiente de la participación activa y ordenada de diferentes poblaciones celulares cardiacas y no cardiacas. Lo anterior hace que este proceso sea sensible a errores que pueden desencadenar una variedad de defectos del desarrollo cardiaco, llamados cardiopatías congénitas, con una incidencia mundial de 8 a 10/1000 nacidos vivos. Para mejorar el diagnóstico y el tratamiento de las cardiopatías congénitas es necesario comprender adecuadamente los eventos implicados en la cardiogénesis normal. En este artículo se revisa el desarrollo cardiaco normal, contrastando la información de los estudios clásicos con la de hallazgos recientes. Se hace hincapié en la información obtenida de los estudios de anatomía descriptiva de cortes histológicos y marcaje selectivo in vivo en embriones de pollo. Adicionalmente, el descubrimiento de los campos cardiogénicos ha estimulado la investigación de eventos cardiogénicos que se creían comprendidos, contribuyendo con propuestas de nuevos modelos del desarrollo del corazón.

Efectos de la obesidad materna sobre el patrón de apoptosis en la cardiogénesis tardía / Effects of maternal obesity on the apoptosis pattern in late cardiogenesis / Efeitos da obsedidade materna sobre o padrão de apoptose na cardiogênese tardia

RESUMEN I: La ganancia excesiva de peso durante la gestación contribuye a un desbalance en el aporte de nutrientes que influyen en la reprogramación de vías moleculares de la organogénesis, aumentando el riesgo de desarrollar anomalías congénitas en el corazón. O: Determinar el efecto de la obesidad materna sobre el patrón de apoptosis en la cardiogénesis tardía. M: Se estableció un modelo de obesidad materna adquirida por dieta de cafetería tipo snack, en ratas Wistar adultas. Las ratas del grupo obeso y del grupo control se aparearon con machos adultos sanos y fueron sacrificadas el día 16.5 de gestación. Los corazones fueron evaluados macroscópicamente y procesados para el estudio de la apoptosis cardíaca mediante la técnica de TUNEL. R: El 25 % de los corazones procedentes de madres obesas presentaron aumento en el tamaño de atrios, ventrículos y tracto de salida. La apoptosis fue menor en los ventrículos de los corazones fetales procedentes de madres obesas vs control (p<0,05). En el atrio derecho la apoptosis fue mayor en los corazones fetales del grupo obeso vs control (p<0,05). De manera importante se observó un aumento significativo en la cantidad de células en apoptosis presentes en el tabique interventricular del grupo control vs el grupo obeso (p <0.000l). C: El proceso de apoptosis se encontró alterado en los corazones fetales que fueron expuestos a condiciones de obesidad materna.

ABSTRACT I: Excessive weight gain during pregnancy contributes to an imbalance in the contribution of nutrients that influences the reprogramming of molecular pathways of organogenesis, increasing the risk of developing congenital anomalies in the heart. O: To determine the effect of maternal obesity on the pattern of apoptosis in late cardiogenesis. M: A model of maternal obesity acquired by snack diet was established in adult Wistar rats. The rats of the obese group and the control group were mated with healthy adult males and were killed on day l6.5 of gestation. The hearts were evaluated macroscopically and processed for the study of cardiac apoptosis using the TUNEL technique. R: 25% of hearts from obese mothers had an increase in the size of atria, ventricles and outflow tract. Apoptosis was lower in the ventricles of fetal hearts from obese mothers vs control (p <0.05). In the right atrium, apoptosis was higher in the fetal hearts of the obese vs control group (p <0.05). Importantly, there was a significant increase in the number of cells in apoptosis present in the interventricular septum of the control group vs the obese group (p <0.000l). C: The process of apoptosis was found altered in fetal hearts that were exposed to conditions of maternal obesity.

RESUMO I: o ganho excessivo de peso durante a gestação contribui a um desequilibrio no aporte de nutrientes que influenciam a reprogramação das vias moleculares da organogênese, aumentando o risco de deselvolvimento de anomalias congênitas no coração. O: determinar o efeito da obesidade materna sobre o padrão de apoptose na cardiogênese tardia. M: estabeleceu-se um modelo de obesidade materna por meio de dieta de lanches tipo snack, em ratos Wistar adultos. Os ratos do grupo obeso e do grupo controle foram acasalados com machos adultos saudáveis, e sacrificados no dia l 6.5 da gestação. Os corações availaram-se macroscopicamente e processaram-se para o estudo da apoptose cardíaca utilizando a técnica do TUNEL. R: 25% dos corações de mães obesas apresentaram aumento do tamanho dos átrios, ventrículos e via de saida. A apoptose foi menor nos ventrículos dos corações fetais de mães obesas vs controle (p <0,05). No átrio direito, a apoptose foi maior nos corações fetais do grupo obeso vs controle (p <0,05). De uma maneira importante, houve um aumento significativo no número de células em apoptose presentes no septo interventricular do grupo controle vs o grupo obeso (p < 0,000l). C: o processo de apoptose encontrou-se alterado em corações fetais que foram expostos a condições de obesidade materna.

Role of kinase insert domain containing receptor (KDR) positive cells in formation of cardiospheric structure, myocardium and vessels / 中国胸心血管外科临床杂志

@#Objective    To investigate the role of kinase insert domain containing receptor (KDR) positive cells in the formation of cardiospheric structure, myocardium and vessels. Methods    Twenty-four Wistar rats weighting 250 g were selected. Cardiosphere-derived cells were isolated by enzymatic digestion of rat hearts, and their immunological phenotypes were analyzed by using fluorescence-activated cell sorting (FACS). The cardiomyogenic and vasculogenic potential was diagnosed by immunohistochemistry. Results    KDR positive cells grew exponentially and formed cell clusters. It also could generate myocardial precursor cells (cardiac troponin T positive). And these cells can develop spontaneous contraction activity in vitro. Meanwhile, KDR positive cells formed many vessel-like structures through a budding process. Conclusion    KDR positive cells form cardiospheric structure in vitro culture, and exhibit differentiation potential towards the cardiac and vascular cells. Therefore, KDR positive cells may have a broad prospect of clinical application as cell donors.

Gene locations may contribute to predicting gene regulatory relationships / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

We propose that locations of genes on chromosomes can contribute to the prediction of gene regulatory relationships. We constructed a time-based gene regulatory network of zebrafish cardiogenesis on the basis of a spatio-temporal neighborhood method. Through the network, specific regulatory pathways and order of gene expression during zebrafish cardiogenesis were obtained. By comparing the order with locations of these genes on chromosomes, we discovered that there exists a reversal phenomenon between the order and order of gene locations. The discovery provides an inherent rule to instruct exploration of gene regulatory relationships. Specifically, the discovery can help to predict if regulatory relationships between genes exist and contribute to evaluating the correctness of discovered gene regulatory relationships.

Thymosin β4 impeded murine stem cell proliferation with an intact cardiovascular differentiation / 华中科技大学学报（医学）（英德文版）

Thymosin β4 (Tβ4) is a key factor in cardiac development, growth, disease, epicardial integrity, blood vessel formation and has cardio-protective properties. However, its role in murine embryonic stem cells (mESCs) proliferation and cardiovascular differentiation remains unclear. Thus we aimed to elucidate the influence of Tβ4 on mESCs. Target genes during mESCs proliferation and differentiation were detected by real-time PCR or Western blotting, and patch clamp was applied to characterize the mESCs-derived cardiomyocytes. It was found that Tβ4 decreased mESCs proliferation in a partial dose-dependent manner and the expression of cell cycle regulatory genes c-myc, c-fos and c-jun. However, mESCs self-renewal markers Oct4 and Nanog were elevated, indicating the maintenance of self-renewal ability in these mESCs. Phosphorylation of STAT3 and Akt was inhibited by Tβ4 while the expression of RAS and phosphorylation of ERK were enhanced. No significant difference was found in BMP2/BMP4 or their downstream protein smad. Wnt3 and Wnt11 were remarkably decreased by Tβ4 with upregulation of Tcf3 and constant β-catenin. Under mESCs differentiation, Tβ4 treatment did not change the expression of cardiovascular cell markers α-MHC, PECAM, and α-SMA. Neither the electrophysiological properties of mESCs-derived cardiomyocytes nor the hormonal regulation by Iso/Cch was affected by Tβ4. In conclusion, Tβ4 suppressed mESCs proliferation by affecting the activity of STAT3, Akt, ERK and Wnt pathways. However, Tβ4 did not influence the in vitro cardiovascular differentiation.

Desarrollo temprano de las cavidades cardiacas ventriculares: Importancia de la Neuregulina 1 / Early development of cardiac ventricles: Importance of neuregulin 1

Introducción. Los defectos septales ventriculares graves, asociados con un ventrículo hipoplásico y otro hiperplásico o con la ausencia total del tabique interventricular, suelen ser incompatibles con la vida embrionaria y fetal. Pese a su gravedad, no se conocen sus causas. Por marcaje in vivo en embriones de pollo se confirmó la importancia de las trabéculas en la morfogénesis del tabique interventricular. Por manipulación genética en embriones de ratón y aves se determinó que la ausencia de la Neuregulina 1 (NRG1) o sus receptores, además de provocar escasa diferenciación de los miocitos ventriculares y deficiente formación de las trabéculas, determina la muerte prematura del embrión. Con base en estos antecedentes, el objetivo fue determinar el papel real de NRG1 en la trabeculogénesis temprana y su importancia en la regulación de la proliferación y apoptosis. Métodos. Se estableció un modelo de órgano cultivo de corazón de embrión de pollo previo al inicio de la trabeculogénesis. Se realizaron ensayos de inhibición total de la actividad de NRG1 endógena y posterior adición de la proteína exógena a diferentes concentraciones, determinando la actividad cíclica de los miocitos ventriculares con el antígeno nuclear de proliferación celular y la apoptosis con lisotraker. Resultados. El suero fetal bovino promueve la proliferación, pero impacta negativamente la trabeculogénesis. La adición de NRG1 a concentraciones bajas y períodos cortos de incubación no induce trabeculogénesis. En contraste, a concentraciones medias y períodos de cultivo no mayores a 24 horas, tiene un efecto positivo sobre este proceso. También promueve la proliferación y evita la apoptosis del miocardio ventricular. El incremento en la concentración de NRG1 posiblemente provoca un desbalance molecular que favorece la proliferación desordenada pero no la trabeculogénesis. Conclusiones. El entendimiento del papel de NRG1 en la trabeculogénesis aporta datos para conocer las redes moleculares involucradas también en el desarrollo del tabique interventricular, información indispensable para entender el origen de los defectos septales ventriculares graves.

Background. Serious ventricular septal defects associated with hypoplastic and hyperplasic ventricles or total absence of the interventricular septum (IVS) are usually incompatible with embryonic and fetal life. Despite the importance of these cardiac diseases, their causes are not yet known. Using in vivo labeling in the chick embryo, the importance of ventricular trabeculation was confirmed in IVS morphogenesis. Using knockout mice and retrovirus in birds, it was determined that lack of function of neuregulin 1 (NRG1) or their ErbB receptors not only causes deficient differentiation of ventricular myocytes and poor formation of trabeculae, but also determines premature death of the embryos. Based on this background, the aim of this work was to determine the actual role of NRG1 in early trabeculogenesis and its importance in proliferation and apoptosis regulation. Methods. An embryonic chicken heart organ culture system at the age prior to the beginning of the trabeculogenesis process was established. Endogenous activity of NRG1 was inhibited in the organ cultures that were then stimulated with NRG1 at different concentrations. Myocyte proliferation was determined using the proliferating cell nuclear antigen and apoptosis with LysoTracker (LTR). Results. Fetal bovine serum promotes proliferation but negatively impacts trabeculogenesis. Low concentration of NRG1 and short periods of incubation do not induce trabeculogenesis. In contrast, average NRG1 concentrations and cultivation periods not exceeding 24 h have a positive effect on the onset of this process. This also promotes myocardial proliferation but avoids apoptosis. Higher concentrations of NRG1 possibly cause a molecular imbalance that favors untidy proliferation but not trabeculogenesis. Conclusions. Understanding of the role of NRG1 on ventricular trabeculogenesis provides valuable information for the molecular pathways also involved in IVS development. This information is essential for understanding the origin of serious ventricular septal defects.

Expression of beta 2 integrin (CD18) in embryonic mouse and chicken heart

Integrins are heterodimeric receptors composed of á and â transmembrane subunits that mediate attachment of cells to the extracellular matrix and counter-ligands such as ICAM-1 on adjacent cells. â2 integrin (CD18) associates with four different á (CD11) subunits to form an integrin subfamily, which has been reported to be expressed exclusively on leukocytes. However, recent studies indicate that â2 integrin is also expressed by other types of cells. Since the gene for â2 integrin is located in the region of human chromosome 21 associated with congenital heart defects, we postulated that it may be expressed in the developing heart. Here, we show the results from several different techniques used to test this hypothesis. PCR analyses indicated that â2 integrin and the áL, áM, and áX subunits are expressed during heart development. Immunohistochemical studies in both embryonic mouse and chicken hearts, using antibodies directed against the N- or C-terminal of â2 integrin or against its á subunit partners, showed that â2 integrin, as well as the áL, áM, and áX subunits, are expressed by the endothelial and mesenchymal cells of the atrioventricular canal and in the epicardium and myocardium during cardiogenesis. In situ hybridization studies further confirmed the presence of â2 integrin in these various locations in the embryonic heart. These results indicate that the â2 integrin subfamily may have other activities in addition to leukocyte adhesion, such as modulating the migration and differentiation of cells during the morphogenesis of the cardiac valves and myocardial walls of the heart.