Heart Tube Formation Research Articles

Mef2c factors are required for early but not late addition of cardiomyocytes to the ventricle

During heart formation, the heart grows and undergoes dramatic morphogenesis to achieve efficient embryonic function. Both in fish and amniotes, much of the growth occurring after initial heart tube formation arises from second heart field (SHF)-derived progenitor cell addition to the arterial pole, allowing chamber formation. In zebrafish, this process has been extensively studied during embryonic life, but it is unclear how larval cardiac growth occurs beyond 3 days post-fertilisation (dpf). By quantifying zebrafish myocardial growth using live imaging of GFP-labelled myocardium we show that the heart grows extensively between 3 and 5 dpf. Using methods to assess cell division, cellular development timing assay and Kaede photoconversion, we demonstrate that proliferation, CM addition, and hypertrophy contribute to ventricle growth. Mechanistically, we show that reduction in Mef2c activity (mef2ca+/−;mef2cb−/−), downstream or in parallel with Nkx2.5 and upstream of Ltbp3, prevents some CM addition and differentiation, resulting in a significantly smaller ventricle by 3 dpf. After 3 dpf, however, CM addition in mef2ca+/−;mef2cb−/− mutants recovers to a normal pace, and the heart size gap between mutants and their siblings diminishes into adulthood. Thus, as in mice, there is an early time window when SHF contribution to the myocardium is particularly sensitive to loss of Mef2c activity.

Retinoic acid signaling in heart development.

Retinoic acid (RA) is a vitamin A metabolite that acts as a morphogen and teratogen. Excess or defective RA signaling causes developmental defects including in the heart. The heart develops from the anterior lateral plate mesoderm. Cardiogenesis involves successive steps, including formation of the primitive heart tube, cardiac looping, septation, chamber development, coronary vascularization, and completion of the four-chambered heart. RA is dispensable for primitive heart tube formation. Before looping, RA is required to define the anterior/posterior boundaries of the heart-forming mesoderm as well as to form the atrium and sinus venosus. In outflow tract elongation and septation, RA signaling is required to maintain/differentiate cardiogenic progenitors in the second heart field at the posterior pharyngeal arches level. Epicardium-secreted insulin-like growth factor, the expression of which is regulated by hepatic mesoderm-derived erythropoietin under the control of RA, promotes myocardial proliferation of the ventricular wall. Epicardium-derived RA induces the expression of angiogenic factors in the myocardium to form the coronary vasculature. In cardiogenic events at different stages, properly controlled RA signaling is required to establish the functional heart.

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence.

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

Excess VEGF‐A disrupts tubular heart formation and cardiac blood flow dynamics

Vascular Endothelial Growth Factor (VEGF) plays a critical role during heart development. Clinical evidence shows that maternal conditions that modify VEGF signaling in utero, such as high altitude hypoxia and maternal diabetes, are correlated with an increased risk of congenital heart defects. While VEGF is essential for vasculogenesis and angiogenesis, its role during early heart tube formation is not fully understood. During early embryogenesis, paired endocardial tubes fuse into a single linear heart tube, which ultimately transforms into a four‐chambered heart. To better understand the role of VEGF in heart tube fusion, we studied how an early transient dysregulation of VEGF, produced by adding exogenous VEGF‐A right before tube fusion, affects heart tube formation and the onset of blood flow in the heart, leading to congenital heart defects.To enable longitudinal studies of heart development, and determine effects on early and late cardiac formation events, we developed an in ovo quail model of early transient VEGF dysregulation. We chose quail due to ease of access and monitoring during development. We applied recombinant human VEGF165 (rhVEGF165; VEGF‐A isoform) to quail embryos in ovo at Hamburger‐Hamilton (HH) stage 8 (~29 hours incubation) just before heart tube fusion (500ng in a 5μL drop on top of the embryo). Control embryos (vehicle applied at HH8) were also prepared. Eggs were then resealed and re‐incubated for an additional 13hrs (approximately HH11, early tubular heart); 22hrs (around HH14, early beating tubular heart); or 10 days (HH38, fully formed heart); to study how a short, transient, early exposure to excess VEGF‐A impacts cardiac formation.We found both early and late cardiac deficiencies after transient VEGF exposure at HH8. Around HH11, the newly formed tubular heart presented three distinct phenotypes when exposed to early VEGF excess: 1. Wider and shorter than normal heart tubes; 2. Heart tubes with multiple ridges (abnormal folds and protrusions); and 3. Lack of heart tube. Total number of malformed hearts accounted for 70% (n = 21/30) in VEGF‐treated embryos versus 27% (n = 6/22) in controls. Around HH14, when the heart is beating, we measured blood flow and cardiac wall mechanics. We found that heart tubes that were significantly wider than normal (~30%) after VEGF‐treatment, had about 30% increase in maximum flow velocity (n = 3), and their heart tubes failed to completely close the lumen to avoid backflow. When the heart was fully formed, at HH38, microCT images revealed four malformation phenotypes: 1. Ventricular septal defect (VSD); 2. Right ventricular hypoplasia; 3. Hearts that were both shorter and wider than normal; and 4. Aortic arch anomalies. Total number of malformed hearts accounted for 28% (n = 9/32) of surviving VEGF‐treated embryos; and 14% (4/28) surviving control embryos. Our data highlight that VEGF‐A is involved in shaping the geometrical characteristics of the early heart tube, and thus the onset of blood flow dynamics, together affecting mature heart formation.Support or Funding InformationNIH R01 HL094570; AHA 16GRNT29840002; OHSU Presidential Bridge Fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Understanding Cardiac Tube Formation in Developing Drosophila Embryos using Light Sheet Microscopy and Cardiac Drug Screening

Heart development begins with the formation of a primitive tube, which is mediated by coordinated cell movements. In Drosophila, the heart is formed from 52 bi-lateral pairs of cardiac precursors that migrate dorsally and medially to join their counterparts. The cells must undergo distinct morphological changes in order to form a lumen. While the genetic pathways that induce cardiac cell specification have been clearly defined, the cellular and molecular mechanisms that regulate collective cell migration during heart tube formation are not well understood. Leveraging the simplicity and pharmacological tractability of Drosophila melanogaster and the ability to perform live imaging of its embryos, we have developed a light-sheet microscopy platform and quantitative image analysis to characterize cell behaviours and molecular rearrangements during heart tube formation in living Drosophila embryos. Automated image analysis allows quantitative comparison of the dynamics of tube formation across embryos. To identify the pathways that regulate collective cell movements during heart development, we are conducting a pharmacological screen for inhibitors of cardiac precursor migration. We are particularly interested in the role of the cytoskeleton as both actin and myosin are important for cell movements in heart development. The kinase Rho-kinase (Rok) phosphorylates and activates the myosin light chain, and thus inhibiting Rho-kinase results in impaired myosin contractility. Preliminary experiments suggest that Rok may be important for the coordinated movement of cardioblasts during Drosophila heart morphogenesis. Embryos injected with water (controls) developed normally but those injected with 10 mM Y-27632, a Rok inhibitor had disrupted coordination of cardioblasts, leading to defects in heart tube formation. Together, our novel tools will allow us to identify pathways critical for cardiac precursor migration, polarization, and cell-cell adhesion.

The heart tube forms and elongates through dynamic cell rearrangement coordinated with foregut extension.

In the initiation of cardiogenesis, the heart primordia transform from bilateral flat sheets of mesoderm into an elongated midline tube. Here, we discover that this rapid architectural change is driven by actomyosin-based oriented cell rearrangement and resulting dynamic tissue reshaping (convergent extension, CE). By labeling clusters of cells spanning the entire heart primordia, we show that the heart primordia converge toward the midline to form a narrow tube, while extending perpendicularly to rapidly lengthen it. Our data for the first time visualize the process of early heart tube formation from both the medial (second) and lateral (first) heart fields, revealing that both fields form the early heart tube by essentially the same mechanism. Additionally, the adjacent endoderm coordinately forms the foregut through previously unrecognized movements that parallel those of the heart mesoderm and elongates by CE. In conclusion, our data illustrate how initially two-dimensional flat primordia rapidly change their shapes and construct the three-dimensional morphology of emerging organs in coordination with neighboring morphogenesis.

Mutations in Hnrnpa1 cause congenital heart defects.

Incomplete penetrance of congenital heart defects (CHDs) was observed in a mouse model. We hypothesized that the contribution of a major genetic locus modulates the manifestation of the CHDs. After genome-wide linkage mapping, fine mapping, and high-throughput targeted sequencing, a recessive frameshift mutation of the heterogeneous nuclear ribonucleoprotein A1 (Hnrnpa1) gene was confirmed (Hnrnpa1ct). Hnrnpa1 was expressed in both the first heart field (FHF) and second heart field (SHF) at the cardiac crescent stage but was only maintained in SHF progenitors after heart tube formation. Hnrnpa1ct/ct homozygous mutants displayed complete CHD penetrance, including truncated and incomplete looped heart tube at E9.5, ventricular septal defect (VSD) and persistent truncus arteriosus (PTA) at E13.5, and VSD and double outlet right ventricle at P0. Impaired development of the dorsal mesocardium and sinoatrial node progenitors was also observed. Loss of Hnrnpa1 expression leads to dysregulation of cardiac transcription networks and multiple signaling pathways, including BMP, FGF, and Notch in the SHF. Finally, two rare heterozygous mutations of HNRNPA1 were detected in human CHDs. These findings suggest a role of Hnrnpa1 in embryonic heart development in mice and humans.

Hey2 regulates the size of the cardiac progenitor pool during vertebrate heart development.

A key event in heart development is the timely addition of cardiac progenitor cells, defects in which can lead to congenital heart defects. However, how the balance and proportion of progenitor proliferation versus addition to the heart is regulated remains poorly understood. Here, we demonstrate that Hey2 functions to regulate the dynamics of cardiac progenitor addition to the zebrafish heart. We found that the previously noted increase in myocardial cell number found in the absence of Hey2 function was due to a pronounced expansion in the size of the cardiac progenitor pool. Expression analysis and lineage tracing of hey2-expressing cells showed that hey2 is active in cardiac progenitors. Hey2 acted to limit proliferation of cardiac progenitors, prior to heart tube formation. Use of a transplantation approach demonstrated a likely cell-autonomous (in cardiac progenitors) function for Hey2. Taken together, our data suggest a previously unappreciated role for Hey2 in controlling the proliferative capacity of cardiac progenitors, affecting the subsequent contribution of late-differentiating cardiac progenitors to the developing vertebrate heart.

Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis.

During vertebrate heart development, two progenitor populations, first and second heart fields (FHF, SHF), sequentially contribute to longitudinal subdivisions of the heart tube (HT), with the FHF contributing the left ventricle and part of the atria, and the SHF the rest of the heart. Here, we study the dynamics of cardiac differentiation and morphogenesis by tracking individual cells in live analysis of mouse embryos. We report that during an initial phase, FHF precursors differentiate rapidly to form a cardiac crescent, while limited morphogenesis takes place. In a second phase, no differentiation occurs while extensive morphogenesis, including splanchnic mesoderm sliding over the endoderm, results in HT formation. In a third phase, cardiac precursor differentiation resumes and contributes to SHF-derived regions and the dorsal closure of the HT. These results reveal tissue-level coordination between morphogenesis and differentiation during HT formation and provide a new framework to understand heart development.

Mapping Heart Development in Flies: Src42A Acts Non-Autonomously to Promote Heart Tube Formation in Drosophila.

Congenital heart defects, clinically identified in both small and large animals, are multifactorial and complex. Although heritable factors are known to have a role in cardiovascular disease, the full genetic aetiology remains unclear. Model organism research has proven valuable in providing a deeper understanding of the essential factors in heart development. For example, mouse knock-out studies reveal a role for the Integrin adhesion receptor in cardiac tissue. Recent research in Drosophila melanogaster (the fruit fly), a powerful experimental model, has demonstrated that the link between the extracellular matrix and the cell, mediated by Integrins, is required for multiple aspects of cardiogenesis. Here we test the hypothesis that Integrins signal to the heart cells through Src42A kinase. Using the powerful genetics and cell biology analysis possible in Drosophila, we demonstrate that Src42A acts in early events of heart tube development. Careful examination of mutant heart tissue and genetic interaction data suggests that Src42A’s role is independent of Integrin and the Integrin-related Focal Adhesion Kinase. Rather, Src42A acts non-autonomously by promoting programmed cell death of the amnioserosa, a transient tissue that neighbors the developing heart.

Matrix Metalloproteinases are required for membrane motility and lumenogenesis during Drosophila heart development.

Matrix Metalloproteinases (Mmps) degrade glycoproteins and proteoglycans of the extracellular matrix (ECM) or cell surface and are crucial for morphogenesis. Mmps and their inhibitors are expressed during early stages of cardiac development in vertebrates and expression is altered in multiple congenital cardiomyopathies such as cardia bifida. Drosophila genome encodes two copies of Mmps, Mmp1 and Mmp2 whereas in humans up to 25 Mmps have been identified with overlapping functions. We investigated the role of Mmps during embryonic heart development in Drosophila, a process which is morphogenetically similar to early heart tube formation in vertebrates. We demonstrate that the two Mmps in Drosophila have distinct and overlapping roles in cell motility, cell adhesion and cardiac lumenogenesis. We determined that Mmp1 and Mmp2 promote Leading Edge membrane dynamics of cardioblasts during collective migration. Mmp2 is essential for cardiac lumen formation, and mutants generate a cardia bifida phenotype. Mmp1 is required for luminal expansion. Mmp1 and Mmp2 both localise to the basal domains of cardiac cells, however, occupy non-overlapping domains apically. Mmp1 and Mmp2 regulate the proteoglycan composition and size of the apical and basal ECM, yet only Mmp2 is required to restrict ECM assembly to the lumen. Mmp1 negatively regulates the size of the adhesive Cadherin cell surface domain, whereas in a complementary fashion, Mmp2 negatively regulates the size of the Integrin-ECM domain and thereby prescribes the domain to establish and restrict Slit morphogen signalling. Inhibition of Mmp activity through ectopic expression of Tissue Inhibitor of Metalloproteinase in the ectoderm blocks lumen formation. Therefore, Mmp expression and function identifies ECM differentiation and remodelling as a key element for cell polarisation and organogenesis.

Physical and Mechanical Forces that Shape Heart Tube in the Chick Embryo

In the embryo, the heart is initially a simple tubular structure that undergoes complex morphological changes. This work focuses on the mechanisms that create the heart tube (HT). The early embryo is composed of three relatively flat primary germ layers called endoderm, mesoderm, and ectoderm. Endoderm and pre-cardiac cells located within bilateral regions of the mesoderm called heart fields (HFs) fold and fuse along the embryonic midline. Then both layers lengthen axially as the anterior intestinal portal (AIP) moves downward. Finally, the fused HFs thicken and expand to create the HT. Here, we combine experiments on chick embryos with computational modeling to explore physical mechanisms of heart tube formation. According to our hypothesis, differential anisotropic growth between mesoderm and endoderm drives ventral folding, and contraction along the AIP generates tension to elongate the HT. We test this hypothesis using biochemical perturbations of cell proliferation and contractility, as well as computational modeling. In embryos exposed to the mitotic inhibitor aphidicolin, little or no folding occurred, confirming that cell proliferation is required to initiate folding of HFs. To determine the effects of actomyosin contraction, we cultured Hamburger-Hamilton stage 5 embryos in media containing the myosin inhibitor blebbistatin. During incubation, folding occurred in treated embryos, but AIP descension proceeded at a progressively slower rate, showing that AIP downward motion is generated mainly by AIP contraction. Simulating these mechanisms in our computational model produces morphology in reasonable agreement with experiments. For the last phase, adding circumferential growth causes the HF to expand, creating the primitive HT, while radial growth increases the thickness of the heart wall. In conclusion, results of our study support our hypothesis for the creation of the heart tube.

A new hypothesis for foregut and heart tube formation based on differential growth and actomyosin contraction.

For decades, it was commonly thought that the bilateral heart fields in the early embryo fold directly towards the midline, where they meet and fuse to create the primitive heart tube. Recent studies have challenged this view, however, suggesting that the heart fields fold diagonally. As early foregut and heart tube morphogenesis are intimately related, this finding also raises questions concerning the traditional view of foregut formation. Here, we combine experiments on chick embryos with computational modeling to explore a new hypothesis for the physical mechanisms of heart tube and foregut formation. According to our hypothesis, differential anisotropic growth between mesoderm and endoderm drives diagonal folding. Then, active contraction along the anterior intestinal portal generates tension to elongate the foregut and heart tube. We test this hypothesis using biochemical perturbations of cell proliferation and contractility, as well as computational modeling based on nonlinear elasticity theory including growth and contraction. The present results generally support the view that differential growth and actomyosin contraction drive formation of the foregut and heart tube in the early chick embryo.

Abstract 247: Rtf1 Controls Sarcomere Integrity and Cardiac Function

Sarcomere integrity must be continuously maintained in cardiomyocytes to ensure contractile function; however, the genetic programs controlling this process are not completely understood. We have previously shown that the RNA polymerase II associated protein Rtf1 directs cardiac progenitor cell formation during early cardiogenesis. Here we report that in mature cardiomyocytes, Rtf1 controls gene programs essential for the maintenance of sarcomere structure and cardiac function. Cardiomyocyte-specific ablation of Rtf1 in mice results in dilated cardiomyopathy characterized by thinning of the myocardial walls, dilation of the ventricular cavity and impaired left ventricle contractile function. Rtf1 knockdown in neonatal rat ventricular myocytes model results in the breakdown of sarcomeres and downregulation of myofibrillar gene programs accompanied by reduced abundance of monoubiquitinated H2BK120 and epigenetic marks associated with actively transcribed genes. In zebrafish embryos, mutation in Rtf1’s histone modification domain arrests cardiomyocyte differentiation and heart tube formation. Together these findings demonstrate that Rtf1 controls cardiomyocyte maturation, myofibril integrity and cardiac function by modulating cardiac epigenetic landscapes.

Gia/Mthl5 is an aorta specific GPCR required for Drosophila heart tube morphology and normal pericardial cell positioning

G-protein signaling is known to be required for cell-cell contacts during the development of the Drosophila dorsal vessel. However, the identity of the G protein-coupled receptor (GPCR) that regulates this signaling pathway activity is unknown. Here we describe the identification of a novel cardiac specific GPCR, called Gia, for “GPCR in aorta”. Gia is the only heart-specific GPCR identified in Drosophila to date and it is specifically expressed in cardioblasts that fuse at the dorsal midline to become the aorta. Gia is the only Drosophila gene so far identified for which expression is entirely restricted to cells of the aorta. Deletion of Gia led to a broken-hearted phenotype, characterized by pericardial cells dissociated from cardioblasts and abnormal distribution of cell junction proteins. Both phenotypes were similar to those observed in mutants of the heterotrimeric cardiac G proteins. Lack of Gia also led to defects in the alignment and fusion of cardioblasts in the aorta. Gia forms a protein complex with G-αo47A, the alpha subunit of the heterotrimeric cardiac G proteins and interacts genetically with G-αo47A during cardiac morphogenesis. Our study identified Gia as an essential aorta-specific GPCR that functions upstream of cardiac heterotrimeric G proteins and is required for morphological integrity of the aorta during heart tube formation. These studies lead to a redefinition of the bro phenotype, to encompass morphological integrity of the heart tube as well as cardioblast-pericardial cell spatial interactions.

Id4 functions downstream of Bmp signaling to restrict TCF function in endocardial cells during atrioventricular valve development

The atrioventricular canal (AVC) connects the atrial and ventricular chambers of the heart and its formation is critical for the development of the cardiac valves, chamber septation and formation of the cardiac conduction system. Consequently, problems in AVC formation can lead to congenital defects ranging from cardiac arrhythmia to incomplete cardiac septation. While our knowledge about early heart tube formation is relatively comprehensive, much remains to be investigated about the genes that regulate AVC formation. Here we identify a new role for the basic helix-loop-helix factor Id4 in zebrafish AVC valve development and function. id4 is first expressed in the AVC endocardium and later becomes more highly expressed in the atrial chamber. TALEN induced inactivation of id4 causes retrograde blood flow at the AV canal under heat induced stress conditions, indicating defects in AV valve function. At the molecular level, we found that id4 inactivation causes misexpression of several genes important for AVC and AV valve formation including bmp4 and spp1. We further show that id4 appears to control the number of endocardial cells that contribute to the AV valves by regulating Wnt signaling in the developing AVC endocardium.

Segregation of Central Ventricular Conduction System Lineages in Early SMA+ Cardiomyocytes Occurs Prior to Heart Tube Formation.

The cardiac conduction system (CCS) transmits electrical activity from the atria to the ventricles to coordinate heartbeats. Atrioventricular conduction diseases are often associated with defects in the central ventricular conduction system comprising the atrioventricular bundle (AVB) and right and left branches (BBs). Conducting and contractile working myocytes share common cardiomyogenic progenitors, however the time at which the CCS lineage becomes specified is unclear. In order to study the fate and the contribution to the CCS of cardiomyocytes during early heart tube formation, we performed a genetic lineage analysis using a Sma-CreERT2 mouse line. Lineage tracing experiments reveal a sequential contribution of early Sma expressing cardiomyocytes to different cardiac compartments, labeling at embryonic day (E) 7.5 giving rise to the interventricular septum and apical left ventricular myocardium. Early Sma expressing cardiomyocytes contribute to the AVB, BBs and left ventricular Purkinje fibers. Clonal analysis using the R26-confetti reporter mouse crossed with Sma-CreERT2 demonstrates that early Sma expressing cardiomyocytes include cells exclusively fated to give rise to the AVB. In contrast, lineage segregation is still ongoing for the BBs at E7.5. Overall this study highlights the early segregation of the central ventricular conduction system lineage within cardiomyocytes at the onset of heart tube formation.

Endoderm convergence controls subduction of the myocardial precursors during heart-tube formation.

Coordination between the endoderm and adjacent cardiac mesoderm is crucial for heart development. We previously showed that myocardial migration is promoted by convergent movement of the endoderm, which itself is controlled by the S1pr2/Gα13 signaling pathway, but it remains unclear how the movements of the two tissues is coordinated. Here, we image live and fixed embryos to follow these movements, revealing previously unappreciated details of strikingly complex and dynamic associations between the endoderm and myocardial precursors. We found that during segmentation the endoderm underwent three distinct phases of movement relative to the midline: rapid convergence, little convergence and slight expansion. During these periods, the myocardial cells exhibited different stage-dependent migratory modes: co-migration with the endoderm, movement from the dorsal to the ventral side of the endoderm (subduction) and migration independent of endoderm convergence. We also found that defects in S1pr2/Gα13-mediated endodermal convergence affected all three modes of myocardial cell migration, probably due to the disruption of fibronectin assembly around the myocardial cells and consequent disorganization of the myocardial epithelium. Moreover, we found that additional cell types within the anterior lateral plate mesoderm (ALPM) also underwent subduction, and that this movement likewise depended on endoderm convergence. Our study delineates for the first time the details of the intricate interplay between the endoderm and ALPM during embryogenesis, highlighting why endoderm movement is essential for heart development, and thus potential underpinnings of congenital heart disease.

Cdo Deficient Mice Display Cardiomyopathy with Alterations in Connexins

Shh signaling is required for various cardiac developmental processes including the looping of heart tube and specification of cardiomyogenesis. Consistently, Shh mutant mice display the abnormalities in heart tube formation and specification of the myocardial and outflow tract. A multifunctional receptor Cdo functions as a Shh coreceptor to fully activate Shh signaling and Cdo deficient mice exhibit multiple defects in embryonic development, associated with the decreased Shh activity. However its role in heart development is entirely unknown. This study examined the role of Cdo in heart development and cardiomyogenesis using Cdo deficient (Cdo-/-) mice. Histological and morphological analyses revealed development of dilated cardiomyopathy (DCM) with a frequent death around 3 weeks of age. Echocardiographic analyses of Cdo-/- mice at 2 weeks confirmed the dilated phenotype of the heart with a significant decrease in the left ventricular fractional shortening. Patch clamp experiments revealed an increase in Ito and reduction in IK1, without any significant changes in action potential duration (APD) in Cdo-/- cariomyocytes. A major gap junctional protein Connexin 43 (Cx43) appears to be localized at the lateral border of the Cdo-/- cardiomyocytes whereas Cx43 remained predominately at the intercalated disk in wildtype cardiomyocytes. Furthermore the phosphorylation status of Cx43 was altered in Cdo-/- hearts. Taken together, these data suggest that Cdo signaling may influence the phosphorylation status and distribution of Cx43 thereby regulating intercellular communication and functionality of cardiomyocytes.

Heart Fields: Spatial Polarity and Temporal Dynamics

In chick and mouse, heart fields undergo dynamic morphological spatiotemporal changes during heart tube formation. Here, the dynamic change in spatial polarity of such fields is discussed and a new perspective on the heart fields is proposed. The heart progenitor cells delaminate through the primitive streak and migrate in a semicircular trajectory craniolaterally forming the bilateral heart fields as part of the splanchnic mesoderm. They switch their polarity from anteroposterior to mediolateral. The anterior intestinal portal posterior descent inverts the newly formed heart field mediolateral polarity into lateromedial by 125° bending. The heart fields revert back to their original anteroposterior polarity and fuse at the midline forming a semi heart tube by completing their half circle movement. Several names and roles were assigned to different portions of the heart fields: posterior versus anterior, first versus second, and primary versus secondary heart field. The posterior and anterior heart fields define basically physical fields that form the inflow-outflow axis of the heart tube. The first and second heart fields are, in contrast, temporal fields of differentiating cardiomyocytes expressing myosin light chain 2a and undifferentiated and proliferating precardiac mesoderm expressing Isl1 gene, respectively. The two markers present a complementary pattern and are expressed transiently in all myocardial lineages. Thus, Isl1 is not restricted to a portion of the heart field or one of the two heart lineages as has been often assumed.