Endocardial Tube Research Articles

MiR-1 modulates HDAC4, calmodulin and MAPK1/Erk2 during early atrial differentiation

Abstract Funding Acknowledgements None. Introduction A large diversity of epigenetic factors, such as microRNAs and histones modifications, are known to be capable of regulating gene expression without altering DNA sequence itself. In particular, miR-1 is considered the first essential microRNA in cardiac development. Purpose The aim of this work is to analyze the miR-1 modulation role in cardiac chamber differentiation through specific signaling pathways. Methods By means of microinjections in both primitive endocardial tubes of chick embryo culture, gain- and loss-of-function experiments were performed with premiR-1 and anti-miR-1, respectively. Subsequently, embryos were subjected to whole mount in situ hybridization with Tbx5, Gata4 and AMHC1 probes, as well as immunohistochemistry with Mef2c, HDAC4, Calmodulin/Calm1 and MAPK1/Erk2 antibodies. Also, we carried out RT-qPCR analysis of control and experimental embryos, including CRABPI, CRABPII and retinoic acid receptors (RAR and RXR). Results Our results reveal that miR-1 increases specific atrial gene expression such as Tbx5, Gata4 and AMHC1, while this microRNA diminishes Mef2c expression. Furthermore, we observed that miR-1 upregulates CRABPII and RARß, and downregulates CRABPI, which are three crucial factors in retinoic acid signaling pathway. Interestingly, we also observed that miR-1 actively interacts with HDAC4, Calmodulin and Erk2/MAPK1, key factors involved in Mef2c regulation. Conclusion All these data suggest that miR-1 functions as an epigenetic factor modulating complementary actions performed by retinoic acid and Mef2c, which are required to properly assign cells as sinoatrial precursors.

EGFR/MAPK signaling pathway acts as a potential therapeutic target for sulforaphane-rescued heart tube malformation induced by various concentrations of PhIP exposure

Background2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyrimidine (PhIP) is a known carcinogen generated mainly from cooking meat and environmental pollutants. It is worth exploring the potential of natural small-molecule drugs to protect against adverse effects on embryonic development. PurposeIn this study, we investigated the potential toxicological effects of PhIP on embryonic heart tube formation and the effect of Sulforaphane (SFN) administration on the anti-toxicological effects of PhIP on embryonic cardiogenesis. Study design and methodsFirst, the chicken embryo model was used to investigate the different phenotypes of embryonic heart tubes induced by various concentrations of PhIP exposure. We also proved that SFN rescues PhIP-induced embryonic heart tube malformation. Second, immunofluorescence, western blot, Polymerase Chain Reaction (PCR) and flow cytometry experiments were employed to explore the mechanisms by which SFN protects cardiac cells from oxidative damage in the presence of PhIP. We used RNA-seq analysis, molecular docking, in situ hybridization, cellular thermal shift assay and solution nuclear magnetic resonance spectroscopy to explore whether SFN protects cardiogenesis through the EGFR/MAPK signaling pathway. ResultsThe study showed that PhIP might dose-dependently interfere with the C-looping heart tube (mild) or the fusion of a pair of bilateral endocardial tubes (severe) in chick embryos, while SFN administration prevented cardiac cells from oxidative damage in the presence of high-level PhIP. Furthermore, we found that excessive reactive oxygen species (ROS) production and subsequent apoptosis were not the principal mechanisms by which low-level PhIP induced malformation of heart tubes. This is due to PhIP-disturbed Mitogen-activated protein kinase (MAPK) signaling pathway could be corrected by SFN administration. ConclusionsThis study provided novel insight that PhIP exposure could increase the risk of abnormalities in early cardiogenesis and that SFN could partially rescue various concentrations of PhIP-induced abnormal heart tube formation by targeting EGFR and mediating EGFR/MAPK signaling pathways.

Inhibition of RhoA and Cdc42 by miR-133a Modulates Retinoic Acid Signalling during Early Development of Posterior Cardiac Tube Segment.

It is well known that multiple microRNAs play crucial roles in cardiovascular development, including miR-133a. Additionally, retinoic acid regulates atrial marker expression. In order to analyse the role of miR-133a as a modulator of retinoic acid signalling during the posterior segment of heart tube formation, we performed functional experiments with miR-133a and retinoic acid by means of microinjections into the posterior cardiac precursors of both primitive endocardial tubes in chick embryos. Subsequently, we subjected embryos to whole mount in situ hybridisation, immunohistochemistry and qPCR analysis. Our results demonstrate that miR-133a represses RhoA and Cdc42, as well as Raldh2/Aldh1a2, and the specific atrial markers Tbx5 and AMHC1, which play a key role during differentiation. Furthermore, we observed that miR-133a upregulates p21 and downregulates cyclin A by repressing RhoA and Cdc42, respectively, thus functioning as a cell proliferation inhibitor. Additionally, retinoic acid represses miR-133a, while it increases Raldh2, Tbx5 and AMHC1. Given that RhoA and Cdc42 are involved in Raldh2 expression and that they are modulated by miR-133a, which is influenced by retinoic acid signalling, our results suggest the presence of a negative feedback mechanism between miR-133a and retinoic acid during early development of the posterior cardiac tube segment. Despite additional unexplored factors being possible contributors to this negative feedback mechanism, miR-133a might also be considered as a potential therapeutic tool for the diagnosis, therapy and prognosis of cardiac diseases.

Evolución filogenética del corazón

In 1859 Charles Darwin in his “The Origin of Species” formulated three facts that explain the principle of evolution: the presence of a common ancestor, the existence of advantageous genetic mutations for survival and the elimination of unfavorable ones or Natural Selection. The embryological development of the mammalian heart improves the need for external oxygen uptake, transport and release in the tissues, which exists in all multicellular living beings from the most primitive and enables the function of all organs and systems. In invertebrates, such as insects, arthropods and molluscs, the open circulatory system reaches a development similar to that of human Carnegie stages 10-13: endocardial tubes, straight tube, or early stages of the bulboventricular loop. In vertebrates, the closed system of fish develops hearts with three and four chambers equivalent to the torsion phase of the loop in mammals, or the fifth week of human embryonic development, with a model closer to that of mammals appearing in reptiles with differences such as the incomplete septation of the ventricles and the development of two aortic trunks in crocodilians. Birds and mammals have reached a similar degree of embryonic development of their hearts, with four septate chambers, the same as the Cono Truncus one, and which separate the systemic and pulmonary circulation. All the embryological development of the human heart is completed between days 20 and 60 of gestation, concluding in the fetal phase its growth together with the rest of the organism.

Transient increase in VEGF-A leads to cardiac tube anomalies and increased risk of congenital heart malformations.

Vascular endothelial growth factor (VEGF) plays a critical role during early heart development. Clinical evidence shows that conditions associated with changes in VEGF signaling in utero are correlated with an increased risk of congenital heart defects (CHD) in newborns. However, how malformations develop after abnormal VEGF exposure is unknown. During embryogenesis, a primitive heart, consisting of an endocardial tube enveloped by a myocardial mantle, is the first organ to function. This tubular heart ultimately transforms into a four-chambered heart. To determine how a transient increase in VEGF prior to heart tube formation affects heart development leading to CHD, we applied exogenous VEGF or a control (vehicle) solution to quail embryos in ovo at Hamburger-Hamilton (HH) stage 8 (28-30 hr of incubation), right before heart tube formation. Light microscopy analysis of embryos re-incubated after treatment for 13 hrs (to approximately HH11/HH12) showed that increased VEGF leads to impaired heart tube elongation accompanied by diameter expansion. Micro-CT analysis of embryos re-incubated for 9 days (to approximately HH38), when the heart is fully formed, showed that VEGF treatment increased the rate of cardiac malformations in surviving embryos. Despite no sex differences in survival, female embryos were more likely to develop cardiac malformations. Our results further suggest that heart tube malformations after a transient increase in VEGF right before heart tube formation may be reversible, leading to normal hearts.

Cardiogenesis with a focus on vasculogenesis and angiogenesis.

The initial intraembryonic vasculogenesis occurs in the cardiogenic mesoderm. Here, a cell population of proendocardial cells detaches from the mesoderm that subsequently generates the single endocardial tube by forming vascular plexuses. In the course of embryogenesis, the endocardium retains vasculogenic, angiogenic and haematopoietic potential. The coronary blood vessels that sustain the rapidly expanding myocardium develop in the course of the formation of the cardiac loop by vasculogenesis and angiogenesis from progenitor cells of the proepicardial serosa at the venous pole of the heart as well as from the endocardium and endothelial cells of the sinus venosus. Prospective coronary endothelial cells and progenitor cells of the coronary blood vessel walls (smooth muscle cells, perivascular cells) originate from different cell populations that are in close spatial as well as regulatory connection with each other. Vasculo- and angiogenesis of the coronary blood vessels are for a large part regulated by the epicardium and epicardium-derived cells. Vasculogenic and angiogenic signalling pathways include the vascular endothelial growth factors, the angiopoietins and the fibroblast growth factors and their receptors.

Early Development of the Heart and Great Vessels

The cardiovascular system is one of the first organ systems to develop in the embryo. By day 21, three germ layers: endoderm, mesoderm, and ectoderm are formed by the gastrulation process. Cells in the mesoderm coalesce through a process called vasculogenesis to form two parallel endocardial tubes. Folding of the gut tube brings these two parallel endocardial tubes towards the midline enabling them to fuse into a single linear‐shaped heart tube. By day 18, the lateral plate mesoderm splits to form two layers – splanchnic (visceral) and somatic (parietal). The cells of the splanchnic mesoderm will develop into the myocardial cells of a Primary (PHF) and Secondary Heart field (SHF). By day 22, the heart begins to beat and lengthen by the addition of cells from the SHF to the PHF. This develops into the outflow tract ‐ aortic and pulmonary vessels, and the right ventricle. By day 28, the linear‐shaped heart tube contains regions called the sinus venosus, primitive atrium, primitive ventricle, and bulbus cordis (outflow). The proximal and distal ends become “fixed” by the pericardium, the bulbus cordis and primitive ventricle grow faster than the other regions of the heart tube, and the heart bends upon itself to form a u‐shape. The primitive atrium and sinus venosus are moved dorsally, and the heart begins septation into 4 chambers. As early as day 14, two actively growing ridges called Endocardial Cushion (of the PHF) nearly divide the single lumen into two separate cavities connected by a singular (atrioventricular) canal. Subsequently, a septum grows from the cushions toward the roof of the common atrium, and from the cushions toward the apex of the primitive ventricle. Continued addition of cushion cells to the atrioventricular canal divides a single canal into a left and right canal, as well as valves attached to the ventricular wall by muscular cords. Abnormalities of Endocardial cushion can affect structures derived from the PHF. For example, Tricuspid Atresia involves the malformation of the right Atrioventricular Canal and is associated with: (i) Atrial, and (ii) Ventricular Septal Defect; (iii) hypotrophy of the right ventricle; and (iv) hypertrophy of the left ventricle. Cells of the SHF are involved in the formation and lengthening of the outflow tract. Interestingly, this growth is regulated by neural crest cells. Insults to neural crest cell development or migration can affect structures derived from the PHF. For example, Tetralogy of Fallot involves an unequal division and laterality of the bulbus cordis associated with: (1) narrow right ventricular outflow; (2) interventricular septal defect; (3) over‐riding aorta directly above the septal defect; (4) hypertrophy of the right ventricular wall because of high pressure in the right side of the heart. Neural crest cells also contribute to craniofacial development leading to facial and cardiac abnormalities in the same individual.

Dullard-mediated Smad1/5/8 inhibition controls mouse cardiac neural crest cells condensation and outflow tract septation.

The establishment of separated pulmonary and systemic circulation in vertebrates, via cardiac outflow tract (OFT) septation, is a sensitive developmental process accounting for 10% of all congenital anomalies. Neural Crest Cells (NCC) colonising the heart condensate along the primitive endocardial tube and force its scission into two tubes. Here, we show that NCC aggregation progressively decreases along the OFT distal-proximal axis following a BMP signalling gradient. Dullard, a nuclear phosphatase, tunes the BMP gradient amplitude and prevents NCC premature condensation. Dullard maintains transcriptional programs providing NCC with mesenchymal traits. It attenuates the expression of the aggregation factor Sema3c and conversely promotes that of the epithelial-mesenchymal transition driver Twist1. Altogether, Dullard-mediated fine-tuning of BMP signalling ensures the timed and progressive zipper-like closure of the OFT by the NCC and prevents the formation of a heart carrying the congenital abnormalities defining the tetralogy of Fallot.

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.

Functional Morphology of the Cardiac Jelly in the Tubular Heart of Vertebrate Embryos.

The early embryonic heart is a multi-layered tube consisting of (1) an outer myocardial tube; (2) an inner endocardial tube; and (3) an extracellular matrix layer interposed between the myocardium and endocardium, called “cardiac jelly” (CJ). During the past decades, research on CJ has mainly focused on its molecular and cellular biological aspects. This review focuses on the morphological and biomechanical aspects of CJ. Special attention is given to (1) the spatial distribution and fiber architecture of CJ; (2) the morphological dynamics of CJ during the cardiac cycle; and (3) the removal/remodeling of CJ during advanced heart looping stages, which leads to the formation of ventricular trabeculations and endocardial cushions. CJ acts as a hydraulic skeleton, displaying striking structural and functional similarities with the mesoglea of jellyfish. CJ not only represents a filler substance, facilitating end-systolic occlusion of the embryonic heart lumen. Its elastic components antagonize the systolic deformations of the heart wall and thereby power the refilling phase of the ventricular tube. Non-uniform spatial distribution of CJ generates non-circular cross sections of the opened endocardial tube (initially elliptic, later deltoid), which seem to be advantageous for valveless pumping. Endocardial cushions/ridges are cellularized remnants of non-removed CJ.

Altered VEGF Signaling Leads to Defects in Heart Tube Elongation and Omphalomesenteric Vein Fusion in Quail Embryos.

Formation of the endocardial and myocardial heart tubes involves precise cardiac progenitor sorting and tissue displacements from the primary heart field to the embryonic midline-a process that is dependent on proper formation of conjoining great vessels, including the omphalomesenteric veins (OVs) and dorsal aortae. Using a combination of vascular endothelial growth factor (VEGF) over- and under-activation, fluorescence labeling of cardiac progenitors (endocardial and myocardial), and time-lapse imaging, we show that altering VEGF signaling results in previously unreported myocardial, in addition to vascular and endocardial phenotypes. Resultant data show: (1) exogenous VEGF leads to truncated endocardial and myocardial heart tubes and grossly dilated OVs; (2) decreased levels of VEGF receptor 2 tyrosine kinase signaling result in a severe abrogation of the endocardial tube, dorsal aortae, and OVs. Surprisingly, only slightly altered myocardial tube fusion and morphogenesis is observed. We conclude that VEGF has direct effects on the VEGF receptor 2-bearing endocardial and endothelial precursors, and that altered vascular morphology of the OVs also indirectly results in altered myocardial tube formation. Anat Rec, 302:175-185, 2019. © 2018 Wiley Periodicals, Inc.

Expression of hLAMP-1-Positive Particles During Early Heart Development in the Chick.

Heart development requires coordinated activity of various factors, the disturbance of which can lead to congenital heart defects. Heart lectin-associated matrix protein-1 (hLAMP-1) is a matrix protein expressed within Hensen's node at Hamburger-Hamilton (HH) stage 4, in the lateral mesoderm by HH stages 5-6 and enhanced within the left pre-cardiac field at HH stage 7. At HH stages 15-16, hLAMP-1 expression is observed in the atrioventricular canal and the outflow tract. Also, the role of hLAMP-1 in induction of mesenchyme formation in chick heart has been well documented. To further elucidate the role of this molecule in heart development, we examined its expression patterns during HH stages 8-14 in the chick. In this regard, we immunostained sections of the heart during HH stages 8-14 with antibodies specific to hLAMP-1. Our results showed prominent expression of hLAMP-1-positive particles in the extracellular matrix associated with the pre-cardiac mesoderm, the endoderm, ectoderm as well as neuroectoderm at HH stages 8-9. After formation of the linear heart tube at HH stage 10, the expression of hLAMP-1-stained particles disappears in those regions of original contact between the endoderm and heart forming fields due to rupture of the dorsal mesocardium while their expression becomes confined to the arterial and venous poles of the heart tube. This expression pattern is maintained until HH stage 14. This expression pattern suggests that hLAMP-1 may be involved in the formation of the endocardial tube.

Temporal expression pattern of hLAMP‐1 and BMP‐2 during early heart development in the chick (542.4)

The heart is the first organ to develop during embryogenesis in mammals and birds. Heart development requires coordinated activity of various factors, the disturbance of which can lead to congenital heart disease. The matrix protein hLAMP‐1 is expressed at Hamburger‐Hamilton stage 4 within the Hensen’s node and subsequently in the lateral mesoderm by stages 5‐6. At stage 7, its expression is enhanced within the left precardiac field. Later, by stages 15‐16, hLAMP‐1 expression has been shown in the atrioventricular (AV) canal and the outflow tract (OT). Also, BMP‐2 expression has been reported in comparable stages of chick embryo. To further elucidate the role of these two molecules in heart development, we examined their expression patterns during stages 8‐14 in chick embryos. In this study, we used antibodies specific to hLAMP‐1 or BMP‐2 to examine the expression pattern of both proteins. We detected hLAMP‐1 at the interface of the splanchnic mesoderm and endoderm at stages 8‐9 where the preendocardial cells delaminate from the precardiac mesoderm and migrate toward the endoderm. In contrast, BMP‐2 was not detected at stages 8‐9. After fusion of the bilateral heart field to form the heart tube at stage 10, both hLAMP‐1 and BMP‐2 expression is restricted to the arterial and venous poles of the heart tube. This expression pattern is maintained until stage 14. This pattern correlates with the time at which the cardiac progenitors being added to both ends of the heart tube. These findings point to a specific role for hLAMP‐1in heart development, possibly in formation of the endocardial tube and the role of BMP‐2 in elongation of the heart tube.

Tal1 regulates the formation of intercellular junctions and the maintenance of identity in the endocardium

The endocardium forms the inner lining of the heart tube, where it enables blood flow and also interacts with the myocardium during the formation of valves and trabeculae. Although a number of studies have identified regulators in the morphogenesis of the myocardium, relatively little is known about the molecules that control endocardial morphogenesis. Prior work has implicated the bHLH transcription factor Tal1 in endocardial tube formation: in zebrafish embryos lacking Tal1, endocardial cells form a disorganized mass within the ventricle and do not populate the atrium. Through blastomere transplantation, we find that tal1 plays a cell-autonomous role in regulating endocardial extension, suggesting that Tal1 activity influences the behavior of individual endocardial cells. The defects in endocardial behavior in tal1-deficient embryos originate during the earliest steps of endocardial morphogenesis: tal1-deficient endocardial cells fail to generate a cohesive monolayer at the midline and instead pack tightly together into a multi-layered aggregate. Moreover, the tight junction protein ZO-1 is mislocalized in the tal1-deficient endocardium, indicating a defect in intercellular junction formation. In addition, we find that the tal1-deficient endocardium fails to maintain its identity; over time, a progressively increasing number of tal1-deficient endocardial cells initiate myocardial gene expression. However, the onset of defects in intercellular junction formation precedes the onset of ectopic myocardial gene expression in the tal1-deficient endocardium. We therefore propose a model in which Tal1 has distinct roles in regulating the formation of endocardial intercellular junctions and maintaining endocardial identity.

Contemporary cardiogenesis: new insights into heart development

Cardiac development is a dynamic process during which progressive specification and differentiation of cardiovascular progenitor cells are intrinsically linked to complex morphogenetic events conferring function to the forming heart.1,2 Cardiogenesis initiates in the mesoderm of the anterior lateral region of the early embryo in response to inductive signals from surrounding tissues. Differentiation of the first cardiomyocytes surrounding an endocardial tube is rapidly followed by formation of a linear heart tube that subsequently elongates, loops to the right and undergoes ballooning morphogenesis to generate distinct atrial and ventricular chambers. Cardiac valve development, septation, and establishment of the coronary vasculature and conduction system follow as the heart acquires its definitive form. Defects in these processes result in a wide range of congenital heart anomalies, including major life-threatening defects such as cardiac arrhythmias and failure to separate systemic and pulmonary circulatory systems. Furthermore, the onset of certain cardiac pathologies in the adult is associated with activation of embryonic transcriptional programmes. Thus, understanding cardiac development, in particular through the study of model organisms, is crucial for both basic and translational research. This is an exciting period in the field of heart development as new molecular, genetic, and cellular approaches accelerate our understanding of the origins and diversification of different cardiac cell lineages as well as the molecular mechanisms and cellular interactions underlying normal and pathological heart development. The collection of reviews in this spotlight issue of Cardiovascular Research aims to provide a broad introduction to aspects of heart development of clinical relevance in addition to highlighting the most recent achievements, as well as the current potential and challenges, in the field of cardiac development. The first step in …

Blood Vessel Tubulogenesis Requires Rasip1 Regulation of GTPase Signaling

Cardiovascular function depends on patent blood vessel formation by endothelial cells (ECs). However, the mechanisms underlying vascular "tubulogenesis" are only beginning to be unraveled. We show that endothelial tubulogenesis requires the Ras interacting protein 1, Rasip1, and its binding partner, the RhoGAP Arhgap29. Mice lacking Rasip1 fail to form patent lumens in all blood vessels, including the early endocardial tube. Rasipl null angioblasts fail to properly localize the polarity determinant Par3 and display defective cell polarity, resulting in mislocalized junctional complexes and loss of adhesion to extracellular matrix (ECM). Similarly, depletion of either Rasip1 or Arhgap29 in cultured ECs blocks in vitro lumen formation, fundamentally alters the cytoskeleton, and reduces integrin-dependent adhesion to ECM. These defects result from increased RhoA/ROCK/myosin II activity and blockade of Cdc42 and Rac1 signaling. This study identifies Rasip1 as a unique, endothelial-specific regulator of Rho GTPase signaling, which is essential for blood vessel morphogenesis.

Tyrosine hydroxylase is expressed during early heart development and is required for cardiac chamber formation

Tyrosine hydroxylase (TH) is the first and rate-limiting enzyme in catecholamine biosynthesis. Whereas the neuroendocrine roles of cathecolamines postnatally are well known, the presence and function of TH in organogenesis is unclear. The aim of this study was to define the expression of TH during cardiac development and to unravel the role it may play in heart formation. We studied TH expression in chick embryos by whole mount in situ hybridization and by quantitative reverse transcription-polymerase chain reaction and analysed TH activity by high-performance liquid chromatography. We used gain- and loss-of-function models to characterize the role of TH in early cardiogenesis. We found that TH expression was enriched in the cardiac field of gastrulating chick embryos. By stage 8, TH mRNA was restricted to the splanchnic mesoderm of both endocardial tubes and was subsequently expressed predominantly in the myocardial layer of the atrial segment. Overexpression of TH led to increased atrial myosin heavy chain (AMHC1) and T-box 5 gene (Tbx5) expression in the ventricular region and induced bradyarrhythmia. Similarly, addition of l-3,4-dihydroxyphenylalanine (l-DOPA) or dopamine induced ectopic expression of cardiac transcription factors (cNkx2.5, Tbx5) and AMHC1 as well as sarcomere formation. Conversely, blockage of dopamine biosynthesis and loss of TH activity decreased AMHC1 and Tbx5 expression, whereas exposure to retinoic acid (RA) induced TH expression in parallel to that of AMHC1 and Tbx5. Concordantly, inhibition of endogenous RA synthesis decreased TH expression as well as that of AMHC1 and Tbx5. TH is expressed in a dynamic pattern during the primitive heart tube formation. TH induces cardiac differentiation in vivo and it is a key regulator of the heart patterning, conferring atriogenic identity.

In vivo imaging of the cyclic changes in cross‐sectional shape of the ventricular segment of pulsating embryonic chick hearts at stages 14 to 17: A contribution to the understanding of the ontogenesis of cardiac pumping function

The cardiac cycle-related deformations of tubular embryonic hearts were traditionally described as concentric narrowing and widening of a tube of circular cross-section. Using optical coherence tomography (OCT), we have recently shown that, during the cardiac cycle, only the myocardial tube undergoes concentric narrowing and widening while the endocardial tube undergoes eccentric narrowing and widening, having an elliptic cross-section at end-diastole and a slit-shaped cross-section at end-systole. Due to technical limitations, these analyses were confined to early stages of ventricular development (chick embryos, stages 10-13). Using a modified OCT-system, we now document, for the first time, the cyclic changes in cross-sectional shape of beating embryonic ventricles at stages 14 to 17. We show that during these stages (1) a large area of diminished cardiac jelly appears at the outer curvature of the ventricular region associated with formation of endocardial pouches; (2) the ventricular endocardial lumen acquires a bell-shaped cross-section at end-diastole and becomes compressed like a fireplace bellows during systole; (3) the contracting portions of the embryonic ventricles display stretching along its baso-apical axis at end-systole. The functional significance of our data is discussed with respect to early cardiac pumping function.

The innateness of coronary artery: Vasa vasorum

Because heart originates from primitive endocardial tube (ventral aorta) embryologically, coronary arteries that supply this tube can be thought as "cardiac vasa vasorum" like arterial vasa vasorum that supply peripheral arteries. Coronary arteries have vasodilative reaction to noradrenaline, different from the most arteries that show vasoconstriction. This phenomenon is similar to vasa vasorum that are insensitive to noradrenaline and has its own vasoregulation. Coronary arteries can supply blood from epicardium, myocardium to endocardium under normal physiological conditions. But heart having a significant myocardial hypertrophy can be susceptible to subendocardial ischemia because the metabolic demands of hypertrophic myocardium cannot be met by a parallel increase of coronary myocardial capillaries. This peculiar characteristic of coronary arteries is similar to vasa vasorum that give rise to ischemic necrosis of media of the host vessel under chronic hypertension. Inherency of coronary arteries is the same endarteries as vasa vasorum. Coronary arteries have many smooth muscle fibers than elastic fibers in media, which protect them to collapse during systole to some extent. This feature also is similar to vasa vasorum. The above similarities support the hypothesis that coronary arteries are a kind of vasa vasorum.

Solitary Intercostal Arterial Trunk

During embryonic life, events may alter the normal growth and fusion of the endocardial tube, the paired dorsal aortae, and the vitelline plexus, leading to anomalies and variants of the mature axial arterial system.1 Many of these are well known, especially those associated with the aorta, heart, and umbilical artery.1,2 Vitelline variants have also been found that are either associated with Meckel diverticulum or caudal regression syndrome.3,4 We present a case of anomalous intercostal arterial supply, which, to our knowledge, has not been reported previously. The aberrant intercostal trunk is associated with aberrant descending thoracic aorta position, a replaced common hepatic artery from the superior mesenteric artery, and an isolated splenic artery origin from the aorta. A 27-year-old man with a medical history of irritable bowel syndrome and stage I hypertension presented for renal vasculature evaluation. A renal arterial magnetic resonance …