Cardiac Morphogenesis: Implications for Congenital Cardiovascular Diseases

Neonatal Cardiology, 3rd Ed. Michael Artman

Chapter 1. Cardiac Morphogenesis: Implications for Congenital Cardiovascular Diseases

■ INTRODUCTION

■ CARDIAC PROGENITORS AND THE CONCEPTS OF FIRST AND SECOND HEART FIELD

■ THE NEURAL CREST

■ THE EPICARDIUM

■ BREAKING SYMMETRY

■ CRITICAL DEVELOPMENTALTIMEWINDOWS

■ CARDIAC MORPHOGENESIS AND DYSMORPHOGENESIS

Incorporation of the Sinus Venosus, Atrial

Septation, and Pulmonary Vein Development Ventricular Inflow and Outflow Tract Septation Pharyngeal Arch Development

Valvulogenesis

Conduction System Development Development of the Epicardium, Myocardium, and Coronary Arteries

■ SUGGESTED READINGS

■ INTRODUCTION

Knowledge of the role of cardiac-specific genes and their modulating factors has increased tremendously over the last decade, although 85% of human congenital cardiovascular disease is still considered to be multifactorial in origin. Advances in the molecular biology of the developing heart have greatly contributed to our understanding of cardiac morphogenesis. Manipulation of conserved genes from a variety of model organisms has increased our understanding of how genetic factors and cellular interactions contribute to cardiac development. Transgenic mouse models have allowed time-specific tracing of cells and their role in heart formation. The problem of embryo-lethality after manipulating “cardiac-specific” genes has been overcome by inducible knockout strategies. Whole genome sequencing programs have also increased understanding of mutations in humans that lead to congenital cardiovascular disease.

This chapter summarizes the initial phases of cardiac development. We then describe in more detail how cardiac morphogenesis leads to formation of the four-chambered heart and how abnormal cardiogenesis contributes to congenital cardiovascular disease.

■ CARDIAC PROGENITORS AND THE CONCEPTS OF FIRST AND SECOND HEART FIELD

Heart development starts with two cardiogenic plates derived from the lateral splanchnic mesoderm. These plates fuse in the midline in the anterior (cranial) region of the embryo. The crescent of the cardiogenic plates is referred to as the first heart field (FHF) and is flanked medially by the second heart field (SHF) mesoderm (Figure 1A). Upon fusion in the midline, the FHF forms the two-layered primary heart tube with myocardium on the outside lined by endocardium on the inside. The myocardium secretes a glycoprotein-rich layer, the cardiac jelly, toward the endocardium. The primary heart tube connects to the arterial pole cranially and to the venous pole caudally (Figure 1-1B) but does not contain all segments of the four-chambered heart. Venous tributaries abut on the small atrial component, followed downstream by the future atrioventricular canal and a primitive left ventricle. Finally, the outflow tract connects to the aortic sac at the arterial pole (Figure 1-1C). The various components can be distinguished soon after, as both the AV canal and the outflow tract contain an increasing amount of cardiac jelly that forms the endocardial cushions. The cushions become even more prominent as they acquire mesenchymal cells, derived from the endocardial lining as a result of endocardial-mesenchymal transition. Subsequently, the primary heart tube starts the developmentally determined rightward looping.

At the same time, the SHF adds progenitor cells to both the venous and the arterial poles, which ultimately form the essential components of the right ventricle (RV) and at least a part of the right side of the interventricular septum (Figure 1-1D). At the venous pole, the SHF forms cardiomyocytes encapsulating the sinus venosus and its tributaries. The sinus venosus is incorporated subsequently into the wall of the right and left atrium. Likewise, the walls of the great arteries, the embryonic pharyngeal arch arteries that connect to the aortic sac, are partly built from SHF-originating cells. Neural crest cells also contribute to formation of the great arteries, as will be explained below in this chapter.

Multiple specific transcription factors and signaling molecules are essential to the early stages of cardiogenesis. These include the earliest markers of the precardiac mesoderm, including the homeobox-containing gene Nkx2.5 and the zinc-finger-containing GATA4/5/6 subfamily. Members of the T-box family, TBx1/5/18/20, also have essential roles in SHF differentiation. Myocardial differentiation is regulated by several myocyte-specific genes, including myosin light and heavy chain, alpha-cardiac actin, and cardiac troponin I.

■ THE NEURAL CREST

The neural crest cells are an ecto-mesodermal derivative arising from the crest of the neural tube, migrating toward various parts of the embryo, including the heart. Cardiac neural crest cells differentiate into cells of the autonomic nervous system and into vascular smooth muscle cells of the pharyngeal arch arteries and contribute to the arterial pole of the heart entering the endocardial outflow tract cushions (Figure 1-1E). Neural crest cells within the heart are involved in modulation and induction of semilunar valve formation and generation of the myocardial component of the outflow tract septum. At the venous pole, where their contribution is less important, a similar effect is observed in the atrioventricular cushions. More recently, cardiac neural crest cells contributing smooth muscle cells to the coronary arteries have been identified.

■ THE EPICARDIUM

The epicardium (splanchnic mesodermal lining of the pericardial cavity) is a secondary layer covering the myocardial tube and the intrapericardial part of the arterial pole. During embryonic development, the epicardium originates from both the venous and the arterial poles (Figure 1-1E-G). The larger epicardial population derives from a protrusion of the coelomic wall, covering the sinus venosus and liver primordium (Figure 1-1F). The cells of the proepicardium spread along the outer wall of the ventricles and atria to the border of the myocardium at the arterial pole. Here, they join the arterial epicardium, which is derived from a much smaller arterial proepicardium exhibiting a slightly different phenotype (Figure 1-1G). On activation, the epithelial epicardium undergoes endocardial-mesenchymal transition, and the resulting mesenchymal cells fill the subepicardial space as epicardium-derived cells. These cells migrate between the myocardial cells of the heart tube, the atrioventricular cushions, and the future fibrous annulus. These epicardium-derived cells, in contrast to neural crest cells, differentiate into several cell lines including the majority of the cardiac fibroblasts and the vascular smooth muscle cells of the coronary vascular system (Figure 1-2). The endothelial lining of the coronary vascular system is derived from the endothelium of the sinus venosus/liver primordium adjacent to the proepicardium (Figures 1-1F, 1-2).

■ BREAKING SYMMETRY

Starting with the developmentally determined rightward looping, it is clear that the heart and its connections to the lungs are not symmetric. Situs inversus and heterotaxy occur in humans; several mouse models have increased our knowledge on essential signaling factors related to determination of situs. It is remarkable that only the atrial situs and its contributing posterior SHF seem to be influenced by these factors, while right ventricle and left ventricle with their specific morphologies do not copy the atrial situs anomalies (eg, inversus or ambiguous).

FIGURE 1-1. Cardiac development. A. Schematic depiction of the precardiac mesoderm in the primitive plate. The brown area reflects the mesoderm of the FHF, whereas the yellow area corresponds to the putative SHF mesoderm. B. Primary heart tube derived of FHF mesoderm. The tube consists of myocardium, lined by cardiac jelly. C. Heart tube after looping. The yellow areas reflect SHF-derived contributions. The SHF contributions to the outflow tract have not been depicted. Note that in this stage the atria are still positioned entirely above the primitive left ventricle, whereas the outflow tract is positioned above the primitive right ventricle. D. Advanced stage of heart development. Septation has now occurred at the level of the atria, ventricles, and outflow tract. E. Sagittal view of an embryo. The (anterior and posterior) SHF mesoderm and its derivatives are depicted in yellow. Contributions from neural crest cells are depicted in blue. A proepicardial organ can be distinguished at the venous pole as well as a smaller proepicardial organ at the arterial pole. F. Scanning electronic microscopic section at the level of the venous pole in a chick embryo, showing the venous pole of the proepicardial organ. G. Scanning electronic microscopic section at the level of the outflow tract in a chick embryo, showing the arterial pole of the proepicardial organ (arrowheads, asterisk). Abbreviations: A, atrium; Ao, aorta; AoS, aortic sac; aPEO, atrial pole of proepicardial organ; AVC, atrioventricular canal; EC, endocardial cushions; FHF, first heart field; HH, Hamburger and Hamilton; IVC, inferior vena cava; LA, left atrium; L, liver; LCV, left cardinal vein; LV, left ventricle; LVV, left venous valve; PAA, pharyngeal arch artery; PC, pericardial cavity; PT, pulmonary trunk; PV, pulmonary vein; RA, right atrium; RCV, right cardinal vein; RV, right ventricle; RVV, right venous valve; SHF, second heart field; SV, sinus venosus; SVC, superior vena cava; VPEO, venous pole of proepicardial organ. B-E: Adapted from: Gittenberger-de Groot AC et al. Ann Med. 2014;46(8):640-652. F and G: Adapted from: Gittenberger-de Groot AC et al. Differentiation. 2012;84(1):41-53.

FIGURE 1-2. Cell lines contributing to the developing heart. Schematic representation of cardiac cell lines (cardiomyocytes, endocardium, epicardium, and endothelium) that are derived from the first and second heart field mesoderm and are the main contributors to the definitive heart and vessels. Epicardium-derived cells (in yellow) as well as inadequate interaction with other cardiac cell types may play a role in some cardiac malformations (green boxes). The second heart field and neural crest cell contribution to the great vessels is not represented. Abbreviations: CMPCs, cardiomyocyte progenitor cells; EPDCs, epicardium-derived cells; ECs, endothelial cells; VSMCs, vascular smooth muscle cells. Adapted from: Gittenberger-de Groot AC et al. Differentiation. 2012;84(1):41-53.

■ CRITICAL DEVELOPMENTAL TIME WINDOWS

In the preceding paragraphs, the cellular building blocks of the cardiovascular system have been presented. Serious disturbances of one or more cell populations can lead to abnormal cardiac development and even early embryolethality. This results in early spontaneous abortion in humans. In homozygous mouse strains, 50% of spontaneous early embryo-lethality related to mutations is caused by cardiovascular abnormalities. In the usually non- homozygous human, the percent of spontaneous abortions caused by congenital cardiovascular disease cannot be determined unequivocally. The FHF lesions are considered to be the most critical for embryonic demise. Most of the cardiac malformations that clinicians encounter in the perinatal period occur during looping and events mediated by the SHF. Spontaneous abortion in the second trimester caused by congenital cardiovascular disease is less common, as most forms of cardiac malformations are compatible with intrauterine survival.

■ CARDIAC MORPHOGENESIS AND DYSMORPHOGENESIS

In the human embryo, the formation of the fourchambered heart occurs by about 8 weeks’ gestation. Thereafter, maturation and remodeling of, eg, the pharyngeal arch arteries and valves are essential for ensuing proper functioning and postnatal survival. The most important elements of cardiac morphogenesis (summarized in Figure 1-3), including septation, valve formation, conduction system maturation, and coronary vascular development, will be presented.

Incorporation of the Sinus Venosus, Atrial Septation, and Pulmonary Vein Development

The precardiac mesoderm of the SHF at the venous pole develops uniquely from an Nkx2.5-negative but myosin light chain positive cell population, surrounding the lumen of the sinus venosus. This myocardial cell lineage is incorporated into the posterior wall of both the right and the left atria. The atrial appendages are probably related to the FHF.

The left and right cardinal veins (the embryonic superior and inferior caval veins) are incorporated into the right atrium and are flanked by the folds of the embryonic right and left venous valves (Figure 1-1C, D). The left inferior cardinal vein (future coronary sinus) and the left superior cardinal vein (regressing in the human heart as the ligament of Marshall) all drain into the cavity of the right atrium. A splanchnic vascular plexus surrounds the developing lung buds. During early developmental stages, the primary route of pulmonary drainage from this plexus is toward the systemic veins; a direct connection of the primitive pulmonary veins to the heart is achieved from different tissue later during development. The anlage of the primitive pulmonary vein, the so-called mid-pharyngeal endothelial strand, initially does not have a lumen and is connected to the sinus venosus.

FIGURE 1-3. Cardiac morphogenesis. Schematic representation focusing on the time line and the major events during cardiac morphogenesis. Many processes essential to heart formation overlap during the 7 to 8 weeks of development, making it difficult to determine the separate molecular pathways or the primary insult that leads to congenital cardiovascular malformations, either isolated or complex. Valvulogenesis occurs relatively late in heart formation, while completion of atrial septation (closure of the foramen ovale) and ductus arteriosus differentiation (closure of the ductus arteriosus) naturally occur after birth because of the unique requirements of the fetal circulation. Source: Jongbloed MRM, et al. Development of the Cardiac Conduction System and the Possible Relation to Predilection Sites of Arrhythmogenesis, TheScientificWorldJOURNAL, vol. 8, pp. 239-269, 2008.

During atrial septation it connects to the dorsal wall of the left atrium. The splanchnic pulmonary venous connections with the systemic cardinal (putative caval) veins gradually disappear during normal development.

During atrial septation, four components deserve attention: the primary septum, the dorsal mesenchymal protrusion, the septum secundum, and the endocardial cushions. The initially two-layered myocardial primary atrial septum is positioned between the right and left atria. It partially disintegrates forming the ostium secundum, which is required for the formation of the embryonic foramen ovale (Figure 1-4A, B), the essential communication between the right and left atria during fetal life. Fusion of a mesenchymal cap on the free rim of the primary atrial septum with the atrioventricular endocardial cushion mass and with the dorsal mesenchymal protrusion closes the ostium primum. This structure is found at the right side of the primary atrial septum as a thick mesenchymal mass (Figure 1-4A, B) and will develop into the muscular base of the atrial septum. At the right side of the primary septum and usually incorporating the left venous valve, a folding process of the atrial myocardial wall forms the crescent ridge of the atrial septum secundum (the limbus). During development, the free edge of the primary atrial septum (the valve of the foramen ovale) and the rim of the septum secundum (the limbus) will overlap, allowing blood to pass via the foramen ovale (Figure 1-4B). At a variable time after birth, these two rims often fuse, resulting in the closure of the foramen ovale, although this structure remains patent in up to 20% of normal adults.

FIGURE 1-4. Atrial septation. A. Early development of the dorsal and cranial wall of the right and left atria. The gold indicates the contribution of the SHF to the incorporated sinus venosus myocardium. The appendages are not depicted. Both the inferior and the superior vena caval veins enter in the right atrium as well as the coronary sinus, which is derived from the left superior cardinal vein. A mesenchymal cap (green) under the rim of the primary atrial septum borders the ostium primum, which connects the right and left atria. The entrance of the primitive pulmonary vein is seen in the left atrium. The infolding of the superior wall of the right atrium that will form the secundum atrial septum is already seen merging with the dorsal mesenchymal protrusion. B. After completion of septation, the ostium primum is closed by fusion of the mesenchymal cap with the atrioventricular cushions, which have now divided the atrioventricular canal into a tricuspid and a mitral orifice. The perforations in the primary atrial septum have enlarged to form an ostium secundum that in combination with the free rim of the secundum atrial septum is part of the foramen ovale (arrow) that closes after birth. Abbreviations: AVC, atrioventricular canal; DM, dorsal mesocardium; IVC, inferior vena cava; LA, left atrium; MC, mesenchymal cap; MO, mitral orifice; OP, ostium primum; OS, ostium secundum; PAS, primary atrial septum; PV, pulmonary vein; RA, right atrium; SAS, secundum atrial septum; SVC, superior vena cava; TO, tricuspid orifice. Used with permission from Gittenberger-de Groot AC, et al., (2011). Normal and Abnormal Cardiac Development. In: Pediatric Cardiovascular Medicine, Second Edition (eds JH Moller and JIE Hoffman), Wiley-Blackwell, Oxford, UK.

Implications for Congenital Cardiovascular Disease

Abnormal pulmonary venous return. As described above, pulmonary drainage is initially via an extensive midsagittal splanchnic vascular network. Disturbance of the SHF can lead to misalignment and faulty incorporation of the primitive pulmonary veins into the dorsal left atrial wall. In case of absence or atresia of the mid- pharyngeal endothelial strand, either the early pulmonary to systemic connections will persist or abnormal connections will develop, leading to abnormal drainage of the pulmonary venous blood to three levels: subdia- phragmatic (eg, scimitar syndrome; Figure 1-5A), cardiac, and supracardiac (Figure 1-5B). The drainage of all the pulmonary veins can be abnormal (total anomalous pulmonary venous connection) or partial, with some pulmonary veins entering in into left atrium and some into either systemic veins (Figure 1-5B) or the right atrium. The few known genetic causes are linked to abnormalities of left/right asymmetry (heterotaxy) caused by mutations of the transcription factor PITx2 or by more downstream signaling abnormalities (eg, PDGFRa).

Atrial septal defects. Several types of atrial septal defects occur. A primum atrial septal defect is caused by a deficient connection of the primary interatrial septum with the atrioventricular cushion complex. This anomaly is often seen in conjunction with an atrioventricular septal defect (see below) in which the dorsal mesenchymal protrusion is also underdeveloped. The most common anomaly of the atrial septum is a secundum atrial septal defect, in which there is deficiency of an atrial septal component. More rarely, a sinus venosus type of atrial septal defect is seen, which is related to the superior or inferior caval vein. Mutations in genes involved in posterior SHF differentiation, such as TBx5 and NKx2.5, are linked to this relatively common group of malformations.

Atrioventricular septal defects. Data from animal models and humans suggest that most cases of atrioventricular septal defect (atrioventricular canal) are caused by a deficiency in the base of the atrial septum resulting from underdevelopment of the dorsal mesenchymal protrusion. Additionally, the posterior inlet ventricular septum is shorter than normal; in combination, this results in partial fusion to absence of the atrioventricular cushions, resulting in a common valve with either one- or two-valve ostia (depending on the amount of fusion). Portions of the atrioventricular cushions develop into the characteristic atrioventricular valve leaflets observed in atrioventricular septal defects (ie, superior and inferior bridging leaflets and left and right-sided mural leaflets).

Ventricular Inflow and Outflow Tract Septation

Viewed from the right, the ventricular septum is divided into several components, including the ventricular inlet septum, an apical trabeculated component, and an anterior (“infolding”) component (Figure 1-6A, B). The outflow tract septum (Figure 1-6C) develops as a separate structure. The different components with their specific developmental history, boundaries, and origin are associated with varying congenital malformations of the ventricular septum.

To understand the process of ventricular septation and the aberrations in development that lead to the most common septal defects, it is helpful to first review several novel findings concerning the contribution of both the anterior SHF and the neural crest cells to the outflow tract septum and the trabecular portion of the right ventricle. Several tracing studies in mouse embryos employing surrogate markers for SHF-derived cells have shown an asymmetric contribution to both the myocardium and the vascular wall of the right ventricular outflow tract and the pulmonary trunk. In this process, which we have termed the “pulmonary push,” the embryonic left (pulmonary) side of the outflow tract is expanded by a relatively large contribution of the SHF as compared to the right (aortic) side, eventually bringing the pulmonary orifice to its normal anterior and cranial position with respect to the aorta. This pulmonary push is responsible for the so-called rotation of the outflow tract and great arteries and also explains the relatively deep position of the aortic orifice in the crux of the heart.

FIGURE 1-5. Anomalous pulmonary venous connection. A. Scimitar syndrome is characterized by a partial or complete right-sided anomalous venous connection to the inferior vena cava. In this case, the right inferior pulmonary vein has an anomalous connection to inferior vena cava. The other veins depicted all drain normally to the left atrium. Other characteristics of Scimitar syndrome include hypoplasia of the right pulmonary artery and lung, resulting in dextroposition of the heart. B. Total anomalous pulmonary venous connection, extracardiac type. The right and left pulmonary veins drain via a vertical vein into a systemic vein (eg, the left innominate vein, which drains into the superior vena cava). The absence of pulmonary venous connections and incorporation into the left atrium lead to a small left atrium that does not contain vessel wall tissue. Abbreviations: Ao, aorta; BV, brachiocephalic vein; IVC, inferior vena cava; LA, left atrium; LAA, left atrial appendage; LPA, left pulmonary artery; LIPV, left inferior pulmonary vein; LPV, left pulmonary vein; LSPV, left superior pulmonary vein; LV, left ventricle; PT, pulmonary trunk; RA, right atrium; RAA, right atrial appendage; RIPV, right inferior pulmonary vein; RPA, right pulmonary artery; RPV, right pulmonary vein; RSPV, right superior pulmonary vein; RV, right ventricle; SVC, superior vena cava; VV, vertical vein. Panel B is Adapted from Douglas YL et al. Int J Cardiol. 2009;134:302-312.

During this process, neural crest cells are incorporated by ingression into the aortic sac, creating the aorto-pulmonary septum, which separates the aorta and pulmonary artery. The aorto-pulmonary septum forms the central condensed mesenchyme, as well as two prongs extending into the septal and parietal endocardial cushions present in the outflow tract (Figure 1-6C). The neural crest cells have an induction effect on the outflow tract, recruiting myocardial cells into the cushions that form the posterior wall of the subpulmonary infundibulum, which also forms the septum between the right ventricular and the

left ventricular outflow tracts. This outflow tract separation complex (distinguishable only as an outflow tract septum in specific cardiac anomalies) fuses by bringing together the septal and parietal cushion with the merged atrioventricular cushions, thereby closing the embryonic interventricular foramen and completing ventricular septation. During this process, an anterior folding septum is formed resulting from the expansion of the right and left ventricles, pushing the two outer faces of both ventricles together and trapping epicardium in between. Of note, the epicardium serves a similar function as the endocardial cushions inside the heart, bringing two myocardial faces together. The trapped epicardial cells will differentiate into epicardium-derived cells, bringing these cells deep into the core of the septum.

FIGURE 1-6. Ventricular septal components. A. Schematic representation showing the components of the interventricular septum including the inlet septum, the anterior folding septum, and the trabecular (apical) septum. The septal band that continues into the moderator band is related developmentally to the inlet septum. The posterior wall of the subpulmonary infundibulum contains the small outflow tract septum (asterisk). B. Postmortem specimen with the above-mentioned septal components. C. Scanning electron microscopic picture of the outflow tract of a chicken embryo. The aorto-pulmonary septum at the level between the aortic and pulmonary trunk orifices, which consists at this stage of condensed mesenchyme of neural crest cell origin, will merge with the distal endocardial outflow tract cushion and extend into the proximal outflow tract cushion. The distal level will remodel into the semilunar valves of the great arteries, while the proximal endocardial cushion will, by induction through the neural crest cell population, transform into myocardium and eventually form the small outflow tract septum. Abbreviations: AFS, anterior folding septum; Ao, aorta; APS, aortopulmonary septum; D, distal (endocardial outflow tract cushion); IS, inlet septum; MB, moderator band; OTS, outflow tract septum; P, proximal (outflow tract cushion); PT, pulmonary trunk; SB, septal band; TS, trabecular septum. A: Adapted from Gittenberger-de Groot, AC, et al., (2012). Normal and Abnormal Cardiac Development, in Pediatric Cardiovascular Medicine, Second Edition (eds JH Moller and JIE Hoffman), Wiley-Blackwell, Oxford, UK. B and C: Used with persimmon from Gittenberger-de Groot, AC, et al., (2012). Normal and Abnormal Cardiac Development, in Pediatric Cardiovascular Medicine, Second Edition (eds JH Moller and JIE Hoffman), Wiley-Blackwell, Oxford, UK.

Implications for Congenital Cardiovascular Disease

Ventricular septal defects. Muscular ventricular septal defects can be found within the anterior folding septum, within the inlet septum, and on the border of the septal band with the anterior folding septum, ie, central muscular ventricular septal defect.

Perimembranous ventricular septal defects and malalignment defects, tetralogy of Fallot, and doubleoutlet right ventricle. These malformations are caused primarily by an abnormal extension and malalignment of the outflow tract septal complex with the atrioventricular cushion mass. Since the fibrous connection between the tricuspid and mitral orifice and valves derives mainly from atrioventricular and outflow tract endocardial cushions, the ventricular septal defect will in part be flanked by fibrous tissue; hence, the term “perimembranous” is often used to describe these defects. The defect can extend more posteriorly toward the inlet septum or, in case of a malaligned or shortened outflow tract septum, toward the orifices of the great arteries. These are generally referred to as subarterial, but more specifically they are subaortic in tetralogy of Fallot and subpulmonary in Taussig-Bing malformation. From a developmental point of view, a double muscular subarterial infundibulum or conus has been proposed to be essential for the anomaly called double-outlet right ventricle.

Given that the “pulmonary push” is an essential element of rotation and extension of the right ventricular outflow tract, anomalies of this region are better understood as resulting from unbalanced contributions from SHF and neural crest cells.

Pharyngeal Arch Development

The pharyngeal arches develop as a bilaterally symmetric system harboring skeletal, muscular, glandular, and vascular elements of the face and neck region. The six pairs of pharyngeal arch arteries begin their development as a cranio-caudal series of endothelial tubes that connect the aortic sac with the bilateral dorsal aortae (Figure 1-7A-D). Stability of the vasculature is provided by a smooth muscle cell layer that is derived from several sources, including the SHF splanchnic mesoderm and cardiac neural crest cells. Remodeling of this system requires a balanced interaction of the SHF and neural crest-derived cells (Figure 1-7E) and differs among species. In fish, five or more branchial arches persist and feed the pairs of gills. In reptiles, pairs of the third (carotid), fourth (aorta), and sixth (pulmonary) pharyngeal arch arteries persist, but in birds and mammals, the arterial system becomes asymmetric. In birds, the right fourth becomes the aortic arch, whereas the left one degenerates during development (Figure 1-7F). In mammals, including humans, the main part of the left fourth develops into the B-segment of the aortic arch (between the origins of the left carotid and left subclavian arteries; Figure 1-7H), while the right fourth becomes part of the right subclavian artery (Figure 1-7G, H). The remodeling process involves apoptosis of the vascular segments that disappear, probably combined with shear stress-invoked signaling. The distal part of the left sixth pharyngeal arch artery persists in mammals as the ductus arteriosus. The proximal part, which can be very short, is connected to the left pulmonary artery. On the right side, the distal part of the sixth pharyngeal arch artery disappears together with the right dorsal aortic segment, to which it joins caudally with the left dorsal aorta. The subclavian arteries are thought to develop from the ipsilateral seventh intersegmental arteries, but how these arteries move superiorly during development is poorly understood (Figure 1-7A-D).

FIGURE 1-7. Remodeling of the pharyngeal arterial arch system. A-D. Schematic representation of the remodeling of the pharyngeal arterial arch system during development. The left arch with principal fourth arch segment becomes dominant, and right-sided structures disappear. The color coding refers to the arterial segments derived from the pharyngeal arches (blue, third arch; purple, fourth arch; green, sixth arch). E. Relative contribution of neural crest cells (blue) in the wall of the great arteries showing that there are mixed wall structures as well as sharp boundaries. F and H. Species variation in pharyngeal arch remodeling in the chicken (right arch), and the mouse and human (left arches). I. Development of a right-sided anomalous subclavian artery occurs when the right fourth arch segment disappears (asterisk) and the right dorsal aortic segment persists. Abbreviations: AoSac, aortic sac; Ao, ascending aorta; CoA, aortic coarctation; DesAo, descending aorta; DA, ductus arteriosus; PA, pulmonary artery; PT, pulmonary trunk; RCA/LCA, right/left carotid artery; RDA, right dorsal aorta; RDAo/LDAo, right/left descending aorta, RSA/LSA, right/left subclavian artery. Adpated from: A-D: From Molin DG, et al. Cardiovasc Res. 2002; 56(2):312-22, by permission of Oxford University Press. Reprinted with permission from Molin DG, et al. Birth Defects Res A Clin Mol Teratol. 2004; 70(12)927-38.

The contribution of SHF and neural crest cells to the pharyngeal arch arterial system is mainly to the differentiation of the smooth muscle layer, whereas the neural crest cells also provide for the innervation of the various segments. The contribution of SHF and neural crest cells differs within the aortic arch system, as there are precise boundaries to the contributions of neural crest cells. The ascending aorta and pulmonary trunk, including their roots, are of mixed origin. The ductus arteriosus is completely derived from neural crest cells with a sharp boundary to the descending part of the thoracic aorta that lacks neural crest cells (Figure 1-7E). The pulmonary and subclavian arteries probably have a complete SHF origin.

Implications for Congenital Cardiovascular Disease

Coarctation and aortic arch malformations. A number of abnormalities may occur during the extensive remodeling of the aortic arch system. However, many are able to sustain the prenatal circulation. Based on variable SHF and neural crest cell contribution and ensuing hemodynamic alterations, some sites are especially vulnerable to develop abnormal anatomy. In humans, the most common site is located in the fourth arch artery, leading to interruption or hypoplasia of the B-segment (Figure 1-7H) resulting in aortic interruption. The causal factor in this anomaly is linked with the SHF and TBxl disturbances, combined with the resulting alterations in blood flow. Dominance of the right-sided fourth arch artery may lead to a right aortic arch with mirror image branching of the carotid and subclavian arteries. The A-segment, or isthmus, located in the aortic arch between the left subclavian and entrance of the ductus arteriosus or its ligament (Figure 1-7H), can also be hypoplastic. The most common abnormality in this segment is the localized aortic coarctation, in which the contribution from the left-sided ductus arteriosus is obvious. Normally, the ductus arteriosus closes after birth, but if the ductal (muscular) tissue extends abnormally into the aortic arch, its constriction at birth can increase the severity of the coarctation (Figure 1-8A, B).

FIGURE 1-8. Aortic coarctation. Schematic depiction of an aortic coarctation involving muscle of the ductus arteriosus A. Ductal tissue with thick intimal cushions (depicted in blue) completely encircles the aortic arch. B. Histological section showing the extension of ductal tissue covering most of the endothelial surface of the aortic wall. Abbreviations: Ao, aorta; DA, ductus arteriosus; PT, pulmonary trunk.

Anomalous origin of the subclavian artery. A relatively common abnormality is seen when the right subclavian artery does not reach its proper cranial position before the right dorsal aortic segment disappears. Rather than arising from the right innominate artery, it arises from the descending aorta, distal to the left subclavian artery. In the presence of an abnormal right-sided aortic arch, the left subclavian artery can arise anomalously (Figure 1-7I).

Valvulogenesis

Both semilunar and atrioventricular valves derive from their respective endocardial cushions. This implies that defective endocardial-mesenchymal transition, essential for proper valve differentiation, can be linked to abnormal valve structure. However, both in humans and in animal models, only a few genes have been linked to deficient valve tissue (eg, NOTCH1, NFATC1). Since the origin of the semilunar and atrioventricular valves differs with respect to SHF and FHF and they receive different contributions from neural crest cells and epicardium-derived cells, the normal development of the respective valves is described below.

Atrioventricular Valves

The fibrous tissue of the atrioventricular valves derives from the endocardial cushions in the atrioventricular canal, including the superior and inferior cushion and a small lateral atrioventricular cushion on each side. The central portions of the superior and inferior atrioventricular cushions fuse, separating the tricuspid and mitral orifices. The tricuspid valve consists of three leaflets (anterior, posterior, and septal), while the mitral valve has two leaflets (aortic and lateral leaflet). The atrioventricular cushions initially adhere to the myocardium of the atrioventricular canal (Figure 1-9A) but separate by a process that is not well understood (Figure 1-9B). The free rims of the developing leaflets remain connected by endocardial cushion-derived chordae tendinae to the papillary muscles of the respective ventricles. The mesenchymal content of the valves is derived by endocardial-mesenchymal transformation from the underlying endocardium. Epicardium-derived cells also migrate from the surface of the heart to the area of the annulus fibrosis, separating the atrial and ventricular myocardium, and into the endocardial valve tissue (Figure 1-9B). In the cushions, epicardium-derived cells are found mainly in the developing parietal (and not septal) leaflets, a pattern potentially leading to the preferential distribution pattern of affected valve leaflets in anomalies like Ebstein malformation and mitral valve prolapse.

FIGURE 1-9. Atrioventricular valve formation. A. The atrioventricular valves develop form the atrioventricular endocardial cushions (depicted in light blue). B. With dissociation of the embryonic atrial and ventricular myocardium, the epicardium-derived cells from the atrioventricular epicardial sulcus (depicted in dark blue) migrate into the endocardial cushions providing the fibroblasts for the annulus fibrosis and a population within the cushions. The cushions form the definitive valve leaflets by delamination from the wall as well as the chordae tendinae that are attached to the papillary muscles. C. Ebstein anomaly of the tricuspid valve. The anterior tricuspid valve leaflet is rather well developed, whereas the septal and posterior leaflets have not delaminated from the ventricular wall. Abbreviations: AL, anterior (tricuspid valve leaflet); AVCu, atrioventricular endocardial cushion; ES, epicardial sulcus; RV, right ventricle.

Semilunar Valves

Both the aortic and the pulmonary semilunar valves derive from the septal and parietal cushions of the outflow tract. Neural crest cells enter the endocardial cushions at the border of the arterial wall and myocardium. At the same time, endocardial-mesenchymal transformation of the endocardial lining is essential to bring in the mesenchymal cells containing the cushion. This process is more prominent in the cusp side facing the aorta. Arterial epicardial cells have recently been identified in the developing valve leaflets. Additionally, two intercalated cushions develop each in the aortic and pulmonary part of the outflow tract (Figure 1-10A). It is not known whether these emerge from the main cushions or whether there are differential contributions of SHF, neural crest cells, and endocardium to the respective cushions and leaflets. The latter is important for understanding malformations like bicuspid aortic valve (Figure 1-10B).

FIGURE 1-10. Outflow tract valve development. A. Schematic representation of the two main outflow tract endocardial cushions (light blue) that line the myocardial outflow tract, showing a saddle-shaped border with the vascular wall of the aortic sac. As a result of inward migration of neural crest cells (indicated as dark blue dots), the cushions are separated into future aortic and pulmonary valve leaflets. In both orifices, the third leaflet arises from an intercalated cushion (green). This process results in two facing coronary aortic leaflets and a nonfacing or noncoronary leaflet. The pulmonary valve leaflets have the same configuration but lack the coronary orifices. B. Postmortem specimen showing a bicuspid aortic valve. Abbreviations: EC, endocardial cushions; IC, intercalated cushion; NCCs, neural crest cells.

Implications for Congenital Cardiovascular Disease

Overriding and straddling atrioventricular valves and double-inlet left ventricle. The tricuspid orifice becomes positioned above the right ventricle during remodeling of the inner curvature of the heart, which is closely linked to the repositioning of the outflow tract. Initially, there is only a slit-like inlet portion of the right ventricle, and the right side of the atrioventricular cushions is contained within this area. If the remodeling or shift of the tricuspid orifice is incomplete, a ventricular septal defect remains. The valve overrides the septal defect; occasionally, there is actual straddling in which the tricuspid valve has chordal attachments into both ventricles. The ventricular septal defect is characteristically in a posterior or inlet position. If more than 50% of the tricuspid valve remains connected to the left ventricle, the resulting anomaly is called doubleinlet left ventricle, which a common form of physiologic single ventricle. It is much less common for the mitral valve to straddle the ventricular septum. This is usually associated with transposition of the great arteries, in which case the ventricular septal defect is located anteriorly.

Stenosis or atresia of an atrioventricular valve. The complete atrioventricular canal is originally connected to the primitive left ventricle (Figure 1-1C). In the absence of a connection of the atrium to the developing right ventricle, the tricuspid orifice is atretic. It is seen as a myocardial dimple in the right atrium. In these cases, the right ventricle has only trabecular and outflow tract portions, lacking the inlet portion.

Ebstein anomaly of the tricuspid valve. This anomaly is caused by deficient delamination of the valve leaflets. The amount of nondelamination is highly variable, from minimal inferior displacement of the hinge point of the leaflet to severe displacement with almost complete atrialization of the right ventricle. The displacement is mainly of the origins of septal and posterior tricuspid leaflets from the atrioventricular junction toward the right ventricular apex. The anterior tricuspid leaflet is usually not displaced. Redundant tissues and fenestrations of the anterior leaflet are frequently observed.

Bicuspid and unicuspid semilunar valves. A bicuspid aortic valve (Figure 1-10) is the most common congenital cardiovascular malformation, encountered in about 1% to 3% of the human population. The valve often functions normally at younger ages. In some cases, this malformation, as well as a unicuspid valve, may also present in the neonatal period because of significant aortic valve dysfunction (usually stenosis).

Conduction System Development

The atrioventricular conduction system differentiates from a cardiomyocyte origin. The atrial part of the conduction system is derived from the posterior SHF- derived myocardium, which is devoid of Nkx2.5 expression. This includes a transient left sino-atrial node and the definitive right sinus node. Expression of markers reflecting a pacemaker phenotype (eg, Tbx3, Podoplanin, and Shox2), as well as histological features, indicates that the developing cardiac conduction system covers the entire sinus venosus area during early embryonic stages, including the internodal myocardium as well as the myocardium surrounding the pulmonary veins. During later development stages, expression is restricted to the definitive elements of the conduction system. The FHF (atrioventricular canal myocardium) and the primitive (left) ventricle appear to contribute to the compact part of the atrioventricular node and the main parts of the ventricular conduction system, including the common bundle of His and the left and right bundle branches. Animal models have shown that proper development of the fibrous annulus requires the presence of epicardium- derived cells. If the epicardial outgrowth is inhibited, a phenotype with accessory pathways and pre-excitation occurs.

Implications for Arrhythmias

Embryonic conduction pathways that normally disappear may persist both in structure and in function. This leads to abnormal atrial automaticity at specific sites, including the terminal crest (formerly the right venous valve), the pulmonary venous myocardium, and the coronary sinus. The occurrence of congenital cardiovascular disease with a combination of nondelamination of the tricuspid valve, noncompaction cardiomyopathy, and accessory pathways with pre-excitation, as is observed in Ebstein anomaly and transposition-transposed great arteries, could be related to abnormal epicardial contributions or deficient epicardial-myocardial signaling during development.

Development of the Epicardium, Myocardium, and Coronary Arteries

Initially, the ventricular myocardium is oxygenated by diffusion from the ventricular lumen. A separate structure for gas exchange becomes necessary as the myocardium grows. In early development, an epicardial covering over the myocardium is essential for normal coronary vascular development. However, the epicardium-derived cells also fulfill an important role in driving compaction of the outer myocardial layers of the ventricle, specifically the anterior part of the folding septum. The coronary endothelial cells arising from the sinus venosus spread in the subepicardial space and invade the compacting myocardium. In the peritruncal area, they form an endothelial ring surrounding the aorta and pulmonary orifice. The endothelial cells also invade the aortic wall and form the main stems and orifices of the coronary arteries in the pulmonary artery-facing (left and right) sinuses of Valsalva. The factors guiding this process as well as those that repel them from entry into the pulmonary wall and from the noncoronary aortic cusp remain to be defined.

Implications for Congenital Cardiovascular Disease

Myocardial thinning and noncompaction. The myocardial thinning based on deficient epicardial-myocardial interaction resembles noncompaction cardiomyopathies. Experiments in both avian and mouse models in which the epicardial cell population has been limited in its outgrowth and endocardial-mesenchymal transition lead to severe thinning of the compact myocardium and a spongy interventricular septum. Whether the basis of these malformations is epicardial or myocardial in humans is unknown.

Coronary vascular anomalies. In anomalies such as transposition of the great arteries, the main coronary stems take the shortest course to the facing sinus of Valsalva, explaining the observed variation in patterning. When epicardial outgrowth and spreading is inhibited, single or pinpoint arterial orifices are observed, suggesting that epicardium-derived cells are necessary for proper connection of the coronary arteries to the aorta. Ventricular- coronary artery connections (Figure 1-11 A) or fistulae develop if there is no connection of the coronary arteries to the aorta. In human congenital cardiovascular disease that includes coronary abnormalities, an epigenetic cause of the supposed link between disturbed epicardium and ventricular-coronary arterial connection is still missing. It has been suggested that the occurrence of connections and coronary orifice pathology in a subgroup of the patients with pulmonary atresia and intact ventricular septum is of epicardial origin (Figure 1-11B, C).

FIGURE 1-11. Ventricular-coronary arterial communications. A. Schematic representation of a heart with pulmonary atresia with intact ventricular septum. The right coronary artery has a pinpoint orifice (arrowhead), and the main subepicardial coronary branches are severely thickened and in part obliterated (arrows). There are ventricular-coronary arterial communications between the diseased coronary arteries and the hypoplastic right ventricular lumen, which is common in this condition. B and C. Sections of a human fetal heart with pulmonary atresia and intact ventricular septum showing that the severe coronary arterial pathology in the subepicardial region is already present in the fetus. The ventriculo-coronary arterial communications are primarily a coronary arterial (epicardium derived) disturbance and not primarily of myocardial origin. Magnifications: Bars, B and C: 1 mm. Abbreviations: CA, coronary artery; SEP, subepicardial region; VCAC, ventriculo-coronary arterial communications. Adapted from Gittenberger-de Groot AC et al. Prog Pediatr Cardiol. 2010;29(1):3-9.