Development of the heart and circulation

Endocardial cushions

The extracellular matrix of the embryonic heart (see Fig. 13.3C–D ), historically termed cardiac jelly, is composed of hyaluronic acid, hyaluronidase and fibronectin among other components. Inductive signals originating from the cardiac myocytes, such as BMP2 and TGF-β, lead to the activation of a subset of endocardial epithelial cells that will line the future atrioventricular canal and the proximal outflow tract; in the future left ventricle, endocardial cells do not undergo such an activation. The activated endocardial cells lose their cell-to-cell connections and undergo an epithelial to mesenchymal transition. Their expression of neural cell adhesion molecule (N-CAM) decreases and expression of substrate adhesion molecules such as chondroitin sulphate and fibronectin increases. Expression of type I procollagen is initiated and cytoskeletal re-arrangements facilitate cell migration into the cardiac extracellular matrix where they continue to proliferate. The cells and the locally accumulated extracellular matrix molecules, form the endocardial or cardiac cushions. Endocardial cushions initially function as valves in the embryonic heart: they give rise to the valves of the atrioventricular canal and the outflow tract and also supply the mesenchyme that is needed to complete the septation of the atria, ventricles and the outflow tract. This particular epithelial to mesenchymal transition may be the only example of a mesenchymal population derived from an endothelial lineage; endothelial to mesenchymal transformation may be relevant to later circulatory pathologies ( , ).

Looping of the heart tube

The early primary heart tube is initially bilaterally symmetric and has the form of an inverted ‘Y’. The caudal ‘legs’ of the Y are positioned on either side of the cranial intestinal portal, where they form the venous pole of the heart ( Fig. 13.4A–C ). The rostral ‘stem’ of the Y connects cranially with the future aortic arch arteries, forming the arterial pole of the heart. From the start, both poles are connected with the systemic vessels that run from the embryo, yolk sac and placenta. The pulmonary veins form later, their initial venous primordium becoming evident in the dorsal mesocardial mesenchyme. Myocardium from the secondary heart-forming field continues to be added to the heart, forming the smooth dorsal wall of the left atrium, and providing the site where the primary atrial septum will form.

The putative cardiac segments are not present in the early linear heart tube. The definitive cardiac chambers are formed by local differentiation and expansion, or ‘ballooning’, of the myocardial walls of the primary heart tube, indicating that cardiac myocyte identity is dependent on genetic, spatial and temporal signals and lacks a fixed ontogeny. Genetic fate map studies have shown that the primary heart tube will contribute to only two compartments: the legs of the inverted Y of the primary heart tube contain precursor cells of the atrioventricular canal, while the stem of the inverted Y contains myocardial precursor cells of the left ventricle ( ). The atrioventricular canal precursors finally reside in the left ventricular free wall and the cells of the original embryonic left ventricle largely end up in the left face of the ventricular septum. The embryonic outflow tract contributes to the right ventricle.

The nodal/pitx2/cited-2 signalling pathway that determines the formation of the morphologically left-sided or right-sided features in the lungs, bronchial tree, liver and spleen is also expressed in the heart outflow tract and atrial appendages ( ). The lengthening heart tube bends ventrally and rightwards, concomitant with the partial breakdown of the dorsal mesocardium. This bend is now named the ventricular loop because the left ventricle will balloon from its outer curvature, the original ventral side of the straight tube; the inner curvature of this loop is the original dorsal side.

At the start of stage 10 the venous pole, the atrioventricular canal, the developing left ventricle and the outflow tract are all positioned symmetrically around the midline ( Fig. 13.5A ). As a result of the subsequent rapid lengthening of the cardiac tube, the atrioventricular canal moves in its entirety to the left ( Fig. 13.5B ), and the ventricular part of the heart tube loops to the right, thus placing the developing left ventricle on the left and the forming right ventricle on the right side of the midline of the body. The atrial floor, including the developing systemic sinus venosus and the atrioventricular canal moves to the right ( Fig. 13.5C ). The developing pulmonary vein remains anchored in the midline and after these manoeuvres, the atrioventricular canal again becomes positioned in the midline; these movements facilitate the appropriate connections of the developing muscular ventricular septum with the atrioventricular and outflow cushions. The orifice of the sinus venosus is now positioned to the right. From the outset, the muscular ventricular septum develops in line with the right side of the dorsal atrioventricular cushion so that the right atrium always has direct access to the developing right ventricle.

Cardiac gene expression, differentiation, contraction, conduction and automaticity

Cardiac differentiation and morphogenesis are intimately related processes, achieved by a still obscure transcriptional network. It is known that the cardiac transcription factor Nkx2–5, which has a homogeneous distribution over the embryonic heart tube, is generally important for cardiac-specific gene expression. In the human, mutations in this factor cause atrial septal defects and disturbances of atrioventricular conduction; in Drosophila , the heart will not form in its absence. The discovery of the involvement of locally expressed T-box transcription factors, provides some evidence of the further complexity of the patterning of the heart ( Fig. 13.6 ). For example, whereas Tbx5 confers caudocranial positional information over the heart tube, determining the regional development of the cardiac chambers along these axes, Tbx2 and Tbx3 prevent regional formation of the cardiac chambers, permitting the myocardium in these regions to differentiate into the conduction system. Tbx18 is involved in the development of the sinus venosus while Tbx1 is necessary for the development of the outflow tract of the heart.

Most cardiac myocytes cease to divide after their differentiation to myocardium ( ). The primary heart tube continues to elongate as additional myocytes are recruited from the secondary heart-forming field. Hypertrophy of cardiac myocytes is not thought to be part of the process in this elongation as in mouse embryos, cardiac myocyte volume remains at approximately 2 ×10 ^-3 μm ³ throughout gestation ( ). Cardiac myocytes share a number of characteristic features that distinguish them from other cells: they are mononuclear, have sarcomeres and a sarcoplasmic reticulum and are joined to adjacent myocytes by the gap-junctional proteins connexin 40 and 43 ( ). The expression of sarcomeric proteins is seen in primitive cardiomyocytes in stage 9 embryos. All cardiac myocytes share the capacity for producing an intrinsic cycle of electrical activity, i.e. the capacity to generate and conduct a depolarizing impulse that results in contraction (the cells are myogenic): the phenomenon is called automaticity, or pacemaker activity. Because the cells are electrically coupled the fastest pacemaking activity will take the lead. In embryonic hearts, the leading pacemaker is always found at the venous pole which corresponds to the sinuatrial node in the adult heart. There is evidence for the presence of a tertiary heart-forming field specific for pacemaker formation ( ).

All of the cardiac conduction system is myocardial in origin. The nodes, sinuatrial and atrioventricular, and the atrioventricular bundle constitute the central conduction system, while the bundle branches and their terminal ramifications, the Purkinje fibres, represent the peripheral ventricular conduction system. The development of the conduction system of the heart is intimately related to overall heart formation and the blood flow through the heart, embryo and placenta. The underlying gene-regulatory networks have been conserved during vertebrate evolution. Each phase of heart development shows different patterns of spontaneous electrical activation and contraction ( ).

Varying degrees of differentiation seen in early populations of cardiac myocytes can be categorized as forming primary, nodal, conducting and working myocardium ( Table 13.1 ). There is a relationship between the degree of apparent differentiation and extent of the coupling between myocytes, and the occurrence of slow or fast wave propagation and corresponding contraction. All regions of the early embryonic heart tube have poorly coupled cells and display intrinsic automaticity ( Video 13.1 ). This produces slow wave propagation of the depolarizing impulse along the heart tube and matching peristaltic waves of contraction that push the blood in an antegrade direction. That part of the early heart tube wall termed primary myocardium retains this phenotype; it shows poorly developed sarcoplasmic reticulum and sarcomeric structures. Poor electrical coupling and slow conduction seen in nodal myocytes is an absolute requirement for effective pacemaking because it allows the cells to build up sufficient electrical charge, which is then propagated through the surrounding myocardium. Cells that differentiate into working myocardium display virtually no automaticity. They have well developed sarcomeres and sarcoplasmic reticular structures, and contiguous cells show well developed gap junctions expressing connexins 40 and 43. The cells of the putative atrioventricular and peripheral ventricular conduction system have an ambiguous phenotype: they are well coupled, thus allowing fast conduction of the depolarizing impulse, but otherwise retain an embryonic phenotype. Myocytes of the atrioventricular bundles and Purkinje fibres have a phenotype between that of working and primary myocardium. Distinguishing conduction myocardium from working myocardium may suggest that the working myocardium does not conduct, while the conduction system conducts rapidly. However, to produce powerful synchronous contractions, the working myocardium must also conduct rapidly. Unambiguous morphological markers are often lacking in the early embryonic heart, which means that it is not possible to distinguish the various cells phenotypically; automaticity and the speed of conduction have therefore become important functional parameters with which to describe the development of the different parts of the heart.

Peristaltic hearts do not need valves, whereas chambered hearts require the presence of one-way valves at the inlets to, and the outlets from, the chambers. During development, specific regions of mesenchyme and cardiac extracellular matrix fulfil this function. The sequential arrangement of slow and fast conduction portions of the early myocardium is key to its early development. The fast conducting working myocardium of the atrial and ventricular chambers permits nearly simultaneous contraction, increasing the velocity of the forward pressure of blood. The slow conducting primary myocardium within the atrioventricular junction and outflow tract is adjacent to endocardial cushions formed from locally accumulated cardiac extracellular matrix. These regions have longer, slower closure and are thus able to function as sphincteric valves, preventing retrograde flow of blood. The longer closure permits discrete and successive contraction of the atrial chambers followed by the ventricular chambers: contraction in the ventricles at these early stages passes from base to apex. Conduction myocytes originate from part of a primary interventricular ring that becomes positioned at the atrioventricular fibrous ring, close to the interventricular septum in the adult heart (see Fig. 13.10 ). For further reading about the electrophysiology of the developing chick heart, see .

Inflow tract

Initially, blood flows into the heart via a sinus venosus, a confluence of bilateral umbilical, vitelline and cardinal venous channels (see Fig. 13.1 ). The sinus enlarges forming right and left sinus horns; a process under the control of the T-box transcription factor Tbx18. The common cardinal veins, the main contributors to the sinus horns, become surrounded by cardiomyocytes ( ). With later development of the septum transversum and hepatic blood vessels, modifications of the cardinal system of veins and the terminal portions of the vitelline and umbilical veins form a main hepatocardiac channel that directs blood from the caudal part of the embryo towards the right horn of the sinus venosus ( Fig. 13.8 ; see Figs 13.17 , 13.21 , 13.22 ). The sinus wall is thickened dorsally where the right and left sinus horns meet: this is the sinus septum that will give rise to the roof of the orifice of the future coronary sinus.

Sinuatrial junction

The sinus venosus becomes incorporated into the right atrium proper (see Fig. 13.8C ), distinguished from the common atrium by a sinuatrial valve. The growth of the sinuatrial valve is slower than that of the surrounding myocardium and the valve leaflets lose their competence over time. The sinuatrial orifice becomes elongated and slit-like because of this differential growth. The two sinuatrial valve leaflets that flank it meet cranially to form the septum spurium. Later, a cranial part of the right sinuatrial valve leaflet, caudal to the septum spurium, becomes continuous with the crista terminalis, a ridge that projects from the atrial roof. More caudally, the right sinuatrial valve leaflet forms the valve of the inferior vena cava (Eustachian valve) and of the original left superior vena cava, later the valve of the coronary sinus (Thebesian valve). When this process is disturbed, a venous network (Chiari’s network) can develop from the right sinuatrial valve leaflet ( ).

As development proceeds, the left-sided systemic venous tributaries diminish in size. The left common cardinal vein (later the left superior vena cava) forms the oblique vein of the left atrium (also known as the oblique vein of Marshall), while the remainder of the left sinus horn is incorporated into the left atrioventricular junction forming the coronary sinus (see Fig. 13.8C ). The coronary sinus retains its own myocardial wall, histologically distinct from the left atrial wall, although some merging may take place.

At the sinus venosus side of the sinuatrial junction, cardiac nodal myocytes form a specialized sinuatrial node able to initiate the electrical impulses that activate the atrial myocardium and heart, and lead to its contraction. This node consists of distinct morphological regions and is supplied by a relatively large artery. It forms under the influence of Tbx18 and its differentiation is regulated by Tbx3. Later, when the dorsal wall of the sinus venosus lies in line with the atrial myocardium, lateral to the sinuatrial valve leaflets, and the sinuatrial valve has lost its competence, this region is termed the sinus venarum: it is smooth-walled and forms the dorsal wall of the right atrium. In adult humans, the sinus venosus myocardium contracts simultaneously with that of the right atrium and can therefore be considered to be incorporated functionally into the right atrium.

Common atrium

The working myocardium of the primitive atrium differentiates at the dorsal and lateral sides of the primary heart tube ( ) (see Fig. 13.4D–G ). The developing atrium then expands, ballooning in dorsal, lateral and cranial directions. The cranial expansion is pouch-shaped and forms the left and right atrial appendage (see Fig. 13.7A–B ). Although it is possible to distinguish the left from the right atrial appendage around stage 14, the right being more extensive than the left, the atrium itself contains only a single cavity at this stage. The onset of primary septum formation is initially visible in the atrial roof. During atrial septation, the sinuatrial junction moves from a symmetric, midline position to a right-sided position (see Figs 13.5 , 13.8 , 13.20 ).

Left atrium

Following the formation of the left atrial appendage and the primary atrial septum, the left atrium takes shape by the incorporation of mediastinal myocardium. While the tributaries of the sinus venosus approach the right atrium caudally and cranially, the differentiating pulmonary veins gain their entrance to the left atrial cavity through the dorsal mesocardium (see Fig. 13.3 ). The definitive topographical relationships between vessels and heart seen postnatally are therefore only established once differentiation of the pulmonary venous portal occurs.

Around stage 14, simultaneously with atrial septation, the pulmonary vein develops as a solitary channel from angiogenic cells derived from the dorsal mesocardium. This channel establishes continuity with the vascular plexus formed in the mediastinal mesenchyme around the developing lung buds. The initially solitary pulmonary vein, guided by Semaphorin 3d signals, later opens into the caudo-dorsal wall of the atrium adjacent to the developing atrioventricular junction, where a depression termed the pulmonary pit, flanked by the pulmonary ridges, has already been established ( ).

The primary atrial septum develops from the right pulmonary ridge, confining the pulmonary venous orifice to the developing left atrium (see Fig. 13.11 ). The pulmonary vein initially branches within the dorsal mediastinal mesenchyme, its tributaries draining blood from the developing lung. With continuing development, the walls of the pulmonary venous channels become surrounded by myocardium; this process occurs up to and beyond the level of the second bifurcation. The veins then expand and their walls become incorporated into the roof of the left atrium, eventually forming the greater part of its cavity. The four pulmonary veins do not achieve their separate opening into the atrial roof until well after septum formation is completed, around postmenstrual week 11. The left half of the primary atrium becomes progressively restricted to the mature appendage. The myocardial sleeves that surround the pulmonary orifices taper off distally and become intermingled with fibrous tissue. In adult life, it is likely that this intermingling of myocardial and fibrous tissues forms the substrate for some forms of atrial fibrillation. Variations in the precise pattern of pulmonary venous drainage are quite common.

Anomalous pulmonary venous connections and cor triatriatum sinistrum

The developing common pulmonary vein migrates towards the heart before connecting to the pulmonary pit, but the correct connection may not occur ( ). In total anomalous pulmonary venous connection, which occurs in about 7/100,000 neonates, no pulmonary veins enter the left atrium ( ) and the more primitive connection between the splanchnic plexus that surrounds the lungs with the right atrial inflow tract persists ( ): these vessels do not become surrounded by myocardium ( ). In case of a partial anomalous pulmonary venous connection, one or more connections with the left atrium will be established alongside this primitive conjunction between cardinal veins and the splanchnic plexus. It has been suggested that these anomalies occur when there is a defect in the Semaphorin 3d signalling that would normally act as a repelling guiding factor ( ).

In cor triatriatum sinistrum, a very rare anomaly, the left atrium is subdivided into two compartments separated by a full or partial membrane. The pulmonary venous confluence is guarded from the rest of the atrial body. There is evidence that mutations in the enzyme HYAL2 can lead to cor triatriatum sinistrum in humans ( ). The membrane might be an outgrowth of a ridge that is naturally found between the left superior pulmonary vein and the atrial appendage. Often this defect coincides with a septal defect, anomalous pulmonary venous connections or a patent ductus arteriosus.

Ventricles

The ventricles develop from the ventral side of the looping primary heart tube (see Figs 13.4 , 13.7 ). The left ventricle develops from the rostral stem of the inverted, Y-shaped primary heart tube. The right ventricle develops later, downstream relative to the left ventricle, after more myocardium has been added to the cardiac tube from the secondary heart-forming field. As a consequence of the heart tube looping, the right ventricle is positioned to the right of the left ventricle, which is a prerequisite for forming the appropriate connections with the expanding atrial component of the heart. Unlike the atrial chambers, the morphological differences between the right and left ventricles are not part of the general asymmetry between the right- and left-sided organs of the body, but under the control of signalling pathways that determine caudocranial differentiation, such as retinoic acid and its downstream transcription factor Tbx5, which play a crucial role in this process. The Hand transcription factors also play a role in left ventricular growth and endocardial cushion maturation ( ).

Formation of the ventricular wall

The myocardium at the inner curvature of the looped heart, which is the original dorsal side of the primary heart tube, remains smooth-walled, whereas the myocardium at the outer curvature of the myocardial tube displays trabeculae as early as stage 10 (see Fig. 13.7C ). By stage 17 the trabeculae have achieved a typical spatial organization, giving a sponge-like appearance to the internal aspect of both ventricles. Embryonic trabeculae are a few tens of micrometres in width, and diffusion with early luminal blood is sufficient to maintain cardiac cellular homeostasis, which means that the increase of cardiac mass by trabecular addition can continue in the absence of a coronary circulation ( Fig. 13.9 ). Trabecular cardiomyocytes proliferate much less and exhibit more mitochondrial activity than compact wall cardiomyocytes, and are likely to be the main contributor to pressure generation. As development proceeds, a portion of the embryonic trabeculae on the luminal side of the ventricles stops proliferating and differentiates into the fast-conducting peripheral ventricular conduction system that includes the bundle branches and the Purkinje fibres ( Fig. 13.10 ). A further portion, from the level of the atrioventricular junction to the apex of the heart, forms thicker adult trabeculae; the trabeculae in the right ventricle are coarser compared to those in the left. Over time the outer layer of ventricular myocardium will become highly proliferative and will form the thick compact layer of the ventricular wall.

Embryonic ventricular myocardium, encompassing both the trabeculae and exterior wall, possesses a specific molecular chamber phenotype: the myocytes express, among other proteins, the fast-conducting gap-junctional proteins connexin 40 and 43, and atrial natriuretic factor. During development, only the peripheral ventricular conduction system retains this molecular phenotype. The ventricular working myocardium loses connexin 40 and atrial natriuretic factor expression while retaining low expression of connexin 43. Changes in the expression of connexin 40 and 43 have been correlated with morphological development of the fetal heart. The increasing complexity of the helical disposition of ventricular myocardial fibres has been studied using high resolution diffusion tensor magnetic resonance imaging ( ).

Left ventricular noncompaction

In left ventricular noncompaction, the left ventricle exhibits particularly prominent trabeculae as compared to the compact wall. Although most cases of left ventricular noncompaction are thought to be developmental in origin, the process behind acquisition of prominent trabeculae is not clear: variation in trabecular prominence is seen during adult life, for example in pregnant women ( ). The condition can reflect failure of the trabeculae to compact, or excessive trabecular formation, or hypoplasticity of the compact wall ( , ) and can be associated with heart failure. Its uncertain aetiology is reflected in the diversity of current diagnostic criteria ( ).

Outflow tract

The outflow tract of the heart (truncus arteriosus) is formed by cells derived from the secondary heart-forming field and is confluent with the right ventricle. Initially, the outflow tract myocardium extends up to the pericardial reflections. As development proceeds, continuous addition of non-myocardial cells changes the position of the myocardium and cardiac extracellular matrix (cardiac jelly) relative to the outflow tract bend, a conspicuous morphological feature in the embryonic heart. The outflow tract is therefore a highly dynamic structure along which cells pass to contribute to the definitive right ventricle. By definition, the outflow tract is downstream of the ventricular loop, irrespective of the fact that, eventually, it will largely become incorporated within the definitive right ventricle (see Figs 13.4 , 13.7 ). The method of incorporation of the outflow tract into the heart is different to that of the sinus venosus, because it involves movement of a structure, whereas the sinus venosus changes identity but otherwise shows no relative movement.

Within the inner curvature of the ventricular loop, the walls of the outflow tract and atrioventricular canal fade into one another without a clear boundary. It is within this ventricular part of the primary heart tube that the cushions of the atrioventricular canal and the outflow tract must achieve appropriate connections with the ventricular septum in order to separate the pulmonary trunk and the aorta.

Septation of the atrial chambers

Atrial septation is a process in which a valvular structure develops that initially directs the flow of placental blood to the body and allows separation of the two atrial chambers immediately after birth. It is typically described in terms of two septa that separate the endocardial and myocardial walls of the common atrium and two foramina that ensure continuous blood flow. Atrial septation starts at about stage 13 with proliferation of a crescentic ridge of mesenchyme in the atrial roof. This ridge, the septum primum, grows by active proliferation from the craniodorsal atrial roof towards the atrioventricular canal, just to the right of the entrance of the pulmonary vein (pulmonary pit) ( Fig. 13.11A,D ). Its leading edge is covered by a mesenchymal cap which is continuous with extracardiac mesenchyme (mediastinal mesenchyme) derived from the dorsal mesocardium that forms a dorsal mesenchymal protrusion. Under the influence of BMP signalling, the ventral horn of the crescentic septum primum will touch the ventral atrioventricular cushion, while the dorsal horn will fuse with the dorsal atrioventricular cushion. The primary atrial foramen (foramen or ostium primum) is therefore entirely surrounded by mesenchyme, subjacent to the atrial endocardium. Caudal to the advancing edge of the septum primum, the developing atrial chambers still communicate through the foramen primum (see Fig. 13.11A,D ). The foramen primum diminishes in size as a result of continuous growth of the muscular primary atrial septum and the cranial part of the septum perforates by apoptosis. The perforations coalesce, producing a secondary atrial foramen (foramen, or ostium, secundum) that allows continued shunting of oxygen-enriched blood from the placenta to the left side of the heart and so to the head and brain. The foramen secundum forms during stages 15–17. The foramen primum is occluded in the median plane by stage 17, when the septum primum mesenchyme merges with the fusing atrioventricular ( Fig. 13.11B,E ). This tissue later differentiates to myocardium and forms a ridge that will become the limbus of the future fossa ovalis.

The secondary atrial septum (septum secundum) is reported to appear during stages 18–21 ( ), signalling the earliest development of the foramen ovale. , however, note that invagination of the roof of the right atrium, forming a myocardial ridge, constitutes the septum secundum, and that this is not present until after stage 23 (see Fig. 13.11B,E ). The free edge of the septum secundum overlaps the foramen secundum forming an oblique channel, the foramen ovale, between the now divided atria. Once these septa have formed, blood passes under the crescentic edge or limbus that is the crista dividens of the septum secundum, and flows obliquely towards and through the foramen secundum to the left atrium. This passage through the foramen ovale, persists throughout intrauterine life (see Video 13.2, Video 23.1 ). In postnatal life, when left atrial pressure exceeds that in the right atrium, the septum primum will close against the septum secundum as a type of flap valve, completing atrial septation ( Fig. 13.11C,F ). On the right side of the foramen ovale, part of the septum primum remains underneath the rim of the septum secundum and becomes the fossa ovalis. Fig. 13.12 summarizes all the anomalies of septation.

Septation and appropriate positioning of the atrioventricular canal

The atrioventricular canal is initially a continuation of myocardium between the common atrium and the primitive ventricle. Four cardiac cushions are formed at the margin of this canal, namely: the left and right cushions, and the much larger ventral and dorsal cushions. The single orifice of the atrioventricular canal is divided by the approximation and later merger of the ventral and dorsal cushions. The dorsal atrioventricular cushion runs along the floor of the atrium. As it extends further dorsally it meets the dorsal mesenchymal protrusion (spina vestibuli) that connects with the mesenchymal cap that runs along the leading edge of the septum primum until it meets the ventral atrioventricular cushion (see Fig. 13.11 ). The dorsal atrioventricular cushion has a ventricular extension to the inner curvature of the heart tube that comes to lie on top of the developing muscular ventricular septum: much of the membranous septum of the ventricles is derived from this extension.

The two atrioventricular cushions fuse during stages 14–17, at which time they become known as the central fibrous body. Relative to the canal itself, the cushions are large, leaving narrow right and left slits for the passage of blood ( Fig. 13.13 ). At the same time as the cushions fuse, the canal expands. This is seen most clearly on the lateral right side where the size of the right atrioventricular opening increases. Relative to the heart, the entire atrioventricular canal also shifts to the right (see Fig. 13.5 ), causing the dorsal cushion to overhang the ventricular septum into the developing right ventricle while the ventral cushion remains mostly on the left side.

Septation of the ventricles

Three distinct structures contribute to the formation of the ventricular septum: the atrioventricular cushions (including the protrusion of the dorsal atrioventricular cushion that lies on the ventricular septal crest), the muscular ventricular septum and the proximal parts of the outflow cushions. Separation of the right and left ventricles is heralded by the appearance of a caudal crescentic ridge within the ventricular loop (see Fig. 13.7 ). This is a persisting part of the Tbx3 and G1N2-positive primary heart tube myocardium, which means that the crest of the septum is the oldest portion of the developing ventricular septum. The ventricles (especially those parts that will become the apex of the heart) become enlarged as if they were expanding like balloons on either side of the muscular ventricular septum (see Figs 13.4 , 13.7 ). Later, the dorsal part of the crest of the septum will become the atrioventricular bundle, while the atrioventricular node will form at the dorsal atrioventricular junction (see Fig. 13.10C ). The ventral parts will largely disappear except the so-called ‘dead-end tract’ ( , ). The crest of the septum, covered by an extension of the dorsal atrioventricular cushion, together with the fused atrioventricular cushions themselves, bounds the ovoid interventricular foramen. The Tbx3 and G1N2-positive myocardium that surrounds this foramen marks a ring composed of primary myocardium which is smooth (see Fig. 13.10 ). Completion of ventricular septation is seen after a rightward shift of the atrioventricular canal and the positioning of the outflow tract between the separating atrioventricular canals so that it too overrides the developing ventricular septum. The foramen finally closes by formation of the membranous septum that completely separates the right from the left ventricle (see Fig. 13.13 ). During ventricular septation, transition from the early ventricular base-to-apex conduction activation to an apex-to-base conduction is initiated. Maturation of the ventricular conduction system, with concomitant development of the fibrous rings of the heart, continues throughout fetal development ( ).

Septation and appropriate positioning of the outflow tract

The appearance of two outflow cushions is key to the separation of the outflow tract (truncus arteriosus). Together with the myocardial wall, the cushions spiral as they extend from their septal and parietal positions at end of the right ventricle to the developing aortic sac (see Fig. 13.13 ; Fig. 13.14 ). Within the developing aortic sac, a transverse wedge of tissue composed of dorsal mediastinal mesenchyme, the aortopulmonary septum, initially separates the origins of the aorta and the pulmonary trunk before their individual walls are formed. Meanwhile, neural crest cells migrate from the pharyngeal mediastinal myocardium into the outflow cushions. They do not populate the septum itself, but form the larger parts of the walls of the intrapericardial portions of the truncus arteriosus ( ).

The outflow cushions start to fuse from stage 15. Fusion starts in the distal part of the outflow tract and continues gradually in the direction of the ventricular septum (see Fig. 13.14 ; Fig. 13.15A ). Early on, the most proximal parts of the cushions remain unfused. The arterial valves and sinuses develop distally within the myocardial sleeve that surrounds this area. When the most proximal parts of the cushions fuse, they are invaded by cardiac myocytes (myocardium) and become continuous with the muscular ventricular septum, so joining the aorta to the left ventricle. Spread of myocardial cells also gives rise to the greater part of the supraventricular crest. In the right ventricle, the wall of the proximal myocardial outflow tract persists as the smooth-walled muscular sub-pulmonary infundibulum. Some myocardium also persists distal to the pulmonary valve. There is no distinct myocardial outflow tract present within the left ventricle in the postnatal heart: the myocardium that initially surrounded the entire aortic root is lost on the side of the non-coronary leaflet because a fibrous continuity develops between the mitral valve and the aorta.

Outflow tract septal anomalies

A number of outflow anomalies may occur if the outflow cushions either fail to develop or fuse in an inappropriate fashion. When the cushions fail to fuse, the result is a persistent truncus arteriosus, represented by an undivided arterial channel, guarded by a common arterial valve, positioned above and astride the free margin of the muscular ventricular septum ( Fig. 13.16 ). This anomaly occurs frequently in DiGeorge syndrome and can be a result of a Tbx1 deletion ( ). Persistent truncus arteriosus is also likely linked to abnormal development of the secondary heart-forming field, which, by means of an overall shorter heart tube, leads to poor alignment of the aorta with the left ventricle. In this condition, there is a coexisting juxta-arterial deficiency of the ventricular septum; right and left pulmonary arteries usually arise via a confluent segment but can also take independent origin from the trunk, which continues as the ascending aorta. The common valve usually has three leaflets but may also have two, four or more.

Transposition of the arterial trunks (also referred to as transposition of the great vessels) is a condition in which the aorta arises from the right ventricle and the pulmonary trunk from the left. Better described as having discordant ventriculoarterial connections, such hearts can coexist with deficiencies of cardiac septation. They can also be found with discordant connections at the atrioventricular junctions, producing a congenitally corrected transposition. The developmental history of discordant connections remains unknown but may have similar signalling pathways that are involved in right atrial isomerism, the nodal/pitx2 pathways ( ).

Double outlet ventricle occurs when the greater parts of both arterial valves fall within the same ventricle, almost always the right. In these cases, in order to continue the circulation of oxygenated blood after birth, it is necessary that either the ventricular septum is deficient, or the ductus arteriosus remains patent with medication; in rare cases, the septal defect can close as a secondary event.