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Experimental and Molecular Cardiology Group, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
ABSTRACT I. INTRODUCTION A. Scope B. The Cardiac Cell Types: Origin and Diversity C. The Different Manifestations of Cardiomyocytes: Contraction, Conduction, and Automaticity II. EVOLUTIONARY ASPECTS OF CHAMBER FORMATION A. Introduction B. Drosophila C. Primitive Chordates 1. Tunicates 2. Amphioxus D. Lower Vertebrates 1. Fishes 2. Amphibians E. The Electrical Configuration of the Chamber Heart F. Tales of the Rings: the Emerging Concept of Ballooning Chambers G. Overview of the Component Parts of the Lower Vertebrate Heart 1. Novel cardiac components 2. Conserved cardiac components III. DEVELOPMENT OF THE PARALLEL-ARRANGED FOUR-CHAMBERED HEART A. Evolutionary Aspects and Terminological Problems B. Cardiac Looping, Changing Blood Flows, and Chamber Formation C. The Primary Ring: Development of the Right Ventricle D. Completion of Septation IV. DEVELOPMENT OF CARDIAC FUNCTION AND CONDUCTION SYSTEM A. Development of Polarity B. Development of the ECG V. LINEAGE ANALYSIS AND CHAMBER DEVELOPMENT A. Formation of the Linear Heart Tube B. The Heart-Forming Regions Include Anterior and Posterior Mesenchyme C. The Inner Curvature Connection D. Origin of the Nodal Tissue E. Fate of the Primary Ring F. Origin of the Ventricular Conduction System G. Overview of the Component Parts of the Higher Vertebrate Heart 1. The atrial and ventricular chambers 2. The conduction system 3. Engrailed-2/lacz transgene expression: speculations on cardiac design VI. ANTERO-POSTERIOR PATTERNING AND CHAMBER FORMATION A. AP Patterning During and After Gastrulation B. Retinoic Acid: a Cardiac AP Patterning Molecule C. Factors Involved in AP Patterning and Morphogenesis 1. Gata factors 2. Tbx5 3. Other factors VII. CARDIAC PATTERNING ALONG THE DORSOVENTRAL AXIS VIII. LEFT-RIGHT SIGNALING IN CHAMBER SPECIFICATION AND MORPHOGENESIS IX. CHAMBER-SPECIFIC AND REGIONALIZED TRANSCRIPTIONAL PROGRAMS A. Chamber-Specific Patterns of Gene Expression B. Regulation of Chamber-Specific Gene Expression 1. Regulation of atrial gene expression: Irx4 and the Smyhc3 gene 2. Regulation of ventricular gene expression: the Mlc2v gene 3. Regulation of chamber-specific gene expression 4. Localized activity of other regulatory DNA fragments 5. Regulation of regionalized transcription factors C. Gene Deficiency Reveals Pathways Involved in Chamber Formation 1. Nkx2-5 2. Hand2 3. Mef2c X. CONCLUSIONS AND PERSPECTIVES
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ABSTRACT |
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Theory without fact is fantasy, but fact without theory is chaos.
C. O. Whitman (334)
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I. INTRODUCTION |
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In birds and mammals, the four chambers of the formed heart are arranged in parallel (Fig. 1, A and C). The right atrium is exclusively connected to the right ventricle and the left atrium exclusively to the left ventricle. The right half of the heart drives blood from the body through the lungs to the left half that drives the blood through the rest of the body. The two halves can only beat in synchrony, the right half pushing the pulmonary circulation and the left half the systemic circulation. This parallel-arranged four-chambered heart develops from a single circuited tubular heart. The current concept is that this heart tube is composed of a linear array of segments (21, 56, 254, 330) (Fig. 1, B and D). In this model the atrium is not connected to the right ventricle and the left ventricle not to the outflow tract. The conversion of such a serial arrangement of segments into the proper parallel arrangement has remained one of the most difficult concepts of heart development (156), for it seems illogical first to make a serial arrangement of cardiac segments and then a parallel one. Based on morphological and flow direction considerations, Steding and Seidl (292) described this concept as "one of the most fatal assumptions." Recent functional and molecular data also challenged the concept (51, 64, 118, 210), and a new concept was formulated, "the ballooning model of chamber formation." In this review we discuss the possibility that during the evolution and ontogenesis of the heart the chambers develop from a linear tube by local differentiation and that this local differentiation and expansion sets the scene for the electrical configuration of the chamber heart. From the outset, the right and left halves of the heart are brought into parallel position by looping of the heart tube. A fundamental question is thus whether indeed all compartments of the formed heart are represented as a linear array in the tubular heart.
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In a stimulating review, Fishman and Olson (90) suggested a model of the vertebrate heart as an assembly of separable genetic modules that were added to the primitive ancestor chordate heart during evolution. The modules are supposed to be component parts or functions of the heart that can be selectively affected by single gene mutations. Examples of such modules would be the cardiac chambers and the localized pacemaker activity. Challenging though these ideas are, this concept can only fully be exploited if the molecular data are integrated morphologically and functionally in a unifying model of chamber formation. The deep evolutionary conservation of molecular pathways in the hearts from fruitfly to human is intriguing and instrumental in finding general developmental mechanisms. On the other hand, evaluation of evolutionary diversity is fundamental to revealing both conserved and novel modules of the building plan of the present-day mammalian heart.
De la Cruz and Markwald (66) wrote an exhaustive review on cardiac development in general. The roles of endocardium (186), epicardium (191), and neural crest (85) in heart development, as well as molecular cardiac genetics (48, 121, 285) were reviewed elsewhere. In this review, we try to define the basic cardiac building blocks and examine their developmental and evolutionary origins. This, in turn, requires evaluation of the identity of the atrial and ventricular chambers and of the conduction system of the avian and mammalian hearts. We need to determine whether these structures are indeed basic cardiac building blocks, or are composed of smaller component parts developed during evolution and integrated during embryonic development. It is our goal to set out a logical model of cardiac chamber formation from these elementary components. In this review we largely restrict ourselves to the myocardial component of the heart.
B. The Cardiac Cell Types: Origin and Diversity
The heart essentially has a mesodermal origin (Fig. 2). The minor contribution of neural crest cells is largely confined to the formation of the aorto-pulmonary septum at the arterial pole of the heart (86, 157), and the endoderm only plays a regulatory role (186). Committment of mesodermal cells to the cardiogenic lineage is established during and shortly after gastrulation (274, 297), upon which the cardiogenic cells are organized in bilateral epithelial sheets that are part of the visceral mesoderm lining the developing coelomic cavity. The cardiogenic cells are in close association with the endoderm. The endocardial cells are formed from the cardiogenic mesoderm in between the endodermal and mesodermallayers. During the subsequent process of folding of the embryonic disc, the bilateral mesodermal cardiac plates migrate into the embryo, fuse in the midline, and form the cardiac tube (64).
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The three principal cardiac cell types comprise 1) the endocardium that forms the endothelial lining of the heart (see Ref. 186 for a review), 2) the myocardium or cardiac muscle cells, and 3) the epicardium that is the source of cardiac fibroblasts and coronary arteries (see Ref. 191 for a review). The endocardium is formed early in cardiogenesis because, after retroviral labeling, endocardial cells were shown to share hardly any lineage relationship with myocardial cells (201). However, the two types of cell must have separated after recruitment of the mesodermal cells into the myocardial lineage, because initially the endocardial cells display a cardiac muscle phenotype as well (62, 184, 317). Upon signals from the myocardium, the endocardial cells undergo a transformation from epithelial to mesenchymal cells, by which the mesenchymal cardiac cushions are formed. These cushions fuse and form part of the cardiac septa (30, 31, 83, 205, 244). The mesenchyme of these cardiac cushions, in turn, can be transformed into cardiac muscle, by which process these parts of the cardiac septa become muscularized (211, 225, 308, 309, 339).
The epicardium develops from the so-called proepicardial organ that is located caudally from the heart. The proepicardial organ forms cauliflower-like mesothelial structures protruding into the coelomic cavity that will cover the entire heart and form the epicardium. Mesenchymal cells derived from the epicardium contribute to the cardiac-cushion mesenchyme, to the cardiac fibroblasts, and to the endothelial and smooth muscle cells of the coronary arteries (72, 203, 238, 242).
Interestingly, endocardium, epicardium, and myocardium share a common lineage as they are all derived from a group of mesodermal cells that express the basic helix-loop-helix transcription factor Mesp1 (266, 267). The role of this transcription factor in the formation of the cardiovascular system remains to be resolved. It is of great interest that endothelial cells proved to be capable of transdifferentiating into cardiac muscle when they were cocultured with cardiomyocytes (54). Preliminary results from studies from the author's lab indicate that proepicardium is able to transdifferentiate into cardiac muscle in vitro as well.
C. The Different Manifestations of Cardiomyocytes: Contraction, Conduction, and Automaticity
The vertebrate heart is myogenic, which implies that all cardiomyocytes are in principle capable of producing an intrinsic cycle of electrical activity that leads to contraction (80). This phenomenon is shared with smooth muscle cells and is called automaticity, intrinsic rhythmicity, or pacemaker activity. Because all cardiomyocytes are electrically coupled, the cells with the fastest intrinsic rate, in the mammalian heart usually the cells of the sinus node, stimulate the whole heart to contract and determine its rate. The cells with the fastest intrinsic rate are called the (leading) pacemakers; the others can be categorized as followers or latent pacemakers. If the pacemakers were to stop, other cardiomyocytes take over, albeit at a slower pace. Automaticity requires poor coupling of the cardiomyocytes, which is achieved by a specific composition and density of gap junctions and ion channels, as well as by the size and the arrangement of the cardiomyocytes involved. Poor intercellular coupling permits loading of the cells to a threshold value of electrical charge, resulting in depolarization of the adjacent myocardium (145). Hence, pacemaker (nodal) cells share automaticity and slow conduction (Table 1).
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Automaticity and slow conduction velocity are also features of the peristaltically contracting hearts of primitive chordates and vertebrate embryonic tubular hearts, because slow conduction of the depolarizing impulse is an absolute prerequisite for peristalsis (see sect. II). The development of synchronously contracting chambers required higher conduction velocities of the depolarizing electrical impulse. For this reason, and because unambiguous morphological criteria are lacking, automaticity and conduction velocity became important characteristics to distinguish the different areas in the developing heart.
In the formed heart, atrial and ventricular working myocardium are conventionally distinguished from the so-called "conduction system" (see Table 1). This notion is supported by the fact that the atrial and ventricular muscle cells have well-developed sarcomeric and sarcoplasmic reticular structures (39) but are also well coupled, allowing fast conduction of the depolarizing impulse and efficient synchronous contractions. On the other hand, and in contrast to the cells of the working myocardium, the cells of the conduction system all share a poorly developed contractile and sarcoplasmic reticular apparatus. In this respect, these cells are reminiscent of the embryonic cells of the tubular heart. In contrast to what might be suggested by its name, the conduction system is not just composed of fast-conducting cells, but also of pacemaking or nodal cells. These cells are slowly conducting and poorly coupled and display high automaticity. Only the cells of the atrioventricular bundle and bundle branches are well coupled, permitting fast conduction of the depolarizing impulse. Because the bulk of the cardiac muscle cells comprise cells of the working myocardium, the embryonic-like cells of the developing conduction system are often called "specialized."
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II. EVOLUTIONARY ASPECTS OF CHAMBER FORMATION |
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The morphologically complex mammalian four-chambered heart has evolved from a simple chordate tubular heart (Fig. 3) (257). Modification of existing elements and the addition of new elements accomplished this evolution. This in turn required continuous adaptation of the genomic regulatory programs that control cardiac development. In general genetic terms, molecular pathways for regional specification of morphological structures had to be selected, which resulted in the activation of sets of downstream genes. Subsequently, cycles of regional specification were required to achieve the final adult configuration (240).
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Many features that seemed unique to vertebrates were already present in the early chordate ancestors, and new functions became associated with modified duplicated sets of genes (114). What we thus need to understand both at the morphological and genomic level is what cardiac elements were added during evolution. It is equally important to integrate conceptual insights derived from the cardiac anatomy and physiology of lower vertebrates that underlie mammalian cardiac development. According to the rules of Von Baer (318), the embryos of different vertebrate groups should be compared rather than the adult stages, because the general features of a vertebrate group appear earlier in development than the specialized features. Consequently, the mammalian embryo heart is never like the heart of an adult lower vertebrate but only like that of its embryo. A practical problem is that most experimental data are from extant adult lower vertebrates and not from embryos. Thus one should be cautious in formulating generalities and specializations derived from these adult specimens.
Recent molecular data indicate that some essential aspects of the regulation of heart development in Drosophila resemble those in vertebrate hearts, despite the long evolutionary distance between both groups (25). Drosophila has become the paradigm for many developmental studies in vertebrates, so we start our description with the Drosophila heart.
The heart of Drosophila lies within a dorsal pericardial cavity and is also called the dorsal vessel. It is an elongated tube that is closed posteriorly and drains into the head anteriorly. The anterior part is narrowed and called the aorta. In fact, the Drosophila heart is a blind sac from which hemolymph is expelled into the body by rhythmic contractions. During relaxation, hemolymph enters the heart tube via ostia in its wall, which are closed when the tube contracts. The wave of contractions initiates caudally and moves rapidly in the anterior direction, indicating localized pacemaker activity (253). The Drosophila heart is myogenic, because the heart continues to beat outside the body for several hours. As yet, no specialized pacemaker cells have been reported, and it is unknown whether the localized pacemaker activity is intrinsic or imposed by innervation.
The wall of the Drosophila heart tube consists of two cell layers. The inner layer lining the lumen is composed of a single type of myocardial cells forming a contractile layer (263). The myocardial cells are not fused but interconnected via adherent junctions where the myofilaments also attach. The outer layer lining the pericardial cavity consists of pericardial cells called nephrocytes. They do not have a contractile phenotype but a filtration and secretion function and should not be confused with the pericardial cells in mammals. The pericardial layer is attached to the body wall via so-called alary muscle connections at the segmental boundaries.
All chordates have a notochord, a neural tube, and metameric lateral muscle blocks. In the early representatives, a closed cardiovascular system has developed. The chordate phylum comprises the subphyla of the urochordates, or tunicates, and related forms, the cephalochordates with Amphioxus as a well-known representative, and the vertebrates (257).
The tunicate heart is a tubular structure that lies in a pericardial cavity. It consists of a single layer of cardiac muscle cells that are electrically coupled and are not lined by endocardial cells (247). These cardiac muscle cells are also called myoendothelial cells. Although all regions of the heart possess intrinsic automaticity, the dominant pacemaker is active at one end of the cardiac tube for a number of beats, after which a pacemaker at the other end of the tube takes over (3). The resulting peristaltic contraction waves travel along the heart tube with velocities of 
0.5 cm/s and propel the blood in either of the two directions (164).
The vascular plan of Amphioxus is basically similar to that of the vertebrates (207), but a unique feature is the lack of a true heart. However, several vessels are capable of contractile movements: the subintestinal vein, the portal (afferent) and hepatic (efferent) veins, and the ventral aorta (endostylar artery), which acts as the principal pump. A myoepithelial layer surrounds the vessels (207). The vessels are not lined by endothelium, although scattered cells called hemocytes are sometimes closely associated with the wall of the vessels (245). These cells might be comparable to the ancestral cell population that gave rise to the vertebrate endothelium or vertebrate blood cells or both (294). The vessels contract in succession, although coordination is poor and a single pacemaker area seems to be lacking (247, 319). Very slow, long-lasting contraction waves (0.03 cm/s) travel along the vessels, which results in movement of the blood in these valveless vessels, indicating some kind of polarity. However, this polarity of pacemaker activity is not well developed because reversal of flow and consequent extinction of the meeting waves of contraction were observed (319).
The vertebrates constitute the principal group of the chordate phylum, and the development of the chambered heart is a new phenomenon in this group (Fig. 4A). It is first seen in the jawless fishes the cyclostomes, such as the hagfish (127, 247, 268). Their hearts consist of a sinus venosus, atrium, ventricle, and conus arteriosus. The common atrial chamber bulges out over the right and the left sides of the ventricle. The conus arteriosus is poorly developed in hagfish but is clearly present in jawed cartilaginous fishes, such as sharks and rays, and also in lungfishes. The conus arteriosus is the most distal part of the primitive fish heart and forms the connection between the ventricle and the ventral aorta.1 At the sinoatrial, the atrioventricular, and the ventriculoconal junctions, valves developed to prevent backflow of blood during relaxation of the preceding compartment. The cardiac tube is S-shaped with a dorsally positioned atrium and a ventrally positioned ventricle pointing caudally. This S-shape, by which the ventricular inlet and outlet become positioned in almost the same plane, is a general feature of all vertebrate hearts. It has dynamic advantages because it redirects the momentum of the bloodstream, which flows from the atrium into the ventricle, toward the arterial pole. In a linear chamber heart, the inflowing bloodstream would be rebounded from the closed semilunar valves toward the atrium (153).
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The sinus venosus became the drainage pool of the venous system to facilitate atrial filling. However, the wall of the sinus venosus is poorly muscularized, and its development as a separate cardiac compartment as well as the development of the sinoatrial valves that physically separate sinus venosus from atrium during contraction have allowed the development of the atrium as the principal driving force for ventricular filling (142, 143). This role has become less important in mammalian hearts where collection of blood is the principle function of the atria. In line with this, in mammals the sinus venosus has become part of the right atrium. Although the lungfish branch does not give rise to the amphibian group, the lungfish heart already has an atrial septum comparable to the amphibian condition. The ventricular septum of the lungfish is considered to be a derived structure that is not homologous to the avian and mammalian ventricular septum (130).
A typical amphibian heart has two atria, a single ventricle, and a bulbus cordis (the amphibian equivalent of the fish conus arteriosus) (127, 143, 342). The sinus venosus drains exclusively to the right atrium. The pulmonary vein bypasses the sinus venosus and directly drains to the left side. The left atrium is often considered to be the evolutionary novel chamber, because it is a component of the pulmonary circulation. Obviously, the atrium of the fish heart has a right and a left atrial myocardial wall as well, but has a common chamber because it has no atrial septum. Hence, rather than the left atrium, one should consider the atrial septum and possibly additional myocardium that surrounds the pulmonary vein as the novel components of the amphibian heart. Despite the presence of a single ventricle, amphibians share a highly effective separation of oxygenated and deoxygenated blood with the related lungfishes (143). Most of the left atrial bloodstream flows to the systemic arteries and most of the right atrial bloodstream to the pulmo-cutaneous arteries. The morphofunctional adaptations for this amazing performance are derived features typical for the extant amphibian specimens. However, these are not dealt with in this review.
E. The Electrical Configuration of the Chamber Heart
The heart tube of tunicates and amphioxus display peristaltic contraction waves, and all regions of the tube display intrinsic automaticity. This is so because peristaltic contraction requires slow propagation of the wave of depolarization, which, in turn, requires poor intercellular coupling. This characteristic also is a prerequisite of automaticity. However, polarity of pacemaker activity is not well developed.
The fish heart displays clear polarity of contraction in a posterior-to-anterior direction. The contraction waves originate in the sinus venosus and terminate in the conus arteriosus. The nodal phenotype persists in the inflow region of the heart, varying from the venosinus to the sinoatrial junctional areas in different species (246, 268). Similar to the mammalian situation, pacemaker tissue with a lower intrinsic rhythmicity is also found at the atrioventricular junction. Interestingly, conus tissue displays pacemaker activity as well (268). In the fish heart, the chamber myocardium has acquired a faster conduction of the depolarization wave (268). For instance, in the ventricular chamber of the shark, the conduction velocities of the depolarization waves are 50 cm/s, whereas velocities of 3 cm/s were measured in the conus myocardium (300). In ECGs these slow depolarization waves of the conus are recognizable as a peak between the QRS complex and the T peaks of the ventricular depolarization and repolarization, respectively (300). The long contraction that results from this slow conduction in combination with the length of the conus supports valve function and prevents backflow of blood from the ventral aorta into the ventricle during diastole (142, 269).
The electrical configuration of the amphibian heart is remarkably similar to that of the fish heart (2, 193). The fastest conduction velocities are observed in the atria and the slowest in the bulbar region. The atrioventricular junction propagates the depolarizing impulse slowly as well. In line with this, ultrastructural analyses of the atrioventricular region display a phenotype of nodal myocardial cells with the typical absence of a transverse tubular system, few myofibrils, few mitochondria, the presence of glycogen particles, small cell diameter, and scarcity of gap junctions (39). The bulboventricular junction would display the slowest conduction time of the impulse between ventricle and bulbus. This might be apparent because of the presence of a sulcus at the bulboventricular junction; the real traveling distance of the impulse over the sulcus myocardium may be longer than the direct distance between the measuring points in ventricle and bulbus. Also in amphibians the myocardium of the bulbus cordis assists valve function (142).
Whereas the peristaltically contracting hearts of the lower vertebrates do not need valves, the synchronously contracting chamber hearts of the lower vertebrates do need one-way valves at both ends of their chambers to prevent backflow from a downstream compartment during relaxation and to an upstream compartment during contraction. The electrical configuration described above shows that the entry and exit of a chamber are flanked by slowly conducting myocardium guaranteeing a sphincter function (prolonged contraction) at both orifices, as has been demonstrated for the myocardium surrounding the semilunar valves in fish and amphibian hearts (142, 269). Because slow conduction is also a prerequisite for nodal function, it may not be coincidental that in these areas nodes have developed, as discussed in section IV.
F. Tales of the Rings: the Emerging Concept of Ballooning Chambers
In the beginning of the previous century, Benninghoff (21) described regions in the hearts of fish, amphibians, reptiles, birds, and mammals that do not participate in the growth of chambers from the heart tube. Because these regions hardly grow, they define the openings of the expanding chambers as rings (Fig. 4B). He ascribed a sphincterlike function to these areas and associated them with the conduction system. As we discussed in the previous section, these areas indeed have a sphincter function and contribute to the slow components of the cardiac conduction system (the nodes, or pacemaking tissues). Later, the rings were retrieved from oblivion in studies on the development of the conduction system in human embryos, but their sphincter function was denied (330) and the rings were not explicitly restricted to the slowly conducting components of the conduction system, but to so-called "cardiac specialized tissue" (7, 330). The latter term does not distinguish unambiguously between the slow and the fast components of the cardiac conduction system. This use of this term is extremely confusing because this term, as well as the term conduction system, is often linked implicitly to fast conduction. Moreover, what is "specialized"? In a paper on the embryonic development of cardiac impulse conduction, Lieberman (179) expresses this ambiguity as follows:
Perhaps, concomitant with an increase in myofibrillar content, a process is triggered in the genetically determined working myocardial cells which causes the myocardial cell membrane to lose this "embryonic" property to pacemake. Implicit in this notion is the idea that only those cells that develop an abundance of contractile proteins (that is, working myocardium) should, in fact, be referred to as the "specialized tissue of the heart." Or, the corollary may also be implied: all cells that lose the ability to synthesize myofibrils and maintain automaticity and conductivity should be considered specialized.
The studies of Benninghoff unfortunately led to the abstractive representation of the cardiac tube composed of a linear array of chambers with rings of conduction tissue at the transitions, which has become the paradigm of the development of the mammalian heart and the conduction system (21, 56, 254, 330) (compare Figs. 1B and 4C). However, the atrial chambers balloon out at the dorsal side and the ventricular chamber at the ventral side of the heart tube and lead to the typical S-folded heart of fishes and amphibians as represented in Figure 4 (127, 254).
This morphological process is typical for the developing mammalian heart as well and has been described in great detail for the developing human heart (59). It also explains why the expanding cardiac chambers are flanked by myocardium of the original heart tube and why this myocardium displays features of the slow components of the conduction system.
G. Overview of the Component Parts of the Lower Vertebrate Heart
The hearts of the lower fish and amphibian vertebrates have often been used as prototypes for the development of the avian and mammalian hearts, particularly to understand the origin of the cardiac conduction system (21, 56, 89, 139, 227, 254). Yet, it has remained a highly controversial topic, clearly illustrating the difficulties in uncovering the essentialities of cardiac design. It is thus of paramount importance to try to assess the essence of the design of the fish and amphibian hearts, because these hearts are considered to be the ancestor avian and mammalian hearts. This basic "prototypic" cardiac building plan will enable us to evaluate the development of the parallel-arranged hearts of birds and mammals in section III. We will not discuss the reptilian heart, as the heart of the extant species is considered to be highly derived. The ventricular septum is considered to have evolved in the primitive reptilian ancestor that gave rise to the mammalian branch on the one hand and to the avian/crocodilian branch on the other hand; its evolution is still controversial (130, 327).
During evolution, the high-volume low-pressure cardiovascular system of the tunicates (blood volume equals 40% of their body weight) developed into a low-volume high-pressure system in vertebrates (blood volume comprises 6% of their body weight). This change was accompanied by a concurrent transformation of a peristaltically contracting straight tubular heart into the more efficient and powerful synchronously contracting looped chamber heart. This type of change allowed reduction of the circulation time of the blood from 6 min in tunicates to 1 min in humans. Vessels covered with a contiguous endothelial sheet replaced the largely leaky vessels in amphioxus.
Three major adaptations, or "novel cardiac components," that were not present in the ancestor chordate heart tube can be distinguished in the lower vertebrate heart: the atrium, ventricle, and possibly the muscular sinus venosus. Furthermore, within the ventricular component a compact outer myocardial component and an interiorly localized extensive trabecular component can be distinguished. The specific activation of the ventricle adds to its complexity as follows. The depolarizing impulse travels rapidly from the atrioventricular node toward the apex and then toward the conal region, achieving activation from apex to base. However, preferential conduction tracts have not been identified histologically as yet (268).
The largest changes in the amphibian heart compared with that of fish occurred at the venous pole (Fig. 5). In evolutionary retrospect, the fish atrial wall can be well described as being composed of a right auricular wall and a left auricular wall that bulge out from the common atrial portion of the heart tube. Similar to the mammalian embryonic condition, a single auricle bulges over the ventricular component at the right and left side. First, the sinus venosus became largely incorporated into the right auricular wall close to the midline. Second, an atrial septum and possibly accompanying pulmonary myocardium developed, by which the single atrial chamber was divided into a right and a left component. The systemic venous system drains to the right (systemic) atrium and the pulmonary system drains to the left (pulmonary) atrium. Thus the atrial myocardial compartment of the amphibian heart is composed of the following components: 1) right and left atrial wall or auricular myocardium; 2) sinus venosus myocardium or caval myocardium; 3) septal myocardium, possibly including pulmonary myocardium that surrounds the entrance of the pulmonary veins; and 4) the myocardium of the atrioventricular canal that forms the so-called atrial vestibulum, which is the smooth-walled lower atrial wall just above the atrioventricular junction. Support for the generality of this description comes from studies on the developing mouse heart, in which these component parts have been recognized on the basis of their unique transcription patterns (91).
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2. Conserved cardiac components
Three components of the lower vertebrate heart might be considered to be minor adaptations, remnants, or "conserved components" of the ancestor cardiac tube. These components are the sinoatrial junction, the atrioventricular junction, and the conus, because these three areas display nodal activity and phenotype. In the absence of clear distinguishing features, sinus node and atrioventricular node remain difficult to identify unambiguously in the lower vertebrates. It is worth noting that nodal cells are virtually devoid of so-called specific myocardial granules, whereas these granules are abundant in atrial and ventricular myocardium (342, 343). In mammals, myocardial granules are the storage sites of atrial natriuretic factor (ANF) precursor (283) and the nodes are devoid of ANF, similar to the fish nodal tissue (282). Because fish hearts do contain ANF, it is assumed that these granules are the storage sites of ANF precursor protein in fish as well. It is of great interest that ANF is not expressed in the early murine embryonic heart tube (51, 347). For that reason its expression has been used to identify the differentiating chamber myocardium (51), as discussed in section IXA.
So far, we tried to extract the essentialities of the body plan of the vertebrate heart from the examination of adult hearts of lower vertebrates, rather than that of their embryos. In section III we examine whether these essentialities can indeed be considered to be unifying features of the vertebrate heart.
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III. DEVELOPMENT OF THE PARALLEL-ARRANGED FOUR-CHAMBERED HEART |
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The parallel-arranged avian and mammalian hearts originate from the heart of primitive fish that gave rise to the lungfishes and the amphibian/reptilian branch (Fig. 3) (257). By that time, the development of the left atrium had already been realized in the lungfishes and amphibians and, therefore, the atrial part of their hearts is already essentially similar to its avian/mammalian counterparts. This subject has been dealt with in the previous sections. The right ventricle is the new component of the four-chambered heart and is therefore the focus of the discussion on the development of the four-chambered heart.
The right ventricle is thought to have developed from the proximal part of the conus arteriosus, which is the homolog of the bulbus cordis of amphibians and of avian and mammalian embryos. Trabecularization of the bulbus "ventricularized" this region that subsequently became easily accessible from the right atrioventricular canal by reduction of the so-called bulbo-auricular ridge at the right side (127). The use of the term bulbo-auricular ridge rather than the term ventriculo-auricular ridge may indicate that the early cardiac embryologists saw no ventricular tissue at the inner curvature. In human embryos, the proximal part of the bulbus cordis becomes trabeculated and is then called primitive right ventricle (314). However, trabecules develop at the ventral, outer curvature side of the bulbus only; at the inner curvature smooth-walled myocardium covered with endocardial cushions remains (73, 314). In our opinion, the question can justifiably be asked, whether these authors thought that the entire proximal bulbus would become the primitive right ventricle or merely the trabeculated part at the outer curvature? Others dispute the origin of the right ventricle in the bulbus cordis and argue that it is not a bulbar structure in its entirety (7). In fact, the latter authors may have called the proximal part of the bulbus mentioned by the authors of references (73, 314), right ventricle. Lack of a clear terminology may have led to misunderstanding. Again, the same question can be asked, whether the entire segment of the heart tube was meant to be the primitive right ventricle or merely the trabeculated ventral part. The conflicting terminology originating from both phylogenetic and ontogenetic considerations (241) has undoubtedly contributed to the confusion in the field, but the severe underexposure of dorsoventral patterning has mystified the field most.
Separate cardiac compartments are clearly recognizable at the outer curvature of the developing four-chambered heart, but one feels uncomfortable when one is forced to distinguish atrioventricular canal, left ventricle, right ventricle, and outflow tract at the inner curvature. Indeed, lumen reconstructions of this area in human embryonic hearts made more than half a century ago do not lend support to true segments at the inner curvature (Fig. 6) (57, 73, 218, 228). Based on the development of trabeculated myocardium, unanimity exists that the right ventricle develops later on in development, downstream and visibly separated from the left ventricle as is clear from Figure 6. These observations indicate that the right ventricle develops from a part of the heart tube, whatever the name of that part, that is downstream from the left ventricle, and argue against development from a common ventricular chamber by septum formation within a single ventricular chamber as occurs in the atrial part of the heart. In addition, the reconstructions clearly showed impressions of the ventricular trabecules at the outer curvature and impressions of smooth-walled myocardium at the inner curvature.
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The significance of the above-mentioned description is that it unequivocally demonstrates dorsoventral (inner-outer curvature) patterning. One way to describe these factual observations of the cardiac configuration at the ventricular part of the heart tube is that the looping heart tube is a single compartment or module, from which two additional modules have grown, the embryonic ventricles. The smooth-walled myocardium is covered with endocardial cushions and ventrally where the endocardium approaches the myocardium, trabeculated myocardium develops. In the past, the smooth-walled myocardium was also called primary myocardium (73, 228), and recently, this name was reintroduced on the basis of functional and molecular characteristics (63, 209).
A comparable description could be made of the development at the venous side of the atrioventricular canal (73). Here, the primary heart tube covered with endocardial cushion tissue also extends toward the venous side. At the dorsolateral sides, where the endocardium approaches the myocardium, the auricular components develop.
The description given above implicitly considers the primary heart tube as a single entity, although in certain aspects the tube is highly patterned, as evidenced by the local differentiation of the atrial and ventricular chambers and the localized pacemaker activity at the intake of the heart (270, 313). Without implying the presence of strict boundaries (in particular not at the inner curvature) but to facilitate dialogue, we propose that the primary heart tube can be divided into an inflow tract part, an atrial part, an atrioventricular canal, a ventricular part, and an outflow tract (bulbus cordis). The patterning of the tube in the ventricular and outflow tract parts deserves additional attention, because it plays a crucial role in the formation of the right ventricle. After the development of this chamber, an anatomically complete parallel-arranged heart had been achieved.
B. Cardiac Looping, Changing Blood Flows, and Chamber Formation
A most conspicuous feature of cardiac looping is that the ventricular chambers that are positioned along the antero-posterior axis of the embryo become positioned in a left-right orientation (32, 190, 291). Looping starts at a stage in which only the ventricular component and part of the atrioventricular canal has been formed (60, 190). The linear heart tube grows at both poles, bends toward the ventral side, and subsequently turns toward the right side (190). By this process, the original left lateral wall becomes the ventral ventricular wall, the dorsal side the inner curvature, and the ventral side the outer curvature (38, 67, 190). The entire dorsal half including the left and right portions of the linear heart tube ends up at the inner curvature of the looped heart in later stages; a label positioned at the ventral midline ends up at the outer curvature (Fig. 7). This indicates that the original ventral side of the heart tube expands and bends toward the right. During this process, the ventricular chambers are formed and the flow of blood reaches its adult pattern (Fig. 8). All experimental evidence so far supports the notion that looping of the heart is intrinsic to the myocardial tube itself (185).
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Because the high viscosity of the embryonic blood results in low Reynolds numbers2 (Re: 0.05-2.0 compared with an adult value of 1,000), flow in the embryonic vascular system is laminar (197). This flow can be considered to consist of numerous parallel streaming layers, which can be marked and followed by application of indian ink. Changing flow patterns have been documented by microcinematography during development (Fig. 8) (292, 293). Watching a beating embryonic heart during an early stage of development gives the impression that two separate bloodstreams flow through the heart tube (344), because contraction is not circular but asymmetric. During contraction, the lumen becomes ovoid, and transitory ridges arise at both long-sided walls of the ovoid, as a consequence of which the central part of the lumen is emptied earlier than the lateral parts (278, 293). At the long-sided wall of the lumen the cardiac cushions develop later. During further development of the ventricles, the decrease in the lumen during systole gradually becomes concentric, and thus the pattern of flow becomes adultlike. The blood-flow pattern changes from an initially right flow at the inner curvature and an initially left flow at the outer curvature to the adult configuration, with the left flow directed toward the inner curvature (Fig. 8) (129, 278, 293). These flow patterns have been meticulously documented during chicken development, and the change proved to take place between Hamburger and Hamilton stages (HH stage, Ref. 119) 26 and 28. This is at about Carnegie stage 16, comparable to about 38 days of human development or 12 days of mouse development. At this stage, the peristaltic contraction form disappears from the ventricle (292).
The change in pattern of flow associated with cardiac looping and formation of the ventricles, as represented in Figure 8, makes immediately clear that during development the antero-posterior serial arrangement of the right and left ventricles at the outer curvature had transformed into a parallel arrangement. Thus, via the primary heart tube, the parallel-arranged ventricles are connected with the atria that were arranged in parallel from the very outset. Usually this is presented as a problem because the atrioventricular canal would be entirely positioned above the left ventricle. However, as we described above, functionally this is not entirely true. Anatomically this is not entirely true either: when describing developing cardiac morphology one is confronted with the changing position of the organ within the body and of the cardiac component parts in relation to each other. This easily results in confusion: people with a medical background tend to use the body axes as the points of reference and those with a biological background use the axes of the organ, while often terminology is used indifferently (156).
Scanning electron microscopic photographs of the atrioventricular canal of the embryonic heart disclose a narrow entrance, not only to the right ventricle but also to the left ventricle (Fig. 9); the atrioventricular cushions separating the right and left blood flows occupy most of the volume of the atrioventricular canal. The scanning electron microphotograph of the atrioventricular canal represented in Figure 9 clearly demonstrates that both the right and the left atrioventricular canal halves have to expand almost equally to achieve the adult configuration. Hence, the expansion of the right atrioventricular canal to the right seems to be overemphasized to match the segmental model in which the common atrium is supposed to be directly connected to the left ventricle only. When the axes of the developing heart tube are taken as prime reference points, this notion is not true. Viewed in the longitudinal (antero-posterior) direction of the primary heart tube, the right side of the atrioventricular canal is directly connected to the right ventricle. Similarly, the left side of the atrioventricular canal is directly connected to the left ventricle. Both sides expand during further development. In line with this view, the so-called dorsal (inferior or caudal) atrioventricular cushion is not limited to the classical atrioventricular canal but extends toward the ventricular septum (Fig. 9) and guides the right blood flow directly toward the right ventricle and the left blood flow to the left ventricle.
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C. The Primary Ring: Development of the Right Ventricle
A crucial landmark in the development of the right ventricle of the four-chambered heart is the so-called primary ring, also called interventricular or bulboventricular ring (Figs. 8, 9, 10) (43, 212, 333). This ring was recognized in the beginning of the previous century and associated with the developing conduction system (see sect. IIF) (7, 21, 330). Lack of molecular markers had prevented unambiguous delineation of its developmental fate and hence full appreciation of its morphological significance. The primary ring comprises the myocardium of the primary heart tube at the position where the ventricular septum develops. In human embryos, the ring is characterized by the expression of a protein epitope called GlN2 (18). In chickens, it exclusively expresses the Drosophila muscle segment related homeobox transcription factor Msx-2 (43). The complex morphological expression patterns of these factors during further development were described in great detail, and three-dimensional reconstructions were made (43, 154, 173). Both in humans and in chickens, remarkably similar patterns of expression appeared. Figure 8 depicts the position of the primary ring relative to the embryonic blood flow and clearly shows that the ring demarcates the entrance to the forming right ventricle and the outlet of the forming left ventricle. Concordantly, during further development the primary ring becomes positioned at the lower rim of the right atrium just above the right atrioventricular junction and at the left ventricular outlet just below the left ventriculoarterial junction (173, 331). Although this morphological analysis is not strictly speaking a lineage analysis, the precise description of the changing patterns of expression of Msx-2 and GlN2 in a large developmental series in two species (chickens and humans) allows assessment of the contiguity of the changing pattern from stage to stage and makes this morphological analysis almost similar to a lineage analysis.
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Considering Msx-2 and GlN2 as lineage markers, their developmental patterns of expression unambiguously demonstrate that right atrial myocardium develops at the inner curvature upstream from the primary ring, and left ventricular outlet myocardium downstream from the ring. The simplest explanation of the previous statement is that these areas develop directly from the primary heart tube rather than from parts of the tube that had already developed a (left and right) ventricular identity, as is assumed in the segmental model. This notion is supported by molecular data (51, 210) (see sect. IX). The ring divides the primary heart tube into two parts, an upstream or atrioventricular part and a downstream or bulboventricular part. For the right blood flow the ring is atrioventricular, demarcating the right ventricular inlet, and for the left flow of blood the ring is bulboventricular, demarcating the left ventricular outlet (Fig. 8). Similarly, one could divide the cushions of the primary heart tube into two types of cushions as well: 1) upstream cushions rather than atrioventricular cushions, and 2) downstream cushions rather than truncus cushions (Fig. 9). We will conform to conventional terminology, but it is important to appreciate that large parts of the cushions are in fact localized in parts of the primary heart tube that are conventionally called right or left ventricle. The atrioventricular cushions also extend into the atrial part of the heart tube upstream from the atrioventricular canal, although this is less pronounced (Fig. 9).
Chamber formation in the avian and mammalian heart is completed with the physical separation of the blood flows (Fig. 9). For the sake of clarity, we only consider septation of the three principal components of the heart: 1) septation of the atrial chamber by formation of the atrial muscular septum, 2) septation of the ventricular chamber by formation of the ventricular muscular septum, and 3) septation of the primary heart tube by formation of the cardiac cushions. For precise descriptions of the changes in these complex morphological processes we refer to detailed accounts from our laboratory and others (59, 64, 73, 154, 171-173, 228, 292, 314). Only some very basic principles are given here.
The atrial chamber is divided by a muscular septum (primary septum) that is formed by local active proliferation of the myocardial cell layer. Its free rim is covered with a mesenchymal cap (100) that fuses with the atrioventricular cushions and the dorsal mesenchymal protrusion (vestibular spine), as shown in Figure 9 (154, 299, 328, 332). Whether the mesenchymal contribution from three distinct origins (atrioventricular cushion, atrial septal cap, and extracardiac mesenchyme) to the formation of the lower rim of the primary atrial septum is of developmental significance is as yet unknown. Folding of the atrial wall at the right side of this primary septum forms the secondary atrial septum (8, 64, 314). Note that the secondary atrial septum is a unique mammalian structure and is not present in avian and amphibian hearts.
The muscular ventricular septum is formed by apposition of cardiomyocytes at the outer side concomitantly with the outgrowth of the ventricles at the outer curvature (314). In vivo labeling studies have shown that the septum is derived from ventricular cells located at the ventral fusion line of the bilateral mesodermal heart epithelia (65).
The primary heart tube is physically separated into left and right components by fusion of the so-called cardiac cushions. In the primary heart tube, the endocardium is separated from the myocardium by an extracellular matrix layer or cardiac jelly (58, 59). The cardiac jelly disappears from the chamber-forming regions of the cardiac tube, but remains covering the smooth-walled myocardial tube. Subsequently, upon myocardial signaling, a subpopulation of endocardial cells overlying the cushions undergoes a transformation from epithelial to mesenchymal cells, by which the cardiac cushions are formed (31, 83, 264, 336). Neural crest cells also contribute to the mesenchyme of the outflow tract ridges (55, 243, 320), while epicardium-derived cells contribute to the atrioventricular cushions (103). However, the specific functions of these cells are still unclear.
Although the molecular mechanisms of the formation of cushion mesenchyme are well-described (30, 31, 83,205, 244) and the position of cushion formation is clear from the contraction pattern mentioned above, the molecular cues that determine the precise anatomical positions and extents of cushion formation are as yet unknown. Upstream from the primary ring, the two atrioventricular cushions (upstream cushions) develop (Fig. 9); note that th
