Introduction

Congenital heart defects (CHDs) affect about 1% of all live births around the world, with variations in reporting depending on birth rates and access to medical care. The care of children with CHDs has increasingly become a neonatal specialty, aided by prenatal detection. The fetal diagnosis of critical CHDs decreases postsurgical morbidity and mortality, and reparative or palliative cardiovascular surgery is commonplace in the first months of life. This success has led to a larger population of adults with CHDs and an expansion of a subspecialty for the care of these individuals. In parallel with these clinical advances, there has been exciting progress in understanding the cellular and molecular basis of normal and abnormal cardiogenesis and the relationship of heart defects to other congenital defects. The global incidence of CHDs has not decreased and has in fact increased as prenatal detection becomes more common worldwide. Yet our knowledge of the pathogenesis of CHDs is far from complete. Understanding normal and abnormal cardiac embryology is critical to understanding the pathology underlying CHDs and ultimately their prevention.

The mature heart is the product of gene expression driven by endogenous and exogenous influences. The developing heart manifests its morphologic and physiologic plasticity under environmental stimuli. A detailed understanding of the factors that drive normal cardiogenesis is necessary for understanding the causes and consequences of abnormal development. Errors in cardiac morphogenesis involved with septation, valve formation, and proper patterning of the great vessels are responsible for most forms of CHDs. Normal heart development requires precise timing for coordination of the complex three-dimensional contortions of tissues; but paradoxically, these tissues also have a remarkable capacity for modification that compensate for mistakes. These adjustments allow abnormal heart structures and resultant function to be compatible with life up to and even after birth, but they complicate identification of the primary causes of cardiac anomalies. This chapter will focus on clinically relevant aspects of cardiac embryology and how disruptions of this process may lead to CHDs.

Overview of Normal Heart Development

The following description of human heart development, especially the earlier events, is synthesized from information provided by studies of animal models and human embryonic and fetal tissues. A timetable of selected events in human heart development is presented in Fig. 71.1 , which depicts the major transitions in early mammalian heart development.

Fig. 71.1, Overview of cardiac embryology in human gestation.

The primordia of the heart are formed by bilaterally symmetric heart fields derived from the lateral plate mesoderm. These primordia migrate through the primitive streak, between the ectoderm and endoderm layers, to become symmetric mesoderm regions on either side of the primitive streak. During body folding, the primary heart fields fuse cranially and ventrally to form a tubular structure comprising an inner layer of endocardium, a thick layer of extracellular matrix, and an outer layer of myocardium. This apparently “simple” tube begins to contract rhythmically and grows differentially so that dilations (primordia of the heart chambers) and constrictions (primordia of the partitions between chambers) appear along its length. Later additions from the second heart field to the distal ends of the tubular heart form the outflow tract (OFT) and the sinus venosus. Dextral looping of the tube brings the venous caudal portion to a more dorsal position and to the left of the arterial cranial portion as septation of the tubular heart begins. The epicardium arises from tissue dorsal to the heart at the level of the atrioventricular (AV) junction and spreads over the outer surface of the myocardium as a layer of squamous epithelial cells and associated connective tissue during the early phase of septation.

The atrial chamber divides into two chambers by the growth of the septum primum, which forms perforations to create the foramen secundum. A second atrial septum subsequently grows to the right of the primary atrial septum and forms a one-way valve (right-to-left blood flow) between the two atria in the fetus. This avenue of blood flow, the foramen ovale, is permanently closed shortly after birth to functionally complete atrial septation. Ventricular septation is not complete at the time the primary atrial septum is formed. The ventricular septum results from the growth and remodeling of trabecular sheets, continues with expansion of the ventricular chambers, and ends with the fusion of several tissues, including endocardial cushions from several sources and the muscular septum, to form the membranous and muscular interventricular septum. The outflow tract septation is the result of growth and fusion of spiraling ridges that eventually divide the truncus into aortic and pulmonary tracts. The venous and arterial vessels (aortic arches) undergo partial incorporation into cardiac chambers, differential degeneration, fusion, and growth to attain the mature structures.

The human heart has completed the major morphogenetic processes 8 weeks after fertilization. What follows is the completion of maturation of structures, growth, accumulation of cellular junctions at the intercalated discs, biochemical and metabolic adjustments, and compensation for the abrupt changes in patterns of blood flow at birth, such as permanent closure of the foramen ovale of the atrial septum and closure and fibrosis of the ductus arteriosus and ductus venosus.

Scientific Basis of Cardiogenesis

Precardiac to Cardiac Tissues: Commitment and Formation of Primary Axes

The tissue regions giving rise to the heart have been mapped by the application of dyes, particles, and radiolabels at stages when the vertebrate embryo comprises two layers: the epiblast (or primitive ectoderm) and the hypoblast (or primitive endoderm). The process of gastrulation, the movement of precardiac epiblast cells through the primitive streak, appears to be important in specification ( Fig. 71.2 A ). These cells migrate laterally to form the left and right heart fields (see Fig. 71.2 B ), which then create the horseshoe-shaped first and second heart fields around the end of the primitive streak, the primitive node. The second heart fields are adjacent to and influenced by cardiac neural crest cells, a process critical to understanding how the later steps in cardiogenesis are affected by neural crest–associated genetic syndromes.

Fig. 71.2, Myocardial progenitors in the chick primitive streak and their fates in the heart tube (A-E) . The craniocaudal organization of progenitor cells (A) transitions as they migrate and assume a mediolateral orientation (B) . Cranial sections marked by asterisks (B) become the ventral portion of the heart tube (C) and later the right-most border of the looping heart (D) . At, Atrium; AVC, atrioventricular canal; C, conus; LV, left ventricle; RV, right ventricle; T, truncus.

Transcription factors are proteins that bind as part of complexes to specific DNA sequences and influence the expression of genes ( Fig. 71.3 ). Such factors influence many aspects of cardiogenesis and include members of the following families: the helix-loop-helix proteins (dHand and eHand), zinc finger proteins (GATAs), homeobox gene proteins (NKX2.5), and MADS box proteins related to serum response factor. Of the zinc finger proteins, GATA4, 5, and 6 are much-studied members of the evolutionarily conserved transcription factor subfamily that recognizes a “GATA” DNA sequence motif. These have been implicated in the early specification of embryonic tissue destined to become heart.

Fig. 71.3, Model of transcription regulation of cardiac chamber and nonchamber genes. NKX2.5, GATA4 , and TBX5 are widely expressed in the developing heart and promote chamber-specific gene expression (chamber), whereas TBX2 and TBX3 are differentially expressed and inhibit chamber-specific gene expression by competing with TBX5 binding (nonchamber). Chambers are the left ventricle ( lv ) and the atrium ( at ). Nonchamber regions include the outflow tract ( oft ), atrioventricular canal ( avc ), and inflow tract ( ift ). These proteins bind to DNA at specific sequences that are involved in controlling gene expression. The combinatorial effect of the binding of these proteins depresses or stimulates the transcription of cardiac-specific genes.

An example from the homeobox gene family demonstrates how the study of fruit flies has advanced our understanding of human heart development. The dorsal vessel pumps hemolymph (insect blood) and is considered the insect heart. It is derived from mesoderm under the influence of homeobox genes. The mutation of one of these genes, tinman , results in absence of the dorsal vessel. Frog and mouse homologues of these homeobox genes were identified and localized in expression to the developing heart. Transgenic mice lacking a tinman homologue ( NKX2.5 ) do not lack hearts but die at 9 days of development with severely abnormal hearts. Humans in whom the tinman homologue NKX2.5 is mutated suffer atrial septal defects with associated AV conduction delay and other complications. The fruit fly findings led to the study of a set of genes critically important to many aspects of human heart development.

The Tubular and Looping Heart

Within each side of the first heart fields primitive endocardial tubes form, come together, and fuse as the lateral body portions fold to create the primitive heart tube. The most caudal cells of the right and left heart fields will form the most ventral portion of the primitive heart tube (asterisks in Fig. 71.2 ). The inflow portion of the heart quickly gains pacemaker activity and initiates slow, unidirectional contractions along the tube. Concurrent with this process, endocardial and myocardial cells—which form the layers of the developing heart—emerge from a common precursor. The myocardium secretes a thick layer of acellular extracellular matrix, also known as cardiac jelly, which creates a distinct layer between the myocardium and the endocardium ( Fig. 71.4 ). This tissue progression requires cell differentiation, sorting, and polarization. Evidence supports both negative and positive influences from the surrounding neural tissue and endoderm on these processes.

Fig. 71.4, In vivo images of a quail heart. The images demonstrate portions of the cardiac cycle in a stage 14 quail embryo. The cardiac jelly is an acellular (black) layer between the myocardium and the endocardium.

The primitive heart tube undergoes three connected steps, looping, convergence, and wedging, all of which are crucial to normal cardiac septation. Looping is the first obvious structural left–right lateralization in the developing embryo ( Fig. 71.5 ). The heart tube elongates and folds into an “S” shape to the right. The direction of looping will determine the ultimate ventricular positions. Looping to the right will give the normal Dextro, or “D,” looping. If the primitive tube loops to the left, this will result in Levo, or “L,” looping and result in the left ventricle on the right side and the right ventricle on the left side. The left–right handedness of looping is directed by monocilia in the primitive node many stages before looping starts. These cilia are angled 40 degrees posterior and rotate in a clockwise direction, resulting in a leftward laminar flow of fluid containing key signaling molecules. Mutations in genes responsible for ciliary ultrastructure result in syndromes such as Kartagener syndrome. Importantly, the primitive atrium is not involved in this portion of looping, so atrial position is not severely disturbed by abnormal looping. Atrial positions are affected by situs abnormalities as seen in heterotaxy syndromes. Left–right embryologic abnormalities have a wide range of known genetic causes in which mutations interfere with early breaking of left–right symmetry, abnormal signal transduction or gene expression in the lateral plate mesoderm, and abnormal morphogenesis of internal organs.

Fig. 71.5, The progression of looping in the tubular heart.

Convergence is the process of proper orientation of the inflow and outflow tracts ( Fig. 71.6 ). The inflow tract rises cranially behind the outflow tract such that the inflow and outflow tracts are aligned, with the outflow tract ventral to the inflow tract. Abnormalities can lead to irregularities in atrial and ventricular septa and outflow tracts. Convergence and looping are dependent on lengthening of the polar ends of the heart tube, which is achieved by the addition of cells from the second heart field. The original primitive heart tube will grow to become the left ventricle and portions of the atrioventricular canal, and additions from the second heat field will direct formation of the outflow tracts, right ventricle, and atria. As cardiac neural crest cells influence the second heart field, it becomes easier to understand how neural crest cell–associated disease such as DiGeorge syndrome, 22q11 deletion syndromes, and velocardiofacial syndromes result in abnormalities of these cardiovascular structures.

Fig. 71.6, The early stages of cardiac development while in the tubular phase include looping, convergence, and wedging. The red circles indicate the changing positions of the future aortic valve. A, Atrium; AVC, atrioventricular canal; LV, left ventricle; OT, outflow tract; RV, right ventricle.

Occurring in parallel with convergence and septation (see Figs. 71.5 and 71.6 ) is wedging ( Fig. 71.7 ). Here, the outflow tract rotates counterclockwise approximately 45 degrees, resulting in the future aorta residing behind the future pulmonary artery (see Fig. 71.7 ). The aortic portion shortens, and the future site of the aortic valve “wedges” between the future tricuspid and mitral valves. This separates the mitral valve from the septum, which is why the mitral valve has no connections to the septal wall. Irregularities in wedging result in conotruncal defects such as tetralogy of Fallot and double outlet right ventricle.

Fig. 71.7, Process of aortic wedging. The outflow tract descends and rotates (a-c) , aligning it with the center of the atrioventricular valves and canal. The area of the future aortic valve then migrates between the future tricuspid and mitral valves (d-e) .

How is the program for cardiac muscle differentiation turned on and regulated? This question has been approached by studying the transcription factors that bind to promoter regions of the cardiac-specific cytoskeletal genes coding for molecules that appear in the early tubular heart. The discovery of MyoD, a helix-loop-helix DNA-binding protein that controls skeletal muscle expression, led investigators to search for similar factors in cardiac muscle. Current findings support the idea that both positive and negative regulators of cardiac genes exist and that they work in complexes that define the spatiotemporal pattern of expression of cardiac genes (see Fig. 71.3 ).

Closely related to the question of cardiac-specific gene regulation is the question of regional specification of the heart tube. Segmentation, although not as obvious in the vertebrate heart as it is in the insect body plan, is important in cardiogenesis. Chamber-specific expression of a number of endogenous genes and promoter-driven reporter genes suggests that the outflow tract, the left and right ventricles, the atria, and the sinus venosus can in some cases be considered separate cardiac segments. However, many of the endogenous cardiac genes do not strictly follow chamber-specific expression but rather appear to be modularly controlled based on their position along the anteroposterior axis (see Fig. 71.2 ).

An understanding of segment specification has been greatly advanced by the study of the homeobox genes in the fruit fly. The identity and differentiation of segments are determined by interactions among homeobox and other genes. These interactions occur in the mesoderm, which gives rise to the heart tissues and the tissues adjacent to the mesoderm.

The vertebrate heart tube normally loops to the right side of the embryo. Sidedness appears to be influenced by factors acting at a time when the heart is not recognizable as a distinct organ structure. Transplantation studies using the early chicken embryo suggest that left and right sidedness are already determined in the precardiac mesoderm stages. Sidedness can also be regulated in part by retinoids. Retinoic acid–soaked bead implants in chicken embryos or application of all- trans retinoic acid in mouse embryos cause transposition of the great arteries. Abnormalities in the direction of cardiac looping also occur in humans. These abnormalities are linked to an autosomal recessive gene defect that has been detected in the Amish population and another gene defect in the X chromosome (Xq24-q27.1) in the general population. Interestingly, abnormalities in genes associated with left–right asymmetry may also be responsible for apparently isolated cases of congenital heart defects. Mutations in the laterality CFC1 gene have manifested as transposition of the great arteries in humans without other obvious laterality defects.

Two mouse mutants have abnormalities in sidedness. The iv/iv mutant mouse develops L-looping and dextrocardia 50% of the time. A large percentage of these mice have a combination of cardiac defects, including persistent sinus venosus, atrial and ventricular septal defects, common AV canal, double-outlet right ventricle, tetralogy of Fallot, and transposition of the great arteries. This gene has been mapped to mouse chromosome 12, which is homologous to human chromosome 14. The inv/inv mutant mouse develops situs inversus close to 100% of the time. Mutation screens using the zebrafish have uncovered a number of genes involved in laterality specification and indicate that there are organ-specific laterality regulators. Identification of the genes involved in these human, mouse, and zebrafish mutations could provide valuable insight into the mechanisms of normal dextral looping of the heart.

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