Molecular Development Of The Heart Tube

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Embryo Patterning

Morphogenesis of the heart begins with the initial patterning of the embryo that determines the three axes of the embryo: anterior-posterior, dorsal-ventral, and left-right. These axes are imprinted onto the cellular program as cell populations expand to form the embryo and extraembryonic tissues. Specific genes have been identified that alter axis determination in a range of species, including the mouse.!2,!3 After determination of the embryo axes, subpopulations of cells are programmed in a segmental body plan. Much of the current understanding of the body plan comes from developmental studies of Drosophila, an insect with a head, thorax, and abdomen.14 In mammals, maternal gene products control the cell through the first two cell cycles and then control switches to the embryonic genome. These patterning (homeobox) genes are arranged along the anterior-posterior axis of the embryo.15 Structural asymmetry is apparent at the blastodisc stage, when the primitive streak defines the anterior-posterior axis and the dorsalventral axis is defined by the position of the yolk sac. Myocyte commitment in the chick embryo occurs in the early blastula stage, followed by clonal expansion in the bilateral heart-forming regions located in the lateral splanchnic folds after gastrulation. Molecular studies also have confirmed the segmental patterning of the cardiac tube, linking gene products with morphologic boundaries between segments that eventually integrate to form the future atria, ventricles, and outflow tract in chick, mouse, and human hearts.!6

The process of mesoderm formation is integral to the organization of the primary axis of the embryo and the differentiation of the right and left sides. At the blastodisc stage of development, there are two primitive germ layers-endoderm and ectoderm-and then the endoderm layer splits into splanchnic and visceral layers with interposed mesodermal cells Fig. 9-2). Mesoderm is formed as ectodermal cells migrate through the primitive streak coursing adjacent to Hensen's node (organizer). Hensen's node contains retinoic acid and serves as an embryonic organizer that may confer positional information on the mesodermal cells.!7 At this critical phase in cell determination, exogenous retinoic acid is extremely teratogenic. Interestingly, retinoic acid has a gradient-like effect on the determination of the heart tube, with the greatest effect at the arterial pole and the smallest effect at the venous pole.18 After migration, this crescent of mesodermal cells forms the precardiac region from which the heart and the precursor cells of the great vessels originate.

Molecular Factors Involved in Cardiogenesis

Defining the molecular basis that underlies the establishment and maintenance of cardiac muscle differentiation has presented a fundamental challenge in developmental biology and molecular genetics. Despite the shared expression of numerous contractile protein genes by both cardiac and skeletal striated muscles, the molecular mechanisms for cell determination, differentiation, and tissue patterning are quite distinct. The following section presents some of the current, relevant information on molecular cardiogenesis, as defects in these molecular mechanisms have been shown to be associated with structural and/or functional heart diseases in children and adults (Fig. 9-3).

Molecular Formation Heart

Figure 9-3: A lateral plate of mesoderm on each side of the midline forms the progenitor for the development of the heart and portions of the great vessels. The NK-Factor homeobox genes, such as Nkx2.5, in combination with multiple other genes, such as the serum response factor (SRF), are responsible for activating commitment of these undifferentiated cells to cardioblasts. Myocyte enhancer factor (MEF) has a binding site in practically all muscle genes and is essential to the development of cardiac myocytes. The CARP gene is downstream of Nkx2.5 in the cardiac lineage. The genes responsible for the fusion of the two lateral mesodermal plates into a single tube are unknown, but experiments show that GATA-4 is necessary, along with SRF and many other genes yet to be identified. Genes involved in laterality include Activin, FGF-9, Shh, and nodal. The cardiac tube that forms a loop to the right requires Nkx genes and the eHAND and dHAND as well as inv (inversion of embryonic turning) and iv genes. The dHAND gene is responsible for the formation of the right ventricle, and the eHAND for that of the left ventricle.

Figure 9-3: A lateral plate of mesoderm on each side of the midline forms the progenitor for the development of the heart and portions of the great vessels. The NK-Factor homeobox genes, such as Nkx2.5, in combination with multiple other genes, such as the serum response factor (SRF), are responsible for activating commitment of these undifferentiated cells to cardioblasts. Myocyte enhancer factor (MEF) has a binding site in practically all muscle genes and is essential to the development of cardiac myocytes. The CARP gene is downstream of Nkx2.5 in the cardiac lineage. The genes responsible for the fusion of the two lateral mesodermal plates into a single tube are unknown, but experiments show that GATA-4 is necessary, along with SRF and many other genes yet to be identified. Genes involved in laterality include Activin, FGF-9, Shh, and nodal. The cardiac tube that forms a loop to the right requires Nkx genes and the eHAND and dHAND as well as inv (inversion of embryonic turning) and iv genes. The dHAND gene is responsible for the formation of the right ventricle, and the eHAND for that of the left ventricle.

Basic Helix-Loop-Helix Factors and Muscle Development

One of the initial, critical discoveries related to muscle development was the observation that a specific transcription factor, Myo-D, expressed in myoblasts19 is sufficient to convert a variety of mesodermal and nonmesodermal cell types to stable myoblasts with active muscle-specific gene expression. Using Myo-D as a probe, several additional regulatory factors that specify skeletal muscle cell lineage in fibroblasts have been identified: myogenin,2021 Myf5,^ MRF4-herculin, and Myf6.23-25 These factors share extensive homology within a basic region and an HLH motif that mediate DNA binding and dimerization, respectively.26 HLH proteins share the ability to recognize the DNA consensus sequence CANNTG, known as an E-box, which first was identified with the immunoglobulin enhancer26 and subsequently was found in regulatory regions of most muscle-specific genes. Thus, the regulatory paradigm for skeletal muscle differentiation is centered on the bHLH myogenic regulatory factors, but Myo-D, myogenin, Myf5, MRF-4, and Myf6 are not expressed in the heart.27

In the heart, there are other basic HLH factors. dHAND and eHAND are two bHLH transcription factors that share high homology in their bHLH regions and show segment-specific expression patterns.28 In the mouse, HAND expression coincides with that of other cardiac transcription factors. dHAND expressed in the endocardium is maintained throughout the straight heart tube but is restricted to the conotruncus and the future right ventricle as the heart tube forms a loop. eHAND expressed in the myocardium rapidly becomes restricted to the conotruncus and the left ventricle.29 Expression of dHAND and eHAND precedes separation of the two ventricles, representing early chamber specification. In addition to cardiac expression, dHAND (HAND1) is expressed in early trophoblast tissue and is required for the nutritional support of the developing mouse embryo.30 It is of interest that Nkx-2.8 has an expression pattern that overlaps eHAND, being restricted to the rostral and caudal regions of the heart tube after looping and being expressed in the endoderm of the pharyngeal arches. The deletion of dHAND by gene targeting showed that dHAND expression is necessary for the formation of the right ventricle.2931 Thus, it appears that dHAND may specify the right ventricle, and eHAND the left ventricle. In addition, dHAND and eHAND specify right ventricle (RV) and left ventricle (LV) specific morphology independent of situs.32 Expression of cardiac-specified genes Ct'MHC, MLC2A, MLC2V, ANE, and Nkx-2.5 was not affected by elimination of the dHAND gene. GATA-4 in the myocardium was downregulated by dHAND-deficient hearts and appears to be a downstream target of dHAND.

Drosophila tinman Is Required for Insect Heart Development

The identification of molecular mechanisms involved specifically in heart development has depended on the investigation of simpler biological models, including the fly Drosophila. Homeotic genes are genes that determine a change in structure and have in common a domain that codes for 60 amino acids. Genes with this sequence, referred to as homeobox (Hox) genes, generally are upregulated during early differentiation and appear in a time-dependent sequence. Homeobox genes have been studied extensively in Drosophila, where they are involved in the commitment of cells to specific developmental pathways and play an important role in pattern formation.33

Recently, the NK homeobox family of genes (NK-1/S59, NK-2/vnd, NK-3/bagpipe, and NK-4/msh-2/tinman and H6) was identified in the mouse.34 Nkx-2 factors are DNA-binding proteins (transcriptional factors) that are capable of activating transcription; their 60-amino-acid homeodomain includes three helices, in which helix II and helix III form a helix-turn-helix motif.35 Helix III fits across the AT-rich major groove of the DNA binding site. Nkx-2.5 has been shown to bind to novel NKE sites,36 certain serum response elements of the cardiac alpha-actin promoter,37 and the NKE sites in the cardiac atrial natriuretic factor promoter.38 Gajewski and associates39 showed that two NKE promoter sites direct Drosophila MEF2(dMEF2) expression in response to tinman. Mutations in the tinman gene result in loss of heart formation in the Drosophila embryo.40 In addition, tinman is known to regulate NK-3/bagpipe expression in the visceral mesoderm41 and the expression of dMEF2.^ These observations suggest that tinman may be involved in cardiac mesoderm patterning and make it a likely marker for cardiac mesoderm induction.

Tinman and Other Related NK-2 Genes Are Required for Vertebrate Heart Morphogenesis

Homeobox genes of the NK class also may function in early cardiac development in vertebrates. The murine NK-2 homeobox gene Nkx-2.5/Csx is expressed in early cardiac progenitor cells before cardiogenic differentiation and continues through adulthood.42,43 Superimposed on the appearance of Nkx-2.5 in cardiac progenitor cells is the sequential expression of the cell type-restricted cardiac alpha-actin and MHC genes.42 The Nkx-2.5 factors identified in other vertebrates, such as zebrafish,44 Xenopus,45 and chickens,46 were highly related in sequence and expression pattern to the mouse gene and to cardiac development.

The similarity in expression patterns between Nkx-2.5, XNkx-2.5, ceh-22, tinman, and bagpipe suggested that the function of these genes might be conserved. Another member of the Nkx-2 family, Nkx-2.8, which was recently isolated from avian species, is closely related to Xenopus, chicken, and zebrafish Nkx-2.5 homeoboxes and is expressed in the developing embryo in the lateral plate mesoderm and underlying pharyngeal endoderm.47 An attractive hypothesis is that these homeodomain factors function in phylogenetically conserved myogenic pathways occurring in muscle types that do not utilize the Myo-D family. Whether the vertebrate Nkx-2.5 or other Nkx-2-related genes expressed in the early heart play a role in heart specification or whether they are downstream regulators of cardiac gene expression remains to be determined. In this respect, it is interesting to note that although it does not inhibit formation of the cardiac tube, homologous recombination knockouts of the endogenous murine Nkx-2.5 gene do result in cardiac dysmorphogenesis at the looping stages of development and embryonic lethality.48

The partially overlapping expression pattern of Hox genes in embryos has led to the concept of a "Hox code."49 The term Hoxcode means that a particular combination of Hox genes is functionally active in a region and thus specifies the developmental fate of this region. The existence of eight Nkx-2 family members, their overlapping DNA-binding specificities, and, most important, their partially overlapping patterns of expression raise the possibility of an "Nkx code."47 Overexpression of Nkx-2.5 in a zebrafish embryo results in an enlarged heart.44 Thus, inactivation of the Nkx genes by homologous recombination and their overexpression as transgenes offer promise in addressing the functional significance of the expression domains and thus also of the Nkx code. As is mentioned below, patients with secundum atrial septal defect have been identified to have specific mutations in the human homolog to the Nkx2.5 gene.49

Cardiac-Restricted Ankyrin Repeat Protein Gene

The cardiac-restricted ankyrin repeat protein (CARP) gene encodes a nuclear coregulator for cardiac gene expression which lies downstream of the cardiac homeobox gene Nkx2.5 and is an early marker of the cardiac muscle cell lineage.50 The expression of the CARP gene is developmentally downregulated and dramatically induced as part of the embryonic gene program during cardiac hypertrophy. A distinct 5' cis regulatory element directs heart segment-specific expression, such as atrial versus ventricular and left versus right. In addition, a 213-base-pair sequence element of the gene confers conotruncal segment-specific expression.50 In addition, an essential GATA-4-binding site is present in the proximal upstream regulatory region of the gene and cooperative transcriptional regulation is mediated by Nkx2.5 and GATA-4. This cooperative regulation is dependent on the binding of GATA-4 to its cognate DNA sequence in the promoter, which suggests that Nkx2.5 controls CARP expression, at least in part, through GATA-4.50

SRF and MEF2, MADS Box Factors Involved with Cardiogenesis

Serum response factor generally was presumed to be a ubiquitous and constitutive trophic factor54 but was later shown to be highly expressed in the embryonic heart.52 Serum response factor (SRF) represents an ancient DNA-binding protein whose relatives shared a highly conserved DNA-

binding/dimerization domain of 90 amino acids, termed the MADS box. SRF-related proteins that are capable of binding to sites found in the regulatory regions of both nonmuscle- and muscle-specific genes also belong to the MADS box family of trophic factors.53 SRF-related proteins are capable of binding MEF2 sites, CTA(A/T)4TAG, which can be found in the regulatory regions of both nonmuscle- and muscle-specific genes.54,55 Like SRF, MEF2 factors contain a MADS box and an adjacent MEF2 box. Expression and mutagenesis studies in Drosophila have shown that MEF2 proteins are necessary for myogenic differentiation during development56,57 and are activated by tinman.39

In the mouse embryo, MEF2 genes are highly expressed in the early heart and skeletal muscle progenitor cells before the induction of cardiac and skeletal muscle structural genes, implicating MEF2 as key regulator of cardiac and skeletal muscle differentiation programs.58-60 Four MEF2 genes have been isolated in vertebrate species and are referred to as MEF2A-MEF2D.6064 The four MEF2 gene products are highly homologous in the MADS box domain but are divergent in the carboxy termini, arising from alternative splicing mechanisms. MEF2C shows a tissue-restricted expression pattern, being expressed exclusively in skeletal muscle, brain, and spleen, and is induced by myogenin in fibroblasts during myogenic differentiation in tissue cultures.62

Transactivation of the Cardiac Alpha-Actin Gene by Nkx-2.5 and SRF

Gilman and coworkers63,64 showed that human SRF interacts with a novel human homeodomain protein, Phox, which shows similarity to the homeodomains of two murine Pax genes. The highest similarity is to a partial murine cDNA termed S865 and to MHox, a novel homeodomain protein expressed in mesoderm.66 Phox interacts with SRF to enhance the exchange of SRF with its binding site in the c-fos gene. It has been shown that Nkx-2.5 transactivates the cardiac alpha-actin gene by binding to SRF, but only after SRF has bound to DNA.67

The Role of the GATA Family in Cardiogenesis

The GATA family of proteins has been subdivided, with GATA-1/2/3 being linked to hematopoiesis and GATA-4/5/6 thought to be involved with cardiac, gut, and blood vessel formation. Each of the six GATA proteins contains a highly specific DNA-binding domain consisting of two C4 zinc fingers that bind to the DNA sequence element (A/T)GATA(A/G) and that may be able to interchange with each other. GATA-4 and 6 have been found to be expressed in a developmentally and lineage-specific pattern within cardiac mesoderm and gut epithelium.68-70 GATA-5 expression is restricted to the endocardium. Experiments have shown that GATA-4 regulates the expression of cardiac-specific genes such as cardiac troponin C74 and alpha-MHC.72 Mice without the GATA-4 gene display a severe defect in the formation of the cardiac tube. Several studies have demonstrated that the GATA-4 transcription factor plays an important role in regulating cardiac-specified genes and appears to be downstream to the Nkx-2.5 gene.

Cardiogenesis, an Nkx-2-Dependent Paradigm

An attractive hypothesis from the analysis of these NK-2 homologues is that these homeodomain factors function in phylogenetically conserved pathways in muscle cell types that do not utilize the Myo-D family. Expression of Nkx-2.5 in fibroblasts demonstrated that downstream targets such as the cardiac alpha-actin gene are not directly activated by Nkx-2.5 alone but require the collaboration of additional factors, such as . Whether the vertebrate Nkx-2.5 or other Nkx-2-related genes with SRF are sufficient to play the primary role in heart specification and serve as regulators of other downstream cardiac genes remains to be determined. It is reasonable to postulate that the vertebrate MEF2C genes and the GATA-4 factor are high in the hierarchical order of regulatory factors that, in combination with Nkx-2.5 and SRF, specify the cardiac cell lineage.

Role for Bone Morphogenic Proteins in Initiating Early Myocardial Cell Differentiation

One type of signaling molecule responsible for cardiogenic commitment was identified to be composed of the bone morphogenic proteins (BMPs), which are members of the transforming growth factor-beta family of signaling molecules. BMP-2 and -4 appear to be capable of inducing the cardiac regulatory factors Nkx-2.5 and GATA-4 when ectopically applied to regions of chick embryos that usually are not specified to become heart tissue.46 In mice with the BMP-4 gene eliminated (knockout mice), there was little or no mesoderm differentiation. Some of the mice deficient for BMP-2 gene that lacked Nkx-2.5 expression also failed to develop beyond the early stages of looping.73 Thus, BMPs appear to have an early influence on cardiogenesis and Nkx-2.5 expression.

Laterality of the Cardiac Tube

Correct laterality is fundamental to the developing embryo, and situs solitus has the lowest risk of congenital cardiovascular malformations.74 The first grossly asymmetric feature to develop is the heart tube, which forms from the fusion of cardiac primordia at the midline (see B-H0i Fig. 9-2). Subsequently, the initially symmetric heart acquires a dextral loop. The tubular heart initiates rhythmic contractions at about day 23 in humans and then undergoes rightward looping. This pattern of left-right asymmetry occurs in all vertebrate internal organs as a result of a signaling cascade present before gastrulation. On the right side of Hensen's node, the secreted morphogen activin represses Sonic hedgehog (Shh) expression and induces expression of the genes for the activin receptor and fibroblast growth factor 8. On the left side, Shh induces Nodal expression in lateral plate mesoderm and subsequent left-sided expression of the bicoid-like homeobox gene Pitx2. The homeobox gene Nkx3.2 is asymmetrically expressed in the anterior left lateral plate mesoderm (LPM) and head mesoderm in the chick embryo.75 Misexpression of the normally left-sided signals Nodal, Lefty2, and Shh on the right side or ectopic application of retinoic acid results in upregulation of Nkx3.2 contralateral to its normal expression on the left. FGF8 is an important negative determinant of asymmetric Nkx3.2, and ectopic application of FGF8 on the left side blocks Nkx3.2 expression, whereas an FGF receptor-1 antagonist implanted on the right side results in bilateral Nkx3.2 expression in the LPM.75

There is a genetic basis for left-right asymmetry, as several types of unlinked mutations affecting left-right laterality exist in mice and humans. For example, in the offspring of iv mice (lacking the iv gene), 50 percent have situs inversus.76 The iv gene has been mapped to mouse chromosome 12 and has been identified to code for the structural protein dynein. The inv (inversion of embryonic turning) gene mapped to chromosome 4 causes complete reversal of left-right symmetry and cardiac looping.7778 Nkx3.2 expression also was found to be asymmetric in the mouse LPM, but unlike in the chick, it was expressed in the right LPM. In the inversion of embryonic turning (inv) mouse mutant, which has aberrant left-right (L-R) development, Nkx3.2 was expressed predominantly on the left side. Thus, Nkx3.2 transcripts accumulate on opposite sides of mouse and chick embryos, although in both the mouse and the chick, Nkx3.2 expression is controlled by the L-R signaling pathways.

Myocardial Expansion and Differentiation

Retrovirus labeling studies have demonstrated that the ventricular myocardium expands by a process of clonal expansion of the epitheliod myocytes of the cardiac tube (see Figure 9-7).7980 The regulation of myocyte specification and differentiation is complex and probably involves cell adhesion molecules, including N-cadherin, extracellular proteases, and morphogenetic signals from the transforming growth factor-beta (TGF-beta) and FGF families of growth and differentiation factors.80-84

Mice Embryo Sections

Figure 9-7: Schematic representation of the localization of remnants of the primary ring in the neonatal human heart. The ring is projected on a superior view of the aortic mitral fibrous unit of the adult heart. The black dots indicate the areas in which remnants of the ring are detected in a series of neonatal hearts. 1 = anterolateral part of the right atrioventricular ring; 2 = posteromedial part of the right atrioventricular ring; 3 = "dead-end" tract of the conduction axis; 4 = lateral part of the retroaortic rootbranch; 5 = posterior part of the retroaortic rootbranch. AVN = atrioventricular node; AoV = semilunar valve of the aorta; LBB = left bundle branch; mi = mitral valve; PuV = semilunar valve of the pulmonary trunk; Rbb = right bundle branch; SB = septal branch; tri = tricuspid valve. (Adapted from Wessels et al.126)

Figure 9-7: Schematic representation of the localization of remnants of the primary ring in the neonatal human heart. The ring is projected on a superior view of the aortic mitral fibrous unit of the adult heart. The black dots indicate the areas in which remnants of the ring are detected in a series of neonatal hearts. 1 = anterolateral part of the right atrioventricular ring; 2 = posteromedial part of the right atrioventricular ring; 3 = "dead-end" tract of the conduction axis; 4 = lateral part of the retroaortic rootbranch; 5 = posterior part of the retroaortic rootbranch. AVN = atrioventricular node; AoV = semilunar valve of the aorta; LBB = left bundle branch; mi = mitral valve; PuV = semilunar valve of the pulmonary trunk; Rbb = right bundle branch; SB = septal branch; tri = tricuspid valve. (Adapted from Wessels et al.126)

The Neural Crest and Cardiac Development

The cardiac neural crest is an important migratory cell population that contributes to cardiovascular morphogenesis. The cardiac neural crest arises from the dorsal margin of the neural tube before fusion and migrates ventrally to form the autonomic ganglia, melanocytes, and Schwann cells. The crest cells move in waves through the branchial arches during the first 4 weeks of human development. The eventual fate of the neural crest cells likely is determined long before the initial phenotypic expression of a heart tube by activation of the cellular gradients of Hox genes and other morphoregulating factors.8586 The cranial neural crest region defines a developmental field that includes the heart, hindbrain, face, and branchial arch derivatives.

Experimental disruption of cranial neural crest produces a spectrum of abnormalities. In a series of elegant ablation and chick quail chimera studies, Kirby and Waldo defined the range of cardiac neural crest that is integral to the septation of the conotruncal region of the heart and branchial arch derivatives, including facial abnormalities, thymus, parathyroid, and autonomic derivatives.85 These neural crest cells are site-specific and carry information for the formation of structures appropriate to their origin rather than being defined at the destination of migration.

Several genes have been identified as important in the proper migration and differentiation of the cardiac neural crest. The Splotch mutant mouse is characterized by a mutation in the Pax-3 gene.87 Homozygote Splotch mutants have a complete neural crest ablation phenotype, including persistent truncus arteriosus and aortic arch anomalies,88 similar to the CV phenotype of neural crest ablation in the chick embryo.86,89 Hox gene abnormalities also are associated with defects in the derivatives of cranial neural crest. A transgenic murine model of Hox 1.1 overexpression has neural crest ectomesenchymal tissue abnormalities, including cleft palate, nonfused pinnae, and open eyes. Hox 1.5-deficient mice have features of DiGeorge's syndrome.86 In humans, DiGeorge's syndrome, velo-cardio-facial syndrome, and conotruncal anomaly face syndrome are associated with chromosomal deletions in the 22q11 region on the long arm of chromosome 22.9093 Recent studies have indicated a number of candidate factors in the pathogenesis of these syndromes (referred to as catch22 syndrome). In addition, retinoic acid is a potent teratogen in humans and produces a syndrome involving all the derivatives of the cranial neural crest.94

Myocyte Differentiation

In the human embryo, the heart begins to contract at day 17 as the machinery of contraction and relaxation becomes functional. These functional units include the sarcomere, composed of the contractile elements; the mitochondria, containing the enzymes for energy production and modulation; and the sarcolemma, the cell envelope with specialized components of the t tubular system linked to the sarcoplasmic reticulum. In the mature myocardium, sarcomeres are organized parallel to the lines of peak systolic stress. In the embryonic myocyte, myofibrils initially appear disarrayed and become aligned as development proceeds.95 Despite this disordered appearance, the contraction pattern of the early embryonic heart is isotropic.96

The temporal and spatial expression of contractile proteins in the developing heart is under intense investigation. At the precardiac tube stage, smooth muscle alpha-actin is the only isoform present. With formation of the cardiac tube, there is progressive expression of the cardiac form of sarcomeric actin with the onset of cardiac pumping. The alpha smooth muscle actin may act as a scaffolding during assembly of the sarcomere.97,98

Mitochondria multiply concurrently with the myofibrils in the differentiating myocyte. In the mature heart, mitochondrial enzymes are the major source of high-energy phosphate necessary for contraction and probably begin this function during embryonic development. In the chick, the mitochondria account for about 10 percent of myocyte volume.95 In the rat embryo, total volume increases from 22 to 34 percent between days 6 and 10, and the mitochondria also change morphologically with development, becoming larger with more cristae and a denser matrix.99 The myocyte mitochondrial volume fraction correlates directly with heart rate and oxygen consumption among animals.!00

Maturation of the sarcoplasmic reticulum and the apparatus for excitation-contraction coupling occurs coincident with the structural morphogenesis of the embryonic heart. The sarcolemma contains ion pumps, channels, and exchangers that maintain chemical and charge differences between extracellular and intracellular spaces.!04 During maturation of the heart, the resting potential increases (becomes more negative) in both birds and mammals 102,103 Ca2+ influx through Ca2+ channels may play a relatively important role in transsarcolemmal Ca2+ influx in the immature heart. However, peak Ca2+ current density is actually decreased compared with that measured in mature cells.104,105 Although Ca2+ influx by way of the Na+-Ca2+ exchanger is less important for excitation-contraction coupling in mature myocardium, Na+-Ca2+ exchange may play an important role in myocytes from relatively immature rabbit hearts.

Relaxation, an active process by which the myocardium returns to steady state after contraction, depends on rapid removal of Ca2+ from troponin C. This is mediated primarily by active transport of Ca2+ back into the sarcoplasmic reticulum (SR). The SR Ca2+ pump ATPase (SERCA2a)

usually couples hydrolysis of adenosine triphosphate (ATP) to active Ca2+ transport. The rate of SR Ca2+ uptake correlates well with the observed rate of myocardial relaxation. Regulation of SR Ca2+ pump activity is mediated by the intrinsic SR protein phospholamban. Ca2+ also is removed from the myofilaments by extrusion across the cell membrane. In the steady state, the amount of Ca2+ removed from the myocyte equals the amount entering through the Ca2+ channels.!06

Segmental Basis of Heart Tube Formation

Formation of the cardiac tube is a complex morphogenetic event. The primitive, bilateral heart tubes each contain an inner layer of endocardium, a middle layer of cardiac jelly, and an outer layer of myocardium. At the cephalic end of the embryo (on each side of the midsagittal plane), myocytes within a section of each heart tube acquire contractile elements, and the position of the heart tubes shifts first to be parallel and close to each other within the cephalic part of the developing body cavity (intraembryonic coelom), ventral to the foregut, followed by fusion of the heart tubes in the ventral midline to form the linear or straight heart tube.4,5107-109

It is important to note that the primitive linear heart tube does not contain all the segments present in the mature heart. During morphogenesis, the proximal portion of the aortic sac is incorporated into the outflow tract of the right ventricle (along with migrating neural crest cells) and the sinus venosus is incorporated into developing atria. Thus, each "segment" of the mature heart arises at a unique time during embryogenesis.ii0 One critical aspect of this segmental assembly and maturation of the heart is that there likely are both temporal and spatial "windows" that are developmentally regulated, and this may explain why similar morphogens such as retinoic acid produce a wide spectrum of teratogenic effects depending on the time in gestation of exposure. Another aspect of this segmental paradigm is the concept that cardiac morphogenesis depends on molecular and cellular as well as mechanical interactions between the respective segments in the developing heart.!!0

Cardiac Jelly

Prior to looping, the acellular space between the myocardium and the endocardium in the heart is filled with a deformable extracellular matrix. This "cardiac jelly" forms before cardiac tube fusion and is closely associated with the primordial myocytes.!!! At the pretubular heart stages, the extracellular matrix contains collagen types I and IV, fibronectin, and laminin. The primordial endothelial cells destined to form the endocardium interact and migrate through this matrix during the establishment of the primitive, bilateral heart tubes. Radioactive labeling has demonstrated that proteins produced in the myocardium flow toward the endocardium and are incorporated into the basal lamina.!!2 The cardiac jelly has a variety of functions related to hemodynamic performance, cardiac looping and cell migration in cardiac septation, and the formation of the endocardial cushion valves at the atrioventricular (AV) junction and outflow tract of the heart.

The protein composition of the cardiac jelly modulates differentiation of the endothelium. Recent information explains the role of genes from the TGF-beta family of peptide growth factors as regulators of morphogenesis.!!3 TGF-beta2 proteins are in the extracellular matrix and are an integral component of the morphogenetic changes at the AV cushion level, acting through second messengers such as protein kinase C.!!4 In addition, fibronectin probably serves to set up migratory pathways in the cardiac jelly. These protein strands are arranged radially in the cushion, presumably along the lines of stress. The fibronectin strands also may serve as a template for the fibrous skeleton of the AV valve leaflets.!!5-!!7 The extracellular matrix proteins stimulate transdifferentiation of the endocardium in these regions, prompting endothelial cells to transform to mesenchymal cells which migrate into the cushion matrix. Laminin and type IV collagen are stabilizing signals or markers, since these compounds are absent in the cushion regions but are present adjacent to the endocardial cells that maintain a typical epithelial integrity.

Endocardial Maturation

The endothelial cells that make up the lining of the embryonic heart initially are arranged as a single sheet. This squamous-like sheet has the morphologic features of an active tissue, including microvilli, ruffles, and intercellular openings.118 The endocardium participates in the formation of endocardial cushions at the AV junction and in the outflow tract.119 Transdifferentiation of the endocardium occurs in the endocardial cushions, where cells round up, produce pseudopodia, and migrate into the cardiac jelly.120 These cells eventually make up a portion of the fibrous skeleton of the cardiac valves. Inductive chemical signals from the myocardium contribute to the endocardial transdifferentiation and regulate the migration of the mesenchymal cells.120 In addition, hemodynamic alterations can influence the orientation of endocardial cells on the endocardial cushions121 and the loci of dead and dying cells in the chick embryo heart.122 This interaction likely is similar to the relation between the endothelium and smooth muscle of the mature vascular bed.123 Finally, expansion of the endocardium is critical to the process of ventricular trabeculation, as is discussed below.

Looping

Following the formation of the straight heart tube, the human embryo is about 2 mm long and 23 days old. At the cephalic (or cranial) end of the myocardial heart tube, the nonmyocardial aortic sac can be recognized. The aortic sac is connected to the first pair of aortic arches and later also to the second, third, fourth, and sixth arches (the fifth pair of aortic arches does not normally develop in mammals or is very rudimentary). The extreme caudal part of the myocardial tube receives the paired confluence of veins that lie extrapericardially and are embedded in mesenchyme. In the early tubular stage, the heart hangs suspended from the ventral foregut by the so-called dorsal mesocardium. This structure disintegrates in the midportion of the tube, leaving the heart connected at the anterior pole at the level of the aortic sac and at the posteriorly located venous pole (atria and sinus venosus). At least three different biomechanical mechanisms may act in combination to generate the characteristic bend to the right of the cardiac tube: locally constrained growth, active cell deformation, and prestressed dorsal mesocardium.124

As the tubular heart continues to grow, it bends to the right and anteriorly Fig. 9-4). This results in a compound sigmoid structure with a so-called d-loop (rightward) configuration. At this stage, it is easy to distinguish the sinus venosus, the common atrium, the atrioventricular canal, the future left and right ventricles, and the outflow segment. Internally, the developing muscular interventricular septum is recognizable, its crest characteristically expressing the molecular marker GLN2/HNK-1.125,126 It is important to note that at this stage, all the future segments of the heart are still basically connected in series and that the common atrium connects, via the atrioventricular canal, completely to the left ventricle [i.e., double-inlet left ventricle (DILV)], while the outflow tract is connected exclusively to the right ventricle [i.e., double-outlet right ventricle (DORY)]. This is schematically depicted in Q-hB; Fig. 9-5.

The transition from a tubular heart in which the future segments are arranged in series (atrium to LV to RV to outflow tract) into a four-chambered heart in which the definitive chambers are arranged in parallel, separated by septa and valves, raises two important questions. The first is how the right atrium becomes connected to the right ventricle, and the second is how the left ventricle gains access to the aortic portion of the outflow tract. The remodeling of the so-called inner curvature of the looping heart tube plays an important role in this process and involves a rightward expansion of the AV canal and a concomitant leftward shift of the aorta. Immunohistochemical studies have demonstrated that this remodeling is intimately related to the development of the so-called primary ring Fig. 9-6).125'126 In the postnatal human heart, derivatives of the primary ring are found in the AV conduction system, in the right AV junction

(the right AV ring), and behind the aorta (the retroaortic root branch) (Fig. 9-7).126

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  • ZAHRA
    What is the genetic development of heart?
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    What is eHAND,dHAND gene and MEF2C tube in heart development in embrology?
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