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Diseases of the cardiovascular system that cause sudden cardiac deaths are often caused by lethal arrhythmias that originate from defects in the cardiac conduction system. Development of the cardiac conduction system is a complex biological process that can be wrought with problems. Although several genes involved in mature conduction system function have been identified, their association with development of specific subcomponents of the cardiac conduction system remains challenging. Several transcription factors, including homeodomain proteins and T-box proteins, are essential for cardiac conduction system morphogenesis and activation or repression of key regulatory genes. In addition, several transcription factors modify expression of genes encoding the ion channel proteins that contribute to the electrophysiological properties of the conduction system and govern contraction of the surrounding myocardium. Loss of transcriptional regulation during cardiac development has detrimental effects on cardiogenesis that may lead to arrhythmias. Human genetic mutations in some of these transcription factors have been identified and are known to cause congenital heart diseases that include cardiac conduction system malformations. In this review, we summarize the contributions of several key transcription factors to specification, patterning, maturation and function of the cardiac conduction system. Further analysis of the molecular programs involved in this process should lead to improved diagnosis and therapy of conduction system disease.
Cardiovascular disease is the leading cause of mortality and sudden cardiac death (SCD) in the United States. SCD prematurely claims the lives of more than 300,000 Americans and some 7 million worldwide1 and is usually caused by the onset of tachyarrhythmias such as ventricular tachycardia and ventricular fibrillation. A subset of lethal arrhythmias may originate from abnormalities associated with the cardiac conduction system. These abnormalities may result from injury, cardiac or neuromuscular disease or from congenital defects in the development of the cardiac conduction system. Although the basic mechanisms of cardiac conduction system development are not well understood, the molecular mechanisms underlying these defects are currently being explored.
Cardiac muscle is intermediate in its histological structure between the intrinsically rhythmic smooth muscle and voluntary skeletal muscle. All cardiac muscle cells can beat rhythmically without an external stimulus. However, the cells of the sinoatrial node (SAN) have the most rapid intrinsic excitation rate, and they establish the rhythmic contractions of the myocardium.2 The electrical impulses originating at the SAN are due to the presence of a specialized subset of pacemaker cells that differentiate from cardiomyocytes. These pacemaking impulses are responsible for initiating and regulating the heartbeat. The SAN, which is a heterogeneous tissue region embedded in the right atrial wall, is one component of the cardiac conduction system (Figure). Transmission of the pacemaking impulses generated at the SAN proceeds from the atrial to ventricular myocardium through a network of cells comprising the cardiac conduction system. In the embryonic and fetal heart, the conduction system is divided into two major components: (1) the central conduction system composed of the SAN, atrioventricular junction including the atrioventricular node (AVN) and the interventricular ring with its derivatives [retroaortic root branch, right atrioventricular ring bundle, atrioventricular bundle (AVB) and proximal bundle branches]; and (2) the peripheral conduction system composed of distal bundle branches and the peripheral ventricular conduction network.3, 4 The components of the cardiac conduction system all have distinct electrophysiological, morphological and transcriptional profiles that differ from the surrounding working myocardium.3, 5 The mechanism of induction and differentiation of cardiomyocytes into pacemaker cells during cardiogenesis is not well defined. However, the process is presumed to involve the expression of specific transcription factors, which will be further discussed in this review, and also hyperpolarization-activated cyclic nucleotide-gated (HCN) channels required for If, the pacemaker current.6
After electrical impulses are initiated by the pacemaker cells of the SAN, these impulses spread across the atrial chamber myocardium to initiate contraction of the atria (Figure). Impulses are delayed at the atrioventricular (AV) junction through specialized slow conducting node cardiomyocytes of the AVN. This delay is necessary in order to allow time for the atrial chambers to fully contract and pump blood across the AV valves prior to the occurrence of ventricular contraction. Direct transmission of electrical impulses from the atrial myocardium to ventricular myocardium is prevented by the presence of the annulus fibrosis, an insulating plane of fibrofatty connective tissue that separates the atrial and ventricular chambers of the heart. After the AV delay, the electrical impulse is rapidly propagated to the ventricular bundle branches via the AV (His) bundle. The ventricular bundle branches divide into left and right segments on either side of the ventricular septum and terminate into a network of Purkinje fibers. The transfer of electrical impulses from the Purkinje fiber network to the working ventricular myocardium to stimulate a ventricular contraction occurs simultaneously from the apex to the base of the heart in a coordinated manner. This apex-to-base contraction allows for efficient ejection of blood from the ventricles into the outflow tract at the base of the heart.
Several genes involved in mature conduction system function have been identified, but few genes required for patterning or specification of differentiated cells of this evolutionarily conserved structure are known. The pattern of cardiac conduction undergoes dramatic changes during normal development of the four-chambered heart. The myocardium of the embryonic tubular heart initially beats irregularly in an uncoordinated fashion, but soon achieves a homogenous slow activation7 and contraction8 sequence. Patten and Kramer8 determined that the first non-rhythmic contractions of the embryonic chick heart occur at the 10-somite stage. Rhythmic base-to-apex electrical activation, which persists until just before ventricular septation, is apparent before Hamburger-Hamilton stage 31 of chick cardiogenesis.9 These contractions allow blood to be propelled in a caudal to cranial direction in the primordial heart with the aid of the endocardial cushions serving as primitive valves.10 It is obvious at this stage of development that a group of specialized cardiomyocytes in the sinoatrial region of the primitive heart tube initiate the heartbeat. During the final stages of outflow septation in the embryonic chick9, and just prior to completion of ventricular septation during mouse cardiogenesis11, the activation sequence is reversed to the apex-to-base pattern of a mature heart. It is not completely clear what makes these pacemaker cells different from the surrounding myocardium, but this process of cardiomyocyte differentiation is known to involve transcription factors. These cells can beat independently, and at different rates when dissociated from cardiac tissue, but they function synchronously with the rate of one cardiomyocyte overriding another when interconnected. This property has been demonstrated in other dissociated cardiac tissues grown in culture as well, but the sinoatrial tissues have always dominated the pulse rate.12
Despite our extensive anatomical and physiological knowledge of the vertebrate cardiac conduction system, the molecular events governing development of this specialized tissue remain vague. Lineage-tracing studies in the chick embryo reveal that the cardiac conduction system is derived from cardiomyocyte progenitors. The cardiac conduction system develops by recruitment of multipotent embryonic myocytes into a node or fiber at specific embryonic stages, starting in the central conduction system and ending with the peripheral network of Purkinje fibers.4, 13 These cells exit the cell cycle after recruitment. The cardiac conduction system is specified by transcription factors. Loss of transcriptional regulation during cardiogenesis has profound effects on heart development and may eventually lead to the formation of arrhythmias. In this review, we will discuss the contributions of several key transcription factors to specification, patterning and maturation of the cardiac conduction system. We will also describe transcription factors that regulate gene expression of ion channels. Although there are several ion channels that provide an essential function in contributing to the electrophysiological properties of the developing conduction system, they are beyond the scope of this review. The transcription factors to be discussed include: Nkx2.5, Shox2, HOP, Irx4, Irx5, Tbx2, Tbx3, Tbx5, Tbx18, and Id2 (Figure). Human genetic mutations in a few of these transcription factors have been identified and are known to cause congenital heart diseases that include cardiac conduction system malformations. Although we focus on these transcription factors in this review, it is clear that many more will be identified, and our current understanding of cardiac conduction system development is only the tip of the iceberg.
Homeodomain proteins are a family of transcription factors defined by the presence of a 60-amino acid domain (the homeodomain). This evolutionarily conserved homeodomain is encoded by a 180-base pair DNA sequence, or homeobox, and is the DNA-binding region of this protein. Homeobox-containing genes play important roles in regulating tissue-specific gene expression that is essential for differentiation. These homeodomain proteins have been extensively characterized as transcriptional regulators controlling a variety of cell fates in both invertebrate and vertebrate species. Mutations in homeobox genes are associated with a number of human genetic diseases.
At least five members of the Nkx2 gene family that are expressed during vertebrate heart development.14 A Drosophila homologue of NKX2.5, tinman, is expressed in the dorsal vessel, the fly analogue of the heart. Mutations in tinman result in the absence of the dorsal vessel. Therefore, tinman is necessary for Drosophila cardiogenesis.15 Schott et al. used linkage analysis and positional cloning to show that mutations in human NKX2.5 cause the Mendelian syndrome of familial atrial septal defects with associated progressive AV block.16 In fact, subsequent genetic screens of patients with other cardiac anomalies revealed that NKX2.5 mutations cause a variety of structural cardiac anomalies associated with conduction system disease, especially conotruncal disorders.16–18
Nkx2.5 is expressed as early as embryonic day (E) 7.5 in the precardiac mesoderm and the adjacent endoderm19 of the developing mouse and expression continues into adulthood.20 Examination of Nkx2.5-haploinsufficient or -null mice has been helpful in gaining insight into its role in development of the heart and, specifically, the cardiac conduction system. Lyons et al. found that targeted disruption of murine Nkx2.5 results in early embryonic lethality; cardiac development is arrested at the linear heart tube stage prior to looping.21 In addition to maintaining a linear conformation, hearts from Nkx2.5−/− mice do not form a proper demarcation between the atrial and ventricular chambers and the atrioventricular canal fails to narrow and elongate. Thus, this potentially impacts the development of cardiac conduction system structures derived from this region. During formation of conduction fibers, Nkx2.5 mRNA and protein expression is elevated relative to the surrounding myocardium in the developing chick, mouse and human hearts.22 In Nkx2.5 knockout mice, Jay et al. demonstrated that the number of cells in the cardiac conduction system is directly related to Nkx2.5 gene dosage.23 Nkx2.5-null embryos lack minimal potassium channel (minK)-expressing cells that give rise to the AVN primordium. Embryonic and adult Nkx2.5+/− mice form smaller central and peripheral conduction systems which are associated with half the normal number of cells being present during development.23
During normal murine cardiogenesis, the AV nodal region contains two cell populations: a proximal, posterior compartment and a distal, anterior compartment which connects to the His bundle. The proximal compartment contains cells that express the gap junction protein connexin (Cx) 45, but not Cx40. The distal anterior compartment contains a small, mixed population of Cx40+/Cx45+ and Cx40−/C45+ cells. Nkx2.5-deficiency causes hypoplasia of the central conduction system and Nkx2.5+/− mice lack the entire proximal population of Cx40−/Cx45+ cells in the AVN. 23 Surprisingly, cellular expression of Cx40 and conduction times through the His-Purkinje network are normal. These studies suggest a restricted role for Nkx2.5 in regulating specification of a cell population necessary for normal central conduction system development. Ventricular-restricted Nkx2.5-deficient mice are viable and the hearts appear structurally normal upon histological examination.24 However, neonatal mice display first degree AV block which progresses to high grade AV block or complete heart block by late adulthood. Progressive AV block is likely due to a hypoplastic AVN present at birth that is followed by selective degeneration of the central conduction system. This degeneration is confirmed by the presence of cardiomyocyte dropout and fibrosis. By contrast, the surrounding myocardium appears unaffected.24
Although progressive AVN degeneration is not consistently observed in all humans with NKX2.5 mutations, investigators have observed progressive AV block in patients heterozygous for NKX2.5 mutations that have necessitated pacemaker implantation.17, 24, 25 Examination of post-mortem cardiac sections from two patients heterozygous for Gln198ter NKX2.5 mutations with progressive AV block reveals marked, preferential degeneration of the conduction system at the level of the AVN and His bundle, while the surrounding myocardium appears normal. In addition, Pashmforoush et al. demonstrated that aberrant expression of several genes predominantly expressed in the conduction system, including Cx40, minK, Hcn1 and homeodomain-only protein (Hop), is detected. These data identify Nkx2.5 as part of a pathway within ventricular myocyte lineages that likely acts in concert with other molecular cues for AVN cell lineage specification within the central conduction system.24
In addition, investigators have also demonstrated a role for Nkx.2.5 in specification of the peripheral conduction system in a time- and dose-dependent manner.26, 27 Nkx2.5 haploinsufficiency causes hypocellularity of the peripheral conduction system. Meysen et al. demonstrated that the ventricular conduction disorders in Nkx2.5+/− mice are due to a hypoplastic Purkinje fiber network that results from a cell-autonomous defect of postnatal cardiomyocyte differentiation and formation into the peripheral Purkinje fiber network.27 Tamoxifen-induced loss of Nkx2.5 in mice demonstrated a perinatal requirement for Nkx2.5 in cardiac conduction system formation.28
Nkx2.5 synergistically acts with the T-box transcription factor, Tbx5, to regulate gene expression during cardiac development. Although the details of this synergistic relationship will be described later, it is important to note that expression of the transcriptional repressor Id2 is cooperatively regulated by Nkx2.5 and Tbx5.29 Compound haploinsufficiency of Tbx5 and Nkx2.5 or Tbx5 and Id2 prevents embryonic specification of the ventricular conduction system. Therefore, Tbx5, Nkx2.5 and Id2 coordinate specification of ventricular myocytes into the ventricular conduction system lineage.
The short stature homeobox (SHOX) gene family of human pseudoautosomal homeobox genes encodes cell type-specific transcription factors involved in cell cycle and growth regulation.30, 31 SHOX2 mRNA is expressed in the heart and other tissues of developing human embryos.32 During murine cardiogenesis, Shox2 is expressed as early as E8.5 in the posterior region of the primitive heart tube. At E10.5, Shox2 expression is detected in the sinus venosus myocardium-derived venous valves. By E11.5, Shox2 mRNA expression expands to include the SAN region as well as the two parallel bundles spanning the longitudinal axis of the atria, the venous valves, which are believed to constitute an integral part of the developing conduction system.11, 33 To date, investigators have not identified human SHOX2 mutations that are associated with disease.
Blaschke et al. showed that Shox2+/− mice do not exhibit any obvious structural defects, altered heart rates, or substantive electrocardiographic abnormalities.34 However, Shox2−/− mice die between E11.5 and E13.5 with several signs of heart failure as well as vascular defects. During murine cardiogenesis, a U-shaped band of myocardium is formed from the wall of the sinus venosus adjacent to the atrial myocardium in E11.5 wildtype mice. This region of myocardium, comprising the SAN primordium, is notable for its expression of myosin light chain (MLC) 2a and for its absence of Nkx2.5 expression. Shox2 mRNA is also expressed in this region34, and sinus venosus myocardium is known to be linked to cardiac conduction system development.35 Recently, Gittenberger-de Groot et al. showed that the sinus venosus myocardium is also demarcated by expression of podoplanin, a mucin-like transmembrane glycoprotein that is expressed in cells lining the pleural and pericardial cavity.36 During murine cardiogenesis, Shox2 expression becomes further restricted to the sinus venosus region, and eventually to the SAN and sinus (venous) valves.37 In E11.5 Shox2−/− embryos, the SAN region is markedly decreased in comparison to wildtype embryos. In contrast to wildtype littermates, this hypoplastic SAN region in Shox2−/− mice displays Nkx2.5 expression and aberrant Cx40 and Cx43 expression. The U-shaped MLC-2a-positive/Nkx2.5-negative region is virtually absent in the Shox2−/− mice. Antisense morpholino-mediated knockdown of Shox2 in the developing zebrafish leads to severe cardiac dysfunction36, characterized by pronounced sinus bradycardia and intermittent sinus exit block. These data demonstrate an essential role for Shox2 in the recruitment of sinus venosus myocardium comprising the SAN region.
Hearts isolated from Shox2−/− mice and maintained in culture display markedly reduced contraction rates. SAN hypoplasia occurs in these mice, and is due to a reduction in cell proliferation, but not apoptosis.37 Concomitant alterations in gene expression are noted within the SAN region of these mice. The SAN region of Shox2−/− mice lacks expression of Tbx3 and Hcn4. However, this region displays ectopic expression of Nppa, Cx40 and Nkx2.5. These findings indicate a failure of the SAN to properly differentiate in response to loss of Shox2.37
In aggregate, these Shox2 findings link this transcriptional pathway to Nkx2.5, which has also been shown to play a role in development and maturation of the SAN region.36 Not only does Shox2-deficiency induce ectopic Nkx2.5 expression, but at least in Xenopus embryos, overexpression of Shox2 represses Nkx2.5 expression.37 Espinoza-Lewis et al. demonstrated that Shox2 represses Nkx2.5 promoter activity in a concentration-dependent manner. The Nkx2.5 promoter includes several Shox homeobox DNA binding sites, and mutagenesis of these sites abrogates the ability of Shox2 to repress Nkx2.5 transcription.37 Thus, it has been suggested that Shox2 operates upstream of Nkx2.5, Tbx3 and Hcn4 to regulate the SAN genetic program.
Homeodomain-only protein, Hop, is the smallest known homeodomain protein. This 73-amino acid homeodomain protein is composed of a divergent homeodomain that, unlike other homeodomain proteins, is incapable of sequence-specific DNA binding.38 It is expressed in the embryonic heart and plays an important role in development of the adult cardiac conduction system. Hop is expressed as early as E8.0 in the mesoderm precursors of the cardiac muscle during murine development.39, 40 In early to mid-gestation, Hop is expressed throughout the myocardium, and its expression persists into adulthood. In adulthood, Hop is most strongly expressed within the cardiac conduction system. Expression of Hop is high in the AVN, His bundle and the left and right bundle branches.
Approximately half of Hop-deficient mouse embryos die between E10.5 and E12.5.39 The Hop−/− mice that survive to adulthood appear to have an anatomically normal cardiac conduction system41 (as marked by histochemical staining for acetylcholinesterase activity42) that exhibits electrophysiological deficits indicative of a disrupted cardiac conduction system. However, surface electrocardiography of these mice is abnormal and displays right-axis deviation, prolonged P-R interval, a widened QRS interval, and a prolonged QT interval suggestive of infra-nodal pathology. An infra-nodal conduction defect and a 30–50% increase in the hisioventricular interval are detected in Hop−/− mice by invasive electrophysiologic study. Thus, the high levels of Hop protein expressed in the His bundle and the left and right bundle branches appear to be required for normal infranodal cardiac conduction system function.41 Hop−/− mice also exhibit a minimal, albeit physiologically insignificant, increase in the atriohisian interval that may reflect some physiologic importance of the high postnatal expression of Hop in the atria and AVN.
Given its inability to bind DNA, how does Hop regulate gene expression and cardiac development? One possible mechanism may reflect interaction between Hop and Nkx2.5. During early cardiogenesis Nkx2.5 directly activates Hop expression, and Hop is markedly downregulated in Nkx2.5-null embryos.28 Activity of serum response factor (SRF), which is a key cardiogenic co-factor of Nkx2.5,43 is antagonized by Hop.40 Therefore, Hop may regulate gene expression during cardiac development by indirectly modulating the activity of Nkx2.5.
Expression of cardiac connexins is dependent upon proper cardiac expression of Nkx2.5 and Hop. Ismat et al.41 demonstrated that Cx40, but not Cx43 mRNA expression is significantly reduced in the whole hearts of Hop−/− mice. In Hop+/− mice, Cx40 protein is expressed throughout the cardiac conduction system. However, Cx40 expression is reduced to a smaller region in Hop−/− mice that does not extend beyond the proximal AVN and His bundle. Cx40 is the predominant connexin isoform in the rodent atrium and cardiac conduction system that contributes to gap junction formation between cardiomyocytes.44 Therefore, it seems likely that loss of Hop in the cardiac conduction system modifies Cx40 expression, and reduced Cx40 may partially explain the electrophysiologic phenotype observed in the Hop−/− mice.
Although Hop is a transcriptional target of Nkx2.5, their functions do not completely overlap during cardiac conduction system development. Nkx2.5, which is highly expressed in the cardiac conduction system, is required for specification of conduction cells during development of the conduction system as well as maintenance of the adult cardiac conduction system. By contrast, Hop does not seem to be necessary for maintenance of conduction tissue because gross anatomic structures of the cardiac conduction system appear to be normal in Hop−/− mice. Instead, Hop regulates the genetic profile of the cardiac conduction system. Loss of Cx40 expression may be a major mechanism underlying the cardiac conduction defects associated with Hop-deficiency, but it is likely that other proteins are involved in mediating cardiac conduction disease in the absence of Hop. Clinically relevant human conduction system disorders associated with downregulation or loss-of-function of Hop have not been identified. However, decreased Hop expression was recently observed in a porcine model of dilated cardiomyopathy.44
The Iroquois homeobox genes (Irx) encode a conserved family of transcription factors in metazoans. Irx factors specify the identity of diverse territories of the body by establishing proper spatiotemporal patterns of target genes during development.45 Members of the Irx family are characterized by a highly conserved homeodomain of the TALE (three amino acid loop extension) superclass as well as two conserved domains of unknown functions: the fourteen amino acid IRO A domain and the thirteen amino acid IRO box.46–49 Six Irx genes have been isolated in mammals. The Irx genes play key roles in development, including neurogenesis and patterning of the heart.45, 49
Irx4 is the earliest marker of ventricular precursors and is expressed in the ventricular myocardium during all stages of embryonic development through adulthood.50, 51 It regulates chamber identity by activating expression of ventricular myosin heavy chain-1 and suppressing expression of atrial myosin heavy chain-1.52 Bruneau et al. showed that Irx4 expression is regulated by Nkx2.5 and dHand.51 Irx4 may not be essential for ventricular chamber formation, but it is required for establishing some components of the ventricle-specific gene expression program. Irx4-deficient mice exhibit normal cardiac morphology during embryonic and postnatal stages of growth but develop adult-onset cardiomyopathy that is characterized by cardiac hypertrophy and impaired contractile function.50
Irx5 is expressed as early as E9 during murine cardiogenesis. It is detected in the endocardium lining of the primitive atrial and ventricular chambers 53 Cardiac morphology and hemodynamics appear normal in adult Irx5−/− mice, but these mice are susceptible to tachyarrhythmias and display signs of abnormal ventricular repolarization.54 Constantini et al. observed an increase in the rapidly inactivating transient outward current (Ito) of Irx5−/− endocardial myocytes due to increased expression of the Kcnd2 gene encoding the voltage-gated potassium channel, Kv4.2.54 Ito is typically greater in epicardial myocytes than endocardial myocytes of normal hearts, and this creates a transmural Ito gradient across the ventricle. The increased endocardial myocyte Ito of Irx5−/− mice effectively abolishes the ventricular transmural Ito gradient and confers epicardial myocardium properties upon the endocardial myocardium. Irx5 recruits the mBop cardiac corepressor to repress transcription of Kcnd2 in neonatal cardiomyocytes, and thus Irx5 deficiency leads to increased Kv4.2 expression and disruption of the Ito gradient.54
In fact, both Irx4 and Irx5 regulate transcription of Kcnd2, the gene encoding Kv4.2.55 Irx4 suppresses Irx5-induced regulation of the Kcnd2 promoter in a dose-dependent manner. The C-terminal region of Irx5 is responsible for mediating regulation of the Kcnd2 promoter and the N-terminal region of Irx4 is responsible for suppressing Irx5-induced regulation of Kcnd2. Thus, maintenance of the Irx4:Irx5 stoichiometry is required to regulate expression of channel proteins.55
These observations suggest that combinatorial interactions between several Iroquois transcription factors refine the spatial distribution of Kv4.2. Although Irx4 and Irx5 may not be responsible for specification of cell lineages during cardiac conduction system development, they play an essential role in establishing region-specific expression of ion channel proteins that contribute to the electrophysiological properties of the conduction system. Mutations of IRX4 and IRX5 in humans have not yet been identified.
T-box (Tbx) transcription factors play critical roles during embryogenesis, and are characterized by their highly conserved T-box DNA-binding domain. Members of the Tbx1 and Tbx2 subfamilies all play specific roles in heart development.56 These T-box genes collectively contribute to cardiac lineage determination, chamber specification, epicardial development and specialization of the conduction system. Multiple T-box transcription factors are important regulators of conduction system specification, differentiation and patterning and also contribute to many other aspects of heart cell maturation and morphogenesis.
The T-box gene family can be divided into subfamilies based upon conservation of protein structure and function. The Tbx2 subfamily includes Tbx2 and Tbx3. Multiple T-box transcription factors of different subfamilies are often coexpressed and can modulate the expression of each other. Tbx2 and Tbx3 are coexpressed in the heart primordia, primitive myocardium and AV canal of chick and mouse embryos and are strong transcriptional repressors.57–59 Although expression of Tbx2 in these regions is much stronger and broader than Tbx3, there is considerable overlap. Tbx2 downregulates expression of the chamber-specific genes, Nppa, Cx40 and Chisel, and represses differentiation and formation of the cardiac chambers.60 Cooperation with Nkx2.5 enables Tbx2 to repress Nppa promoter activity in vitro.59 Similar to Tbx2, Tbx3 is able to repress Nppa and Cx40 promoter activity.61 Tbx2 and Tbx3 are required for controlling cell proliferation in the AV canal during zebrafish cardiogenesis.62 Together, Tbx2 and Tbx3 repress transcription in the AV canal of Bmp10,62 a cardiac chamber-specific gene required for chamber growth and maturation.63
Tbx2 and Tbx3 also contribute to cardiac conduction system development. They are differentially expressed in the peripheral and central conduction system where they inhibit cell proliferation and regulate lineage-specific gene expression.64, 65 Aanhaanen et al. demonstrated that Tbx2-expressing myocardial cells contribute to the maturation of the primary myocardial AV canal.66 Tbx2+ AVC myocardium contributes to the AVN and AV ring bundle. Despite a structural continuity of the nascent conducting myocardium throughout development, Tbx2+ AVC myocardium does not contribute to the AV bundle or proximal left and right bundle branches.67 These data suggest that progenitors of the AV conduction system components become specified in early cardiogenesis through segregation and exposure to distinctive molecular signals.
In Wolff-Parkinson-White syndrome (WPW) 68, patients present with symptomatic tachyarrhythmias. A WPW phenocopy is caused by a mutation in the gene encoding the γ2 regulatory subunit of AMP-activated protein kinase (PRKAG2).69 These patients can have intermittent preexcitation because transmission of electrical impulses through the conduction system occurs in a retrograde fashion from ventricles to atria due to an accessory excitatory pathway that seems to be a consequence of either abnormal expansion of the AV ring70 or failure to maintain an insulating AV ring3. However, the genetic etiology of classic WPW remains elusive, and Tbx2-dependent pathways that regulate AV ring maturation may be a ripe source of candidate genes in WPW.
During embryogenesis, Tbx3 is involved in several key developmental processes including regulation of cell proliferation, senescence, apoptosis, cell cycle exit and developmental patterning.56, 71 Mutations in TBX3 cause Ulnar-Mammary syndrome, a human genetic disorder distinguished by upper limb defects, mammary gland hypoplasia and also dental and genital abnormalities.72, 73 Cardiac defects and conduction system disease are not common manifestations of Ulnar-Mammary syndrome, although ventricular septal defect, right ventricular hypertrophy and pulmonary stenosis have been reported.72, 73 Tbx3 is expressed in the SAN, AVN, AV bundle and proximal bundle branches during cardiac development,61 and Tbx3 regulates the formation, maturation and function of the SAN.64 Hoogaars et al.64 observed lineage segregation of Tbx3-negative atrial and Tbx3-positive SAN precursor cells as soon as the cardiac cells turn on the atrial gene expression program. These findings suggest that the SAN is formed by proliferation of Tbx3-positive precursor cells and not by recruitment of adjacent myocytes designated for the atrial gene program. Also, Cx40 repression and activation of the pacemaker channel, Hcn4, occurs when TBX3 is ectopically expressed in atrial cardiomyocytes.64 Atrial working myocardium genes, including Cx40, Cx43, Nppa and Smpx (Chisel), are expressed in the SAN domain of Tbx3−/− mice. Thus, Tbx3 is required for induction and maintenance of the distinct SAN genetic program while simultaneously preventing the expansion of atrial gene expression into the domain of the SAN. Moreover, overexpression of Tbx3 is sufficient to alter gene expression patterns in atrial cells to promote the induction of functional pacemaker cells that exhibit a SAN genetic signature. Expression of Tbx3, then, serves as a major factor in determining whether embryonic atrial cells differentiate into pacemaker cells or working myocardium.64
Bakker et al.65 showed that Tbx3 is required for specification of the AV conduction tissue and the proximal bundle branches during formation of the conduction system. Tbx3 prevents cardiomyocytes within the ventricular conduction system from differentiating into working myocardium. It suppresses both atrial and ventricular myocardial gene expression in the conduction system by repressing transcription of the chamber myocardial-specific genes, Cx40, Cx43 and Nppa, and by inhibiting proliferation in this cell population.61 The muscle segment homeobox proteins, Msx1 and Msx2, cooperate with Tbx3 to suppress Cx43 promoter activity and regulate its expression in the conduction system.74 Cooperative interaction between Tbx3 and Tbx18, as discussed later in detail, is also important in regulation of SAN development. To date, TBX3 mutations have not been associated with isolated conduction or arrhythmia disorders in humans.
Multiple T-box transcription factors of different subfamilies are often co-expressed during development and can modulate the expression of one another. An important example of this is the relationship between Tbx18 and Tbx3 expression during SAN formation. The biological role of Tbx18 in the primitive proepicardial organ75 has been extensively studied. Although there is an ongoing debate of whether cardiomyocyte lineages derive from Tbx18-positive epicardial progenitors76, 77, far less is known about the role of Tbx18 in the development of the cardiac conduction system. Wiese et al. showed that Tbx18 and Tbx3 control distinct programs in SAN formation which lead to regionalization of gene expression in the SAN during normal cardiogenesis.78 The mammalian SAN is nonuniform in cell content, function and shape. Although morphological studies reveal that the SAN is composed of three distinct cell types (nodal, intermediate and elongated cells), the nodal cells are believed to drive the pacemaking function of the SAN. The head and tail regions of the SAN give the structure a unique comma shape, and all SAN cells express Hcn4, the pacemaker channel gene.79 However, the expression patterns of Tbx3 and Tbx18 in the developing SAN head and tail differ. In the E14.5 mouse heart, the SAN head expresses Tbx3, Tbx18 and Hcn4, but lacks expression of Nkx2.5.78 By contrast, the SAN tail expresses high levels of Tbx3 and Hcn4, but weakly expresses Nkx2.5 and lacks expression of Tbx18. Thus, Tbx18 is a critical marker of SAN compartmentalization. In Tbx18-deficient embryonic mouse hearts, the SAN tail is visible as early as E10.5 – E12.5, and Tbx3 and Hcn4 are detectable. However, Tbx18-deficient embryos exhibit failure of SAN head formation. By E14.5, delayed formation of a SAN head-like structure begins, but it is significantly shortened along the longitudinal axis of the right superior caval vein. Investigators speculate that, in the wildtype mice, Tbx18+ mesenchymal precursors within this region of the superior caval vein expand and differentiate to form the myocardium of the future SAN head.78 A similar requirement of Tbx18 for SAN head formation has been reiteratively demonstrated in Tbx18−/−/Tbx3−/− mice that also fail to form a SAN head despite formation of a SAN tail. These data further demonstrate a requirement for Tbx18 in formation of the SAN head from mesenchymal precursors. Although Tbx3 is not required for SAN formation, it imposes the pacemaker gene program and function in atrial myocardium.78 Genetic mutations in TBX18 have not yet been identified in humans.
As a member of the highly conserved T-box transcription factor family, Tbx5 contributes to many aspects of cardiovascular development. Mutations in human TBX5 cause Holt-Oram syndrome80, an autosomal dominant disorder characterized by cardiac defects in the setting of upper limb deformities.81, 82 About 75% of all affected individuals exhibit structural cardiac malformations that typically include atrial and/or ventricular septal defects with isomerism. Cardiac rhythm disturbances frequently occur in Holt-Oram syndrome individuals including sinus bradycardia and variable degrees of AV block and atrial fibrillation.81, 83 Often these individuals require implantation of permanent pacemakers. Although such arrhythmias can occur in the setting of structural cardiac abnormalities, patients with Holt-Oram syndrome may exhibit isolated conduction disease without any evidence of structural heart disease.
During cardiogenesis, TBX5 is expressed in all chambers of the heart and higher levels of protein are expressed in the atrial chambers.84 Expression of TBX5 in the AVN provides a molecular basis for the progressive AV conduction block seen in individuals with Holt-Oram syndrome. Targeted mutagenesis of Tbx5 in mice results in severely hypomorphic development of the primitive heart tube in null embryos that do not survive past E10.5.85 Adult Tbx5−/− mice exhibit various degrees of electrophysiological deficits including AV block and sinus node dysfunction, and 50% of these animals exhibit 2° AV block.85 Moskowitz et al. demonstrated that Tbx5 is expressed throughout the central conduction system, including the AVN, AV bundle and bundle branches in the newborn mouse heart.29 Furthermore, Tbx5 has specific roles within the AV canal in postnatal morphological and functional maturation of the AV node and AV bundle. The bundle branches are defined by a broad sheet of specialized conduction cells that form postnatally into discrete fascicles in the mature left and right bundle branches during normal murine cardiogenesis. Tbx5 cardiac conduction system expression levels are usually maintained at birth, and postnatal levels in the murine conduction system structures are higher than in the surrounding myocardium. At birth, the developing left bundle branch initially appears to be unaffected by Tbx5 deficiency. However, in Tbx5+/− mice, the left bundle branch fails to mature into a narrow fascicle by adulthood, and the immature branch retains the appearance of a broad band of specialized conduction cells without a discrete AV bundle. Severe patterning defects are visible in the right bundle branch in both newborn and adult Tbx5+/− mice. In the right ventricle of Tbx5+/− mice, a paucity of cells results in the foreshortening or complete absence of a discrete right bundle branch. This results in the functional consequence of electrocardiographic abnormalities including right bundle branch block. Cx40 mRNA is expressed in the immature AV bundle and left bundle branch of Tbx5+/− mice, but is absent from the region adjacent to the membranous septum where the right bundle normally resides. These findings demonstrate a requirement for Tbx5 in postnatal maturation of the AV bundle and left bundle branch as well as an essential requirement for Tbx5 in patterning and function of the right bundle branch.29
Development of the ventricular conduction system also requires cooperation between Tbx5 and Nkx2.5. Their activity is, at least in part, mediated by Id286, a member of a gene family encoding helix-loop-helix-containing transcriptional repressors.87 Expression of Id2 mRNA is detected in the nascent AV bundle beginning at E12.5 of murine cardiogenesis and in the bundle branches beginning at E16.5.86 Id2-null newborn and adult mouse hearts display various interventricular conduction delays, including left bundle branch block. Moskowitz et al. reported that a discrete AV bundle, which physically separates the AVN from the left bundle branch in adult wildtype mice, is absent in adult Id2−/− mice. Instead, the left bundle branch is unusually broad, and the morphology of the AV bundle and bundle branch appear similar to that observed in adult Tbx5+/− mice.86 Despite the morphological similarities of the left bundle branch in Id2−/− mice and Tbx5+/− mice, Id2−/− mice develop left bundle branch block compared with the right bundle branch block reported in Tbx5+/− mice. The explanation for this discrepancy is unclear and may reflect incomplete overlap of Tbx5 and Id2 expression and function. Id2 expression is only partially dependent upon Tbx5, and Moskowitz and colleagues demonstrated that Nkx2.5 and Tbx5 cooperatively interact with the Id2 promoter to regulate conduction system-specific expression of Id2.86 Examination of compound Tbx5+/−/Nkx2.5+/− mice and Tbx5+/−/Id2+/− mice reveals genetic interactions between Tbx5, Nkx2.5 and Id2 during ventricular conduction system development and function. The AV bundle and proximal bundle branches are present and well-formed in wildtype/minKlacZ/+ as well as Tbx5+/−/minKlacZ/+, Nkx2.5+/−/minKlacZ/+ and Id2+/−/minKlacZ/+ mutant hearts. However, MinK:lacZ expression is completely absent from the AV bundle and bundle branches in Tbx5+/−/Nkx2.5+/−/minKlacZ/+ and Tbx5+/−/Id2+/−/minKlacZ/+ mutant hearts. Despite these findings, a well-formed AVN is present in both the Tbx5+/−/Nkx2.5+/−/minKlacZ/+ mice and Tbx5+/−/Id2+/−/minKlacZ/+ mice. The deficiency in the synergy between these factors does not appear to impair specification or differentiation of the AVN.
Cx40 expression is absent from the region of the AV bundle and bundle branches of Tbx5+/−/Nkx2.5+/− mice, and these compound heterozygous mice exhibit ventricular conduction system delays. Cell lineage tracing studies show that presumptive ventricular conduction system precursor cells are present, but fail to adopt a conduction system fate in mice haploinsufficient for both Tbx5 and Nkx2.5.86 Ventricular conduction delays are observed in compound Tbx5+/−/Id2+/− mice that are similar to delays observed in Tbx5+/−/Nkx2.5+/− mice. Therefore, haploinsufficiency of either Tbx5 and Nkx2.5 or Tbx5 and Id2 abrogates ventricular conduction system development and function. Together, these data define a required molecular pathway involving Nkx2.5, Tbx5 and Id2 for ventricular conduction system development.
Synchronized contractions of the atria and ventricles are essential for normal cardiac function. Cardiac conduction system components work together as a functional unit to provide the rhythmic activity of the heart. Transcription factors, including homeodomain proteins and T-box proteins, are at the core of pathways specifying the components of the cardiac conduction system. They are essential in activating or repressing a constellation of regulatory genes, most of which still remain unidentified. Together, the transcription factors and regulatory genes specify and maintain the cardiac conduction system in a normally functioning state. Mutations in genes encoding some of these transcription factors produce human disorders defined by the presence of congenital heart defects as well as associated or isolated conduction system abnormalities. In addition to the transcription factors that specify cell lineages destined to become part of the cardiac conduction system, several transcription factors regulate expression of genes encoding the ion channel proteins. Ion channels are essential in contributing to the electrophysiological properties of the conduction system by maintaining the membrane potential of myocardial cells and controlling the release of ions necessary for eliciting a muscle contraction. Dysregulation of these ion channels due to alterations in expression of their modulatory transcription factors can affect proper functioning of the conduction system and lead to the manifestation of arrhythmias. Further characterization of the molecular programs involved in cardiac conduction system specification, maintenance and function, and ion channel expression should lead to improved diagnosis and therapy of conduction system disease.
We are grateful to Dr. Jon Seidman and Dr. Ivan Moskowitz for their contribution of the figure. The figure was reproduced and adapted with permission from the Development journal.29 C.T.B. is grateful for support from Raymond and Beverly Sackler.
Sources of Funding
C.J.H. is supported by NIH K01 HL080948. C.T.B is supported by NIH R01 HL80663 and R01 HL61785, and the Snart Cardiovascular Fund.