Specification of the Cardiac Conduction System by Transcription Factors

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 2 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 wild-type mice. This region of myocardium, comprising the SAN primordium, is notable for its expression of myosin light chain 2a and for its absence of Nkx2.5 expression. Shox2 mRNA is also expressed in this region,³⁴ 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 wild-type embryos. In contrast to wild-type littermates, this hypoplastic SAN region in Shox2^−/− mice displays Nkx2.5 expression and aberrant Cx40 and Cx43 expression. The U-shaped myosin light chain 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 dysfunction,³⁶ 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 caused by 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 natriuretic precursor peptide A (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-aa 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 midgestation, 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 infranodal pathology. An infranodal conduction defect and a 30% to 50% increase in the hisio-ventricular interval are detected in Hop^−/− mice by invasive electrophysiological 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 physiological 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, which is a key cardiogenic cofactor 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 on proper cardiac expression of Nkx2.5 and Hop. Ismat et al⁴¹ 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 electrophysiological 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 (3-aa loop extension) superclass, as well as 2 conserved domains of unknown functions: the 14-aa IRO A domain and the 13-aa 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 Costantini et al observed an increase in the rapidly inactivating transient outward current (I_to) of Irx5^−/− endocardial myocytes resulting from increased expression of the Kcnd2 gene encoding the voltage-gated potassium channel, voltage-activated potassium channel (K_v)4.2.⁵⁴ I_to is typically greater in epicardial myocytes than endocardial myocytes of normal hearts, and this creates a transmural I_to gradient across the ventricle. The increased endocardial myocyte I_to of Irx5^−/− mice effectively abolishes the ventricular transmural I_to gradient and confers epicardial myocardium properties on the endocardial myocardium. Irx5 recruits the mBop cardiac corepressor to repress transcription of Kcnd2 in neonatal cardiomyocytes, and thus Irx5 deficiency leads to increased K_v4.2 expression and disruption of the I_to gradient.⁵⁴

In fact, both Irx4 and Irx5 regulate transcription of Kcnd2, the gene encoding K_v4.2.⁵⁵ 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 K_v4.2. 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.

The T-box gene family can be divided into subfamilies based on 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 electric impulses through the conduction system occurs in a retrograde fashion from ventricles to atria because of 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 ring.³ 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 al⁶⁴ 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 al⁶⁵ 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 (muscle segment homeobox 1 and 2), 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 coexpressed 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 progenitors,^76,77 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 that 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 3 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 to 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 wild-type 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 syndrome,⁸⁰ an autosomal dominant disorder characterized by cardiac defects in the setting of upper limb deformities.^81,82 Approximately 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^o 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 AVN 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 Id2,⁸⁶ 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 wild-type 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 on 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 wild-type/minK^lacZ/+ as well as Tbx5^+/−/minK^lacZ/+, Nkx2.5^+/−/minK^lacZ/+, and Id2^+/− /minK^lacZ/+ mutant hearts. However, MinK:lacZ expression is completely absent from the AV bundle and bundle branches in Tbx5^+/−/Nkx2.5^+/−/minK^lacZ/+ and Tbx5^+/−/Id2^+/− /minK^lacZ/+ mutant hearts. Despite these findings, a well-formed AVN is present in both the Tbx5^+/−/Nkx2.5^+/−/minK^lacZ/+ mice and Tbx5^+/−/Id2^+/− /minK^lacZ/+ 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.