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. Author manuscript; available in PMC: 2007 Aug 1.
Published in final edited form as: Trends Cardiovasc Med. 2006 Aug;16(6):193–198. doi: 10.1016/j.tcm.2006.03.009

Role of Homeodomain-Only Protein in the Cardiac Conduction System

Fang Liu 1, Fraz A Ismat 2, Vickas V Patel 3,*
PMCID: PMC1615917  NIHMSID: NIHMS12904  PMID: 16839862

Abstract

Diseases of the cardiac conduction system (CCS) are a significant health issue in adult patients where few therapeutic options exist outside of expensive, device-based procedures. An evolving paradigm pointing toward several key transcription factors required for CCS development and maintenance may be a group of potential targets for reversing or treating degenerative conduction system disease. Recently, a small homeodomain-only protein (Hop) involved with regulating cardiac development has been identified, which is highly expressed in the adult murine CCS. Targeted disruption of the Hop locus leads to infra-nodal conduction defects with downregulation of connexin40 expression within the confines of the CCS. Loss of Hop does not appear to affect the size or distribution of the mature murine CCS and further studies will be required to determine whether Hop is associated with conduction system disease in humans.

As early as the fourth century BC, the Greek physicians Herophilus and Erasistratus were among the first physician-scientists to document that the heart beats with regularity and consistency (Lonie 1973). However, more than two and a half millennia passed before Gaskell (1883) demonstrated, in the tortoise heart, that impulse propagation from the atria to the ventricles was an electrical event mediated through the myocardial tissue and not the nerve. Still another 20 years passed before Tawara (1906) clearly identified and reported the anatomy of the cardiac conduction system (CCS). Today, based upon contributions from many investigators, we know that the mammalian CCS depends upon the sinoatrial node to generate a reliable impulse which depolarizes the atrial myocardium. This impulse is then appropriately delayed within the atrioventricular node (AVN), conducted through the His bundle to the bundle branches, and distributed to the working ventricular myocardium via the Purkinje network. This highly coordinated pattern of conduction repeats itself over 85,000 times a day in the average adult, where failure in any one component of the CCS could induce significant morbidity or even be fatal. Dysfunction of the CCS is one of the leading cardiovascular diagnoses prompting hospital admission and intervention among adult patients in whom pacemaker implantation is often the only effective treatment (Hreybe and Saba 2004). Although permanent pacemakers are highly effective for treating CCS disease, they are expensive and associated with potentially serious complications. Therefore, understanding the basic mechanisms underlying the function of the adult CCS has important implications for developing alternative therapies for CCS disorders.

Recent insights into the development of the CCS suggest the ventricular specialized conduction tissue is locally derived from multipotent progenitor cells within the ventricular myocardium and AV canal, which can contribute either to cardiac chamber formation or to nonchamber myocardium (Gourdie et al. 1995, Cheng et al. 1999, Christoffels et al. 2004). Endothelin, neuregulin-1, Wnts, and bone morphogenic proteins have each been implicated as contributors to the transition between chamber forming and conductive cell lineages in animals (Rentschler et al. 2002, Gourdie et al. 1998, Patel and Kos 2005, Bond et al. 2002). In addition, recent insights are evolving supporting the role of transcription factors in CCS development. Although the contribution of transcription factors to cardiac development has been recognized for some time now, their contribution to CCS development has only recently been widely appreciated (Fishman 2005). When the transcription factor Nkx2-5 was first identified, germline deletion studies demonstrated their importance for cardiac development and chamber specification (Lints et al. 1993, Lyons et al. 1995, Tanaka et al. 1999). However, it was only later that mutations within Nkx2-5 were linked to congenital heart disease and conduction system dysfunction in humans, which brought to light their contribution to CCS development and function (Schott et al. 1998, Benson et al. 1999). Yet although many exciting insights are emerging about the mechanisms of CCS development, we still know relatively little about the factors and molecules involved with preserving function of the mature mammalian CCS or the mechanism by which these molecules contribute to CCS function (Fishman 2005). As previously mentioned, the transcription factor Nkx2-5 is known to be important for CCS development, but just as importantly it appears that preserved Nkx2-5 function is required during the postnatal period for proper maintenance of the adult CCS architecture (Jay et al. 2004, Pashmforoush et al. 2004). Similarly, it appears that the T-box transcription factor Tbx5 is necessary for normal development of AV nodal and sinus node conduction (Bruneau et al. 2001), whereas heterozygous deletion of Tbx5 has been shown to induce mispatterning and underdevelopment of the right bundle branch in mice (Moskowitz et al. 2004). Evidence also suggests that two additional members of the T-box family of transcription factors, Tbx2 and Tbx3, are expressed in the myocardium of the AV canal during development which then go on to form critical regions of the proximal CCS (Christoffels et al. 2004, Hoogaars et al. 2004). In addition, there also appears to be a contribution to CCS dysfunction from altered cardiac sarcolemma ion channels in the postnatal period. It has been demonstrated that loss-of-function mutations in the cardiac sodium channel, SCN5A, are linked to degenerative conduction system function in Lenègre’s disease (Probst et al. 2003) and this disease state has just been faithfully recapitulated in a murine model (Royer et al. 2005).

Recently, we have shown that a small protein known as the homeodomain-only protein (Hop) is highly expressed within the adult murine CCS, and that targeted deletion of Hop induces postnatal conduction system defects without altering the gross anatomic structure of the CCS (Ismat et al. 2005). Hop knockout mice display infra-nodal conduction defects with specific loss of the gap junction protein connexin40 within the CCS. Therefore, Hop null mice represent one of the few mammalian models of infra-nodal conduction system defects that are available to investigate this relatively common human syndrome. Here we will make an overview of the general features and developmental expression pattern of Hop in the heart. We will then describe the functional role of Hop in the adult heart and the mechanisms by which Hop it is thought to contribute to CCS function.

Hop in the Heart

Hop is a small, 73-amino acid protein that was identified independently by two groups and shown to be important for the regulation of cardiac development (Chen et al. 2002, Shin et al. 2002). As its name implies, Hop contains a 60-amino acid homeodomain sequence that is similar to the classic homeodomain motifs of Hox or homeobox genes which encode transcription factors. Hop has about 40% identity and 57% similarity to the homeodomain proteins Pax6 and goosecoid within the homeodomain-like regions of these proteins at the amino acid level (Chen et al. 2002). The homeodomain motif binds DNA and is composed of three α-helices, where the second and third helices are predicted to adopt a helix-turn-helix structure (Billeter et al. 1993). The third α-helix contains 17 amino acids and appears to be the one primarily responsible for interacting with DNA through its binding in the major groove (Kissinger et al. 1990). Other contacts with DNA are made by the amino terminal region of the homeodomain, which just precedes the first helix and projects into the minor groove (Lewin 2004). Hop is an atypical homeodomain protein in that it does not bind DNA. There are several amino acids in the Hop homeodomain which differ from those that are conserved between other homeodomains. For example, in the Hop homeodomain a leucine and glutamic acid residue occupy positions 53 and 55, respectively, whereas in other homeodomains these residues are usually occupied by an arginine and lysine which are involved in making contact with DNA (Chen et al. 2002, Shin et al. 2002). In addition, some of the residues that usually make electrostatic interactions with DNA in other homeodomains are missing from the homeodomain sequence of the Hop protein. Therefore, it was not surprising that experimental evidence from mobility shift and binding site assays confirmed the hypothesis that Hop does not bind DNA (Chen et al. 2002, Shin et al. 2002). However, the question still remained as to how Hop may regulate gene expression and regulate cardiac development if it does not directly bind DNA.

Homeobox transcription factors regulate downstream gene expression, either directly by binding to promoter or enhancer regions of the target genes or by interacting with other transcription cofactors. In the developing heart, the mammalian homolog of the Drosophila tinman gene, Nkx2-5, plays an important role in the early stages of cardiogenesis (Lints et al. 1993, Lyons et al. 1995, Tanaka et al. 1999). Nkx2-5 is a paired-type homeodomain-containing protein that functions in association with serum response factor (SRF) to synergistically activate SRF-dependent genes in the heart (Chen and Schwartz 1996). The expression of Hop can be found in myocardial precursors just after that of Nkx2-5, and in Nkx2-5 null embryos Hop expression is downregulated (Chen et al. 2002, Pashmforoush et al. 2004). Evidence from transient transfection assays shows that Nkx2-5 directly activates Hop expression and suggests that Hop is a downstream target of Nkx2-5 during early cardiogenesis (Chen et al. 2002). In Hop null mice, SRF-dependent genes are upregulated, such as atrial naturetic factor and myosin light chain 2v, suggesting that Hop may have an inhibitory effect upon SRF-dependent transcription (Chen et al. 2002, Shin et al. 2002). Whereas Hop does not bind DNA, it is expressed within the nucleus of cardiomyocytes and appears to modulate cardiac growth and proliferation by inhibiting SRF function (Chen et al. 2002, Shin et al. 2002). Transgenic mice engineered to over-express Hop under a cardiac-specific promoter develop cardiac hypertrophy and fibrosis, which is reversed by the administration of histone deacetylase (HDAC) inhibitors (Kook et al. 2003). These results were interpreted to imply that Hop inhibits SRF-dependent transcriptional activation by recruiting HDAC activity (Kook et al. 2003). Therefore, Hop appears to function as a regulator of cardiac development by balancing myocardial proliferation and differentiation through negative feedback with Nkx2-5 and inhibition of SRF via HDAC activity. This is somewhat similar, but not identical with, the putative role of Tbx5, which is also preferentially expressed in the CCS and thought to negatively regulate proliferation during cardiogenesis (Hatcher et al. 2001). This phenomenon may partially explain why cells within the CCS seem to have diminished proliferation during embryonic and fetal stages compared with cardiomyocytes of the working myocardium (Sedmera et al. 2003, Rentschler et al. 2002).

Expression of Hop in the heart

Hop is expressed in the heart during both embryonic development and into adulthood (Chen et al. 2002, Shin et al. 2002). Hop expression is first seen in mesodermal precursors of the cardiac muscle around postcoitum day 8.0 during murine development and by postcoitum day 9.5 Hop can be found in the myocardium and in regions of the pharyngeal arches (Chen et al. 2002, Shin et al. 2002). As late as postcoitum day 9.5, there are no detectable phenotypic abnormalities in Hop-deficient embryos; however, by postcoitum day 10.5 approximately 50% of the Hop-deficient embryos develop a phenotype with thinned myocardium, gross pericardial effusions, and die between postcoitum days 10.5 and 12.5 (Chen et al. 2002). On the other hand, Hop null embryos that survive to birth go on to develop a hypercellular phenotype in the immediate postnatal period, which further emphasizes the role of Hop as a regulator of proliferation and differentiation in the heart. During early to mid-gestation, Hop is expressed throughout the myocardium; however, it appears to be more robustly expressed in the inner trabecular layer compared with the outer compact zones and there is no detectable expression in the endocardium or endocardial cushions. By postnatal day 1, Hop is most strongly expressed in the endocardium and interventricular septum with less robust expression throughout the rest of the myocardium (Ismat et al. 2005). In adulthood, Hop is expressed throughout the atrial and ventricular myocardium, but is most strongly expressed within the CCS (Figure 1) where its pattern of expression closely mirrors that of CCS-lacZ mice (Rentschler et al. 2001) and Mink-lacZ mice (Kupershmidt et al. 1999). The fact that Hop expression is more restricted to the CCS in the mature mouse heart suggests that it plays an important role in regulating the CCS in postnatal life.

Figure 1.

Figure 1

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Hop expression in the CCS of adult mice. (A and B) β-Galactosidase staining of Hop−/− hearts, demonstrating staining of the ventricular myocardium with strong staining in the CCS. (A) There is marked staining in the AVN and left bundle branches (arrowheads) in this particular heart. The left and right ventricles are delineated for reference. (B) Right ventricular septal view of another Hop−/− heart, showing strong staining of the right bundle branch (arrowheads). (C and D) Microscopic evaluation of the CCS in Hop−/− (C) and Hop+/− (D) mice. (C) β-Galactosidase staining of the AVN and His bundle in a Hop−/− mouse heart. (D) β-Galactosidase staining of the His bundle and right and left bundle branches of a Hop+/− mouse heart. (E and F) Hop expression compared with AchE activity. (E) Proximal His bundle (arrowhead) of the heart from an adult Hop−/− mouse stained for AChE activity (brown). (F) β-Galactosidase expression (blue) in an adjacent section of the heart shown in panel (E). There is subtle staining of the muscle and very strong staining of the His bundle (arrowhead). (Adapted with permission from Ismat et al., Homeobox protein Hop functions in the adult cardiac conduction system, Circulation Research; 96(8):898–903.)

Hop in the CCS

Hop null mice that survive to maturity appear have a completely intact and anatomically well-developed CCS (Figure 1). These mice also have structurally normal hearts with normal function, as assessed by invasive hemodynamic monitoring (Kook et al. 2003) and cardiac-gated magnetic resonance imaging (Ismat et al. 2005). Surface electrocardiogram analysis reveals right-axis deviation, QRS-complex widening, a longer QT interval, and prolongation of the p-wave in adult Hop knockout mice (Figure 2). The surface electrocardiogram findings of a right-axis deviation and increased QRS-complex duration suggest the predominant conduction delay is below the AV node in Hop null mice. Invasive electrophysiologic analysis confirmed the presence of an infra-nodal conduction defect with ~30% to 50% increase in the hisioventricular (HV) interval (Figure 2). The electrophysiologic phenotype found in Hop null mice is consistent with the strong expression of Hop in the His-bundle and bundle branches. Similarly, there is prolongation of the p-wave, atrial refractory period, and a minor increase in the atriohisian interval, which is also consistent with the predominant expression of Hop in the atria and AV node during the postnatal period. This pattern of electrophysiologic findings is similar, but not exactly identical, to those described in connexin40-deficent mice (Verheule et al. 1999, Bevilacqua et al. 2000). Connexin40 is the predominant connexin isoform contributing to gap junction formation between cardiomyocytes of the rodent atrium and CCS, so it seems reasonable that deletion of Hop would affect the expression of connexin40 in the CCS. As was predicted, immunocytochemical analysis of connexin40 expression in Hop null mice revealed a loss of connexin40 within the CCS (Figure 3). Therefore, it appears that Hop contributes to postnatal function of the CCS by regulating connexin40 expression, the absence of which largely explains the electrophysiologic phenotype observed in Hop null mice. Whereas connexin40 is a well-documented transcriptional target of both Nkx2-5 and Tbx5 (Bruneau et al. 2001), deletion of Hop should increase connexin40 expression and not decrease it as is seen in Hop null mice. This should be the case if Hop acts via negative feedback upon both Nkx2-5 and Tbx5 activity, as the latter two factors function synergistically in the heart (Hiroi et al. 2001). This suggests that Hop may regulate connexin40 expression by modulating the transcriptional activity of other factors independent of Nkx2-5 or Tbx5. In addition, Hop does not appear to be involved with patterning or maintenance of conduction system tissue, as the CCS develops normally and is functionally preserved in adult Hop null animals. This is in contrast to the role of Nkx2-5 and Tbx5, where both Nkx2-5 and Tbx5 null mice develop hypoplasia of the CCS (Jay et al. 2004, Pashmforoush et al. 2004, Moskowitz et al. 2004), suggesting that Nkx2-5 and Tbx5 can activate sufficient target genes to direct conduction system development in the absence of Hop. Another possibility is that the expression pattern of Hop-lacZ in the mature murine CCS does not accurately reflect the true expression of Hop protein. This is the case with the expression pattern of MinK-lacZ in the adult mouse CCS, which does not match the expression of MinK mRNA during later development in the heart, where in situ hybridization shows complete loss of MinK mRNA from the intraventricular septum and region of the CCS (Franco et al. 2001). A similar situation may exist with the Hop-lacZ mice, which can be addressed by immunocytochemical or in situ hybridization studies of Hop expression in the heart, as this has important implications for understanding the biological role of Hop in CCS development.

Figure 2.

Figure 2

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Conduction defects in adult Hop null mice. Intracardiac electrophysiologic recording, with surface ECG leads I, II, aVF, the right atrial electrogram and His-bundle electrogram (HBE) from a Hop−/− and Hop+/+ mouse demonstrating prolongation of the HV interval in the absence of Hop. The Hop−/− mouse has an HV interval of 15 ms versus 10 ms in the Hop+/+ mouse (large arrowheads). Vertical scale bar = 1 mV; Horizontal scale bar = 100 ms. (Adapted with permission from Ismat et al., Homeobox protein Hop functions in the adult cardiac conduction system, Circulation Research; 96(8):898–903.)

Figure 3.

Figure 3

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Immunohistochemistry of the CCS in Hop mutant mice. Serial frozen sections of newborn Hop+/− (A through C) and Hop−/− (D through F) hearts were stained for β-galactosidase activity (A and D) and connexin40 (B and C, E and F). Areas of the CCS are outlined. Although there was strong expression of connexin40 seen in the Hop+/− CCS (B), the Hop−/− CCS showed expression of connexin40 in a much smaller area that did not extend beyond the proximal AVN and His bundle (E). This pattern was noted on several serial sections. (C and F) Connexin40 staining in Hop mutant atria. Frozen sections of newborn Hop+/− (C) and Hop−/− (F) mice stained together show reduced intensity of staining in the Hop−/− atrium as compared with Hop+/− mice. This finding was observed throughout the atria. All sections were photographed at the same magnification (×20 CCS, ×10 atria). (Adapted with permission from Ismat et al., Homeobox protein Hop functions in the adult cardiac conduction system, Circulation Research; 96(8):898–903.)

Future Directions

Hop is a recently described homeodomain protein that was first identified as a regulator of cardiac development, through its inhibition of SRF-dependent transcription, which acts to balance myocardial proliferation and differentiation. Although Hop transcript is expressed during both embryogenesis and into adulthood, its role in the mature mammalian heart is only now becoming clearer. We have shown Hop is highly expressed in the mature CCS, where targeted deletion of the Hop locus induces cardiac conduction defects with downregulation of connexin40, whereas the gross anatomic structure of the CCS remains preserved. Our studies investigating the role of Hop in the mature murine CCS will hopefully stimulate further investigation to define the role of Hop in the CCS and perhaps help identify novel targets for the therapy of conduction disorders in patients. Of course, these findings will first need to be confirmed in human disease, because at this time no clinically relevant conduction system disorders have been linked to downregulation or loss-of-function in Hop. However, recent evidence suggests that Hop expression is downregulated in end-stage human heart failure and correlates with conduction defects in this patient population (Cappola et al. unpublished). In addition, although loss of connexin40 within the CCS is probably the major mechanism underlying conduction defects induced by deleting Hop, it is possible that other proteins involved with mediating cardiac conduction may also be altered in the absence of Hop. Identification of additional downstream targets of Hop involved with CCS function may someday improve therapy for symptomatic bradycardia, either in conjunction with permanent pacemakers or as a replacement for device-based therapies.

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