Published online before print January 18, 2007, doi: 10.1161/01.RES.0000258019.74591.b3
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© 2007 American Heart Association, Inc.
Molecular Medicine |
Molecular Pathway for the Localized Formation of the Sinoatrial Node
From the Center for Heart Failure Research (M.T.M.M., W.M.H.H., C.d.G.-d.V., C.W., D.E.W.C., A.F.M.M., V.M.C.), Academic Medical Center, Amsterdam, The Netherlands; the Victor Chang Cardiac Research Institute (O.W.J.P., R.P.H.), Darlinghurst, Australia; the Faculties of Life Sciences and Medicine (R.P.H.), University of New South Wales, Kensington, Australia; Department of Genetics and Development (V.E.P.), College of Physicians and Surgeons of Columbia University, New York; and the Division of Basic Medical Sciences (N.A.B.), St. George’s, University of London, UK.
Correspondence to Vincent M. Christoffels, Center for Heart Failure Research, Academic Medical Centre, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands. E-mail v.m.christoffels{at}amc.uva.nl
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Abstract |
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The sinoatrial node, which resides at the junction of the right atrium and the superior caval vein, contains specialized myocardial cells that initiate the heart beat. Despite this fundamental role in heart function, the embryonic origin and mechanisms of localized formation of the sinoatrial node have not been defined. Here we show that subsequent to the formation of the Nkx2-5–positive heart tube, cells bordering the inflow tract of the heart tube give rise to the Nkx2-5–negative myocardial cells of the sinoatrial node and the sinus horns. Using genetic models, we show that as the myocardium of the heart tube matures, Nkx2-5 suppresses pacemaker channel gene Hcn4 and T-box transcription factor gene Tbx3, thereby enforcing a progressive confinement of their expression to the forming Nkx2-5–negative sinoatrial node and sinus horns. Thus, Nkx2-5 is essential for establishing a gene expression border between the atrium and sinoatrial node. Tbx3 was found to suppress chamber differentiation, providing an additional mechanism by which the Tbx3-positive sinoatrial node is shielded from differentiating into atrial myocardium. Pitx2c-deficient fetuses form sinoatrial nodes with indistinguishable molecular signatures at both the right and left sinuatrial junction, indicating that Pitx2c functions within the left/right pathway to suppress a default program for sinuatrial node formation on the left. Our molecular pathway provides a mechanism for how pacemaker activity becomes progressively relegated to the most recently added components of the venous pole of the heart and, ultimately, to the junction of the right atrium and superior caval vein.
Key Words: heart development • sinus node • sinoatrial node • Nkx2-5 • Pitx2c • Tbx3 • Cx40 • transgenic mice
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Introduction |
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The sinoatrial node (SAN) is the pacemaker of the heart that initiates the heartbeat and controls its rate of contraction. It is a specialized myocardial structure positioned at the junction of the right atrium and the superior caval vein. Disease, ageing, or gene defects may cause SAN dysfunction (sick sinus syndrome), a common cardiac disorder that currently requires the implantation of a pacemaker. SAN function depends on a complex tissue architecture and the expression and function of a set of ion-channel, sarcomeric and gap junction proteins expressed at levels distinct from the atrial working myocardium.1,2 Hyperpolarization-activated pacemaker channel Hcn4 is expressed in the SAN and plays an important role in pacemaker activity in vivo.2,3 Furthermore, the SAN expresses very low levels of connexin 40 (Cx40) and Cx43 gap-junctional subunits that are essential for the fast conduction of the electrical impulse in the atrial and ventricular working myocardium and ventricular conduction system.4,5 The T-box transcription factor Tbx3, which, when mutated, causes ulnar-mammary syndrome of congenital malformation in human,6 is expressed in the developing nodal tissues of the heart of mammals and chicken and is able to suppress promoter activity of atrial chamber myocardial genes including natriuretic peptide precursor type A (Nppa, encoding atrial natriuretic factor) and Cx40 in cell culture.7
Despite its fundamental role in heart function, the embryonic origin of the SAN and the mechanisms of its patterning and formation are still enigmatic. In the present study we investigated the spatio-temporal patterns of genes relevant to the developing inflow tract, SAN, and atrium and provide evidence for roles for Nkx2-5 (NK2 transcription factor related, locus 5 [Drosophila]), Pitx2c (paired-like homeodomain transcription factor 2), and Tbx3 in the patterning and formation of the SAN.
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Materials and Methods |
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Mice
The R26R,8 Nkx2-5–,9 Nkx2-5ires-Cre,10 Pitx2c–,11 and Tbx3–12 transgenic mouse lines have been described previously. A cDNA fragment encoding the full-length human TBX3 protein13 was cloned in a construct containing a 5-kb regulatory fragment of the murine ß-Mhc gene and the human growth hormone pA signal,14 kindly provided by Dr J. Robbins (Children’s Hospital Research Foundation, Cincinnati, Ohio). As a control, a cDNA fragment encoding TBX3 with a point mutation in the T-box DNA-binding region (L143P)13 was cloned in the same construct. Vector sequences of these constructs were removed, and the DNA was microinjected into nuclei of FVB zygotes, which were subsequently implanted into fosters to generate transgenic embryos. At embryonic day 9.5 (E9.5), embryos were isolated for analysis. Embryos and fetuses were dissected in PBS and fixed in 4% paraformaldehyde overnight. Amnion or tail biopsy genomic DNA was used for PCR assays to detect the Cre, lacZ, GFP, or TBX3 transgenes.
ß-Galactosidase Activity Detection and Immunohistochemical Analyses
Detection of ß-galactosidase activity on 20-µm cryostat sections was performed as described.15 For immunohistochemistry on 10-µm embryo sections, the following primary antibodies were used: polyclonal antibody against cardiac troponin I (cTnI) (1:1000; Hytest Ltd); polyclonal antibody against Nkx2-5 (1:250; Santa Cruz Biotechnology); polyclonal antibody against Hcn4 (1:200; Chemicon); polyclonal antibody against Cx40 (1:200; Chemicon); and monoclonal antibody against desmin (1:50; Monosan).
Nonradioactive In Situ Hybridization
Nonradioactive in situ hybridization of 12-µm embryo sections was performed as described.16 The probes for Isl117 and Hcn418 were kindly provided by S. Evans (Skaggs School of Pharmacy, University of California, San Diego) and B. Santoro (Center for Neurobiology and Behavior, Columbia University, New York). Other probes have been described previously.16 All patterns in wild-type embryos were observed in at least 3 independent embryos.
Three-Dimensional Reconstructions
Three-dimensional visualization and geometry reconstruction of patterns of gene expression determined by in situ hybridization was performed as described previously.19 Files with reconstructions are available on request.
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Results |
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Expression and Genetic Lineage Analysis Reveal a Mechanism of SAN Development
To investigate formation of SAN in development, we assessed the expression patterns of Hcn4, a SAN marker1; Nppa and Cx40, atrial chamber myocardial markers4; Tbx3, a developing conduction system marker and putative repressor of Nppa and Cx407; Nkx2-5, a key cardiac homeobox gene expressed in the first and second heart fields20 and essential for the activation of Cx40 and Nppa21,22; and cTnI, desmin, and myosin light chain 2a (Mlc2a), which mark all differentiated myocardium. "Inflow tract" refers to the myocardial intake at the caudal end of the heart tube until E9.5. "Venous pole" refers to the myocardial components of the systemic venous return that are added to the heart from E9.5 onward, including the SAN (this study), venous layer of the bilayered venous valves, and left and right sinus horns.16
The E9.5 heart tube coexpressed myocardial marker genes and Nkx2-5. The inflow tract in addition expressed Hcn4 in a pattern that initially overlaps that of Nkx2-5 (Figure 1A and 1B). Expression of Cx40 had been initiated at this stage in the Nkx2-5–positive inflow tract (Figure 1B and 1C). After E9.5, Nkx2-5–negative myocardial sinus horns are formed from cardiac progenitor cells adjacent to the inflow tract.16 At E10.5, Cx40 and Nppa were expressed in the Nkx2-5–positive myocytes, which at this stage have contributed to the atrial chambers and atrial layer of the venous valves. Hcn4 expression was downregulated in this myocardium to become confined to the newly formed Nkx2-5–negative venous pole (Figure 1B and 1C). Thus, an early pattern in which Nkx2-5 and Cx40 expression overlaps that of Hcn4 resolves into a mutually exclusive pattern by E10.5.
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From E10.5 on, a thickening of myocardial cells was formed in the right sinus horn bordering but exclusive of the Nkx2-5/Cx40/Nppa-positive atrial cells, corresponding to the primordial SAN23 (Figure 1C). This structure also expressed Tbx3. Three-dimensional reconstructions show that the expression domains of Tbx3 and of Cx40 were mutually exclusive (Figure 2A). At the atrial side, the Tbx3 expression domain bordered the Cx40-positive domain, but neither Tbx3 nor Cx40 were expressed in the myocardium of the sinus horns (Figures 1C and 2
A). Together, these data indicate the existence of 3 bordering domains at the sinuatrial junction, an atrial domain, a SAN domain, and a sinus horn domain (Figure 2B through 2E).
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To address whether the Nkx2-5–negative SAN primordium is derived from Nkx2-5–positive inflow tract myocardium, which subsequently loses Nkx2-5 expression, or, like the sinus horns, from adjacent Nkx2-5–negative mesenchyme,16 we crossed Nkx2-5ires-Cre mice10 with R26R mice8 to reveal all cells derived from Nkx2-5–positive cells. These data showed that the Tbx3-positive, Cx40-negative SAN primordium was devoid of Nkx2-5–Cre–positive cells and therefore never expressed Nkx2-5 (Figure 3A and 3C). We conclude that the primordial SAN myocardium is formed de novo from Nkx2-5–negative cells.
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LIM homeodomain transcription factor islet-1 (Isl1) is reported to be a marker of the second heart field precursors that is switched off when these mesenchymal precursors differentiate to myocardium.17 However, probably because of the half-life of its transcripts, Isl1 expression remains detectable shortly after differentiation of the precursors into myocardium.16 Isl1 expression was still observed in the Hcn4-positive primordial SAN (Figure 3B), providing support for de novo differentiation of the SAN from second heart field progenitors.
Nkx2-5 Suppresses Hcn4 and Tbx3 and Establishes the Sinoatrial Boundary
The boundary between the SAN and atrial myocardium could be established at least in part if Nkx2-5 acted as a repressor of Hcn4 and Tbx3. We therefore tested the expression of Hcn4 and Tbx3 in Nkx2-5–deficient mice. In these embryos, heart development is arrested during looping, chamber formation does not occur, and Cx40 and Nppa are not activated.21,22 We found a marked ectopic expression of both Hcn4 and Tbx3 throughout the heart tubes of Nkx2-5—deficient embryos (Figure 4A), showing that Nkx2-5 is required to repress these genes in the heart tube. These observations indicate a critical role of Nkx2-5 in the confinement of Hcn4 and Tbx3 to the Nkx2-5–negative venous pole domain (Figure 4C).
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Because Nkx2-5 mutant mice die at E10, this model does not allow the analysis of the SAN region in a more advanced state of development. We investigated fetal mice compound heterozygous for the Nkx2-5ires-Cre allele and for a null allele. These mice express 
25% of the normal level of Nkx2-5 expression in wild type, because the ires-Cre insert in the 3' untranslated region of the Nkx2-5 gene creates a hypomorphic allele (O.W.J.P., R.P.H., submitted manuscript, 2006). Nkx2-5ires-Cre/– mice die shortly after birth showing a range of cardiac malformations, including atrial and ventricular septal defects, much more severe than haploinsufficient mice. As controls, Nkx2-5ires-Cre/+ mice were used and appeared completely normal. In Nkx2-5ires-Cre/– E14.5 fetuses, the Hcn4 and Tbx3 expression domains of the SAN were much broader and extended into the atrium (Figure 4B). Thus, the expression border between the SAN and atrium was blurred. Cx40 expression was reduced in these hearts, consistent with the notion that Nkx2-5 is required for Cx40 expression,22 but was particularly weak in the enlarged Hcn4 and Tbx3 domain around the SAN (Figure 4B). These observations indicate a critical role of Nkx2-5 in the regulation and maintenance of the atrial chamber gene program at the border with the SAN domain (Figure 4C).
Tbx3 Suppresses Chamber Myocardial Differentiation and Gene Expression
Our analysis revealed that Tbx3 could be involved in the suppression of atrial chamber differentiation and repression of chamber gene expression, thus contributing to the chamber-negative phenotype of the SAN. We analyzed Tbx3 mutant embryos, which die from between E11 and E13.12 Embryos that clearly survived until E11.5 had formed a primordial SAN-like structure (Figure 5A). Moreover, Nkx2-5, Cx40, and Nppa were not ectopically activated in this primordium, and Hcn4 expression was maintained (Figure 5A). Notably, because Nkx2-5 is strictly required for Nppa and Cx40 gene activity,21,22 maintenance of the Nkx2-5 boundary at the junction between the atrium and SAN/venous pole at this developmental stage provides a likely explanation for the maintenance of the SAN gene expression signature in the Tbx3 mutants.
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Nonetheless, Tbx3 may provide a tuning mechanism to ensure stabilization of SAN gene expression, in particular, to ensure the absence of the atrial differentiation program. To investigate whether Tbx3 is able to suppress chamber myocardial differentiation and Nppa and Cx40 in vivo, transgenic embryos were generated that express the human Tbx3 cDNA under the control of the ß-Mhc promoter, which drives expression in the tubular heart before the differentiation of chamber myocardium.14,24 To evaluate possible nonspecific effects of Tbx3 overexpression, eg, by competing for limiting cofactors, we also generated embryos that express Tbx3-LP, which contains an L-to-P substitution at position 143 in the T-box domain that has been found in ulnar-mammary syndrome patients.6 Tbx3-LP and Tbx3 protein showed comparable stability in transfected cells,7,13 but Tbx3-LP is not able to bind to its DNA binding sites.7 Transgenic embryos were analyzed at E9.5, after the initiation of chamber differentiation and Nppa and Cx40 expression. These embryos were smaller but not obviously abnormal with respect to general body patterning (Figure 5B). Hearts of these embryos were linear, or looped to some extent, and partially or completely failed to form chambers (Figure 5B and 5C). Section in situ hybridization showed that Nppa and Cx40 were not expressed in these hearts, indicating that their expression was suppressed by Tbx3 (Figures 4C and 5
C). However, Hcn4 was not precociously activated in the Tbx3-overexpressing hearts (data not shown), supporting the data obtained in the Tbx3 mutant suggesting that Tbx3 does not directly regulate Hcn4 (Figure 4C). Tbx3-LP embryos were normal, and even though Tbx3-LP was robustly expressed in the hearts, the morphology and the expression of Cx40 and Nppa expression were not affected (Figure 5C). We conclude that Tbx3 is able to specifically block chamber myocardial differentiation and the expression of Nppa and Cx40 in vivo in a DNA-binding–dependent manner.
Pitx2c Suppresses SAN Formation in the Left Side of the Venous Pole
The Pitx2 homeobox factor is essential for late aspects of left–right asymmetric morphogenesis.25 Pitx2c-deficient mutant mice have right isomerism of the atria and venous pole.11 In situ hybridization showed that the primordial SAN does not express Pitx2c (Figure 6A). We investigated whether Pitx2c is also involved in the placement of the primordial SAN. Pitx2c mutant embryos of E9.5, shortly before the primordial SAN is formed and the asymmetry of the sinus horns becomes apparent,26 have apparently normal inflow tracts (not shown). However, after E10.5, shortly after initiation of the formation of the primordial SAN, we noted that the atria had 2 sets of venous valves, and 2 short sinus horns, indicating right isomerism of the left venous pole (Figure 6B). Moreover, a second SAN primordium was invariably observed at the left side. This left-sided SAN primordium showed an appropriate Hcn4-positive, Cx40-negative SAN gene program. At E15.5, the left-sided SAN contained the typical nodal artery, and the gene expression profile of this SAN was indistinguishable from that of the right-sided SAN in the mutant, and of the SANs of wild-type littermates. We conclude that the default morphogenetic pathway of the venous pole leads to the formation of a right-sided sinus horn, SAN and venous valves, and that the Pitx2c pathway suppresses SAN formation.
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Atrialization of the Venous Pole
From the above, we conclude that after establishment of an Nkx2-5–expressing heart tube, the sinus horn myocardium and SAN are formed from cardiac field cells via a novel, Nkx2-5–independent pathway and run a nodal gene program. However, from E14.5 onward, expression of Cx40 was upregulated in the sinus horn myocardium, whereas that of Hcn4 was gradually lost from the sinus horn myocardium (Figure 7A through 7D). Furthermore, the sinus horn myocardium gradually became positive for Nkx2-5 expression (Figure 7C through 7D). Moreover, regions within the SAN initiated Nkx2-5 expression, which now for the first time overlapped with Tbx3 expression (Figure 7E through 7G). However, Tbx3 and Hcn4 were not downregulated and the SAN remained free of Cx40 expression (Figure 7F and 7G). Taken together, the data suggest that in the fetal period, the venous pole myocardium obtains an atrial gene program, except for the SAN, which, despite the upregulation of Nkx2-5, still runs the Tbx3/Hcn4-positive, Cx40-negative nodal gene program (Figure 3C).
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Discussion |
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In this study, we demonstrate that the SAN initially forms from Nkx2-5–negative precursors and remains largely free of Nkx2-5 expression until the onset of a second phase of atrial myogenesis in the venous pole beginning at E14.5. Nkx2-5 is a cardiac homeobox gene critical for heart development in all species examined thus far and is at the core of transcriptional regulatory mechanisms that control gene expression and differentiation in the heart.21 Heterozygous mutations in Nkx2-5 in humans cause congenital atrioventricular conduction system defects.27 Moreover, the atrioventricular node, which expresses relatively high levels of Nkx2-5, is hypoplastic in heterozygous mutant mice.28 Therefore, although the SAN and the atrioventricular node share many phenotypic features, the differential expression of Nkx2-5 is a fundamental difference between the 2 nodes, implying that distinct regulatory mechanisms control their formation and function.
Early Localization of Pacemaker Activity and Patterning of the Venous Pole
All embryonic cardiomyocytes of the early developing heart possess intrinsic pacemaker activity, which is followed by contractility slightly later. However, dominant pacemaker activity is always found at the intake (inflow tract, venous pole) of the developing heart tube.29,30 Hcn4 is required for pacemaker activity in mouse embryos, whereas humans with a mutation in HCN4 have bradycardia.2,3 The steep caudocranial expression gradient of Hcn4 observed from E7.5 in the cardiac crescent31 and subsequent expression in the entire venous pole (SAN and sinus horns; Figures 1B, 2E, and 3C) may well explain the observation that dominant pacemaker activity is always localized at the intake.
The embryonic heart tube elongates by recruitment of cardiac field cells to the heart tube and inflow complex.20 In the inflow tract region, the atria are formed first, followed after E9.5 by the sinus horns16 and the primordial SAN (this study). Expression of Hcn4 initially overlaps with that of Nkx2-5 but subsequently becomes restricted to the newly recruited Nkx2-5–negative venous pole components. This indicates that Hcn4 expression is extinguished in the Nkx2-5–positive myocardium (Figures 2E and 3C), which is fated to form the atria and atrial layer of the venous valves.16 In line with these patterns of expression, Nkx2-5–deficient embryos showed a dramatic ectopic expression of Hcn4 in the heart tube, explaining previous observations that a large fraction of Nkx2-5–deficient embryos initiate beating from the embryonic ventricular region rather than from the inflow tract.22 Together, these data indicate that Hcn4 expression is initiated by unknown factors in newly formed venous pole myocardium and that as the heart matures, Nkx2-5 represses Hcn4, thereby confining its expression to the Nkx2-5–negative venous pole (Figures 3C and 4C). This repressive activity for Nkx2-5 on Hcn4 provides a mechanism for the progressive shift of Hcn4 expression as well as pacemaker activity to the newly formed venous pole components in normal embryos (Figures 3C and 4C). This mechanism provides an explanation for the longstanding observation that during heart development, dominant pacemaker activity is always localized at the caudal end (intake) of the heart.29,30
Patterning and Formation of the SAN
Nkx2-5–deficient embryos showed ectopic expression of Hcn4 and Tbx3 in the whole-heart tube. Furthermore, in Nkx2-5 hypomorphic fetuses, the otherwise sharp expression boundary between the atria and SAN was blurred. The expression of Tbx3 and Hcn4 extended into the right atrium, whereas Cx40 expression was specifically downregulated in the extended Tbx3-positive area. These observations indicate that Nkx2-5 is required to establish the boundary between the atria and SAN and, in a dose-dependent manner, to prevent the SAN phenotype from invading the atria or the atrial phenotype invading the SAN (Figures 3C and 4C).
A next step involves asymmetrical gene expression and morphogenesis after E9.5, which leads to the formation of a Tbx3-expressing SAN primordium at the right sinoatrial junction. Human patients and iv/iv mice with right isomerism have 2 sinoatrial nodes, whereas in patients and mice with left isomerism the sinoatrial node is hypoplastic or absent.32,33 Therefore, the SAN is an integral component of the right side–specific morphogenesis program that is repressed by Pitx2c. We found that Pitx2c-deficient embryos form 2 SANs running the SAN signature gene program. Therefore, the complete right-sided morphogenesis program, including Tbx3 expression, is the default program, which in the left sinoatrial region is masked and converted to a left-sided program without a SAN by Pitx2c. These observations also indicate that Pitx2c is a critical factor for the entire left morphogenetic program in the sinuatrial region. None of the genes studied here was deregulated in other sites of Pitx2c expression of Pitx2c mutants, including the atrioventricular canal (Tbx3) or atria (Nkx2-5, Nppa, Cx40) (Figure 6B and data not shown), indicating that Pitx2c does not directly control expression of these genes. Asymmetry of the venous pole is first observed after E9.5,26 when the sinus horns are being formed from their progenitors.16 Pitx2 was found to control asymmetric morphogenesis at least in part by regulation of cell type–specific proliferation.34 It is expressed in the left-sided myocardium from very early stages on and, therefore, may control proliferation of the left venous pole myocardium as soon as it is recruited from the progenitor pool.
Finally, after E14.5 the nodal gene program becomes confined to the SAN domain. The sinus horns, at the end of gestation recognizable as the myocardial sleeves surrounding the right and left caval veins and sinus venarum, gradually switch to a Cx40-positive, Hcn4-negative atrial gene program (Figure 3C). Nkx2-5 expression gradually increased toward the end of gestation, indicating that it may be responsible for the switch to the atrial gene program, although the involvement of other factors cannot be excluded at this point. However, despite the upregulation of Nkx2-5 expression in the SAN, Cx40 was not activated here, and Hcn4 was not repressed. Therefore, we hypothesize that in the SAN primordium, Tbx3 expression is, or has become, insensitive to Nkx2-5 and, furthermore, that Tbx3 shields the SAN domain from atrial gene expression and differentiation once Nkx2-5 is being activated in the SAN (Figure 4C). Although this putative function of Tbx3 remains to be demonstrated, it is consistent with the found ability of Tbx3 to suppress Nppa and Cx40 and to block chamber differentiation in the embryonic heart tube.
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Acknowledgments |
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We thank Sameer Rana for his laboratory assistance.
Sources of Funding
This work was supported by grants from the Netherlands Heart Foundation (96.002 to V.M.C. and A.F.M.M.); the Netherlands Organization for Scientific Research (Vidi grant 864.05.006 to V.M.C.); European Union FP6 contract "Heart Repair" (LSHM-CT-2005-018630 to V.M.C., A.F.M.M., and N.A.B.); the National Health and Medical Research Council (Australia) (354400), the Network for Genes and Environment in Development (Australia), the National Institute of Child Health and Human Development, NIH (HD047858), and the Johnson and Johnson Focused Giving Program (to R.P.H. and O.W.J.P.); the NIH (RO1 HD33082 to V.E.P.); and the British Heart Foundation (RG RG-03-012 to N.A.B).
Disclosures
None.
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Footnotes |
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Original received September 21, 2006; resubmission received November 16, 2006; revised resubmission received December 21, 2006; accepted January 4, 2007.
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