Download Development of the Pacemaker Tissues of the Heart

This article is part of a new thematic series on Mechanisms of Pacemaking in the Heart, which includes the following
articles:
Be Still, My Beating Heart—Never! [2010;106:238 –239]
Development of the Pacemaker Tissues of the Heart
Mapping Cardiac Pacemaker Circuits: Methodological Puzzles of the Sinoatrial Node Optical Mapping [2010;106:255–271]
The Role of the Funny Current in Pacemaker Activity
Ca2⫹ Cycling in the Mechanism of Pacemaking
Cardiac Pacemaking: Historical Overview and Future Directions
Dennis Noble, Guest Editor, and Brian O’Rourke, Editor
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Development of the Pacemaker Tissues of the Heart
Vincent M. Christoffels, Gertien J. Smits, Andreas Kispert, Antoon F. M. Moorman
Pacemaker and conduction system myocytes play crucial roles in initiating and regulating the contraction of the
cardiac chambers. Genetic defects, acquired diseases, and aging cause dysfunction of the pacemaker and
conduction tissues, emphasizing the clinical necessity to understand the molecular and cellular mechanisms of
their development and homeostasis. Although all cardiac myocytes of the developing heart initially possess
pacemaker properties, the majority differentiates into working myocardium. Only small populations of
embryonic myocytes will form the sinus node and the atrioventricular node and bundle. Recent efforts have
revealed that the development of these nodal regions is achieved by highly localized suppression of working
muscle differentiation, and have identified transcriptional repressors that mediate this process. This review will
summarize and reflect new experimental findings on the cellular origin and the molecular control of
differentiation and morphogenesis of the pacemaker tissues of the heart. It will also shed light on the etiology of
inborn and acquired errors of nodal tissues. (Circ Res. 2010;106:240-254.)
Key Words: conduction system 䡲 pacemaker 䡲 sinus venosus 䡲 atrioventricular canal 䡲 development
T
he contractions of the heart are initiated and coordinated by electric signals from pacemaker tissues. At
the entrance of the right atrium, sinus node (sinoatrial node
[SAN]) myocytes generate the impulse to activate the
atrial myocardium. After rapid propagation through the
atria, the impulse is delayed in the atrioventricular node
(AVN) and further propagated to the fast-conducting
atrioventricular bundle (AVB), bundle branches (BB), and
Purkinje fiber network, from which the mass of the
ventricular working myocardium is activated. The components of the conduction system contain cardiomyocytes
with pacemaker activity and other specific nodal properties
that discriminate them from atrial and ventricular working
myocardium (Figure 1). The SAN serves as the primary
pacemaker, whereas the AVN and ventricular conduction
system act as secondary (accessory) pacemakers to secure
Original received July 20, 2009; revision received October 12, 2009; accepted October 15, 2009.
From the Heart Failure Research Center (V.M.C., A.F.M.M.), Academic Medical Center, Amsterdam, The Netherlands; Swammerdam Institute
for Life Sciences (G.J.S.), University of Amsterdam, The Netherlands; and Institut für Molekularbiologie (A.K.), Medizinische Hochschule
Hannover, Germany.
Correspondence to Vincent M. Christoffels, Heart Failure Research Center, Academic Medical Centre, University of Amsterdam, Meibergdreef 15,
1105 AZ Amsterdam, The Netherlands. E-mail [email protected]
© 2010 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.109.205419
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Non-standard Abbreviations and Acronyms
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a
as
AV
AVB
AVC
avj
AVN
BMP
E
ev
ift
la
laa
lbb
lv
oft
pr
pv
pvcs
ra
raa
ran
ravrb
raw
rbb
rv
rvv
SAN
scv
sv
vv
WM
atrium
atrial septum
atrioventricular
atrioventricular bundle (bundle of His)
atrioventricular canal
atrioventricular junction
atrioventricular node
bone morphogenetic protein
embryonic day
embryonic ventricle
inflow tract
left atrium
left atrial appendage
left bundle branch
left ventricle
outflow tract
primary ring
pulmonary vein
peripheral ventricular conduction system
right atrium
right atrial appendage
retroaortic atrioventricular node
right atrioventricular ring bundle
right atrial wall
right bundle branch
right ventricle
right venous valve
sinus node/sinoatrial node
superior caval vein
sinus venosus
venous valves
working myocardium
repetitive ventricular contraction in conditions of SAN failure
or AV block.1 In addition, the lower rim of the atrial
myocardium, just above the AV junction, and the myocardium surrounding the systemic venous return contain nodal-like
cells, which, under pathological conditions, can acquire
ectopic pacemaker activity.2–5
Disease, congenital malformations, aging, or somatic gene
defects may cause dysfunction of the pacemaker tissues
resulting in severe arrhythmias.6,7 Although the electrophysiological properties, structure and cellular composition of the
nodes have been extensively studied,1,6,8 –10 pathologies associated with the nodes are still far from understood. SAN and
AVN development is cellularly and molecularly intertwined
with that of the chambers. Therefore, the elucidation of the
mechanisms underlying heart development in general will
make a crucial contribution to our understanding of the
functional regulation of the nodes. These insights may provide leads for the therapeutic intervention of pacemaker and
conduction system diseases.
The mode of initiating and propagating the depolarizing
impulse through the simple hearts of lower vertebrates, hearts
Figure 1. Schematic overview of heart development in higher
vertebrates. A, The early heart tube has a primitive phenotype
(gray) and sinusoidal ECG. Chamber myocardium (blue)
expands from the outer curvatures of the primary heart tube,
whereas nonchamber myocardium (gray) of the sinus venosus
(sv), AVC (avc), outflow tract (oft), and inner curvatures does not
expand. The chamber heart has a mature type of ECG. B, The
basic configuration of slow conducting pacemaker and fast conducting chamber components is found in the adult fish and
human heart. C, The sinus node (san) forms in the sinus venosus, the AVN (avn), and atrioventricular junction (avj) in the atrioventricular canal. The ventricular septum crest part of the primary ring (pr) will form the AVB. a indicates atrium; ev,
embryonic ventricle; ift, inflow tract; la, left atrium; lbb, left bundle branch; lv, left ventricle; pvcs, peripheral ventricular conduction system; ra, right atrium; rv, right ventricle; scv, superior
caval vein; rbb, right bundle branch.
of vertebrate embryos, and multichambered hearts of adult
higher vertebrates (including human) is essentially identical
(Figure 1). In fact, even in the absence of discernible nodal and
conduction system components in simple hearts, a mature ECG
can be derived. Thus, the coordinated generation and propagation of the depolarizing impulse is deeply rooted in the basic
design of the vertebrate heart, indicating that the cellular and
molecular mechanisms that drive the formation of pacemaker
tissues are evolutionary conserved. This review discusses current
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February 5, 2010
insights into the cellular origin of the nodal pacemaker tissues,
their formation and function in relation to other cardiac components, and the molecular mechanisms controlling nodal tissue
development. The formation of the AV bundle, BBs, and
Purkinje network has been reviewed elsewhere.11–13
Development of the Heart and Its Basic
Electric Configuration
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A simple heart tube is formed subsequent to the formation of
a cardiac crescent by fusion of the 2 heart-forming regions of
the lateral plate mesoderm in gastrulation stage mammalian
embryos. Not all cells of the (pre)cardiac mesoderm differentiate immediately. Cells located medially in the cardiac
mesoderm are kept behind and become positioned dorsally
and caudally to the arising heart tube. These cells, referred to
as second heart field,14 proliferate rapidly and serve as a
progenitor pool that continuously provides myocardium to
both poles of the heart tube.15 Before their differentiation to
myocardium and addition to the heart tube, these cells
drastically decrease their proliferation rate.14,15 Labeling studies have indicated that the cardiac crescent and early heart
tube only represent the outer curvature (apex) of the left
ventricle and parts of the AVC and atria, whereas the
remainder of the heart derives from cells added later.14,16 –18
The initial embryonic heart tube myocardium possesses a
phenotype that resembles that of the nodal tissues in displaying automaticity, poor contraction, and slow transmission
of the depolarizing impulse.19 –21 Sarcomeres and sarcoplasmic reticulum are not well-developed in this primary
myocardium.19,20,22 Caudal pacemaker activity in this slowconducting heart tube results in sluggish, unidirectional
peristaltic contractions that are reflected in a sinusoidal ECG.
During further elongation of the heart tube, the developing
ventricular and atrial chambers acquire a working myocardial
phenotype and rapidly expand by highly increased levels of
proliferation (Figure 1A).20,23 The underlying molecular programs involve upregulation of genes for high conductance
gap junctions that contribute to the rapid transmission of the
electric impulse, of mitochondrial genes associated with the
increase in mitochondrial number and activity, and of genes
for sarcomere components.19,20,24
Cardiac regions including the sinus venosus, the AVC,
inner curvatures, and the outflow tract do not differentiate
into chamber myocardium, retain low proliferation rates and
will consequently form constrictions.19,20 Dominant pacemaker activity remains localized at the intake, the sinus
venosus.25 The AVC retains its embryonic mode of conduction, which is much slower that that of the newly formed
chambers. Thus, the impulse between the rapidly propagating
atrial and ventricular chambers is effectively delayed.19,21
Concomitant with chamber formation, the initially peristaltic
contraction mode of the embryonic heart tube is replaced by
a pattern of serial and rapid contractions of the atrial and
ventricular compartment(s), and the derived ECG starts to
resemble that of a mature heart (Figure 1A).26 This basic
configuration of alternating slow-conducting and poorly contracting pacemaker components (sinus venosus, AVC, out-
flow tract) and fast-conducting myocardial components (atria
and ventricles) can be found in embryos and adults of all
vertebrates with multi-chambered hearts, including human
(Figure 1B).20 Higher vertebrates, such as mammals, will
develop a morphologically distinctive SAN in the sinus
venosus, an AVN within the AVC, and an AV bundle and
ventricular conduction network to efficiently and coordinately activate the ventricles. The structural differentiation of
these components varies widely between species. They are
hardly present or not discernable in fish, they are very
well-developed in large mammals, and in the mouse they are
in-between. Thus, species-specific morphological adaptations
are likely to have arisen during evolution.
The Sinus Node and Its Markers
The mature SAN is located in the intercaval region at the
entrance of the right atrium.6 The SAN has an elongated
structure described as “comma-shaped” with a “head” at the
superior caval vein-atrial border and a “tail” along the
terminal crest.6,27 However, the shape, position and composition of the SAN varies considerably between species.6,9 The
SAN core is surrounded by an area that displays a myocardial
phenotype and expression profile approximately in-between
that of the working atria and the SAN,6,28 and an outer ring of
connective tissue and arteries.9,22 This SAN periphery plays a
role in the functional protection of the SAN from the resistive
load of the atrium, allowing the SAN to activate the atrium
through narrow superior and inferior SAN exit pathways.29
Classically, the SAN has been delineated using histological
properties associated with nodal myocytes, including pale glycogen rich cells, poorly developed myofibrils, connective tissue,
acetylcholine esterase activity, and HNK-1 expression.1,9 In
addition, biosynthetic enzymes for acetylcholine and catecholamines are transiently expressed in the embryonic and fetal SAN
and other parts of the conduction system,30 providing useful
immunohistochemical markers for this tissue.
More recently, a panel of SAN-specific molecular markers
has been established that relate to the distinct electrophysiological properties of this tissue (Figure 2).1,6,10,27,28,31–35 Pacemaker activity requires a relatively high intercellular resistance (low conductivity) to protect it from the suppressing
hyperpolarizing influence of the atrium.1 Because gap junctions play an important role in the intercellular conduction
velocities, it is not surprising that the SAN expresses a set of
Cx45, Cx30.2, and Cx30 subunits32,36 that form gap junction
channels with very low conductance. In contrast, the adjacent
fast-conducting atrial working myocardium mainly expresses
Cx40 and Cx43 subunits that assemble high-conductance gap
junction channels.6,28,32,35 Furthermore, Scn5a, encoding
Nav1.5, the main sodium channel responsible for rapid
depolarization and conduction, is low in the SAN compared
to the adjacent atrial myocytes.10,34 Thus, the Cx30.2/Cx45high, Cx40/Cx43/Scn5a-low gene expression profile provides
a useful set of markers for both fetal and adult SAN tissue
(Figure 2A). Hcn4 is a member of the family of channels
responsible for the hyperpolarization-activated current, If,
that plays a key role in the pacemaker potential of the
SAN.1,37 Hcn4 is enriched in pacemaker tissues, including the
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Figure 2. Molecular pathways regulating
sinus node development. A, Serial section in situ hybridization and immunohistochemistry reveal that Cx40 and Nav1.5
are ectopically expressed in the Cre⫹
and Hcn4⫹ SAN domain, respectively, of
Tbx3 mutants. Note complementary
expression in wild-type hearts. B, Model
for SAN and sinus horn formation in the
right wall of the venous pole. Yellow
shows mesenchymal precursors; gray,
sinus horn myocardium; red, nodal myocardium; light blue, primitive myocardium; and dark blue, atrial “chamber-type”
myocardium. Depicted transcription factors regulate formation of boundaries
and zones of expression and differentiation of atrium, SAN and cardinal vein. C,
Scheme depicting roles and genetic
interactions of factors involved in sinus
venosus and SAN formation. raw indicates right atrial wall; rvv, right venous
valve; scv, superior caval vein; WM,
working myocardium.
SAN,6,37–39 and serves as an excellent positive nodal marker
in the mature heart. The gene for atrial natriuretic peptide,
Nppa, is expressed in atrial myocytes, not in the SAN or other
nodal cells, providing a complementary negative marker. In
rabbit, neurofilament is frequently used to mark the nodal
tissues including SAN.10 Finally, the T-box transcription
factor Tbx3 is specifically expressed in the developing and
the mature conduction system, including the SAN, of mammals and chicken.39,40 Further information on structure, function, composition and “molecular architecture” of the mature
SAN can be found in some excellent reviews.1,6,9
E10 (Figures 2B and 3A).39 This initial Tbx3⫹ thickening in
the sinus venosus most likely represents the primordium of
the SAN extension along the right venous valve, the
“tail.”27,39 This SAN primordium continues to grow by
proliferation and myocardial differentiation of mesenchymal
progenitors (see below) to form a “head” component that will
extend into the right sinus horn. The SAN and the venous side
of the bilayered venous valves express Tbx3 and Hcn4, whereas
the atrium and the atrial aspect of the venous valves express
Cx40 and Nppa in a complementary manner (Figure 4A).27,39
Formation of the Sinus Venosus and SAN
Until E9 to E9.5, the homeobox transcription factor Nkx2.5 is
expressed in all myocardium derived from the first and second
heart field but not in mesenchymal cells at the caudal-ventrallateral side of the inflow tract.44 These cells express the T-box
transcription factor Tbx18 in a strictly complementary pattern.27,44 Intriguingly, the myocardium of the sinus venosus and
SAN, which differentiates after E9 to E9.5, expresses Tbx18
but not Nkx2.5, suggesting that the sinus venosus and SAN
myocardium differentiates de novo from Tbx18⫹/Nkx2.5negative mesenchymal precursors (Figures 2B and 3A).
Indeed, genetic lineage analyses (Nkx2.5⫹/Cre;R26RlacZ and
Tbx18⫹,Cre;R26RlacZ) provided strong evidence for the origin
of the entire sinus venosus from these noncardiac progenitors
(Figure 4A and 4B).27,44 When isolated and cultured, the
Tbx18⫹ noncardiac precursors differentiated into Hcn4⫹/
Nkx2.5-negative pacemaker myocardium that was beating
much faster than cultured age-matched embryonic ventricular
myocardium.27 Taking into account that these cells are
precursors of the sinus venosus, this finding is consistent with
Cellular Origin of the Sinus Venosus and SAN
The sinus venosus comprises the SAN, the venous side of the
bilayered venous valves and the right and left sinus horns
(Figures 2B and 3). Based on histological sections, a SAN
primordium was observed at embryonic day (E)10.5 in
mouse.41 Virágh and Challice described that sinus muscle
cells, including the SAN primordium, form from loose
mesenchymal cells of the pericardial wall from E11.5.42 From
that stage onward, a nodal structure can be easily observed in
the right sinus horn at the junction with the atrium. In the left
sinus horn of the mouse41,42 and human heart, the transient
development of a small SAN was noted (A Sizarov, AFM
Moorman, unpublished observations, 2009). This structure
probably degenerates, but may develop to a full SAN in case
of atrial right isomerism,43 a process that is repressed by the
transcription factor Pitx2c (see below).39
Recent molecular analyses revealed that a Tbx3⫹ SAN
primordium develops within the right side of the Hcn4⫹/
Cx40-negative sinus venosus myocardium as early as E9.5 to
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Figure 3. Development of the sinus
venosus and sinus node. A, Schematic
representation of the patterns of expression and lineages until E9.5 (left),
between E9.5 and E14.5 (middle) and
after E14.5 (right). Thin dotted line represents the border of the Pitx2c expression domain; heavy dotted line, the border of the Tbx18⫹ lineage. Except for
the yellow-colored mesenchyme, only
myocardium is depicted (see legend
beneath illustrations). B, Same as in A,
with the shown (left and right) and probable (middle) region where dominant
pacemaker activity resides. Note the
developmental shifts of activity. as indicates atrial septum; laa, left atrial
appendage; pv, pulmonary vein; raa,
right atrial appendage; vv, venous
valves. cs, coronary sinus; tc, terminal
crest. For additional abbreviations, refer
to the Non-standard Abbreviations and
Acronyms box.
the much older observation that the elongating heart tube
shows an increase in beat rate.45,46 Furthermore, Tbx18deficient mice fail to form the large head component of the
SAN (Figure 4C), demonstrating that Tbx18 is required for
the recruitment of these mesenchymal precursors to the
cardiac lineage.27,44 Myocardial differentiation of the sinus
venosus and SAN occurs between E9.5 and approximately
E11 or later in mouse. After this period, the sinus venosus and
SAN continuously gain size by slow proliferation.27,47 The
Tbx18⫹ sinus horn precursors do not express the transcription
factor Isl1, an operational marker of second heart field cells,
before their differentiation.44 However, both the precursors of
the SAN and the SAN itself coexpress Isl1 and Tbx18,
indicating that the SAN progenitors have features of both
second heart field (Isl1⫹) and sinus venosus progenitors
(Tbx18⫹).39,44,48
SAN growth may me mediated by recruitment of surrounding
myocardial cells that acquire the SAN phenotype (Figure 4D).
However, when emerging atrial cells (Nppa⫹) directly adjacent
to the developing SAN (Tbx3⫹) were labeled, the SAN remained
unlabeled (Figure 4E). This observation suggests rather that
SAN growth is achieved by proliferation of a subpopulation of
specified SAN primordial cells. In turn, daughters of Tbx18⫹
sinus horn and SAN cells, which can be traced from approximately E8.5 to E9 onward in a Tbx18⫹/Cre;R26RlacZ mouse, did
not contribute to the atria (Figure 4B).27 Therefore, atrial and
SAN lineages segregate at least as soon as they initiate the
expression of these atrial and SAN markers.
Tbx3 Transcriptional Repression and
SAN Development
Mature SAN cells resemble embryonic myocytes,9,19,20,22,42
contrasting the phenotype of atrial working myocytes with
their well-developed contractile apparatus, as recognized as
early as in 1907 by Keith and Flack,49 who described the SAN
cells as “palely stained undifferentiated fibers.” It is thus
conceivable that prospective SAN cells are actively inhibited
from differentiation into working myocardium. Indeed, the
transcriptional repressor Tbx3 has recently emerged as the
molecular executor of this developmental restriction. Tbx3 is
expressed selectively in the SAN primordium from E9.5 to
E10 onward in the mouse.39,40 The SAN expression of Tbx3
is conserved in human and chicken. Tbx3 deficiency results in
loss of Lbh expression and expansion of working myocardial
gene expression (Cx40, Cx43, Nppa, Scn5a) into the SAN
domain (Figure 2A).34 In contrast, conditional atrial misexpression of Tbx3 in mice led to repression of working
myocardium genes, and activation of SAN genes (eg, Hcn4,
Cx30.2, Cav3.1) but did not change the expression of panspecific myocardial genes.34 Gene expression profiling identified another 1500 transcripts representing direct or indirect
targets of Tbx3 in the atria (WM Hoogaars, VM Christoffels,
unpublished observations, 2009). Intriguingly, forced atrial expression of Tbx3 led to the ectopic development of functional
pacemaker tissue identifying Tbx3 as a key regulator of the SAN
phenotype (Figure 2C).34
Molecular Mechanisms of SAN Development
Because of the potential of Tbx3 to confer the pacemaker
gene program, restriction of Tbx3 expression to the developing SAN is mandatory. The expression of Hcn4 becomes
restricted to the sinus venosus as well. A Shox2-Nkx2.5
repressor pathway was shown to regulate this process (Figure
2B and 2C). Hcn4 is initially expressed in the caudal end of
the Nkx2.5⫹ heart tube (Figure 3A).37,38 It is subsequently
downregulated in this domain, and activated in newly differentiated Tbx18⫹/Nkx2.5-negative myocardial cells of the
sinus horns, effectively shifting its expression domain into the
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Figure 4. SAN develops from nkx2-5⫺, Nppa⫺, and
Tbx18⫹ precursor cells. A, E12.5 nkx2-5⫹/Ires-Cre;
R26RlacZ embryo stained with X-gal and the corresponding area stained for Cx40 (atria) and Tbx3
(SAN). B, Serial section in situ hybridization of an
E18.5 Tbx18⫹/Cre;R26RlacZ/lacZ fetus stained as
indicated. Black arrowhead indicates the Tbx18⫹
SAN head, the white arrowhead depicts the
Tbx18⫺ SAN tail. Red arrowheads indicate atrial
myocardium free of lacZ⫹ cells. C, Threedimensional reconstructions of the lumen of the
right superior caval vein (rscv) (yellow) and the
Tbx3-positive SAN head and tail (blue) of E12.5
wild-type and Tbx18GFP/GFP embryos from serial
section in situ hybridization analysis for Tbx3. D,
Models depicting specification versus recruitment.
E, The adult SAN (Hcn4⫹) does not receive contributions from the Nppa⫹ atrial lineage (GFP⫹).
NppaCre labels the atrial lineage as early as E10
and robustly at E12.5. For additional abbreviations,
refer to the Non-standard Abbreviations and Acronyms box.
newly added sinus venosus (Figure 3A).39 Because Tbx3 is
activated in the Nkx2.5-negative SAN primordium within the
sinus venosus as well,39 Nkx2.5 may repress these genes,
whereas it is required to activate Cx40, Nppa, and many other
genes associated with atrial myocardial differentiation.50
Indeed, Nkx2.5-deficient embryos show ectopic expression of
Hcn4 and Tbx3 in the heart tube,39 and fail to activate Cx40,51
suggesting that Nkx2.5 represses Hcn4 and Tbx3, thereby
confining their expression to the forming Nkx2.5-negative
sinus venosus (Figure 3). Intriguingly, in Nkx2.5-deficient
embryos, pacemaker activity originated from the early ventricular region, indicating derepression of a functional pacemaker program in the heart tube.51
Analyses of the Shox2 gene in mouse and frog have
pointed to a role for the paired-related homeodomain transcription factor as a negative upstream regulator of Nkx2.5
expression in the sinus venosus (Figure 2B and 2C).52,53
Shox2-deficient mice have a hypoplastic sinus venosus and
show upregulation of Nkx2.5, Cx40, and Cx43 and downregu-
lation of Hcn4 and Tbx3 in the presumptive SAN primordium. Shox2-deficient mouse and zebrafish embryos display
bradycardia, consistent with the ectopic expression of Nkx2.5
in the sinus venosus, subsequent loss of the pacemaker
program, and ectopic activation of the chamber myocardium
program.52,53
The T-box transcription factor gene Tbx5 is expressed in a
caudal-high gradient in the developing heart and is crucial for
the formation of the early inflow/atrial region54 and for the
expression of Shox2 and Tbx3 (Figure 2C).55 Tbx5 expression
in all atrial and venous myocardium (or the formation of the
Tbx5-positive cell population therein) is controlled by retinoic acid signaling,56 indicating that a retinoic acid-Tbx5 axis
activates, rather than spatially restricts, Shox2 and Tbx3
expression in the SAN region. The transcription factor Pitx2
is known to control asymmetrical morphogenesis of the
heart.57 In Pitx2c-deficient fetuses, SANs form at both the
right and left sinuatrial junction. These SANs have indistinguishable molecular signatures, including Tbx3 expression,
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suggesting that Pitx2c functions within the left/right pathway
to suppress a default program for SAN formation in the left
sinus venosus (Figure 2B).39
Evolutionary Conservation of Sinus
Venosus Development
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Given the functional importance of pacemaker activity, it is
not surprising that the molecular programs underlying sinus
venosus and SAN formation appear highly conserved. In
Drosophila, cardioblasts fated to differentiate into working
myocytes express tinman, the Drosophila homolog of Nkx2.5,
whereas the 6 pairs of cardioblasts that surround the inflow
ostia, resembling the vertebrate sinus venosus, do not express
tinman but the dorsocross T-box factor homologs instead.58
Inactivation of tinman results in ectopic expression of dorsocross, and loss of expression of Sur, encoding a K⫹ channel
subunit, in tinman-positive cardioblasts. Tinman itself is
repressed in ostia precursors by seven-up (svp), the homolog
of the orphan nuclear receptor Nr2f2 (Coup-TF2), which in
mouse is selectively expressed in the early inflow tract.59
Similar to the mouse, the caudal (inflow) pole of the
zebrafish heart tube is formed by differentiation of isl1⫹
cells.60 Loss of isl1 results in failure to differentiate and
lengthen the inflow pole from progenitors and to increase
beat rates during development (relative bradycardia). The
formation of the definitive sinus venosus was not addressed
in this study. However, expression of Tbx18 in the sinus
venosus is conserved in zebrafish,61 Xenopus,62 chicken,63
mouse,64 and human (A Sizarov, VM Christoffels, AFM
Moorman, unpublished observations, 2009), suggesting the
existence of a Tbx18-controlled module for sinus venosus
development in vertebrates.
Confinement of Pacemaker Activity to the
Sinus Venosus and SAN
Whereas pacemaker activity is tightly associated with the
SAN in the adult heart, pacemaker activity initiates more
broadly and is only gradually confined to the developing
SAN. First pacemaker activity can be recorded before the
onset of contractions of the just-differentiated myocardium in
the embryo.25,65 In the initial heart tube the myocytes at the
caudal end, the intake, possess the fastest cycle, dictating the
beat rate.25,46 Pacemaker activity then shifts to the left side
of the inflow tract,25,65 for reasons yet unknown. With
myocytes added to the inflow tract, the sinus venosus will
form (Figure 3).44 Notably, during the lengthening of the
heart tube, dominant pacemaker activity remains associated
with the caudal end,25 implying that pacemaker activity
continuously shifts to the cardiac cells most recently differentiated from the progenitor pool at the systemic venous side
(Figure 3B). Indeed, once formed, the sinus venosus (sinus
horns and SAN primordium) acts as pacemaker in the embryo
and early fetus,25 consistent with the expression of Hcn4 and
repression of working myocardial “high conductance” genes
Cx40, Cx43, and Scn5a at this site. The mechanism underlying the shift of dominant pacemaker activity to the newly
added sinus venosus cells may involve the repressive activity
of Nkx2.5 as discussed above.
At some as yet undefined prenatal stage, pacemaker
activity becomes confined to the actual SAN structure (Figure
3B). The reason for this is unclear, but it is conceivable that
the sinus horn myocardium, with the exception of the SAN
itself, matures to obtain a phenotype comparable to the atrial
working myocardium.39,42 This “atrialization” of sinus horns
is confirmed by characteristic shifts of marker gene expression. The sinus horns activate Cx43 and Scn5a and, after
approximately E12, also Cx40.39 Expression of these genes
continues to be low or absent in the SAN, which maintains
the expression of Cx45 and Cx30.2. Moreover, the expression
of Hcn4 in the entire sinus venosus becomes restricted to the
SAN during fetal stages.38,39
Sites of Ectopic Pacemaker Foci and
Arrhythmias: Remnants of
Pacemaker Development?
Although the sinus venosus acquires atrial properties and
looses pacemaker activity, the extent to which this occurs is
unknown. It is possible that atrialization of sinus venosusderived structures (superior caval vein [right sinus horn],
crista terminalis [right venous valve], coronary sinus ostium
[atrial entrance of the left sinus horn], and ligament of
Marshall [remnant of the left sinus horn]) does not occur
completely in some individuals. As a result, pacemaker or
focal automatic activity may persist and give rise to nonpulmonary vein paroxysmal atrial fibrillations.66,67
HNK-1/Leu-7 immunohistochemistry revealed several
strands of internodal tracts between the SAN and AVN in the
dorsal atrial wall,68,69 the function of which have not been
defined. These tracts express Tbx3, CCS-lacZ and other
conduction system markers (Figure 5A),40,70 suggesting they
may have a nodal phenotype. These tracts may be remnants of
the original Tbx3⫹/Cx40-negative nodal cell at the connection
of the sinus venosus and atrium that connects the SAN and
AVN. When SAN and AVN separate during expansion of the
venous region, the intervening tissue forms strands between
the venous entrance and the dorsal atrial septum (Figure 5A).
Pulmonary vein myocardium represents another major site
of ectopic foci that initiate paroxysmal atrial fibrillation, and
its formation has been discussed in the context of conduction
system development. However, the pulmonary myocardium
forms independently from the sinus venosus, and displays an
atrial working myocardial phenotype and gene program from
the outset (eg, Nkx2.5⫹/Cx40⫹ and Tbx18/Hcn4-negative).
Hence, the mechanisms underlying arrhythmias from the
pulmonary myocardium are, in all likelihood, different from
those underlying arrhythmias from the residual nodal tissues
of the sinus venosus and AV junction.
The AVN and Its Markers
The second major site in the heart with potential pacemaker
activity is the AV junction, which includes the AVN. The
adult AVN is a complex and heterogeneous structure, consisting of multiple components and different cell types with
Christoffels et al
Pacemaker and Conduction System Development
247
Figure 5. Tbx3 expression delineates the conduction system. A, Reconstruction of the Tbx3 expression domain (red) delineates the san, AVC, AVN,
internodal tracts, AVB, and BBs. The myocardium
has been removed revealing the lumen of the
chambers. The san and AVC/AVN express Tbx3
throughout development. B, Tbx3BAC-Egfp
expression in the E9.5 AVC is progressively
restricted to the AVN, right atrioventricular ring
bundle (ravrb), and retroaortic AVN (ran) derived
from the AVC. Stages shown are E11.5, E17.5,
and 3 weeks after birth.
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distinctive gene expression profiles.1,8,71 The region of AV
nodal–like myocytes extends from the AVN to encircle the
vestibule of the tricuspid valve (right AV ring bundle) and the
mitral valve (left AV ring) to contact anteriorly and form
what has been called a “retroartic node” (Figure 5B).5,40
Similar to SAN cells, AVN and AV junction cells are smaller
and more primitive (poorly developed myofibrils and sarcoplasmic reticulum, lack of complex intercalated discs, high
glycogen content, lower mitochondrial content, etc) than
working myocytes.19 Their expression profile is in agreement
with their pacemaking and conduction-slowing properties,
Cx40/Cx43/Scn5a-negative and Cx45/Cx30.2(mouse)/Hcn4/
Tbx3-positive.8,32,40,72–74 In addition, several transgenes have
been identified that mark the AVN and junction, including
minK-lacZ, cGata6-lacZ, CCS-lacZ, cTnI-promoter-lacZ, and
BAC(Tbx3-GFP) (Figure 5).17,24,75–77 Collectively, these genetic markers can be used to distinguish the AV nodal
myocardium from the surrounding working myocardium. The
AVN/junction has distinctive subdomains, which may express the markers to a different extent.8,71,78 Furthermore, the
frequently used marker Cx45 seems to be AVN restricted
only at the protein level, as Cx45 mRNA has been found at
similar levels in nodal and working myocardium.33 The AVN
is connected to the fast-conducting (Cx40/Scn5a-positive)
proximal AVB.32,74,79
Formation of the AVN and AV Junction
Myocardium From the Embryonic AVC
Functional and morphological studies initially suggested de
novo formation of the AVN and proximal AVB in the lower part
of the atrial septum,80 but derivation from the left sinus horn
(coronary sinus), from the “AV ring,” AVC, or from multiple
tissues has also been discussed (reviewed elsewhere78). Current
studies, however, support the view that the embryonic AVC
contains the precursors of the AVN, of the AV ring bundle(s), of
the support of the AV valves, and of the lower rim of the atrium
(Figure 5).17,18,78,81,82,82 In general, markers robustly expressed in
the embryonic AVC become restricted to the AVN, AV valvesencircling myocardium of the AV junction and anterior node.
After birth, the expression domains of minK-lacZ, Tbx3 and
BAC(Tbx3GFP) become further restricted to the AVN and small
parts of the right AV junction only (Figure 5B).5,40,83 Together,
these molecular findings argue that the mature AVN and nodal
AV junction myocardium are derivatives, or remnants, of the
prenatal AVC myocardium.13,82
The initial heart tube consists of precursors of the left
ventricle and most of the AVC.14,17,18 LacZ expression driven
by an AVC-specific chicken Gata6-enhancer at early stages
marks the limbs of the cardiac crescent.17 Similarly, Tbx2 is
expressed in the limbs of the cardiac crescent (E7.5), inflow
tract (E8), and, subsequently, in the early AVC (E9).18
Classic studies and Tbx2Cre-mediated fate mapping indicated
a lineage relation between these embryonic tissues,16,18 suggesting that the early AVC itself is largely derived from the
cardiac crescent, which is derived from the first heart field. In
the embryo, the electric impulse traverses the myocardium of
the AVC on its way from the atria to the ventricles,19,21,84,85
with the dorsal AVC region being the preferential site of
conduction.84,85 In agreement, AVN primordial cells (high
glycogen content) are observed in the dorsal AVC as early as
E9.5, shortly after chamber differentiation (Figure 6A).78,81,86
They form a (Tbx3⫹) inner layer that faces the dorsal AV
cushion and extends deeply into the left ventricle, where it
interconnects with both the (Cx40⫹) trabeculae and the
emerging interventricular septum (Figure 6A through 6C).
The dorsal outer wall myocardium disappears, probably by
apoptosis. The ventral and lateral walls of the AVC harbor
loose spongy myocardium that becomes penetrated by connective tissue to form the annulus fibrosus. From E11 onward
until completion of septation, the AVN primordial cells at the
distal end proliferate and expand into the dorsal AV cushion
and form the definitive AVN node. The cells of the ridge of
the interventricular septum form the AV bundle and remain in
contact with the AVN (Figure 6D an 6E).86
Lineage Relations Between the AVC and the
Definitive AV Conduction System
Based on morphological studies, the AVN has been proposed
to forms in its entirety from a small subpopulation of
proliferating AVC cells.78 However, cellular contributions
from outside the AVC could not be excluded. Retrospective
clonal analyses in chicken represented the first lineage
analyses of the AV conduction system.11,87 These studies
settled the debate regarding a neurogenic versus cardiac
248
Circulation Research
February 5, 2010
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Figure 6. Development of the AVN and AV bundle.
A, Schematic drawing of a sagittal section of an
E10 mouse heart, modified from Virágh and Challice,81 showing the dorsal AVC region and the AV
node primordium (green arrow), “necrotic AV cells”
(light green arrow), and connection between the
AV nodal cells and the trabeculae/crest of interventricular septum (red arrow). B, Sagittal sections
of an E9.5 mouse heart of the same area as in A,
showing expression of Tbx3 in the AV nodal cells
and necrotic AV cells and expression of Cx40 in
the ventricular myocardium connected to the AV
nodal cells. C, An overview of the mouse heart,
the boxed region is enlarged in B. D, Drawing of
sagittal section of an E12 mouse heart modified
from Virágh and Challice,86 showing the AVC components and connected AVB/interventricular septum crest. E, Right-sided view of a 3D reconstruction of the Tbx3 pattern at this stage. All other
components have been removed, except for the
lumen of the left ventricle. Colored arrows point to
corresponding structures in D and E. ec indicates
endocardial cushion; ivf, interventricular foramen;
ivs, interventricular septum; ofc, outflow tract
cushion; tr, trabeculae. For additional abbreviations, refer to the Non-standard Abbreviations and
Acronyms box.
origin of the conduction system components by showing that
conduction system myocytes share a common origin with
other myocytes.11,87 In these studies, rare single cell– derived
clones in the AV conduction system, including the right AV
ring bundle, were found to always extend into the adjacent
working myocardium.87 The myocardium of the forming
conduction system proliferates much less compared to the
adjacent chamber myocytes.23,47,87 These data can be rationalized in 2 models. First, the AV conduction system components grow by ongoing accretional recruitment of developing working myocytes to an initial framework represented by
the early AVC,11 a process that continues until the end of
septation. Alternatively, the early AVC may not only form
the AV conduction system components, but may additionally
provide cells to the adjacent chambers. This model has
recently gained support by a genetic lineage tracing experiment: mouse AVC cells labeled between E8 and E9.5 by
Tbx2-driven Cre, corresponding to the early AVC, give rise
to the definitive AVN and AV ring bundles, but they also
provide an extensive contribution to the adjacent working
myocardium.18 Because AV myocardial cells do proliferate
significantly,18,47 their increase in number is likely to be
sufficient to generate cells of both the AV conduction system
components and the developing working myocardium.
The AVB is formed from the crest of the developing
ventricular septum, the left and right BBs develop from the
subendocardial myocytes along the ventricular septum.86 In
contrast to the AVN, the AVB and BB are not derived from
the Tbx2⫹ AVC, indicating that the precursors of these AV
conduction system components segregate very early in development (mouse E8) and have been exposed to distinctive
regulatory signals.18
Differentiation and Maturation of the AVN
and Junction
Although gene expression and cell lineage studies have
uncovered that the AVN develops from embryonic myocardium within the AVC, the molecular programs that drive the
localized formation of a structurally complex AVN have
remained elusive. Because the developing AVN and AV ring
bundles always face the cushion mesenchyme,82,86 signals
from the cushion mesenchyme may mediate or participate in
the maintenance and further specialization of the pacemaker
phenotype. Support for this hypothesis comes from zebrafish
studies that showed simultaneous ectopic induction of AVC
myocardium and cushion formation in Apc mutants88 and loss
of AVC in cloche mutants that do not develop endocardium.89
Furthermore, immigrating epicardial and neural crest cells
may influence the maturation of these components.70 The
expression of minK-lacZ, Tbx3, Tbx3GFP and other markers
in the embryonic AVC becomes restricted to the AVN and
small portion of the AV junction (Figure 5B).24,40,83 This
finding indicates disappearance of AVC myocardium by
apoptosis81 or by differentiation into working myocardium.18
Ectopic beats may originate from the remaining AV junction
myocardium, an Hcn4⫹/Tbx3⫹/Cx43-negative nodal tissue,
around the tricuspid and mitral valve.2–5,40 Other than that, not
much is known regarding regulatory mechanisms of postnatal
maintenance or degree of interindividual variations in the
extent of these tissues. During development, the conduction
velocity rapidly increases in the chambers, but also changes
in the AVC, implying further specialization of the AVC
phenotype.21,89 We recently performed genome-wide expression analyses of the developing AV nodal and working
myocardium.24 The profiles were compatible with a more
embryonic, less working muscle–typical phenotype of the
Christoffels et al
AVC and AVN. Moreover, AV nodal cells express an
extensive neurogenic gene program. Cross-comparison of the
different data sets revealed that the majority of differentially
expressed transcripts in the E10.5 embryonic AVC maintained differential expression in the E17.5 late fetal AVN.
These data indicate maintenance of properties during AVN
development. However, the profile of E17.5 AVN substantially differs from that of the E10.5 AVC, revealing that the
AVN substantially specializes during development.
Atrioventricular Insulation and
Accessory Pathways
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Soon after the establishment of the AVC, connective tissue
from the AV cushion and subepicardial mesenchyme starts to
invade the AV myocardium to form the annulus fibrosus,
which physically separates and insulates atria from ventricles.
The AVB remains as only connection between the AVN at
the atrial side of the fibrous body with the ventricles.
However, this process of insulation is gradual and often not
completed before birth. AV reentrant tachycardias are caused
by the presence of abnormal accessory myocardial bundles
connecting the atrial and ventricular myocardium.90 Usually,
these bundles conduct rapidly, such as the Kent bundles in
Wolff–Parkinson–White syndrome, suggesting they are of a
working myocardial phenotype. Accessory bundles with AVnodal properties (slow), as found in Mahaim tachycardia, are
rare and usually occur at the tricuspid side. These bundles
probably derive from the remnants of prenatal AVC myocardium, which is more extensive around the tricuspid valve
(Figure 5B).82,91
The formation of accessory bundles probably involves
abnormalities in both the formation of the fibrous insulation
and the differentiation of the original slow-conducting AVC
myocardium to fast-conducting working myocardium. Mutations in PRKAG2 have been associated with ventricular
preexcitation (Wolff–Parkinson–White syndrome). Expression of a mutant isoform leads to disruption of the fibrous
insulation and direct contact between the atrial and ventricular myocardium, allowing the electric signal to by-pass
the AVN-AVB connection.7 Furthermore, inhibition of
epicardium formation results in disturbed formation of the
fibrous insulation and prenatal ventricular preexcitation.92
These data provide evidence for the importance of the
fibrous insulation to prevent peri- and postnatal ventricular
preexcitation arrhythmias.
Regulatory DNA Sequences Reveal
AVC-Specific Transcriptional Modules
Analysis of the molecular pathways involved in AVC specification and formation has greatly benefited from the dissection of regulatory DNA elements conferring AVC-restricted
patterns of activity, including the chicken Gata6-enhancer, a
cardiac troponin I--promoter, an Nppa regulatory module, a
Cx30.2-enhancer, and a large genomic fragment of
Tbx3.17,24,75,93–95 Cardiac troponin I and Gata6 are not specifically expressed in the AVC, indicating that these genes
have multiple regulatory modules that define their complete
Pacemaker and Conduction System Development
249
pattern. In contrast to Tbx3 itself, the Tbx3 genomic fragment
was not active in the AVB (Figure 5B), showing that gene
expression in the AVC and the AVB depends on distinct
regulatory sequences and pathways.
The activity of the chicken Gata6-enhancer and Cx30.2enhancer depends on GATA elements to which Gata4 (and
other Gata family members) binds. The Cx30.2-enhancer in
addition requires binding sites for T-box genes.93,95 The
Cx30.2-enhancer and Cx30.2 itself are less active in
Gata4⫹/⫺ and Tbx5⫹/⫺ embryos. Moreover, Gata4⫹/⫺ mice
display shortened P-R intervals, consistent with Cx30.2
downregulation.32,95 Together, these data indicate Tbx5 and
Gata4 are responsible for Cx30.2 activity in the AVC. Tbx5
and Gata4 are expressed in both the AVC and working
myocardium,24,95 suggesting the existence of additional cofactors for AVC-restricted activity of Cx30.2 and the chicken
Gata6-enhancer.
Nppa expression marks the differentiating chambers, and
has been a very useful negative marker for the AVC.94 The
proximal Nppa promoter contains a small regulatory module
that is activated cooperatively by Tbx5 and Nkx2.5,54 and that
is active in the chambers but inactive in the AVC in vivo.94
Unexpectedly, mutation of either the T-box element or
Nkx2.5-binding site in the Nppa promoter resulted in loss of
repression in the AVC, whereas chamber activity was maintained.94 This study suggested that chamber-specific activity
is mediated by active transcriptional repression by a T-box
factor in the AVC.
T-Box Factors and Nkx2.5 in
AVC Development
Tbx2, a T-box factor that acts as a repressor, is selectively
expressed in all primary (nonchamber) myocardium, including the AVC.18,94,96 –98 Tbx2 competes with Tbx5 for binding
of T-box elements in Nppa and Cx40 promoters and for
interaction with Nkx2.5, thus repressing Nppa in the AVC.94
Subsequent gain- and loss-of-function studies showed that
Tbx2 is a general repressor of the chamber-specific gene
program in the AVC (Figure 7).94,97–99 In zebrafish, tbx2b
expression is restricted to the AVC. Morpholino knockdown
of this gene resulted in loss of the AVC and of AV conduction
delay.100
The AVC also expresses Tbx3 (Figures 5A and 6B), which
is equivalent to Tbx2 in its ability to inhibit chamber
differentiation and gene expression.39,40,101 Tbx2-deficient
embryos develop a limited AVC defect,99 whereas Tbx3deficient embryos have a normal AVC and display cardiac
defects only at sites where Tbx2 is not expressed (SAN,
AVB).18,98,102 Simultaneous inactivation of both Tbx2 and
Tbx3 results in loss of the AVC phenotype (VM Christoffels,
A Kispert, unpublished observations, 2009), confirming functional redundancy of the 2 T-box genes in AVC development.
Msx2 (Drosophila muscle segment homeobox related)
expression marks the AVC in chicken and mouse.103 Its
function and ability to form a complex with Tbx2 and Tbx3
that represses Cx43 expression render Msx2 a possible
component of the regulatory pathway controlling AVC for-
250
Circulation Research
February 5, 2010
Figure 7. Molecular pathways regulating the
development and boundary establishment of the
AVC and chamber myocardium.
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mation.104,105 Nevertheless, mice that are deficient in Msx2 do
not display conduction defects.106
Nkx2.5 and Tbx5 are broadly expressed in the embryonic
heart, with Tbx5 displaying a more restricted caudocranial
graded pattern of expression that is maintained in the atria
and AV conduction system.54,83,107 Both factors are crucial
activators of chamber-specific genes and essential for chamber differentiation.54,107 Nkx2.5 and Tbx5 play important
roles in AV conduction system development.54,83,95,108,109
Dominant mutations in NKX2.5 and TBX5 (Holt–Oram syndrome) cause congenital heart defects and AV conduction
defects (AV block).110 –112 In Nkx2.5-haploinsufficient mice,
the Cx40-negative AVN and Cx40⫹ AVB and BBs are
hypoplastic,108,109 even though their development appeared
unaffected.102 Tbx5⫹/⫺ mice display postnatal failure in
maturation of the AV conduction system, AVB and BB
defects, and prenatal reduction of expression of Tbx3 and
Cx30.2 in the AVC.83,95 Transcriptional repressors Id2 and
Tbx3 suppress proliferation and ventricular differentiation of
the AVB and BBs.102,108 Nkx2.5 and Tbx5, in turn, are
required for the transcriptional activation of Id2 in the crest of
the ventricular septum.102 Furthermore, Tbx5 is possibly
required for the expression of Tbx3 in the AVB, and Tbx3
and Nkx2.5 interact physically.95,102,113 Taken together, a
small network of more broadly expressed activators and
spatially restricted repressors has been identified regulating patterning, differentiation, and maintenance of the AV
conduction system (Figure 7).
Bone Morphogenetic Protein Signaling
Controls Tbx2 Expression and AVC
Development: Implications for
Ventricular Preexcitation
AVC-restricted expression of Tbx2 (and possibly Tbx3) is
mediated by bone morphogenetic protein (Bmp) signaling
(Figure 7). Bmp2 that is expressed remarkably early and
specifically in the embryonic AVC, is required for AVC
specification and Tbx2 expression, and is sufficient to activate
Tbx2 and Tbx3.96,114 Tbx2 expression in the AVC in vivo
relies on a small Smad-dependent enhancer upstream of the
transcription start site.115 In the zebrafish heart, the transcription factor sli/foxn4 is required for AVC formation. Foxn4
and Tbx5 have been identified as activators of tbx2b expres-
sion that act through conserved T-box element and Foxn4
sites.100 In mouse, these sites are not required for Tbx2
enhancer activity in the AVC, suggesting that AVC restriction of Tbx2 has been achieved by different molecular
pathways in vertebrate evolution.115
Tbx20 is required for heart tube formation and chamber
development.50 Importantly, Tbx20 deficiency leads to widespread ectopic expression of Tbx2 in the entire heart tube. The
phenotype of Tbx20 mutants is reminiscent of that of embryos
in which Tbx2 is ectopically expressed in the entire primitive
heart tube,97 indicating that Tbx20 represses Tbx2. In addition
to this function, Tbx20 was found to be required for chamber
formation independently of Tbx2.115 Although Tbx20 may
repress Tbx2 directly,116 putative T-box elements conferring
repression were not required for Tbx20-mediated repression
in vivo. Instead, Tbx20 was found to interact with Smad1/5,
thereby interfering with Bmp-Smad activation of this enhancer,
thus providing an indirect mechanism to deactivate Tbx2 in the
early heart tube and confine its expression to the AVC.115
Notch-Hey signaling in the chambers delimits the Bmp2Tbx2 pathway, hence AVC formation.117,118 In forming
chamber myocardium of chicken, Serrate-Notch2 signaling
activates the expression of Hey1. Hey1 together with Hey2
then repress Bmp2. Bmp2-activated Tbx2 represses Hey1 and
Hey2 in the AVC, in turn, generating a feedback inhibition
loop that sharpens the chamber–AVC border.117 Similarly, in
mouse, ventricular Hey2 represses Bmp2 and Tbx2, whereas
atrial Hey1 represses Tbx2, thereby confining the expression
of Bmp2 and Tbx2 to the AVC.118 In contrast to the situation
in chicken, mouse Hey1 and Hey2 are not activated by
Notch2 and not repressed by Tbx2.118
Abrogation of BMP signaling in the AVC by cGata6-Cre–
mediated inactivation of Bmpr1a/Alk3, the type IA receptor
for BMP, resulted in the disruption of the fibrous insulation.
Myocardial AV connections expressing Cx43 developed that
were sufficient to cause preexcitation, and AVN morphology
was changed.119,120 BMP signaling in the AVC is essential for
the formation of AV cushions and the expression of Tbx2
(Figure 7).114 Because inactivation of Tbx2 causes the formation of abnormal accessory myocardial bundles and preexcitation (W Aanhaanen, B Boukens, VM Christoffels, unpublished observations, 2009), the BMP-Tbx2-pathway in the
AVC may play an important role in coordinating AV insula-
Christoffels et al
tion. Intriguingly, a 20p12.3 microdeletion that includes
BMP2 was found to predispose to Wolff–Parkinson–White
syndrome.121
Conclusions
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Until very recently, our understanding of the etiology of
cardiac arrhythmias has been compromised by the lack of
insight into the molecular and cellular mechanisms underlying development and function of the conduction system.
Insight into ion channel gene regulation was scarce, the
development of the tissue architecture, cellular composition,
and innervation of the nodes and bundle not well understood,
and the spatial and temporal integration of conduction system
development with that of the heart in toto is insufficiently
explained. Fortunately, this unsatisfying situation has started
to change by the identification of progenitor cell sources of
the SAN and AVN, the definition of their lineage contributions, and the revelation of key transcriptional circuits that
control specification and differentiation of the conduction
system components. We can now explain the “primitive”
phenotype of conduction system myocytes as a consequence
of local transcriptional repression of working myocardial
differentiation. These transcriptional pathways provide a link
between cellular differentiation, morphogenesis, and the regulation of expression of ion channels and gap junctions that
define the electrophysiological function of the mature tissues.
Finally, the important interaction between heart function and
cardiac developmental gene regulation has been uncovered.
Genome-wide analyses of tissue-specific transcription factor–
DNA interactions and their dependence of coregulatory
factors within the heart will hopefully bring us closer to
understanding the complexity of the regulatory mechanisms
underlying normal and aberrant development and function of
the cardiac conduction system.
Sources of Funding
This work was supported by grants from The Netherlands Heart
Foundation (2005B076 to V.M.C.); The Netherlands Organization
for Scientific Research (VIDI grant 864.05.006 to V.M.C.); the
European Community’s Sixth Framework Programme contract
(“HeartRepair” LSHM-CT-2005-018630 to V.M.C., A.F.M.M., and
A.K.); and the German Research Foundation (DFG) for the Cluster
of Excellence REBIRTH (from Regenerative Biology of Reconstructive Therapy) at Hannover Medical School (to A.K.).
Disclosures
None.
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Development of the Pacemaker Tissues of the Heart
Vincent M. Christoffels, Gertien J. Smits, Andreas Kispert and Antoon F. M. Moorman
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Circ Res. 2010;106:240-254
doi: 10.1161/CIRCRESAHA.109.205419
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