Download Development of the cardiac conduction system

Seminars in Cell & Developmental Biology 18 (2007) 90–100
Review
Development of the cardiac conduction system
Takashi Mikawa ∗ , Romulo Hurtado 1
University of California San Francisco, Cardiovascular Research Institute, Box 2711, Rock Hall Room 384D,
1550 4th Street, San Francisco, CA 94158-2324, United States
Available online 4 January 2007
Abstract
The cardiac conduction system (CCS) is a specialized tissue network that initiates and maintains a rhythmic heartbeat. The CCS consists of several
functional subcomponents responsible for producing a pacemaking impulse and distributing action potentials across the heart in a coordinated
manner. The formation of the distinct subcomponents of the CCS occurs within a precise temporal and spatial framework; thereby assuring that as
the system matures from a tubular to a complex chambered organ, a rhythmic heartbeat is always maintained. Therefore, a defect in differentiation
of any CCS component would lead to severe rhythm disturbances. Recent molecular, cell biological and physiological approaches have provided
fresh and unexpected perspectives of the relationships between cell fate, gene expression and differentiation of specialized function within the
developing myocardium. In particular, biomechanical forces created by the heartbeat itself have important roles in the inductive patterning and
functional integration of the developing conduction system. This new understanding of the cellular origin and molecular induction of CCS tissues
during embryogenesis may provide the foundation for tissue engineering, replacement and repair of these essential cardiac tissues in the future.
© 2007 Elsevier Ltd. All rights reserved.
Keywords: Pacemaker; Purkinje fibers; AV-Node; SA-Node; Connexin; Endothelin; Neuregulin
Contents
1.
2.
3.
4.
5.
6.
Functional subcomponents of the CCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. Pacemaker component . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2. Slow impulse conduction component . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3. Rapid impulse conduction components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Topological changes in the conduction pathway of pacemaking impulses during heart development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
The myogenic versus the neurogenic origin of the CCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
In-growth versus out-growth mechanism of CCS development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cellular, molecular and biophysical mechanisms of CCS differentiation and patterning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. SA-node development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. AV-node formation and maturation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3. Induction and patterning of fast conduction Purkinje fiber network: inductive interactions between cardiac endothelial cells
and myocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Functional subcomponents of the CCS
Abbreviations: AV, atrio-ventricle; CCS, cardiac conduction system; Cx,
connexin; ET, endothelin; If , pacemaking current; SA, sinoatrium
∗ Corresponding author. Tel.: +1 415 476 3230; fax: +1 415 476 3892.
E-mail address: [email protected] (T. Mikawa).
1 Present address: Department of Physiology and Biophysics, Cornell University Medical College, NY 10021, New York.
1084-9521/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.semcdb.2006.12.008
The vertebrate heart produces a coordinated contraction by
the precisely timed initiation and transmission of action potentials through the cardiac conduction system (CCS) [1]. The
CCS consists of several subcomponents, each of which plays a
T. Mikawa, R. Hurtado / Seminars in Cell & Developmental Biology 18 (2007) 90–100
distinct role in coordinating a rhythmic heartbeat (Fig. 1A). The
functional subcomponents of the CCS include: the sinoatrial
node (SA-node, also known as the pacemaker) which generates a
pacemaker impulse; the atrioventricular node (AV-node) which
delays the impulse, thus separating the contraction of atrial and
ventricular heart chambers; and the His–Purkinje system which
rapidly transmits the impulses to, and throughout, the ventricles.
Each subcomponent of the conduction system utilizes a distinct set of ion channels [2,3], channel-associated proteins [4],
connexins (Fig. 1B; [5–8]), cytoskeletal components [9,10],
and transcriptional regulators [11,12]. For example, conduction
cells express genes typical of both neural tissues and skeletal
muscle [13], and upregulate both Nkx2.5 and GATA4 [14–19].
Expression of other genes marks distinct subpopulations of the
conduction system. In particular, the transcription factors Msx2 and Tbx2/3 are expressed in the slow conducting AV-node,
but are absent from the fast conducting AV-bundle and Purkinje
fibers [20,21]. In the mouse, several promoters and enhancers
mark conduction cells [13]: mink-nlacZ in the proximal ventricular conduction cells [4], GATA6-lacZ in the AV-node [11], and
Engrailed2-lacZ in both atrial and ventricular conduction cells
[22]. This section provides a brief overview on characteristics
of each subcomponent.
1.1. Pacemaker component
Pacemaking impulses are generated at the SA-node, which is
a heterogeneous tissue embedded in the right atrial wall. While
all heart muscle cells can rhythmically beat without external
stimulus, the cells of the SA-node are those with the most rapid
intrinsic rate of excitation and they set the beating rhythm of the
rest of the myocardium, thereby functioning as the “pacemaker”
of the heart. The pacemaking action potential is produced by a
slow, diastolic depolarization that is regulated by several different ion currents, including T- and L-type calcium currents and
the sustained inward current [3,23], which can be modulated
by autonomic neurons. In particular, the hyperpolarization-
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activated (“pacemaker” of “funny”) If current plays a major role
in generating a pacemaking action potential in the SA node. If
is conducted by the gene family of hyperpolarization activatedand cyclic nucleotide-gated channels which are directly controlled by the activity of ␤-adrenergic and muscarinic receptors
[24].
1.2. Slow impulse conduction component
Transmission of the pacemaking impulse from atrial to ventricular myocardium is slowed at the AV-node located at the
base of the interatrial septum [1] (Fig. 1). This AV delay assures
an effective ejection of blood from the atrial chambers before
the initiation of ventricular contraction. While the superior and
right margins of the AV-node contain loosely connected fibers,
the inferior region of the node consists of cells that are more
regularly aligned. The peripheral cells in the medial portion of
the node contain few myofibrils. Many avian species develop
an AV-ring which plays a role similar to that of the mammalian
AV-node [25].
Several models have been proposed for cellular and molecular mechanisms responsible for a slow conduction velocity at the
AV-node/ring. For example, a multiple-layered arrangement of
nodal cells may play a role in delaying the impulse conduction
through the AV-node. In addition, cell shape, tissue architecture,
and gap junction-mediated intercellular coupling, can define the
direction and rate of impulse propagation. Interestingly, cells
of the AV-node do not express the fast conduction type gap
junction channels, such as Cx40 and Cx43 [6], although they
express Cx45, as do other cells of the CCS [8]. The differential
expression of the Cx gene family members in the AV-node may
be directly related to conduction velocities, as Cx45 is known
as a high voltage-sensitive and low conductance gap junction
channel [26]. Further experimental data on conductances of the
various gap junction channels will firmly verify the exact mechanism responsible for delaying the impulse conduction by the
AV-node/ring system.
Fig. 1. (A) Diagram of functional organization and electrophysiological phenotyes of individual subcomponents of the cardiac conduction system. The CCS consists
of the sinoatrial (SA) node, the atrioventricular (AV) node, bundle branches, and the most distal component, Purkinje fibers (from Pennisi et al. [12], Mikawa et al.
[89] and Gourdie et al. [8]). Action potentials representative of different conduction components and cardiomyocytes are presented in the left column. Red lines:
depolarized membrane potential level, AO, aorta; LV, left ventricle; RV, right ventricle. (B) In situ hybridization for mRNA of a fast gap junction protein Cx40
visualizes the subendocardial Purkinje fiber network of the chick embryonic heart (from Hall et al. [92]). ivs, interventricular septum.
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1.3. Rapid impulse conduction components
From the AV-node, the propagation of the pacemaking
impulse accelerates along the AV-bundle and bundle branches,
finally activating ventricular muscle via the Purkinje fiber
network system. Thus, the main function of the ventricular conduction network is to rapidly propagate and transmit impulses
to the ventricular muscle. The fast conduction cells are scattered
throughout the myocardium, but can be distinguished from ventricular muscle cells by their distinct electrophysiological and
molecular characteristics. They exhibit faster action potential
upstroke, prolonged action potential duration, higher membrane
diastolic potential and greater electrical restitution properties.
Also, several heart muscle-specific genes are expressed at considerably lower levels or not at all in conduction cells [27–29],
while expression of genes usually associated with neural tissues or skeletal muscle are often found in subpopulations of
conduction cells [13,30]. Cx40, a gap junction protein for fast
intercellular conductance of actionpotentials, was identified as
the predominant connexin of the His–Purkinje system in all
higher vertebrates thus far examined (Fig. 1B) [5–7,31–34].
2. Topological changes in the conduction pathway of
pacemaking impulses during heart development
One of the more challenging questions in heart development
has been to understand how the functionally distinct subcomponents of the CCS are precisely integrated within the heart
during embryogenesis, especially as they are forming within
distinct regions of the organ. When the primitive heart begins
beating, the conduction system has not yet developed and all
epithelioid myocytes are electrically active. Optical mapping
studies in chick embryos have shown that pacemaking impulses
are evoked predominantly at the posterior inflow tract (the presumptive sinus venosus and atrium) well before heart begins
beating (Fig. 2A) [35–37]. These impulses spread anteriorly
and unidirectionally towards the outflow tract, through gap junc-
tions between the epithelioid myocytes (Fig. 2A), and without
any local changes in velocity. This indicates that except for the
pacemaker cells, no other subcomponents of the CCS are present
at this stage.
As the heart tube begins to loop, three significant changes in
the impulse propagation pattern along the myocardium become
evident (Fig. 2B). First, the impulse velocity is significantly
slowed at the AV junction, coinciding with a morphogenic division between atrial and ventricular chambers [38–40]. This AV
delay together with formation of the AV-cushion gives rise to
an effective peristaltic wave of myocardial contraction, thereby
increasing the pumping efficiency of the embryonic heart.
Second, as heart development proceeds further, any direct electrical coupling between atrial and ventricular chambers through
myocyte–myocyte gap junctions is dissociated by the forming
interventricular septum. Third, during ventricular chamber separation by septation, the propagation of pacemaking impulses
in the ventricle dramatically changes from base-to-apex and
becomes to apex-to-base (Fig. 2C and D) [22,40–42]. The striking topological shift of the impulse-transmission pathway is
essential to ensure effective pumping of the four-chambered
heart, and depends upon the differentiation and patterning of the
ventricular conduction system, including the AV-bundle, bundle
branches, and Purkinje fiber network.
3. The myogenic versus the neurogenic origin of the
CCS
In the 19th century, competing “myogenic” and “neurogenic”
theories explained the initiation of heartbeat as a result of intrinsic heart muscle activity and electrical stimuli from extracardiac
neurons or intracardiac ganglions, respectively [43]. While there
had been several experimental demonstrations for heartbeat
without influence of extracardiac neurons, the discovery of intraventricular neurons in the early 19th century tipped the balance
in favor of a “neurogenic” source of stimulation. The ultimate
closure of this debate occurred in the early 20th century after
Fig. 2. Impulse-conducting pathways during looping and septation stages of embryonic chick hearts (based on electrophysiological and optical mapping studies,
including Kamino [37], De Jong et al. [40], Chuck et al. [41], Mikawa [30], Pennisi et al. [12], Hall et al. [92] and Sedmera et al. [126]). (A) The primitive
“pacemaker”: the pacemaker (p, yellow area) begins to generate a pacemaking action potential mainly at the left sinoatrium (sa) soon after tubular heart tube forms.
The pacemaking impulses (red arrows) propagate unidirectionally through the ventricular (v) myocardium toward the outflow tract (ot). Isochrones indicate a relative
velocity within the myocardium at a given developmental stage. At this developmental stage, no significant change of impulse propagation rate is detectable along
the heart tube. (B) Differentiation of AV-delay and ventricular fast conduction: during heart looping, the impulse propagation velocity is significantly delayed at
the AV-junction (avj), then accelerates in the outer curvature of the presumptive ventricle. (C) Unidirectional impulse propagation during interventricular septation:
pacemaking impulses still propagate unidirectionally from the left ventricle (lv) to the right ventricle (rv) via the ventricular apex, although the velocity of impulse
propagation continuously increases. (D) A dramatic topological shift in the impulse propagation pathway as the fast ventricular conduction network is established
close to completion of septation, activation of the myocardium spreads from the apex toward the base of the ventricle.
T. Mikawa, R. Hurtado / Seminars in Cell & Developmental Biology 18 (2007) 90–100
the discovery of the excitation–conduction system [1] and the
SA-node [44].
Interestingly, a similar debate at the end of the 20th century argued whether cells of the CCS arose from “myogenic”
or “neurogenic” precursor population [10,13,45,46]. Because
of neural cell-type gene expression found in cells of the CCS,
a neural crest origin was proposed as the parental population
for this specialized tissue [47–52] rather than a myogenic origin as suggested in previous studies [53]. While studies probing
expression of marker genes/proteins have provided important
insights into the development of specialized myocardial tissues,
the approach is inadequate to determine the cellular ontogeny
of the CCS or other cardiac cell types. Many marker genes for a
given cell type are often found to be expressed dynamically in
cells of unrelated cell lineages during embryogenesis. The confusion regarding the cellular origin of the CCS has been settled
by retroviral and adenoviral cell lineage studies in the chicken
embryonic heart [30,45,54–57].
Fate map and cell lineage studies in the chicken embryo have
identified that all cardiac cells arise from three distinct embryonic origins [30,58]: First, mesodermal cells localized bilaterally
to Hensen’s node [59] and in an anterior cardiac field [60] differentiate into the myocardial and endocardial cell populations
during double-walled tubular heart formation at neurula stages;
Second, cardiac neural crest cells migrate from the hindbrain
to the beating heart tube, differentiating into smooth muscle of
the great vessels and cardiac ganglia [61–63]; third, cells of the
proepicardium (PE) migrate from the mesothelium towards the
looping, beating heart tube, and form the epicardium [64–67]
and coronary vessels [68–70].
A series of retroviral cell lineage studies [30,71–73] on the
three embryonic sources have revealed that the CCS originates
from cardiomyocytes and no other cardiac cell types (Fig. 3)
[54–57,68,69,74–78]. The proof is based on the observation that
individual myocyte precursor cells produce a series of daughter
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myocytes that proliferate more perpendicularly than horizontally relative to the plane of the myocardial wall. This directional
growth results in a myocyte clone that spans the full thickness
of the myocardium, i.e. from epicardial to endocardial surfaces
of the muscle wall, forming a column (Fig. 3A) [30,45,54,55].
Transmural myocyte clones have recently been confirmed in the
heart of mice chimeric for lacZ [77] or EGFP [78]. In lineage
studies, cells of the CCS, such as the His bundle and Purkinje
fibers (Fig. 3A), are exclusively and frequently found in these
myocyte clones. That is, they are genealogically related to the
myocytes in the columnar clones. In contrast, no conduction
cells are produced from cardiac neural crest or proepicardial
cells, although their derivatives can be found adjacent to the
CCS network. Studies on neural crest derivatives in the mouse
embryo have independently confirmed the non-neural crest origin of the conduction system [79,80], except for one recent study
in which the presence of neural crest-derived conduction cells
is suggested [81]. The results from these studies suggest that
the CCS cells predominantly differentiate from a subset of contractile myocytes, not from neural crest as previously suggested
(Fig. 3B).
4. In-growth versus out-growth mechanism of CCS
development
While lineage studies have unambiguously identified the
myogenic origin for the CCS, they still do not explain how
the entire CCS network, particularly the complex pattern of
the His–Purkinje network, is formed in the developing heart.
One model proposes an out-growth mechanism. This model is
based upon the observed progression of marker expression in the
ventricle during heart development (Fig. 4A) [13,20,22,82,83].
As soon as the looping heart tube initiates interventricular septum formation, conduction cell markers become detectable in
a ring-like cluster of cells, referred as “the primary conduction
Fig. 3. (A) Myocyte clone expansion and Purkinje fiber differentiation during formation of the ventricular myocardium (modified from Mikawa [30] and Mikawa et
al. [89]). Lineage tracing experiments where cardiomyocytes were “tagged” using replication-incompetent retroviral vectors demonstrated that a clonal population
derived from a virally tagged parental cardiomyocyte contains ordinary contractile cardiaomyocytes and conducting Purkinje fibers (pf) of the CCS. Importantly,
these studies demonstrated that Purkinje fibers were recruited locally, indicating that the conduction system did not expand simply by out-growth and branching.
Green represents “tagged” cardiomyocytes and clonally related daughter cells. Blue represents the ventricular conduction network including the atrioventricular
bundle (avb), bundle branches (bb), and Purkinje fibers. ao, aorta; avj, atriovetricular junction; lv, left ventricle; ot, outflow tract; ra, right atrium; sa, sinoatrium;
v, ventricle. (B) A model for Purkinje fiber differentiation within the cardiomyocyte lineage during chick heart development (from Mikawa [30]). A subpopulation
of clonally related contractile myocytes juxtaposed to arteries and subendocardium with the ability to differentiate into Purkinje fibers (green). Within a clonal
population, cells that have differentiated into Purkinje fibers withdrawal from the mitotic state, while those that remain as contractile cardiomyocytes continue to
proliferate throughout heart development (based on Gourdie et al. [56] and Cheng et al. [57]).
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Fig. 4. Schematic illustration of competing “in-growth” and “out-growth” models for CCS network formation.
ring” [84], at the junction of presumptive right and left ventricles
[13,20,22,83,85]. The expression of CCS marker genes progressively expands to the entire His–Purkinje network. Based on the
proximal–distal wave of marker expression, an “out-growth”
model (Fig. 4A) has been proposed wherein the distal elements
of the CCS network are successively derived from proximal cell
populations [13,82,83].
The “out-growth” model has been challenged by the fact
that absent/significantly lower DNA-synthesis rates (i.e. few
mitotic cells) have been mapped to cells of the developing CCS
[57,86–88]. Another problem with the “out-growth” model is
the fact that conduction cell differentiation occurs within individual myocyte growth columns, which occupy only a segment
of the myocardium, as discussed in the cell lineage studies
above [56,57]; they do not produce progeny that extend beyond
their myocyte columner clone. The proximal–distal wave of
conduction system development can be explained by an alternative model [30,45,89] wherein conduction cells are recruited
locally within each clonal domain and linked together in situ to
establish the Purkinje fiber network (Fig. 4B). This “in-growth”
model is consistent with birthdating studies showing that differentiation of the proximal conduction components, such as
the AV-node/rings, His-bundle and bundle branches, is complete at ventricular septum formation [57,87], while Purkinje
fibers (the most distal subcomponent) are continuously recruited
throughout embryogenesis [88]. The “in-growth” model is also
consistent with an in situ linkage between subendocardial and
periarterial Purkinje fibers (Fig. 5). This model is further supported by other studies demonstrating independent formation of
the AV-bundle (a proximal component derived from the primary
conduction ring) and the bundle branches (a distal conduction
component derived from the interventricular septum) and their
connection later in development [90].
5. Cellular, molecular and biophysical mechanisms of
CCS differentiation and patterning
The definitive elucidation of the myocyte origin of the conduction cell lineage has led to many studies on the mechanisms
that convert cardiac myocytes from a muscle to a conducting
phenotype. Several potential signaling and transcriptional cascades have been implicated in the induction, maturation, and
patterning of the CCS [12]. These signaling factors include
endothelin (ET) [28,29,91–94], neuregulin [95,96], Notch [96]
and Wnt [97]. The transcription factors involved in CCS development are Msx [20], Nkx [14,17,18,98], Hop [99], Tbx and
GATA gene families [21,100,101]. This section will discuss
what is known about the cellular and molecular mechanisms
that specify the different subcomponents of the CCS.
5.1. SA-node development
The pacemaker activity is the first element to function in
the CCS, but little is known about the mechanisms underlying induction and differentiation of the pacemaker cells during
development and maturation of the heart. Genetic studies in
human and mouse have revealed that HCN channels are required
for If , the normal pacemaker current [102–104], but it is not clear
how the complex expression of HCN channels is induced and
regulated at the specific site of the developing heart. In contrast to pacemaker cells, ordinal beating myocytes express the
Kir2 gene. This channel family is responsible for inward-rectifier
potassium current IK1 that stabilizes a strongly negative resting
potential. Interestingly, dominant-negative inhibition of Kir2encoded inward-rectifier potassium channels in the ventricle
resulted in phenotypic conversion of myocytes into pacemaker
cells, suggesting that the crucial factor for inducing pacemaker
phenotype is the absence of the strongly polarizing IK1 , rather
than the expression of certain genes [105]. These studies provide
an important hint as to how to create biological pacemakers for
therapeutic purposes.
It has recently been demonstrated that pacemaker cell phenotypes, such as a higher spontaneous beating rate, faster
If current activation and larger If current densities, can be
induced spontaneously in embryonic stem (ES) cell-derived
cardiocytes. Furthermore, exposure to endothelin-1 (ET-1) significantly increases the percentage of pacemaker-like cells
without affecting their electrophysiological properties [93].
Conversely, treatment of the ES-derived cardiocytes with
neuregulin-1 exhibited no effect on differentiation, suggesting that endothelin-1 specifically promotes the development
of ES cell-derived cardiocytes to a pacemaker-like phenotype.
However, since these ES cell-derived pacemaker-like cells also
express Cx40 and Cx45, known markers for the ventricular fast
T. Mikawa, R. Hurtado / Seminars in Cell & Developmental Biology 18 (2007) 90–100
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Fig. 5. In situ linkage between subendocardial and intramyocardial Purkinje fibers during heart development of the chick embryo. First row: whole-mount in situ
hybridization analysis of E6 (A–C) and E10 embryonic chick heart (D–F), probes for ventricular myosin heavy chain mRNA (vmhc-1), detecting the ventricular
myocardium (A) and Cx40 mRNA identifying subendocardial Purkinje fibers (sepf) and intramyocardial Purkinje fibers (impf) (B, C, E and F). C and F are higher
magnification of boxed areas in B and E, respectively. Note that in the E6 heart, Cx40+ subendocardial Purkinje fibers are already present but no intramyocardial
Purkinje fibers are detectable. In the E10 heart, recruitment of intramyocardial Purkinje fibers become evident along coronary arterial branches (+a) which penetrate
from the subepicardial space toward the endocardium. Second and third rows: Cx40 in situ hybridization on transverse sections of basal and medial regions of the
ventricle, respectively. Hearts were examined at E10 (G–J), E12 (K–N), and E14 (O–R). H, J, L, N, P and R are high magnification of boxed areas in G, I, K, M, O
and Q, respectively. At E10, Cx40+ subendocardial Purkinje fibers (sepf) are evident at a tip of the trabeculae. In addition, the initiation of Cx40+ intramyocardial
Purkinje fiber recruitment along forming arteries (impf+a) can be seen in the compact myocardium (H and J). By E12, some intramyocardial/periarterial Purkinje
fibers at the basal region of the ventricle expand through the entire thickness of the myocardium (red asterisks) and reach to the subendocardial Purkinje fibers (K
and L). No linkage between subendocardial and intramyocardial Purkinje fibers has been established yet in medial region of the ventricle at this developmental stage
(M and N). By E14, many intramyocardial Purkinje fibers have reached to the subendocardial Purkinje fibers at both basal (P) and medial (R) regions of the ventricle
(red asterisks).
conduction system, such as His–Purkinje system, it remains to be
seen whether His–Purkinje cells are also induced in ET-treated
ES-derived cardiocytes.
It is unknown how the action potential from the SA-node
propagates into the atrial myocardium in a non-radial manner
or how the leading pacemaker site is blocked from shifting into
atrial muscle as the heart matures. Significant heterogeneity has
been detected amongst cells of the SA-node in regards to both
structural and functional aspects, such as cell shape, production
of pacemaker action potential, densities of ionic currents, and
composition of gap junction complexes [106]. The complexity
of the SA node architecture has been proposed to account for
the heterogeneity of electrical activity throughout the SA node.
There are two possibilities to describe how the cellular heterogeneity of the SA node could form. The first proposal is that
a gradual change in the properties of node cells occurs from
the periphery to the center of the tissue; basically, a “gradient”
model. The second possibility, a “mosaic” model, predicts a variable mix of atrial and SA node cells from the periphery to the
center. It still remains to be determined which method is used
to achieve the heterogeneity, which appears to be necessary for
numerous reasons, including protection of the SA node from
the hyperpolarizing influence of the surrounding atrial muscle
and assistance to the SA node to drive the surrounding atrial
muscle.
5.2. AV-node formation and maturation
Similarities have been identified between nodal cells and
embryonic “primary” heart muscle cells [46,13], including the
small cell size, irregular cell shapes, poorly developed myofibrils, expression of embryonic myocyte-type contractile and
cytoskeletal proteins, shape of action potential, slower conduction velocities, and a lower level of gap junctions. Based on
these phenotypic similarities, a model has been proposed for
AV-node development, wherein a subpopulation of the primary
myocardium is inhibited from differentiating into the mature
myocardium and instead forms the slow conducing AV-node
[13,21,83]. This hypothesis is consistent with the expression
pattern of a few transcriptional repressors, such as Tbx2 and
Tbx3, which are excluded from the region expressing marker
genes namely Cx40 and ANF [107] for the fast conduction cells
and atrial muscle cells.
However, many other phenotypes characteristic of the AVnode are not completely reminiscence of those of the embryonic
“primary” myocytes. It is evident that mature nodal cells
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T. Mikawa, R. Hurtado / Seminars in Cell & Developmental Biology 18 (2007) 90–100
have functional phenotypes distinct from not only other subcomponents of the CCS but also from embryonic/primary
myocardial cells. For example, cells of the AV-node exhibit
unique expression patterns of specific membrane channels,
receptors, and Ca2+ regulatory proteins, which are distinguishable from embryonic/primary myocardial cells. Unique
transcriptional mechanisms, including Nkx2.5-dependent regulation of the post-natal survival/function of nodal cells, have
also been documented in the AV-node as compared to embryonic/primary myocardial cells both in human and animal models
[14,18,98]. A recent study further suggests that the AV-delay of
impulse conduction in the zebrafish embryo requires Notch-1b
and neuregulin, indicating that additional signaling mechanisms play a role in AV-nodal cell specification [96]. Studies
of connexin localization in the rodent AV-node have revealed
significant heterogeneity within this unique conduction tissue,
as well as a particularly complex expression patterns at the
interface between the node and adjacent myocardial cells [26].
These studies suggest that AV-node formation may be regulated
by a multiple-step mechanism that may be more intricate than
previously thought.
5.3. Induction and patterning of fast conduction Purkinje
fiber network: inductive interactions between cardiac
endothelial cells and myocytes
While the presence and distribution of the intramyocardial
branch of the fast conduction network is highly variable among
species, its proximal components run subendocardially regardless of species [10,13]. As discussed above, the subendocardial
Purkinje fiber recruitment within individual myocyte clones
[56,57] has provided an important insight into mechanisms of
this inductive event in the embryonic heart. A series of studies
using chick and mouse models have demonstrated that paracrine
interactions between embryonic myocytes and cardiac endothelial cells play a key role in local recruitment of conduction cells
from beating myocytes [12,30,45].
In avian hearts, in addition to the subendocardial Purkinje
fibers, there are also intramyocardial Purkinje fibers. Importantly, intramyocardial Purkinje fibers penetrate along coronary
artery branches [12,29,56,108,109]. Cell lineage studies have
shown that Purkinje fiber recruitment from contracting myocytes
takes place at two restricted sites, periarterially and subendocardially, supporting the model in which paracrine interactions
of myocytes with endocardial and arterial cells may play an
inductive role in conduction cell differentiation [30,45]. Indeed,
suppression of coronary vessel development resulted in a significant loss of intramural Purkinje fiber differentiation, indicating
the necessity of coronary arterial beds for intramural conduction
cell differentiation [110]. Furthermore, ectopic Purkinje fibers
developed along arteries that were ectopically induced in the
myocardium [110]. These data show that coronary arterial beds
are not only necessary but also sufficient for recruiting adjacent
myocytes to differentiate into conduction cells.
Since the endothelial cell is the only cell type commonly
present in both endocardium and arteries along which adjacent
myocytes differentiate into Purkinje fibers, it was suspected that
endothelial cell-derived signal(s) may play an inductive role in
recruitment of conduction cells. Consistent with this idea, the
expression of a conduction cell marker gene in presumptive ventricular conduction cells of the mouse embryonic heart appears
to be dependent upon the co-presence of cardiac endothelial cells
[12].
It is well demonstrated that vascular bed-specific phenotypes
are critically regulated by environmental cues [111]. Shearstress is one vascular bed-specific mechanism that regulates the
expression and/or secretion of vascular cytokines [112]. Evidently, endothelial cells of the endocardium and arterial branches
are exposed to higher shear-stress than those of venous and
capillary networks. Indeed, it has been shown [28] that cultured embryonic myocytes can be induced to express conduction
cell markers by a shear-stress-induced cytokine, endothelin-1
(ET-1) [113,114]. This ET-1-dependent conversion is dosedependent, inhibited by specific antagonists of ET receptors and
not observed following treatments with other cytokines known
to be prominent in vascular development, including FGF, VEGF,
and PDGF.
ET-1 was originally identified as a potent vasoconstrictor
derived from endothelial cells [113]. ET ligands are secreted
through two steps of post-translational processing from its precursor, preproET [115]; The first step is cleavage by non-specific
furin proteases into big-ET, and the second step is further
Fig. 6. (A) Whole-mount in situ hybridization for Cx40 mRNAs (purple) in an E8.5 heart cut in half frontally. Cx40+ subendocardial Purkinje fibers (sepf) are
indicated by white arrows. (B) A transverse section of an E8.5 heart stained for Cx40. (C) High magnification of the boxed area in B. (D) As in B but for ECE-1. (E)
High magnification of the boxed area in D. at, atrium; ec, endocardium; ivs, interventricular septum; lv, left ventricle; rv, right ventricle; tb, trabeculum (modified
from Hall et al. [92]).
T. Mikawa, R. Hurtado / Seminars in Cell & Developmental Biology 18 (2007) 90–100
processing by the ET-specific metalloprotease, Endothelin Converting Enzyme (ECE-1) into biologically active ET ligand
[115,116]. Binding of ET ligands to G protein-coupled receptors triggers ET signaling [117,118]. In the embryonic heart,
ET-receptors are expressed by all myocytes and absent from
cardiac endothelial cells [29,119]. In contrast, ECE-1 expression is present in a portion of the endocardium and in coronary
arterial endothelium, and absent from myocytes and endothelia
of cardiac veins and capillaries [29,113].
Since the expression pattern of ECE-1 in the embryonic heart
coincides with the timing and location of endogenous Purkinje
fiber differentiation (Fig. 6), localized ET-dependent induction
of embryonic myocytes in vivo might be explained by the distribution of endogenous ECE-1. Indeed, viral-mediated ectopic
co-expression of ECE-1 and PreproET-1 in the embryonic
ventricular myocardium results in the ectopic and precocious
differentiation of Purkinje fibers [29]. Furthermore, gadolinium,
an antagonist for stretch-activated cation channels, downregulates the expression of ECE-1 and a conduction cell marker,
Cx40, in ventricular chambers, concurrently with delayed maturation of a ventricular conduction pathway [92]. Conversely,
pressure-overload in the ventricle by conotruncal banding results
in a significant expansion of endocardial ECE-1 expression and
Cx40-positive putative Purkinje fibers [92]. Coincident with this,
an excitation pattern typical of the mature heart is precociously
established. These in vivo data suggest that biomechanical forces
acting on, and created by, the cardiovascular system during
embryogenesis play a crucial role in Purkinje fiber induction
and patterning [42,92], and support the model (Fig. 7) wherein
induction of conduction cells is localized in the ventricular
myocardium by the site-specific cleavage of Big-ET-1 by ECE-1.
The expression of many Purkinje fiber marker genes as
well as the downregulation of heart muscle-specific genes can
be induced by ET-1 both in vitro and in vivo [12,29,91].
97
There is a report, however, that a subset of genes that are
upregulated in bona fide Purkinje fibers are not significantly
induced in ET-1-induced Purkinje fibers in culture [16], suggesting involvement of an ET-independent pathway in regulation
of a unique gene expression pattern in Purkinje fibers [16].
Indeed, potential involvement of other paracrine interactions
such as neuregulin has been suggested [94,95]. In the heart,
expression of neuregulin is confined to the endocardium, while
erbB2 and erbB4 receptors are expressed in the myocardium.
A loss-of-function mutation in the neuregulin or erbB gene in
the mouse results in the loss of trabeculae formation, while
the compact zone of the myocardium is apparently normal
[120–122]. These mutant embryos exhibit irregular heartbeats
and eventually die. Although the cause of the embryonic death is
currently unknown, conduction disturbances may be associated
with insufficient contractile capacity [13]. Neuregulin-signaling
appears to play multiple roles in the heart, promoting myocyte
survival and growth [123], and regulating cardiac cushion formation [124]. Neuregulin expression is known to be regulated by
ET-1 signal [123]. Like ET-1, neuregulin can induce myocytes
to upregulate some conduction cell markers, such as atrial natriuretic factor (ANF) and skeletal muscle protein [123]. However,
recent studies have also detected some differences in roles of
ET and neuregulin signals in CCS development. As discussed,
neuregulin plays a role that is more prominently in specification of slow conduction cells than fast conduction cells [96].
While shear-stress/stretch can regulate both ET-1 signaling and
fast conduction cell differentiation, it is unknown whether the
neuregulin signal cascade is regulated in a shear-stress-induced
manner [125]. Furthermore, expression of fast conduction cell
markers such as Cx40 in ES cell-derived cells are promoted by
ET-1 but not by neuregulin. Continued investigation into the
interplay between different paracrine signaling cascades and
transcriptional regulatory networks will facilitate our understanding of molecular mechanisms involved in conduction cell
differentiation and patterning.
6. Concluding remarks
Fig. 7. Model for biophysical and molecular cascades involved in local conversion of embryonic myocytes into Purkinje fibers (from Hall et al. [92]).
Biomechanical force generated by heartbeat and/or blood flow (step 1) triggers
shear-stress and/or pressure-sensitive mechanism(s) (step 2), that includes an
endothelial cell subpopulation to express ECE-1 (step 3) in the embryonic heart.
Only endothelial cells expressing ECE-1 can produce active ET peptide (step
4), thereby restricting the ET-induced Purkinje fiber recruitment from working
myocytes (step 5) within the embryonic heart. Differentiated Purkinje fibers
transmit a pacemaking impulse to sister cardiomyocytes to trigger their contraction (step 6) ppET: preproendothelin, bigET: big endothelin, ETR: endothelin
receptor.
Lineage and fate map studies of cardiac cells have identified
the timing and location of differentiation and patterning of the
CCS and have revealed the plasticity of embryonic myocytes
in their cell fate diversification. Identification of signaling factors and transcriptional regulations has significantly enhanced
our understanding of the molecular and cellular mechanisms
involved in these processes. These include a dynamic role for
vascular-derived signaling cues, such as hemodynamic-induced
paracrine factors secreted from cardiac endothelia, in the patterning and formation of the CCS. The new data underscore the
need for a better understanding of the biophysical forces acting
on, and created by, the cardiovascular system during embryogenesis, particularly to address a longstanding question of how
the disparate subcomponents of the CCS integrate into a functional entity during embryogenesis. A better understanding of
these processes will provide a solid basis for future therapeutics
to allow for the regeneration of damaged cardiac tissues or the
construction of biologically engineered heart tissues.
98
T. Mikawa, R. Hurtado / Seminars in Cell & Developmental Biology 18 (2007) 90–100
Acknowledgement
We thank to Drs. Y. Ishii, I. Oda, R. Garriock, and J. Timmer and Ms. A. Nevetta for their valuable comments on this
manuscript. Supported in part by the NIH-NHLBI.
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