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of 56 in PresS. Am J Physiol Heart Circ Physiol (September 7, 2007). doi:10.1152/ajpheart.00750.2007
Electrophysiology and pacemaker function of the developing sinoatrial node
Mirko Baruscotti1 and Richard B. Robinson2
1
Laboratory of Molecular Physiology and Neurobiology, Department of Biomolecular
Sciences and Biotechnology, University of Milano, Italy and
2
Department of
Pharmacology and the Center for Molecular Therapeutics, Columbia University, New
York, New York 10032, USA
Running title: Electrophysiology of the developing SAN
Corresponding author:
Richard B. Robinson
Department of Pharmacology
Columbia University,
630 W. 168th St., New York, NY 10032.
Tel.: 212-305-8371
Fax: 212-305-8780
E-mail: [email protected].
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Copyright © 2007 by the American Physiological Society.
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Abstract
The sinoatrial node performs its task as a cardiac impulse generator throughout the life of
the organism, but this important function is not a constant. Rather, there are significant
developmental changes in expression and function of ion channels and other cellular
elements, which lead to a postnatal slowing of heart rate and may be crucial to the
reliable functioning of the node during maturation. In this review we provide an overview
of current knowledge regarding these changes, with the main focus placed on maturation
of the ion channel expression profile. Studies on the sodium and pacemaker currents have
shown that their contribution to automaticity is greater in the newborn than in the adult,
but this age-dependent decrease is at least partially opposed by an increased contribution
of the L-type calcium current. Whereas information regarding age-dependent changes in
other transmembrane currents within the sinoatrial node are lacking, there are data on
other relevant parameters. These include an increase in the nodal content of fibroblasts
and in the area of non-expression of connexin 43, considered a molecular marker of nodal
tissue. Although much remains to be done before a comprehensive view of the
developmental biology of the node is available, important evidence in support of a
molecular interpretation of developmental slowing of intrinsic sinoatrial rate is beginning
to emerge.
Keywords: SAN, cardiac pacemaker, development, Na current, Ca current
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Introduction
The anatomical identification of the sinoauricular junction (now named sino-atrial node,
SAN) was originally reported in 1907 by two physiologists/anatomists, Keith and Flack
(58). This finding, tog4ether with the earlier identification of other structures of the
cardiac conduction system (104, 110, 123), prompted further studies which soon yielded
the concept that the heartbeat in mammals originates in the SAN (70, 124) and then
spreads to the rest of the cardiac tissue in a temporally and anatomically orderly manner.
The impact on the cardiac scientific community was immediate and led to continuing
effort to unravel the working mechanisms of this biological impulse generator. As a
result, automaticity mechanisms in adult SAN have been extensively studied and have
been the subject of numerous reviews (21, 31, 48, 98, 99, 120). Despite this, the basis of
automaticity, and the issue of the relative importance of different currents to pacemaking,
remains controversial. However, a few generalities can be stated. Pacemaking will
depend on current flow during diastole, as well as the presence and characteristics of the
major depolarizing currents that take the cell to and beyond threshold. Multiple currents
are likely to contribute to both diastolic depolarization and threshold, and the mix varies
with species, with disease and regionally within the node. In addition to the
characteristics of the channels at the cellular level, pacemaking also will be influenced by
characteristics of the tissue, including connections to surrounding atrial myocytes and
fibroblasts.
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Despite the extensive literature devoted to the ionic basis of automaticity in the adult
SAN, and the fact that age-dependent differences in intrinsic heart rate have been
recognized for some time (52, 53, 87, 114, 127, 128), there has been far less research on
the basis of automaticity in the newborn or immature node. There is, however, sufficient
evidence to state categorically that the ionic basis of automaticity in the newborn is
distinct from that of the adult. These differences can have important implications for the
diagnosis and treatment of cardiac rhythm disorders in the immature, adult and aged
individual. In this review we will focus specifically on the young versus adult SAN, but
will include data on the aged heart where appropriate. We will not describe the adult
characteristics in detail, but will refer to published reviews and simply highlight key
aspects for the purpose of comparison to the young node. Our intent is, in part, to educate
the reader to the fact that the basis of SAN automaticity is quantitatively and qualitatively
distinct in the newborn and adult. However, our additional purpose is to highlight how
much remains to be determined in this field and thereby to encourage additional research
on the topic.
General properties of the SAN
In mammals the pacemaker region of the heart (the SAN) is located in the wall of the
right atrium, but its precise location can vary slightly in different species. In rabbits, a
long standing model for heart automaticity studies, it extends along its major axis
between the ostii of the superior and inferior venae cavae and along its minor axis
between the interatrial septum and the crista terminalis (33), while in human, murine and
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canine hearts the pacemaker cells are more concentrated in the proximity of the sinus
node artery at the junction of the superior vena cava and the right atrium (12, 94, 99).
Studies on the cytological organization of the SAN have revealed a structure composed
of a dense array of connective tissue in which a small number of specialized cells, all
electrically connected, are sparsely, but not homogeneously embedded. Although the
SAN as a whole is normally recognized as a uniform functional pacemaker unit, only a
small portion of it consists of primary pacemaker cells which are the early generators of
the excitatory depolarizing waves that first spread to the rest of the nodal tissue and then
to the rest of the myocardium. Indeed several studies at the single cell level have verified
that the properties of SAN cells are not identical throughout the entire node, but present
noticeable differences that are relevant to their specific role in the context of generation,
modulation, and early spread of the impulse (14, 118). For example isolated central nodal
cells have a slower rate of contraction and are normally more depolarized than peripheral
or secondary pacemaker cells. Furthermore, autonomic stimulation induces a shift in the
position of the leading pacemaker, thus revealing an inherent lack of homogeneity within
the central nodal region itself (88). The above observations suggest that understanding of
intrinsic SAN function must proceed along two lines: one is the elucidation of the ionic
and modulatory properties of a single pacemaker cell, and the other is the electrical
interaction between nodal cells and nodal-atrial cells. A detailed analysis of the ionic
mechanisms generating automaticity requires studies at the single cell level, but to fully
understand the complexity of the SAN physiology it is important to consider that each
cell exists as an element of a complex network and this network in turn strongly
influences the functional characteristics of its component cells (13, 54, 133). For example
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several papers (13, 55) have shown that central nodal cells must be adequately protected
from the more hyperpolarized surrounding atrial tissue since the electrotonic influence of
the atrium can profoundly influence SAN activity. This protection, on the other hand,
must not be so extreme as to isolate the SAN cells, or the consequence would be that
spontaneous activity would be confined within the node, and fail to propagate to the rest
of the heart.
Molecular markers of the SAN
Until recently the only way to precisely identify the SAN region was electrical mapping
(26, 126). This technique continues to be utilized in basic and clinical research since it
can be applied to both in vivo and in vitro preparations and permits a precise description
of the progression of the depolarizing wave. More recently molecular-based
identifications have been suggested, based on comparative studies of adult sinoatrial and
atrial myocytes that rely on the presence or absence of specific molecular markers.
Combined efforts of several groups have determined that adult SAN cells express
connexin 45 (Cx45) and neurofilament M (NF-M), but do not express connexin 43
(Cx43) and Atrial Natriuretic Peptide (ANP). This expression profile is completely
reversed in atrial cells. Further, crucial differences also exist at the level of ionic currents;
for example SAN cells are characterized by the pronounced expression of the pacemaker
current, If (see the section below on ionic currents in the developing node), while they
express little or no inwardly rectifying IK1 current (24, 32, 48, 85). These data have been
largely collected in adult preparations; therefore an important question that should be
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asked is whether the presence or absence of these molecular markers is stable during the
entire lifetime of the organism. If not, this would raise the possibility that variations in
the expression of these proteins could be linked to selective and specific age-dependent
functional differences in the node which could eventually lead to the occurrence of agingrelated pathologies. Further, a strong developmental regulation of any of these proteins
would impact their utility as markers in the young or perhaps aged SAN.
Expression of Neurofilament M
Studies have explored the expression of NF-M in the conduction system of the adult
heart, as well as during embryonic development in rabbits (39, 121). Positive staining for
NF-M is considered an appropriate hallmark for the identification of this specialized
tissue (33, 35). NF-M can be considered an early marker of the developing conduction
system of the embryo since it is already detectable at E9.5 just before heart looping has
completed (121), and its expression profile is complete by birth (40). However, in
considering the relevance of NF-M as a tool to identify and investigate the properties of
the pacemaker region, it is important to note that there may be marked species
differences, since there has been no evidence for the presence of NF-M in humans (121)
and rats (5).
Expression of connexin isoforms
Connexins can serve both as molecular markers and as important contributors to the
electrophysiological properties of the node. Connexins are proteins that when properly
assembled give rise to gap-junctions that allow the intercellular exchange of ions and
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small molecules. In the human and mouse genome the family of connexins includes at
least 20 members (106). For several years the isoforms known to be expressed in cardiac
tissue were Cx40, Cx43, and Cx45. This list has been recently updated by Kreuzberg et
al. (64) who also reported the presence of Cx30.2. The distribution of these isoforms is
not uniform throughout all heart regions. Indeed Cx40 and Cx43 are mostly present in the
working atrial and ventricular myocytes, while conduction tissue is characterized by the
presence of Cx45 and Cx30.2 (the mouse homolog of human Cx31.9). In particular,
Cx30.2 seems to be selectively expressed in the SA and atrioventricular (AV) nodes (64).
This pattern of expression has been particularly useful in studies of the SAN since the
absence of Cx43, originally observed in several studies (28, 29, 86, 112, 116) has been
utilized to identify the SAN region from the surrounding Cx43-rich atrial tissue. In terms
of functionality, Cx45 has a somewhat lower unitary conductance than either Cx40 or
Cx43 (17, 18). Further, Cx30.2 has the lowest unitary conductance of all gap junctional
proteins, and knocking out this connexin results in a 25% decrease in the PQ interval
(63). Thus, the nodal specific connexin expression pattern may be critical to its property
of slow conduction.
FIGURE 1 NEAR HERE
Jones et al. (52) have investigated the expression profile of Cx43 in the SAN throughout
the lifetime of the guinea-pig. They found Cx43 to be uniformly present throughout the
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node in newborn animals, but progression from youth to senescence was accompanied by
a 14-fold increase in the nodal area lacking Cx43 and a corresponding decrease in the
level of Cx43 protein expression (Fig 1). Interestingly, and in agreement with previous
data, the disappearance of the Cx43 initiated in the central nodal area and then extended
to more peripheral nodal regions with further aging. Furthermore there was no increase in
Cx40 and Cx45 to compensate for the loss of Cx43. Two key questions regarding this
example of developmental regulation of Cx43 are (1) what is its influence on functional
aspects of the pacemaker site (i.e. does the disappearance of Cx43 relate to the intrinsic
heart rate and/or nodal propagation)? (2) What are the modulatory signals that control
Cx43 expression? While the latter is still unanswered, some work has been done
regarding the former. In particular Yamamoto et al. (126) reported that the nodal area in
which Cx43 is absent is characterized by a 5-fold decrease in conduction velocity, and
this is consistent with the concept that a dependence solely on Cx45 and Cx30.2 would
lead to slower conduction. Furthermore Jones et al. (52) observed that the distance that
the impulse generated at the center of the node must travel before reaching atrial tissue
(identified by the crista terminalis) increases with age. Taken together these findings
provide a molecular correlation to the functional observation of a decrease in the overall
intrinsic heart rate and conduction time with age.
Fibroblast content of the developing node
The fibroblast component is one of many factors differentiating SAN structure from that
of other cardiac regions. For example, in humans the fibroblast content of the adult
working myocardium is about 5-6% of the total volume, but this proportion is much
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higher in the SAN, where it reaches values of 45%-75% (19, 27, 103). Studies on humans
and lower mammals also have highlighted that the fibroblast, and therefore collagen,
content of the heart is subject to strong developmental regulation: it is low during initial
embryogenesis, and increases during late embryogenesis and postnatal growth (3, 19, 27,
103). After this period of expansion, the proliferation halts but can be triggered by the
occurrence of pathological states. The apparent lack of a symmetrical organization of the
myocellular component of the node is also true for the fibroblasts since they are found
both adjacent to (and thus in contact with) sinoatrial myocytes and as isolated groups
(20). It is interesting to note that these two separate populations also display different
types of connexins. Fibroblasts interacting with nodal cells preferentially express Cx45,
while those that do not contact SAN cells express Cx40; Cx43 has not been found in
sinoatrial node fibroblasts (20). The contribution of the fibroblast population to the
physiology of the node is complex, but important roles certainly include mechanical
support and regulation of local inflammation (105). Since fibroblasts are non-excitable,
their dense presence in the SAN, and the observation that they are at least partially
coupled via functional gap junctions to nodal cells, pose the intriguing question of a
possible electrical consequence. In this regard an interesting observation is that fibroblast
resting potential is influenced by stretch as a direct consequence of the presence of
stretch-activated ion channels on their cell membrane (56, 60). Whether changes in the
membrane potential of fibroblasts directly influence the chronotropism of coupled nodal
cells is an interesting question that awaits investigation.
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In addition to the observations above concerning fibroblast distribution during early
development, changes in the structural properties of the SAN associated with aging result
in slow conduction and reduced voltage amplitude on eletroanatomic mapping (59, 95).
Interestingly these functional changes display some similarities with those observed in
pathological states such as sinoatrial node dysfunction (SND) (43, 59, 95). Finally,
besides their above mentioned roles, it is possible that fibroblasts contribute to the
metabolic homeostasis of the node (19).
Ionic channels in the developing node
Pacemaker (If) current
Pacemaker cells lack a stable resting potential, instead exhibiting a slowly rising
depolarization that encompasses the interval from the end of action potential
repolarization to the upstroke of the subsequent action potential. The events underlying
this diastolic depolarization are the main determinants of cardiac pacemaker rate. Since
its early discovery If has been identified as a contributor to diastolic depolarization (15),
and indeed its activation upon hyperpolarization and its inward and time-dependent
Na+/K+ mixed nature are well-suited to drive cell depolarization during the diastolic
interval. The vast majority of information on the biophysical and molecular properties of
this current derives from studies in single SAN myocytes isolated from adult animals (6,
25, 31, 93, 108). In addition to its contribution to basal pacemaking, If is a preferential
target in the autonomic modulation of cardiac chronotropism due to the presence of a
cAMP binding domain on the channel’s C-terminus. The channels responsible for If (f
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channels) are modulated by both the sympathetic and parasympathetic branches of the
autonomic nervous system; activation of the muscarinic cascade affects f channels by
reducing the cAMP bound to the channel, thereby inducing a hyperpolarizing shift of the
activation curve, while the stimulation of K-adrenoceptors elicits an increase in cAMP
available for binding to the channel and thus increases f current during diastole due to a
depolarizing shift of the activation curve. To assess whether the pacemaker current
undergoes developmental changes that could at least partially explain the slower heart
rate observed in adult animals Accili et al. (2) carried out comparative studies of If in
SAN myocytes isolated from newborn (9-10 day old) and adult rabbits. They found that
newborn SAN cells had both a greater If density (newborn: 0.244 pS/pF, adult: 0.158
pS/pF), and a significantly steeper slope of the activation curve (slope factor: newborn, 9.6 mV; adult, -11.3 mV) when compared to adult cells, but no difference in the midpoint
of activation (newborn, -66.7 mV; adult, -66.3 mV). These results, in addition to the
observation that the capacitance of SAN cells does not vary with development (7),
indicate that the amount of If that flows at pacemaker potentials is greater in newborn
animals than in adults and thus could contribute to the higher intrinsic heart rate of rabbit
newborn SAN (114).
The intrinsic variation of SAN rate during development has also been studied by Yang et
al. (127, 128). These authors carried out radioimmunoassay experiments to evaluate agedependent changes in the basal concentration of cAMP in sinoatrial cells and reported a
marked decrease (0.31 LM at 2 weeks, vs 0.025 Lm at 12 weeks). They also reported a
negative shift of the If threshold potential (M45.5 vs M51.1 mV in 2 week vs 4 week
hearts, respectively). Based on these findings the authors proposed that heart rate slowing
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during development can be partially attributed to a reduced contribution of the pacemaker
current secondary to a decrease in cellular cAMP content. The observation that cAMP
levels change during development is interesting, but any extrapolation from these data
must be done with caution since there is increasing evidence that membrane proteins such
as ion channels exist in highly controlled microdomains where the concentration of
cytoplasmic molecules such as cAMP can be different from those of the bulk cytoplasm.
Furthermore the data of Yang et al. (127, 128) differ from those of Accili et al. (2), who
reported no differences in either the midpoint or the acetylcholine- and isoproterenolinduced shifts of the If activation curves between newborn and adult animal. Although
Accili et al. (2) did not observe an age-dependent change in the cAMP responsiveness of
f channels, this does not preclude an age-dependent difference in the modulation of the
current or heart rate. The developmental differences in the biophysical properties of the
channels (higher conductance and steeper activation relation in the newborn) means that
an identical shift in the position of the activation curve would per se lead to a greater
current variation and thus to a more marked effect of autonomic control of heart rate in
the newborn, as indeed has been observed both for vagal (30) and K-adrenergic (44)
stimulation. This is illustrated in Fig 2, where the activation parameters of Accili et al.
(2) have been used to plot the relative activation relations of newborn and adult SAN
cells under basal conditions and under the influence of high concentrations of
acetylcholine and isoproterenol, to represent maximal vagal and sympathetic tone,
respectively. As shown, at a potential of -55 mV, which is the approximate maximum
diastolic potential in the SAN, the amount of current contributed by If under maximal
vagal tone is approximately the same in newborn and adult. However, with maximal
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sympathetic tone the current increases only 2.8-fold in adult, compared to 4.5-fold in
newborn, representing a 60% greater change in current at the younger age.
FIGURE 2 NEAR HERE
Molecular identification of pacemaker channels, either at the message or protein level,
has been carried out in several species, including rabbits, mice, rats, and dogs (38, 46, 49,
71, 72, 73, 96, 97, 100, 102, 115, 135). There are convincing indications that in the adult
SAN each functional channel is composed of 4 subunits belonging to the family of
Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels. The major
component of the native SAN pacemaker current is generally the HCN4 isoform (49, 71,
79, 81, 102), although a recent study using laser capture and RT-PCR unexpectedly found
that in rat SAN the major isoform at the message level is HCN2 (46). While these data on
transcript expression need to be extended to the protein level, this same study also
reported changes in message as a function of age. Both HCN2 and HCN4 transcript
expression progressively decreased with development, and consistent with this
observation, the non-selective f-channel blocker cesium became less effective in slowing
rate with development (46). To our knowledge this is the only study of pacemaker
currents in the developing rat SAN, and so the results await confirmation by other groups
and extension to other species.
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The presence of HCN4 signal is generally considered a marker for the early identification
of the SAN region during embryonic development (80). Although when singularly
expressed in heterologous system all members of the HCN family display similar
properties, the most distinguishing characteristic of HCN4 is its slow kinetics (typical
time constants of activation at -95 mV are: 0.11, 1.13, and 2.52 s for HCN1, HCN2 and
HCN4, respectively) (1, 4). These kinetics can be modified by specific interactions with
physiological modulators such as PIP2 or MiRP1 (MinK Related Protein 1), which
profoundly increase the current density and speed up the activation process of some HCN
isoforms (89, 92, 131). Although to our knowledge no specific data are available on the
molecular identification of the newborn f currents in rabbit heart, the observation by
Accili et al. (2) that its properties are similar to those of adult animals would tend to
suggest a similar molecular assembly. Further evidence in support of a prevalent HCN4
composition of newborn pacemaker channels comes from embryological data in mice. In
a pivotal study Stieber et al. (107) generated both global and cardiac-specific HCN4
knock-out mice and showed that these mice die at about day E9.5-E11.5 due to severe
functional anomalies of the cardiac conduction system. Analysis carried out in this period
confirmed that mice lacking HCN4 were characterized by a severe bradycardia (88 bpm
for HCN4-/- vs 139 bpm for wt HCN4+/+) likely caused by a 75%-90% reduction in the If
current. Interestingly, cardiac rate of knockout mice could not be modulated by a cAMPdependent mechanism, thus strengthening the evidence that cAMP-dependent rate
modulation is mostly induced by an If-dependent mechanism. Given the molecular
composition and biophysical properties of the pacemaker currents during embryological
and post-natal development, it is reasonable to conclude that HCN4 subunits are central
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elements of cardiac pacemaker myocytes at all ages. Thus, an important aspect of future
studies will be the analysis of the genetic mechanisms that regionally and temporally
control or repress HCN4 channel expression. In this respect, Kuratomi et al. (65) recently
have investigated some aspects of the molecular mechanism controlling expression of
HCN4 and have identified both the promoter region responsible for basal activity and the
presence within the first intron of a conserved Neuron-Restrictive Silencer Element
(NRSE) sequence. NRSE is a sequence that when bound to the Neuron-Restrictive
Silencing Factor (NRSF), inhibits the transcriptional processing of neural genes in non
neuronal cells. Interestingly, Kuratomi et al. (65), have found that the NRSE-NRSF
system plays a crucial role in controlling the known progressive decrease of HCN4
channels during embryological development of the working myocardium (129). Indeed,
the levels of NRSF signal increases during embryological development thus leading to a
substantial inhibition of the transcriptional activity of the HCN4 gene. It is worth noting
that the same mechanism operating in the opposite direction controls the increased
expression of HCN4 channels induced by cardiac hypertrophy (36, 65, 66). Finally, the
role of the NRSF-NRSE system in controlling channel expression is not limited to HCN4,
but is also believed to control transcription of T-type calcium channels (66, 130). The
extent to which these regulatory mechanisms are active in the developing sinoatrial node
remains to be determined.
Information on the mechanism controlling the differential fate determination of cardiac
embryonic cells bound to become sinoatrial node or atrial cells are also currently
becoming available. As shown by Hoogaars et al., (45) the transcriptional repressor Tbx3
seems to be the key element inducing the formation of the SAN from the rest of the
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(Tbx3-free) future working atrial myocardium. Whether or not the presence of Tbx3 is
directly involved in the expression of HCN4 channels is not known. However, it is likely
that the elucidation of the molecular events controlling expression of the pacemaker
phenotype will have practical implications for understanding arrhythmogenic cardiac
diseases and innovative therapeutic strategies.
Sodium currents
Until a decade ago it was commonly accepted that there was little or no functional
contribution of Na current to the activity of primary sinoatrial pacemaker cells. This
conclusion was mostly derived from the observation that the Na channel blocker
tetrodotoxin (TTX) did not affect the spontaneous rate of either the intact SAN (61, 62,
69, 125) or single cells from adult rabbits (82, 83). In more recent years the view that Na
current is irrelevant to pacemaker activity has been profoundly modified by
developmental studies in the rabbit and by studies of adult SAN in other species (7, 24,
34, 68, 75).
FIGURE 3 NEAR HERE
In 1996 we reported that a Na current was present in rabbit primary nodal cells at birth.
Its developmental regulation was evident from the observation that by the 40th postnatal
day the current had fully disappeared (7). The functional role of the current was assessed
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by challenging the spontaneous activity of newborn cells with the Na channel blocker
TTX (3 LM). Somewhat unexpectedly, TTX modified all action potential parameters
rather than only the upstroke velocity. Particularly intriguing was the decrease in the
slope of the diastolic depolarization (from 0.035 to 0.015 V/s) and the consequent
slowing of spontaneous rate from 90 to 33 beats per minute. Similar investigations of
adult SAN cells failed to demonstrate INa and, as expected, TTX did not affect rate. This
marked age-dependent difference in the effect of TTX on spontaneous rate is illustrated
in Fig 3. Whole-cell and single channel kinetic analysis demonstrated that a slow
inactivation process due to multiple single channel re-openings at pacemaker potentials
was the cause for the small but significant inward current that, in addition to If,
contributed to diastolic depolarization (8, 9). The contribution of this current to diastolic
depolarization is best seen under ramp clamp conditions, where the membrane potential
is slowly depolarized and the net current recorded. When TTX is used to block the
contribution of any Na current, one can determine the TTX-difference current. As shown
in Fig 4 (panels A and B), there is a marked TTX-sensitive inward current during this
membrane depolarization in a newborn, but not an adult SAN cell.
A further characterization of this developmentally regulated newborn SAN Na channel
unexpectedly revealed a pharmacological profile (i.e. high TTX, and low cadmium
sensitivity) that did not correspond to that of the cardiac Na channel isoform (Nav1.5),
but was rather more typical of a neuronal isoform. The neuronal character of the Na
channel in the newborn SAN was confirmed by in situ hybridization experiments that
revealed the expression of Nav1.1 in the newborn SAN (10). This explained the
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functional contribution of the channel, since inactivation of neuronal Na channel isoforms
typically requires greater depolarization than the cardiac isoform. Thus Na current
derived from neuronal channel isoforms would have greater availability at the typical
diastolic potentials of the SAN.
The results with TTX also made it apparent that the Na current could not provide the only
difference between newborn and adult pacemaker activity. As evident in Fig 3, the Naindependent automaticity of newborn cells unveiled by the use of TTX (33 bpm) was
significantly slower than that of adult cells (68 bpm). Based on these results it appears
that the presence of a Na current during early postnatal development is a consequence of
the immature state of other sinoatrial currents, which alone are not sufficient to maintain
a cardiac rate compatible with metabolic requirements. This critical situation resolves
during the first 40 days of life in the rabbit, thus removing the necessity for the current. It
is noteworthy that while most currents contributing to pacemaker activity in the adult
such as If, Ca current and Na/Ca exchanger are highly modulated by autonomic agonists,
with cAMP dependent processes enhancing the currents, (11, 16, 119, 132) this is less
true for INa. Modulation of Na channels is isoform specific, and protein kinase A can be
associated with either an increase or decrease in current (22, 122). For this reason
understanding of the functional contribution of Na channel modulation to the control of
pacemaker mechanisms is still unclear. However, when taken together these observations
lead to the highly speculative hypothesis that during the early postnatal period the
contribution of INa ensures a failsafe mechanism protecting the animal from a potential
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parasympathetic/sympathetic imbalance, and that the necessity for this fades during
development as the autonomic nervous system reaches its mature state.
Investigation of the presence of Nav1.1 and Nav1.5 transcripts in the adult rabbit by
Tellez et al. (111) found Nav1.5 in the periphery and low levels of Nav1.1 in the center,
compatible with the almost negligible contribution of INa to adult automaticity as
previously suggested by Baruscotti et al. (7). Interpretation of these data together with
those obtained in newborn rabbit could suggest that the expression of Na channel in the
center of the node is regulated differently than the expression in the periphery. In fact, it
would be expected that Nav1.5 channels could only participate in peripheral regions due
to the more negative inactivation relation of this isoform, and thus are more involved in
the propagation of the impulse out of the node rather than to determination of rate. These
conclusions were further validated by experiments in mice having a targeted disruption of
the SCN5A (Scn5a+/-) gene. Peripheral SAN cells isolated from these animals displayed a
reduced presence of Na currents and reduced spontaneous rate; furthermore experiments
on intact SAN preparations confirmed that in Scn5a+/- mice there was a significant
slowing of SA conduction time and a propensity to sino-atrial block (67).
To our knowledge, thorough comparative studies of SAN Na currents in newborn and
adult have not been carried out in any other species, and thus the progressive
disappearance of Na currents during development could be a process typical of some, but
not all species. Indeed, studies carried out in adult mice show significant differences with
those from rabbits. Recordings from single SAN cells revealed that pacemaking rate in
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adult mice is extremely sensitive to TTX, and in fact Na currents were recorded in almost
all cells investigated (68, 75). In 2004 Lei et al. (68) concluded that although Na currents
are common throughout the whole node of adult mice, their molecular substrates are not
the same in all regions. Cardiac Nav1.5 channels are absent from the center of the SAN,
but densely populate the periphery, while Nav1.1 are homogeneously distributed
throughout the node. It is possible that the persistence of Nav1.1 expression in the adult
mouse SAN, compared to the rabbit, reflects the need to maintain a markedly higher heart
rate in the adult mouse heart. While the mechanism(s) controlling the differential
regulation of Nav1.1 and Nav1.5 are unknown, a report (101) of developmentally
regulated splice variants of the murine SCN5a gene with distinct cardiac specific
enhancer regions, and the presence of homologous human splicing, suggests a direction
for future research.
Calcium currents
Two types of calcium currents are expressed in SAN cells: the low voltage activated
(ICaL) and the high voltage-activated (ICaT) (42, 117). The relative abundance of these
two currents seems to be species-specific since, for example, in rabbits ICaL is the
predominant current while in mice the opposite situation occurs. Although several details
still need to be fully understood there is general agreement that L-type calcium current
sustains the action potential upstroke (phase 0), either alone or in combination with Na
current, and contributes to the later part of diastolic depolarization (76, 117, 132). The
specific role of ICaT is more controversial since original electrophysiological studies on
pacemaker cells were carried out in rabbits, a species in which the T-current is poorly
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expressed. With the more recent focus on pacemaking mechanisms in mice, studies have
revealed that in this species T-current participates in diastolic depolarization (78). A
further role for ICaT has been associated with its ability to locally trigger Ca2+ release
from the sarcoplasmic reticulum in atrial cells (47), although this specific action has not
yet been proved in primary pacemaker cells (77).
FIGURE 4 NEAR HERE
Given their importance, the obvious question is whether calcium currents are subjected to
developmental regulation in the SAN. As mentioned earlier, when the contribution of Na
currents in rabbit newborn cells is removed by TTX, the remaining automaticity is slower
than that of the adult (33 vs 68 bpm, (7)). A logical explanation for this observation is
that other ionic components crucial for nodal automaticity are still not fully mature in the
newborn, and the calcium currents are obvious candidates. Protas et al. (91) carried out a
detailed investigation of developmental changes in nodal ICaL and ICaT. Comparative
analysis of ICaT in newborn and adult rabbit SAN cells did not show any difference, thus
excluding it as a major player in the developmental regulation of SAN rate in the rabbit.
Taken at face value the absence of differences in newborn and adult T-type currents, in
spite of the differences in rate in the presence of TTX, might indicate that in rabbits ICaT
is not a critical determinant of rate. However, the only conclusion that can be definitively
drawn from these data is that, to the extent that ICaT contributes to automaticity in the
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rabbit SAN, it is a developmental constant. Again, this may reflect species differences
since it has been shown that spontaneous rate of Cav3.1 (one of 3 T-type isoforms)
knockout mice is significantly slower (~10%) than that of control animals (78). In
contrast, when Cav3.2 knockout mice were investigated for chronotropic performance
and atrioventricular conduction of the impulse, no alterations were observed (23).
Interestingly Niwa et al. (84) have shown that in the mouse heart Cav3.2 expression is
high during embryogenesis. Opposite to Cav3.2, Cav3.1 increases during late
embryogenesis and at birth to become the only expressed isoform found in the heart after
birth and during adulthood (37, 84). These molecular data are consistent with the
observation by Protas et al. (91) that in the rabbit the properties of newborn and adult
sinoatrial ICaT current are not different.
More interesting are the results comparing L-type Ca currents in adult and newborn (91).
In particular, development acts on ICaL currents by inducing a decrease in current
density (newborn, 17.6 pA/pF; adult, 12.1 pA/pF), a rightward shift of the inactivation
curve (midpoint: newborn, -33.4 mV; adult, -28.3 mV), and a leftward shift of the
activation curve (midpoint: newborn, -17.3 mV; adult, -22.3 mV). Recovery from
inactivation was also slower in the newborn. The decreased density of the current
observed in the adult would suggest a diminished contribution of this current during
diastole, but the opposite is likely true when one considers that the overlapping area
delimited by the activation/inactivation curves is increased in the adult due to the
opposite shift of the curves. This is illustrated in Fig 4 (panels C and D), where the
nifedipine sensitive current is shown during a slow ramp clamp to mimic diastolic
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depolarization. While the use of nifedipine during a ramp clamp can isolate currents in
addition to ICaL, such as the sustained inward current (see below and (24, 41)), it is
evident that the threshold of the nifedipine sensitive current is more negative in the adult,
so that it contributes earlier in diastole. Mean data confirm this, with the measured
threshold shifted 4 mV negative in the adult (n=6-8; P<0.05). To the extent that this
reflects L-type current it is thus likely that even though maximal current is higher in the
newborn, the more negative activation threshold, and the faster recovery from
inactivation observed in the adult could explain why in the presence of TTX the rate of
newborn cells is slower than that of adult.
The mechanisms underlying the differences observed in newborn and adult ICaL could
arise from a developmental switch between channel isoforms. Four R subunits of the Ltype calcium channels have been identified and those expressed in the heart are Cav1.2
and Cav1.3, with the latter being mainly expressed in the atrium, in the SAN and
conduction system (12, 76, 79, 109). When Cav1.3 knockout mice were generated SAN
dysfunction and disturbances of AV conduction were observed (76, 90, 134). Analysis of
sinoatrial ICaL current in Cav1.3 knockout mice revealed significant differences with
those recorded in wild type; in particular in the absence of Cav1.3 the voltage
dependence is shifted ~ 5 mV positive (midpoint from -16.6 to -11.4 mV in (134)) and
this shift demonstrates that in the SAN two different isoforms of L-type calcium channels
coexist. In particular since Cav1.3 is selectively present in supraventricular regions, and
since cardiac rate is slower when this isoform is absent, it is possible that Cav1.3 in the
SAN mostly contributes to diastolic depolarization, while contractility is contributed to
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by the Cav1.2 isoform. Interestingly when the derivative of the action potential was
measured at three different potentials (-50, -45 and -40 mV) during diastolic
depolarization in wild type and Cav1.3 deficient mice, differences were observed only for
the two more positive voltages (134). These data indicate that the contribution of Cav1.3
is indeed critical during the pacemaker phase, but not in early diastole where other
currents contribute. Whether Cav1.2 and Cav1.3 are subjected to developmental
regulation remains to be established. Based on the 5 mV developmental leftward shift in
activation observed by Protas et al. (91) and the 4 mV shift in threshold of the nifedipine
sensitive current reported above, when considered in comparison to the 5 mV shift in the
activation relation in the Cav1.3 knockout (134), it could be speculated that the immature
SAN expresses a prevalence of Cav1.2 channels, and postnatal development favors the
expression of Cav1.3. This hypothesis also agrees with the evidence that while Cav1.3 is
not necessary during heart embryogenesis, the lack of Cav1.2 is lethal. Additional
support for this hypothesis comes from the finding of Jones et al. (51) of a progressive
loss of Cav1.2 with aging in the guinea pig SAN, however it was not reported if a
concomitant increase in Cav1.3 occurred.
Other currents
Other currents have been suggested to contribute to SAN pacemaking in the adult by
various researchers, including the deactivation of the delayed rectifier current IK (48), a
nifedipine-sensitive sustained cation current called, Ist (24, 41) and Na/Ca exchange
current (74). We are not aware of any studies in the immature sinoatrial node that indicate
any of these currents vary developmentally, but this is clearly a relevant question. In
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particular the Na/Ca exchange mechanism merits study, as this is intimately associated
with other components of Ca homeostasis such as sarcoplasmic reticular Ca stores and
release mechanisms, both of which change with postnatal development in other cardiac
regions (50, 57, 113).
Conclusions
We have attempted to provide a framework for considering the developmental maturation
of the SAN in mammals. While any conclusions must be qualified because of the
possibility of substantive differences among species, it appears the SAN undergoes
substantial rearrangements both in its anatomical extension, as shown by the decrease in
the Cx43 area, and in its electrical aspects, as shown by profound modifications of the
ionic channel expression profile. All these aspects combine to determine an agedependent slowing of intrinsic heart rate and conduction time. Interestingly we have
observed that while the contribution of If and INa to pacemaking are down-regulated
during postnatal life in the rabbit SAN, the opposite is true for ICaL. Certainly these
events are controlled by an overall homeostatic developmental plan, but identification of
the common regulators is a goal that remains for future investigations, as is extension to
other species including human. What can be stated at this time is that although some
aspects are beginning to be clarified, much remains to be done before a comprehensive
view of the developmental biology of SAN function will be complete.
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Acknowledgments
Supported by NIH HL-28958. We thank Drs. Dario DiFrancesco and Michael R. Rosen
for helpful comments on the manuscript.
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Figure legends
Figure 1:
Sinoatrial node development and aging are accompanied by a progressive reduction
of Connexin 43
Distribution (top) and expression (bottom) of the junctional protein connexin 43 in the
sinoatrial node of Duncan-Hartley guinea pigs of different ages (<1 day up to 38 months)
were evaluated by immunofluorescence analysis. The area lacking Cx43 signal,
calculated from a two-dimensional reconstruction of the whole SAN obtained from
sequential slices, increased linearly with the age of the animal. The quantification of the
expression of connexin 43 was accomplished by western-blot and densitometric analysis
and normalized to desmin expression, which is not influenced by development or aging.
Data are reproduced, with permission, from panels 3B and 4B of (52).
Figure 2: The range of If current availability under autonomic control is greater in
the newborn than in the adult.
Activation curves of the pacemaker current obtained in control (continuous line) and in
the presence of maximal sympathetic (isoproterenol) or vagal (acetylcholine) activation.
The range of fractional activation at -55 mV, a voltage near the maximal diastolic
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potential, is 60% greater in the newborn than in the adult. The parameters of the
activation curves were taken from Accili et al., 1997 (2).
Figure 3: Contribution of INa to spontaneous activity decreases with post-natal
development.
Action potentials recorded in single cells isolated from an adult and a newborn rabbit
sinoatrial node exhibit a different sensitivity to the Na channel blocker TTX (3 LM).
Drug perfusion induces no significant effect (-10.6 % beats per minute, n=5, p>0.05) on
spontaneous rate in the adult animal, while it profoundly (-66.7 % bpm, n=5) alters rate in
the newborn. (modified from (7)).
Figure 4: TTX- and nifedipine-sensitive currents elicited by ramp depolarization
differ in newborn and adult SAN cells.
A, B: Whole-cell current traces obtained in control and in the presence of TTX (3 LM) by
applying a voltage ramp (from -60 mV to -20 mV) to single SAN cells at a depolarizing
rate (35 ms/s) similar to that of spontaneous diastolic depolarization (top). TTX-sensitive
current (A, B bottom) is present in the newborn, but not in the adult. C, D: Similar
protocols allow the identification of nifedipine-sensitive currents (bottom) both in the
newborn and in the adult. On average, the threshold of the nifedipine-sensitive current is
4 mV less negative in the newborn.
52
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Cx43 protein expression (%)
Area absent of Cx43
protein expression (mm2)
Page 53 of 56
50
40
30
20
10
0
Neonate
Young
Adult
Old
Senescent
70
60
50
40
30
20
10
0
Neonate
Young
Adult
Age of animal
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Old
Senescent
Normalized Activation
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0.0
0.5
1.0
1.5
-120
2.8x
-80
-60
Voltage (mV)
-100
ADULT
Normalized Activation
(Relative to Adult)
-40
0.0
0.5
1.0
1.5
-120
-80
-60
Voltage (mV)
-100
4.5x
NEWBORN
-40
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Voltage (mV)
Voltage (mV)
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-60
-40
-20
0
20
40
-60
-40
-20
0
20
40
Control
NEWBORN
0.5
Control
ADULT
1.0
Time (s)
TTX
TTX
1.5
2.0
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Current (pA/pF)
Current (pA/pF)
Current (pA/pF)
Copyright Information
Current (pA/pF)
-20
-1.5
-1.0
-0.5
0.0
0.5
-1.5
TTX
Nifedipine-sensitive
0.5
0.0
-0.5
-1.0
-1.5
-2.0
-2.0
0.0
-1.0
4.0
6.0
2.0
+Nifedipine
-1.5
-1.0
-0.5
0.0
0.5
-1.5
-1.0
-0.5
-0.5
0.5
-2.0
-60
0.0
C
-30
TTX-sensitive
-40
Voltage (mV)
0.0
0.5
-2.0
-60
0.0
Control
4.0
6.0
-1.5
-50
+TTX
2.0
A
-1.0
-0.5
0.0
NEWBORN
D
B
-50
+TTX
Control
TTX
-20
Nifedipine-sensitive
+Nifedipine
TTX-sensitive
-40
-30
Voltage (mV)
ADULT
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