Genesis and Regulation of the Heart Automaticity

Abstract

The heart automaticity is a fundamental physiological function in higher organisms. The spontaneous activity is initiated by specialized populations of cardiac cells generating periodical electrical oscillations. The exact cascade of steps initiating the pacemaker cycle in automatic cells has not yet been entirely elucidated. Nevertheless, ion channels and intracellular Ca(2+) signaling are necessary for the proper setting of the pacemaker mechanism. Here, we review the current knowledge on the cellular mechanisms underlying the generation and regulation of cardiac automaticity. We discuss evidence on the functional role of different families of ion channels in cardiac pacemaking and review recent results obtained on genetically engineered mouse strains displaying dysfunction in heart automaticity. Beside ion channels, intracellular Ca(2+) release has been indicated as an important mechanism for promoting automaticity at rest as well as for acceleration of the heart rate under sympathetic nerve input. The potential links between the activity of ion channels and Ca(2+) release will be discussed with the aim to propose an integrated framework of the mechanism of automaticity.

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88:919-982, 2008. doi:10.1152/physrev.00018.2007 Physiol Rev
Matteo E. Mangoni and Joël Nargeot
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Genesis and Regulation of the Heart Automaticity
MATTEO E. MANGONI AND JOE
¨
L NARGEOT
Institute of Functional Genomics, Department of Physiology, Centre National de la Recherche Scientifique
UMR5203, Institut National de la Sante´ et de la Recherche Me´dicale U661, University of Montpellier I and II,
Montpellier, France
I. Introduction 920
II. Automaticity in Cardiac Cells and Distribution of Pacemaker Activity in the Heart Tissue 922
A. Molecular determinants of SAN formation 922
B. Automaticity in the sinoatrial node 923
C. Properties of isolated SAN pacemaker cells 925
D. Pacemaker shift and extranodal supraventricular automaticity 926
E. Automaticity in the atrioventricular node 927
F. Automaticity in Purkinje fibers 929
III. Molecular Determinants of Ion Channels in Automatic Heart Cells 930
A. f-Channels 930
B. Voltage-dependent Ca
2⫹
Channels 931
C. St-channels 932
D. Voltage-dependent Na
⫹
channels 932
E. Voltage-dependent K
⫹
channels 932
F. G protein-activated, ATP-dependent, and inward rectifier K
⫹
channels 933
G. Cl
⫺
channels, volume-activated channels, and stretch-activated cationic channels 934
IV. Pumps and Exchange Currents 934
A. The Na
⫹
-K
⫹
pump current I
p
934
B. The Na
⫹
-Ca
2⫹
exchanger current I
NCX
and the Na
⫹
-H
⫹
exchanger 935
V. Patterns of Gene Expression in Adult Pacemaker Tissue 936
VI. Genesis of Cardiac Automaticity: Mechanisms of Pacemaking 937
A. Concepts 937
B. Ion channels and cardiac automaticity: general considerations 937
C. Role of I
Kr
and I
Ks
in automaticity 938
D. Role of I
f
in automaticity 939
E. Role of I
Ca,L
in automaticity 942
F. Role of I
Ca,T
in automaticity 945
G. Role of N- and R-type channels in heartbeat regulation 946
H. Role of I
Na
in automaticity 947
I. Role of I
st
in automaticity 948
L. SR Ca
2⫹
release and automaticity 948
VII. Autonomic Regulation of Pacemaker Activity 951
A. Principles 951
B. Sympathetic regulation of pacemaker activity 952
C. Parasympathetic regulation of pacemaking 955
VIII. Cardiac Automaticity as an Integrated Mechanism: Numerical Modeling of Pacemaker Activity 957
A. General models of automaticity 957
B. Dedicated models of automaticity 958
IX. Additional Regulators of Cardiac Automaticity 960
A. Neuropeptides 960
B. Adenosine 961
C. Hormones 961
D. Mechanical load and atrial stretch 962
E. Electrolytes and temperature 962
X. Genetic and Acquired Diseases of Cardiac Automaticity 963
A. Inherited dysfunction of SAN automaticity 963
B. Automaticity in heart failure and cardiac ischemia 965
XI. Heart Automaticity and Cardioprotection 965
A. Heart rate and cardiac morbidity 966
Physiol Rev 88: 919–982, 2008;
doi:10.1152/physrev.00018.2007.
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B. Automaticity in engineered “biological pacemakers” 966
XII. Concluding Remarks 968
Mangoni ME, Nargeot J. Genesis and Regulation of the Heart Automaticity. Physiol Rev 88: 919-982, 2008;
doi:10.1152/physrev.00018.2007.—The heart automaticity is a fundamental physiological function in higher organ-
isms. The spontaneous activity is initiated by specialized populations of cardiac cells generating periodical electrical
oscillations. The exact cascade of steps initiating the pacemaker cycle in automatic cells has not yet been entirely
elucidated. Nevertheless, ion channels and intracellular Ca
2⫹
signaling are necessary for the proper setting of the
pacemaker mechanism. Here, we review the current knowledge on the cellular mechanisms underlying the
generation and regulation of cardiac automaticity. We discuss evidence on the functional role of different families
of ion channels in cardiac pacemaking and review recent results obtained on genetically engineered mouse strains
displaying dysfunction in heart automaticity. Beside ion channels, intracellular Ca
2⫹
release has been indicated as
an important mechanism for promoting automaticity at rest as well as for acceleration of the heart rate under
sympathetic nerve input. The potential links between the activity of ion channels and Ca
2⫹
release will be discussed
with the aim to propose an integrated framework of the mechanism of automaticity.
I. INTRODUCTION
The heart pacemaker activity sets the rhythm and
rate of cardiac chamber contraction. Autonomic regula-
tion of heart rate plays a fundamental role in the integra-
tion of vital functions and influences animal behavior and
capability to respond to changing environmental condi-
tions. Heart rate has also been recently linked to the
overall risk of cardiovascular mortality and morbidity
(232).
In the adult heart of higher vertebrates, pacemaking
is generated by specialized “pacemaker” cells having low
contractility and generating a periodical electrical oscil-
lation (Fig. 1). Gene expression in cardiac pacemaker
cells seems to be qualitatively similar to that of working
myocytes, yet a quantitatively different level of expres-
sion of some ion channels, connexins (Cx), and transcrip-
tion factors generates the distinctive phenotype of spon-
taneously active myocytes (179, 317, 477). In spite of the
physiological importance of cardiac automaticity, some
aspects of the pacemaker mechanism have not been elu-
cidated and are still under debate. Indeed, different views
of the ionic and cellular basis of the pacemaker mecha-
nism and its regulation by the autonomic nervous system
have been proposed [see previous articles in this Journal
(63, 215) and Refs. 74, 120, 307].
Unraveling the mechanisms of pacemaking in spon-
taneously active cells is a fascinating and complex task.
Experimental and theoretical approaches, such as in vitro
and in vivo electrophysiology, pharmacology, and genet-
ics, as well as numerical modeling of pacemaking are
necessary. Automatic cells of the adult heart are charac-
terized by the presence of the diastolic depolarization, a
depolarizing phase that drives the membrane voltage at
the end of repolarization to the following action potential
threshold (Fig. 1B).
The pacemaker mechanism has been intensively
studied for more than 40 years. The diastolic depolariza-
tion is an electrical phenomenon, so pacemaking has been
first interpreted in terms of activation of specific ionic
currents (see Refs. 63, 215). Between 1960 and 1980,
pacemaker activity has been investigated by intracellular
recording of automaticity and ionic currents on sponta-
neously active tissue strips coming from the Purkinje fiber
network (118, 192, 485, 495) and the sinoatrial node (SAN)
(62, 364, 533). During these pioneering years, some ionic
currents involved in the generation of automaticity have
been described. Key breakthroughs were the discovery of
the hyperpolarization activated current (I
f
) in SAN (64)
and Purkinje fibers (118, 122) and the description of
the role of dihydropyridine (DHP)-sensitive Ca
2⫹
current
(I
Ca
) in the SAN action potential generation (364). Very
low inward rectifier current (I
K1
) density is found in the
rabbit SAN. In contrast, automatic tissue expresses strong
I
f
(366). The SAN action potential is predominantly con-
trolled by I
Ca
so that the action potential upstroke veloc-
ity is much lower in the SAN than in the ventricle. The
SAN repolarization phase also differs from that of the
working myocardium. Indeed, as the SAN expresses low
levels of the transient outward current (I
to
), the repolar-
ization phase is predominantly controlled by the delayed
rectifier current (I
K
) (62). Finally, the SAN action poten-
tial also lacks the plateau phase. These electrophysiolog-
ical properties confer to the SAN action potential its
typical form (Fig. 1B).
The patch-clamp technique has become popular for
studying the ionic basis of pacemaker activity on individ-
ual pacemaker cells (215). Different families of ion chan-
nels have been described (58). Two distinct I
Ca
compo-
nents have been identified (172): the L-type Ca
2⫹
current
(I
Ca,L
) and the T-type current (I
Ca,T
). A fast (I
Kr
) (451) and
a slow (I
Ks
) (278) delayed rectifier has been reported.
Furthermore, application of the patch-clamp technique
has improved the study of the sensitivity of these chan-
nels to autonomic agonists. Two major insights into the
physiological role of ion channels in the generation and
920
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¨
L NARGEOT
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regulation of pacemaker activity have been obtained: the
relevance of f-channels in the regulation of heart rate at
low parasympathetic tone (124) and the physiological
significance of the heterogeneity of ion channel expres-
sion in the SAN (58). The importance of the Na
⫹
-Ca
2⫹
exchanger (NCX) in pacemaking in the amphibian sinus
venosus (227, 228) and mammalian SAN (44) has been
recently highlighted, and the contribution of spontaneous
intracellular Ca
2⫹
release in the generation of automatic-
ity has been emphasized (510).
New important insights into the pacemaker mecha-
nism are now coming from the study of genetically mod-
ified mouse strains in which specific ion channels have
been inactivated or modified. This genetic approach con-
stitutes a necessary implementation to electrophysiologi-
cal and pharmacological evidence, since selective inhibi-
tors of ion channels are not always available. Gene tar-
geting in the mouse has generated interesting models of
dysfunction of pacemaker activity (393, 467, 522). The
possibility to study pacemaker activity in mouse models is
very recent (312, 313) and has provided exciting insights
into the specific functional role of L-type Ca
v
1.3 (309, 546)
and T-type Ca
v
3.1 (314) channels in the generation of SAN
automaticity. Because of the tiny size of the dominant
pacemaker region in the mouse heart (498), isolation of
mouse SAN cells is technically challenging. Our group has
been the first to obtain recordings of automaticity and ion
channels from isolated mouse SAN cells (312, 313). Other
groups have successfully employed this new preparation
to study pacemaker activity in wild-type (86, 93, 279) and
genetically modified mouse strains (196, 294, 309, 314,
546). Limitations of the use of the mouse for the study of
cardiac automaticity are correlated with the very high
basal heart rate of this species. We can thus expect that
pacemaker mechanisms may assume a different role in
mice than in larger mammals and humans. Nevertheless,
the new possibility of isolating mouse SAN tissue and
primary pacemaker cells has created a new interest into
the study of the ionic basis of pacemaker activity gener-
ation and regulation.
Until the last decade, research on the physiology of
heart automaticity was limited to the domain of basic
science. However, there is currently a renewal of interest
on cardiac pacemaking, and an increasing number of
laboratories are now focusing their efforts on the regula-
tion of heart rate. Indeed, the pharmacological control of
cardiac automaticity is now becoming an important issue
in the management of ischemic heart diseases (123). Iden-
FIG.1.A: the mammalian heart with the cardiac conduction system.
The sinoatrial node (SAN) is located at the entry of the superior vena
cava (SCV) in the right atrium (RA). The atrioventricular node (AVN)
extends in a region delimited by the inferior vena cava (ICV), the central
fibrous body (CFB), and the tricuspid valve (TV). The atrioventricular
bundle (AVB) divides in the bundle branches (BB) and originates the left
and right Purkinje fibers network (PFN). Other abbreviations are: LA,
left atrium; PV, pulmonary veins; MV, mitral valve; RV, right ventricle;
LV, left ventricle. [Adapted from Moorman and Christoffels (341).]
B: recordings of automaticity and action potential waveforms of isolated
mouse SAN, AVN, and PFN cells. Cl, cycle length; APD, action potential
duration; E
th
, action potential threshold; LDD, linear part of the diastolic
depolarization; EDD, exponential part of the diastolic depolarization.
(From Marger Nargeot and Matteo Mangoni, unpublished observations.)
GENESIS AND REGULATION OF THE HEART AUTOMATICITY
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tification of ion channels involved in the generation of
automaticity also constitutes the basis for the develop-
ment of putative genetic and cellular therapies for dys-
functions of pacemaking (94, 423, 424). Finally, knowl-
edge of pacemaker mechanisms is fundamental for under-
standing the basis of inherited diseases of the heart
rhythm.
In this review, we will focus on the cellular mechanisms
underlying heart automaticity and its regulation by the au-
tonomic nervous system in the adult heart. Different views
of automaticity will be compared and commented. To this
aim, we will try to give an overview of how ion channels and
Ca
2⫹
signaling can influence pacemaking under different
physiological conditions. Finally, we will provide the reader
with a short discussion on how ion channels involved in
pacemaker activity are becoming useful targets in the devel-
opment of new therapies for heart diseases.
II. AUTOMATICITY IN CARDIAC CELLS AND
DISTRIBUTION OF PACEMAKER ACTIVITY
IN THE HEART TISSUE
In the mammalian heart, three major structures are
endowed with automaticity and are able to drive the
heartbeat: the SAN, which constitutes the physiological
pacemaker, the atrioventricular node (AVN), and the Pur-
kinje fibers network (Fig. 1A). Functional evidence in
favor of a supraventricular dominant pacemaker was ob-
tained by Gaskell in the slowly beating tortoise heart (154,
155). Anatomically, the SAN was identified a century ago
by Keith and Flack (237), shortly after Tawara’s descrip-
tion of the atrioventricular junction (476; see Ref. 460 for
review). The intrinsic SAN beating rate is normally faster
than that of the cardiac conduction system and sup-
presses pacemaking in the AVN and Purkinje network.
However, automaticity in AVN can become dominant in
case of SAN block or failure (220). Purkinje fibers can
also generate a viable rhythm in conditions of atrioven-
tricular block. For these reasons, the SAN region is indi-
cated as the primary pacemaker, while the AVN and Pur-
kinje fibers are indicated as secondary (or accessory)
pacemakers. In this section, we review basic principles of
automaticity in primary and secondary pacemaking re-
gions. Some aspects of the structure and function of SAN,
AVN, and the His-Purkinje system are commented on.
A. Molecular Determinants of SAN Formation
In the early embryonic heart, all cardiomyocytes dis-
play automaticity. The embryonic myocardium of the pri-
mary heart tube is characterized by slow conduction ve-
locity, and even if dominant pacemaker activity is present
at the venous pole, automaticity can be originated at any
point of the heart tube (341). The differentiation of the
cardiac chambers (heart ballooning) and the maturation
of myocytes forming the working myocardium of the late
embryo are associated with the disappearance of this kind
of “diffuse” automaticity. Working myocytes of the ma-
ture cardiac chambers have completely lost automaticity
and display fast conduction velocity (see Refs. 341, 386
for review). In the adult heart, pacemaker activity is con-
fined to the SAN and AVN, two regions in which the
intercellular conduction velocity is relatively slow. The
mechanisms underlying the confinement of pacemaker
activity and the patterning of the SAN and AVN during
development are mostly unresolved. However, progress
has been recently obtaining in the identification of tran-
scription factors involved in the differentiation of cell
lineages contributing to the SAN formation (207, 208, 338,
341).
Mommersteeg et al. (338) reported that the SAN de-
velops in the inflow tract region of the embryonic heart
(Fig. 2). This region is characterized by expression of the
T-box transcription factor Tbx3 and the Hcn4 ion channel
gene (coding for f-channels). This SAN primordium is also
devoid of Cx40, which is typical of fast-conducting work-
ing myocardium (Fig. 2). Mommersteeg et al. (338) have
also provided evidence that the Nkx2–5 transcription fac-
tor (NK2 transcription factor related to locus 5 of Dro-
sophila) may act as a repressor of the SAN lineage gene
program, since mice lacking Nkx2–5 show ectopic expres-
sion of Tbx3 and Hcn4 throughout the heart tube. Intact
activity of Nkx2–5 is also required to suppress Tbx3 ex-
pression outside the developing SAN and to trigger the
atrial gene expression program (338). If Nkx2–5 appears
to suppress SAN formation through inhibition of HCN4
expression and other markers, Tbx3 constitutes an acti-
vator of the SAN gene expression program (208) and a
repressor of heart chamber formation. Expression of Tbx3
in the whole heart tube suppresses chamber formation
(207). Mouse embryos in which Tbx3 has been inactivated
show that this gene is essential to activate the SAN related
gene expression program, and to prevent expression of myo-
cardial markers in the SAN region. Ectopic expression of
Tbx3 in the adult atria of transgenic mice is sufficient to
reactivate focal automaticity and some SAN markers (208).
Hoogars et al. (208) have proposed that SAN forma-
tion (at least starting from E10) is due to proliferation of
Tbx3-expressing cells rather than to recruitment of myo-
cytes having initiated the atrial gene program. Also, label-
ing of cell lineages suggests that the SAN is to be consid-
ered as a remnant of cell populations that did not enter
the myocardial expression program (338). Blaschke et al.
(40) have reported that the homeodomain transcription
factor Shox2 is involved in the formation of the SAN
region. Particularly, Shox2 knockout mice die around E12
and show severe SAN hypoplasia. Furthermore, zebrafish
embryos lacking Shox2 are bradycardic, an observation
922
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¨
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which is consistent with the involvement of Shox2 in the
determination of the pacemaker tissue (40).
B. Automaticity in the Sinoatrial Node
In this section, we aim to provide a concise overview
of automaticity in the SAN. Some fundamental structural
aspects of the SAN cellular and electrophysiological or-
ganization will be discussed. Because the rabbit has been
extensively used as an animal model to study the mech-
anisms of pacemaking, we will focus on results obtained
from the rabbit atrial-SAN preparation. Further discus-
sion of the SAN structure and variations between species
can be found in some excellent reviews by Opthof (374)
and Boyett and co-workers (57, 58).
SAN pacemaker tissue is located in the intercaval
region (Fig. 3A) and extends towards the endocardial side
of the crista terminalis (Fig. 3, B–D). Spontaneously ac-
tive cells are found in the area delimited by the crista
terminalis, the left and right branch of the sinoatrial ring
bundle and the interatrial septum (Fig. 3, B–D). Beside
pacemaker cells, the SAN also contains atrial cells, fibro-
blasts, and adipocytes (130). The collagen content of the
SAN region is relatively high (377). However, collagen
does not seem to have an adverse effect on SAN conduc-
tion, because species having different SAN collagen con-
tent display similar conduction times (377). During aging,
the size and position of SAN do not change, but its struc-
ture undergoes remodeling associated with an augmenta-
tion of collagen content (6).
The overall electrical coupling within the SAN is
weak (see Ref. 58 for review). Two lines of evidence
indicate that SAN automaticity is in fact partially sup-
pressed by the electrotonic load imposed by the atrium.
First, removal of the right atrium results in an accelera-
tion of SAN pacing rate (241, 248). Second, direct cou-
pling of a SAN cell to an atrial cell suppresses pacemaker
activity (224, 482). However, a relatively low electrical
coupling between pacemaker cells seems to be necessary
for the SAN to be able to drive the atrium. Indeed, by
employing numerical modeling, Joyner and van Cappele
(225) have indicated that the SAN should have a minimal
size to drive the atrium, but also that intranodal cellular
coupling should be relatively weak to protect pacemaking
from the hyperpolarizing electrical load imposed by the
atrium. Watanabe et al. (514) have also used numerical
modeling to show that stronger electrical coupling be-
tween SAN and atrium would stop pacemaking.
The SAN is structurally heterogeneous. Two views of
the cellular organization of the SAN have been proposed.
In the “gradient” model of SAN (58), there is a progressive
transition in the size and electrical properties of pace-
maker cells between the SAN center and the periphery.
Cells in the center (labeled in red in Fig. 3) are small and
have intrinsically slower pacing rate and upstroke veloc-
ity compared with that at the periphery (labeled in blue in
Fig. 3), which have faster pacing rate, upstroke velocity,
and intermediate properties between “pure” pacemaker
and atrial cells. Verheijck et al. (499) have proposed that
the SAN region is constituted by automatic cells having
FIG.2.A: computer-based 3-dimensional reconstructions (465) of the mouse embryonic heart at 14.5 post coitum (E14.5). In this reconstruction,
the left ventricular myocardium has been removed to show the blood-filled lumen (orange), from the dorsal (top panels) and the right side (bottom
panels). The Tbx3-positive myocardium is in red, and the Cx40-negative myocardium is in grey. Note that expression of Tbx3 is restricted to areas
in which Cx40 expression is absent. B: schematic representation which shows the patterns of expression of critical markers in the developing fast
conducting myocardium or SAN primordia (see sect. IIA). Left panel delineates the expression pattern until E9.5, and middle panel shows the pattern
between E9.5 and E14.5. Dotted lines identify borders of Nkx2–5 expression domains. Except for the yellow-colored mesenchyme, only myocardium
is depicted (see legend in right panel). Abbreviations are as follows: as, atrial septum; avc, atrioventricular canal; ift, inflow tract; la/ra, left and right
atrium; laa/raa, left and right atrial appendage; lsh/rsh, left and right sinus horn; lv/rv, left and right ventricle; pv, pulmonary vein; san, sinoatrial node;
vv, venous valves. [From Mommersteeg et al. (338).]
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variable intrinsic pacing rate and action potential config-
uration. Contrary to the “gradient” model, there would be
no preferential distribution of small pacemaker cells in
the SAN center. Indeed, pacemaker cells having different
degrees of automaticity and action potential configuration
are supposed to be uniformly distributed in the SAN
region. Verheijck et al. (499) have indicated that atrial
cells are present in the SAN, their density being maximal
in the SAN periphery and minimal in the center. This
latter view of the SAN structure has been called the
“mosaic” model.
Consistent with the gradient model of SAN is the
observation that tissue balls isolated from the rabbit SAN
periphery display a distinct electrophysiological profile
compared with that from the center (57, 60, 248, 250, 355,
370) (Fig. 4A). More importantly, a “central” and “periph-
eral” SAN can be defined by using electrophysiological
and histochemical criteria (57, 130). Consistently with the
mosaic model, atrial cells have been unambiguously iden-
tified in the peripheral SAN by using a Cx40
EGFP⫹/⫺
knock-in mouse line (331) and by direct staining with
Cx40 antibodies (130). Verhejick et al. (498) have re-
corded atrial action potential activity in the intact mouse
SAN (Fig. 4B). However, atrial cells seem to be absent in
the rabbit central SAN. In conclusion, even if the gradient
model seems to explain better SAN electrophysiological
behavior, certain elements of the mosaic model are to be
included in future structural SAN models.
Dobrzinsky et al. (130) have recently proposed the
first comprehensive structural computer model of the
rabbit SAN (see Fig. 3). In this model, peripheral SAN
tissue constitutes the SAN impulse exit pathway. Periph-
eral cells can be defined by membrane expression of Cx43
and middle neurofilament (NF-M) protein. SAN peripheral
cells are large and predominantly arranged in parallel.
Cells in the SAN center are smaller and express the
low-conductance Cx45 and NF-M. Central pacemakers
are arranged in a mesh and wrapped around bundles of
connective tissue. The mouse SAN seems to share some
common features with the rabbit SAN (289). In the central
mouse SAN, cells are packed in a compact structure
(compact node) and are oriented perpendicularly to the
crista terminalis (289). Cells at the periphery are loosely
packed and are oriented parallel to the crista. Numerical
simulations of impulse propagation from the leading
pacemaker site to the atrium have indicated that the SAN
could not sustain atrial beating if cellular coupling and
conduction were uniformly low throughout the SAN
(224). Expression of different connexins in the SAN cen-
ter and periphery contributes to create a gradient in im-
pulse conduction velocity from the leading pacemaker
site to SAN periphery and exit zone, so that conduction
velocity in central SAN is lower than that measured in the
periphery (see Ref. 61 for review). Beside Cx45, expres-
sion of Cx30.2 has been also reported in the mouse SAN
FIG. 3. A 3-dimensional computer model of the rabbit SAN as
proposed by Dobrzynski et al. (130). In all panels, central SAN tissue is
labeled in red, while peripheral SAN tissue is depicted in green. The
yellow dot indicates the leading SAN pacemaking site, and the blue line
indicates the SAN ring. A: topographical organization of the rabbit SAN,
as viewed in an ideal intact heart diagram. Note the extension of
peripheral SAN tissue around the superior vena cava (see also Fig. 6C).
Ao, aorta; PA, pulmonary artery; CS, coronary sinus. Other abbreviations
are as in Fig. 1. B and C: endocardial (left) and epicardial (right) view of
SAN central and peripheral tissue. In B, SAN myocytes are shown in a
simple framework model of the right atrium. In C, myocyte orientation
is shown. Note the mesh organization of central SAN tissue. Peripheral
SAN myocytes are predominantly oriented perpendicularly to the crista
terminalis and extend toward the interatrial septum (SEP). D: model
section of the SAN cut at the level of the white line in C. Note that
peripheral SAN tissue extends onto the endocardial side of the crista
terminalis. Central SAN myocytes are embedded in connective tissue.
[From Dobrzynski et al. (130).]
924 MATTEO E. MANGONI AND JOE
¨
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and AVN (257). Cx30.2 has smaller conductance than
Cx45 (70). It has been proposed that Cx30.2 contributes to
the intrinsically slow conduction velocity in the SAN and
in the AVN (258). This view is supported by the observa-
tion that mice in which Cx30.2 has been inactivated have
shortened P-Q intervals (256).
Expression of Cx43 in the SAN periphery enhances
cell-to-cell electrical coupling thereby forming a “transi-
tional” zone between the slow-conducting leading pace-
maker site and the fast-conducting atrial tissue (130).
Kodama et al. (248) have shown that small tissue
balls from the SAN periphery have intrinsically faster
pacing rate than balls from the central SAN (Fig. 3A).
However, as reported by Bleeker (41), the central SAN
can lead pacemaker activity in spite of its lower beating
rate, because cells at the periphery are electrotonically
inhibited by the right atrium. Differences in intrinsic firing
rates of central and peripheral SAN are due to heteroge-
neous expression of ion channels (60, 250, 355) and pro-
teins involved in Ca
2⫹
homeostasis (267, 349). Particu-
larly, larger cells from SAN periphery express higher I
f
and I
Na
densities than small cells in the SAN center.
Enhanced expression of I
f
and I
Na
can explain the faster
pacing rate and upstroke velocity recorded in tissue from
the SAN periphery. The stronger pacemaker activity in
cells of the SAN periphery can help overcome the sup-
pressive effect of the right atrium on the overall SAN
automaticity (58). It would be important to develop a
comprehensive model of SAN activity including both
structural and electrophysiological data. Theoretically,
such a model could be integrated in an anatomical and
biophysically detailed three-dimensional model of the
heart (102) and would be an important step for under-
standing some integrative properties of heart automatic-
ity at the organ level.
The physiological role of fibroblasts in the SAN has
not been completely elucidated. Fibroblasts form an ex-
tensive network in the SAN (73, 105). Camelliti et al. (73)
have shown that fibroblasts can functionally connect with
SAN myocytes, possibly by Cx45-mediated gap junctions.
Apparently, SAN fibroblasts do not participate to intran-
odal conduction (105). However, they may act in the
transduction of the atrial wall stress to SAN myocytes. In
this respect, Kohl et al. (252) have proposed that fibro-
blasts participate in the mechanoelectrical regulation of
SAN rate.
C. Properties of Isolated SAN Pacemaker Cells
Isolated SAN pacemaker cells are a widespread ex-
perimental model for studying the pacemaker mecha-
nism. Several research groups have described the electro-
physiological properties of enzymatically isolated calci-
um-tolerant (216) pacemaker cells from a variety of
mammals including the rabbit (114, 125, 492), guinea pig
(12, 333), pig (372), rat (457), and mouse (86, 313). The
gross morphology of spontaneously active cells is con-
served between species. Cells from the rabbit and mouse
SAN have been empirically classified in three distinct
morphologies, namely, “spindle,” “elongated,” and “spi-
der” (125, 313, 499) (Fig. 5, A and B). In the rabbit and
mouse, spindle-shaped cells are generally smaller than
elongated and spider cells, their capacitance varying be-
tween ⬃15 and 30 pF. Under Nomarski optics, the cyto-
plasm of spindle cells appears transparent and poor in
myofilaments. Elongated cells have a longer longitudinal
axis and larger capacitance (between 35 and 50 pF) than
spindle-shaped cells. In some elongated cells, the cyto-
plasm is darker than in spindle cells. This is possibly due
FIG.4.A: pacemaker activity of isolated tissue balls from the SAN center, periphery and transitional zone. Tissue from the SAN center has
intrinsically slower automaticity and more positive maximum diastolic potential, while peripheral and transitional tissues are intrinsically faster and
have a more negative maximum diastolic potential. [Original recordings from Boyett et al. (59).] B: intracellular microelectrode recordings of
automaticity in an isolated mouse SAN. The leading pacemaker site is indicated by asterisk. Note the presence of the diastolic depolarization phase
in this recording site (right panel) and suppression of automaticity in a location in the vicinity of the leading site (left panel), and in the SAN
periphery close to the interatrial septum (IAS). Atrial-type action potentials are recorded in the mouse SAN (right panel). Automaticity is not
recorded in the crista terminalis (CT) and in the right branch of the sinoatrial ring (SARB). [Original recordings from Verheijck et al. (498), with
permission from Elsevier.]
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to a higher myofilament density than in spindle cells (Fig.
5A). Spider cells have branched cytoplasm (see Fig. 5C).
The functional significance of such a peculiar morphology
is not known. In the rabbit, small spindle-shaped cells are
considered to be leading pacemaker cells (presumably
from the SAN center), while larger elongated cells come
from the SAN periphery (204). However, this may not be
an absolute rule, since small clusters of mouse SAN cells
containing both spindle and elongated cells have been
observed in the mouse SAN (499) (Mangoni and Nargeot,
unpublished observations).
D. Pacemaker Shift and Extranodal
Supraventricular Automaticity
The position of the leading pacemaker site is not
fixed. The pacemaker initiation site can shift in different
zones of the SAN. Pacemaker shifts can be induced by
autonomic regulatory inputs, temperature, atrial stretch,
as well as by pharmacological interventions (Fig. 6A) (58,
442). Experimentally, pacemaker shift has been studied in
the rabbit SAN (374), but naturally occurs also in dogs
(47) and humans. Variability in the position of the leading
pacemaker site in human subjects has been shown by
Boineau et al. (46). It is possible that pacemaker shift
underlies dynamic changes in the morphology of the P-
wave observed in ECG recordings (442). Schuessler et al.
(442) have reported that pacemaking can be located at
any point of the intercaval region as well as, in some
cases, in the left atrium. Interestingly, cells from the
sleeves of pulmonary veins have recently been shown to
display spontaneous activity (83, 205).
The leading pacemaker site is situated where local
automaticity is faster. We can thus expect that sympa-
thetic input will shift the leading site where automaticity
is most sensitive to adrenergic stimulation, while para-
sympathetic activity shifts pacemaking where automatic-
ity is less inhibited by cholinergic transmitters (300).
Boyett et al. (58) have attributed pacemaker shift to the
heterogeneous expression of ion channels in the SAN
center and periphery. Accordingly, a given channel blocker
will shift pacemaking to a site where automaticity is less
dependent from the targeted channel. This hypothesis has
been experimentally verified by using Ca
2⫹
(250), K
⫹
(249),
and f-channel inhibitors (355). In the rabbit, shifts induced
by epinephrine and acetylcholine roughly correspond to
SAN zones in which I
f
block has very moderate effect on
pacing rate, an observation that matches the framework
proposed for channel blockers (58). Yamamoto et al. (531)
have shown that application of isoproterenol to intact atria
can shift the leading pacemaker site from the central SAN, to
a location between the superior vena cava and the interatrial
groove. Under basal conditions, automaticity in this site is
less robust than in the SAN, but under isoproterenol, it can
be as fast as in the SAN. These observations are indicative of
the existence of multiple functional pacemaker sites outside
the “classical” SAN region (Fig. 6B).
There is now substantial evidence indicating that
myocytes capable to drive pacemaking are present out-
side the SAN. Indeed, spontaneously beating cells can be
isolated from the right atrium (408, 427) and from the
bundle branches of the mouse heart (Mangoni, unpub-
lished observations). Automatic cells are also present
around the tricuspid valve (11). These cells are very likely
to be part of an extranodal extension of the atrial con-
duction system. Importantly, the existence of such an
extension has been recently demonstrated by staining of
FIG. 5. Morphology of isolated mouse (A) and rabbit (B) SAN cells.
Atrial cells can be isolated from the SAN in both species. C: a simple
structural model of a rabbit SAN spider cell. These cells have branched
cytoplasm (arrows) and are mononucleated. [Photographs in A from
Mangoni and Nargeot (312), with permission from Elsevier; pictures in B
and C from Verheijck et al. (499).]
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rabbit and rat atria with antibodies directed against Cx45
and HCN4 (Fig. 6C) (531). Particularly, it has been shown
that cells having “nodal” phenotype extend from the rear
of the superior vena cava to the interatrial groove. Nodal-
like cells with high expression of both Cx45 and HCN4 are
present around the atrioventricular valves (the atrioven-
tricular ring bundles) and reach the AVN (531). The pres-
ence of an atrial conduction system extension may be due
to common embryonic origin and/or development of
these cells with that of SAN and AVN (411).
The physiological significance of automaticity in la-
tent pacemaker cells of the right atrium is unclear. These
cells have been shown to be capable of assuming the
control of the heartbeat under conditions of SAN failure
and to play a role in the generation of atrial arrhythmias
(426). In the left atrium, cells generating focal discharge
have been found at the boundary between the left atrium
and the pulmonary veins in human subjects affected of
paroxystic atrial fibrillation (532). The key role of these
cells in triggering and sustaining atrial fibrillations has
been clearly recognized (175, 176). Tissue sleeves from
rabbit pulmonary veins display transient automaticity
triggered by rapid pacing, application of ryanodine (205,
531), and stretch (83). A diastolic depolarization phase
can be recorded in these cells, but automaticity is insen-
sitive to I
f
block by Cs
⫹
(531). The expression of ionic
currents in these cells resembles much more that of non-
automatic atrial than pacemaker cells (141). The ionic
mechanism responsible for pacemaking will need inves-
tigation in the near future.
In conclusion, one can wonder why some automatic
sites outside the SAN (e.g., the interatrial groove exten-
sion) can effectively sustain normal pacemaking while
others can trigger atrial arrhythmias. In this respect, it is
worth noting that pacemaking in these regions is intrinsi-
cally less robust than that of the SAN (or less sensitive to
autonomic regulation). Consequently, in case of activa-
tion of these sites, atrial activation may not have the
correct space- and frequency-dependent properties.
E. Automaticity in the Atrioventricular Node
The AVN sets the appropriate frequency-dependent
conduction delay between the atria and ventricles. It also
limits ventricular activation during atrial tachyarrhyth-
mias, thereby protecting ventricular rhythm. The AVN has
a dual electrical input from the atrium, namely, the “fast”
FIG. 6. Pacemaker shift and extranodal supraventricular automa-
ticity in the rabbit heart. A: shift of the leading pacemaker site inside the
rabbit SAN region. In the left panel, SAN activation after impulse gen-
eration in a central leading pacemaker site (marked as the point acti-
vating at zero time) is depicted. Isochronal curves indicate the spread of
impulse conduction toward the crista terminalis. After activation of the
crista terminalis, the impulse propagates in the right atrium (30 ms
isochronal) and to the right and left branches of the SAN ring bundles
(RBSARB and LBARB). Note the block of impulse conduction in the
direction of the interatrial septum. [From Boyett et al. (59).] The right
panel indicates the shift of the leading pacemaker site under the influ-
ence of different ionic and pharmacological agents. [From Boyett et al.
(58).] B: variability of the position of the leading pacemaking site in
isolated rabbit hearts. The leading site can be found inside and some-
times outside the “classical” SAN region. Asterisks indicate the position
of the impulse origin in each of the 18 preparations tested. C: dorsal view
of the left and right atria showing the extension of the atrial conduction
system in the rabbit heart. Extension is defined according to extranodal
distribution of HCN4 (red) and Cx45 (yellow) immunoreactivity (see
text of sect. IIC). Positively stained cells are found around the mitral and
tricuspid valves, as well in the interatrial groove. [Modified from
Yamamoto et al. (531).]
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and “slow” pathways of conduction (336, 356). However,
the AVN is also endowed of automaticity. AVN automa-
ticity can effectively drive the heart after SAN failure or
block.
The intact AVN is a complex and highly heteroge-
neous structure. For a recent review on the AVN structure
and conduction, the reader is referred to Efimov et al.
(140). Here, we will discuss the principal aspects of the
atrioventricular junction and automaticity. In spite of a
growing knowledge about the histology and electrophys-
iology of AVN, a detailed correlation between the differ-
ent anatomical components and action potential configu-
rations recorded from the AVN is not yet available. De-
bate still exists between morphologists and physiologists
about which structures of the atrioventricular junction
should be included in an ideal “true” AVN (140). In this
review, we will endorse the physiological definition of the
AVN, as indicated by Billette (39). This includes all struc-
tures contributing to the atrial-His conduction interval.
We thus define the AVN as the region comprised in the
Koch’s triangle, with the heterogeneous structure in-
cluded in the central fibrous body, as well as the posterior
nodal extension (PNE) projecting in the isthmus below
the coronary sinus (Fig. 7A). The central fibrous body
contains the enclosed part of the AVN with the compact
node (CN) and lies at the apex of the triangle. The action
potential configuration and expression of ion channels in
the AVN are heterogeneous. Three types of action poten-
tial waveforms have been identified in early studies (see
Ref. 140 for review): atrionodal (AN), nodal (N), and
nodo-His (NH). The N-type action potential is character-
ized by slow upstroke velocity and action potential am-
plitude, while AN and NH action potentials have interme-
diate properties between nodal and atrial or His action
potentials, respectively. Billette (38) has presented a mi-
croelectrode mapping study of the AVN and defined six
cell types based on action potential morphology and re-
fractoriness. It would be very important to build a com-
prehensive model of the functioning of the AVN. How-
ever, it is still difficult to correlate action potential types
with the structural organization of the atrioventricular
tissue. Histologically, the AVN is composed by a “super-
ficial” (subendocardial) layer and “middle” and “deep”
innermost layers (37a). Based on simultaneous microelec-
trode recording and optical mapping of rabbit AVN acti-
vation under SAN rhythm, Efimov and Mazgalev (139)
have supported the hypothesis that the superficial layer is
predominantly composed by AN-type cells, while the mid-
dle layer is composed of cells having N-type properties.
The AVN superficial layer is rich in Na
⫹
channels (391)
and Cx43, while the intermediate layer forming the CN is
essentially poor in Na
⫹
channels (391, 441). Furthermore,
the intermediate layer expresses the low-conductance
Cx45 rather than Cx43 or Cx40 (61, 140). Consistently,
lower conduction velocities are recorded in the middle
N-type layer than in the superficial AN-type layer (139).
FIG.7.A: a computer model of the rabbit atrioventricular junction
shows the basic structural organization of the AVN. The AVN is a hetero-
geneous structure delimited by the tricuspid valve, the tendon of Todaro,
and the coronary sinus. The posterior nodal extension (PNE) is depicted in
red and constitutes a major site of origin of junctional AVN automaticity. It
can also be part of the slow pathway of atrioventricular conduction. The
enclosed node is shown in purple and is continuous to the PNE. The green
zone corresponds to AVN tissue composed by loosely packed atrial cells. It
is possible that these atrial cells are part of the fast AVN conduction
pathway. [From Boyett et al. J Electrocardiol 38 Suppl: 113-120, 2005, with
permission from Elsevier.] B: mapping of automaticity in the rabbit AVN.
Pacemaking originates in the PNE and spreads to the atrial muscle and to
the enclosed node (top panel). Optical recording of automaticity in the PNE
is shown in the bottom panel.
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The location of the leading AVN pacemaker site has
been a matter of debate. Initiation of automaticity has
been identified in the N-NH part of the node (140, 515).
Recently, Dobrzynski et al. (131) attempted to locate the
site of origin of pacemaker activity in isolated rabbit AVN
preparations. These authors have employed optical map-
ping with voltage-sensitive dyes to study the spread of
excitation from the automatic site to the fast and slow
AVN conduction pathways. In 14 preparations studied,
pacemaker activity originated in the PNE in 10 prepara-
tions (Fig. 7B). Pacemaking originated in the N/NH region
of AVN in four other preparations. Dobrzynski et al. (131)
also reported that the PNE pacemaker site expresses high
density of neurofilament 160 (NF160), Cx45, and the
HCN4 protein which codes for “pacemaker” f-channels.
Finally, the PNE can constitute a region of slow conduc-
tion during AVN reentry and premature beats (131).
In conclusion, the PNE extension of AVN is able to
generate pacemaking and can effectively pace the atrio-
ventricular junction. It is thus possible that the leading
pacemaker site in AVN more frequently originates in the
PNE, which is also part of the slow AVN conduction
pathway. It is possible that in vivo both the PNE and
NH-CS region can generate junctional automaticity. It
would be interesting to test if pacemaker shift exists in
the AVN. Indeed, it cannot be excluded that the dominant
site can shift between the PNE and the NH-CS region
depending on the physiological conditions.
AVN cells have been successfully isolated from rab-
bit (187, 347), guinea pig (539), and mouse (314). Individ-
ual rabbit AVN cells display two different phenotypes:
“ovoid” cells resembling spindle SAN cells and “rod-
shaped” cells. Munk et al. (347) have reported action
potential waveforms having AN, N, and NH properties in
isolated rabbit AVN cells. These authors reported that N
and NH action potential waveforms were typical for ovoid
cells, while AN waveforms were associated with rod-
shaped cellular morphology. It seems a consistent finding
that the majority of ovoid cells are spontaneously active
(347, 539). Some rod-shaped cells are also automatic
(187), but pacemaker activity is present in a more nega-
tive diastolic range (347). As to ionic currents, ovoid cells
express relatively high densities of I
f
and I
Ca,L
(347, 539).
However, rod-shaped cells seem to lack I
f
(187, 347).
F. Automaticity in Purkinje Fibers
The intrinsic conduction velocity of ventricular myo-
cardium is not sufficient to achieve synchronized contrac-
tion of the cardiac chambers. The Purkinje fiber network
ensures a proper propagation of the cardiac impulse along
the ventricular myocardium. Purkinje fibers are very fast
conducting. The upstroke velocity in Purkinje fibers (429)
and cells (72) can approach 1,000 V/s, a value at least
threefold that of ventricular muscle (203). Quick conduc-
tion in Purkinje fibers is due to high expression of both
Na
⫹
channels (101, 191) and Cxs (245). Furthermore, the
intercellular resistance to impulse conduction in the Pur-
kinje network of different mammals can be as low as 100
⍀/cm, approaching that of the intracellular milieu (72,
335).
The Purkinje fibers network can pace the heart, in
case of complete atrioventricular block. Automaticity in
Purkinje fibers can become important, because conduc-
tion in the AVN and bundle branches is prone to block
under different genetic and physiopathological condi-
tions. Automaticity in Purkinje fibers is slower than in the
SAN and AVN. Depending on the species considered,
pacemaking rate in Pukinje fibers varies between 25
and 40 beats/min, which is sufficient to set a viable
cardiac output. Action potentials in Purkinje fibers are
similar but not identical to those of ventricular myo-
cardium and display longer action potential duration
and a more negative diastolic potential than SAN and
AVN cells (Fig. 8B) (13, 72, 331, 501). Verkerk et al.
(501) have shown heterogeneity in the duration of re-
polarization in sheep Purkinje cells (PCs). According to
these authors, sheep PCs can display either prominent
phase I repolarization and short-lasting plateau phase
or absence of phase I associated with longer plateau
phase. Early afterdepolarizations (EADs) are often ob-
served in intact Purkinje fibers (13) and isolated PCs
(Fig. 8D) (331). Spontaneous EADs in the Purkinje
network can constitute an important trigger of ventric-
ular arrhythmias (13).
Compared with pacemaker cells from the SAN and
AVN, spontaneously beating PCs have distinct morphol-
ogy and electrophysiological properties (Fig. 8, C and D).
After enzymatic isolation, PCs can be distinguished in
vitro from ventricular and SAN cells by their shape and
size (72, 331). PCs are mainly rod-shaped and larger than
SAN and AVN cells, but generally smaller than ventricular
myocytes (72, 331) (Fig. 8C). PCs have been successfully
isolated from the mouse heart by using a transgenic
mouse line in which the enhanced green fluorescent pro-
tein (EGFP) has been knocked in the gene coding for
Cx40 (Cx40
EGFP⫹/⫺
mouse, see Fig. 8, A and C) (331).
Similar to what is observed in intact fibers, spontaneously
beating PFCs have a large action potential overshoot and
a long-lasting plateau phase (Figs. 1B and 8D). EADs can
also be recorded during repolarization (Fig. 8D), a phe-
nomenon that has been observed also in paced intact
fibers (13). The maximum diastolic potential can be close
to ⫺90 mV in calf Purkinje fibers (127). In contrast, mouse
Purkinje fibers seem to have more positive diastolic po-
tentials (⫺75 mV) (13). The negative diastolic potential in
Purkinje fibers is attributed to I
K1
. Indeed, in calf Purkinje
fibers, I
K1
density is high enough to drive the maximum
diastolic potential close to the equilibrium of K
⫹
(72, 118).
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Consequently, automaticity needs to be generated in a
more negative voltage interval than in the SAN or AVN.
However, automaticity of Purkinje fibers can be increased
by stretch induced by ventricular filling (233). The pres-
ence of a large I
K1
can partly explain the relatively slow
intrinsic beating rate of Purkinje fibers. Ionic currents
underlying SAN pacemaking have been found also in Pur-
kinje fibers (72, 501, 502). The presence of I
f
has been
extensively described in isolated Purkinje fibers by Di-
Francesco (118, 122) and others (72) as that of I
Ca,T
. Other
ionic mechanisms as the NCX can participate to automa-
ticity in Purkinje fibers (307).
III. MOLECULAR DETERMINANTS OF ION
CHANNELS IN AUTOMATIC HEART CELLS
A. f-Channels
Almost all spontaneously active cells coming from
heart rhythmogenic centers express f-channels (21). Di-
Francesco and co-workers (20, 120, 121) have proposed
that I
f
is the predominant ionic mechanism underlying
cardiac automaticity. SAN pacemaker cells robustly ex-
press I
f
(125, 313). In the rabbit, I
f
density is higher in the
SAN periphery than in the center (355). I
f
can be recorded
also in the myocardium, where it can be activated below
the physiological resting potential (535). It has been
shown that disease states such as atrial fibrillation and
heart failure enhance I
f
expression and shift positively the
current voltage dependence (see Ref. 79 for review). I
f
is
a mixed cationic current carried by Na
⫹
and K
⫹
(2).
Permeability to Na
⫹
is predominant, but K
⫹
activates I
f
conductance (115, 122, 125). Ca
2⫹
permeability of f-chan-
nels has been reported in HEK cells expressing recombi-
nant HCN2 channels (538) and in rat ventricular cells
(537). In the SAN and AVN, I
f
activates upon membrane
hyperpolarization with variable threshold between ⫺50
and ⫺65 mV (21). The threshold of I
f
is substantially more
negative in Purkinje fibers (118).
Several factors can influence the activation of f-chan-
nels.
␤
-Adrenergic receptor activation stimulates (21) while
muscarinic agonists inhibit I
f
(129). This regulation is medi-
ated by direct activation of f-channels by cAMP (50, 128),
which facilitates channel opening (126). DiFrancesco and
Tortora (128) failed to observe a direct regulation of SAN
FIG.8.A: structural organization of mouse Purkinje fibers (PF). In this mouse line, the EGFP has been knocked in the gene encoding for Cx40.
Asymmetry of the PF network is then visualized by epifluorescence. In the right ventricle, PF originate from a common trunk (RBB) that ramificates
in secondary branches, which laterally terminate in contact with papillary muscle (APM). PF in the left ventricle arise from the left bundle branches
(LBB) in continuity with the His bundle (HB) and form a dense network on the left ventricular free wall (LVW). Dotted lines indicate the axis of
cutting along the right (RS) and left (LS) ventricular septum to expose the ventricular free walls. B: evoked action potentials recorded using the same
preparation as in A. Records are from the ventricular working myocardium (VWM), the right (RBB) and left (LBB) bundle branches. Note the longer
action potential plateau phase in the bundle branches composed by Purkinje fibers. C: line shows the morphology of a spontaneously active mouse
Purkinje cell under visible light (right panel) and EGFP epifluorescence (left panel). D: automaticity and action potential configuration in an isolated
Purkinje cell as shown in C. Spontaneous early afterdepolarizations (EADs) are often observed during the action potential plateau phase. [From
Miquerol et al. (331), with permission from Elsevier.]
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f-channels by purified protein kinase A (PKA) (128). In SAN
cells, direct regulation of voltage dependence by cAMP can
quantitatively account for I
f
regulation by autonomic ago-
nists (126). The existence of a PKA-mediated regulation of I
f
has been suggested by Chang et al. (80) in canine Purkinje
cells and in murine stem cells derived from cardiomyocytes
(1). Intracellular Ca
2⫹
have been reported to positively reg-
ulate I
f
by either increasing current conductance (171) or by
positively shifting the current voltage dependence (416). The
mechanisms by which Ca
2⫹
regulate I
f
has not been eluci-
dated. Inside-out patch-clamp experiments seem to exclude
a direct Ca
2⫹
effect on f-channels (540).
Four gene isoforms, named HCN1–4, code for f-chan-
nels and have been cloned from the mouse (295, 435),
rabbit (217), and human (296, 444). These isoforms dis-
play different activation threshold, kinetics, and sensitiv-
ity to cAMP (234). Particularly, HCN1 channels show the
more positive threshold for activation, the fastest activa-
tion kinetics, and the lowest sensitivity to cAMP (295, 435),
while HCN4 channels are slowly gating and strongly sensi-
tive to cAMP (217, 296, 444). HCN2-mediated channels have
intermediate properties between HCN1- and HCN4-medi-
ated channels (234). It has been proposed that the trans-
membrane protein KCNE2 stimulates the surface expres-
sion and accelerates the kinetics of HCN channels in heter-
ologous expression system (536) and in cardiomyocytes
(404). It has also been proposed that KCNE2 can switch
HCN2-mediated currents from time dependent to time
independent (302).
HCN4 is the predominant f-channel isoform ex-
pressed in the SAN (317, 342, 450) and AVN (317). The
HCN1 and HCN2 isoforms are also expressed in these
rhythmogenic centers (317, 342, 344, 450).
However, the molecular composition of native car-
diac f-channels has not been elucidated. The strong ex-
pression of HCN4 mRNA in the SAN and AVN and the high
sensitivity of native f-channels to cAMP (126) suggest that
HCN4 is a major determinant of native I
f
. Remarkably,
native I
f
has faster activation kinetics than I
f
mediated by
HCN1-HCN4 heterotetramers. Such a difference cannot
be accounted for the presence of basal cAMP or KCNE2 in
intact SAN cells (7). Recent studies have described a
“context-dependent” regulation of native f-channels. Qu
et al. (403) overexpressed HCN2 channels in neonatal and
adult ventricular myocytes and showed that I
f
voltage
dependence was dependent on the developmental state of
the cells. Furthermore, transfection of HCN2 and HCN4
channels in HEK cells and neonatal ventricular myocytes
yields I
f
currents that activated more positively in cardi-
omyocytes than in HEK cells, irrespectively of the trans-
fected isoform (402). These observations indicate that
cellular factors other than cAMP and KCNE2 contribute
to the native SAN I
f
kinetics and voltage dependence.
Barbuti et al. (19) have reported that in rabbit SAN cells,
HCN4 channels interact with caveolin-3 and are concen-
trated in caveolar membrane lipid rafts. Chemical disor-
ganization of caveolar structures positively shifted the I
f
activation curve and slowed the current deactivation ki-
netics (19). In a follow-up study, the same group showed
that
␤
2
-receptors colocalize with f-channels in cavelolar
structures and that
␤
2
-dependent regulation of I
f
also
requires caveolar membrane compartimentalization (20).
It thus seems very likely that cAMP-dependent regulation
of f-channels takes place locally, in a subcellular space
delimited by lipid rafts. Pian et al. (392) have reported that
phosphatidylinositol 4,5-bisphosphate (PIP
2
) prevents
rundown of both recombinant HCN2-mediated and native
rabbit SAN I
f
. It is thus possible that a multiplicity of
factors other than cAMP can participate in the regulation
of I
f
in the SAN. Regulation of HCN2 channels by stretch
has been reported by Li et al. (287). These authors have
studied HCN2-mediated I
f
expressed in Xenopus oocytes.
In this preparation, stretch accelerated both activation
and deactivation kinetics of I
f
(287).
B. Voltage-Dependent Ca
2ⴙ
Channels
Voltage-dependent Ca
2⫹
channels (VDCCs) are an
important pathway of Ca
2⫹
entry in pacemaker cells. L-
and T-type VDCCs have been consistently recorded in
spontaneously active SAN and AVN cells (145, 172, 314,
401, 539). L-type VDCCs are expressed throughout the
myocardium, are sensitive to dihydropyridines (DHPs)
such as nifedipine and BAY K 8644, and are stimulated by
PKA-dependent phosphorylation (491). For further dis-
cussion on the physiology and pharmacology of cardiac
L-type VDCCs, the reader is referred to some recent re-
views (470, 491). In the heart, L-type VDCCs initiate con-
traction (475) and contribute to pacemaker activity (309,
497, 546).
I
Ca,L
in the SAN and AVN activates upon depolariza-
tion at a variable threshold between ⫺50 and ⫺30 mV
(309, 314, 497, 539, 546) and displays Ca
2⫹
- and voltage-
dependent inactivation (172, 311). Expression of I
Ca,L
in
the rabbit SAN is heterogeneous; larger cells (presumably
from the SAN periphery) express less current density than
smaller cells (349). In the SAN, I
Ca,L
is regulated by PKA
(390) and by activated Ca
2⫹
/calmodulin-dependent pro-
tein kinase II (CaMKII) (509), which regulates the current
activation and reactivation kinetics (509). Similarly, in
ventricular cells, CaMKII facilitates opening of L-type
channels in response to Ca
2⫹
permeation, with conse-
quent stimulation of I
Ca,L
amplitude and slowing of cur-
rent inactivation (135, 528).
L-type VDCCs are multisubunit complexes, consti-
tuted by a pore-forming
␣
1 subunit in association with
different accessory subunits (
␣
2-
␦
,
␤
, and
␥
) (470). Four
L-type
␣
1-subunits have been cloned and classified in the
Ca
v
1 gene family (78). The Ca
v
1.1
␣
1-subunit is responsi-
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ble for excitation-contraction coupling in skeletal muscle
(474). Ca
v
1.4 is expressed in the retina, spinal cord, and
immune cells (324). Ca
v
1.2 and Ca
v
1.3 are expressed in
neurons, as well as in cardiovascular and neuroendocrine
cells (77). In the ventricle, the Ca
v
1.2
␣
-subunit is the
predominant molecular determinant of I
Ca,L
. This
␣
-sub-
unit is expressed in the SAN, AVN, and the atria (45, 309,
317, 472). Recombinant and native Ca
v
1.3-mediated I
Ca,L
displays a more negative activation threshold and slower
inactivation kinetics than Ca
v
1.2-mediatd I
Ca,L
.Ca
v
1.3
channels have also reduced sensitivity to DHPs (254). In
contrast, SAN Ca
v
1.2-mediated and Ca
v
1.3-mediated I
Ca,L
seem to be similar as to their sensitivity to
␤
-adrenergic
agonists (309).
T-type VDCCs are activated at more negative volt-
ages than L-type VDCCs. The kinetic hallmark of native
and heterogously expressed I
Ca,T
is slow activation and
fast voltage-dependent inactivation. The single-channel
conductance of T-type VDCCs is also smaller than that of
L-type VDCCs (388). Three genes coding for T-type
␣
1-
subunits have been cloned (103, 339, 340, 387) and named
Ca
v
3.1, Ca
v
3.2, and Ca
v
3.3. The Ca
v
3.1 isoform is function-
ally expressed in neonatal rat atrial myocytes (282), in the
AT-1 cell line (438), in the developing mouse heart (103),
and in mouse SAN and AVN (314). In the adult SAN,
expression of the Ca
v
3.1 isoform is higher than that of
Ca
v
3.2 (45, 314). The Ca
v
3.2 isoform has been cloned from
a human heart library (103), and possibly constitutes the
predominant I
Ca,T
isoform in the rat embryonic heart
(146). In contrast, expression of Ca
v
3.1 channels in-
creases during the perinatal period and is maximal in
adulthood (358). To date, expression of the Ca
v
3.3 iso-
form has not been found in the myocardium, the SAN, and
AVN (314, 340). No specific drugs able to discriminate
between Ca
v
3.1 and Ca
v
3.2 channels are presently avail-
able. However, Ca
v
3.2 channels are much more sensitive
to Ni
2⫹
than Ca
v
3.1 channels, the concentration for half-
block (EC
50
) being ⬃5 and 150
␮
M for Ca
v
3.2 and Ca
v
3.1
channels, respectively (265).
C. St-Channels
The I
st
current has been recorded by Guo et al. (166)
in rabbit SAN and AVN (165). This current has also been
found in SAN cells from guinea pig, rat, and mouse (86,
457). I
st
activates at about ⫺70 mV, peaks at about ⫺50
mV, and is positively regulated by
␤
-adrenergic agonists
(166). I
st
is carried by Na
⫹
, but is clearly distinct from I
Na
,
since it is insensitive to tetrodotoxin (TTX), blocked by
DHP antagonists, and inhibited by divalent cations such
as Ca
2⫹
,Mg
2⫹
, and Ni
2⫹
(166). Single st-channels have a
unitary conductance similar to that of L-type VDCCs (⬃13
pS) and are facilitated by BAY K 8644 (333). Mitsuye et al.
(334) have reported that I
st
expression is restricted to
spontaneously active SAN and AVN cells, since no I
st
is
recorded in quiescent cells from these two regions.
The molecular determinants of I
st
have not yet been
identified. I
st
could be mediated by a new subtype of
L-type VDCCs (166), or by a still unidentified splice vari-
ant. This hypothesis would be consistent with the phar-
macological sensitivity of I
st
to agonist and antagonist
DHPs.
D. Voltage-Dependent Na
ⴙ
Channels
A significant fraction of rabbit SAN pacemaker cells
expresses I
Na
in culture (348, 351). I
Na
is heterogeneously
expressed in the adult rabbit SAN (204, 250). Honjo et al.
(204) reported that I
Na
expression is higher in large cells
(presumably from the SAN periphery) than in small cells
(from the SAN center). A high percentage of small cells is
devoid of I
Na
(204). Consistently, pacemaking in SAN
periphery is sensitive to TTX, while automaticity in tissue
balls from the SAN center is insensitive to TTX applica-
tion (250, 255). In contrast, SAN cells of newborn rabbits
show high I
Na
expression (24). In the newborn, expres-
sion of I
Na
is maximal during the first 3 wk after birth and
then declines irrespectively of cell size (24). High expres-
sion of I
Na
is present also in adult mouse SAN cells (279,
312, 313). The SAN I
Na
is more sensitive to TTX than I
Na
of the working myocardium. TTX-sensitive I
Na
has been
reported in rabbit neonatal (25) and in adult mouse SAN
cells (279). TTX in the nanomolar range reduces the beat-
ing rate of isolated mouse hearts (304) and SAN pace-
maker cells (279).
Na
⫹
channels are related to a large family of genes
coding for 10
␣
-subunit isoforms (see Ref. 534 for review).
The electrophysiological properties of the
␣
-subunit can
be modulated by accessory
␤
1- and
␤
2-subunits (534). By
using in situ hybridization, Baruscotti et al. (25) have
shown that the newborn SAN expresses the neuronal
TTX-sensitive Na
v
1.1
␣
-subunit isoform. Expression of
both the TTX-sensitive Na
v
1.1 and TTX-resistant “cardiac”
Nav1. 5 isoforms has been reported in the mouse SAN at
the protein level (279).
E. Voltage-Dependent K
ⴙ
Channels
Voltage-dependent K
⫹
channels (VDKCs) control the
action potential repolarization phase in spontaneously
active cells (58) and in the working myocardium (353,
422). In SAN and AVN, three VDKC-mediated currents
have been recorded: the fast (I
Kr
) and slow (I
Ks
) delayed
rectifiers and the transient outward current (I
to
).
I
Kr
is activated upon depolarization from a threshold
of ⫺50 mV. In voltage-clamp experiments, I
Kr
fully acti-
vates at about ⫺20 mV and displays strong inward recti-
fication (93, 218, 496). At positive voltages, inactivation of
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I
Kr
counterbalances activation, thereby generating a re-
gion of negative slope conductance (93). When the mem-
brane voltage is switched back to negative potentials, I
Kr
slowly deactivates to generate tail currents, which contrib-
ute to set the maximum diastolic potential (see sect.
VI). Tail
currents are due to channel reopening followed by time-
dependent closure at negative membrane potential (452).
Shibasaki (452) has reported that the single-channel con-
ductance of I
Kr
channels in 150 mM K
⫹
is 11 pS, giving an
estimate of ⬃1,000 channels in a typical rabbit SAN cell.
I
Kr
is sensitive to class III methanesulfonanilide com-
pounds E-4031 and dofetilide (UK-68798), as well as to a
plethora of unrelated compounds used in medical prac-
tice (473).
I
Kr
channels are coded by the ERG gene family,
which includes three members named ERG1-ERG3. The
ERG1 gene is expressed in the heart (398). Mutations in
the human ERG1 gene or in its accessory subunit KCNE2
can impair ventricular repolarization and lead to long-QT
syndrome (92, 236). In the mouse SAN, mRNAs of the
three known ERG1 splicing variants 1a, 1a⬘, and 1b have
been found (93). However, the composition of native I
Kr
channels in SAN, AVN, and Purkinje fibers has not yet
been elucidated. Lees-Miller et al. (273) have proposed
that both ERG1a and -1b are able to form recombinant
channels with properties similar to those of native I
Kr
, but
to date, only ERG1a immunoreactivity has been identified
in the ventricle (397).
The I
Ks
current is kinetically and pharmacologically
distinct from I
Kr
. I
Ks
has slower activation and faster
deactivation kinetics than I
Kr
. I
Ks
is sensitive to the 293B
channel blocker, which is currently employed for study-
ing the physiological role of this current (275, 278). Ex-
pression of I
Kr
and I
Ks
in the rabbit SAN is heterogeneous.
Indeed, high I
Kr
and I
Ks
densities have been recorded in
large SAN cells, while smaller cells seem to express only
I
Kr
(278). Species-dependent differences in I
Kr
and I
Ks
expression seem to exist. I
Kr
has not been recorded in
isolated pig SAN cells, which express only I
Ks
(372). I
Ks
has not yet been directly recorded in mouse SAN cells
(Mangoni, unpublished observations). However, Temple
et al. (478) have reported expression in the mouse SAN of
the KCNQ1 accessory subunit KCNE1. On the other hand,
mice lacking KCNE1 have susceptibility to spontaneous
atrial fibrillation, but do not show alterations in SAN
pacemaker activity (478).
Three genes named KCNQ1-KCNQ3 encode for I
Ks
,
but only KCNQ1-related expression is found in the heart
(352). The sensitivity of I
Kr
and I
Ks
to
␤
-adrenergic ago-
nists in pacemaker cells would need further investigation.
Voltage-clamp studies on intact SAN tissue preparations
have reported stimulation of I
K
by epinephrine (62).
These observations have been confirmed in isolated rab-
bit SAN cells (277). Sensitivity of I
Kr
to
␤
-adrenergic
agonists has been reported in ventricular myocytes (194),
but this issue has not been addressed in SAN cells.
The I
to
current is characterized by fast activation and
inactivation kinetics (see, for example, Ref. 277). Pharma-
cologically, I
to
can be identified thanks to its sensitivity to
4-aminopyridine (4-AP) (422). In working myocytes, at
least two components of I
to
have been identified and
named I
to,f
and I
to,s
, according to their fast and slow
inactivation kinetics, respectively (353). Expression of I
to
in rabbit SAN cells is heterogeneous (60, 206, 277). In-
deed, pacemaker activity of SAN tissue balls isolated from
the SAN periphery is more sensitive to block of I
to
by 4-AP
than that from the center of the SAN (60). In rabbit SAN
cells, I
to
density is positively correlated with the cell size
(206, 277).
I
to
channels are coded by the K
v
1 and K
v
4 gene family
(354). The composition of native I
to
channels in the SAN
and conduction system is not yet known. In the mouse
heart, inactivation of Kv1.4 channels abolishes I
to,s
(291).
The channel complex mediating the I
to,f
component is
formed by heteromultimers of K
v
4.2 and K
v
4.3 channel
subunits (168). The KChIP2 protein is also associated
with the I
to
channel complex (8). KChIP2 regulates cur-
rent inactivation and channel targeting to the cell mem-
brane (8). Ventricular cells isolated from mice lacking
KChiP2 have no I
to
and display prolonged action potential
duration. Episodes of ventricular tachyarrhythmias are
recorded in KChiP2 knockout mice (260).
In conclusion, I
to
appears to play a role in the action
potential repolarization phase of peripheral SAN cells, but
is probably less important in the SAN center. However,
mice lacking both I
to,s
and I
to,f
have no apparent dysfunc-
tion of the SAN rhythm (290). Further studies will be
needed to elucidate the subunit composition and the
physiological role of I
to
in spontaneously active cells.
F. G Protein-Activated, ATP-Dependent,
and Inward Rectifier K
ⴙ
Channels
The acetylcholine-activated current (I
KACh
)is
strongly expressed in the SAN, atria, and AVN (124, 159,
367). In these tissues, I
KACh
is activated by muscarinic and
adenosine receptors by direct binding of G protein
␤␥
-
subunits to the I
KACh
channel complex (for review, see
Refs. 520, 521). Four genes named Kir3.1-Kir3.4 underlie
I
KACh
channels (520). Kir3.1 and Kir3.4 are expressed in
the heart (521). Cardiac I
KACh
channels are tetrameric
complexes containing Kir3.1 and Kir3.4 channels (521).
However, mice lacking Kir3.4 channels have no cardiac
I
KACh
(522), because Kir3.1 channels require Kir3.4 chan-
nels to be targeted at the cell membrane (238).
ATP-dependent K
⫹
channels have been described in
rabbit SAN cells (184). These channels underlie I
K,ATP
and
are open when the intracellular concentration of ATP is
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lowered. Van Wagoner (493) has shown that I
K,ATP
chan-
nels are activated by stretch. Cell swelling by a hypotonic
solution was used in this work to demonstrate that I
K,ATP
can generate a significant whole cell current under me-
chanical stress. Activation of I
K,ATP
by stretch has been
reported also in ventricular cells (251), but these obser-
vations have not yet been confirmed in pacemaker tissue.
In rabbit SAN cells, I
K,ATP
slows pacemaker activity and
hyperpolarizes the cell maximum diastolic potential
(184). Han et al. (184) have proposed that the slowing of
heart rate induced by I
K,ATP
can be important for protect-
ing the myocardium from ischemic damage or stunning.
This interesting hypothesis has not yet been experimen-
tally verified.
Rabbit SAN and AVN cells express very low densities
of inward-rectifier K
⫹
current I
K1
(62, 215, 350, 363, 366).
In contrast, I
K1
is present in the working myocardium
(422) and in Purkinje fibers (118). I
K1
is responsible for
the negative diastolic potential of PFCs. However, I
K1
can
be moderately expressed in rat (457) and mouse (86) SAN
cells, even if at a much lower density than the working
myocardium. In the heart, I
K1
channels are coded by the
Kir2.1 and Kir2.2 channel subunits (352).
G. Cl
ⴚ
Channels, Volume-Activated Channels,
and Stretch-Activated Cationic Channels
The first evidence for the presence of chloride cur-
rent (I
Cl
) in spontaneously active cells came from Pur-
kinje fibers. In this preparation, De Mello (106) reported
that I
Cl
represents a substantial fraction of the total mem-
brane conductance during the action potential. I
Cl
has
been reported by Seyama who estimated that Cl
⫺
are
responsible for ⬃9% of the total membrane conductance
and that I
Cl
underlies a significant part of the membrane
inward-going rectification in the SAN (449). Hagiwara
et al. (174) have characterized a stretch-activated back-
ground I
Cl
in rabbit SAN cells. This current is distinct
from the voltage- and Ca
2⫹
-dependent, cAMP-sensitive
[I
Cl(Ca)
] present in ventricular myocytes (16, 471), and
probably belongs to the family of volume-activated anion-
selective currents (VAC
Cl
) (27), because the current was
activated by cell inflation (174). Hagiwara et al. (174)
reported that SAN background I
Cl
is insensitive to intra-
cellular Ca
2⫹
depletion and cAMP. Bescond et al. (37)
have reported that angiotensin II (ANG II) activates back-
ground I
Cl
in SAN cells via a protein kinase C (PKC)-
dependent signaling pathway. Verkerk et al. (504) have
tested the possibility that I
Cl(Ca)
is present in SAN cells
and its possible role in the action potential repolarization.
They recorded I
Cl(Ca)
in about one-third of SAN cells
tested. I
Cl(Ca)
activated from about ⫺20 mV, peaked be-
tween ⫹30 and ⫹40 mV, and was augmented by norepi-
nephrine. The kinetic behavior of SAN I
Cl(Ca)
(504) was
similar to I
Cl(Ca)
in atrial, Purkinje, and ventricular myo-
cytes (210). Verkerk et al. (504) have tested the role of
I
Cl(Ca)
in SAN automaticity, by employing action potential
clamp experiments and numerical modeling (504). They
found that I
Cl(Ca)
activates late during the action potential
upstroke phase and gives moderate contribution to the
repolarization phase. No contribution of I
Cl(Ca)
to the
maximum diastolic potential or diastolic depolarization
rate was observed (504).
VAC
Cl
are activated by an increase in cell volume or
by agents that alter membrane tension and mechanical
stretch (27). VAC
Cl
open with a quite slow response to cell
volume change (464). I(VAC
Cl
) is time independent, out-
wardly rectifying, and reverses between the cell resting
potentials and action potential plateau phase. Conse-
quently, VAC
Cl
shortens the action potential duration and
depolarizes the membrane resting potential, thereby act-
ing to decrease cell volume (27). In the heart, VAC
Cl
play
a role in the ischemic response and seem to be overex-
pressed in the hypertrophied myocardium (27). The exis-
tence of mechanosensitive VAC
Cl
in SAN has been di-
rectly proposed firstly by Arai et al. (14), who showed that
the positive chronotropic response induced by strong
stretch stimuli in SAN tissue is inhibited by Cl
⫺
channel
blockers. However, Cooper and Kohl (99) have failed to
observe an effect of a Cl
⫺
channel blocker under condi-
tions of moderate mechanical load.
SAC are cationic nonselective or K
⫹
selective. Com-
pared with VAC
Cl
, SAC activate fast upon mechanical
stimulation. Kohl et al. (251) have suggested that fast
activation of SAC can be involved in the beat-by-beat
regulation of heart rate. SAC are inhibited by Gd
3⫹
(383),
streptomycin (151), and the spider toxin GsMTx-4 (42).
Cooper and Kohl (99) reported that the chronotropic
response of isolated pacemaker cells is inhibited by
GsMTx-4, thus indicating functional expression of SAC in
SAN cells.
IV. PUMPS AND EXCHANGE CURRENTS
A. The Na
ⴙ
-K
ⴙ
Pump Current I
p
The electrogenic role of Na
⫹_
K
⫹
pump is well estab-
lished. Under physiological ionic concentrations, three
Na
⫹
are extruded and two K
⫹
are transported in the
intracellular milieu for each pump cycle. Consequently,
the Na
⫹_
K
⫹
pump generates a net outward current that
influences cellular pacemaking. In Purkinje fibers, I
p
stim-
ulation hyperpolarizes the membrane potential and slows
spontaneous activity (149). Noma and Irisawa (362) have
recorded I
p
-mediated membrane hyperpolarization in rab-
bit SAN multicellular preparations, by rapidly switching
perfusion from K
⫹
-free Tyrode’s solution to one contain-
ing 5.4 mM K
⫹
(362). A similar hyperpolarization of the
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membrane potential has been observed in atrioventricular
cells (261). I
p
properties can be studied by varying the
intracellular K
⫹
concentration after block of time- and
voltage-dependent currents, according to the protocol de-
scribed by Gadsby et al. for ventricular cells (150). I
p
can
also be isolated pharmacologically by employing ouabain
or strophantidine (261, 362, 431). Sakai et al. (431) have
studied the properties of I
p
in isolated rabbit SAN cells.
They reported that I
p
carries a significant steady-state
outward current in physiological extracellular K
⫹
and 50
mM intracellular Na
⫹
. Similarly to I
p
recorded in ventric-
ular cells (150), SAN I
p
displays voltage-dependent behav-
ior (431). I
p
voltage dependence is evident in the pace-
maker range with a relative half activation voltage of
about ⫺50 mV. Furthermore, I
p
is very sensitive to
changes in intracellular Na
⫹
so that the current doubles
when changing Na
⫹
from about 15 to 25 mM Na
⫹
(431).
In SAN cells, I
p
contributes to the setting of the
maximum diastolic potential in the range of ⫺60 mV
(362). In this respect, Sakai et al. (431) have estimated
that the net outward I
p
-mediated current in intact SAN
cells is ⬃14 pA in the experiments by Noma and Irisawa
(362) and ⬃20 pA in their recordings. However, since
automaticity is essentially a periodical process, it is also
possible that I
p
varies cyclically during the pacemaker
cycle according to its intrinsic voltage and Na
⫹
depen-
dency. Furthermore, one may wonder if I
p
can respond to
transient changes in intracellular Na
⫹
induced by activa-
tion of ion channels such as I
Na
and I
f
. Choi et al. (90)
have measured changes in intracellular Na
⫹
activity (a
i
Na
)
in multicellular and isolated SAN cell preparations under
application of isoproterenol, carbachol, and I
f
blockers.
These authors reported that isoproterenol-induced accel-
eration of the pacing rate was accompanied by an eleva-
tion of a
i
Na
. Application of Cs
⫹
and ZD-7288 reduced (but
did not completely abolished) the rise in a
i
Na
, thus sug-
gesting a link between I
f
stimulation by isoproterenol and
a
i
Na
. In contrast, both carbachol and ZD-7288 reduced a
i
Na
in spontaneously beating myocytes but had no effect on
quiescent cells. These data are strongly suggestive of a
significant influence of I
f
on intracellular a
i
Na
. It is thus
tempting to speculate that f-channels can be functionally
coupled to I
p
in SAN pacemaker cells. This hypothesis can
be particularly relevant when considering that f-channels
are concentrated in membrane lipid rafts. It is possible
that opening of f-channels induces a highly localized rise
in intracellular Na
⫹
concentration. The presence of the
Na
⫹_
K
⫹
pump near f-channels is important for maintain-
ing the ionic homeostasis.
B. The Na
ⴙ
-Ca
2ⴙ
Exchanger Current I
NCX
and the
Na
ⴙ
-H
ⴙ
Exchanger
The NCX is a major actor intervening in cardiac cell
Ca
2⫹
homeostasis. Indeed, Ca
2⫹
entry during the diastole
and action potential upstroke increases intracellular Ca
2⫹
concentration ([Ca
2⫹
]
i
) stimulating NCX activity. Stoichi-
ometry of Na
⫹
-Ca
2⫹
transport through NCX is electro-
genic, one Ca
2⫹
is extruded for three Na
⫹
entering the cell
(454). The result is a net inward current (I
NCX
) which
depolarizes the membrane voltage. In the working myo-
cardium, NCX is responsible for Ca
2⫹
efflux during action
potential repolarization (35, 142). In the rabbit myocar-
dium, I
NCX
extrudes ⬃30% of the total Ca
2⫹
required to
activate contraction, while the sarcoplasmic reticulum
Ca
2⫹
-ATPase (SERCA) actively removes Ca
2⫹
from the
cytoplasm to replenish the SR (142). Regardless of the
species considered, Ca
2⫹
extrusion by NCX quantitatively
matches Ca
2⫹
influx triggering Ca
2⫹
-induced Ca
2⫹
release
(CICR), while SERCA transport must match ryanodine
receptor (RyR)-dependent Ca
2⫹
release. The ventricular
I
NCX
contributes to action potential plateau as well as to
contractile relaxation (35). Positive regulation of NCX
activity by
␤
-adrenergic receptor activation is linked to
positive inotropism in ventricular and Purkinje myo-
cytes (35).
Early evidence of the presence of I
NCX
in pacemaker
tissue was obtained by Brown et al. (65) using multicel-
lular SAN preparations. These authors showed that the
slow inward current had a second late component that
could be attributed to Na
⫹
-Ca
2⫹
exchange. Similarly,
Zhou and Lipsius (547) recorded I
NCX
in cat latent atrial
pacemaker cells by applying depolarizing voltage steps
eliciting I
Ca,L
which, in turn, activated I
NCX
. NCX activity
closely follows changes in [Ca
2⫹
]
i
. For example, in cane
toad (Bufo marinus) sinus venosus cells, an increase in
I
NCX
precedes the measured systolic Ca
2⫹
transient, thus
suggesting that I
NCX
is significantly stimulated by an in-
crease in subsarcolemmal [Ca
2⫹
]
i
(228). Furthermore, in-
hibition of NCX activity by Ni
2⫹
slows the decline of the
caffeine-induced release, indicating that NCX is a major
controller of [Ca
2⫹
]
i
in toad sinus venosus cells (228). The
physiological role of I
NCX
in pacemaking can be tested by
fast removal of extracellular Na
⫹
or substitution with Li
⫹
.
In these conditions, [Ca
2⫹
]
i
quickly rises and pacemaker
activity stops (44, 228, 433). Intact NCX activity is thus
necessary for maintaining automaticity of pacemaker
cells in both amphibians (228) and mammals (44, 433).
The importance of NCX in the generation of cardiac au-
tomaticity will be discussed in a following section of this
review.
The Na
⫹
-H
⫹
exchanger (NHE) is a pH-regulatory
protein present in the plasma membrane of all cardiac
myocytes. In response to intracellular acidosis, the pro-
tein removes one intracellular H
⫹
in exchange for an
extracellular Na
⫹
(305). NHE is regulated by several hu-
moral factors including ANG II and endothelin (305).
NHE1 is the predominant isoform expressed in cardiac
myocytes (305). In isolated ferret hearts, NHE1 is respon-
sible for ⬃50% of the total H
⫹
efflux (163). NHE1 has been
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implicated in cardiac pathological states including ische-
mia (see Refs. 198, 305 for review). Indeed, strong activa-
tion of NHE1 following intracellular acidosis leads
to increased intracellular Na
⫹
concentration and conse-
quent [Ca
2⫹
]
i
build up through activation of I
NCX
(198).
We can thus expect that NHE and, more generally, pro-
teins involved in H
⫹
handling (198) can be involved in the
SAN chronotropic response to ischemia and acidosis (see
sects.
IXE and XB). However, we currently lack a detailed
characterization of NHE-mediated current in SAN.
V. PATTERNS OF GENE EXPRESSION IN
ADULT PACEMAKER TISSUE
In this short section, we review evidence on the
differential expression pattern of ion channels in the
heart’s rhythmogenic centers and in working myocar-
dium. Han et al. (179) have studied the expression of ion
channels potentially involved in automaticity in canine
Purkinje fibers and compared it with ventricular myocar-
dium. This study combined RT-PCR with Western blotting
of membrane proteins. Results have shown that HCN4
is highly expressed in canine Purkinje fibers, while it
seemed undetectable in ventricular muscle. HCN2 expres-
sion was comparable in Purkinje fibers and the ventricle.
Expression of Ca
v
1.2 and NCX was higher in the ventricle
than in Purkinje fibers. Genes coding for I
Ca,T
(Ca
v
3.1,
Ca
v
3.2, and Ca
v
3.1) were all more expressed in Purkinje
fibers. Similar peculiarities in the pattern of expression of
Purkinje fibers have been recently reported by Gaborit
et al. (148) in nondiseased human heart.
Marionneau et al. (317) have employed a high-
throughput RT-PCR approach (112) to assess the ion
channel expression pattern in the mouse SAN and AVN.
Indeed, real-time RT-PCR array technology allows han-
dling of small tissue samples that contain low quantities
of mRNA. These authors have compared the expression
of 71 channels and related genes in the SAN, AVN, right
atrium, and left ventricle (Fig. 9A). Transcripts coding for
HCN1 and HCN4 show the highest expression level in the
SAN. The SAN and AVN are distinguished by high expres-
sion of Ca
v
1.3 and Ca
v
3.1 mRNAs. HCN2 mRNA is present
in the SAN, but at lower levels than HCN1 and HCN4
mRNA. The AVN is characterized by expression of Nav1.7,
Kv1.6, as well as the K
⫹
␤
-subunit Kv
␤
1. These transcripts
appear to be specific of the AVN (317). Significant expres-
sion of Nav1.1 has been found in the AVN. The Ca
v
␣
2
␦
2
may constitute a potential accessory subunit for VDCCs in
the SAN and AVN, because mRNA encoding for this sub-
unit is highly expressed in the nodes and in Purkinje
fibers. Mouse pacemaker tissues show predominant ex-
pression of the Na
⫹
channel
␤
-subunits Nav
␤
1 and Nav
␤
3.
It is possible that the association of Nav
␤
1 and Nav
␤
3to
Nav
␣
-subunits speeds up inactivation of I
Na
. Finally,
FIG.9.A: a combined quantitative PCR and two-way hierarchical
clustering approach is used to compare the expression pattern in six
mouse SAN (SAN1– 6), AVN (AVN1– 6), atria (A1– 6), and ventricles
(V1–6). Seventy-one mRNAs encoding ion channel subunits and Ca
2⫹
handling proteins are considered. The expression of each gene is rep-
resented by a colored row, and each column defines a tissue sample. A
false color scale starting from dark green (lowest expression) to bright
red is applied. Four gene clusters (A–D) are shown at the left of the
panel. In the figure, cluster B identifies genes that are highly expressed
in working myocardium, while cluster C identifies groups of genes that
are highly expressed in the SAN and AVN. [From Marionneau et al. (317),
with permission from Wiley-Blackwell.] B: quantitative RT-PCR analysis
of 10 representative gene transcripts in rabbit central SAN (blue), pe-
ripheral SAN (green), right atrium (red), and SAN ring bundle (RSARB,
purple). Symbol (⫹⫹) stands for abundant expression, (⫹) stands for
expression, (⫹/⫺) indicates expression in some cells, and (⫺) indicates
absence of detectable expression. [From Tellez et al. (477).]
936 MATTEO E. MANGONI AND JOE
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Kv1.1, Kv1.4, Kv1.6, Kv
␤
1, and Kv
␤
3 are highly expressed
in the SAN and AVN (317). Electrophysiological investi-
gation of mouse SAN and AVN cells will be needed to
elucidate the functional role of these K
⫹
channels in
pacemaking and conduction.
By combining quantitative RT-PCR and immunohis-
tochemistry, Tellez et al. (477) have investigated the re-
gional expression of genes involved in automaticity and
conduction in the rabbit central and peripheral SAN. The
atrial muscle was used as a reference of nonpacing myo-
cardium (Fig. 9B). In this work, a quantitative switch
between channel isoforms has been highlighted: moving
from the atrium to the SAN center, Ca
v
1.2 is substituted
by Ca
v
1.3, Nav1.5 by Nav1.1, and Kv1.4 by Kv4.2. As to
RyRs, RyR2 is gradually downregulated, while expression
of RyRs augmented in the SAN center. The SAN periphery
shows an intermediate expression pattern between atrial
muscle and central SAN (477).
In conclusion, cells endowed with automaticity are
characterized by enhanced expression of some channels
including HCN4, Ca
v
1.3, and Ca
v
3.1. Furthermore, the
central rabbit and mouse SAN express the TTX-sensitive
Na
v
1.1 subunit. Differences in the expression pattern of
ion channels in spontaneously active tissues versus the
working myocardium are essentially quantitative, rather
than qualitative. Numerical modeling work has suggested
that quantitative changes in ionic channels can induce
pacemaking (264). Overexpression of HCN (143) channels
or silencing of I
K1
(327) has been shown to induce auto-
maticity in myocardial cells. However, pacemaker cells
have also a peculiar morphological phenotype. It is thus
likely that factors other than ion channels contribute to
the specific phenotype of primary SAN pacemaker cells.
VI. GENESIS OF CARDIAC AUTOMATICITY:
MECHANISMS OF PACEMAKING
A. Concepts
In the following sections, we will review the current
knowledge on the ionic and intracellular signaling mech-
anisms underlying the generation of pacemaker activity.
We aim to discuss published evidence on how different
families of ion channels, pumps, exchangers, and intra-
cellular Ca
2⫹
signaling generate and regulate pacemaking.
Many research groups have focused on primary SAN
pacemaker activity so that the bulk of evidence discussed
here is obtained from isolated pacemaker cells or intact
SAN tissue. There is currently no general agreement as to
which mechanism is necessary for initiating automaticity
or, in other words, which ionic and/or cellular mechanism
specifically confers automaticity to pacemaker cells.
Different groups have proposed alternative views of
the mechanisms underlying pacemaker activity and em-
phasized the importance of in situ catecholamines pro-
duction (136, 395, 396), ionic currents such as I
f
(120,
121), I
Ca
(172, 309), I
Kr
(93, 371), I
st
(334), or spontaneous
diastolic Ca
2⫹
release (307).
From a conceptual point of view, searching for pace-
maker mechanisms requires the description of the elec-
trophysiological, signaling, and transcriptional processes
that are active (or specifically inactive) in pacemaker
cells. These mechanisms underlie the morphological and
functional differences between spontaneously active cells
and contractile myocytes and/or would be responsible for
the commitment of cardiac precursors toward the pace-
maker function in the adult SAN and in the cardiac con-
duction system. Knowledge of the pacemaker mecha-
nisms would help confer automaticity to target regions of
the working myocardium or to undifferentiated cardiac
precursors. Such a concept has now been applied with the
aim to create “biological pacemakers” in stem cells or in
the cardiac conduction system by in situ gene transfer
(see sect.
XI). We will first discuss evidence involving ion
channels in the generation of automaticity and then re-
view recent work showing that pacemaking also involves
the activity of NCX and spontaneous diastolic Ca
2⫹
re-
lease. I
f
is considered as a key ion channel underlying
automaticity. The relevance of I
f
in the genesis of pace-
maker activity is supported by different lines of electro-
physiological, pharmacological, and genetic evidence.
The importance of spontaneous Ca
2⫹
release in the gen-
eration of pacemaker activity is indicated in experiments
involving pharmacological inhibition of RyRs in rabbit
SAN cells. When reviewing the current knowledge of the
I
f
-based pacemaking and that mediated by spontaneous
Ca
2⫹
release, we will try to highlight differences as well as
similarities between these two proposals. Particularly,
these views are not mutually exclusive and, more impor-
tantly, experiments performed in other laboratories (in-
cluding ours) from genetically modified mouse strains
lacking Ca
v
1.3, Ca
v
3.1, and HCN channels indicate that
the generation of automaticity requires the intervention of
more than one individual mechanism. Also, it has been
shown that the influence of a given ion channel or RyRs
on pacemaker activity can vary regionally in the SAN.
B. Ion Channels and Cardiac Automaticity:
General Considerations
Automaticity in pacemaker cells of the adult heart
stops when the cell membrane potential is depolarized by
addition of KCl in the extracellular solution. Automaticity
in adult pacemaker cells thus differs from that of early
embryonic myocytes which maintain spontaneous con-
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tractions even at depolarized membrane potentials (326,
505). This observation demonstrates that mature pace-
maker cells require voltage-dependent ion channels to
generate automaticity. From a theoretical point of view,
pacemaker activity can be considered as an oscillator
generated by a time-varying outward current with a con-
stant or voltage-dependent inward current activated dur-
ing the repolarization phase. The generation of the dia-
stolic depolarization requires the flow of a net inward
current at the end of the repolarization phase. Several ion
channels with distinct biophysical and pharmacological
properties are potentially involved in the diastolic depo-
larization phase. I
Kr
and I
Ks
are activated during the up-
stroke phase of the action potential and then deactivate
quite slowly during the end of the repolarization and
diastolic depolarization. As I
Kr
and I
Ks
drive outward
currents, a net inward current must overcome outward
components to initiate membrane depolarization. As to
the relevance of membrane ion channels, we will base our
discussion on the working hypothesis that, in isolated
pacemaker cells, such an inward current is generated by
the association of I
f
with I
st
,I
Ca,T
, I
Ca,L
, and I
Na
. The
importance of I
NCX
will be reviewed in a separate section.
A substantial amount of data on ion channels in SAN
pacemaker activity comes from rabbit and guinea pig
SAN. However, new insights have been obtained from
genetically altered mouse strains.
C. Role of I
Kr
and I
Ks
in Automaticity
In rabbit SAN pacemaker cells, the cardiac I
Kr
cur-
rent sets the position of the maximum diastolic potential
and controls action potential repolarization (371, 496).
The physiological significance of I
Kr
in rabbit SAN auto-
maticity has been studied in spontaneously beating cells
using E-4031. In spontaneously active cells, partial inhibi-
tion of I
Kr
by nanomolar concentrations of E-4031 posi-
tively shifts the maximum diastolic potential and de-
creases the action potential amplitude and rate of rise.
E-4031 also prolongs the action potential repolarization
phase (371, 496). The overall physiological effect is a
slowing of the pacing rate in isolated hearts and pace-
maker cells (Fig. 10, A and B). Slowing of pacemaker
activity is due to a reduction in the recruitment of other
currents contributing to the diastolic depolarization, such
as I
f
, I
Ca,L
, and I
Ca,T
. Indeed, the positive shift of the
maximum diastolic potential will reduce I
f
activation and
increase voltage-dependent inactivation of I
Ca,L
and I
Ca,T
during the diastolic depolarization (93). In isolated SAN
cells of rabbit (371) and guinea pig (320), complete block
of I
Kr
by micromolar concentrations of E-4031 quickly
terminates automaticity, and the cell resting potential
settles between ⫺30 and ⫺40 mV. Similar effects of I
Kr
block by E-4031 have been reported in mouse pacemaker
cells by Clark et al. (93). Consistent with this pharmaco-
FIG. 10. A: I
Kr
inhibition by 5
␮
M E-4031 slows the pacing rate of isolated Langendorff-perfused mouse hearts. [From Clark et al. (93).]
B: atrio-sinus electrical interactions influence SAN pacemaking and action potential repolarization phase. Effect of 1
␮
M E-4031 on automaticity
recorded from rabbit SAN tissue strips. Two independent experiments are shown. Asterisks indicate location of the leading pacemaking site. I
Kr
inhibition depolarizes the maximum diastolic potential and slows pacemaking in all tissue strips connected to the crista terminalis. However, E-4031
abolishes pacemaker activity in strip B after that it has been cut off the crista terminalis. Filled circles indicate the site of action potential recording.
[From Verheijck et al. (500).] C: currents from 20 consecutive action potential cycles are averaged before and after application of 2.5
␮
M E-4031.
E-4031-sensitive difference current is obtained by subtraction. Cell has been voltage-clamped with a simulated ideal SAN action potential waveform
(b). c: Current-to-voltage plot of mean E-4031-sensitive current from six different SAN cells voltage-clamped with the action potential waveform in
b. Arrows show direction of current trajectory. The ideal diastolic interval (DD) of simulated action potential is indicated by shadowed grey vertical
box at ⫺65 and ⫺55 mV. [From Clark et al. (93).]
938 MATTEO E. MANGONI AND JOE
¨
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logical evidence is the observation that mice lacking the
ERG1b-mediated channels have no fast I
Kr
component
and are prone to develop episodes of bradycardia (272). It
is interesting to note that I
Kr
blockers induce negative
chronotropism also in the zebrafish heart (329). This ob-
servation indicates that the function of ERG1/KCNH2
channels in pacemaking may be shared between fishes
and mammals. Clark et al. (93) have studied I
Kr
density
and kinetics during pacemaking by pharmacological sub-
traction of the E-4031-sensitive current from the total
systolic and diastolic current. These recordings have
shown that I
Kr
reaches a maximum during the repolariza-
tion phase at about ⫺25 mV and then progressively de-
clines throughout the diastolic interval to reach almost
the zero level before the following action potential thresh-
old (Fig. 10C). However, Zaza et al. (541) reported that I
Kr
in rabbit SAN cells does not completely deactivate during
the pacemaker cycle and can be present as a sustained
current component throughout the diastolic depolariza-
tion phase.
The role of I
Kr
in rabbit pacemaking has also been
studied by a numerical modeling approach (369, 371).
Calculation results are consistent with the view that I
Kr
is
the key repolarizing current in rabbit SAN cells. I
Kr
in-
ward rectification results in a decrease in the membrane
resistance during the repolarization phase, relative to pla-
teau phase (369). I
Kr
deactivation contributes to the net
current change during the early diastolic depolarization
phase (369). Consequently, I
Kr
activation and kinetics
during the pacemaker cycle are critical factors for deter-
mining activation of ion channels during diastolic depo-
larization. It has also been suggested that I
Kr
dependency
from the extracellular K
⫹
concentration ([K
⫹
]
o
)iniso-
lated pacemaker cells may account for the sensitivity of
pacing rate to elevated [K
⫹
]
o
in the intact SAN (369).
I
Kr
thus plays an obligatory role in action potential
repolarization of isolated rabbit and mouse (93) SAN
cells. However, block of I
Kr
by E-4031 significantly slows,
but does not completely suppress, pacemaking in isolated
Langendorff-perfused mouse hearts (93, 371) (Fig. 10A)or
in intact rabbit right atrial preparations (500) (Fig. 10B).
These observations may be explained by the presence of
the electrotonic load imposed to the SAN by atrial tissue,
which has a more negative diastolic potential. Atrial elec-
trical influence partially compensates for I
Kr
block to
drive SAN repolarization (500). In conclusion, I
Kr
also
plays an important role in the way in which SAN automa-
ticity is coupled to the surrounding atrial tissue. The
regional distribution of I
Kr
expression needs to be taken
into consideration for understanding the physiological
relevance of this current. As previously indicated, I
Kr
is
expressed regionally in the rabbit SAN (249). Small cells
display a lower I
Kr
density than larger cells (278). It has
been proposed that low I
Kr
expression is responsible for
the less negative maximum diastolic potential in central
SAN cells and could explain the enhanced sensitivity of
pacemaking in the SAN center to E-4031 (278).
If I
Kr
appears to be the predominant delayed rectifier
current in the rabbit and rodents, primary SAN automa-
ticity in pigs has been reported to depend from I
Ks
(372).
In this preparation, block of I
Ks
by 293B stops automatic-
ity the same way E-4031 does in rabbit SAN cells. As no
detectable I
Kr
has been found in pig SAN cells, we should
conclude that I
Ks
effectively replaces I
Kr
for pacemaking
in the pig. The presence of I
Ks
rather than I
Kr
in pig SAN
can be a form of adaptation to a slower heart rate in large
mammals compared with rodents (372). Inhibition of I
Ks
has a significant effect on pacemaker activity in guinea pig
(320) and peripheral rabbit (275) SAN cells. As these cells
express both I
Kr
and I
Ks
, I
Kr
can still drive pacemaking
under inhibition of I
Ks
.
D. Role of I
f
in Automaticity
The physiological relevance of the I
f
current in pace-
making has been a matter of debate for many years.
However, a consistent amount of data now shows that I
f
plays a key role in the generation of adult SAN pacemaker
activity. For completeness, we will briefly summarize
some useful aspects of the debate on the role of I
f
in SAN
pacemaking, because this point is still discussed in some
recent reviews (2, 94, 121, 307).
In the SAN, I
f
is the only voltage-dependent current
to be activated upon membrane hyperpolarization. By the
biophysical point of view, f-channels can open during the
late phase of repolarization close to the maximum dia-
stolic potential. According to the current view, I
f
would
initiate the first part of the diastolic depolarization until
the activation threshold of T- and L-type channels is
reached (94, 117). It has been questioned whether I
f
size
and kinetics at diastolic voltages would be compatible
with the amount of inward current needed to initiate a
depolarizing phase in the presence of an outward current
(113, 173). Arguments for this were based on the observed
variability of the I
f
activation threshold in isolated SAN
cells and on the relatively slow I
f
activation kinetics at
positive voltages (114). These kinds of uncertainties have
led Denyer and Brown (114) and Hagiwara et al. (173) to
propose an alternative view of the initiation of the dia-
stolic depolarization in SAN. These authors have mea-
sured a voltage-independent, time-independent “back-
ground” current (I
b
) carried by Na
⫹
(173). The size of this
current would be theoretically sufficient to drive the dia-
stolic depolarization upon I
Kr
decay (173). According to
this view, the diastolic depolarization is initiated by the
decay of an outward current (I
Kr
) which unmasks a volt-
age-independent constant depolarizing current (I
b
) (173).
These results have been directly challenged by Di-
Francesco (116) who showed that, at least in some beat-
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ing SAN cells, the instantaneous time-independent cur-
rent on hyperpolarization flows in the outward direction
(rather than inward) in the full range of the diastolic
depolarization. This work has also shown that the I
f
size
at weak hyperpolarizing potentials can be substantially
underestimated in patch-clamp experiments due to cur-
rent run-down and wash-out phenomena of the cytosolic
environment (116). However, the reversal potential of I
b
can vary in preparations of isolated SAN cells as reported
by different authors (114, 116, 173, 492, 496). Noble et al.
(359) have used a numerical modeling approach to infer
the effects of varying I
b
and I
f
densities on rabbit SAN
pacemaking. These authors have suggested that I
b
and I
f
may play a reciprocal role in pacemaking, because an
increase in one of these current induces a reduction in the
amount of the other current. As for I
b
, the size of I
f
at
voltages positive to the maximum diastolic potential
would be sufficient to drive the diastolic depolarization.
In this respect, Van Ginneken and Giles (492) and Zaza
et al. (541) have also reported that the size of I
f
can
increase significantly in the diastolic depolarization range.
Particularly, Zaza et al. (541) have shown that the net
inward Cs
⫹
-sensitive current during the diastolic depolar-
ization is almost fourfold that theoretically necessary to
drive this phase.
In conclusion, available electrophysiological data
show that I
f
is in fact activated during the diastolic depo-
larization. Both time-independent background currents
and I
f
may show variability between preparations and
recording conditions employed (113, 116, 173, 496). At
least part of SAN background currents can be attributed
to either I
Cl
(37) or TRPM4-mediated I
b
described by
Demion et al. (109) However, their relevance to pacemak-
ing is not yet firmly established. Discussions on the role of I
f
in pacemaking are now focused on whether this current is
necessary for automaticity and on its relative contribution to
the
␤
-adrenergic modulation of heart rate (121, 307).
The relevance of I
f
to pacemaker activity is now
supported by pharmacological and genetic evidence. In-
deed, pharmacological inhibition of f-channels slows
pacemaker activity in vitro in isolated pacemaker cells
and SAN tissue, as well as in vivo in experimental animal
models.
Sensitivity of f-channels to Cs
⫹
has been used to infer
the quantitative contribution of f-channels to SAN pace-
maker activity (113, 365). When tested on spontaneously
beating cells, 2–5 mM Cs
⫹
significantly slows the pacing
rate (113). Cs
⫹
induces negative chronotropism even in
the absence of any evident additional effect on I
Kr
or I
Ca,L
(113). The fact that Cs
⫹
slows but does not stop pace-
maker activity has been interpreted as an indication that
I
f
contributes to pacemaking without being an absolute
prerequisite for automaticity (113, 365). This interpreta-
tion has been questioned by DiFrancesco (120), who high-
lighted that Cs
⫹
block would be partially relieved at pos-
itive voltages. According to DiFrancesco (120), unblocked
f-channels would still be able to drive the diastolic depo-
larization at rest and to mediate control of pacemaking by
autonomic agonists (420). In conclusion, the action of
Cs
⫹
on pacemaker activity demonstrates that I
f
partici-
pates to the generation of pacemaker activity. However,
no definitive quantitative insights about the importance of
f-channels can be obtained by using this ion.
The negative chronotropic action of organic f-chan-
nel blockers such UL-FS 49 (480, 488), ZD-7228 (53, 318),
and ivabradine (480) provides further evidence on the
relevance of I
f
in the control of the diastolic depolariza-
tion rate. These inhibitors of native cardiac I
f
are all open-
and use-dependent f-channel blockers (see Ref. 21 for
review). The mechanism of channel block is essentially
similar for all these drugs. Indeed, the drug binds to the
channel from the intracellular side and probably stays in
its binding site when the channel shuts off during deacti-
vation. When the channel opens during the following
pacemaker cycle, current flow in the inward direction will
promote channel unblock (66, 162, 489, 490).
The most specific organic blocker of I
f
is ivabradine
(21). When tested at concentrations that are selective for
I
f
, ivabradine slows pacemaker activity by specifically
reducing the slope of the linear part of the diastolic
depolarization (68, 479) (Fig. 11A). This drug blocks the
native rabbit SAN I
f
with an EC
50
of ⬃2–3
␮
M (48). At
these concentrations, ivabradine slows automaticity in
isolated rabbit pacemaker cells by ⬃20% (479). Consis-
tently, ivabradine reduces the heart rate in vivo in con-
scious dogs (461) and mice (132, 280) (Fig. 11B). The
observation that ivabradine leads to heart rate slowing,
but does not block pacemaking, suggests that pharmaco-
logical inhibition of f-channels in these conditions can
regulate pacemaking without impairing automaticity per
se. However, the relative fraction of I
f
blocked during
pacemaker activity in these experiments is difficult to
estimate since, as previously discussed, the blocking ac-
tion of ivabradine on f-channels is both use and current
dependent (66). Consequently, current block will be more
pronounced at positive potentials such as during action
potential repolarization and late diastolic depolarization.
The fraction of I
f
blocked for a given concentration of
ivabradine measured at potentials negative to the maxi-
mum diastolic potential is thus likely to represent an
underestimation of the real current block during pace-
maker activity. On the other hand, slowing of pacing rate
induced by ivabradine will favor unblock of the channel.
Reduction of heart rate by ivabradine at a given rate is
thus the equilibrium between f-channel block and un-
block aided by the reduced firing rate. Further investiga-
tion is needed to clarify the exact quantitative relationship
between I
f
block by heart rate reducing agents (as ivabra-
dine) and the slowing of pacemaker activity. The hetero-
geneous distribution of I
f
will also influence the sensitiv-
940
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ity of pacemaker activity to different f-channel blockers in
the center versus the periphery of the SAN. Indeed, the
fractional slowing of pacemaker activity by Cs
⫹
, UL-FS
49, and ZD-7228 is larger in the SAN periphery than in the
center (355).
In addition to electrophysiological and pharmacolog-
ical data, there is now substantial genetic evidence of the
relevance of f-channels in the generation of automaticity.
In animal models, insights are coming from a spontaneous
zebrafish mutant (18) as well as from genetically modified
mice lacking HCN2 and HCN4 channels in the heart (196,
294, 466). The first genetic evidence associating f-chan-
nels with the generation of cardiac pacemaking came
from a large-scale random mutagenesis study aimed to
identify new genes involved in zebrafish cardiovascular
development (Fig. 12A). The mutant line slow mo is as-
sociated with a reduced heart rate in embryos (18) and
adult (513) zebrafish. Isolated heart cells from slow mo
embryos have no fast-activating I
f
component and show
strongly reduced I
f
density (18) (Fig. 12A). Similarly, adult
slow mo fishes have reduced heart rate and low I
f
expres-
sion in the atrium and ventricle (513). The association
between low I
f
expression and bradycardia in slow mo
mutants constitute strong evidence of the importance of
f-channels for automaticity in zebrafish.
Inactivation of HCN2 channels in the mouse heart
induces SAN dysrhythmia and a 30% reduction of I
f
in
isolated SAN cells (294) (Fig. 12B). HCN2 gene inactiva-
tion slows the kinetics of I
f
activation, suggesting that
HCN2 channels may contribute to the fast kinetic compo-
nent of I
f
. Interestingly, maximal I
f
current stimulation by
cAMP is not changed in SAN cells from HCN2 knockout
mice (294) (Fig. 12B). HCN2 knockout mice do not show
reduction of the mean heart rate (294). However, SAN
dysrhythmia of HCN2 knockout mice indicates that these
channels play a role in stabilizing the heart rate. Global or
heart-specific inactivation of HCN4 channels provokes
lethality in mouse embryos. Embryos lacking HCN4 chan-
nels die between day 9 and 12 post coitum (466). Younger
embryos lacking HCN4 channels have slow heart rate and
show almost complete suppression of I
f
. Furthermore,
cAMP regulation of heart rate is abolished in developing
hearts (466). Stieber et al. (466) have also reported an
absence of pacemaker cells having “mature” action poten-
tial characteristics in HCN4-deficient embryonic hearts.
Indeed, only cells characterized by early “embryonic”
pacemaker activity (308) are found in HCN4-deficient
hearts. To overcome the problem of embryonic lethality
in mice lacking HCN4 channels, Hermann et al. (196) have
developed a temporally inducible deletion of Hcn4
(HCN4
C
mouse). SAN cells from HCN4
C
mice show a 75%
reduction of I
f
with a depressed response to isoprotere-
nol. Most HCN4
C
cells are quiescent, but normal automa-
ticity can be restored to normal pacing rates by superfu-
sion of saturating doses of isoproterenol (196). In vivo,
HCN4
C
mice are characterized by the presence of SAN
pauses, yet the maximal heart rate measured under exer-
cise, or after administration of isoproterenol, does not
differ from that of wild-type counterparts (196). Hermann
et al. (196) reported that the frequency of SAN pauses is
significantly increased during transitions for elevated to
low heart rates. These authors have thus proposed that
HCN4 channels are not required for acceleration of heart
rate, but rather constitute a backup mechanism that is
FIG. 11. A: pharmacological inhibition of I
f
by 0.3
␮
M ivabradine
slows pacemaker activity of a rabbit SAN pacemaker cell (top panel).
Note that I
f
inhibition reduces the cell pacing rate by selectively reduc-
ing the slope of the linear part of the diastolic depolarization, without
affecting any other action potential parameter. Ivabradine block of I
f
current is shown in the bottom panel. [From DiFrancesco (121).]
B: in vivo heart rate reduction in mice treated by an intraperitoneal bolus
of increasing doses of ivabradine. (A⫹P) indicate coinjection with iv-
abradine of atropine and propranolol to inhibit pharmacologically, the
autonomic input (see sect. VIIA). [Original data from Marger et al. (316).]
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important for stimulating and stabilizing pacemaker initi-
ation in conditions of lowered heart rate.
In summary, results obtained in mice lacking HCN2
and HCN4 channels suggest a predominant role of HCN4
channels in mediating the adrenergic stimulation of I
f
.
However, results obtained from HCN4
C
mice (196) indi-
cate that HCN4 channels are not the only mechanism
involved in the
␤
-adrenergic regulation of heart rate, at
least in the adult mouse.
E. Role of I
Ca,L
in Automaticity
L-type Ca
2⫹
channels are important contributors to
the upstroke phase of the pacemaker action potential and
play a role in the generation of the SAN diastolic depo-
larization (for a recent review, see Ref. 310). Block of I
Ca,L
by nifedipine stops pacemaker activity in primary central
pacemaker cells (250). However, application of nifedipine
to SAN tissue strips from the periphery of the node slows
pacemaking but does not block automaticity. These ob-
servations have been accounted for by the presence in the
SAN periphery of I
Na
driving the action potential upstroke
under I
Ca,L
blockade (250). The differential effect of I
Ca,L
inhibition in the center and in the periphery of the rabbit
SAN underlines the problem of separating the possible
contribution of I
Ca,L
to the diastolic depolarization from
that of the upstroke phase of the action potential. This
problem is particularly difficult to solve, since pharmaco-
logical block of I
Ca,L
during the action potential upstroke
can have “knock on” effects on the recruitment of ionic
currents during repolarization as well as the following
diastolic depolarization. Furthermore, I
Ca,L
is regulated by
PKA and CaMKII (390, 509). Consequently, the contribu-
tion of I
Ca,L
to automaticity can be influenced by the
phosphorylation state of the channel. CaMKII activity is
required for pacemaking, since CaMKII inhibitors termi-
nate automaticity in isolated cells (509). The necessity of
CaMKII in automaticity can be explained, in part, by the
regulation of I
Ca,L
activation and reactivation kinetics by
CaMKII (509). However, CaMKII can also have other un-
known targets in pacemaker cells. Thus sensitivity of
pacemaking to inhibition of CaMKII should not be taken
as evidence of the importance of I
Ca,L
in the genesis of
automaticity per se.
There is now substantial genetic and pharmacolog-
ical data supporting the view that I
Ca,L
contributes to
both the action potential upstroke and diastolic depo-
larization. Indeed, Ca
v
1.2 channels seem to be involved
in the action potential upstroke phase, while Ca
v
1.3
channels substantially contribute to the diastolic depo-
larization (309, 310).
Verheijck et al. (497) have been the first to propose
that the I
Ca,L
activation threshold is more negative in SAN
than in the ventricle (497). These authors recorded the
FIG. 12. A: Slow mo zebrafish mutant embryos have reduced heart
rate and lack the fast kinetic component of I
f
. Heart rate slowing is
consistently found throughout the embryo development and is indepen-
dent from the water temperature (top panel). I
f
current density is
strongly downregulated in cardiac myocytes isolated from slow mo
embryos. Residual I
f
in Slow mo hearts displays slower activation kinet-
ics than in wild-type (WT) counterparts (bottom panel). [From Baker
et al. (18), copyright National Academy of Science, USA.] B: telemetric
electrocardiograms in freely-moving HCN2 knockout (HCN2
⫺/⫺
) mice
show SAN arrhythmia. Note that the basal heart rate is similar in WT and
HCN2
⫺/⫺
mice, but the interbeat interval is more variable in knockout
animals. The density of I
f
is reduced by ⬃30% in HCN2
⫺/⫺
SAN cells, but
current responsiveness to cAMP is unaltered. [From Ludwig et al. (294),
with permission from Macmillan Publisher Ltd.]
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DHP-sensitive current during the diastolic depolarization
and showed that I
Ca,L
is activated as early as in the linear
part of the pacemaker potential. I
Ca,L
current density then
increases at the action potential threshold and becomes
fully activated during the upstroke. This work demon-
strated that the kinetics of SAN I
Ca,L
do not behave as its
ventricular counterpart but is activated at more negative
voltages to support the pacemaker potential.
Involvement of L-type channels in pacemaking in vivo
is also strongly suggested by in vivo ECG recordings
showing that DHPs induce bradycardia in anesthetized
mice (268). The unexpected observation that mice lacking
L-type Ca
v
1.3 channels had pronounced bradycardia and
SAN arrhythmia was the first genetic indication of the
importance of these channels in pacemaker activity (393).
In these mice, bradycardia persists after pharmacological
block of the autonomic nervous system, and intact atria
from Ca
v
1.3
⫺/⫺
mice have slower pacing rate than wild-
type counterparts (393) (Fig. 13). Two studies have dem-
onstrated that Ca
v
1.3 channels play a major role in auto-
maticity of isolated SAN (546) (Fig. 14A) and in pace-
maker cells (309) (Fig. 14B). Indeed, pacemaker cells
from Ca
v
1.3 knock-out mice have erratic and intermittent
pacemaking, leading to an overall lower degree of au-
tomaticity than that of wild-type cells (309) (Fig. 14B).
These studies have also shown that Ca
v
1.3 channels
generate I
Ca,L
with different properties than that of
Ca
v
1.2-mediated I
Ca,L
. Indeed, native Ca
v
1.3 channels
activate at negative potentials from about ⫺50 mV (see
Fig. 15A). Inactivation of Ca
v
1.3 channels shifted the
activation of I
Ca,L
to more positive potentials, thereby
abolishing I
Ca,L
in the voltage range corresponding to
FIG. 13. Electrocardiograms of freely moving Ca
v
1.3
⫺/⫺
mice show
bradycardia and slowing of atrioventricular conduction. Note that in
Ca
v
1.3
⫺/⫺
mice, the interbeat interval is irregular and significantly longer
than in WT mice (SAN arrhythmia, top panel). Association of bradycar-
dia and arrhythmia is typical of resting Ca
v
1.3
⫺/⫺
mice with unaltered
autonomic regulation of heart rate (ANS⫹). When the input of the
autonomic nervous system is blocked postsynaptically by combined
administration of atropine and propranolol (ANS⫺), arrhythmia is in
part compensated, but bradycardia and prolongation of the PR interval
are still evident. (Mangoni and Nargeot, unpublished recordings.)
FIG. 14. A: intracellular microelectrode recordings of automaticity in isolated SANs from WT, Ca
v
1.3
⫹/⫺
, and Ca
v
1.3
⫺/⫺
mice. Note slowing of
automaticity and pauses in a SAN from Ca
v
1.3
⫺/⫺
mouse. [Data from Zhang et al. (546).] B: isolated SAN pacemaker cells from Ca
v
1.3
⫺/⫺
mice have
slower pacemaker activity than WT cells (top panel). Slow pacemaking in cells lacking Ca
v
1.3 channels is associated with irregular interbeat interval
(bottom panel). [Data from Mangoni et al. (309).]
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that of the diastolic depolarization. Inactivation of
Ca
v
1.3 channels reduces I
Ca,L
density by ⬃70% in pace-
maker cells. Residual I
Ca,L
is attributable to Ca
v
1.2-
mediated I
Ca,L
. Consistent with this hypothesis, I
Ca,L
in
pacemaker cells from Ca
v
1.3 knockout mice is more
sensitive to DHPs (309) and has faster inactivation
kinetics (546). Activation of
␤
-adrenergic receptors by
norepinephrine shifts negatively the threshold for acti-
vation of Ca
v
1.3-mediated I
Ca,L,
to about ⫺55 mV (309).
Interestingly, this threshold is comparable to that ob-
served for the nifedipine-sensitive I
Ca,L
measured in
spontaneously beating rabbit SAN cells (497). This sug-
gests the existence of Ca
v
1.3 channels also in rabbit
SAN cells. The maximal pacing rate in Ca
v
1.3 knockout
hearts in the presence of isoproterenol is slightly
slower than that of wild-type hearts (322). This obser-
vation can be explained by taking into consideration
that stimulation of I
Ca,L
by saturating concentrations of
norepinephrine cannot compensate for the lack of I
Ca,L
in the diastolic depolarization range (309).
In conclusion, insights gained from mice lacking
Ca
v
1.3 channels are indicative of a distinction in the func-
FIG. 15. A: voltage-dependent Ca
2⫹
cur-
rents in pacemaker SAN cells from WT and
Ca
v
1.3
⫺/⫺
mice. Currents are evoked from a
holding potential of ⫺80 mV at the indicated
test potentials. Current stimulation by BAK K
8644 is used to detect activation of I
Ca,L
at a
given test voltage. In Ca
v
1.3
⫺/⫺
SAN cells, BAY
K 8644 has no effect at test potentials negative
to ⫺30 mV. This observation indicates that
inactivation of Ca
v
1.3 channels abolishes a
I
Ca,L
component which has intermediate acti-
vation threshold between I
Ca,T
, and the “clas-
sical” I
Ca,L
.Ca
v
1.3-mediated I
Ca,L
is activated in
the diastolic depolarization range (DD, shaded
gray box). [Data from Mangoni et al. (309).]
B: inactivation of Ca
v
3.1 channels suppresses
I
Ca,T
in spontaneously active SAN and AVN
cells isolated from Ca
v
3.1
⫺/⫺
mice. Note reduc-
tion of the total Ca
2⫹
current density and the
slowing of the total I
Ca
inactivation. The resid-
ual Ca
2⫹
current is attributed to Ca
v
1.3- and
Ca
v
1.2-mediated I
Ca,L
. [Data from Mangoni
et al. (314).] C: current-to-voltage relationships
(left) and steady-state inactivation (right)of
native SAN Ca
v
3.1, Ca
v
1.3, and Ca
v
1.2 chan-
nels. [Adapted from Mangoni et al. (310).]
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tional role of Ca
v
1.3 channels contributing to automaticity
and Ca
v
1.2 channels triggering myocardial contraction.
Indeed, suppression of Ca
v
1.3-mediated I
Ca,L
impacts on
cardiac automaticity and conduction, but has no effect on
myocardial contractile performance (322). The differen-
tial roles of Ca
v
1.3 and Ca
v
1.2 channels in the heartbeat
has also been shown pharmacologically by employing a
knock-in mouse strain in which Ca
v
1.2 channels are in-
sensitive to DHPs (Ca
v
1.2DHP
⫺/⫺
mouse) (462). The in
vivo bradycardic effect induced by DHPs is not changed
in Ca
v
1.2DHP
⫺/⫺
mice, indicating that the dominant L-
type VDCC isoform participating to automaticity is in fact
Ca
v
1.3 (462).
As stated above, Ca
v
1.3 knockout mice also display a
slowing of atrioventricular conduction (see Fig. 13) (322,
393, 546). Particularly, Ca
v
1.3 knockout mice show I and
II degree atrioventricular blocks, an observation which
has been found in freely moving (393) and anesthetized
mice (546), as well as in isolated Ca
v
1.3 knockout hearts
(322). It will be interesting to test the hypothesis that
Ca
v
1.3 channels are also involved in automaticity of the
AVN.
Inactivation of the Ca
v
1.2
␣
1-subunit induces lethal-
ity of the late mouse embryo. Heart development in early
Ca
v
1.2 knockout embryos is paralleled by overexpression
of the Ca
v
1.3
␣
1-subunit (529), as well as by upregulation
of a distinct L-type Ca
2⫹
current that is also present in
Ca
v
1.3 knockout embryonic hearts (445). Overexpres-
sion of Ca
v
1.3 channels can be a compensatory mecha-
nism for the loss of Ca
v
1.2 channels, even if Ca
v
1.3 chan-
nels cannot ensure viability of the late embryo. Embry-
onic hearts from a spontaneous zebrafish mutant strain
lacking the Ca
v
1.2
␣
1 subunit (Isl mutant) show de-
pressed and erratic atrial beating (425). Consistent with
the role of Ca
v
1.2 in mediating cardiac contraction, hearts
from Isl mutants have no ventricular contraction. It is not
known whether Ca
v
1.2 channels can contribute to auto-
maticity in embryonic fish atria. Jones et al. (222) re-
ported progressive loss of Ca
v
1.2 protein in SAN tissue
from aging guinea pigs, concomitantly with a reduction in
automaticity and an augmentation of SAN rate sensitivity
to DHPs. These data are suggestive of a participation of
Ca
v
1.2 channels in pacemaking, at least during the aging
process.
F. Role of I
Ca,T
in Automaticity
I
Ca,T
is expressed in the SAN (145, 172), AVN (140),
and Purkinje fibers (199, 484). T-channels can be found in
the automatic mouse and zebrafish embryonic myocar-
dium (18, 104). I
Ca,T
has also been described in the am-
phibian sinus venosus (49).
The role of I
Ca,T
in cardiac automaticity has long
been uncertain. Native I
Ca,T
has low steady-state availabil-
ity at diastolic membrane voltages typical of leading SAN
pacemaker cells. However, it has long been known that
I
Ca,T
inhibitors such as Ni
2⫹
and tetrametrine slow pace-
maker activity of SAN cells (172, 439).
The role of T-channels in automaticity has been re-
cently investigated by targeted inactivation of the Ca
v
3.2
(84) and Ca
v
3.1 (314)
␣
-subunit isoforms. Mice lacking
Ca
v
3.2 channels have no ECG alterations (84), indicating
that the lack of this isoforms either has no impact on the
generation and conduction of the cardiac impulse, or that
a compensatory mechanism is established during heart
development. It is not still possible to discriminate be-
tween these hypotheses, because SAN ionic currents and
automaticity have not yet been studied in Ca
v
3.2
⫺/⫺
mice.
A striking finding coming from Ca
v
3.1
⫺/⫺
mice is that I
Ca,T
cannot be detected in SAN and AVN cells (Fig. 15B).
Indeed, as both Ca
v
3.1 and Ca
v
3.2 mRNA are expressed in
mouse SAN, one would expect to find residual I
Ca,T
in
Ca
v
3.1
⫺/⫺
pacemaker cells. The absence of Ca
v
3.2-medi-
ated I
Ca,T
in Ca
v
3.1
⫺/⫺
SAN cells has not yet been ex-
plained, but recent studies have reported that only Ca
v
3.1-
mediated I
Ca,T
is recorded in rat atria after birth (146,
358). These findings are suggestive of a developmental
switch between Ca
v
3.2 and Ca
v
3.1 channels in the heart.
According to this hypothesis, Ca
v
3.2 channels are func-
tionally expressed in the developing myocardium, while
Ca
v
3.1 channels are predominant in the adult heart. It
would be important to investigate the differential role of
Ca
v
3.1 and Ca
v
3.2 channels to automaticity during the
cardiac development. Inactivation of Ca
v
3.1 channels in-
duces moderate bradycardia and slowing of atrioventric-
ular conduction in Ca
v
3.1
⫺/⫺
mice (314) (Fig. 16). Moder-
ate bradycardia is observed in Ca
v
3.1
⫺/⫺
mice even after
pharmacological block of the autonomic nervous system,
indicating slowing of SAN automaticity (Fig. 16C). Ac-
cordingly, spontaneous activity in isolated SAN pace-
maker cells is slowed by ⬃30% (Fig. 16D).
Despite pharmacological and genetic data showing
the involvement of I
Ca,T
in pacemaking, we presently lack
a precise description of how T-channel activity contrib-
utes to the diastolic depolarization. The existence of a
I
Ca,T
-mediated “window” current component in the dia-
stolic depolarization range has been proposed for rabbit
SAN cells (401). However, this is not a consistent finding
between different authors (see, for example, Ref. 172).
The relatively negative threshold for activation of Ca
v
1.3-
mediated I
Ca,L
can possibly interfere with measurement of
I
Ca,T
, leading to the false impression of residually avail-
able I
Ca,T
at about ⫺50 mV (M. Mangoni, unpublished
observations; see also Fig. 15, A and C). As the absolute
density of I
Ca,T
available at pacemaker potentials is low,
one may wonder whether contribution of T-channels to
pacemaking can be due to coupling with intracellular
Ca
2⫹
signaling. Such a mechanism has been proposed by
Huser et al. (211) in cat latent atrial pacemaker cells.
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These authors have recorded intracellular Ca
2⫹
release
during pacemaking and found that 40
␮
MNi
2⫹
reduced
Ca
2⫹
release from the sarcoplasmic reticulum (SR) and
the slope of the late phase of the diastolic depolarization.
Ni
2⫹
did not affect Ca
2⫹
release during the action poten-
tial upstroke (systolic Ca
2⫹
), but only Ca
2⫹
released dur-
ing the diastolic depolarization phase (diastolic Ca
2⫹
). In
latent pacemaker cells, I
Ca,T
activation during diastolic
depolarization triggers local Ca
2⫹
release, generating an
inward current through stimulation of I
NCX
(211). Accord-
ing to Lipsius et al. (288), there exists a local control of
Ca
2⫹
release involving T-type channels, RyRs of the SR,
and NCX (288). Such a functional coupling between T-
type channels and SR could explain earlier observations
indicating that prevention of SR Ca
2⫹
release with ryan-
odine reduces I
Ca,T
(285). No direct evidence for I
Ca,T
-
induced SR Ca
2⫹
release in primary SAN pacemaker cells
has yet been reported. Vinogradova et al. (506) have re-
ported that 50
␮
MNi
2⫹
failed to inhibit subsarcolemmal
diastolic Ca
2⫹
release in spontaneously beating rabbit
SAN cells, as well as spontaneous Ca
2⫹
release in arrested
pacemaker cells (510). However, these data do not ex-
clude the possibility that Ca
v
3.1-mediated T-channels can
contribute to SR Ca
2⫹
release since native SAN Ca
v
3.1
channels are resistant to this concentration of Ni
2⫹
(314).
The more negative diastolic potential in latent pacemaker
cells can favor T-channel opening during the diastolic
depolarization. The role of I
Ca,T
in automaticity of the
conduction system is still unknown. Ca
v
3.1
⫺/⫺
mice have
slowed atrioventricular conduction (314) (Fig. 16). Mea-
surements of atrioventricular conduction times indicate
that the AVN is the site where impulse propagation is
slowed (314). It will be interesting to test if automaticity
of AVN cells is affected by inactivation of Ca
v
3.1 channels.
Ca
v
3.1 channels do not appear to be involved in conduc-
tion in mouse Purkinje fibers. Indeed, neither the intra-
ventricular conduction time nor the QRS are changed in
Ca
v
3.1
⫺/⫺
mice (314).
G. Role of N- and R-type Channels in
Heartbeat Regulation
Beside L- and T-type channels, an involvement of
VDCCs belonging to the Ca
v
2 gene family in the regulation
of heart rhythm and rate has been recently proposed.
Mouse lines lacking N-type Ca
v
2.2 and R-type Ca
v
2.3 chan-
nels have been developed (214, 293, 516). No expression
of Ca
v
2.2 channels in the heart has been reported. How-
ever, these channels contribute to the activity of sympa-
thetic nerve terminals (214). Ca
v
2.2
⫺/⫺
mice have in-
creased heart rate and mean blood pressure (214). These
effects are possibly due to reduced activity of the sympa-
thetic nerve terminals (214). Parasympathetic input is not
affected in these mice (214).
Expression of Ca
v
2.3 isoforms in the mouse heart has
been reported in atrial tissue by in situ hybridization
(332), and in the heart at the protein level (293). Enhanced
sympathetic tonus, ventricular arrhythmias, and dysfunc-
tion in ventricular conduction have been found in mice
lacking Ca
v
2.3 channels (293, 516). Disturbances of atrial
activation and alterations of the QRS complex are ob-
FIG. 16. Ca
v
3.1
⫺/⫺
mice have moderate heart rate reduction and prolongation of the PR interval. A: telemetric electrocardiograms of WT and
Ca
v
3.1
⫺/⫺
mice. B: circadian variability of heart rate in WT and Ca
v
3.1
⫺/⫺
mice. Continuous recordings lasting 24 h provide evidence that the mean
heart rate is lowered in Ca
v
3.1
⫺/⫺
mice (dotted lines). C:Ca
v
3.1
⫺/⫺
mice have intrinsically slower heart rate than WT mice. D: pacemaker activity
of isolated SAN cells is slower in Ca
v
3.1 than in WT mice. [Data from Mangoni et al. (314).]
946 MATTEO E. MANGONI AND JOE
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served even after administration of atropine and propran-
olol (516). Overexpression of Ca
v
3.1 mRNA has been
reported in adult Ca
v
2.3
⫺/⫺
hearts (516), but direct evi-
dence supporting functional expression of Ca
v
2.3 chan-
nels in the mouse SAN and conduction system is lacking.
H. Role of I
Na
in Automaticity
Primary pacemaker cells are characterized by a rel-
atively slow action potential upstroke. The concept that
the action potential of pacemaker cells is mainly driven by
Ca
2⫹
rather than Na
⫹
channels is still valid, although new
studies on the rabbit SAN have indicated that a distinction
has to be made between small pacemaker cells from the
SAN center and larger cells from the periphery of the
node. Two different I
Na
components have been function-
ally identified in SAN, a TTX-sensitive “neuronal” I
Na
pre-
dominantly coded by the Na
v
1.1 (24, 25, 279, 304) isoform,
and a TTX-resistant I
Na
, coded by the Na
v
1.5 isoform (276,
279). TTX-sensitive I
Na
contributes to pacemaking in new-
born rabbits (24) (Fig. 17A) and in the adult mouse (279,
304). Na
v
1.5-mediated TTX-resistant I
Na
is involved in
SAN intranodal conduction (276) (Fig. 17, B and C).
The contribution of TTX-sensitive I
Na
to automaticity
has been studied in neonatal rabbit SAN by Baruscotti
et al. (24). In newborn SAN cells, application of 3
␮
M TTX
slows pacemaker activity by 63%. Baruscotti et al. (24)
have reported slowing by TTX of the late phase of the
diastolic depolarization, reduction of the action potential
threshold, and overshoot. These observations indicate
that TTX-sensitive I
Na
contributes to SAN pacemaking
essentially by quickening the late diastolic depolarization
phase and by shifting the action potential threshold neg-
atively to that of VDCCs. The mechanism of contribution
of TTX-sensitive I
Na
to the diastolic depolarization in
neonatal rabbit SAN cells has also been investigated by
these authors in two distinct studies (23, 24). In the first
study (24), they reported the presence of a significant I
Na
“window” component that declines with increasing age of
the animal. In a follow-up (23) study, these authors have
shown that the amount of TTX-sensitive current recorded
by applying ramp depolarizations mainly depends on the
ramp slope. This observation suggests that incomplete
inactivation of TTX-sensitive I
Na
during the upstroke
phase underlies persistent I
Na
during the diastolic depo-
larization (23). The expression of the Na
v
1.1 isoform is
downregulated in the adult (Fig. 17A) (23). The physio-
logical significance of the expression of Na
v
1.1-mediated
I
Na
in newborn SAN has not been completely elucidated,
but it is likely that TTX-sensitive I
Na
constitutes a mech-
FIG. 17. A: differential effect of TTX on pacemaker activity in SAN pacemaker cells from newborn (top, left) and adult (top, right) rabbits.
Inhibition of I
Na
by TTX blocks pacemaking in newborn cells, while it has no effect on automaticity of adult cells. A voltage-clamp ramp protocol
shows the presence of a TTX-sensitive current component in pacemaker cells from newborn rabbits (bottom, left). Such a component is not recorded
in cells from adult rabbits, indicating a developmental regulation of the expression of TTX-sensitive I
Na
. [From Baruscotti et al. (23).] B:a
TTX-sensitive I
Na
contributes to the exponential phase of the diastolic depolarization in the mouse SAN. The figure depicts an “action potential
clamp experiment” in which a mouse SAN cell has been clamped with its own action potential (top, left) so that the net clamp current equals zero.
The cell has then been exposed to TTX and the current necessary to maintain the membrane voltage recorded. This net TTX-sensitive I
Na
is plotted
versus the time (bottom, left) and versus the membrane voltage (right). From this experiment, it is apparent that TTX-sensitive I
Na
activates in the
exponential phase of the diastolic depolarization between ⫺45 and ⫺40 mV, quickly reaches its peak at ⫺20 mV, and switches off at about ⫹20 mV.
[From Lei et al. (279).] C: electrical mapping of SAN intranodal conduction in WT and Scn5a
⫹/⫺
mice. The change in the SAN activation sequence
in Scn5a
⫹/⫺
mice indicates that TTX-resistant Scn5a-mediated I
Na
is important in impulse conduction within the SAN and from the SAN to the right
atrium. [From Lei et al. (276), with permission from Wiley-Blackwell.]
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anism for increasing the basal heart rate in newborn
animals (24).
During the last years, it has become apparent that I
Na
significantly contributes to mouse pacemaking. Pharma-
cological inhibition of I
Na
by lidocaine reduces the heart
rate of adult mice (268). The pacing rate of Laghendorff-
perfused mouse hearts is slowed by low doses of TTX
(50–100 nM) (304). Adult mouse pacemaker cells show
constitutive expression of I
Na
(86, 312). Both TTX-sensi-
tive and -resistant I
Na
have been functionally identified in
mouse SAN cells (279). Similar to newborn rabbits, ex-
pression of Na
v
1.1
␣
-subunit has been reported in SAN
sections from adult mice (279, 304). These in vivo and in
vitro experiments have demonstrated the involvement of
TTX-sensitive, neuronal I
Na
in mouse cardiac pacemak-
ing. The action of TTX on cellular pacemaking has been
directly investigated in spontaneously beating mouse SAN
cells by Lei et al. (279). These authors have combined
voltage-clamp recordings of I
Na
with staining of SAN tis-
sue with antibodies directed against Na
v
subunits. TTX-
sensitive and TTX-resistant I
Na
have been recorded. TTX-
sensitive I
Na
is associated with expression of the Na
v
1.1
␣
-subunit, while TTX-resistant I
Na
is associated with
Na
v
1.5 expression. Block of TTX-sensitive I
Na
by 50 nM
TTX slows automaticity of intact SAN and isolated pace-
maker cells (279). TTX-sensitive I
Na
has been recorded in
action potential clamp experiments and shown to be
present during the late phase of the diastolic depolariza-
tion and upstroke (Fig. 17B) (279). Na
v
1.5 channels un-
derlie TTX-resistant I
Na
(276, 279). The role of Na
v
1.5
channels in SAN pacemaking has been described in het-
erozygous Scn5a
⫹/⫺
mice (276). Scn5a
⫹/⫺
mice display
major age-dependent dysfunction in atrioventricular con-
duction and a moderate reduction of the mean heart rate
(381). Intact atrial-SAN preparations from Scn5a
⫹/⫺
mice
have shown normal pacemaking in the SAN center, but
demonstrated slower intranodal SAN conduction and exit
block (276) (Fig. 17C). Pacemaking in small leading SAN
cells from Scn5a
⫹/⫺
mice is not different from that re-
corded in wild-type mice. In conclusion, available evi-
dence indicates that Na
v
1.5-mediated I
Na
does not partic-
ipate in the generation of automaticity per se (in the
central SAN), but can influence heart rate by contributing
to impulse propagation within the SAN and from the SAN
to the atrium. (276).
I. Role of I
st
in Automaticity
Limited knowledge exists on the role of I
st
in the
generation of cardiac automaticity. Indeed, we presently
lack selective pharmacological agents targeting the I
st
current. Genetic manipulation of st-channels is also im-
possible, since their molecular basis is still unknown.
Consequently, it is difficult to directly investigate the
significance of I
st
in pacemaking. A significant contribu-
tion of I
st
in controlling the rate of the diastolic depolar-
ization has been proposed by Shinagawa et al. (457) on
the basis of numerical simulations of pacemaking in the
rat SAN. Modeling suggests the possibility that I
st
contrib-
utes to pacemaking by virtue of its low threshold of
activation and slow inactivation rate. These properties
would allow I
st
to be present throughout the pacemaker
cycle (457). The potential contribution of I
st
compared
with that of other currents involved in pacemaking has
also been investigated by Zhang et al. (544). This study
suggests that I
st
will affect pacemaking depending on its
relative size compared with other currents contributing to
pacemaking such as I
f
or I
Na
. Consequently, in rabbit
central SAN pacemaker cells, a significant contribution of
I
st
to pacemaking is predicted. According to Zhang et al.
(544), pacemaking at the periphery of the SAN would be
less sensitive to I
st
, because of the larger density of I
Na
and I
f
(544). Elucidation of the molecular nature of I
st
is
needed to gain new insights into the physiological role of
this current.
L. SR Ca
2ⴙ
Release and Automaticity
Two studies by Rubenstein and Lipsius (428) and Li
et al. (285) supplied initial evidence that Ca
2⫹
release and
I
NCX
were involved in the generation of pacemaker activ-
ity. Rubenstein and Lipsius (428) observed that ryanodine
slowed automaticity in atrial subsidiary pacemaker cells
by reducing the slope of the late diastolic depolarization
(the exponential phase), while Cs
⫹
(presumably by block-
ing I
f
) reduced the first fraction of the diastolic depolar-
ization. They concluded that multiple mechanisms were
involved in automaticity of subsidiary cells and that Ca
2⫹
release from the SR was important in the generation of
the late phase of the diastolic depolarization (428). Slow-
ing of automaticity by ryanodine was also reported by
Rigg and Terrar in guinea pig SAN preparations (417) and
by Li et al. (285) in cultured rabbit SAN cells. The latter
report also described a reduction of intracellular Ca
2⫹
transients (417) and abolition of I
NCX
by ryanodine and
BAPTA-AM (285). These studies were indicative of a role
of intracellular Ca
2⫹
signaling in maintaining automatic-
ity, yet they did not establish a link between a specific
Ca
2⫹
signal and pacemaking. Particularly, we can wonder
if spontaneously active cells posses an intracellular Ca
2⫹
signal coupled to the diastolic depolarization phase. Ju
and Allen (226) have described distinct types of Ca
2⫹
signals in toad sinus venosus cells: a cytosolic transient
(predominantly driven by the action potential), a delayed
Ca
2⫹
signal linked to a change in nuclear Ca
2⫹
content,
and a third signal possibly due to subsarcolemmal local
Ca
2⫹
-induced Ca
2⫹
release (LCICR).
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During the last five years, the group led by E. Lakatta
has extensively investigated the cellular mechanism gen-
erating LCICR and emphasized its relevance in determin-
ing the chronotropic state of rabbit SAN cells. Bogdanov
et al. (44) have studied intracellular Ca
2⫹
release in beat-
ing SAN cells and documented “preaction potential” Ca
2⫹
signals due to LCICR. These signals precede the Ca
2⫹
transient evoked by the action potential upstroke phase
(Fig. 18, A–C). These signals are initiated at the cell edge
and are due to Ca
2⫹
release from the SR, since they are
abolished by ryanodine (Fig. 18D). During spontaneous
activity, preaction potential signals are generated by
LCICR. LCICR stimulates I
NCX
activity (44). Using ramp
protocols mimicking the diastolic depolarization, Bog-
danov et al. (44) have estimated LCICR-mediated I
NCX
density to be ⬃1.7 pA/pF (Fig. 18F). The size of I
NCX
would thus be sufficient to quicken the exponential frac-
tion of the diastolic depolarization. LCICRs observed in
rabbit SAN cells are similar to diastolic Ca
2⫹
release
described in cat latent pacemaker cells (211). Vino-
gradova et al. (510) have supplied evidence indicating that
LCICRs in rabbit primary SAN can be generated in the
absence of a change in the membrane voltage. Indeed,
LCICRs can still be observed in quiescent permeabilized
SAN cells. LCICR size depends on extracellular Ca
2⫹
concentration. (Fig. 19, A and B). Furthermore, if sponta-
neously beating SAN cells are arrested by voltage clamp-
ing at the maximum diastolic potential or at a positive
voltage (⫺10 mV), LCICRs do not disappear, but persist
for some seconds in the absence of action potentials (Fig.
19C) (510). Under these conditions, LCICRs display sto-
chastic “roughly periodical” (510) behavior and maintain
similar periodicity as during spontaneous activity. These
results support the view that LCICRs are not directly
linked to the activity of membrane ion channels but re-
flect the existence of an independent intracellular “Ca
2⫹
clock” mediated by spontaneous Ca
2⫹
release from SR.
LCICRs eventually disappear in arrested cells possibly
after SR depletion. We can expect that during pacemak-
ing, Ca
2⫹
stores of the SR are cyclically refilled by open-
ing of VDCCs (510) or by store-operated Ca
2⫹
influx as
described by Ju et al. (229).
Cellular mechanisms underlying this internal “Ca
2⫹
clock” are not understood, but Vinogradova et al. (508) have
recently shown that LCICRs are abolished by inhibition of
PKA activity and stimulated by cAMP (Figs. 20 and 21). They
also reported that basal PKA activity is almost 10 times
higher in SAN cells than in atrial and ventricular myocytes
and that high PKA activity seems to be a prerequisite for
pacemaking. Indeed, PKA inhibitors can stop cellular auto-
maticity (Fig. 20) (508).
In conclusion, SR-mediated LCICR is involved in the
generation of the exponential fraction of the diastolic
depolarization. The two key elements of this mechanism
are pre-action potential RyR-mediated LCICR and NCX,
FIG. 18. Characteristics of LCICR in rabbit SAN pacemaker cells. A: line scan image of Ca
2⫹
release (bottom panel) and corresponding
normalized fluorescence (top panel) as a function of time and position within the scan line. The scan line lays perpendicularly to the cell long axis
(inset cell drawing). Colored arrows indicate areas where fluorescence is averaged. B: pacemaker activity (black line) and normalized fluorescence
at the cell edge (red line) and middle (green line). Arrows indicate the time interval corresponding to the 3-dimensional plot in A. Asterisks indicate
a peak of Ca
2⫹
release at the cell edge. This Ca
2⫹
release occurs locally, during the exponential part of the diastolic depolarization. C: here, the line
scan image is oriented parallel to long cell axis and visualizes the cell edge where LCICR is triggered. White lines indicate the propagation of
the [Ca
2⫹
]
i
wave. D:3
␮
M ryanodine (ryan) slows pacemaker activity and abolishes preaction potential LCICR in rabbit SAN cells. E: dose-response
curve of pacemaker activity inhibition by ryanodine. F:Ca
2⫹
release (top panel) and total membrane current (middle panel) during voltage clamping
of a SAN cell with a waveform simulating the SAN pacemaker cycle (bottom panel). In each panel, the black line indicates recording in control
conditions, and the red line is plotted after application of 3
␮
M ryanodine. Ryanodine blocks diastolic LCICR and reduces the inward current
recorded during the simulated diastolic depolarization (see enlarged middle inset). Asterisks indicate LCICR; the symbol (#) indicates the residual
inward current in presence of ryanodine. [From Bogdanov et al. (44).]
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which converts subsarcolemmal Ca
2⫹
release to an inward
current contributing to diastolic depolarization. In a recent
work, Lyashkov et al. (299) have reported colocalization of
NCX and RyRs in rabbit SAN cells and proposed that the
proximity between NCX and RyRs permits quick conversion
of LCICR into oscillations of the membrane voltage.
FIG. 19. LCICR in intact (A) and permeabilized (B) rabbit SAN pacemaker cells. A: simultaneous recordings of pacemaker activity and confocal
line scan images of LCICR as in Fig. 18C. B: images of Ca
2⫹
release in a quiescent permebilized SAN cell under different extracellular Ca
2⫹
concentrations as indicated. LCICR is present even in quiescent SAN cells, and its amplitude is a function of extracellular Ca
2⫹
concentration. C:
voltage clamping to the cell maximum diastolic potential does not immediately block LCICR in SAN cells. Note LCICR events (wither arrows) in
the absence of changes in membrane voltage. Membrane voltage oscillations, possibly due to I
NCX
activated by LCICR, are recorded. These
observations indicate LCICR are a spontaneous voltage-independent phenomenon. [From Vinogradova et al. (510).]
FIG. 20. Basal PKA-dependent phosphorylation is necessary for maintaining pacemaker activity in rabbit SAN pacemaker cells. A: superfusion
with 15
␮
M of protein kinase inhibitor (PKI) reversibly suppresses pacemaking. B: a line scan image shows strong reduction of spontaneous LCICR
by PKI in permebilized SAN cells. C and D: simultaneous recordings of pacemaker activity (top panel), line scan images (middle panel), and
normalized Ca
2⫹
release averaged over the line scan image (bottom panel). In C, control conditions are established by voltage clamping a
spontaneously beating SAN cell to ⫺30 mV to record spontaneous Ca
2⫹
release. In D, note the reduction in both frequency and size of spontaneous
Ca
2⫹
release by PKI. [From Vinogradova et al. (510).]
950 MATTEO E. MANGONI AND JOE
¨
L NARGEOT
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VII. AUTONOMIC REGULATION OF
PACEMAKER ACTIVITY
A. Principles
In this section, we review the current knowledge on
the cellular mechanisms underlying the autonomic regu-
lation of cardiac automaticity.
The autonomic nervous system is the major extracar-
diac determinant of the heart rate. Sympathovagal control
of cardiac automaticity is a complex phenomenon. Multi-
ple interactions occur between sympathetic and parasym-
pathetic centers in the central nervous system, and pre-
synaptic peripheral interactions exist also (223, 284, 375).
In the adult heart, the sympathetic branch of the auto-
nomic nervous system accelerates heart rate, while the
parasympathetic branch slows it. Exceptions to this gen-
eral rule can be observed experimentally, but a detailed
discussion of these mechanisms is beyond the scope of
this review. The terminal part of the cardiac autonomic
nervous system is constituted by the intrinsic cardiac
neuronal plexus (ICNP), which plays a pivotal role in
regulating the heart rate, conduction, and contractile
force (15). Autonomic fiber projections to the heart rhyth-
mogenic centers are abundant. The canine SAN is densely
innervated by postganglionic fibers of the ICNP, which is
formed by nerve fibers entering the epicardium and form-
ing ⬃400 ganglia around the junction between the right
atrium and the superior vena cava (384). Pauza et al. (384)
have estimated that the canine SAN can be innervated by
more than 54,000 intracardiac neurons residing in the INP.
A similar organization of SAN innervation by the INP has
been found in humans (385). The rat AVN and common
bundle are innervated by a dense network of thin fibers
projecting from a neuronal cluster adjacent to the inter-
atrial septum and the right pulmonary sinus (26). The
distribution of sympathetic and parasympathetic fibers in
the SAN is heterogeneous (375) and can slightly vary
between individuals (385), as well as on an age-dependent
way (26, 385). Vagal and sympathetic activation induce a
shift of the leading pacemaker site (54). Compared with
the atrial myocardium, the SAN is enriched in adrenergic
and muscarinic receptors (29). Similar to SAN ion chan-
nels, adrenergic and muscarinic receptor densities vary
regionally and may contribute to pacemaker shift during
autonomic stimulation (300, 375). SAN nerves contain
between 5 and 15 axons and terminate as naked terminals
with varicosities containing the neurotransmitter release
site (419). Adrenergic and nonadrenergic release sites are
in close vicinity to each other (375, 419). Choate et al. (88)
reported that synaptic varicosities at cholinergic termi-
nals in the guinea pig SAN are in close proximity with the
membranes of SAN myocytes. According to Hirst et al.
(200), cholinergic terminals form specialized “neuromus-
cular” junctions with SAN myocytes.
Autonomic control of pacemaker activity in vivo is
based on concomitant input from sympathetic and para-
sympathetic limbs. However, the ratio between vagal and
sympathetic input varies in a species-dependent way
(375). The impact of sympathovagal balance on the heart
rate can be appreciated by comparing basal rates in dif-
ferent mammalian species in the presence and in the
absence of autonomic input. In mammals, basal heart
rates are inversely correlated with the body weight.
Smaller mammals such as mice and bats have fast heart
FIG. 21. Stimulation of the PKA-dependent signaling pathway by
cAMP or 0.1
␮
M isoproterenol (ISO) stimulates intracellular Ca
2⫹
re-
lease. A: this line scan image shows the increase in the frequency of
Ca
2⫹
release in a permebilized rabbit SAN cell superfused with 10
␮
M
cAMP. B: recordings of pacemaker activity (top panel) line scan image
(middle panel) and fluorescence (bottom panel, same protocol as in Fig.
20) in control conditions (B) and during superfusion with ISO (C). [From
Vinogradova et al. (510).]
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rate, ranging from ⬃500 beats/min in mice during daytime
(157) to 800–1,000 beats/min in flying bats (269). In con-
trast, the heart rate in medium-sized and larger mammals
can vary between 60 and 70 beats/min in humans and ⬃20
beats/min in whales (see Ref. 375 for review). Isolated
hearts and SAN pacemaker cells also show a wide range
of basal rates and maintain the same rate-to-animal
weight ratio as in the presence of an autonomic input
(375). The intrinsic properties of pacemaker cells in dif-
ferent species are one of the bases of the variability of
heart rate in mammals. However, the species-dependent
balance between sympathetic and parasympathetic input
also contributes to this wide range of pacemaking fre-
quencies (375). The two branches of the autonomic ner-
vous system interact to generate an adaptable equilibrium
so that SAN automaticity can be under dominance of the
sympathetic or parasympathetic limb. Sympathovagal
dominance is generally assessed in vivo by pharmacolog-
ical inhibition of the autonomic input by combined injec-
tion of atropine and propranolol. Even if propranolol does
not block
␣
-adrenergic receptors, the heart rate measured
under these conditions constitutes a reliable estimate of
the intrinsic pacing rate of a “denervated” heart (29).
In animals, the beating frequency of the isolated SAN
is also an index of the intrinsic heart rate. In the mouse,
the intrinsic SAN rate is significantly lower than the
in vivo heart rate, indicating the existence of a significant
sympathetic tone in this species (157). In contrast, it can
be shown that dogs and humans are under prominent
vagal tone, since pharmacological block of the autonomic
input significantly accelerates the basal heart rate (223,
375). However, adrenergic dominance does not demon-
strate the absence of a vagal tone. For example, the
presence of vagal tone in small rodents can be easily
demonstrated by injection of atropine in freely moving
mice (157).
The effect of parasympathetic input on pacemaking
is greater when the SAN is under sympathetic tone. This
phenomenon has been named “accentuated antagonism”
by Levy (284). At the organ level, presynaptic regulation
of catecholaminergic terminals by the vagus nerve and
pacemaker shift contribute to accentuated antagonism
(300). In this respect, the functional inhomogeneity of
SAN tissue can be an important factor underlying accen-
tuated antagonisms (300). In SAN and AVN cells of the
rabbit, accentuated antagonism has been explained by the
regulation of cAMP levels by nitric oxide (NO) (see be-
low). Beside the basal regulation of the heart rate exerted
by the autonomic balance, cardiac automaticity is modu-
lated on a beat-by-beat basis so that pacemaking is
quickly adapted to the physiological state of the organism.
In this respect, the high-frequency (HF) and low-fre-
quency (LF) spectra of the heart rate variability (HRV)
have been used to study the dynamic regulation of pace-
making (157). However, it is becoming clear that intrinsic
factors are also involved in the beat-by-beat regulation of
heart rate. Indeed, it has been shown that “nonautonomic
mechanisms” may contribute to HF spectra of HRV in
some physiological conditions (34, 76, 463). Bernardi et al.
(34) reported that, at peak exercise, in both healthy hu-
man subjects and transplanted patients the HF spectra of
the HRV is almost completely generated by a “nonauto-
nomic” mechanism that is synchronized with ventilation.
These results were then confirmed by Casadei et al. (76),
who reported that under exercise, the “nonneuronal”
component of heart rate regulation increases by ⬃35%.
Slovut et al. (463) have suggested that this phenomenon
is due to SAN stretch during diastolic atrial filling (see
sect. IXD).
B. Sympathetic Regulation of Pacemaker Activity
Activation of the
␤
-adrenergic receptor underlies the
positive chronotropic effect induced by catecholamines
on automaticity. Catecholamines enhance the activity of
ion channels as well as intracellular Ca
2⫹
release. The
relative importance of sarcolemmal ion channels and
LCICR in the
␤
-adrenergic regulation of pacemaker activ-
ity is still debated. In this section, we will present and
compare evidence linking these mechanisms to stimula-
tion of pacemaker activity by catecholamines.
I
f
and I
Ca,L
have been proposed to constitute impor-
tant mechanisms in heart rate acceleration by cat-
echolamines (64, 67, 120, 364). The open probability of
f-channels increases even for a small augmentation of
intracellular cAMP. A rise in cAMP positively shifts the I
f
activation curve, thereby supplying more inward current
during the linear part of diastolic depolarization (120)
(Fig. 22). DiFrancesco (120) has proposed that I
f
is the
predominant mechanism for increasing the slope of the
diastolic depolarization at low adrenergic tone. This is
based on the observation that low doses of the
␤
-adren-
ergic agonist isoproterenol (which is supposed to mimic
weak adrenergic activity) increase the slope of the dia-
stolic depolarization without affecting the action poten-
tial waveform (120). Remarkably, specific regulation of
the diastolic depolarization slope is a common property of
low doses of autonomic agonists and selective I
f
blockers
(21). Another strong line of experimental evidence for the
capability of f-channels to stimulate pacemaker activity is
the acceleration by cAMP analogs of pacing rate in SAN
cells. Bucchi et al. (67) have shown that isoproterenol and
Rp-cAMP similarly increase the diastolic depolarization
slope in rabbit SAN cells. Stieber et al. (466) also gave
strong evidence for the ability of f-channels to accelerate
embryonic heart rate. These authors have shown that
cAMP cannot accelerate the heartbeat of HCN4
⫺/⫺
em-
bryos. Overall, different lines of direct and indirect evi-
dence are in favor of an important contribution of f-
channels in sympathetic regulation of pacemaker activity.
952
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The functional role of I
Ca,L
in adrenergic regulation of
heart rate is still unresolved. Three lines of indirect evi-
dence are suggestive of a role of I
Ca,L
in the sympathetic
regulation of heart rate. First, catecholamines robustly
enhance I
Ca,L
in the same concentration range as pace-
maker activity (542). Zaza et al. (542) have shown that I
f
and I
Ca,L
have similar sensitivity to isoproterenol in rabbit
SAN cells. Second, Choate and Feldman (87) have re-
ported that DHPs reduce the positive chronotropic re-
sponse of mouse atria to stimulation of the stellate gan-
glion. Third, the contribution of Ca
v
1.3 channels in the
generation of the diastolic depolarization in mouse SAN
cells suggests that I
Ca,L
can constitute an important mech-
anism for accelerating the diastolic depolarization slope
upon activation of
␤
-adrenergic receptors (309, 310). Con-
sistent with this hypothesis, the positive chronotropic
response to isoproterenol of isolated Ca
v
1.3
⫺/⫺
hearts is
moderately reduced (321). However, the in vivo heart rate
of mice lacking Ca
v
1.3 channels is comparable to that of
wild-type mice (393). The maximal heart rates of wild-
type and Ca
v
1.3
⫺/⫺
mice are significantly reduced by se-
lective I
f
block by ivabradine (Mangoni et al., unpublished
observations). Taken together, these observations are
suggestive of a distinct role of f-channels and Ca
v
1.3
channels in the autonomic regulation of heart rate (see
also Ref. 196).
The relevance of TTX-sensitive and TTX-resistant I
Na
in the sympathetic regulation of heart rate is of interest,
yet no reports have specifically studied the autonomic
regulation of I
Na
in the SAN. The cardiac TTX-resistant
Scn5A-mediated I
Na
is sensitive to phosphorylation by
PKA and PKC (286, 407). Taking into consideration the
relevance of Scn5A channels in SAN conduction, we can
speculate that sympathetic stimulation of TTX-resistant
I
Na
can accelerate heart rate by accelerating impulse con-
duction within the SAN as well as from the SAN to the
atrium. In contrast, the effects of autonomic agonists on
TTX-sensitive I
Na
are not easily predictable. Maier et al.
(304) have hypothesized that TTX-sensitive Na
⫹
channels
can be negatively regulated by the sympathetic nervous
system as in neurons. If this hypothesis is valid, the
contribution of TTX-sensitive I
Na
to the diastolic depolar-
ization may be higher in basal conditions than under
strong adrenergic activation.
In pacemaker cells of amphibians (227) and mam-
mals,
␤
-adrenergic receptor activation stimulates SR Ca
2⫹
release and NCX activity (415, 506). In spontaneously
active rabbit SAN cells, isoproterenol robustly enhances
the amplitude and frequency of RyR-dependent LCICR
(415, 506). Vinogradova et al. (506) have reported that
ryanodine abolishes the augmentation of subsarcolemmal
Ca
2⫹
release and LCICRs. This effect is accompanied by a
strong reduction in the isoproterenol-induced positive
chronotropic effect, particularly at low agonist doses.
Vinogradova et al. (506) have thus proposed that SR Ca
2⫹
release is the major effector of the positive chronotropic
effect of the
␤
-adrenergic receptor pathway. Partial inhi-
bition of the isoproterenol chronotropic effect in rabbit
SAN has been qualitatively confirmed by other authors
(67, 267, 415), although it shows quantitative variability.
FIG. 22. A: low doses of isoproterenol and ACh selectively modulate the slope of the diastolic depolarization in rabbit SAN cells (a) and shift
the I
f
voltage dependence (b, c). These experiments have led DiFrancesco (see Ref. 121 and sect. VII for discussion) to propose that the predominant
mechanism by which the autonomic nervous system controls the heart rate is I
f
.[a from DiFrancesco (120), with permission from the Annual Review
of Physiology; b and c are from Accili and DiFrancesco (4).] B: a cAMP analog (R
p
-cAMPs) stimulates pacemaker activity by increasing the slope
of the diastolic depolarization under control conditions and in the presence of ryanodine (Ry). This observation suggests that impairment of Ca
2⫹
release from the SR does not prevent regulation of pacemaker activity by direct binding of cAMP analogs to f-channels. [From Bucchi et al. (67),
with permission from Elsevier.]
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Indeed, in a previous study using intact SAN, ryanodine
reduced
␤
-adrenergic stimulation of pacemaking by ⬃40%
(415). Pacemaking in the intact SAN is also less sensitive
to ryanodine than in isolated pacemaker cells. Musa et al.
(349) have indicated that such a difference can be due to
heterogeneous expression of proteins regulating Ca
2⫹
handling in the center versus the periphery of the SAN.
Functionally, the central leading pacemaking site is less
sensitive to inhibition of SR Ca
2⫹
release (267). However,
Lyashkov et al. (299) have reported that, in isolated rabbit
SAN cells, the effects of ryanodine, BAPTA, and Li
⫹
on
pacemaker activity are similar irrespective of the cell size.
Discrepancy between results obtained in intact SAN tis-
sue and isolated cells may be attributed to the network
organization of pacemaker cells in the SAN.
In Figure 23, we attempt to summarize the down-
stream targets of the
␤
-adrenergic receptor-dependent
signaling pathway in SAN pacemaker cells. This model is
based on evidence from rabbit SAN cells and is comple-
mented by recent insights from genetically modified
mouse strains. For our discussion, we can distinguish two
distinct pacemaker mechanisms: an “ion channel clock”
formed by voltage-dependent ion channels and the intra-
cellular SR-dependent “Ca
2⫹
clock” (307). Activation of
␤
-adrenergic receptors stimulates adenylyl cyclase (AC)
activity, which converts ATP in cAMP. Elevated cAMP
promotes voltage-dependent opening of f-channels and
activates PKA (126). The catalytic subunit of PKA en-
hances the activity of different ion channels of the mem-
brane-delimited pacemaker mechanism by channel phos-
phorylation. These include Ca
v
1.3, Ca
v
1.2 channels (309),
as well as st-channel complexes (334). Vinogradova et al.
(508) have reported that basal PKA activity is higher in
rabbit SAN cells than in working myocytes. High phos-
phorylation of PLB and RyRs generates periodical LCICR
causing I
NCX
-mediated membrane voltage oscillations,
which contribute to the control of the chronotropic state
of the cell (43, 508).
␤
-Adrenergic receptor activation
further elevates PKA activity, thereby increasing the fre-
quency and number of LCICRs and stimulating I
NCX
.
The “I
f
-based” pacemaker mechanism (121) and that
of the spontaneous SR-dependent “Ca
2⫹
clock” (307) have
been placed as opposed to each other. However, they can
be qualitatively reconciled in a general framework. Both
models have a common major messenger that is cAMP.
cAMP can control the chronotropic state of the cell by at
least three distinct effectors: 1) f-channels by direct chan-
nel opening, 2)Ca
v
1.3 channels, and 3) RyRs by channel
FIG. 23. This cartoon summarizes the ionic mechanisms contributing to the diastolic depolarization in a SAN pacemaker cell. Voltage-dependent
ion channels as well as the proposed “Ca
2⫹
clock” (307) are represented together. Possible interactions between these mechanisms are indicated.
In pacemaker cells, high basal cAMP-mediated PKA-dependent phosphorylation stimulates a perpetual “free running” Ca
2⫹
cycling by pumping Ca
2⫹
into the SR via SERCA2 and LCICR via RyRs. PKA also stimulates Ca
2⫹
entry through Ca
v
1.3-mediated I
Ca,L
. The thick red line indicates the
persistent spontaneous Ca
2⫹
cycling. The possibility that Ca
v
3.1-mediated I
Ca,T
can contribute to replenishment of SR Ca
2⫹
stores is suggested.
Spontaneous LCICR from SR is linked to the diastolic depolarization via Ca
2⫹
activation of inward I
NCX
current. Direct cAMP-dependent activation
of HCN channels or cAMP-mediated, PKA-dependent phosphorylation of Ca
v
1.3 channels and I
st
(dashed lines) strongly stimulates the pacemaker
cycle driven by the “membrane ion channels clock” (MCC). It is conceivable that the MCC can entrain the intracellular Ca
2⫹
clock because of the
dependency of SR Ca
2⫹
content from VDCCs. On the other hand, the Ca
2⫹
clock may trigger oscillations of the membrane voltage and initiate normal
pacemaking via the MCC. It is thus likely that under physiological conditions the MCC and the Ca
2⫹
clock mutually entrain one another. [Adapted
from Vinogradova et al. (508).]
954 MATTEO E. MANGONI AND JOE
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phosphorylation. Experimentally, maneuvers altering in-
tracellular cAMP levels will affect the activity of all these
effectors. One may wonder why the “ion channels clock”
and the “free-running Ca
2⫹
clock” (307) share a common
messenger. The answer probably resides in the necessity
to synchronize the replenishment of SR Ca
2⫹
stores to
Ca
2⫹
release in the late phase of the diastolic depolariza-
tion. Spontaneous LCICR ceases in arrested cells, since
Ca
v
1.3 channels are probably the predominant source of
Ca
2⫹
entry during the action potential. Consequently, as
much as the “Ca
2⫹
clock” accelerates under the action of
catecholamines, the sarcolemmal pacemaker mechanism
mediated by f- and Ca
v
1.3 channels will also speed up and
shorten diastolic depolarization.
However, as highlighted by Maltsev et al. (307), the
“Ca
2⫹
clock” hypothesis of the genesis of SAN automatic-
ity assumes that spontaneous LCICR is the predominant
mechanism that controls the chronotropic state of SAN
cells in both basal conditions and under
␤
-adrenergic
receptor stimulation. These authors have proposed that I
f
plays only a minor role in mediating the effect of cat-
echolamines on heart rate. This view is contradicted by a
substantial set of experimental results, such as the obser-
vation that ryanodine does not prevent acceleration of
pacemaker activity in rabbit SAN cells by cAMP analogs
(67, 68) and that specific f-channel inhibition by ivabra-
dine can reduce the heart rate of freely moving mice by up
to 26% (Mangoni et al., unpublished observations). Fur-
thermore, the importance of f-channels in the autonomic
regulation of heart rate is strongly supported by inherited
dysfunction of pacemaking in humans harboring muta-
tions in the HCN4 gene (see sect. XA) and related murine
models (see sect. VID).
C. Parasympathetic Regulation of Pacemaking
The parasympathetic regulation of cardiac automa-
ticity is mediated by the activation of muscarinic recep-
tors following release of acetylcholine (ACh) from vagal
nerve endings. Cholinergic agonists induce a potent neg-
ative chronotropic effect on cardiac automaticity and
atrioventricular conduction in vivo as well as in isolated
heart preparations (215, 325). Signaling and ionic mecha-
nisms underlying the muscarinic regulation of heart rate
have been studied for more than 30 years, yet it is not
completely understood which mechanisms determine re-
sponses in heart rate under various physiological condi-
tions, such as high sympathetic tone or parasympathetic
discharge.
Hutter and Trautwein (212) observed that vagal stim-
ulation stops pacemaking in the frog sinus venosus. As
suppression of automaticity was due to an increase in
membrane K
⫹
conductance, Hutter and Trautwein (212,
213) concluded that the vagal control of the heart rate was
due to activation of a K
⫹
current. Early observations by
Hutter and Trautwein are now accounted for by the acti-
vation of I
KACh
channels via vagally released ACh (432).
However, other authors have failed to observe hyperpo-
larization of the membrane potential after vagal stimula-
tion. For instance, studies by Toda and West (481, 519)
reported that vagal stimulation slowed automaticity by
reducing the slope of diastolic depolarization in atrial and
AVN preparations. This has been confirmed by Shibata et
al. (453) on isolated rabbit SAN preparations. Both hyper-
polarization of the maximum diastolic potential and a
decrease in the slope of diastolic depolarization are ob-
served when the muscarinic regulation of pacemaker ac-
tivity is studied using isolated spontaneously active cells.
Indeed, in isolated rabbit SAN cells, high ACh doses (1
␮
M) hyperpolarize the maximum diastolic potential and
eventually stop pacemaking due to maximal I
KACh
activa-
tion (124, 507). In contrast, low ACh doses (1–10 nM) slow
pacemaker activity by decreasing the slope of diastolic
depolarization in the absence of other changes in action
potential parameters and maximum diastolic potential
(Fig. 22A). This is because binding of ACh to muscarinic
receptors activates different signaling pathways in pace-
maker cells. These pathways involve direct activation of
I
KACh
channels, negative regulation of cAMP production,
and positive regulation of cAMP hydrolysis.
The muscarinic M
2
receptor activates the inhibitory
G protein
␣
-subunit (
␣
i
) which negatively couples to AC
activity (283, 440). Downregulation of cAMP can affect
the activity of voltage-dependent ion channels involved in
pacemaker activity and reverse the signaling processes
involved in sympathetic stimulation of heart rate (Fig. 24).
Moreover,
␤␥
-subunits of
␣
i
directly open K
ACh
channels
(520, 523). The role of I
KACh
, I
f
, and I
Ca,L
in mediating the
negative chronotropic effect of ACh has been studied in
isolated SAN pacemaker cells. Recently, insight into the
importance of I
KACh
in the regulation of heart rate has
been obtained using genetically modified mice lacking
K
ir
3.4 channels (522). Wickmann et al. (522) have shown
that K
ir
3.4
⫺/⫺
mice lack I
KACh
in the atrium and show
prominent reduction autonomic regulation of heart rate in
the HF and LF spectrum of the HRV. Consistent with
these observations, Gehrmann et al. (158) have shown
that transgenic mice expressing low amounts of
␤␥
have
a reduced negative chronotropic response to cholinergic
agonists.
DiFrancesco et al. (124) have studied the relative
sensitivity to ACh of I
f
and I
KACh
in rabbit SAN cells.
These authors have compared the ACh dose dependency
of the shift in the I
f
activation curve, the I
KACh
density,
and the slowing of pacemaker activity. In this study, low
ACh doses significantly shifted the I
f
activation curve in
the negative direction and slowed pacemaking in a con-
centration range that did not activate I
KACh
. Using a sim-
ilar approach, Zaza et al. (542) have compared the sensi-
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tivity of I
Ca,L
and I
f
to ACh. In this study, I
f
inhibition was
observed at lower doses of ACh than that required to
inhibit “basal” I
Ca,L
(542). These results have led Di-
Francesco (119) to propose that I
f
is the predominant ion
channel which underlies the muscarinic control of heart
rate at low vagal tone. This view is consistent with recent
evidence obtained by Yamada in isolated beating mouse
hearts (530). In these experiments, heart rate reduction
induced by low ACh doses was reported to be insensitive
to I
KACh
inhibition by tertiapin. Caution should be used
when transposing in vitro observations from isolated
pacemaker cells and denervated hearts to in situ heart
rate regulation. Due to the accentuated antagonism phe-
nomenon, different ionic mechanisms can participate to
muscarinic regulation of pacemaker activity, according to
their relative sensitivity in cholinergic agonists as well as
to intracellular levels of cAMP. The cardiac I
Ca,L
has been
reported to be remarkably “insensitive” to muscarinic
regulation in basal conditions, because relatively high
ACh doses are required to inhibit this current (190, 323).
However, Petit-Jacques et al. (390) have shown that mus-
carinic regulation of SAN I
Ca,L
depends on the previous
␤
-adrenergic stimulation and cAMP levels. Indeed, mod-
erate doses of ACh can significantly inhibit I
Ca,L
if previ-
ously stimulated by
␤
-adrenergic agonists (390). PKA in-
hibition or cell dialysis with a nonhydrolyzable cAMP
analog abolishes I
Ca,L
regulation by ACh (390). ACh can
thus act as an “antiadrenergic” agent. Interestingly, accen-
tuated antagonism of ACh after
␤
-adrenergic receptor
activation has been observed also on I
f
in canine Purkinje
fibers (81). It is thus important to distinguish between
“direct” and “indirect” cholinergic regulation of I
Ca,L
,to
distinguish between basal current inhibition and that ob-
served after adrenergic stimulation. Thus it cannot be
excluded that indirect (or antiadrenergic) inhibition of
I
Ca,L
can be relevant in vagal regulation of heart rate in
conditions of tonic sympathetic activation, when the ac-
centuated antagonism phenomenon is present. The regu-
lation of Ca
v
1.3 channels by ACh has not yet been inves-
tigated. In this respect, the relatively positive holding
potentials (about ⫺30 mV) employed thus far to study the
regulation of I
Ca,L
by autonomic agonists (186, 542) would
inactivate most of Ca
v
1.3 channels (309).
Han et al. (186) have demonstrated that an NO sig-
naling pathway is involved in the muscarinic regulation of
I
Ca,L
in conditions of accentuated antagonism, when in-
FIG. 24. Summary of the signaling pathways involved in the muscarinic regulation of pacemaker activity. In the SAN and AVN, ACh and Ado
share the same pathways for signal transduction. ACh binds to the muscarinic M
2
receptor, which is coupled to a “direct” G protein-dependent
pathway activating Kir3.1/Kir3.4 (GIRK1/GIRK4) channel complexes. Beside this direct pathway, two other “indirect” channel regulation pathways
lead to downregulation of intracellular cAMP. In the first cascade of events, the
␣
G
i
protein subunit inhibits AC activity, thereby reducing the
synthesis of cAMP. Inhibition of AC synthesis is viewed as the predominant pathway by which ACh regulates the voltage dependence of f-channels
(HCN). The second muscarinic pathway is initiated by the stimulation of the NOS activity and production of NO. NOS activates the enzyme guanylate
cyclase (GC), which converts GTP in cGMP. Elevation of cGMP concentration stimulates PDE II-dependent hydrolysis of cAMP, thereby reducing
PKA activity. This NOS-dependent pathway for regulation of PKA activity has been proposed to be the major pathway for the muscarinic regulation
of I
Ca,L
during pacemaking. In the figure, negative NOS-dependent regulation of Ca
v
1.3-mediated I
Ca,L
and I
st
is suggested. It is not known if the SAN
I
Kr
can also be negatively regulated by the NOS-dependent pathway.
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¨
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tracellular cAMP is increased by stimulation of
␤
-adren-
ergic receptors. In cardiac myocytes, muscarinic receptor
activation is coupled to NO synthesis, which stimulates
guanylyl cyclase activity. Elevated cGMP production pro-
motes phosphodiesterase activity that inhibits I
Ca,L
by
cAMP breakdown (323). Han et al. (186) have provided
evidence that the NO signaling pathway plays an obliga-
tory role in the muscarinic regulation of rabbit SAN I
Ca,L
(186). In these cells, block of NO synthesis prevents I
Ca,L
inhibition by cholinergic agonists such as carbamylcho-
line (185). This signaling pathway regulating I
Ca,L
is
present also in the AVN (170). The NO signaling pathway
in rabbit SAN seems to be primarily mediated by a con-
stitutive endothelial NO oxide synthase (eNOS) isoform
(182). Also, specific pharmacological inhibition of pos-
phodiesterase II (PDEII) blocks NO-dependent inhibition
of I
Ca,L
, indicating that this PDE isoform plays a dominant
role in downstream regulation of cAMP level (182). In
contrast, the role of intracellular [Ca
2⫹
]
i
in mediating
NO-dependent inhibition of I
Ca,L
would need further in-
vestigation. Han et al. (186) have shown that buffering
[Ca
2⫹
]
i
with BAPTA blocks eNOS activity. However, in
the same study, depletion of SR Ca
2⫹
stores by ryanodine
and thapsigargin did not affect regulation of I
Ca,L
, suggest-
ing that Ca
2⫹
involved in modulation of eNOS activity
comes from an independent intracellular compartment.
The relevance of the eNOS/PDEII signaling pathway in
mediating accentuated antagonism in the canine heart has
been shown by Sasaki et al. (437). These authors have
also shown that NO can partially modulate accentuated
antagonism but does not play an important role in the
absence of previous
␤
-adrenergic receptor stimulation.
Beside the eNOS/PDEII signaling pathway, protein
phosphatases can also regulate the activity of ion chan-
nels under basal conditions or under the action of auto-
nomic agonists. Ke et al. (235) have reported that the
p21-activated protein kinase (Pak1) is functionally ex-
pressed in guinea pig SAN cells. In pacemaker cells, when
cellular Pak1 activity is enhanced by expressing a consti-
tutively active form of the protein, I
Ca,L
and I
K
densities
are reduced and the positive chronotropic response of
cells to isoproterenol is blunted. The regulatory role of
active Pak1 on I
Ca,L
and I
K
is likely attributable to in-
creased activity of the protein phosphatase PP2A (235). It
remains to be established if Pak1 can be functionally
associated with the vagal regulation of heart rate.
The importance of NO signaling pathway in the vagal
regulation of heart rate in mice has not been clearly
demonstrated. Mice lacking eNOS or the G
␣
o
protein
isoforms lack muscarinic regulation of I
Ca,L
(183, 487).
However, Vandecasteele et al. (494) failed to observe
reduction of both adrenergic and muscarinic regulation of
I
Ca,L
in another eNOS-deficient mouse line. Consistent
with these results, Mori et al. (343) have reported that
inhibition of eNOS does not change accentuated antago-
nism in isolated mouse atria.
VIII. CARDIAC AUTOMATICITY AS AN
INTEGRATED MECHANISM: NUMERICAL
MODELING OF PACEMAKER ACTIVITY
A. General Models of Automaticity
Modeling of cardiac automaticity integrates the be-
havior of channels and ionic homeostasis in a calculating
environment to predict pacemaking under different con-
ditions. Numerical models are used to gain insight into the
roles of ion channels and/or Ca
2⫹
handling proteins, when
biophysical or pharmacological approaches are limited or
impossible. The growing knowledge on the cardiac pace-
maker mechanism is reflected by the increasing complex-
ity of numerical models of automaticity. A detailed com-
parison of all the numerical models of automaticity that
have been developed in the past is beyond the scope of
this review. For further discussion on the historical and
technical aspects of this topic, the reader can consult the
recently published manuscript by Wilders (525). Here, we
will discuss some predictions of numerical models on
the importance of ion channels and Ca
2⫹
signaling in the
generation and regulation of automaticity.
Recent cellular models of SAN automaticity have
been developed from the DiFrancesco and Noble model
of Purkinje fibers automaticity (127). In this model, auto-
maticity is based on I
f
. Suppression of I
f
abolishes auto-
maticity (127). The DiFrancesco and Noble model in-
cluded calculations of intracellular systolic Ca
2⫹
tran-
sients and I
NCX
activity. Noble and Noble (360) have
adapted the DiFrancesco and Noble model to the central
and peripheral SAN automaticity, by scaling current den-
sities to approximate that of SAN cells. Noble et al. (359)
have used this model to predict the possible roles of I
f
and
that of Na
⫹
background current [I
b(Na)
] and proposed that
I
f
is a major mechanism for stabilizing automaticity and to
protect SAN pacemaking from hyperpolarizations.
Wilders et al. (526) have also assessed the role of I
f
and
I
b(Na)
in the generation of the diastolic depolarization.
These authors have predicted that the association of I
f
and I
b(Na)
is necessary for pacemaking of isolated rabbit
SAN cells, since joint abolition of both currents stops
automaticity (526). Block of I
f
slows pacemaking by spe-
cifically reducing the slope of the diastolic depolarization
(526). Wilders et al. have suggested that their modeling
results are consistent with the experimental work by
Denyer et al. who proposed that I
b
can sustain pacemak-
ing under I
f
block (113, 526) (see sect. VID). Demir et al.
(111) have developed a model of automaticity of periph-
eral SAN cells, by associating voltage-dependent ion chan-
nels activity with intracellular Ca
2⫹
release and handling
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by intracellular Ca
2⫹
buffers. In this model, different ionic
currents contribute to the generation of the diastolic de-
polarization. Particularly, block of I
f
and I
Ca,T
almost
equally slow pacemaker activity, while inhibition of I
Ca,L
mainly affects the late diastolic depolarization and action
potential upstroke (111). Predictions obtained by the
Demir et al. model are generally consistent with many
physiological and pharmacological results on several
ionic currents involved in automaticity, including I
Ca,T
,
I
Ca,L
, I
Kr
, and I
f
(111). The Demir et al. model can suc-
cessfully reproduce certain aspects of the parasympa-
thetic regulation of pacemaker activity. Particularly, the
negative regulation of pacemaking by specific reduction
of the slope of the diastolic depolarization, and the phase-
resetting SAN response upon bursts of vagal activity can
be computed by this model (110). More generally, predic-
tions obtained by the Wilders et al. and Demir et al.
models are qualitatively consistent with experimental re-
sults in rabbit SAN cells. However, they have some limi-
tations when inferring the role of I
Ca,L
and intracellular
Ca
2⫹
release. These limitations are justified as far as these
models have been developed when experimental data on
Ca
v
1.3 channels and LCICR were not available. Kurata
et al. (262) have been the first to develop a model of
SAN automaticity in which the subsarcolemmal Ca
2⫹
([Ca
2⫹
]
sub
) and the SR Ca
2⫹
content were included in
calculations. Furthermore, buffering of [Ca
2⫹
]
i
by tropo-
nin, calsequestrin, and calmodulin were also included
(262). The Kurata et al. model (262) thus aims to combine
the advantages of calculating Ca
2⫹
buffering and Ca
2⫹
exchange between the subsarcolemmal space and the SR.
Subsarcolemmal Ca
2⫹
controls Ca
2⫹
-dependent inactiva-
tion of I
Ca,L
(262). In the Kurata et al. model, block of SR
Ca
2⫹
release has only minor effects on pacemaking, es-
sentially because Ca
2⫹
release is limited to the action
potential upstroke phase.
B. Dedicated Models of Automaticity
Recently developed models of SAN pacemaking con-
sider specific SAN properties, such as the regional varia-
tions of ion channel expression (545), regulation of auto-
maticity by LCICR (306), and automaticity in genetically
modified mice (314). These models will be referred to
here as “dedicated.”
Zhang et al. (545) have modeled pacemaker activity
in the periphery and center of the SAN. This model is
based on experimental results on the heterogeneity of cell
size and ionic current densities observed in the rabbit
SAN (58) (see sects.
II and III). Numerical simulations by
Zhang et al. (545) show that it is possible to reproduce the
slower pacemaker activity, action potential upstroke ve-
locity, and the more positive maximum diastolic potential
in the SAN center by setting lower densities of I
Ca,L
, I
f
, I
Kr
,
and I
Na
in the central than in the peripheral SAN model. In
the central model, I
Na
is set to zero so that the action
potential upstroke phase is entirely controlled by I
Ca,L
.In
the peripheral model, the diastolic depolarization is car-
ried by I
Ca,T
and I
f
, while in the central model I
Ca,T
pre-
dominates on I
f
. The model predicts the higher effect of I
f
blockers on automaticity in the SAN periphery versus the
center. Suppression of I
Ca,L
in the Zhang et al. model stops
pacemaking in the center and sets the membrane poten-
tial to ⫺35 mV (545). In the peripheral model, block of
I
Ca,L
moderately accelerates automaticity possibly be-
cause of the shortening of the action potential duration.
I
Kr
suppression stops automaticity in both the peripheral
and central SAN model. However, it is possible to set a
20% block of I
Kr
without stopping pacemaking. According
to Zhang et al. (545), the moderate slowing of pacemaking
induced by partial inhibition of I
Kr
is consistent with what
is observed in peripheral SAN tissue balls (249). In the
peripheral SAN model, I
to
has higher density than in the
central model. Consistent with experimental results (60),
suppression of I
to
in the peripheral model prolongs the
action potential duration, while has limited effects on the
central SAN model (545). Garny et al. (152) have adapted
the Zhang et al. model in an attempt to reproduce atrio-
sinus interactions and calculate the predicted position of
the SAN leading pacemaker site in the presence and ab-
sence of the sorrounding atrium (Fig. 25). In this model, a
gradual transition between cells matched with the central
and the peripheral model was established via a distribu-
tion function. A variable ratio of intercellular conductivity
was also implemented (see legend of Fig. 25). This cellu-
lar array was connected to cells matched with a rabbit
(197) or human (368) atrial model. The position of the
leading pacemaker site settled in the SAN center when a
greater intercellular coupling between cells in the SAN
periphery than in the center was established. The leading
pacemaker site shifted to the periphery when intercellular
conduction was uniform in the SAN center and periphery.
Finally, the leading pacemaker site was located in the
periphery when the SAN cellular array was electrically
disconneted from the atrium (Fig. 25). In conclusion, this
atriosinus model successfully reproduced the electro-
tonic inhibition of atrial cells on SAN pacemaking. Garny
et al. (152) have interpreted the electrotonic suppression
of pacemaking in the SAN periphery in terms of an “op-
posing depolarization,” rather than a simple hyperpolar-
izing inhibition (152).
In its first version, the model by Zhang et al. did not
include calculations of intracellular Ca
2⫹
handling. Simi-
larly [Na
⫹
]
i
and [K
⫹
]
i
were kept constant (58). In a follow
up manuscript, these authors have included calculations
of Ca
2⫹
handling in both the central and peripheral SAN
model (56). Available experimental data on Ca
2⫹
-depen-
dent effects on I
Ca,T
, I
Ca,L
, I
Kr
, and I
f
were included.
Boyett et al. (56) have then compared the effects of
958
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¨
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blocking Ca
2⫹
transients in this model and that by Noble
and Noble (360), Wilders et al. (526), and Demir et al.
(111). In all models tested, abolition of Ca
2⫹
transients
variably slowed pacemaking, from a minimum of 9% in the
Wilders et al. model and a maximum of 41% in the Demir
et al. model. Calculations by Boyett et al. suggested that
this slowing of automaticity was predominantly due to
changes in the Ca
2⫹
-dependent activity of ion channels
rather then I
NCX
. These calculations are very useful for
interpreting the possible physiological consequences of
the Ca
2⫹
dependency of ion channel activities, but do not
include LCICR and its functional coupling with I
NCX
(see
sect.
VIL).
Maltsev et al. (306) have modified the Kurata et al.
(262) model to include spontaneous preaction potential
LCICR and activation of I
NCX
. These authors have inter-
preted spontaneous LCICR in terms of a periodical global
cellular release phenomenon of a variable phase (and
amplitude), occurring during the diastolic interval. In this
model, a global LCICR eventually induces the exponential
part of the diastolic depolarization. Shortening of the
interval between successive LCICRs is predicted to aug-
ment the frequency of pacemaking of rabbit SAN cells, by
accelerating the onset of the exponential late phase of the
diastolic depolarization. Vinogradova et al. (508) and Bog-
danov et al. (43) further developed this model by dividing
the global LCICR into the sum of finite release elements of
which the individual phase of a given release event is set
by a random generator (Fig. 26A). In this model, a release
event can occur at any time comprised between the mean
and the standard deviation of the phase of LCICRs ob-
served experimentally. This modification allows one to take
into account the “roughly periodical” nature of LCICR in
basal conditions and to simulate synchronization of Ca
2⫹
release units under activation of the
␤
-adrenergic recep-
tor (508). Bogdanov et al. (43) have used this model to
calculate fluctuations of I
NCX
under basal conditions and
stimulation of pacemaking by isoproterenol (Fig. 26, A
and B). In conclusion, numerical modeling predicts that
subsarcolemmal Ca
2⫹
sigaling may have little effect on
pacemaking when only systolic SR Ca
2⫹
release is con-
sidered (262). However, models including diastolic preac-
tion potential Ca
2⫹
release show that LCICR significantly
accelerates pacemaking (43, 306, 508). At present, no
model including both LCICR and Ca
2⫹
dependency of ion
channels is available.
Mangoni et al. (314) have adapted the Zhang et al.
(545) central model to predict the activation of Ca
v
1.3
and Ca
v
3.1 channels during the diastolic depolarization
in mouse SAN pacemaker cells (Fig. 26C). Simulations
obtained using this model suggest that, in the mouse
SAN, I
Kr
controls the action potential repolarization
phase and is present as a relatively large outward com-
ponent throughout the diastolic depolarization (see
sect.
VIC). I
f
is the first time-dependent current to be
FIG. 25. Numerical modeling of SAN-atrium interactions using a
modified version of the Zhang et al. model of rabbit central and periph-
eral SAN automaticity (152). The membrane voltage of the leading
pacemaker site is depicted in green, the “follower” SAN membrane
voltage in red, and the atrial membrane voltage is depicted in blue. In A,
the central SAN coupling conductance is set 10 times lower than the
peripheral conductance (37.5 vs. 375 nS, see Ref. 152). Note that the
leading pacemaker site locates in the SAN center. In B, a uniformly low
coupling conductance has been set throughout the SAN model (37.5 nS).
In these conditions, the leading pacemaker site shifts to the SAN pe-
riphery. C: when the SAN and atrium are disconnected (no SAN-driven
action potential in the atrium), the leading pacemaker site shifts to the
SAN periphery, where the intrinsic cell beating rate is faster than in the
SAN center. D: simulation parameters has been set as in A, but a
numerical model of a human atrial cell has been employed. [From Garny
et al. (153), with permission from Elsevier.]
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activated at the end of the repolarization phase. Ca
v
3.1
channels activate upon depolarization before Ca
v
1.3
and TTX-sensitive Na
⫹
channels. According to this
model, Ca
v
3.1 channels can contribute to the linear part
of the diastolic depolarization. Ca
v
1.3 channels activate
close to the exponential fraction of the diastolic depo-
larization and may constitute the predominant voltage-
dependent mechanism contributing to this phase. This
prediction is consistent with experimental observations
indicating that DHPs mainly affect the late phase of the
diastolic depolarization (319). Ca
v
1.3 channels also
contribute to the upstroke phase of the action poten-
tial. Consistent with experimental observations in
mouse SAN cells (279), TTX-sensitive I
Na
appears to
contribute to the exponential fraction of the diastolic
depolarization, as observed by Lei et al. (279). I
st
is
present throughout the pacemaker cycle as a sustained
component.
IX. ADDITIONAL REGULATORS OF
CARDIAC AUTOMATICITY
A. Neuropeptides
Neuropeptides are released with ACh or norepineph-
rine at autonomic nerve terminals (297, 468). The vasoac-
tive intestinal polypeptide (VIP) is coreleased with ACh
and stimulates the synthesis of cAMP (91). VIP acceler-
ates heart rate in different species including monkeys
(107), dogs (412), and rats (418). It has been proposed
that VIP can contribute to postvagal tachycardia, because
at the end of vagal discharge ACh will be removed faster
from the synaptic space than VIP (195). Shvilkin et al.
(458) have reported that in Langendorff-perfused rat
hearts, vagally released VIP moderates the negative chro-
notropic effect induced by high-frequency vagal stimula-
tion. A direct positive chronotropic effect of VIP has been
FIG. 26. A: simulated effects of
␤
-adrenergic receptor activation (ISO) on I
NCX
and membrane voltage noise during the diastolic depolarization.
Families of simulated LCICR events, total I
NCX
(I
NaCa
), and membrane voltage (V
m
) in control conditions (top panel), under ISO (middle panel), and
ryanodine (no LCRs, bottom panel). Predicted beating rates are shown in beats per min (bpm). B: overlapped representative cycles from simulations
as in A, showing changes in cycle length and I
NCX
amplitude and fluctuations. Inset depicts fluctuating I
NCX
at the initiation of the exponential phase
of the diastolic depolarization (indicated as boxes). The symbol ⌬
i
indicates initial changes in the NCX current related to individual LCRs. [From
Bogdanov et al. (43)] C: voltage-dependent “ion channels clock” generating automaticity is proposed here. The “clock” has been represented by using
a numerical computation of the mouse SAN automaticity according to Mangoni et al. (314). For each ionic current considered, the underlying ion
channel or channel gene family has been indicated. See the text (sect.
VIIIB) for further discussion.
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¨
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reported by Rigel and Lathrop in dogs (413). These au-
thors have observed stimulation of pacemaker activity of
SAN and atrioventricular junctional pacemakers (414). A
positive shift of I
f
activation curve by VIP (5– 6 mV) has
been described by Chang et al. (82) in canine Purkinje
cells, and by Accili and DiFrancesco (3) in rabbit SAN
cells. This shift of the I
f
voltage dependence is probably
the predominant mechanism of action of VIP (3, 82).
B. Adenosine
Adenosine (Ado) is a paracrine and autocrine factor
regulating a plethora of cellular functions in the cardio-
vascular system. These include automaticity, coronary
circulation, cell adhesion and migration, angiogenesis,
and metabolism (346). The cardiovascular effects of Ado
have been intensively studied, due to its cardioprotective
effects in ischemic preconditioning and cardiac stunning
(345). Three distinct Ado receptors (AAR), namely, the
A1, A2, and A3AR are expressed in heart tissue (346).
Transgenic mice overexpressing A1ARs have significant
bradycardia and constitutive slowing of atrioventricular
conduction (242). Indeed, Ado is a potent negative regu-
lator of SAN pacemaker activity (517, 518) and AVN con-
duction (31). This latter property has led to the clinical
use of Ado for termination of reentrant supraventricular
tachycardias (32). In spontaneously active cells, AARs
share the signaling pathway of muscarinic receptors (Fig.
24). Ado mimics downstream effects of muscarinic recep-
tor activation, such as downregulation of cAMP synthesis
and activation of Kir3.1/Kir3.4 channels (I
KAdo
) (30, 543).
Under conditions of accentuated antagonisms, Ado re-
duces automaticity in guinea pig Purkinje fibers (281) and
rabbit SAN cells (30). Belardinelli et al. (30) have studied
the effects of Ado on I
f
and I
Ca,L
in rabbit SAN cells. They
reported that Ado consistently activated I
KAdo
, but inhib-
ited I
Ca,L
and I
f
only after stimulation by isoproterenol
(30). Shimoni et al. (456) have proposed that the indirect
inhibitory effect of Ado on I
Ca,L
is mediated by a NO-
dependent signaling pathway (Fig. 24). However, Zaza,
Rocchetti, and DiFrancesco (543) have reported that Ado
and ACh can shift the activation of “basal” I
f
(without
previous stimulation by isoproterenol) by ⬃5and9mV,
respectively. In this study, low doses (0.03
␮
M) of Ado
slowed automaticity of SAN cells without hyperpolarizing
the maximum diastolic potential. Reasons for the discrep-
ancy between these two studies are not known. Zaza et al.
(543) have emphasized that Ado can slow the heart rate
by specific regulation of I
f
(543).
C. Hormones
Some hormones can influence pacemaking by regu-
lating ion channels involved in automaticity. The SAN and
AVN are enriched with ANG II receptors (430). Habuchi
et al. (169) have shown that ANG II can slow pacemaker
activity in rabbit SAN cells by inhibiting I
Ca,L
. These au-
thors have reported that maximal I
Ca,L
inhibition by ANG
II is ⬃30%. Slowing of pacemaker activity is characterized
by depolarization of the maximum diastolic potential and
a reduction of the action potential velocity. These effects
on the pacemaker cycle are consistent with inhibition by
ANG II of I
Ca,L
(169). Relaxin (RLX), a reproduction hor-
mone, accelerates heart rate in vivo in animal models.
Han et al. (181) have studied the effects of RLX in rabbit
SAN cells. In this study, RLX accelerated pacemaker ac-
tivity of isolated cells and dose-dependently enhanced
I
Ca,L
amplitude. The RLX signaling pathway is dependent
on cAMP and PKA, because cAMP analogs and PKI abol-
ished the effects of RLX (181).
Thyroid hormones (TH) are involved in many physi-
ological processes. The TH triiodothyronine (T
3
) has a
major role in long-term regulation of heart rate, cardiac
output, and tissue lipid content (246). Hyperthyroidism is
associated with tachycardia, arrhythmias, and elevated
cardiac output in animals and humans (28). In contrast,
hypothyroidism is associated with reduced heart rate and
cardiac output (28). THs act through nuclear hormone
receptors (TRs), which are ligand-dependent transcrip-
tion factors (28). Changes in the expression of cardiac ion
channels have been observed in hyperthyroid and hypo-
thyroid animals. Shimoni et al. and Guo et al. have shown
that I
to
density is robustly augmented in ventricles (455)
and neonatal cardiomyocytes (167) isolated from hyper-
thyroid rats. Renaudon et al. (410) have studied the ef-
fects of incubation of isolated rabbit SAN cells with T
3
.In
this study, T
3
significantly increased the density of I
f
in
the absence of an effect on its current-voltage depen-
dence. This observation suggests an augmentation of f-
channels expression in the cell membrane. Renaudon
et al. (410) have proposed that an increase in f-channel
expression underlies the increase in heart rate in hyper-
thyroid states. This hypothesis is partially supported by
three studies measuring the amount of mRNAs coding for
HCN2 channels in the heart of hyperthyroid and hypothy-
roid rats and mice (161, 270, 380). Indeed, Pachucki et al.
(380) have reported a fourfold increase in HCN2 mRNA
when T
3
was administrated to hypothyroid rats. Le Bouter
et al. (270) have found complex remodeling of ion channel
gene expression patterns in ventricles of hypothyroid
mice and reported a significant reduction in HCN2
mRNAs. In mammals, two genes encode for TRs, namely,
the TR
␣
and TR
␤
receptors with their isoforms (28). Mice
lacking the TR
␣
isoform show ⬃20% heart rate slowing
even after stimulation by T
3
. Gloss and co-workers (161,
524) have produced two distinct mouse lines lacking TR
␣
and TR
␤
.TR
␣
⫺/⫺
mice have mild bradycardia and a re-
duction in HCN2 and HCN4 mRNAs in the heart. These
effects were mimicked by hypothyroidism. In contrast,
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TR
␤
⫺/⫺
mice were euthyroid and showed no significant
alterations in HCN4 and HCN2 mRNAs. Consequently,
Gloss et al. (161) proposed that TR
␣
is the target for T
3
in
the heart and is responsible for the regulation of heart
automaticity by TH. However, the functional expression
of ion channels in SAN cells of TR
␣
⫺/⫺
mice has not been
studied. It is possible that T
3
affects the expression of
proteins other than HCN in the SAN. For instance, down-
regulation of NCX and concomitant augmentation of Na
⫹
-
K
⫹
-ATPase in hypothyroid hearts has been reported
(303).
Parathyroid (PTH) hormones accelerate heart rate.
Hara et al. (189) have studied the effects of PTH on
pacemaker activity in rabbit SAN and canine Purkinje
fibers. PTH reversibly accelerated pacing rate in both
preparations and augmented the I
f
maximal conductance
by 68% (189). The effect of PTH was due to an increase in
the slope of diastolic depolarization. In isolated rabbit
SAN cells, Cs
⫹
inhibited the effects of PTH much better
than the VDCC blocker verapamil. Hara et al. (189) have
thus proposed that I
f
is the predominant mechanism by
which PTH increases heart rate.
D. Mechanical Load and Atrial Stretch
The cardiac cycle is associated with periodical filling
of the atrial chambers. Changes in the venous return thus
affect the diastolic atrial dimensions. An increase in the
right atrial filling stretches the atrial wall, as well as the
SAN. Atrial stretch due to venous return can have a direct
effect on pacemaker activity. Bainbridge (17) reported
that injection of fluids into the jugular vein of anesthetized
dogs elevated venous return and increased the heart rate.
This phenomenon is still referred to as the “Bainbridge
reflex.” Other investigators repeated Bainbridge’s experi-
ments and observed a positive or negative in vivo chro-
notropic response under various experimental conditions
(see Refs. 177, 251 for review). In vivo, this variability has
been accounted for by a dependence of the Bainbridge
reflex from the initial heart rate (95, 99).
Even if Bainbridge had attributed the positive chro-
notropic response to a reduced vagal tone upon increased
venous return, there is now substantial evidence indicat-
ing that at least part of the Bainbridge reflex is due to an
intrinsic control of SAN pacemaking by the mechanical
load of the atrium. Indeed, it is possible to reproduce a
positive chronotropic response to stretch in SAN tissue
(108), and on Purkinje fibers automaticity (233). These
results strongly suggest that the Bainbridge effect is, at
least in part, an intracardiac phenomenon. In this respect,
Wilson and Bolter have excluded that the Bainbridge
reflex can be due to a stretch effect on intracardiac neu-
rons (527). Furthermore, and consistent with early in vivo
evidence (95), Cooper and Kohl (99) have reported that
the direction of the chronotropic effect (positive or neg-
ative) induced by cell stretch depends on the initial cell
pacing rate. At the species level, the chronotropic effect
will be positive or negative depending on the mean resting
cellular rate of the species considered (99).
Cooper et al. (100) have shown that pacemaker ac-
tivity of SAN cells can be intrinsically mechano-modu-
lated via activation of SACs, because application of the
GsMTx-4 toxin, which blocks SACs, prevents the positive
chronotropic response to stretch in guinea pig SAN tissue
strips (99). The action of stretch on SAN cells can explain,
in part, the differences in pacing rate of isolated pace-
maker cells and in vivo heart rate, because electrophysi-
ological experiments are generally performed on mechan-
ically unloaded myocytes (251). Beside SACs, it is likely
that the mechanosensitivity of other ion channels can also
be involved in the chronotropic response to stretch of
SAN cells. For example, Lin et al. (287) have reported that
HCN channels are mechanosensitive. Regulation of pace-
maker activity by atrial filling can be relevant for the fine
beat-by-beat modulation of heart rate. Cooper et al. (99)
have highlighted that regulation of the diastolic phase by
mechanical load is useful for initiating a new heartbeat
when the venous return is elevated (99). Somewhat con-
flicting evidence exists as to the physiological relevance
of Bainbridge’s reflex in humans. Slovut et al. (463) have
proposed that stretch-induced modulation of SAN auto-
maticity can be responsible of the respiratory sinus ar-
rhythmia in cardiac allograft recipients (see also sect.
VIIA). This suggestion is based on the observation that in
⬃23% of patients studied, the heart rate oscillated with
arterial pulse pressure (463). These authors have also
shown that intrinsic cardiac neurons are not involved in
this phenomenon so that HRV could be related to Bain-
bridge’s reflex. On the other hand, Casadei et al. (76) have
reported that, in normal subjects, the nonneuronal com-
ponent of HRV is negligible at rest and that this compo-
nent augments only in conditions of ganglionic block
during strong exercise. It is thus possible that, in humans,
Bainbridge’s reflex can become relevant under particular
physiological conditions (e.g., under reduced or blocked
autonomic tone), while under basal conditions, the auto-
nomic nervous system dominates the beat-by-beat regu-
lation of heart rate.
E. Electrolytes and Temperature
By affecting SAN pacemaker activity, the extracellu-
lar ionic environment (89, 447, 448) and the body temper-
ature can influence the heart rate.
In conditions of cardiac ischemia or intense exercise,
there is a significant augmentation of the extracellular K
⫹
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and of the sympathetic tone (75). In the SAN, an increase
in the extracellular K
⫹
concentration shifts the leading
pacemaker site (292) (Fig. 6) and depolarizes the maxi-
mum diastolic potential (240, 248). It has been proposed
that increased extracellular K
⫹
will reduce the contribu-
tion of I
f
to the diastolic depolarization (240), but such an
effect would be difficult to demonstrate, because K
⫹
will
also enhance the I
f
Na
⫹
conductance (89). The negative
chronotropism associated with increased extracellular
K
⫹
may thus be attributed to a reduction of I
Kr
current
(240). Choate et al. (89) have reported that in intact SAN
preparations, the positive chronotropic effect observed
after stimulation of the sympathetic ganglion is reduced
when the extracellular K
⫹
is raised. These authors have
proposed that such a mechanism may protect the isch-
emic heart from deleterious effects of an excessive sym-
pathetic input (89). Ca
2⫹
and Mg
2⫹
also affect SAN chro-
notropism and can induce pacemaker shift (373, 378).
Ca
2⫹
has a positive chronotropic effect, while Mg
2⫹
in-
duces negative chronotropism (378). Mg
2⫹
also interferes
with autonomic regulation of SAN pacemaking, possibly
by preventing pacemaker shift associated with norepi-
nephrine and ACh (378) (see Fig. 6). Acidosis of the
extracellular environment induces negative chronotro-
pism (399, 469). This mechanism can be responsible for
bradycardia during parturition (469) or under ischemic
conditions (164). Changes in heart rate following alter-
ations in the ionic concetrations of blood plasma have
been reported also in humans. For example, Severi and
co-workers (447, 448) have shown that even moderate
(within the physiological range) changes in the plasma
concentration of Ca
2⫹
,K
⫹
, and H
⫹
induce significant
changes in the heart rate of human subjects, thus high-
lighting the importance of plasma electrolyte compositi-
tion in regulating cardiac automaticity.
The effects of temperature on pacemaking can be
roughly distinguished between short-term effects (e.g.,
transient changes in pacing rate) or long-term effects that
represent adaptation to low temperatures. In vivo, lower-
ing the temperature induces reduction of the SAN rhythm
and conduction time (247, 271). In the intact SAN, the
effects of temperature on pacemaking can also be asso-
ciated with pacemaker shift (271). Le Heuzey et al. (271)
reported that lowering the temperature in isolated rabbit
SAN preparations induces a caudal shift of the leading
pacemaker site. Hof et al. (202) have shown that a drop in
the temperature below the physiological range can also
interfere with the chronotropic effect of extracellular
Ca
2⫹
, a phenomenon which seems also to be due to
pacemaker shift.
It has been recently demonstrated that pacemaking
can show long-term adaptation to low environmental tem-
peratures (193). Haverinen and Vornanen (193) reported
that rainbow trout bred at low water temperature have
increased heart rate. These authors have shown increased
I
Kr
density in isolated sinus venous cells of low-tempera-
ture bred fish. These results demonstrate that ionic cur-
rents involved in pacemaking can be modulated on a
long-term basis to adapt to environmental constraints.
SAN tissue seems also particularly resistant to long-
term exposure to low temperatures. Nishi et al. (357) have
shown that SAN automaticity can be recovered unscathed
after several days in cold Tyrode’s solution (5°C), while
the surrounding atrial tissue becomes completely unex-
citable. Similarly, Furuse et al. (147) reported that SAN
pacemaking is resistant to cold cardioplegia. Resistance
of SAN pacemaking to low temperatures has been ex-
ploited clinically for cardioprotection during heart sur-
gery, grafting and, more generally, for clinical hypother-
mia (259, 382, 409). Indeed, heart rate slowing induced by
low temperatures increases coronary blood flow, via an
improvement of diastolic atrial filling (10). Clinical hypo-
thermia can be very important in the treatment of ische-
mic heart disease, because elevated body temperature is
associated with worsening of ischemia-induced myocar-
dial necrosis (178).
Increased body temperature can directly affect the
heart rate (51, 98, 188, 239). Core temperature elevation
can significantly increase SAN rate during exercise (51).
Fever also induces a direct positive chronotropic re-
sponse (98, 188, 239). In a clinical study by Kiekkas et al.
(239), it was noted that increase of core temperature
during fever episodes was followed by a significant in-
crease in heart rate and a decrease in arterial blood
pressure. Alterations of heart rate and arterial blood pres-
sure were significantly affected by magnitude of fever
(239).
X. GENETIC AND ACQUIRED DISEASES OF
CARDIAC AUTOMATICITY
A. Inherited Dysfunction of SAN Automaticity
Propagation of the heartbeat requires coordination
between impulse generation and the spread of cardiac
excitation through the conduction system, which delivers
the impulse to the working myocardium. Genetic or ac-
quired dysfunction of cardiac ion channels can lead to
debilitating and life-threatening arrhythmias or heart
block. Arrhythmias are generally linked to desynchroni-
zation of the sequential activation of the heart, or to
defects in myocardial repolarization. Acquired arrhythmo-
genic diseases can be secondary to medical interventions,
including cardiac surgery and/or drug administration. Fur-
thermore, cardiac ischemia and heart failure can favor the
genesis of arrhythmias and sudden death (266, 315). Sev-
eral mutations in genes coding for cardiac ion channels
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have been identified and shown to be linked to either
inherited dysfunction of the heartbeat or enhanced sus-
ceptibility to drug-induced arrhythmias (92, 236). During
the last few years, alterations in ion channels contributing
to the genesis and regulation of pacemaker activity have
been found, and some insight into possible physiopatho-
logical mechanisms underlying inherited dysfunction of
automaticity is now available.
SAN dysfunction (SND) accounts for half of the num-
ber of patients requiring implantation of an electronic
pacemaker device (266). SND is characterized by a com-
bination of symptoms including fatigue and syncope. Bra-
dycardia, SAN arrest, or exit block are typical features of
SND (315). In some cases, alternating periods of brady-
cardia and tachyarrhythmias can be observed in SND
patients (36). In a substantial number of cases, SND is
associated with acquired cardiac conditions, such as
heart failure, ischemia, cardiomyopathy, or administra-
tion of antiarrhythmic drugs. However, in a significant
percentage of patients, SND is unrelated to structural
abnormalities of the heart, but shows familial legacy (274,
301, 436).
To date, three mutations underlying SND have been
identified in the human HCN4 gene (hHCN4) (330, 443,
486). In a recent case report, Schultze-Bahr et al. (443)
have identified a hHCN4 mutation that caused sinus bra-
dycardia with intermittent atrial fibrillation and loss of
exercise-dependent increase in heart rate in a patient.
This mutation named (hHCN4–573X) generates a trunca-
tion at the protein COOH terminus upstream to the chan-
nel cAMP-binding domain (CNBD). Recombinant hHCN4
and hHCN4 –573X channels have similar voltage depen-
dence. However, hHCN4 –573X channels are insensitive to
cAMP. Coexpression of hHCN4–573X with normal recom-
binant hHCN4 channels indicates that mutant channels
have a dominant-negative effect so that coexpressed f-
channels cannot be facilitated by cAMP. Insensitivity of
hHCN4–573X channels to cAMP can explain SAN brady-
cardia and autonomic incompetence in patients carrying
the hHCN4–573X mutation (443). It is possible that atrial
fibrillation could be secondary to bradycardia. Ueda et al.
(486) have searched for mutations in ion channel genes in
a series of 25 unrelated patients having SND, conduction
disturbances, and idiopathic ventricular fibrillation. A
missense mutation in hHCN4 (hHCN4-D553N) has been
identified in this survey. This mutation is located in the
linker region between the transmembrane core and the
intracellular COOH terminus of hHCN4. The D553N mu-
tation seems to affect cellular trafficking of hHCN4 chan-
nels. In coexpression experiments, hHCN4-D553N chan-
nels reduced I
f
current mediated by normal HCN4 chan-
nels, suggesting a dominant negative suppression of
HCN4. In a patient carrying the hHCN4-D553N mutation,
severe bradycardia with associated prolongation of the
Q-T interval was present. Also, a long sinus pause, fol-
lowed by polymorphic ventricular tachycardia, has been
recorded (486).
A mutation named hHCN4-S672R has been recently
identified by Milanesi et al. (330) in members of a large
Italian family. Compared with the other two mutations
discussed above, hHCN4-S672R is associated with con-
genital asymptomatic bradycardia and is inherited as an
autosomal dominant allele. Affected individuals have mild
bradycardia (mean heart rate is 52 beats/min) without
other associated arrhythmias. The hHCN4-S672R maps in
the CNBD, yet affected f-channels are normally respon-
sive to cAMP. However, coexpressing normal hHCN4
with hHCN4-S672R channels yields currents that activate
at voltages ⬃10 mV more negative than wild-type hHCN4-
mediated currents (330). We can infer that such a negative
shift in the I
f
activation curve results in diminished I
f
-
mediated inward current in the diastolic depolarization
range, leading to slowed basal heart rate. Milanesi et al.
(330) have emphasized the similarity between the moder-
ate reduction of heart rate in patients carrying hHCN4-
S672R mutation and the effect of low ACh doses (10–30
nM) on pacemaking of isolated rabbit SAN cells. Another
hHCN4 familial mutation, namely, G480R, has been re-
cently identified by Nof et al. (361). The hHCN4-G480R
maps in the pore domain. Affected f-channels activate at
more negative voltages than wild-type counterparts and
have defective regulation of intracellular trafficking. Indi-
viduals carrying hHCN4-G480R have asymptomatic bra-
dycardia (361).
SAN bradycardia has also been shown in association
with congenital heart block (CHB) (209). CHB is charac-
terized by progressive complete atrioventricular block
affecting fetuses and newborns (see Ref. 55 for review).
CHB is generally detected just before or immediately after
birth (512). CHB is due to production of autoantibodies
against intracellular soluble ribonucleoproteins named 48
kDa SSB/La, 52 kDa SSA/Ro, and 60 kDa SSA/Ro (512). Hu
et al. (209) have reported inhibition of I
Ca,L
and I
Ca,T
by
IgGs isolated from mothers having CHB-affected children.
It is thus tempting to speculate that downregulation of
Ca
v
1.3 and Ca
v
3.1 channels by maternal antibodies under-
lies SAN bradycardia in CHB (209). This hypothesis has
been recently supported by Qu et al. (406), who showed
that the Ca
v
1.3 channel protein is expressed in the human
fetal heart and that anti-Ro/La antibodies can effectively
inhibit Ca
v
1.3-mediated I
Ca,L
expressed in tsA201 cells.
Mutations in Na
v
1.5 channels can also cause SND.
Benson et al. (33) have recently described two mutations
in the Na
v
1.5 gene leading to recessive disorders of car-
diac conduction, characterized by bradycardia progress-
ing to atrial unexcitability during infancy. It is unclear
whether bradycardia is due to dysfunction of the impulse
generation in the SAN or to partial block of intranodal
conduction.
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B. Automaticity in Heart Failure and
Cardiac Ischemia
Heart failure (HF) is a major risk factor for life-
threatening ventricular arrhythmias and sudden death. HF
carries a poor prognosis; mortality rates at 5 years for
untreated or poorly treated HF are close to 60% (231).
ECG recordings in HF patients show decreased mean
heart rate and heart rate variability (223, 375). Remodel-
ing of intrinsic SAN function and regulation by the auto-
nomic nervous system has been observed in HF. For
example, Sanders et al. (434) have mapped SAN activation
and conduction reserve in 18 HF patients. They have
shown complex rearrangements of SAN excitability, in-
cluding alteration of intranodal conduction, prolongation
of the SAN recovery time, and a caudal shift of the leading
pacemaker site. Experimental animal models of HF fairly
reproduce clinical observations (see the recent review by
Janse, Ref. 221). Opthof et al. (376) have investigated SAN
function in a rabbit model of HF associated with sudden
death. Isolated right atrial preparations of HF rabbits
(376) show a decrease in the intrinsic SAN rate and an
increase in the sensitivity to ACh. However, in spite of
such an enhancement of negative chronotropic factors,
sudden death is still observed in these animals at accel-
erated heart rates. Opthof et al. (376) have suggested that
the reduction of intrinsic SAN rate constitutes an adapta-
tion mechanism to counteract an augmentation in sympa-
thetic tone and its arrhythmogenic potential, and to opti-
mize myocardial oxygen consumption. Verkerk et al.
(503) have reported that isolated SAN cells from HF rab-
bits have an intrinsically longer cycle length and show a
40% reduction of I
f
(and 20% of I
Ks
) compared with con-
trol cells. The slope of the diastolic depolarization ap-
pears to be specifically affected by HF. Verkerk et al.
(503) have thus proposed that the intrinsically slower
SAN rate observed in HF animal models (and possibly in
humans) is due to I
f
downregulation. Consistent with
these findings, a strong reduction in HCN4 and HCN2
mRNA and protein in the SAN has been reported by Zicha
et al. (548) using a canine model of congestive HF. Dis-
ease seems also to have differential effects on the SAN
and the atria, since HF induced an increase in HCN4
expression in atrial tissue. It is possible that HCN upregu-
lation can contribute to the development of supraventric-
ular arrhythmias associated with HF (548).
Cardiac ischemia can induce SND. Bradycardia is
commonly observed upon resuscitation after cardiac ar-
rest or ventricular fibrillation (379). Severe bradycardia
following cardiac ischemia is a major cause of poor sur-
vival after cardiac arrest (156, 379). SAN artery stenosis or
occlusion is another factor leading to bradycardia in pa-
tients (5) and dogs (85). Heart rate slowing can be ob-
served in Langendorff-perfused hearts upon ischemia-
reperfusion (337). Considerable interest exists therefore
in elucidating the mechanisms of ischemia-induced
bradycardia for managing cardiac ischemic disease and
cardiac arrest. The cellular basis of ischemia-dependent
bradycardia is not entirely understood. Slowing of dia-
stolic depolarization, reduction in action potential ampli-
tude, and depolarization of the maximum diastolic poten-
tial have been consistently observed in isolated rabbit
atria during hypoxia (446) or metabolic inhibition (253).
Similar changes in the pacemaker cycle parameters have
been recently reported in isolated rabbit SAN cells by
Gryshenko et al. (164), using a low pH (6.6) “ischemic”
Tyrode’s solution to mimic acidosis of the extracellular
environment in ischemic conditions (see Ref. 75 for re-
view). These effects were reversible when switching back
to normal Tyrode’s solution. Metabolic inhibition of rabbit
SAN cells attenuates I
Ca,L
, I
Kr
, and I
f
(180) and activates
I
K,ATP
(184). In contrast, Gryshenko et al. (164) have
observed an augmentation of the instantaneous net in-
ward current upon hyperpolarization, an effect attributed
to acidosis-dependent reduction of I
KACh
(even if the in-
duction of an inward background current cannot be ex-
cluded). Furthermore, reduction in I
Ca,T
and I
NCX
has
been reported by Du and Nathan (133). It is a surprising
finding of this work that I
Ca,L
was augmented by ischemic
Tyrode’s solution. However, as suggested by Du and
Nathan (133), such an augmentation may not lead to a
positive chronotropic effect, since the more positive max-
imum diastolic potential, induced by ischemic Tyrode’s
solution, can accentuate I
Ca,L
inactivation. From the
above results, it is difficult to attribute ischemia-induced
slowing of cardiac automaticity to a single ionic mecha-
nism. Furthermore, it is possible that a given ionic current
can be more or less affected depending on the experimen-
tal conditions (e.g., acidosis or direct metabolic inhibi-
tion). Future investigations will further elucidate the cel-
lular mechanisms of ischemia-induced bradycardia and
SND.
XI. HEART AUTOMATICITY AND
CARDIOPROTECTION
In the preceding sections, we have discussed the
mechanisms underlying automaticity and its regulation by
the autonomic nervous system, hormones, and different
physiopathological conditions including cardiac ischemia
and HF. We will now review recent pharmacological and
gene therapy approaches aiming to control pacemaker
activity and heart rate in cardiac disease. These new
therapeutic strategies are currently focused on f-channels
and reduction in I
K1
. Indeed, the capability of I
f
to mod-
ulate the slope of the diastolic depolarization renders
f-channels suitable targets for pharmacological regulation
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of heart rate and for creating “biological pacemakers” in
the diseased myocardium.
A. Heart Rate and Cardiac Morbidity
The mean resting heart rate is correlated with all-
cause cardiovascular mortality and morbidity. The Fra-
mingham epidemiological study has highlighted the asso-
ciation between elevated heart rate and all-cause mortal-
ity (134, 160, 232). The mechanistic link between heart
rate and mortality is still unclear. Perski et al. (389)
presented evidence supporting the hypothesis that ele-
vated heart rate contributes to the progression of athero-
sclerosis in postinfarct patients. In subjects with ischemic
cardiac disease such as stable angina pectoris, elevated
heart rate can cause an imbalance between myocardial
oxygen supply and the actual physiological demand.
Heart rate is inversely correlated with the degree of filling
of the left ventricle with oxygen-rich blood (10). A better
irrigation of the coronary system is achieved by a reduc-
tion of the heart rate (10). Andrews et al. (9) have con-
firmed this prediction at the clinical level by showing that
lowering heart rate with propranolol effectively prevents
cardiac ischemic episodes. Development of therapeuti-
cally active molecules, able to reduce heart rate, is thus of
outstanding interest. Targeting of ion channels specifi-
cally involved in the regulation of diastolic depolarization
rate constitutes a natural approach toward this goal.
␤
-Blockers (201) and I
f
inhibitors (123) are effective in
reducing ischemic episodes and cardiac mortality by vir-
tue of their heart rate-reducing action. Specific reduction
of heart rate is now considered as a new therapeutically
effective approach to manage cardiac ischemia (123, 201).
Among I
f
inhibitors, the only molecule that has been
clinically developed for treatment of stable angina pecto-
ris is ivabradine (123). In animal models, ivabradine limits
exercise-induced tachycardia and improves the balance
between myocardial oxygen supply and demand (97). To
date, no inotropic (461) or dromotropic effects (96) have
been reported for ivabradine. A phase II clinical study,
conducted on patients with stable angina, has confirmed
specific heart rate reduction at rest and during exercise
and shown the efficacy of ivabradine as an anti-ischemic
and antianginal agent (52). The in vivo pharmacological
properties of ivabradine are consistent with a specific
action on I
f
in the SAN. Compared with
␤
-blockers, iv-
abradine seems more effective in preserving myocardial
recovery and performance after cardiac stunning (52).
The f-channel is the first ion channel underlying pacemak-
ing to be targeted by a therapeutically active drug. It is
possible that other channel classes, such as Ca
v
1.3 and
Ca
v
3.1 channels, will constitute future pharmacological
targets for the development of new molecules to regulate
heart rate without negative inotropism.
B. Automaticity in Engineered “Biological
Pacemakers”
1. Direct gene transfer of “pacemaker” channels in
cardiac cells
Identification of ion channels involved in cardiac au-
tomaticity harbors the attractive perspective of engineer-
ing “biological” pacemakers to stimulate automaticity in
defined regions of the heart (Fig. 27). Biological pacemak-
ers have been proposed as a possible alternative to elec-
tronic devices (424), even if a biological pacemaker can
also work in combination with an electronic pacemaker
(421). Several up-to-date reviews have been recently pub-
lished on this topic (421, 423, 424). Here, we focus on
some fundamental aspects of biological pacemakers. The
first gene therapy approach to increase pacemaker activ-
ity in the working myocardium came from experiments in
which a cDNA encoding for the
␤
2
-adrenergic receptor
was injected in the right atrium of mouse (137) and pig
(138). Overexpression of
␤
2
-receptors increased the basal
heart rate in both animal models (137, 138).
A gene therapy approach was employed by Miake
et al. (327) to generate pacemaker activity in the ven-
tricular myocardium of guinea pigs. In this work, ad-
enoviral gene transfer of a dominant negative Kir2.1
subunit (Kir2.1AAA) has been employed (327). This con-
struct downregulated I
K1
by 80% in ⬃20% of ventricular
myocytes tested (328). Depolarization of the resting po-
tential to ⫺60 mV generated a diastolic depolarization
phase in ventricular myocytes and triggered premature
ventricular beats in vivo (327). On the basis of these
results, Miake et al. (327) have suggested that the working
myocardium is also endowed with automaticity, but pace-
making in ventricular cells is naturally repressed by I
K1
expression. This proposal challenges the view that auto-
maticity depends on the expression of particular “pace-
maker” ion channel. Silva and Rudy (459) and Kurata et al.
(263) have studied the possible mechanisms of automa-
ticity in ventricular cells expressing Kir2.1AAA by em-
ploying modified versions of the Luo and Rudy model of
ventricular action potential (298). Numerical simulations
suggest that automaticity in these cells can be generated
by I
NCX
(459), or by I
Ca,L
and I
NCX
(263). Particularly,
CICR triggered by I
Ca,L
during the action potential would
stimulate I
NCX
, which depolarizes the cell membrane to
begin a new cycle (459), while I
Ca,L
can be responsible for
a membrane voltage instability which promotes automa-
ticity (263). Silva and Rudy have proposed also that
␤
-ad-
renergic stimulation of the ventricular I
NCX
-driven pace-
maker mechanism could not exceed 25%. Consequently,
the automaticity in Kir2.1AAA-expressing cells probably
lacks the sensitivity to autonomic regulation needed to
sustain everyday life. Viswanathan et al. (511) have fur-
ther assessed, by numerical modeling, the problem of
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automaticity in directly transfected ventricular cells.
These authors have suggested that to obtain stable auto-
maticity, both downregulation of I
K1
and expression of
HCN channels are required (511).
Overexpression of HCN channels by gene transfer
constitutes another strategy for creating biological pace-
makers (421). This approach is based on the experimental
observation that expression of HCN2 or HCN4 channels in
cultured neonatal ventricular myocytes robustly improves
cellular automaticity (143, 403). Expression of a dominant
negative HCN2 subunit abolished automaticity (143).
These observations indicate that HCN channels are able
to confer automaticity to myocardial cells upon gene
transfer. Qu et al. (405) took advantage of this new con-
cept and employed gene transfer of HCN2 channels
in vivo in the canine left atrium. Qu et al. (405) have
shown that I
f
expression in atrial myocytes generated
viable atrial rhythms upon inhibition of SAN activity.
Plotnikov et al. (394) have employed gene transfer of
HCN2 channels to stimulate automaticity in the left bun-
dle branch of the Purkinje fiber network (Fig. 27). In this
work, adenoviral gene transfer of HCN2 channels im-
proved the rate of ventricular rhythms observed after
inhibition of SAN activity by transient vagal stimulation.
To evaluate the sensitivity of these ventricular rhythms to
autonomic agonists, radiofrequency ablation of the AVN
was performed to dissociate the atrial rhythm from that
originating from the left bundle branch (421). In these
conditions, ventricular rhythms were significantly respon-
sive to epinephrine (421).
Bucchi et al. (69) have employed gene transfer of a
mutant HCN2 channel having a positively shifted activa-
tion curve midpoint (⫺46 versus ⫺66 mV for wild-type
HCN2 channels). Compared with rhythms generated by
wild-type HCN2 channels, expression of mutant HCN2
channel significantly improved the responsiveness of ven-
tricular rhythms to catecholamines. Expression of HCN4
channels in the ventricle also seems to create
␤
-adrener-
gic modulation of ventricular escape rhythms (71). Mod-
ification of HCN voltage dependency to favor channel
opening at positive voltages has been employed also by
Tse et al. (483). These authors have shown that focal
FIG. 27. The primary SAN pacemaker cell is the natural pacemaker (top panel). In SAN cells, high expression of native HCN channels (black
channels) contributes to SAN pacemaker dominance and to the autonomic regulation of heart rate. The SAN pacemaker cycle (top, left) drives the
right atrium at a physiological suitable rate. In case of SAN failure or AVN block, the intrinsic pacing rate of the Purkinje fibers network could be
enhanced by gene transfer of recombinant HCN channels (gray channels) in the bundle branches. The ideal Purkinje cell (middle panel) will thus
drive the ventricular cell at a faster frequency than under normal conditions, thereby generating a physiologically suitable ventricular basal rate. The
sensitivity of HCN channels to cAMP is also potentially important for creating a ventricular pacemaker that can be regulated by the autonomic
nervous system. A stem cell-derived biological pacemaker (bottom panel) is based on overexpression of recombinant HCN channels in unexcitable
stem cells implanted in a target region of the working myocardium. Stem cells connect to ventricular cells by forming gap junctions with ventricular
cells. The positive membrane potential of stem cells will cyclically drive ventricular cells to the action potential threshold. [Adapted from Robinson
et al. (421).]
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expression of a mutant form of HCN1 in the left atrium
reduces the dependence on electronic pacemakers of
SAN-ablated pigs (483).
2. Stem-cell based gene transfer
An alternative approach for creating biological pace-
makers consists in overexpressing HCN channels in un-
excitable human mesenchymal stem cells (hMSCs). These
cells have the natural capability of coupling to cardiac
myocytes via gap junctions (400). When unexcitable hM-
SCs connect to myocytes, their membrane potential will
become more negative due to the electrotonic influence of
myocytes. Membrane hyperpolarization will activate HCN
channels, thereby generating inward current that is fed
back to the myocyte to trigger an action potential [Fig. 27,
see Robinson et al. (421) for further discussion]. Action
potential repolarization will then tend to shut off HCN
channels in the hMSC before the beginning of a new cycle.
Potapova et al. (400) have reported that HCN2-mediated
currents expressed in hMSCs are sensitive to isoprotere-
nol, indicating that the signaling pathway necessary for
HCN channel regulation is present in hMSCs. For a more
detailed discussion on the clinical and experimental prob-
lems associated with stem cell transfection and implan-
tation in the heart, the reader is referred to the review by
Rosen et al. (423). Downregulation of I
K1
in ventricular
cells could also be employed to achieve robust stem cell-
mediated pacemaking (230). Gene transfer of stem cells
may not constitute an exclusive approach to create bio-
logical pacemakers based on cell therapy. Indeed, it is
possible that also fibroblasts can be genetically modified
to express ion channels (144). Fibroblasts are spontane-
ously capable of connecting with cardiomyocytes in vitro
and can affect conduction between myocytes (144). In
cell culture, Kizana et al. (243) have obtained functional
electrical coupling between myotubes differentiated from
MyoD-Cx43 coexpressing fibroblasts. Interestingly, these
authors have reported that coexpression of Cx43 with
MyoD was critical for observing electrical coupling and
uniform threshold for excitation between adjacent myo-
tubes (243). Cx43 expression also enhances electrical
coupling between neonatal cardiomyocytes and fibro-
blasts in cell culture (244). Expression and modulation of
appropriate Cxs together with ion channels can thus be an
important step for overcoming the problem of poor elec-
trical coupling between stem cells and myocytes in situ.
This potential approach is supported by a numerical mod-
eling study by Jacquemet (219), who emphasized that
fibroblasts can significantly affect myocardial conduction
only if the degree of coupling between fibroblast and
myocytes is sufficiently high.
In conclusion, establishment of biological pacemak-
ers in defined regions of the heart is a fascinating per-
spective for the cellular therapy of the heartbeat. A bio-
logical pacemaker may also necessitate the coexpression
or modification of more than one type of ion channel to
reach a suitable degree of intrinsic automaticity and au-
tonomic regulation of pacemaking (263, 511). The obser-
vation that the Tbx3 transcription factor can reprogram
nonpacing atrial cells to a phenotype similar to that of
automatic cells may also open new perspectives into the
creation of biological pacemakers (208).
XII. CONCLUDING REMARKS
Our understanding of cardiac automaticity has pro-
gressed considerably during the last 10 years. Indeed,
at the beginning of the 1990s, the study of the genera-
tion of pacemaker activity was somewhat limited to the
electrophysiological description of ion currents in
spontaneously active cells. Molecular cloning of gene
families coding for ion channels has since allowed in-
vestigation of gene expression in cardiac tissue and the
establishment of genetically engineered mouse lines
that show specific dysfunctions of cardiac automatic-
ity. These lines are now yielding precious insights into
the generation and regulation of pacemaking and will
probably constitute new animal models of heart rhythm
pathologies. We can expect that the molecular basis of
many inherited diseases of cardiac automaticity will be
elucidated in the near future and that different ion
channels and proteins regulating Ca
2⫹
handling will be
involved in such diseases.
Several questions on how the heart rhythm is gener-
ated and controlled in physiological and pathological con-
ditions remain unanswered. It is not completely under-
stood, for example, which ion channels are essential for
generating the diastolic depolarization in the SAN, AVN,
and the Purkinje fibers network, and which mechanisms
play a dominant role in the autonomic regulation of au-
tomaticity in humans. The interactions between the mem-
brane “ion channels clock” and the intracellular “Ca
2⫹
clock” are not completely elucidated. Nevertheless, these
interactions are of fundamental importance for under-
standing the integration of pacemaker mechanisms at the
cellular level.
From the above discussion, it also appears that car-
diac automaticity at the organ level is a very complex
phenomenon and that, beside cellular mechanisms, inte-
grative factors are involved in cardiac pacemaking. These
factors include the heterogeneity of SAN and AVN tissue
organization and the regulation of heart rate by mechan-
ical load and by several humoral, environmental, and
pathophysiological states.
Finally, ion channels involved in automaticity, such
as f-channels, are now becoming potential new drug tar-
gets. In this respect, the development of specific heart
rate-reducing agents underlines the importance of pursu-
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¨
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ing the effort of obtaining further insight into the cardiac
pacemaker mechanisms. The engineering of “biological
pacemakers” is currently based on expression and manip-
ulation of K
ir
and f-channels in native myocardium or in
stem cells. Other complementary approaches can be the
use of fibroblasts and, possibly, manipulation of Cxs. It
will also be interesting to see if future approaches will
also include coexpression of other ion channels involved
in “native” automaticity.
NOTE ADDED IN PROOF
After acceptance of this manuscript, Li et al. (284a)
have proposed a detailed anatomical model of the rabbit
AVN. Numerical simulations of impulse conduction
through AVN are consistent with the hypothesis that au-
tomaticity originates in the PNE of the AVN.
Harzeim et al. (190a) have shown that abolition of
cAMP sensitivity of HCN4 channels by knock-in of a point
mutation in the channel CNBD prevents
␤
-adrenergic
stimulation of heart rate in mouse embryos and lethality
as in HCN4 knockout embryos.
ACKNOWLEDGMENTS
We thank Patrick Atger for excellent technical skills in creat-
ing and editing the manuscript figures. We are indebted to Elodie
Kupfer and Anne Cohen-Solal for breeding and managing mouse
lines in our group. We thank Peter Kohl and Etienne Verheijck for
helpful discussion. We also thank Halina Dobrzynski, Mark Boyett,
and Dario DiFrancesco for sharing figure originals.
Address for reprint request and other correspondence:
M. E. Mangoni, Institute of Functional Genomics, Dept. of Phys-
iology CNRS UMR5203, INSERM, U661, Univ. of Montpellier I,
Univ. of Montpellier II, Montpellier, F-34094 France (e-mails:
matteo.mangoni@igf.cnrs.fr; joel.nargeot@igf.cnrs.fr).
GRANTS
Work in our laboratory has been supported by grants from
the CNRS, the Action Concerte´ e Incitative in Physiology and
Developmental Biology of the French Ministry of Education, the
INSERM National Program for Cardiovascular Diseases, the
Fondation de France and the Agence Nationale pour la Recher-
che (ANR). We also thank the International Research Institute
Sevier (IRIS) for having supported research activity in our
group.
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