Download The cardiac pacemaker current Journal of Molecular and Cellular

Journal of Molecular and Cellular Cardiology 48 (2010) 55–64
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Journal of Molecular and Cellular Cardiology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y j m c c
Review article
The cardiac pacemaker current
Mirko Baruscotti ⁎, Andrea Barbuti, Annalisa Bucchi
Department of Biomolecular Sciences and Biotechnology, Laboratory of Molecular Physiology and Neurobiology, Università degli Studi di Milano; Centro Interuniversitario di Medicina
Molecolare e Biofisica Applicata (CIMMBA), via Celoria 26, 20133 Milano, Italy
a r t i c l e
i n f o
Article history:
Received 29 April 2009
Received in revised form 15 June 2009
Accepted 26 June 2009
Available online 8 July 2009
Keywords:
Pacemaker current
SAN
HCN clones
HCN knockout mice
HCN-associated pathologies
a b s t r a c t
In mammals cardiac rate is determined by the duration of the diastolic depolarization of sinoatrial node
(SAN) cells which is mainly determined by the pacemaker If current. f-channels are encoded by four
members of the hyperpolarization-activated cyclic nucleotide-gated gene (HCN1–4) family. HCN4 is the most
abundant isoform in the SAN, and its relevance to pacemaking has been further supported by the discovery of
four loss-of-function mutations in patients with mild or severe forms of cardiac rate disturbances. Due to its
selective contribution to pacemaking, the If current is also the pharmacological target of a selective heart
rate-reducing agent (ivabradine) currently used in the clinical practice. Albeit to a minor extent, the If current
is also present in other spontaneously active myocytes of the cardiac conduction system (atrioventricular
node and Purkinje fibres). In working atrial and ventricular myocytes f-channels are expressed at a very low
level and do not play any physiological role; however in certain pathological conditions over-expression of
HCN proteins may represent an arrhythmogenic mechanism. In this review some of the most recent findings
on f/HCN channels contribution to pacemaking are described.
© 2009 Elsevier Inc. All rights reserved.
Contents
The mechanism of cardiac pacemaking and the If current .
Basic biophysical properties of the pacemaker current . .
Molecular structure of pacemaker channels . . . . . . .
Pacemaker channels during embryonic development . . .
Pacemaker channels in the adult heart . . . . . . . . .
5.1.
SAN . . . . . . . . . . . . . . . . . . . . . .
5.2.
Atrioventricular node (AVN) . . . . . . . . . . .
5.3.
Purkinje fibres (PFs) . . . . . . . . . . . . . . .
5.4.
Working myocardium . . . . . . . . . . . . . .
6.
Basis of functional heterogeneity of pacemaker currents .
6.1.
MiRP1 . . . . . . . . . . . . . . . . . . . . .
6.2.
PI(4,5)P2 . . . . . . . . . . . . . . . . . . . .
6.3.
Caveolin 3 . . . . . . . . . . . . . . . . . . .
7.
Genetics of HCN: HCN knockout mice and HCN-associated
8.
Biological pacemaker . . . . . . . . . . . . . . . . . .
9.
f-channels blockers. . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . .
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pathologies in
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1. The mechanism of cardiac pacemaking and the If current
Cardiac pacemaking originates in the sinoatrial node (SAN) as a
consequence of spontaneous firing of rhythmic action potentials
⁎ Corresponding author. Tel.: +39 02 5031 4931; fax: +39 02 5031 4932.
E-mail address: [email protected] (M. Baruscotti).
0022-2828/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.yjmcc.2009.06.019
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generated by specialized myocytes. Although the electrical behavior of
a typical SAN cell differs in several aspects from that of a working
myocyte, the functional hallmark can be precisely identified in the
events that take place during the diastolic interval. During this phase
atrial and ventricular myocytes rest in a standby-like condition at a
stable voltage (∼−80 mV); a quite different situation characterizes
SAN cells, where the cell potential slowly creeps up from the
M. Baruscotti et al. / Journal of Molecular and Cellular Cardiology 48 (2010) 55–64
56
maximum diastolic potential of about −60 mV to the threshold for
the ignition of a new action potential. Since this time interval sets the
pace of the heart, this phase is named “pacemaker depolarization”.
Given the large spectrum of heart rates observed in mammals the
duration of this phase can vary substantially, however the voltage
range encompassed is extremely constant and roughly extends from
−60 to −40 mV [1–3]. To sustain this phase several ionic currents and
pumps enter in action at variable times and voltages [4–6], and this
complexity allows for a highly flexible system since the chronotropic
fine tuning operated by neuro-hormonal regulators can target
different effectors.
In this review we will focus on the If current which is responsible
for initiating the diastolic depolarization of SAN cells. Due to its
fundamental role and its unusual characteristics of being activated in
hyperpolarization, this current was named “pacemaker current” or
“funny” (If) current [7–9]. The unique property of a reverse voltage
dependence, together with the inward nature of the current at
diastolic potentials, makes this current apt to initiate and support the
diastolic depolarization. In addition, the direct modulation of the
current operated by the second messenger cAMP, represents one of
the main pathways by which the autonomic nervous system controls
cardiac chronotropism [10]. Two recent clinical findings further
confirm the role of f-channels in setting the cardiac rate: one is the
evidence of a causative link between the presence of loss-of-function
mutations found in these channels and the arrhythmic state of
individuals carrying the mutations, and the other is the specific heart
rate reduction observed in patients treated with ivabradine, a drug
that at therapeutic doses selectively reduces the If current (see
specific sections in this review).
Although originally discovered in the heart, the If current is also
abundantly present in a large fraction of neuronal elements, where it
contributes to rhythmic firing, synaptic integration, and dendritic
integration [11].
2. Basic biophysical properties of the pacemaker current
The If current is carried by Na+ and K+ ions and its reversal
potential is between − 10 and −20 mV, and permeation occurs
according to a multiion, single-file mechanism [9,12,13]. Interestingly,
recent single channel experiments carried out in rat and human
working myocytes have also reported a weak permeability to Ca2+
ions [14]. Voltage-clamp studies have shown that activation is a
complex event determined initially by an intrinsic rearrangement of
the closed structure of the channel, which originates a “shoulder-like”
delay, and followed by the proper close-to-open transition which
proceeds according to an exponential relaxation [15,16]. The quantitative aspects of this activation process, i.e. the time-course of the
current activation, and the steady-state current level reached at the
end of each pulse, are finely controlled both by the membrane voltage
and by a direct interaction with the second messenger cAMP [17,18].
Although variable data have been reported, the voltage threshold for
sinoatrial If activation is compatible with a functional presence of the
current at diastolic potentials (Table 1 in [19]); for example, recent
experiments carried out on human SAN cells indicate that threshold
for If activation occurs between −50 and − 60 mV, a voltage range
well comprised in the diastolic range [3].
3. Molecular structure of pacemaker channels
The molecular constituents of pacemaker channels belong to the
hyperpolarization-activated cyclic nucleotide-gated (HCN) channel
family which is part of the superfamily of K+ channels. In mammals
four HCN isoforms are known (HCN1–4), and heterologous expression
and in vivo investigations have shown that they can assemble both as
homotetramers and heterotetramers (with the exceptions of HCN2–
HCN3 heteromers) to yield functional channels. Currents resulting
from homomeric and heteromeric assembly have biophysical and
modulatory properties qualitatively similar to the native If [20]; but
when quantitative aspects are taken into account noticeable differences are evident in terms of kinetics, voltage dependence, and cAMP
sensitivity [21–26]. Each HCN isoform is composed of three large
macrodomains: a cytoplasmic N-terminus, a 6 transmembranespanning “core” region, and a cytoplasmic C-terminus. Each of these
regions mediates important functional aspects. The N-terminus
contains a conserved stretch of residues which appears to be
important in mediating channel trafficking [27]. The core transmembrane region is the more conserved region among isoforms and
contains both the voltage sensor (S4 segment) and residues involved
in pore formation (S5-P-S6 regions), and therefore constitutes the
proper gating module of the channel. All HCN isoforms exhibit within
the permeation pathway the GYG triplet which, as in pure K+
channels, constitutes the selectivity filter. It is not immediately
apparent why HCN channels are also permeable to Na+ ions, but it
is now believed that the inner pore of HCN channels is somewhat less
rigid than that of K + channels, and for this reason it also
accommodates partially hydrated Na+ ions [28]. Further information
Table 1
Effects of “heart rate-lowering” agents on electrical properties of cardiac pacemaker cells.
If reduction
IK reduction
ICa reduction
DDS
APD50
Rate reduction
Refs
Alinidine
Zatebradine
(UL-FS49)
Cilobradine
ZD7288
Ivabradine
∼80%
30 μM
rbSAN cells
30%
30 μM
rbSAN cells
11%
30 μM
rbSAN cells
↓
30 μM
rbSAN cells
+ 23%
30 μM
rbSAN cells
22%
30 μM
rbSAN cells
[146]
∼65%
1 μM
rbSAN cells
∼20%
1 μM
rbSAN cells
No effect
1 μM
rbSAN cells
− 42%
3 μM
rbSAN tissue
+ 29%
3 μM
rbSAN tissue
28%
3 μM
rbSAN tissue
[147–149]
∼60%
1 μM
mSAN cells
∼22%
5 μM
mSAN cells
Not tested
78%
1 μM
gpSAN cells
No effect
1 μM
gpSAN cells
14%
1 μM
gpSAN cells
− 54%
1 μM
gpSAN tissue
+ 8%
1 μM
gpSAN tissue
∼35%
1 μM
gp right atrium
[150,151]
∼60%
3 μM
rbSAN cells
No effect
3 μM
rbSAN cells
No effect
3 μM
rbSAN cells
−67%
3 μM
rbSAN tissue
+ 9%
3 μM
rbSAN tissue
∼24%
3 μM
rbSAN tissue
[10,13,148,149]
− 70%
1 μM
mSAN cells
+ 60%⁎
1 μM
mSAN cells
∼55%
1 μM
mSAN cells
[139]
DDS: diastolic depolarization slope; APD50: action potential duration measured at 50% of repolarization (⁎APD measured from the threshold to the following maximum diastolic
potential); rb, rabbit; m, mouse; gp, guinea-pig.
M. Baruscotti et al. / Journal of Molecular and Cellular Cardiology 48 (2010) 55–64
on the structural organization of the pore comes from the observation
that the pore blocker ZD7288 directly interacts with residues of the S6
segment, and that closing of the channel entraps the drug into the
inner vestibule of the HCN1 channel [29]. Taken together, these data
and the homology model based on the KcsA K+ channel crystal [30],
suggest that the four S6 segments line the inner wall of HCN pore, and
that access to the S6 residues involved in ZD7288 binding is guarded
by a gating structure likely to comprise part of the S6 segment. The Cterminus region comprises three separate structural elements: the Clinker, which is organized in 6 α-helices, the cyclic nucleotide binding
domain (CNBD), and the proper C-terminus. Collectively, the CNBD
and the C-linker act as a functional unit that modulates the open
probability of a pacemaker channel. Detailed information on several
aspects of the HCN channel structure–function relation is now
available [21,31,32] and here we will only provide some general
concepts. It is now clear that voltage-dependent ion channels do not
behave like rigid structures and therefore the rotational/translational
movement of the S4 segments imposed by changes in the membrane
electrical field does not induce a general rearrangement of the protein
leading to the opening of the pore [33]. Due to its direct interaction
with the S4 segment a particular focus was placed on the role of the
S4–S5 linker, which indeed was shown to undergo a spatial
reorganization upon S4 movement [33,34]. Single alanine substitution
of aminoacids E324, Y331, and R339 of the HCN2 S4–S5 linker was able
to disrupt channel closing thus indicating that this linker plays a
crucial role in gating [34]. There is now evidence that the S4–S5 linker
functionally interacts with the C-linker region; for example in HCN2
channels the electrostatic interaction between R339 of the S4–S5
linker and D443 of C-linker stabilizes the closed state of the channel
[35]. These data indicate that the interaction between the S4–S5 linker
and the C-linker mediates the coupling of voltage sensing and channel
opening and closing. In addition to the voltage, the HCN channel open
probability is also controlled by the second messenger cAMP through
a direct binding to the channel [17]. Crystallization data indicate that
in the presence of cAMP the A′ and B′ helices of the C-linker of each
subunit interact with the C′ and D′ helices of the neighboring subunit,
and this tetrameric arrangement favors the open state of the channel.
In the absence of cAMP, the tetrameric assembly is lost and this favors
the closed channel conformation [21,36,37]. This mechanism thus
represents the final molecular event in the modulation of the If
current exerted by the autonomic nervous system.
4. Pacemaker channels during embryonic development
Pacemaker activity characterizes the developing heart at a very
early stage; for example, in chick embryos electrical pacemaker
activity is present even before the onset of regular contractions of the
primitive heart tube [38]. In mammals, the linear heart tube starts to
beat around embryonic day 8.5 (ED8.5), a stage at which all
cardiomyocytes are autorhythmic and express the If current [39].
With further development (ED18) only 33% of myocytes present
spontaneous but irregular action potentials and the If current is
decreased by 82% [39]. In agreement with functional data the same
authors also reported a significant down-regulation of HCN1 and
HCN4 mRNAs and a moderate increase of HCN2; HCN3 was not
detected [39]. A detailed analysis of HCN4 localization in the
developing heart, by in situ hybridization, indicates that the HCN4
mRNA is already detectable in the cardiac crescent at ED7.5, and its
expression remains confined to the venous pole, the cardiac region
from which develops the mature SAN [40]. Specific expression of
HCN4 mRNA in the SAN and conduction system has also been
demonstrated in a series of studies addressing the molecular pathway
leading to the formation of these regions. Tbx3 is a transcriptional
repressor whose expression during heart development specifically
delineates the SAN/conduction system and match the HCN4 expression [41,42]. Interestingly, ectopic expression of Tbx3 in the atria
57
generates foci of autorhythmic cells in which HCN4 results upregulated [43]. Also, constitutive deletion of the transcription factors
Shox2 causes embryonic lethality at mid-gestation (ED11.5–ED12.5),
and this genetic alteration prevents the expression of both HCN4 and
Tbx3 and causes formation of an underdeveloped SAN and significant
bradycardia [44]. Taken together these data indicate that HCN4 is a
major component of the f-channel in the developing SAN, but they
leave some uncertainty regarding its presence in the embryonic
working myocytes. The embryological lethality of HCN4 knockout
mice further indicates that the presence of the If current is necessary
for proper cardiac development [45]. Despite the observation that
Tbx3 and Shox2 are important in the modulation of HCN4 expression
during normal heart development, they do not account for basal HCN
gene transcription. Indeed, the lack of Tbx3 does not influence HCN4
expression [43], and the lack of Shox2 does not modify HCN4
expression until ED10 [44]. Two conserved sequences accounting for
HCN4 gene transcription have recently been found in the non-coding
region of HCN4 [46]. The first, an 846 bp long sequence is located at
the 5′UTR, the second sequence is a neuron-restrictive silencer
element (NRSE) located in the intronic region between exons 1 and
2, which specifically represses the promoter activity of the first
sequence [46].
A different approach in studying early events of cardiac development consists of the in vitro differentiation of embryonic stem cells
(ESC) through the formation of embryoid bodies (EBs), which are
three-dimensional cell aggregates able to recapitulate early embryonic developmental events. 7–8 days after cell aggregation mouse EBs
start to display foci of spontaneous contraction generated by early
cardiomyocytes [47–49]. The If current has been found both in human
and in mouse ESC-derived cardiomyocytes and its direct involvement
in the generation and modulation of rate has been assessed by
applying specific If inhibitors [48,50–54]. In mouse ESC-derived
cardiomyocytes, ivabradine (3 μM) slowed beating rate by 25% and
reduced the If current by 50%, while ZD7288 (0.3 μM) slowed beating
rate by approximately 50% and reduced the If current by 15% [52,53].
Similarly, zatebradine, another If blocker, slowed spontaneous rate of
human ESC-derived cardiomyocytes [50]. Patch-clamp investigations
have shown that the If current is already present in cells isolated from
10 day-old mouse EBs, and that its density increases significantly with
the progression of differentiation [52–54].
There is some variability concerning the mRNA and protein
expression of HCN isoforms in mouse ESC-derived myocytes. While
two studies reported the expression of mRNAs of all four isoforms
[52,55], another study showed that mouse ESC-derived myocytes only
express HCN1 and HCN4 [56]. At the protein level, western blot
experiments revealed the expression of HCN2 and HCN3 isoforms
[53], while immunofluorescence experiments reported that HCN1 and
HCN4 are the only isoforms expressed on the membrane of pacemaker
cells [52,57]. Investigations of human ESC-derived cardiomyocytes
revealed a high and stable expression of HCN2 mRNA while HCN1 and
HCN4 appear to be present in undifferentiated human ESC and their
expression decreases with differentiation [50,51].
Despite this variability, likely originating from differences in ES
clones, species and in the techniques employed, it is clear that the If
current and the underlying HCN channels are expressed very early
during cardiac development and significantly contribute to the
pacemaking mechanism.
5. Pacemaker channels in the adult heart
The If current has been described in all tissues of the heart,
however its functional contribution in non pathological conditions is
limited to the cardiac conduction system. Here we provide an
overview of the characteristics of the If current in various heart
regions.
58
M. Baruscotti et al. / Journal of Molecular and Cellular Cardiology 48 (2010) 55–64
Fig. 1. Molecular and functional properties of SAN myocytes. (A) Spontaneous action potentials (left) and If current traces (right) recorded from typical rabbit SAN myocytes; currents
were elicited by hyperpolarizing voltage steps in the range − 45 to − 75 mV. (B) Immunofluorescence analysis of rabbit SAN tissue slice labelled with anti-connexin 43 (Cx43, red)
and anti-HCN4 (green) antibodies. HCN4 is strongly expressed in the central region of the SAN, while the opposite staining is observed for Cx43; crista terminalis (CT), interatrial
septum (IS). (C) HCN4 labelling of single myocytes isolated from CT, SAN and IS (top), and representative current traces recorded at − 125 mV from myocytes isolated from the same
regions (bottom). Both If current density and HCN4 labelling are more abundant in the central nodal area. (Panels B and C from [61] with permission).
5.1. SAN
In agreement with the leading role of the SAN in pacing, this tissue
presents the highest level of If current density and HCN expression
[58–62]. As previously mentioned, the presence of the If current has
been documented in adult SAN myocytes of many species including
humans [3,19], and its activation threshold (∼− 50.8 mV, n = 9, from
Table 1 in [19]) falls within the voltage excursion encompassed by SAN
cells during the diastolic depolarization. In SAN tissues of lower
mammals and humans, the predominant molecular constituent of fchannel is the HCN4 isoform (Fig. 1); HCN1 and HCN2 have also been
detected, but at low to moderate levels depending on the species [58–
66]. HCN3 is absent from the SAN [58,67].
5.2. Atrioventricular node (AVN)
The AVN is able to pace the heart in the absence of proper sinus
rhythm, and the If current has been recorded in the majority of cells
isolated from the AVN of rabbit, mouse and guinea-pig [68–70]. A
more detailed investigation on ovoid- and rod-shaped cells isolated
from rabbit AVN indicates that in ovoid cells the pacemaker current is
abundantly present (− 5.18 pA/pF at − 100 mV) and its activation
threshold is about −60 mV, while in rod-shaped cells the current is
nearly absent [68]. Studies on mRNA distribution indicate that in the
AVN, as well as in the SAN, the HCN4 isoform is the most abundant
isoform [71], and, at least in mice, the overall HCN4 protein expression
is about one third of that found in the SA node [72].
5.3. Purkinje fibres (PFs)
Isolated PFs are able to beat spontaneously, and for this reason they
were largely used in early electrophysiological experiments investigating the nature of the pacemaker currents responsible for the
diastolic depolarization [8,9,73,74]. The diastolic depolarization of
these fibres develops between −90 mV and − 70 mV, thus in a range
of potential more negative than that of a typical SAN cell. Interestingly,
the threshold for If current activation in these cells is also negatively
shifted (∼ −80 mV), and this suggests an active role of the current in
the generation of the spontaneous activity [8,9]. The molecular
composition of f-channels in PFs is extremely dependent on the
species investigated. For example, canine PFs, which exhibit a
significant automaticity and a large If current, have high levels of
HCN mRNA (∼ 35% of the HCN signal recorded in the SAN), with ∼90%
of the transcripts constituted by HCN4 and the remaining by HCN2
M. Baruscotti et al. / Journal of Molecular and Cellular Cardiology 48 (2010) 55–64
[75,76]; interestingly, at the protein level HCN2 is the major isoform
expressed [76]. In contrast, rabbit PFs, which tend not to be automatic
and exhibit little If current, show minimal levels of HCN messenger
(∼4% of SAN) with an equivalent presence of HCN4 and HCN1, and a
minor contribution of HCN2 (∼10% [65]). In human PFs HCN4 is the
predominant isoform expressed [77].
5.4. Working myocardium
The presence of the If current in the working myocardium is well
documented but its range of activation is much more negative than in
the SAN. Reported values of thresholds are extremely variable and
range from −60 to − 120 mV [78–83]. Furthermore, even when
assessed at −130 mV the current densities are extremely low; for
example in human atrial and ventricular myocytes the reported values
are −0.8 pA/pF and −0.47 pA/pF, respectively [79,81]. For comparison at − 130 mV the If density measured in human SAN cells is
− 8 pA/pF [3]. Taken together these data indicate that under
physiological conditions the If current is not expected to play a
functional role in atria and ventricles. The molecular composition of fchannels in working myocytes is extremely complex due to interspecies variability and sometimes due to contrasting results between
transcriptional and protein data. In general, it can be stated that the
major protein isoform both in atria and ventricles is HCN2. HCN4 is
only occasionally reported, while HCN1 does not appear to be present
[62,71,76,84,85]. Despite the fact that pacemaker currents do not play
a role in the healthy atria and ventricles, growing evidence in humans
and animals demonstrates that the over-expression of the If current in
these tissues is often associated to some cardiac diseases, and this
over-expression may represent an important arrhythmogenic source
[82,83,85–90]. For example, in human ventricular myocytes isolated
from failing hearts of patients with ischemic cardiomyopathy, the If
current is over-expressed by approximately two-fold and its threshold
of activation is shifted positively by 9.6 mV [82]. In agreement with
these observations up-regulation of HCN2 and HCN4 mRNAs/proteins
was found in both atria and ventricle myocytes obtained from failing
hearts explanted from patients with end-stage ischemic cardiomyopathy [85]. A regulatory mechanism controlling the expression of HCN
isoforms may consist of post-translational events. One such mechanism, known to control the functional expression of both HCN2 and
HCN4 is operated by two muscle-specific micro RNAs, miR-133 and
miR-1 [91,92]. For example, during cardiac development, up-regulation of miR-133 and miR-1 decreases the ventricular If current by
reducing HCN2 and HCN4 proteins without significantly affecting
mRNA levels. The opposite event occurs in hypertrophied hearts
where miR-133 and miR-1 are decreased and this down-regulation
causes the re-expression of HCN2 and HCN4 channels [91,92]. This
same mechanism could also explain the variability between mRNA
and protein expression data found in different systems.
6. Basis of functional heterogeneity of pacemaker currents
The identification of the exact molecular composition of native
pacemaker channels was initially challenged by the attempt to
reproduce the native kinetic and modulatory properties of the native
f through heterologous expressions of HCN isoforms. Unfortunately,
this approach proved unsuccessful. Although HCN4 and HCN1 are the
main isoforms of the SAN, their expression either alone or in
combination failed to reproduce the sinoatrial If current. For example,
even though homomeric HCN4 channels retain a cAMP modulation
similar to that observed in native channels, both the activation and
deactivation kinetics and the voltage dependence are much different.
On the contrary HCN4–HCN1 heteromers generate currents with
kinetics approaching those of native SAN f-channels, but they do not
reproduce the same voltage dependence of activation [25]. Furthermore, it has been shown that even when the same isoform is
59
expressed in different cells, the resulting currents do not display
identical properties. To this regard Qu et al. [93] have shown that
currents resulting from HCN2 expression in neonatal and in adult
ventricle myocytes have different biophysical properties such as a
different position of the voltage-dependent curve (V1/2, − 76 mV and
−96 mV, respectively). These data clearly suggest that, in addition to
the heterogeneity provided by different HCN isoform and cAMP
modulation, the large phenotypic variation of If properties observed
both in the healthy heart and in pathological conditions likely reflects
the presence of additional modulatory factors. Recent evidence
indicates that protein–protein and protein–phospholipid interactions,
and modulatory cytoplasmic factors play a relevant role in modulating
the pacemaker current. Taken together all these regulatory elements
are defined as “context dependence”. Some of the most relevant
modulatory factors are MiRP1, phosphatidylinositol 4,5-bisphosphate
(PI(4,5)P2), and caveolin 3.
6.1. MiRP1
Mink-related protein 1 (MiRP1 or KCNE2) is a single transmembrane-spanning protein that acts as a β-subunit of HCN channels.
Although its modulatory actions depend on the HCN isoform and on
the expression system used, the most relevant effect is to increase
current density [94–97].
6.2. PI(4,5)P2
Recent data indicate that the voltage dependence of native
sinoatrial f- and HCN1/2 channels is also regulated by local pools of
PI(4,5)P2 [98,99]. Further experiments suggested that stimulation of
receptors coupled to phospholipase C (PLC), such as bradykinin BK2
receptor and the muscarinic M1 receptor, can modulate the gating of
both recombinant and native HCN channels via an increase of PI(4,5)
P2 [100]. Specifically, activation of the BK2 receptor induces a positive
shift of the voltage dependence of HCN2 (∼ 20 mV), HCN1 (∼ 6 mV),
and sinoatrial f-channels (∼8 mV), and also affects time constants of
activation and deactivation. Noteworthy is the fact that basal levels of
PI(4,5)P2 may change with development, stress, and pathological
conditions [101–103].
6.3. Caveolin 3
It has also been shown that in rabbit SAN cells HCN4 localizes in
caveolin-rich membrane microdomains (caveolae) and interacts with
caveolin 3 [104,105]. Disorganization of caveolae strongly affects fchannel kinetics, shifting the activation curve toward more positive
potentials and slowing the deactivation kinetics. Similar effects were
observed for HCN4 current after lipid raft disruption in HEK cells
[106].
Post translational modification events may also contribute to
modulate HCN properties: both native and HCN channels can indeed
be modulated by phosphorylation processes and particularly by
tyrosine-kinase phosphorylation, but a clear understanding of the
functional role of this regulatory mechanism remains in part elusive
[107–115].
7. Genetics of HCN: HCN knockout mice and HCN-associated
pathologies in humans
Genetically manipulated knockout mice and genetic analysis in
human patients affected by severe or mild cardiac rhythm alterations
have brought new information on the role and relevance of HCN4
channels to cardiac pacemaking. In recent years four HCN4 knockout
mouse models have been developed to evaluate the functional
contribution of this isoform to pacemaking in vivo [45,116,117]. Both
global and cardiac specific constitutive knockouts determine the
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M. Baruscotti et al. / Journal of Molecular and Cellular Cardiology 48 (2010) 55–64
premature death of mice at mid-gestation (ED9.5–ED11.5), period in
which a regular contractile activity of the developing heart normally
appears [45]. Hearts isolated from knockout embryos at day ED9.5
exhibited a decreased spontaneous rate (−36.7%) and If current
(−75/−90%). Interestingly, the knockout process also led to the
complete loss of cAMP-mediated β-adrenergic chronotropic modulation in both intact hearts and single cells; this finding is consistent
with the observation that the residual If current could be carried by
the HCN1 and HCN3 isoforms which are known to be mildly or not
affected by cAMP modulation [67,118,119]. Taken together these data
indicate that expression of HCN4 is a necessary element for proper
embryonic heart development, but unfortunately they do not provide
any hint on the role of the HCN4 current in the adult animal. In order
to overcome this limitation, temporally-controlled global [116], and
cardiac specific [117] HCN4 knockout models were developed. ECG
recordings in freely moving knockout animals showed the presence of
sinus pauses (mean duration = 321 ms, mean frequency = 8.1/min) in
otherwise normal rhythmic activity. Interestingly, β-modulation of
rate was not lost in the adult knockout animals, and any acceleration
in heart rate led to a reduction of sinus pauses. When the pacemaking
activity was investigated at the single cell level, it was found that 90%
of sinoatrial node myocytes of the global knockout and 50% of the
cardiac specific knockout were quiescent [116,117], and that the If
current was reduced by about 75%–80%. Interestingly, β-adrenergic
stimulation was able to rescue spontaneous activity in knockout cells
[116]. As pointed out by the same authors, the presence in vivo of a
sympathetic tone and of additional network properties may explain
the persistence of spontaneous activity in vivo, however it does not
fully explain the presence of sinus pauses [116].
The members of the HCN channel family were among the last ion
channel genes to be cloned; for this reason research aiming at
identifying a link between alterations in the HCN genes and cardiac
pathologies has only recently begun. During the course of independent screenings of patients affected by various forms of rhythm
disturbances, four mutations/deletions of the HCN4 open reading
frame have been identified and directly correlated to the phenotypes
[120–123]. Although all the identified mutations determine a reduced
contribution of the pacemaker If current to the diastolic depolarization
(loss-of-function), the functional mechanisms by which this reduction is achieved are quite different (Fig. 2).
The first evidence of a mutation of the HCN4 in humans was found
by Schulze-Bahr et al. [120] who reported the presence of the
heterozygous 1-bp deletion (1631delC) in a single patient affected by
idiopathic sinus node dysfunction (SND) with severe bradycardia
(41 beats per minute, bpm), intermittent atrial fibrillation, and
chronotropic incompetence. The resulting HCN4 protein (573X) had
a shorter C-terminus, lacking the CNBD, caused by the presence of an
earlier stop codon. In vitro heterologous expression of the 573X
channels revealed that the major alteration in the current was a
dominant negative loss of cAMP modulation, which could explain the
chronotropic incompetence of the patient both at rest and during
maximal workload [120]. Although these data are extremely intriguing, the causative relation between the altered function of the
channel and the pathological phenotype of the patient is not
conclusive since the study is based on a single-case report. In another
study Ueda et al. [121] reported the case of arrhythmic patients
affected by SND with recurrent syncope, severe bradycardia, QT
prolongation, polymorphic ventricular tachycardia. The study, based
on a two generation family, suggested a possible linkage between the
presence of the D553N mutation in the C-linker region and the disease
since members carrying the mutation (n = 3) also presented the
clinical phenotype. Patch-clamp and immunofluorescence analysis
showed that this mutation exerts a dominant negative effect and
severely impairs channel trafficking to the membrane leading to an
almost complete loss of the pacemaker current. Despite the strong
suggestion of a link between the mutation and the disease, the
Fig. 2. hHCN4 mutations associate to cardiac rhythm alterations. Schematic topology of
a single HCN4 subunit with the 6 transmembrane segments, and the N- and C-termini.
The C-terminal region is composed by the C-linker (A′ to F′ helices), the CNBD (A helix,
β-roll, B and C helices) and the proper C-terminus. Orange dots indicate the position of
the point mutations (G480R, D553N, and S672R), while the orange X indicates the site
where the mutant protein (573X), originated by the 1631delC, is truncated. Insets show
the main effects on If properties induced by the mutations. Top insets show bar-graphs
indicating the reduction in current densities observed for mutant G480R (at −100 mV)
and D553N (at − 120 mV) proteins (measured by eye from [121,123]). Bottom left inset
shows that cAMP does not modulate the position of the voltage-dependent curve of the
mutant 573X (reproduced from [120]), while bottom right inset shows that S672R
mutant channels display a negative shift of the voltage-dependent curve (reproduced
from [122]). For clarity data relative to homomeric channel expressions have been used.
F.A., fractional activation.
complexity of the clinical manifestations cannot be easily explained by
in vitro results.
A direct correlation between a heterozygous mutation and cardiac
rhythm alteration has been demonstrated for the S672R mutation
which induces a form of familial asymptomatic bradycardia [122]. An
extensive familial analysis carried out on 27 members allowed the
conclusion that the mutation co-segregated with the bradycardic
phenotype according to an autosomal dominant pattern (LOD score
N5). The mean heart rate of affected individuals (52.2 bpm) was
reduced by 29% when compared to unaffected members (73.2 bpm) of
the same family. In vitro heterologous expression of mutant and wild
type channels demonstrated that the half-activation voltage (V1/2) of
homomeric mutant channels was 8.4 mV (heteromeric channels:
4.9 mV) more negative than that of wild type channels; a slower
kinetics of deactivation was also observed. Despite the fact that this
mutation is localized in the CNBD region, cAMP retained its normal
M. Baruscotti et al. / Journal of Molecular and Cellular Cardiology 48 (2010) 55–64
modulatory action. Interestingly, the quantitative and qualitative
effects of the mutation (4.9 mV shift and 29% heart rate reduction)
completely resemble those of a low dose (30 nM) of acetylcholine.
This study not only identifies a tight linkage between the mutation
and bradycardia but it is also the first one that fully describes the
underlying molecular mechanism.
A form of familial sinus bradycardia was also found to co-segregate
with the mutation G480R which is located in the pore region of the
HCN4 channel [123]. In vitro studies seem to indicate both an extreme
negative shift (∼ 40 mV) of the voltage dependence and trafficking
defects.
Taken together studies on HCN4 channelopathies seem to suggest a
tight relation between the amount of current suppressed by the
presence of the mutation and the severity of the associated disease.
Indeed, while the limited reduction of the If current caused by the
S672R mutation only determines asymptomatic bradycardia, progressively more severe clinical manifestations appear with the deletion
1631delC which eliminates the cAMP modulation, and with the D553N
mutation which suppresses the presence of functional channels.
8. Biological pacemaker
The search of new therapeutic tools consisting of gene- and/or
cell-based intervention aimed to restore compromised cardiac
functions has prompted researchers to exploit the use of HCN
channels to alter cellular electrical activity in order to generate, in
normally quiescent substrates, stable rhythmic activity similar to that
of native pacemaker myocytes. The specific features of pacemaker
channels and in particular the fact that they are activated only at
diastolic potentials and do not contribute to other phases of the action
potentials, make them particularly suitable for such purpose. Early in
vitro studies demonstrated that virus-mediated over-expression of
HCN2 channels induced a significant increase in the rate of
spontaneously beating neonatal ventricular myocytes by causing an
If-mediated increase of the diastolic depolarization slope [93]. This
approach was later confirmed in vivo by showing that direct injection
of the HCN2-adenovirus in the left atrium or into the ventricular
conduction system of dogs, was able to induce ectopic regular
spontaneous activity after AV block [93,124–126]. Similarly, adenovirus-mediated over-expression of HCN1 or HCN4 was sufficient to
induce a regular rhythm in quiescent cardiomyocytes [127,128].
Alternative cell-based strategies, aimed to avoid the use of viruses,
have been developed by engineering cells in order to express high
levels of HCN channels. Engineered human mesenchymal stem cells
(hMSCs) expressing either HCN2 or HCN4 have been shown in vitro to
properly connect to neonatal cardiomyocytes and to increase their
intrinsic spontaneous rhythm [129,130]. HCN2-expressing hMSCs
have also been successfully transplanted in canine left ventricular
wall where they were able to induce stable ectopic beats [129].
Furthermore, spontaneously beating heterokarion cells generated by
the fusion of HCN1 expressing lung fibroblast and ventricular
myocytes were shown to induce in vivo ectopic beats at the site of
injection [131].
A different approach to generate a biological pacemaker would be
to use a cellular substrate as close as possible to native pacemaker
myocytes. Both murine and human embryonic stem cells differentiate
into spontaneously beating cell aggregates (EBs) which contain
myocytes with functional and molecular properties typical of pacemaker cells [50,52–54,132–135]. Two separate studies have demonstrated that spontaneously beating portions of human EBs are able to
pace either cultures of neonatal rat cardiac myocytes in vitro or the
whole heart in vivo [136,137]. Although neither the If current nor HCN
expression was directly addressed in these studies, the increase in rate
by β-adrenergic stimulation and, more importantly, the decrease by
the f-channel blocker ZD7288 [136,137] strongly suggest a role of the
pacemaker current in the generation of the rhythmic activity.
61
9. f-channels blockers
The specific and restricted contribution of If to the generation and
modulation of the sinoatrial diastolic depolarization phase has long
made this current a crucial target for pharmacological applications. In
principle, a selective reduction of the If current should cause a slowing of
heart rate devoid of undesired side effects. For this reason, drugs able to
block f-channels are expected to have the potential for treatment of
heart diseases characterized by a deficiency of oxygen supply to the
working myocardium such as angina pectoris, heart failure and ischemic
heart disease. Several f-channel blockers, called specific “heart ratelowering” agents, have been developed and extensively characterized in
in vitro and in vivo studies. This family includes alinidine (ST567),
zatebradine (UL-FS49), cilobradine (DK-AH26), ZD-7288, and ivabradine (S16257) [19]. The effects of these agents on the action potential
parameters and membrane currents of pacemaker cells/tissue are
shown in Table 1. The only If blocker extremely selective at therapeutic
doses and with mild side effects is ivabradine [19]. The action of
ivabradine on native f-channels displays both a marked use-dependence, since the drug can only access its binding site when channels are
in the open state, and current-dependence since the direction of current
through the channel affects the stability of the binding [13]. The “usedependence” is the mechanism responsible for block accumulation
during repetitive channel opening–closing cycles with the possible
consequence that the higher the initial cardiac rate the more effective is
the ivabradine block [66]. Experiments investigating the affinity of
ivabradine for homomeric HCN channels do not reveal any substantial
isoform-specificity although the drug was shown to act with a higher
degree of co-operativity in binding to HCN1 than to HCN4 and native fchannels [138,139]. A striking difference however exists between HCN4
and HCN1 in the state-dependence of channel block: while ivabradine
behaves as an open-channel blocker of HCN4 and native channels, it can
only block HCN1 channels when they are in the closed state [138].
Currently, ivabradine is marketed for treatment of chronic stable angina
in patients with normal sinus rhythm who have a contraindication or
intolerance to β-blockers; clinical studies of patients with chronic stable
angina have shown that ivabradine acts as a pure heart rate-reducing
agent and has anti-ischemic and anti-anginal properties equivalent to βblockers and Ca2+ channel blockers and presents a good safety and
tolerability profile even during long-term treatment [140–143]. Mild
visual symptoms (phosphenes) were occasionally reported, but were
generally well tolerated [140,143]. Additional information comes from
results from a recent large clinical trial (BEAUTIFUL) which indicate that
ivabradine treatment of patients with stable coronary artery disease
(CAD) and heart rate ≥70 bpm can reduce the incidence of some CAD
outcomes such as hospitalization for myocardial infarction and coronary
revascularization [144,145].
Acknowledgments
This work was supported by grants from the Ministero dell'
Istruzione dell'Università e della Ricerca (Cofin 2007WB35CW) to MB
and by the European Union (Normacor) grant. We would like to thank
Prof. DiFrancesco for his helpful suggestions and discussions.
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