Journal of Molecular and Cellular Cardiology 46 (2009) 343–351
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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
Original article
Molecular composition and functional properties of f-channels in murine embryonic
stem cell-derived pacemaker cells
Andrea Barbuti ⁎, Alessia Crespi, Daniela Capilupo, Nausicaa Mazzocchi, Mirko Baruscotti, Dario DiFrancesco
Department of Biomolecular Sciences and Biotechnology, Laboratory of Molecular Physiology and Neurobiology, University of Milano, via Celoria 26, 20133 Milano, Italy
a r t i c l e
i n f o
Article history:
Received 17 September 2008
Received in revised form 14 November 2008
Accepted 3 December 2008
Available online 11 December 2008
Keywords:
Hyperpolarization-activated cation channel
Embryonic stem cells
Pacemaker channels
β-adrenergic receptors
Muscarinic receptors
f-channels
a b s t r a c t
Mouse embryonic stem cells (mESCs) differentiate into all cardiac phenotypes, and thus represent an
important potential source for cardiac regenerative therapies. Here we characterize the molecular
composition and functional properties of “funny” (f-) channels in mESC-derived pacemaker cells. Following
differentiation, a fraction of mESC-derived myocytes exhibited action potentials characterized by a slow
diastolic depolarization and expressed the If current. If plays an important role in the pacemaking
mechanism of these cells since ivabradine (3 μM), a specific f-channel inhibitor, inhibited If by about 50% and
slowed rate by about 25%. Analysis of If kinetics revealed the presence of two populations of cells, one
expressing a fast- and one a slow-activating If; the two components are present both at early and late stages
of differentiation and had also distinct activation curves. Immunofluorescence analysis revealed that HCN1
and HCN4 are the only isoforms of the pacemaker channel expressed in these cells. Rhythmic cells responded
to β-adrenergic and muscarinic agonists: isoproterenol (1 μM) accelerated and acetylcholine (0.1 μM) slowed
spontaneous rate by about 50 and 12%, respectively. The same agonists caused quantitatively different effects
on If: isoproterenol shifted activation curves by about 5.9 and 2.7 mV and acetylcholine by −4.0 and −2.0 mV
in slow and fast If-activating cells, respectively. Accordingly, β1- and β2-adrenergic, and M2-muscarinic
receptors were detected in mESC-derived myocytes. Our data show that mESC-derived pacemaker cells
functionally express proteins which underlie generation and modulation of heart rhythm, and can therefore
represent a potential cell substrate for the generation of biological pacemakers.
© 2008 Elsevier Inc. All rights reserved.
1. Introduction
The pacemaker of the heart is located in the sinoatrial node (SAN),
a region structurally and functionally different from the rest of the
myocardium. Its function is to generate spontaneous action potentials
and drive the heartbeat with a mechanism allowing fine tuning of
rate by the autonomic nervous system [1–3]. Sinoatrial myocytes and
autorhythmic cells in general (like embryonic ventricular myocytes)
exhibit action potentials with a slow diastolic depolarization, which
at the termination of an action potential drives the membrane
potential to the threshold for the next one. It is established that an
important role in initiating the diastolic depolarization and modulating its rate, hence the rate of spontaneous activity, is played by the
pacemaker If current [4,5]. If flows through Hyperpolarizationactivated Cyclic Nucleotide-gated (HCN) channels, the molecular
correlates of f-channels; four HCN isoforms have been identified so
far which are all expressed with different densities in different
regions of the heart [6–8].
⁎ Corresponding author. Tel.: +39 02 50314931; fax: +39 02 50314932.
E-mail address: [email protected] (A. Barbuti).
0022-2828/$ – see front matter © 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.yjmcc.2008.12.001
Evidence confirming the basic function of HCN channels in the
generation, development and modulation of pacemaker activity has
recently been provided in mice and humans [9–14].
The specific role of f/HCN channels in pacemaking makes their
properties essential in the development of new tools aiming to heart
rate control, such as the biological pacemakers [15]. Overexpression of
HCN channels can indeed provide a depolarizing stimulus sufficient to
induce, in vitro and in vivo, quiescent myocytes to beat spontaneously
[16–20]. An alternative approach proven to be effective in pacing the
heart has employed human embryonic stem cell-derived autorhythmic agglomerates [21,22]. Although the mechanism by which ES cells
can pace silent cardiac tissue has not been fully elucidated, it is long
known that ES cells can differentiate toward a cardiac pacemaker
phenotype [23–26].
While it is well established that ESC-derived cardiomyocytes
express the If current [23,26,27] and that HCN channels, whether
native or overexpressed, underlie generation of spontaneous activity
[28,29], a detailed analysis of the HCN isoforms expressed in ESCderived cells is still lacking. In this work, we have analyzed the HCN
composition of both spontaneously beating embryoid bodies (EBs)
and isolated autorhythmic cells. Further, we have analyzed the basic
properties of If, its involvement in pacemaking and its modulation by
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neurotransmitters and which type of G-protein-coupled receptors
underlie the If-mediated autonomic response.
2. Materials and methods
2.1. Cell culture
Mouse ES cells (D3 line, ATCC) were cultured on a feeder layer of
mitomycin C (0.01 mg/ml, Sigma) -treated mouse embryonic
fibroblasts (STO, ATCC). ES cells culture, differentiation and isolation
protocols are detailed in the online Supplementary Methods.
2.2. Electrophysiology
Spontaneous action potentials and If current were recorded by the
patch-clamp technique in the whole-cell configuration. For details on
solution and for data analysis, see Supplementary Methods.
2.3. Immunofluorescence and video-confocal analysis
For immunofluorescence experiments, EBs, and single cells, at
various stages of differentiation were plated onto coverslips coated
with poly-L-lysine solution (0.01% v/v, Sigma). For details see
Supplementary Methods. Fluorescence staining was analyzed by
Video Confocal microscopy (ViCo Nikon). Control experiments with
secondary antibodies only were carried out for all types of antibodies
used, and resulted in no staining.
3. Results
It is known that ESCs can differentiate spontaneously into cardiac
myocytes with electrical properties typical of either the working
myocardium or the conduction system [23–25]. In order to characterize the functional and molecular features of autorhythmic, pacemakerlike cells derived from murine ESC, we induced cell differentiation by a
procedure based on the formation of compact cell aggregates known as
embryoid bodies (EBs), as described in the Methods. During the
differentiating process, portions of the EBs started to contract
spontaneously, suggesting that some of the cells had differentiated
toward a cardiac pacemaker phenotype. The number of contracting EBs
increased with time and by day 7 + 7 of differentiation, around 80% of
EBs presented one or more foci of spontaneously contracting cells. The
number of contracting EBs started to decrease at day 7 + 13 and by day
7 + 22 only 20% of them was still beating (Fig. 1A). To study the electrical
properties of spontaneously active cells, we dissected and plated the
contracting portions of the EBs at various stages of differentiation
(from day 7 + 3 to day 7 + 20) [24]. In Figs. 1B, C the activity recorded
from a spontaneously contracting portion of an EB dissociated at day
7 + 8 is shown (expanded scale in the lower panel see also
Supplementary Online Video 1).
Repetitive action potentials were characterized by a pronounced
diastolic depolarization phase and the absence of a plateau phase,
distinctive features of action potentials of cardiac pacemaker
myocytes [1]. In n = 16 contracting portions of EBs, the mean
maximum diastolic potential (MDP) was −60.4 ± 1.8 mV and the
mean spontaneous rate was 264 ± 38 beats per minute (bpm). These
values are similar to those previously reported in isolated murine
sinoatrial myocytes [30,31].
HCN channels are the molecular correlates of native pacemaker fchannels, whose main role in underlying generation of diastolic
depolarization and control of spontaneous rate is well established [4].
Of the four known isoforms (HCN1-4), HCN1, HCN2 and HCN4
contribute to different degrees to the If current in heart, HCN4 being
the most highly expressed isoform in pacemaker SAN myocytes [32].
We checked for the presence in ESC-derived cells of the mRNA of
all four known HCN subunits. RT-PCR analysis revealed that
Fig. 1. Electrical activity of a spontaneously beating EB-derived cell aggregate. (A) Bar
graph plotting the mean fractions of contracting EBs at various times during
differentiation. Data are mean ± SEM with n varying from 3 to 13. (B) Top, spontaneous
and regular action potentials recorded from a cell cluster dissociated from a 7 + 8-day EB
(see also Supplementary Online Video 1) in control Tyrode solution at T = 36 °C. B,
bottom, the same recording plotted on an expanded time scale reveals a marked slow
“pacemaker” (or “diastolic”) depolarization phase between consecutive action
potentials.
undifferentiated ES cells expressed the mRNA of all HCN subunits;
we also found expression of all HCN subunits in EBs at day 7 + 8 of
differentiation (Supplementary Fig. 1).
Since RT-PCR analysis cannot quantify levels of mRNA, nor directly
correlate them with the level of protein expressed, we carried out
immunofluorescence experiments in whole EBs, at the various stages
of differentiation, using isoform-specific HCN antibodies. In order to
identify portions of EBs rich in myocytes, we co-labelled EBs with
antibodies against muscular/cardiac specific proteins such as caveolin
3. We found that HCN1 and HCN4 were the only HCN subunits
detectable at early (day 7 + 3 Figs. 2A, B), intermediate (day 7 + 8; Figs.
2C, D) and late stage (day 7 + 20, Figs. 2E, F) of differentiation. HCN
staining was detected exclusively in portions of EBs positive for
caveolin 3, except for the early stages where caveolin 3 was never
detected (Figs. 2A, B, right)
The HCN3 subunit was detected rarely and only in caveolin 3negative portions of EBs at day 7 + 8 (Supplementary Fig. 2B); we never
detected HCN3 in EBs at day 7 + 3 (data not shown) and at day 7 + 20
(Supplementary Fig. 2D). The HCN2 signal was never detected
independently of the differentiation stage (Supplementary Figs. 2A,
C). In control experiments aimed to verify antibody functionality, clear
staining was detected in CHO cells transiently transfected with the
mHCN2 isoform (Supplementary Fig. 3).
We evaluated the electrical properties of single spontaneously
active cells isolated from contractile portions of EBs from early (day
7 + 3 to 7 + 6), intermediate (day 7 + 7 to 7 + 10) and late (day 7 + 12 to
7 + 21) stage of differentiation. Figs. 3A–C illustrates the properties of
a representative autorhythmic cell (see Supplementary Online Video
2). Spontaneous action potentials recorded from this cell had a
pronounced slow diastolic depolarization typical of pacemaker cells
expressing If (panel A). To investigate the expression of If, we carried
out voltage clamp experiments by applying hyperpolarizing voltage
steps from the holding potential of −35 mV to the range −45/
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345
Fig. 2. HCN1 and HCN4 expression in whole EBs. Single confocal sections of whole EBs double-labelled with specific anti-HCN antibodies (left panels, red) and caveolin 3 (right panels,
green) at day 7 + 3 (A, B), 7 + 8 (C, D) and 7 + 20 (E, F) of differentiation. Nuclei were stained with DAPI. Calibration bars 40 μm. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
−125 mV. These steps elicited a large If component (conductance of
0.036 pS/pF, panel B) which was typically almost fully blocked on
hyperpolarization by 5 mM CsCl (panel C).
We next studied the expression of the various HCN isoforms at the
single cell level, using caveolin 3 as a muscular/cardiac differentiation
marker. In agreement with the whole EBs data, we found that HCN1
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Fig. 3. Spontaneous activity, If current and HCN distribution in isolated ESC-derived beating cells. (A) Spontaneous action potentials recorded from an isolated ESC-derived cell (see
Supplementary Online Video 2), displaying features of a typical SAN action potential. (B) Family of If traces recorded from the same cell during an activation curve protocol consisting
of voltage steps to the range − 45/−125 (20 mV increments) followed by a fully activating step to −125 mV. (C) If traces elicited by a two-pulse protocol to −75/−125 mV from a holding
potential of −35 mV before and during perfusion with 5 mM CsCl to block If. (D, E) Single confocal sections of isolated ESC-derived cells double-labelled with anti-HCN antibodies, as
indicated (D, red) and caveolin 3 (E, green). Nuclei stained with DAPI. Calibration bars 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
and HCN4 signals (Fig. 3D, red) were readily detected on the
membrane of cells expressing caveolin 3 (Fig. 3E, green); HCN3 was
detected in a few cells, none of which co-expressed caveolin 3, while
HCN2 was never detected in either caveolin 3 positive or negative cells.
Immunofluorescence experiments were then performed to check
whether the isoforms HCN1 and HCN4 were co-expressed in the same
cells by a double-staining procedure with anti-HCN1 and anti-HCN4
antibodies. The left panels of Figs. 4A to C show a portion of a 7 + 8 EB
in which both isoforms HCN4 (A, red) and HCN1 (B, green) were
detected; merging of the two signals (C, yellow) indicates that some of
the cells expressed both isoforms, while other cells expressed mostly
HCN4 or HCN1.
Similar experiments were then performed in isolated cells. A
representative cell (from a 7 + 12 EB) showing partial co-localization
(yellow) of HCN4 (red) and HCN1 (green) is shown in the right panels
of Figs. 4D to F. Taken together, these data show that in ESC-derived
myocytes, the only isoforms expressed up to detectable levels are
HCN4 and HCN1, i.e. the same isoforms expressed in the SAN of
different species [6–8,33,34].
In a series of similar experiments carried out onto undifferentiated
ES cells, we only detected HCN4 at a low level of expression in a small
fraction of cells (7 out of 64); accordingly, we did not record any
substantial If current in undifferentiated cells (n = 30, see also
Supplementary Fig. 4).
To evaluate the contribution of If current to spontaneous activity
we investigated the action of the highly selective f-channel inhibitor
ivabradine. In Fig. 5A, spontaneous action potentials recorded from a
single cell in control solution and during perfusion with 3 μM
ivabradine are shown. The drug slowed activity from 264 bpm to
174 bpm (−34%) by specifically reducing the steepness of the diastolic
depolarization. On average ivabradine reduced rate of ESC-derived
pacemaker myocytes by 24.9 ± 4.7% (n = 7).
Fig. 5B shows representative If traces recorded at −95 mV before
and during application of 3 μM ivabradine, when current reduction
had reached steady-state. On average ivabradine blocked If current by
50.0 ± 8.3% (n = 4).
The identification of HCN1 and HCN4 prompted us to investigate in
greater detail the kinetic properties of If recorded from isolated ESCderived autorhythmic cells. For all stages of differentiations, we found
that in some cells If activated with slow kinetics, while in others the
kinetics were clearly faster. In Fig. 6A representative recordings from
two such cells are superimposed after scaling.
A. Barbuti et al. / Journal of Molecular and Cellular Cardiology 46 (2009) 343–351
347
Fig. 4. Membrane co-localization of HCN4 and HCN1 isoforms. Left, confocal images of an EB double-labelled with anti-HCN4 (A, red) and anti-HCN1 (B, green) primary antibodies.
(C), merged images showing signal co-localization (yellow). Right panels, single sections of an isolated ESC-derived cell showing the specific signals for HCN4 (D, red) and HCN1 (E,
green) and after merging to see the extent of signal co-localization (F, yellow). Calibration bars 20 μm. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
Fig. 5. Action of ivabradine on spontaneous action potentials and If. (A) Spontaneous
action potentials recorded from an isolated ESC-derived cell before and during
superfusion of 3 μM ivabradine, when rate slowing had reached steady-state. (B) If
traces elicited by stepping to − 95 mV from a holding potential of −35 mV during control
and after steady state block by 3 μM ivabradine.
Plotting in Fig. 6B the time constants from all cells analyzed (n = 20)
suggested the presence of two separate families of cells with
activation constants converging toward two distinct curves, a fast(n = 10, open circles) and a slow-kinetic curve (n = 10, filled circles). The
mean time constant curves from these two families were significantly
different at all voltages (not shown, p b 0.05).
Although immunofluorescence data show three populations of
cells, this is not necessarily in contrast with the above kinetic analysis.
Our electrophysiological data show indeed that cells tend to cluster
around two distinct populations, with predominance of either the
slow or the fast kinetic component, but within each population there
is some variability in the time constants which suggests the existence
of cells with a mixed HCN1 and HCN4 expression.
The bar graph in Fig. 6C shows the fraction of cells with a fast(blank) or slow-activating (diagonal pattern) If current at various
stages of differentiation; while the fast If component is predominant
at early stages, the slow one predominates at later stages. The
existence of two distinct cell populations was further supported by the
difference between mean activation curves of cells from the same two
families, as shown in Fig. 6D. The mean half activation voltages (V1/2)
were −72.1 ± 1.3 mV (n = 14, open circles) and −79.4 ± 1.6 mV (n = 11,
filled circles) for cells with fast and slow activation kinetics,
respectively (significantly different, p b 0.05); inverse slope factors
were not significantly different (7.2 ± 0.5 mV and 8.0 ± 0.8 mV for fast
and slow If-kinetics cells, respectively). As shown in Fig. 6E, the V1/2 of
fast and slow If did not change significantly with the differentiation
stage. On the contrary, the If conductance density did change, increasing
from 0.013 ± 0.002 pS/pF to 0.049 ± 0.007 and 0.077 ± 0.009 pS/pF at
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Fig. 6. Two types of ESC-derived pacemaker cells expressing different If kinetics. (A) If
traces recorded at −75 mV in two different cells normalized to maximal size to
highlight the different activation kinetics. (B) Plot of the voltage dependence of time
constants recorded from cells with fast-activating (empty circles) and slow-activating
If (filled circles); lines are through points. (C) Bar graph showing the fraction of cells
expressing a fast- (blank) or slow-activating If (line pattern) at different stages of
differentiation. (D) Plot of mean activation curves for the fast-activating (empty circles)
and the slow-activating If (filled circles). Full lines represent best fittings by the
Boltzmann equation (see text for best-fitting parameters). (E) Plot of the mean V1/2 of
fast- and slow-activating If during differentiation. (F) Bar graph of the mean If
conductance density at different times; the asterisk indicate a significant difference by
one-way ANOVA.
early, intermediate and late stages of differentiation, respectively (Fig.
6F, significant, p b 0.05).
In isolated beating cells, we measured spontaneous rate as the
mean from at least 20 second recordings and averaged values for the
three stages of differentiation. Mean rates were 270 ± 67 bpm (n = 7),
292 ± 44 bpm (n = 10) and 281 ± 89 bpm (n = 4) at early, intermediate
and late stages of differentiation, respectively, and were not
significantly different (p N 0.05, one-way ANOVA). Since the observed
increase in If density should give rise to increased rate, we speculate
that development may also be associated with increased density of
other ion currents and specifically of K+ currents, which would
counteract the acceleratory action of If density increase.
Among cells dissociated at all stages, we found myocytes that fired
action potentials only when stimulated (see Supplementary Fig. 5A).
Action potentials from these cells differed substantially from those of
autorhythmic cells and looked more like those of atrial/ventricular
myocytes (mean resting potential = −73.5 ± 7.7 mV). Correspondingly,
there was essentially no If in these cells (mean conductance density
was 3.9 10− 4 ± 1.6 10− 4 pS/pF, n = 6; Supplementary Fig. 5B).
An important property of pacemaker cells is their If-dependent
rate modulation by autonomic neurotransmitters. β-adrenergic
agonists accelerate spontaneous rate by steepening the phase 4
depolarization through a positive shift of If current activation curve,
while muscarinic agonists slow rate by the opposite process [1,5].
To investigate this mechanism in ESC-derived autorhythmic cells,
we first checked if they responded to autonomic transmitters. Either
cell aggregates or single cells were superfused with the β-adrenergic
agonist isoproterenol (Iso, 1 μM) and/or the muscarinic agonist
acetylcholine (ACh, 0.1 μM) while recording spontaneous activity.
The activity of two representative cells recorded in the control
solution and during superfusion with Iso (left) or ACh (right) is shown
in Fig. 7A. Iso accelerated, and ACh slowed rate. In both cases the rate
change was due to a modification in the slope of the slow pacemaker
depolarization, where If plays an important role, with little or no
modifications of action potential shape and duration.
On average, Iso (1 μM) accelerated and ACh (0.1 μM) slowed rate by
50.0 ± 7.7% (n = 4) and 12.3 ± 5.5% (n = 4), respectively (see Supplementary
Online Video 3).
We next studied the effects of the same agonists on If kinetics in
the two populations of cells with either fast or slow If-activation
kinetics.
In Figs. 7B, C, typical fast- (B) and slow-activating (C) If traces
recorded at −75 mV are shown in control conditions and during
superfusion with Iso (1 μM, left) and ACh (0.1 μM, right). We measured
the shift of If activation curve induced by the agonists according to a
method described previously [35]. The two populations showed, on
average, significantly different responses to agonist stimulation. Iso
induced shifts of +2.7 ± 0.6 mV (fast, n = 7) and +5.9 ± 0.4 mV (slow,
n = 14) while ACh induced shifts of −2.0 ± 0.7 mV (fast, n = 6) and −4 ±
0.5 mV (slow, n = 10) (p b 0.05).
The response to autonomic neurotransmitters implies a functional
signal transduction pathway involving activation of specific G proteincoupled receptors. We therefore investigated if ESC-derived autorhythmic cells express the same types of receptors that in native
pacemaker myocytes initiate β-adrenergic and M2 muscarinic
modulatory pathways. Typical results of immunofluorescence analysis
of single cells performed with antibodies against β-adrenergic and
muscarinic receptors are shown in Fig. 8. We found that most caveolin
3-positive cells reveal a strong expression of β1 adrenergic (panel A)
and M2 muscarinic receptors (panel C), while β2-adrenergic receptors
are expressed less frequently and in general more weakly (panel B).
4. Discussion
We have investigated the molecular and functional properties of
the funny (If) current expressed in murine ESC-derived autorhythmic
myocytes.
The observation that pacemaker myocytes are formed during the
development of embryoid bodies was established in early studies of
embryonic stem cell differentiation. Pacemaker cells can be identified
by the fact that they generate spontaneous action potentials with
features similar to those of SAN cells, including the “pacemaker”
depolarization phase of the action potential [23–25]. In native
mammalian tissue, generation of pacemaker activity in the SAN is
Fig. 7. Modulation of rate and the If current by autonomic transmitters. (A) Spontaneous
action potentials recorded from representative cell aggregates in Tyrode solution (ctr)
and during perfusion with isoproterenol 1 μM (Iso, left) or acetylcholine 0.1 μM (ACh,
right). (B, C) Cells expressing fast- (B) or slow-activating If (C) were exposed first to Iso
1 μM (left) and then to ACh 0.1 μM (right); If traces recorded during hyperpolarization to
−75 mV from a holding potential of −35 mV.
A. Barbuti et al. / Journal of Molecular and Cellular Cardiology 46 (2009) 343–351
349
Fig. 8. Distribution of β-adrenergic and muscarinic receptors in single ESC-derived cells. (A-C) Single confocal sections of isolated ESC-derived cells labelled with anti-β1 (A, red) or
anti-β2 (B, red) adrenergic receptor antibodies (β-AR), or with anti-M2 muscarinic receptor antibody (C, red; M2-mAChR); cells were also co-imunolabelled with anti-caveolin 3
antibody (right panels, cav3, green). Nuclei stained with DAPI. Calibration bars 10 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
under control of funny channels [1,32], whose molecular correlates are
mainly HCN4 subunits, with a limited species-dependent contribution
of HCN1 and/or HCN2 subunits [6–8,33,34]. However the mRNAs of
the four known HCN isoforms are by no means exclusive of sinoatrial
pacemaker cells, and are differently distributed in various cardiac
regions tissues as well as in other excitable cells [36,37].
Our RT-PCR data indicate expression of all four HCN isoforms both
in undifferentiated ES cells and in differentiated EBs. Since EBs
recapitulate embryonic development, this result is not surprising; it is
known that cells derived from all three embryonic germ layers are
present in differentiating EBs, including neuronal cells and other cell
types which possibly express HCN channels [38–41].
There is some variability in the HCN expression in ESC-derived
myocytes according to previously published work. In one study, ESCderived cardiomyocytes were reported to express all four isoforms,
with HCN2 and HCN3 representing the main isoforms [29,42], while in
another study differentiating ESC were shown to express only HCN1
and HCN4 [43]. Variable results have also been published on the
expression of HCN isoforms in undifferentiated ESCs. According to one
report HCN1 and HCN4 are the major isoforms [43], while in another
report only HCN2 and HCN3 were found [44]; in this latter study a Cs+sensitive hyperpolarization-activated current was also recorded in
about 30% of undifferentiated ES cells.
In our study, PCR data indicated the presence of the mRNA of all
four HCN isoforms in undifferentiated cells. We failed however to
record any substantial If-like current in these cells, indicating lack of
functional channels correctly inserted into cell membranes. Lack of a
functional role of HCN channels in undifferentiated ES cells is
consistent with the observation that these cells have resting voltage
levels around −20 mV, at which no HCN channel is functional under
normal conditions. The mRNA of all HCN isoforms could simply reflect
the presence of a small number of cells undergoing early stages of
differentiation among the undifferentiated ones, which were detected
due to the high resolution power of the PCR technique. The
discrepancy between expression of mRNA and functional proteins
can also result from post-translational regulatory mechanisms such as
control by microRNAs (miRNAs); it is known for example that
expression of HCN2 is controlled by two muscle-specific miRNAs
(mir-I33 and miR-I), the latter controlling also expression of the HCN4
isoform [45,46].
Since RT-PCR data did not provide evidence discriminating
between HCN expression at various stages of ES cell differentiation,
we choose to investigate protein expression. It has been reported
recently that ESC-derived flk-1+ colonies display a diffuse staining for
HCN1 and HCN4 when differentiated into cardiomyocytes [28].
Our present data provide the first evidence for membrane
localization of HCN channels on the plasma membrane of isolated
ESC-derived cardiomyocytes and of whole EBs. We have found a clear,
membrane-delimited expression only of HCN1 and HCN4 isoforms in
caveolin 3-positive ESC-derived cardiomyocytes. Caveolin 3 is a
structural protein of muscular/cardiac-caveolae abundantly expressed
in SAN cells; we have recently shown that in isolated rabbit SAN
myocytes HCN4 co-localizes and interacts with caveolin 3 and that
this interaction is critically important for both proper channel
function and modulation [3]. Our present data support the hypothesis
that localization and interaction of HCN channels with caveolin 3 may
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be important during development/maturation of pacemaker myocytes. In the ESC-derived cells HCN3 was detected with a very low
frequency only in caveolin3-negative cells, while HCN2 was never
detected. Given that HCN2 is the predominant isoform in adult
ventricular myocytes [6], our results indicate that the level of
expression of this isoform is below the detectable threshold during
early stages of cardiac differentiation, in agreement with previous
reports [47].
HCN1 and HCN4 are the isoforms most highly expressed in the SAN
of various species [8,33,34]. HCN4 in particular is considered to be a
specific marker of pacemaker cells of the SAN, as inferred from in situ
hybridization experiments showing that this isoform is expressed
very early during cardiac embryogenesis and is confined to the
pacemaker region that will form the mature SAN, while it is absent
from atria and ventricles [48–50]. Correspondingly, there are several
indications for the role of HCN channels in pacemaker activity. For
example, mice specifically lacking cardiac HCN4 die during embryogenesis [9] or have an unstable heart rhythm when the gene is
reported to be turned off in adulthood according to recent work [10];
also, mutations of the HCN4 gene were reported to be linked to
rhythm disturbances [11,12] and have been shown more recently to
cause sinus bradycardia in humans [13,14]. Expression of HCN4 and
HCN1 in ESC-derived myocytes together with caveolin3 suggests
therefore that these cells have acquired a pacemaker-like phenotype
typical of SAN cells.
The presence of the If current in ESC-derived cardiomyocytes has
been reported previously [23,27]. Recently, the role of If in the
generation of spontaneous activity during development of embryoid
bodies has been investigated [28,29,51]. To assess a direct role of If in
the generation of spontaneous activity of ESC-derived cells we have
used the specific f-channel inhibitor ivabradine. The extent of
inhibition of If and rate reduction caused by ivabradine 3 μM were
similar to values reported in the SAN tissue and single SAN myocytes
[52–56].
We have also found two distinct populations of ESC-derived,
spontaneously beating cells expressing If currents with different
kinetic properties: in one cell type If activated relatively fast (τ = 0.5 s
at −75 mV) with a half-activation voltage of −72.1 mV, while in
another cell type it activated more slowly (τ = 1.6 s at −75 mV) with a
more negative half-activation voltage (−79.4 mV). Differences in If
kinetics between the two cell populations could arise from a variety of
factors, among which different subunit composition, specific interactions with ancillary subunits, differences in phosphorylative states
and in the basal concentration of cAMP. Clearly however, different
cAMP levels cannot be the only cause, since this would cause similar
shifting effects on activation curve and τ curve. On the contrary, while
V1/2 values differ by 7.3 mV (Fig. 6D), a much larger shift would be
necessary for superimposing the τ curves plotted in Fig. 6B
(approximately 25 mV on mean τ curves, data not shown).
The existence of two populations of pacemaker cells expressing
either fast- or slow-activating If has been previously reported in
myocytes isolated from murine SAN [30], which are also known to
express higher levels of HCN1 and HCN4 than HCN2 and HCN3 [7,8].
The comparison between our data and previous data suggests
therefore a close similarity between ESC-derived autorhythmic cells
and mature murine pacemaker cells concerning If expression.
An important characteristic of functional pacemaker cells is their
ability to interact with the modulatory pathways controlling heart
rate. Since in mature pacemaker cells a fundamental mechanism of
rate modulation involves the control of the If voltage activation range
by autonomic neurotransmitters [1,32], we checked if a similar
mechanism also operates in ESC-derived pacemaker cells. Indeed we
found that in these cells, too, isoproterenol and acetylcholine shift the
If activation curve to more positive and to more negative voltages, and
are correspondingly able to accelerate and slow spontaneous rate,
respectively.
Interestingly, cells with fast activating If had a significantly smaller
response to adrenergic and muscarinic agonists than cells with a slow
activating If. This, and the different activation kinetics illustrated in
Fig. 7, suggest that the two pacemaker cell populations identified
could correspond to cells with different levels of expression of the
HCN1 and HCN4 isoforms. It is indeed well established that HCN1
channels, when expressed individually, have faster kinetics and lower
cAMP sensitivity than HCN4 channels [32,57,58].
Comparing our data with data from HCN1/HCN4 channel expression
in heterologous system at 35 °C [59] indicates a close similarity between
the kinetics of the slow-activating If with those of homomeric HCN4
channels, and of the fast-activating If with those of heteromeric HCN1–
HCN4 channels. For example, time constants of activation at −75 mV are
1.57 and 0.5 s for slow- and fast-activating If and about 1.5 and 0.5 s for
HCN4 homomers and HCN1–HCN4 chimeric channels, respectively
[59]. Thus, our data suggest that ESC-derived cells with slow-activating
If express predominantly homomeric HCN4 channels, while cells with
fast-activating If express heteromeric HCN1–HCN4 channels.
In mature pacemaker myocytes, rate of spontaneous activity is
controlled by autonomic transmitters via modulation of the cAMPdependent pathway through specific membrane receptors, such as β1
and β2 adrenergic receptors and M2 muscarinic receptors. Our results
provide the first evidence for the expression of these receptors in ESCderived caveolin 3-positive myocytes.
We have recently shown that in rabbit SAN myocytes, β2-ARs are
largely responsible for the If-mediated increase in rate, while β1-ARs
activation modulates rate and the If current to a lesser extent [3].
According to our present data in ESC-derived myocytes, on the other
hand, β2-ARs appear to be expressed at a low level and in only a few
cells. This agrees with previous observation that the isoproterenolinduced acceleration of rate in spontaneously beating EBs is
diminished upon perfusion of the selective β1-AR antagonist CGP
20712A, but is unchanged upon perfusion of the specific β2-reverse
agonist ICI 118,551[60].
In conclusion, our results indicate that ES cells, even at very early
stages, differentiate into cardiomyocytes which comprise a subpopulation of cells with functional and molecular characteristics typical of
cardiac pacemaker cells. This includes ion channels required for
pacemaker activity, as well as the biochemical pathways needed for
neurotransmitter-mediated frequency modulation. Following proper
enrichment through more stringent culture and/or differentiating
conditions aimed to increase the yield of selected pacemaker
myocytes, isolation of these cells could provide a substrate suitable
for generation of ESC-based biological pacemakers.
Acknowledgements
This work was supported by European Union (Normacor), CARIPLO
2004.1451/10.4878 and MIUR-FIRB (RBLA035A4X) grants to DD.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.yjmcc.2008.12.001.
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