© 2001 American Heart Association, Inc.
Integrative Physiology |
If Current and Spontaneous Activity in Mouse Embryonic Ventricular Myocytes
From the Department of Circulation (K.Y., W.L., T.O., K. Kada, J.-K.L., K. Kamiya, I.K.), Division of Regulation of Organ Function, Research Institute of Environmental Medicine, Nagoya University, Japan and Department of Medical Physiology (T.O.), University Medical Center Utrecht, The Netherlands.
Correspondence to Kenji Yasui, MD, Department of Circulation, Division of Regulation of Organ Function, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. E-mail kenji{at}riem.nagoya-u.ac.jp
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Abstract |
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Abstract—Knowledge of the initiation of electrical and contractile activity in the embryonic heart relies to a large extent on data obtained in chicken. In recent years, molecular biological techniques have raised an interest in mouse physiology, including early embryonic development. We studied action potentials and the occurrence of one of the pacemaker currents, If, by the whole-cell voltage and current-clamp technique at the earliest stage at which a regular heartbeat is established (9.5 days postcoitum) and at 1 day before birth. We show, first, that at the early stage there is a prominent If in mouse embryonic ventricles, which decreases by 82% before birth in concert with the loss of regular spontaneous activity of ventricular cells. Second, the decrease in If current is associated with a slight change in channel gating kinetics and a decrease in total mRNA expression of the genes encoding for If current. Third, the most prevalent mRNA subtype is switched from HCN4 to HCN2 during the second half of embryonic development. Fourth, the If current may be modulated by the ß-adrenergic cascade, although the coupling to the ß-adrenoceptor in the sarcolemma itself is not yet mature. We conclude that If current of the sinus node type is present in early embryonic mouse ventricular cells. In association with a loss of If current, the ventricle tends to lose pacemaker potency during the second half of embryonic development.
Key Words: embryo • development • If current • HCN gene • ß-adrenoceptor modulation
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLimitationsReferences |
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Knowledge about the onset of electrical activity in the embryonic heart is restricted to chicken, and information available for mammalian species is still limited.1 2 3 4 Recent progress of genetic engineering has raised an interest in the early development of the mouse heart. It is not known when first electrical activity occurs, although first contractions may be seen at 8.5 days postcoitum (dpc), with regular beating at 9 dpc.5 The order by which membrane currents appear in the embryo has started to be determined from studies in mouse embryos6 and in cell cultures derived from mouse embryonic stem cells.7 The slow inward calcium current (ICa-L) has been demonstrated at 9.5 dpc8 and increases steadily until birth at 19 dpc,6 8 whereas the fast inward sodium current (INa) becomes prominent at a later stage.6 With respect to the repolarizing currents, the transient outward current (Ito) develops first6 7 with higher atrial than ventricular density.6 Other outward potassium currents develop later with different regional densities.6 Thus, cells at 11 to 13 dpc depend on ICa-L for the upstroke and on Ito for repolarization of their action potentials, in line with the observation that these currents also develop first in cells derived from mouse embryonic stem cells.7 However, it is not clear which currents are responsible for diastolic depolarization in embryonic cells.
The membrane current If plays a prominent role in pacemaking in the adult sinus node and Purkinje system.9 10 If at the pacemaker potential range has been shown to be enhanced by catecholamine, whereas it is attenuated by acetylcholine via positive or negative shifts of its activation curve.11 Whatever the role of If for basic pacemaker function,12 it is at least important for the autonomic modulation of heart rate. The pacemaker current If has not been investigated in cells from mouse embryos. Because this current is present in early pacemaker cells derived from mouse embryonic stem cells and because all cells with If current showed spontaneous activity,7 we investigated whether this current is associated with early electrical activity in the mouse embryo. The genes encoding for If current (HCN1 through HCN4) were analyzed by mRNA measurement.
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Materials and Methods |
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Isolation and Culture of Cardiac Myocytes
Single myocyte cultures were prepared from ventricles of 9.5- and 18-dpc mouse embryonic hearts by methods previously described.8 Briefly, pregnant mice were killed, and ventricles were dissected from the exposed embryos. All animal procedures were approved by the Animal Care and Use Committee, Research Institute of Environmental Medicine, Nagoya University. After incubation in collagenase-containing solution, single myocytes were isolated. Myocytes were cultured on collagen-coated glass coverslips in MEM including 10% FBS and 10 µg/mL gentamycin for 18 to 24 hours before current recording.8 To exclude important effects of rundown of channel density during the culture period,13 14 15 we also performed experiments 2 to 4 hours after plating at both the 9.5- and 18-dpc stages.
Electrophysiological Experiments
A coverslip with attached ventricular myocytes was
placed in the chamber of an inverted microscope and superfused with
normal Tyrode’s solution containing (in mmol/L) NaCl 146.9, KCl 5.4,
CaCl2 1.8, MgCl2 0.5,
NaH2PO4 0.33, and HEPES 5
(pH 7.35). Whole-cell patch clamp was performed using Axopatch 200B
(Axon Instruments). Internal solution contained (in mmol/L) KOH 60, KCl
80, aspartate 40, HEPES 5, EGTA 10, MgATP 5,
Na2-phosphocreatinine 5, and
CaCl2 0.65 (pH 7.2, pCa 7.96). Action potentials
were recorded from 9.5- and 18-dpc ventricular myocytes in a
current-clamp mode in normal Tyrode’s solution. To study
If
current, the external solution was modified to reduce the interference
of other components by adding to the normal Tyrode’s solution (in
mmol/L) BaCl2 2, NiCl2 2,
and 4-aminopyridine 0.5. In some experiments, 30 µmol/L tetrodotoxin
was added to the solution. The amplitude of
If was
determined as the difference between the instantaneous current at the
beginning of hyperpolarizing pulse and the steady-state current
recorded at the end of the pulse and normalized to cell capacitance.
Cell capacitance was measured by the application of a ramp voltage
pulse of 0.5 V/s at a potential ranging between -50 and +70
mV.
The If activation curve was constructed from the amplitude of time-dependent inward current during hyperpolarizing pulses (-140 to -60 mV) from a holding potential of -50 mV. The values were normalized by the If amplitude at -140 mV and fitted by the Boltzmann equation: If=1/{1+exp[(Vm-V1/2)/k]}, where Vm is the membrane voltage, V1/2 is the voltage at half-maximal activation, and k is the slope factor. All experiments were carried out at 37°C.
Reverse Transcriptase–Polymerase Chain
Reaction and Identification of HCN Subtype
Total RNA of heart (ventricles) or brain was
extracted with RNeasy Mini Kit (Qiagen) from 9.5-dpc mouse embryo and
acid guanidinium thiocyanate-phenol-chloroform method from 18-dpc mouse
embryo and adult mouse. Single-stranded cDNA synthesis was performed
with total RNA using oligo d(T) primer and SuperScript II RNase H
reverse transcriptase (Gibco BRL).
Polymerase chain reaction (PCR) was carried out by 2
different methods. The GAPDH gene was used as an endogenous control.
The first method was to obtain the PCR product for all HCN1 through
HCN4 genes by using degenerated primers
(Table 1
) and AmpliTaq Gold (Roche Molecular Systems, Inc).
For identification of the HCN subtype, the PCR product of degenerated
primers was digested by specific restriction enzymes
(ClaI for HCN1,
ScaI for HCN2,
MluI for HCN3, and
Tth111I for HCN4).
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The second method is to quantify the expression of each HCN
gene by a real-time fluorogenic 5'-nuclease PCR assay (Perkin-Elmer ABI
Prism 7700). The respective primers and TaqMan probes are listed in
Table 1
. The threshold cycle (Ct)
from the baseline to reach a statistically significant increase in
fluorescence signal was measured. The Ct value
predicts the quantity of target cDNA in the sample. PCR products for
HCN1 through HCN4 genes were subcloned using TA cloning (pGEM-T Easy,
Promega) and were verified by sequencing. cDNA standards were obtained
by digesting plasmid by EcoRI.
Five different molecules of cDNA standards for HCN1 through HCN4 genes
(1x107, 1x106,
1x105, 1x104,
1x103) were amplified to determine the
linear relationship between Ct and log starting
molecule number of cDNA standards. Slope factors of HCN1, HCN2, HCN3,
HCN4, and GAPDH were -3.283, -3.355, -3.382, -3.582, and
-3.525 cycles/log decade, respectively.
Data Analysis
Electrophysiological data were analyzed using
Clampfit of the pCLAMP program (Axon Instruments). Data are presented
as mean±SEM. Statistical analysis of data were performed using paired
and nonpaired Student’s t test
(patch-clamp data) or ANOVA (quantitative PCR). Differences were
considered significant at
P<0.05.
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Results |
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Action Potentials at 9.5 and 18 dpc
Recordings were made from 13 isolated ventricular cells of 9.5-dpc mice and 18 cells of 18-dpc mice. Representative records are shown in Figures 1A
and 1B. All 13 cells from 9.5-dpc mice showed
regular spontaneous activity with prominent diastolic depolarization.
In contrast, only 6 myocytes (33%) from the 18-dpc mice were
spontaneously active, albeit at irregular cycle length. Maximum
diastolic potential, maximum upstroke velocity, diastolic
depolarization rate, and cycle length are summarized in
Table 2. Spontaneous action potentials at 18 dpc had slower
diastolic depolarization rate and higher maximum upstroke velocity than
those at 9.5 dpc
(P<0.05).
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Identification of
If
Current
Figures 1C and 1D show membrane currents in response to
hyperpolarizing voltage steps from a holding potential of -50 mV to
test potentials ranging from -60 to -130 mV at 9.5 and 18 dpc. At
9.5 dpc, hyperpolarizing pulses induced substantial inward currents
(Figure 1C). Current density of the time-dependent current
was -7.9±4.0 pA/pF at -130 mV (n=11). At 18 dpc,
hyperpolarization-induced inward current recorded from ventricular
myocytes was minimal, with density at -130 mV of only -1.4±0.4
pA/pF, which constitutes an 82% decrease
(Figure 1D; n=6). There was no significant difference of cell
capacitance (28±3.6 pF at 9.5 dpc and 31±3.3 pF at 18 dpc). The
decrease in
If
current might be attributable to greater rundown during the 18 to 24
hours after plating in the cell cultures from the 18-dpc ventricles
than that in the cell cultures from the 9.5-dpc ventricles. Therefore,
we did 2 subsets of 8 experiments each in cell cultures of 2 to 4 hours
derived from 9.5- and 18-dpc ventricles. A similar reduction of
If
density (88%) from 9.5 dpc to 18 dpc was observed in the experiments
with earlier cell cultures.
Figure 2 shows the voltage dependence of
If
activation. The cells were hyperpolarized to various test potentials
(-60 to -140 mV) from the holding potential of -50 mV to elicit
the time-dependent inward current and then clamped to 10 mV to observe
positive tail currents. The duration of hyperpolarization pulses was
longer at less negative voltages, where the time-dependent inward
current activates slowly. Representative current traces at 9.5 and 18
dpc are shown in Figures 2A and 2B, respectively. The tail currents at
18 dpc are not presented, because they were interrupted by a
substantial inward
INa
despite the presence of 30 µmol/L tetrodotoxin. Normalized amplitude
of the time-dependent inward current was plotted as a function of the
hyperpolarizing potential
(Figure 2C). The activation curves were S-shaped, with a
threshold potential between -60 and -70 mV. Fitting with a
Boltzmann function yielded
V1/2 at
-89.5±2.7 mV with k of
12.3±0.9 mV at 9.5 dpc (n=6) and
V1/2 at
-88.9±3.6 mV with k of
7.8±1.1 mV at 18 dpc (n=6). There was no significant difference in
V1/2
between 9.5 and 18 dpc, whereas the slope factor
k was significantly higher at
18 than at 9.5 dpc
(P<0.05).
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, at 9.5 dpc; 
, at 18 dpc) were obtained from the time-dependent inward current amplitude during hyperpolarization and fitted by Boltzmann equation (C). V1/2 and k were -89.5±2.7 and 12.3±0.9 mV, respectively, at 9.5 dpc (n=6) and -88.9±3.6 and k=7.8±1.1 mV, respectively, at 18 dpc (n=5).
The reversal potential of
If of
ventricular myocytes at 9.5 dpc was measured at 2 different
extracellular potassium concentrations
([K+]o) (5.4 mmol/L
and 25 mmol/L)
(Figures 3A and 3B). After a hyperpolarizing pulse to -130
mV with maximal activation of inward current, the clamp potential was
changed in the range from -50 to +10 mV and the tail currents were
measured. The time-dependent inward current was larger at the higher
[K+]o.
Figure 3C shows the averaged current density of tail
currents as a function of tail pulse potential (n=4). The reversal
potential of the I-V
relationship was -24±1.5 mV at 5.4 mmol/L and -16±1.8 mV at 25
mmol/L [K+]o. The
slope of the I-V relationship
was steeper at 25 mmol/L
[K+]o. The
characteristics of the time-dependent inward current induced by
hyperpolarization in ventricular myocytes at 9.5 dpc are comparable to
those of
If found
in sinoatrial node and Purkinje
fibers9 10
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The hyperpolarization-induced inward current was blocked by Cs+, and its blocking action was voltage-dependent16 (data not shown). The application of 3 mmol/L CsCl completely abolished the current activated by the hyperpolarizing pulse, whereas the tail current at the more positive potentials than the reversal potential was unaffected. We conclude that If is well developed in mouse ventricular cells at 9.5 dpc but decreases by >80% before birth.
HCN Gene Expression in Mouse Embryonic Cardiac
Ventricles
The apparent loss of
If with
development was additionally substantiated by investigating mRNA
expression of HCN genes that encode
If
channels. In the first series of PCR experiments, the primers were
designed to amplify cDNAs of all HCN1 through HCN4 genes.
Figure 4A shows a strong signal in 9.5-dpc ventricles and a
weak signal in 18-dpc ventricles, with no expression in adult
ventricles. In adult brain, a strong band was detected in line with the
presence of the mRNAs of all HCN1 through HCN4 genes, as reported
previously.17 This suggests
that in mouse cardiac ventricles the expression of the
If
channel decreases with development. To identify the subtypes of the HCN
gene expressed in cardiac ventricles at 9.5 dpc, the PCR product was
digested by a restriction enzyme for each of them
(Figure 4B). In the adult brain, all HCN1 through HCN4 genes
were detected after the enzyme digestion. In 9.5-dpc cardiac
ventricles, the restriction enzyme for HCN4, but not those for HCN2 and
HCN3, digested the PCR product. The restriction enzyme for HCN1
produced a small effect. Similar findings were obtained in 2 other
experiments.
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In the second series of experiments, each subtype of HCN
genes in the ventricles of mouse embryo was quantified by real-time
PCR. The amounts of each subtype mRNA (normalized to GAPDH) are
summarized in
Figure 4C. Major HCN gene subtypes detected at 9.5 dpc were
HCN4 and HCN1 (HCN4>HCN1,
P<0.05), but HCN2 was also
detectable with minimal amount. The HCN genes detected at 18 dpc were
HCN2, HCN4, and HCN1, in order of their amounts (HCN2>HCN4 or HCN1,
P<0.05), although the total
amount was much less than that at 9.5 dpc. Thus, at 9.5 dpc, HCN4 was
the predominant subtype. At 18 dpc, HCN2 was more expressed than the
other subtypes. For HCN4 and HCN1, there was a clear-cut downregulation
between 9.5 and 18 dpc. The opposite was seen for
HCN2.
ß-Adrenergic Modulation of
If
If
increases in response to ß-adrenergic stimulation in adult
myocytes.11
Figure 5A shows how we tested ß-adrenergic modulation of
If in
9.5-dpc ventricular myocytes. From a holding potential of -50 mV, we
applied a hyperpolarizing pulse to -70 mV, followed by another
hyperpolarizing pulse to -130 mV. This protocol was also performed in
the presence of either 3 µmol/L isoproterenol, a ß-adrenoceptor
agonist, or 10 µmol/L forskolin, a direct activator of adenylate
cyclase. Obviously, isoproterenol did not affect maximum
If
current at -130 mV, although there was a minimal effect at -70 mV.
In contrast, forskolin (10 µmol/L) induced a larger increase of
If at
-70 mV, although it did not affect maximal conductance at -130 mV.
Isoproterenol increased the averaged current density at the end of the
step pulse to -70 mV from 0.94±0.25 to 1.10±0.35 pA/pF (+17%; n=6,
not significant). For forskolin we observed a significant increase from
1.44±0.14 to 2.34±0.25 pA/pF (+62%; n=5,
P<0.05).
Figure 5B shows the effect of isoproterenol (n=8) and
forskolin (n=5) on the activation curve, which was obtained from
normalized
If
amplitude during hyperpolarizing pulses. Isoproterenol shifted the
half-maximum activation voltage by 2 mV (not significant) in the
positive direction, whereas this shift with forskolin was 7.8 mV
(P<0.05). This suggests that
in mouse ventricular myocytes, the signaling cascade to stimulate
If
channel downstream from the adenylate cyclase is already evolved at 9.5
dpc, but that ß-adrenergic receptors or their transducing mechanisms
via G proteins are still immature.
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indicate control. Forskolin substantially increased If current at -70 mV. B, Shifts of If activation curve by ß-adrenergic agonists. Activation curves were obtained from the time-dependent inward current amplitude during hyperpolarization and fitted by Boltzmann equation. Right and left panels show activation curves in the absence ( |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionLimitationsReferences |
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In this study, we demonstrated substantial If current in spontaneously beating 9.5-dpc ventricular myocytes, whereas the current decreased by 82% in 18-dpc ventricular myocytes in association with the loss of regular spontaneous activity. As early as at the 9.5-dpc stage, forskolin dramatically increased If current at physiological voltage, although isoproterenol had only a minor effect.
If
in the Embryonic Heart: Presence and Decrease
In embryonic hearts,
If has
thus far only been demonstrated in chicken ventricular
myocytes,18 19
where it has a comparable density as in the present study. The current
has also been observed in cell cultures derived from mouse embryonic
stem cells.7 In a previous
study on mouse embryonic heart, however,
If was
not detected; no explanation was
provided.6
The decrease in density of If current that we observed during embryonic development of the mouse ventricles was previously also demonstrated in the chicken heart.18 19 Because the activation curve was more or less similar at 9.5 and 18 dpc, it is suggested that downregulation of the If channel underlies the 82% loss of current density. In the chicken heart, a change in the activation kinetics of the If current (negative shift of the activation curve) was reported in one study at room temperature,19 whereas in another study at 37°C,18 no change in activation kinetics was observed. We additionally demonstrated a decrease of mRNA expression of the If encoding genes during development. These findings together strongly suggest downregulation of the channel protein as explanation for the observed decrease in current density during the second half of embryonic development.
Cerbai et al20 observed that in rat ventricular myocytes during the neonate stage, If current decreased without a shift of the activation curve, and that the number of cells in which the current could be elicited decreased from nearly 100% at 1 to 2 days to only 32% at 28 days. In contrast, Shi et al21 reported a negative shift of the activation curve of If current with aging in rat ventricle from the neonate to the adult stage. They demonstrated HCN2 and HCN4 but no HCN1 gene expression, with a ratio of HCN2 through HCN4 of 4.7 in the neonate stage and 13.7 in the adult stage. The change in the balance of HCN gene expression may explain the more negative I-V relationship of If in ventricle22 23 24 compared with the sinus node.10 In the present study, we show that the isoforms of HCN gene expressed in mouse embryonic ventricle are HCN4 (dominant) and HCN1 at early stage (as in the rabbit sinus node) but that HCN2 is predominant at the late embryonic stage. The switching of the most predominant isoform from HCN4 to HCN2 during the embryonic development seems consistent with that observed in rat ventricle after birth.21 However, there was no appreciable negative shift of the If activation curve from the early to the late stage of mouse embryo.
ß-Adrenergic Modulation
We demonstrated that the
If
current at 9.5 dpc may be increased >60% at physiological membrane
potential by forskolin, which directly activates adenylate cyclase,
although the effect of isoproterenol was much smaller (17%). An even
larger increase in
If in
response to isoproterenol has been reported in 3-day-old chicken
cardiac myocytes.22 By and
large, both the appearance and regulation of
If
current are early events in embryonic development. We previously
demonstrated ß-adrenoceptor mediated modulation of the L-type
Ca2+ current at the same stage of
development of the mouse embryonic
heart.8
Implications
Early pacemaking starts in the sinus venosus in the
chicken heart even before the heart starts to
beat.1 In mammals, the
ventricles are the first contracting parts of the embryonic
heart.2 It is not obvious
whether this reflects early pacemaking in the ventricles or early
excitation-contraction coupling. We have shown previously that in the
mouse embryo, isolated ventricular myocytes beat faster than isolated
ventricles or intact hearts.8
This observation together with the present data on the sinus-node type
of mRNA of the genes encoding for
If in
the mouse embryonic ventricle raises the question of whether pacemaking
may start in the ventricle rather than in the sinus venosus in
mouse.
If has been demonstrated in ventricular myocytes of the adult rat,25 guinea pig,23 and dog24 but not in rabbit.21 The presence of the current in these adult cells probably is without functional consequence, because the threshold for activation seems to be far more negative from the normal resting membrane potential. Recently, it was demonstrated in the adult rat that the HCN2 mRNA isoform gene product probably encodes for this adult type of If channel.21 Interestingly, If current has been reported in ventricular cells of patients with ischemic26 27 or dilated cardiomyopathy27 and also in atrial cells of patients who underwent a coronary bypass graft procedure or open heart surgery.28 The relevance of this increase has previously been discussed.12 In the hypertensive rat, the If current has also been shown to increase as a function of the degree of hypertrophy caused by the hypertension,29 and this density becomes even larger if heart failure is involved.30 It has been speculated that this increase in If density in hypertrophy and heart failure may involve a recapitulation of a fetal gene program.26 27 28 29 30 Our data on If current in the mouse embryonic heart are relevant to this hypothesis. The threshold for activation of If in atrial28 and ventricular cells26 27 from human patients and in rats with hypertrophy29 or heart failure30 seems more negative than in the mouse embryonic heart (this study) but far more positive than in the normal adult rat,25 guinea pig,23 and dog ventricular myocyte.24 The molecular characterization of the type of HCN mRNA underlying the fetal If current and the upregulated If currents under pathophysiological conditions within the same species shall establish whether this fetal recapitulation hypothesis is correct.
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Limitations |
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In this study, we recorded If only by the ruptured whole-cell patch-clamp technique. We selected cells showing little rundown of If for data analysis. Still, we cannot neglect the possibility that rundown could distort the results.
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Acknowledgments |
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This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan. We thank Drs M. Takano and T. Ishii (Kyoto University, Japan) for technical advice for the molecular study.
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Footnotes |
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Original received December 14, 1999; resubmission received January 3, 2001; revised resubmission received February 6, 2001; accepted February 6, 2001.
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References |
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