Download Sympathetic innervation modulates repolarizing K currents in rat

Sympathetic innervation modulates repolarizing K1
currents in rat epicardial myocytes
QIN-YUE LIU,1 MICHAEL R. ROSEN,1 DAVID MCKINNON,2 AND RICHARD B. ROBINSON1
of Pharmacology and Pediatrics, College of Physicians and Surgeons of Columbia
University, New York 10032; and 2Department of Neurobiology and Behavior,
State University of New York at Stony Brook, Stony Brook, New York 11794-8661
1Departments
transient outward potassium current; inwardly rectifying
potassium current; nerve growth factor; potassium ion
of sympathetic innervation occurs concurrently with a decrease in action potential
duration (APD) in rat and dog ventricular muscle (32).
We have previously demonstrated that daily injection
of newborn rats for 10 days with nerve growth factor
(NGF) or NGF antibody (Ab), respectively, accelerates
or delays cardiac sympathetic innervation (29). In this
earlier study, alteration in the time course of cardiac
sympathetic innervation was confirmed using tissue
catecholamine determinations, tyrosine hydroxylase
assays, and the release of norepinephrine by tyramine
infusion. With this method, we previously found that
acceleration of the time course of innervation consistently shortens the electrocardiographic Q-T interval
and ventricular APD (29, 36).
K1 carries the major outward currents that determine repolarization, thereby controlling the duration of
the action potential. The progressive shortening of APD
POSTNATAL EVOLUTION
in rat heart during postnatal development (26) has
been ascribed to evolution of the transient outward K1
current (Ito ) and the inwardly rectifying K1 current (IK1;
see Refs. 37 and 38). In addition, K1 currents are
important targets for the actions of many neurohumors
and intracellular second messengers that modulate
repolarization (18). Therefore, we used protocols for
sympathetic modulation similar to those employed
previously (29) to determine the relationship between
sympathetic innervation and the electrocardiogram
and action potential and the K1 currents Ito and IK1 in
rat epicardial myocytes.
METHODS
Treatment of rats. The method of injection with NGF,
placebo, or Ab was described by Malfatto et al. (29). Littermate Wistar rats born in our animal care facility were
randomly separated into three groups (3–4 animals/group
per litter). Each rat was injected subcutaneously from days 0
through days 13–15 of life. One group was treated with 1.0 µg/day
NGF on days 0–5, 1.5 µg/day on days 6–10, and 2 µg/day on
days 11–15. A second group was administered 10 µl/day NGF
Ab on days 0–5, 15 µl/day on days 6–10, and 20 µl/day on days
11–15. The third group received normal saline (the solvent for
NGF and Ab) as a placebo. All solutions were administered in
a final volume of 100 µl. NGF and Ab were purchased from
Collaborative Research (Bedford, MA). Electrocardiograms of
the animals in the three groups were recorded on days 0, 3, 6,
9, and 11, as reported in previous studies, and manifested
comparable results (data not shown; see Refs. 29 and 36).
Preparation of myocytes. Epicardial myocytes were isolated
on days 13–15 using a method modified after Liu et al. (28) .
On the day of study, two to three rats were narcotized with
CO2 and decapitated, and the hearts were rapidly excised.
Only the apical two-thirds of the ventricles were used to avoid
contamination by atrial cells and the great vessels. The
ventricles were cut open, and the endocardium and septum
were removed. The remaining epicardium was washed three
times in a solution (dissociation solution) containing the
following (in mM): 110 NaCl, 5.4 KCl, 4 NaHCO3, 1.6
MgCl2 · 6H2O, 1.8 NaH2PO4, 20 N-2-hydroxyethylpiperazineN8-2-ethanesulfonic acid (HEPES), 5 glucose, 4 L-glutamine,
and 10 taurine. The epicardium was then chopped into small
pieces that were bathed in the dissociation solution, also
containing 3 mg/ml trypsin (Sigma Chemical, St. Louis, MO)
and 5 mg/ml bovine albumin (Sigma), and placed in a
Gyrotory water bath at 37°C for 15 min. The first incubation
solution was discarded to remove connective tissue and
debris. There were four successive changes of enzyme solution under these conditions, and the tissue was triturated
with a Pasteur pipette after each incubation. After the
remaining tissue pieces were removed, the enzyme solution
was collected and centrifuged. The cells were then resuspended using the dissociation solution containing 5 mg/ml of
bovine albumin. All data are based on studies of at least three
0363-6135/98 $5.00 Copyright r 1998 the American Physiological Society
H915
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Liu, Qin-Yue, Michael R. Rosen, David McKinnon,
and Richard B. Robinson. Sympathetic innervation modulates repolarizing K1 currents in rat epicardial myocytes. Am.
J. Physiol. 274 (Heart Circ. Physiol. 43): H915–H922, 1998.—
During postnatal development, sympathetic innervation of
the heart evolves, and repolarization accelerates. Our goal in
this study was to test whether sympathetic innervation
modulates the ion channels that regulate repolarization. We
studied action potentials and repolarizing K1 currents in
epicardial myocytes from rats in which sympathetic innervation was accelerated or delayed, respectively, by subcutaneous injection of nerve growth factor (NGF) or NGF antibody
(Ab) for the first 15 days of life. A placebo group was included
as well. Action potential duration (APD) to 90% repolarization was greater in the Ab (158 6 18 ms)-treated than the
NGF (106 6 10 ms)-treated animals (P , 0.05); the APD at
90% repolarization for the placebo group was intermediate
(125 6 30 ms). The transient outward (Ito ) and inward
rectifier (IK1 ) K1 currents were recorded in freshly dissociated
cells using the whole cell patch-clamp technique. Ito was
decreased in density at potentials positive to 140 mV in
Ab-treated rats when compared with rats treated with NGF
(P , 0.05). In addition, the inactivation curve of Ito in
Ab-treated rats was shifted 13 mV positive to that of NGFtreated rats. IK1 also decreased in the Ab-treated group
compared with the NGF group in the potential ranges of
2100 to 290 mV (P , 0.05). However, the channel transcript
abundance (RNA) in NGF-, Ab-, or placebo-treated rat hearts
did not differ. Our results suggest that sympathetic innervation contributes to the developmental differences in K1
currents and APD postnatally in the rat.
H916
SYMPATHETIC MODULATION OF REPOLARIZING K1 CURRENTS
of the prepulse voltage V, V1/2 is the voltage at which 50% of
the current is inactivated, and S is the slope factor. This
allowed calculation and comparison of the mean midpoint of
the inactivation curve (V1/2 ) and the steepness of the curve (S)
between the treatment groups.
Preparation of RNA. The lower two-thirds of the left and
right ventricle after 15 days of treatment with NGF, Ab, or
placebo was rapidly dissected out and quick-frozen in liquid
N2. Tissue samples were then homogenized in guanidinium
thiocyanate. Total RNA was prepared by pelleting the homogenate over a CsCl step gradient. All RNA samples were
quantified by spectrophotometric analysis.
Ribonculease protection assay. DNA templates for the
preparation of RNA probes were prepared as described previously (10, 11). In all cases, a significant amount of nonhybridizing sequence (<50 bp) was included in the probe to allow
easy distinction between the probe and the specific protected
band. The specificity of the assay was such that there was no
evidence for unwanted cross-reaction between any probe and
another nonspecific K1 channel transcript.
Ribonuclease (RNase) protection assays were performed as
described previously (10). For each sample point, 10 µg of
total RNA were used in the assay. A probe for the rat
cyclophilin gene (9) was included in the hybridization as an
internal control to confirm that the sample was not lost or
degraded during the assay. Five micrograms of yeast tRNA
were used as a negative control to test for the presence of
probe self-protection bands. RNA expression was quantified
directly from dried RNase protection gels using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
RESULTS
APD and membrane potential measurements. Current-clamp experiments were carried out using dissociated ventricular epicardial myocytes from rats treated
with NGF, Ab, or placebo. The cells were stimulated via
the patch electrode using suprathreshold constantcurrent pulses at 0.2 Hz. Figure 1 shows action potential records from three individual cells treated, respectively, with Ab, placebo, or NGF. The table in Fig. 1
shows the data from the three sets of cells. This limited
number of action potentials was sufficient to confirm
consistency with our previously reported results from
intact myocardium (29, 36), i.e., hearts from rats treated
with Ab have significantly longer APD at 90% repolarization (APD90 ) compared with NGF-treated animals,
and results from placebo-treated rats are intermediate.
Although the decrease that we report in maximum
diastolic potential in Ab-treated rats is not significant
with this small n value (Fig. 1, P . 0.05), it is of the
same magnitude as that described by us previously in a
larger series of animals, in which the change was
significant (36).
Ito. To test the role of Ito in determining APD in young
rats, we studied the effect of the Ito blocker 4-AP (0.5
mM) on APD of myocytes from rats treated with
placebo. We limited the concentration used in these
action potential studies because we had previously
demonstrated the importance of nonspecific effects of
high 4-AP doses on APD in a prior study (27). Because
0.5 mM 4-AP may not fully block Ito, these experiments
provide a lower limit on the contribution of Ito to APD.
Figure 2 demonstrates that 0.5 mM 4-AP reversibly pro-
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to four separate litters of animals. Only rod-shaped, quiescent cells were used for electrophysiological experiments. The
cells from the three treatment groups were morphologically
similar at the light microscopic level, although the Ab-treated
cells had a smaller mean membrane capacitance (see RESULTS).
Recording techniques. Epicardial myocytes were transferred to a 0.5-ml Lucite bath on the stage of an inverted
microscope (Nikon Diaphot; Nikon Instrument, Tokyo, Japan). The cells were allowed to settle onto a glass coverslip
coated with poly-L-lysine to facilitate adhesion of the cells to
the glass. The cells were superfused at a rate of 2 ml/min with
Tyrode solution (see below) for action potential recording. The
standard gigaohm seal, whole cell recording method (17) was
used to measure the action potentials and the ionic currents.
The voltage-current clamp circuit was provided by an Axopatch
1C patch-clamp amplifier (Axon Instruments, Foster City,
CA). Patch electrodes were made using borosilicate glass
capillary tubes and a micropipette puller (model P 80/PC;
Sutter Instrument, Novato, CA). The pipettes had a resistance of 3–4 MV. Data were sampled using pCLAMP software (5.5) and a TL1 analog-to-digital interface (Axon Instrument) and were stored on disk using a Dell computer. Data
were analyzed using pCLAMP 6.0. The experiments were
performed at 31°C.
Recording solutions. The action potentials were recorded in
Tyrode solution containing (in mM) 131 NaCl, 1.8 NaH2PO4,
18 NaHCO3, 5.5 dextrose, 0.5 MgCl2 · 6H2O, 4 KCl, and 2.7
CaCl2 and were gassed with 95% O2-5% CO2. The recording
pipette solution contained (in mM) 120 potassium aspartate,
30 KCl, 1 MgCl2 · 6H2O, 5 MgATP, 10 HEPES, and 5 glucose.
Ito was measured in a bath solution (5) containing (in mM)
144 N-methyl-D-glucamine chloride, 5.4 KCl, 1 MgCl2 · 6H2O,
2.5 CaCl2, 10 HEPES, and 0.3 CdCl2. N-methyl-D-glucamine
was used to replace Na1 in the bath solution, and CdCl2 was
employed to block Ca21 current. The pipettes were filled with
a solution containing (in mM) 140 KCl, 1 MgCl2 · 6H2O, 5
ethylene glycol-bis(b-aminoethyl ether)-N,N,N8,N8-tetraacetic acid (EGTA), 5 MgATP, 5 Na2-creatine phosphate, 0.2 GTP,
and 10 HEPES. In voltage-clamp experiments, 4-aminopyridine (4-AP) at a concentration of 2 mM was used to define Ito
as the 4-AP-sensitive current (5). In the action potential
studies, a lower concentration of 0.5 mM was employed to
avoid potential nonspecific actions of 4-AP (27).
The IK1 measurements were carried out in a bath solution
containing (in mM) 140 NaCl, 5.4 KCl, 2 MgCl2 · 6H2O, 10
glucose, 10 HEPES, 1.8 CaCl2, and 0.3 CdCl2. In some
experiments, BaCl2 (2 mM) was dissolved in this solution to
block IK1. The composition of pipette solution for IK1 was as
follows (in mM): 120 potassium aspartate, 30 KCl, 1
MgCl2 · 6H2O, 5 MgATP, 11 EGTA, 1 CaCl2, and 10 HEPES.
All solutions were filtered (0.22 µm) before use. pH was
adjusted to 7.2 with KOH for pipette solutions and 7.4 for
bath solutions with HCl. None of the data have been corrected
for liquid junction potential, which was determined to be 8 6
0.5 mV (n 5 4) and 4 6 0.4 mV (n 5 4) for IK1 and Ito solutions,
respectively.
Data analysis and curve fitting. Ito and IK1 were expressed
as current density. Curves were compared using nested
analysis of variance unless otherwise indicated in the text,
and individual data points were subsequently compared
using a Bonferroni t-test for multiple comparisons. The
significance level was determined at P , 0.05. In addition, the
inactivation curves for Ito in the three treatment groups were
fit by the Boltzmann equation, I 5 (Imax 2 Imin )/51 1 exp[(V 2
V1/2 )/S]6 1 Imin, where Imax and Imin are maximal and minimal
currents, respectively, I is the current expressed as a function
SYMPATHETIC MODULATION OF REPOLARIZING K1 CURRENTS
H917
Fig. 1. Action potentials recorded from epicardial myocytes of rats treated with nerve growth
factor (NGF) antibody, placebo, or NGF. Action
potentials in single cells were evoked at 5-s
intervals by a suprathreshold depolarizing current pulse delivered through the recording pipette. Action potential duration at 50% (APD50 )
and 90% (APD90 ) repolarization and maximum
diastolic potential (MDP) are displayed in the
table. APD90 in antibody-treated epicardial myocytes is significantly longer than the NGF group.
The placebo group is intermediate. Data obtained
from 4 litters.
To study Ito density, myocytes were initially perfused
with Tyrode solution to form a gigaohm seal and the
membrane ruptured. After equilibrium was reached
between the pipette and the cytoplasm (,5 min), cell
capacitance was measured. In cells treated with placebo, capacitance was 36 6 1 pF (n 5 39). This is the
same as the cell capacitance, 36 6 1 pF (n 5 54),
measured from control cells (without any treatment)
and the NGF-treated group (36 6 1 pF, n 5 67),
suggesting that the size of the myocytes in the three
groups is comparable. In contrast, the cell capacitance
was 32 6 1 pF (n 5 58) in the Ab-treated group (P ,
0.05), consistent with decreased myocyte size and suggesting that NGF Ab may delay cell development.
Once the cell capacitance was measured, the myocytes were perfused with Ito bath solution. As soon as
Na1 current disappeared, the full current-voltage (I-V)
relationship for Ito was recorded as presented in Fig. 4.
Data were normalized to the capacitance of each cell.
The highest Ito density was observed in epicardial
myocytes treated with NGF, whereas Ito density in the
Ab-treated group was the smallest (despite the smaller
capacitance). Using the current at 160 mV as the
maximum, we generated normalized I-V curves for
NGF and Ab (Fig. 4, inset). The curves superimposed,
indicating that the threshold for activation of this
current is not changed. The results suggest that an Ito
Fig. 2. Effect of 4-aminopyridine (4-AP) on
action potential duration in epicardial myocytes treated with placebo. Left: action
potential recorded from a single epicardial
myocyte before, during, and after administration of 0.5 mM 4-AP. Action potentials
were evoked as described in Fig. 1. Right:
table of APD90 values from a group of cells
before, during, and after 4-AP. APD90 was
significantly increased by 0.5 mM 4-AP,
and this effect was readily reversed on
washout. Data are expressed as means 6
SE. * P , 0.05 cf. control (paired Student’s
t-test); n value is 5 for control and 4-AP
and 3 for washout.
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longed the APD by 26% (P , 0.05), suggesting that Ito is
critically involved in action potential repolarization.
Because the action potential studies confirmed that
variations in Ito could potentially account for the differences in APD90 observed between NGF- and Ab-treated
animals, we next investigated the nature and the
kinetics of ventricular epicardial Ito in NGF-, Ab-, and
placebo-treated animals. Ito was measured as the peak
current relative to steady state at the end of a 250-ms
test pulse. To confirm that this was Ito, we first tested its
sensitivity to 4-AP. In these experiments, we employed
a higher concentration of 4-AP (2 mM) than in the
action potential studies, because the possible nonspecificity of the higher concentration is not a concern given
the control of external and internal solutions and
voltage provided by the voltage-clamp procedure. That
4-AP reduces Ito comparably under all three treatment
conditions is demonstrated in Fig. 3. Results from two
individual cells are shown in Fig. 3, top, for a test
voltage of 150 mV; one cell was from a rat treated with
NGF, and the other was from a rat treated with Ab. In
this and all subsequent experiments, the current was
measured as the peak current minus the steady-state
current. Mean data are provided in Fig. 3, bottom, and
clearly indicate that the transient current component is
indeed Ito in all three treatment groups, i.e., 4-AP block
was equivalent in the three groups.
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SYMPATHETIC MODULATION OF REPOLARIZING K1 CURRENTS
Fig. 3. Sensitivity of transient outward current (Ito ) to
4-AP in the different treatment conditions. Top: records
from 2 cells of animals treated with antibody or NGF.
Cells were depolarized to a test potential of 150 mV
from a holding potential of 280 mV. j, Control, i.e.,
before application of 4-AP; r, effect of 2 mM 4-AP. Ito
was almost completely blocked by 4-AP. Bottom: data
from groups of cells are expressed as means 6 SE at the
150 mV test potential. Percentage of inhibition was
used to compare the extent of blockade by 4-AP in the 3
treatment groups.
Fig. 4. Density change of Ito in epicardial
myocytes from rats treated with antibody,
placebo, and NGF. Cells were depolarized
to various potentials from a holding potential of 280 mV at a rate of 0.1 Hz. Current
(I)-voltage (V) relationship was plotted
after the currents were normalized to their
capacitance. Density was decreased in the
myocytes treated with antibody compared
with those treated with NGF at potentials
positive to 140 mV. * P , 0.05, antibody
group vs. NGF group. Placebo group did
not differ from the treated groups. Normalized I-V relations for NGF and antibody
(AB) are plotted in inset and superimpose,
indicating that the voltage dependence for
Ito activation was not shifted. Current at
160 mV was used as maximal current
(Imax ).
mV, and S was 8.6 6 1.0 mV (n 5 7). However, at the
normal resting potential of these myocytes (261 to 267
mV; Fig. 1), Ito is fully available under all treatment
conditions. Therefore, the data for voltage-dependent
inactivation demonstrate that the reduced Ito density
(Fig. 4) and the greater APD (Fig. 1) in the Ab group are
not due to a voltage shift in steady-state inactivation.
IK1. The method for determination of IK1 is depicted in
Fig. 6A. The voltage was stepped from 2100 to 0 mV
from a holding potential of 240 mV, evoking a timedependent inwardly rectifying current that decayed to
a sustained, apparently steady-state level at the end of
the 250-ms pulse. This current was completely blocked
by 2 mM Ba21. It manifests significant inward current
at negative potentials and marked inward rectification
at potentials positive to 280 mV. A small negative slope
was observed in some of the cells, which was also
reported by Wahler (37). Characteristics such as Ba21
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density change may explain, at least in part, the longer
APD in the Ab-treated animals.
We then constructed steady-state inactivation curves
(13) to determine whether or not the change in Ito
density in the three groups was in part due to an effect
of the treatment conditions on the voltage dependence
of inactivation of the channel (Fig. 5). In the placebo
group, the currents were almost completely inactivated
at around 110 mV, V1/2 was 223.3 6 0.8 mV, and S was
6.7 6 0.4 mV (n 5 5). Similar results were obtained
from the NGF-treated group as follows: V1/2 was 222.1 6
0.8 mV and S was 7.6 6 0.6 mV (n 5 5). In contrast, in
the Ab treatment group, the curve was shifted toward
more depolarized potentials and was not completely
inactivated at 110 mV. The 50% inactivation potential
was shifted to more positive potentials (13 mV, P , 0.05
cf. the NGF group). The calculated V1/2 was 29.7 6 1.5
SYMPATHETIC MODULATION OF REPOLARIZING K1 CURRENTS
H919
sensitivity, inward rectification, inactivation of inward
current, and negative slope indicate that the current is
indeed IK1 (Fig. 6A).
The current densities of both peak and steady-state
IK1 (defined as the Ba21 difference current) were compared (Fig. 6, B and C). At potentials of 2100 to 290
mV, both peak and steady-state IK1 were significantly
smaller in the Ab-treated group compared with that
treated with NGF. The values for placebo were between
the others. The slope conductance (fitted from the
linear portion of the curve from 2100 to 280 mV) was
significantly different. The slope conductance of peak
current for NGF versus Ab was 0.52 6 0.03 nS/pF (n 5
11) and 0.38 6 0.03 nS/pF (n 5 9); for steady-state
current, NGF versus Ab was 0.45 6 0.05 nS/pF (n 5 11)
and 0.28 6 0.03 nS/pF (n 5 9; P , 0.05). The reversal
Fig. 6. Determination of peak and steady-state inward rectifier K1 current (IK1 ) in an epicardial myocyte. A:
current records before (control) and in the presence of 2 mM Ba21. Difference records, i.e., Ba21-sensitive current,
are at right. Peak currents were measured at the beginning of the pulse, and the steady-state I-V relationship was
measured at the end of the pulse Vh , holding potential. B: I-V relationship of peak IK1 in the 3 groups. C: I-V
relationship of steady-state IK1 in the 3 groups. Currents were normalized to their capacitance. Myocytes in
antibody-treated group had the smallest density of both peak and steady-state IK1 when compared with NGF (* P ,
0.05). Placebo group did not differ from either treated group.
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Fig. 5. Effects of NGF and antibody on
steady-state inactivation of Ito. Protocol
used is shown in inset. A 500-ms prepulse to a range of potentials was followed by a 300-ms test pulse to a fixed
potential to elicit Ito. These paired pulses
were delivered at 0.1 Hz from a holding
potential of 280 mV. Amplitude measurements were normalized to the value
obtained at the most negative potential
in each experimental condition. Data
from groups of cells are expressed as
means 6 SE, and the curves are fit by
the Boltzmann equation. Voltage at
which 50% of the current is inactivated
(V1/2 ) and slope factor were calculated
from the equation described in METHODS. V1/2 of the inactivation curve from
the cells treated with antibody was
shifted 13 mV when compared with
NGF and placebo (* P , 0.05).
H920
SYMPATHETIC MODULATION OF REPOLARIZING K1 CURRENTS
cannot be ruled out at this stage, although IK is
relatively small in rats (38).
Studies of transcript abundance. It has been suggested previously that the voltage-gated K1 channels
(Kv) 4.2 and Kv4.3 underlie the Ito in rat heart (11). To
determine whether the changes in Ito levels that we
have observed in the NGF-treated and Ab-treated
animals could be produced by changes in gene expression, we examined the expression of Kv4.2 and Kv4.3
mRNA in rat heart (Table 1). No significant changes in
either Kv4.2 or Kv4.3 mRNA were observed in either
experimental group relative to the control.
The inward rectifier in heart is a ‘‘strong’’ inward
rectifier (20) that is thought to be encoded by members
of the inward rectifying K1 channal (Kir) 2.0 subfamily
of inward rectifier channels (12). There are three
members of this subfamily of inward rectifier genes
[Kir2.1, Kir2.2, and Kir2.3 (12)]. Both the Kir2.1 and
Kir2.2 mRNA are abundantly expressed in rat heart,
whereas Kir2.3 mRNA is not expressed (12). There was
no significant change in the level of Kir2.1 and Kir2.2
mRNA in either of the experimental groups compared
with control (Table 1).
DISCUSSION
In our previous work (29), we found that 1) norepinephrine level in heart is highest in NGF-treated rats
and lowest in Ab-treated rats when compared with
placebo; 2) tyrosine hydroxylase-positive material in
the ventricles of NGF-treated animals is greater than
in placebo-treated animals; and 3) positive chronotropy
in response to tyramine is greatest in NGF-treated
animals and smallest in Ab-treated animals. These
data, together, support our contention that NGF treatment increases and Ab attenuates the functional sympathetic innervation of the heart. The similarity in
APD measurements in our previous and present results confirm that treatment with Ab in these animals
is sufficient to retard the decrease in APD that is seen
with normal development and with NGF treatment. In
our earlier work, we also noted that the membrane
potential of Ab-treated rats was lower than that of
placebo- or NGF-treated rats. Both the lower memTable 1. Effects of NGF antibody and NGF on K 1
channel transcript abundance in the rat heart
Kv4.2
Kv4.3
Kir2.1
Kir2.2
Cyclophilin
Placebo
NGF
Antibody
NGF
100 6 2
100 6 7
100 6 2
100 6 2
99 6 2
92 6 11
100 6 23
100 6 2
90 6 5
100 6 4
103 6 15
97 6 4
101 6 5
99 6 8
101 6 2
Data are means 6 SE from 4 independent experiments with pooled
results from 6 to 8 animals for each experimental group. NGF, nerve
growth factor; Kv, voltage-gated K1 channel; Kir, inward rectifying
K1 channel. Data were normalized to the amount of cyclophilin
transcript, which was not significantly different between experimental groups. Values for the experimental groups are expressed as a
percentage of the placebo values. There was no statistically significant difference between either of the experimental groups and the
placebo group for any of the 4 mRNAs tested.
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potential (calculated from the steady-state curve) for
IK1 was 277.4 and 274.3 mV for Ab- and NGF-treated
myocytes, respectively (P . 0.05).
Predicted relation between repolarization of action
potential and Ito and IK1 current density. The current
density of Ito in Ab-treated rats is ,70% of that in
NGF-treated rats, as shown in the Ito I-V relationship
curves (Fig. 4). The difference between these two groups
is greatest as potentials become more positive and only
reaches statistical significance at 140 mV (Fig. 4), a
voltage not within the physiological range. A similar
consideration applies to the IK1 data. Nonetheless,
there are significant differences in repolarization of
action potentials between the two treatment groups
(Fig. 1). These observations led us to measure the slope
of action potential repolarization at two voltages, 0 and
240 mV, to represent ranges at which Ito and IK1,
respectively, would be expected to contribute to repolarizing current. The location of these voltages during
action potential repolarization can be appreciated by
examining Fig. 2, which displays a voltage axis to the
left of the action potentials. If the action potentials
generated are membrane action potentials, then the
current flowing across the cell membrane at any time
can be calculated according to the equation I 5 CdV/dt,
where C is capacitance; therefore, I/C 5 dV/dt. I/C is the
current density and the unit of measurement is pA/pF,
and dV/dt is the slope of action potential repolarization.
The predicted current density (calculated from the
action potential repolarization) around 0 mV is 3.15
pA/pF (n 5 5) in NGF-treated rats and 1.92 pA/pF (n 5
4) in Ab-treated rats. Therefore, the predicted current
density around 0 mV in Ab-treated rats is 61% that of
NGF-treated rats. The actual Ito current densities at 0
mV obtained from our studies are 2.01 (n 5 16) and 1.47
pA/pF (n 5 14) in NGF- and Ab-treated rats, respectively, indicating that the Ab-treated group has an Ito
density that is 73% of the NGF group. It has been
reported that Cd21 (100 µM) shifts the availability of Ito
positive by 10 mV (1). Because Cd21 was present in our
recording solution, we therefore also calculated Ito at
110 mV. The ratio of Ab and NGF is 77% (2.66 vs. 3.44
pA/pF). The good agreement between the current measurements and slope calculation suggests that the
difference in actual Ito density in these two groups
probably is a significant contributor to the observed
difference in early action potential repolarization.
A similar approach was taken to measure the slope of
repolarization around 240 mV to determine whether or
not IK1 is involved. The predicted current density for
repolarization around 240 mV is 0.7 pA/pF (n 5 5) in
NGF-treated rats and 0.26 pA/pF (n 5 4) in Ab-treated
rats (i.e., Ab current 5 37% of NGF current). The actual
measured IK1 densities at 240 mV are 1.24 (n 5 9) and
0.73 (n 5 11) pA/pF in NGF- and Ab-treated rats,
respectively, (i.e., Ab IK1 5 58% of NGF IK1 ). Again, the
reasonable agreement between the predicted and calculated changes in current density at 240 mV suggests
that IK1 may play a role in the developmental regulation of the later phase of repolarization of the action
potential. However, other K1 currents, such as IK,
SYMPATHETIC MODULATION OF REPOLARIZING K1 CURRENTS
and 10 and then remains constant through adulthood
(30, 37). Such developmental changes may reflect a
variety of influences on the current, including that of
the sympathetic nervous system. The changes seen in
IK1, in turn, could reflect differences in channel number,
conductance, and/or open probability (30, 37).
Changes in IK1 impact on the action potential by
influencing the resting potential (35) and the final
phase of repolarization (14, 19). Hence, an increase in
IK1 can shorten APD and hyperpolarize the membrane.
Although we did not observe a significant change in
maximum diastolic potential in our three treatment
groups, the membrane potential was lower in the Ab
than in the placebo or NGF groups. In an earlier report,
we also found a lower membrane potential in the Ab
group, consistent with the present observation of reduced IK1 (36). In this respect, the magnitude of membrane potential difference was comparable to that in
the present study, but the n was larger, and the result
was statistically significant.
Our data clearly suggest that the densities of Ito and
IK1 are lower in rats treated with NGF Ab than those
treated with NGF for the first 15 days of life and that
the reduced densities of Ito and IK1 may contribute to
the prolongation of APD. Our investigation of K1
channel transcript abundance in hearts from the three
treatment groups showed no significant differences in
message levels. Given the modest difference in current
seen among the three groups, this result is not surprising. The change in transcript level may have been
below our limit of resolution. Alternatively, it may be
that innervation or NGF regulates current level by
affecting the channel posttranscriptionally or otherwise modifying current density.
Finally, although we have focused on Ito and IK1, this
does not rule out additional changes in other currents
during development and cardiac innervation, which
thereby impact on action potential configuration. For
example, it has been reported that L-type Ca21 current
density increases developmentally in the rat ventricle
(15), and nerve-muscle coculture experiments suggest
that sympathetic innervation may play a role (31).
However, due to additional developmental changes in
channel kinetics (as measured in rabbit), the contribution of the current during normal activity may not
increase and may even decrease with age (39). Another
current worthy of future consideration is the Na1/Ca21
exchange current, which has been reported to contribute an inward current that would slow terminal repolarization in the rat ventricle (34). Furthermore, the
message levels of the Na1/Ca21 exchanger decrease
developmentally in both rat and rabbit as the sarcoplasmic reticulum develops (4), which would be consistent
with a developmental reduction in the slowing of
repolarization and thus a shorter APD in the adult
ventricle. However, this developmental decrease in
message level of the exchanger has been attributed to
the postnatal surge in thyroid hormone level (3) rather
than the ontogeny of sympathetic innervation.
We express gratitude to Dr. Ira Cohen for thoughtful comments
during the performance of these studies and for critique of the
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brane potential and the long Q-T interval seen may be
explained, at least in part, by the differences we have
found in the density of IK1 and Ito.
The current that we identified as Ito was recorded in
Na1-free solution and in the presence of Ca21 channel
blockade, with 0.3 mM CdCl2. It was an outward current
with a time-dependent inactivation consistent with an
earlier description of Ito (23). The external application
of 2 mM 4-AP rapidly reduced the peak amplitude of
the inactivating component of the outward current
without significantly affecting the sustained current
component, as observed in other studies (5, 6). The
4-AP sensitivity of this current further confirms that it
is Ito. That Ito is the major repolarizing current responsible for the alteration of APD after injection of NGF or
Ab to the neonatal rat is suggested by the following: 1)
there is a lower Ito density in Ab-treated rats than
NGF-treated animals; 2) the magnitude of reduction at
0 mV is consistent with the change in repolarization
slope at this voltage; 3) APD of epicardial myocytes
from rats treated with placebo is prolonged by low
concentrations of 4-AP, which preferentially blocks Ito;
and 4) Ito plays an important role in the shortening of
APD in rat heart during development (38).
That Ito density increases severalfold during the
postnatal development of heart has been reported in
freshly dissociated rat ventricular myocytes (38), cultured rat ventricular myocytes (25), and human atrial
myocytes (8). Jeck and Boyden (22) reported that
neonatal canine ventricular myocytes completely lack a
definable Ito until ,2 mo of age. Hence, the majority of
earlier studies are consistent with a developmental
increase in Ito, the timing of which roughly parallels
that of sympathetic innervation. This is consistent with
our results with NGF and Ab, although we did not see
the severalfold change in Ito density reported developmentally by others. This may be due to the fact that
NGF and its Ab modulate sympathetic development
only to a degree and/or that sympathetic innervation,
in its own right, may be a modulator rather than the
prime determinant of Ito changes during development.
No shift of Ito activation was observed by us. However, the Ito inactivation curve shifted toward more
positive potentials in the Ab-treated groups, which is in
contrast to reports of no change in inactivation during
development in human atrial (16) and rabbit ventricular myocytes (33). This could arise from species differences and/or regional variations in the ventricular wall
(2, 7) and/or a nonspecific action of Ab. However, this
shift is unlikely to impact on the contribution of Ito to
action potential repolarization, since, in all treatment
groups, the inactivation was fully relieved at the resting potential.
That the density of peak and steady-state IK1 was
smaller in the Ab-treated animals than in animals
treated with NGF suggests a role for sympathetic
innervation in the evolution of the current. This finding
is consistent with reports that IK1 increases developmentally in embryonic chick ventricular myocytes (24),
young rabbit ventricular myocytes (21), and freshly
isolated rat ventricular cells between neonatal days 3
H921
H922
SYMPATHETIC MODULATION OF REPOLARIZING K1 CURRENTS
manuscript. We also express gratitude to Drs. Irina Golyakhovsky
and Natalia Egorova for assistance in the performance of the
experiments and to Eileen Franey for careful attention to the
preparation of the manuscript.
Address for reprint requests: R. B. Robinson, Dept. of Pharmacology, College of Physicians and Surgeons of Columbia Univ., 630 West
168 St., PH 7W-318, New York, NY 10032.
Received 3 April 1997; accepted in final form 1 December 1997.
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