Vol. 277, Issue 1, H211-H220, July 1999
Electrophysiological mechanisms by which hypothyroidism delays
repolarization in guinea pig hearts
Ralph F.
Bosch1,
Zhiguo
Wang1,2,
Gui-Rong
Li1,2, and
Stanley
Nattel1,2,3
1 Department of Medicine and
2 Research Center, Montreal Heart
Institute and University of Montreal, Montreal H1T 1C8; and
3 Department of Pharmacology
and Therapeutics, McGill University, Montreal, Quebec, Canada H3G 1Y6
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ABSTRACT |
Thyroid hormone is known to exert important
effects on cardiac repolarization, but the underlying mechanisms are
poorly understood. We investigated the electrophysiological mechanisms
of differences in repolarization between control guinea pigs and
hypothyroid animals (thyroidectomy plus 5-propyl-2-thiouracil).
Hypothyroidism significantly prolonged the rate-corrected Q-T interval
in vivo and action potential duration (APD) of isolated ventricular
myocytes. Whole cell voltage-clamp studies showed no change in current
density or kinetics of L-type Ca2+
current, inward rectifier K+
current, or Na+ current in
hypothyroid hearts. Dofetilide-resistant current
(IKs) step
current densities were smaller by ~65%, and tail current densities
were reduced by 80% in myocytes from hypothyroid animals compared with
controls. The ratio of delayed rectifier step current at +50 mV to tail
current at 
40 mV was significantly larger in hypothyroid cells
for test pulses from 60- to 4,200-ms duration, reflecting a smaller
IKs.
Dofetilide-sensitive current
(IKr) densities were not significantly changed.
IKs
half-activation voltage shifted to more positive voltages in
hypothyroidism (29.5 ± 2.2 vs. 21.3 ± 2.7 mV in control,
P < 0.01), whereas
IKr voltage
dependence was unchanged. We conclude that hypothyroidism delays
repolarization in the guinea pig ventricle by decreasing
IKs, a novel and
potentially important mechanism for thyroid regulation of cardiac electrophysiology.
electrocardiogram; action potential; biophysics; cardiac
arrhythmias; antiarrhythmic drugs; ion channels
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INTRODUCTION |
THYROID HORMONES DISPLAY a variety of
potentially important effects on cardiac electrical function. These
include a positive chronotropic and inotropic effect and a shortening
in repolarization (22). Hyperthyroidism is a clinically important cause
of atrial fibrillation, almost certainly caused in large measure by
accelerated atrial repolarization. A typical electrocardiographic
feature in hypothyroid patients is a marked prolongation of the Q-T
interval (32), reflecting delayed ventricular repolarization.
Experimental hypothyroidism prolongs the Q-T interval (33) as well as
action potential duration (APD) measured with fine-tipped
microelectrodes (11, 28).
The ionic and molecular mechanisms of thyroid hormone effects on
repolarization remain poorly understood. Rubinstein and Binah (4, 23)
found that, although hypothyroidism increased guinea pig ventricular
APD by 31-44%, the only associated change in membrane current was
a 41% reduction in L-type Ca2+
current (ICa)
amplitude, which should, if anything, have accelerated repolarization.
Hypothyroidism has been found to decrease transient outward current
(Ito) density
and slow its recovery in rat ventricle (30) but does not appear to
alter Ito density
in rabbit atrium or ventricle at physiological temperatures (29).
The present investigation was designed to determine the ionic basis of
delayed repolarization in hypothyroid guinea pigs. We sought to
establish whether hypothyroidism causes changes in the density or
kinetics of currents flowing during the action potential plateau
(particularly K+ and
Ca2+ currents) that could account
for concomitant alterations in repolarization.
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MATERIALS AND METHODS |
Experimental groups.
Adult male Hartley albino guinea pigs (500-600 g) were assigned to
a control (n = 25) or a hypothyroid
(n = 9) group. The procedures followed
were in accordance with the guidelines of the Montreal Heart Institute
and the Canadian Council on Animal Care. Animals assigned to the
hypothyroid group were thyroidectomized by Charles River (St. Constant,
PQ, Canada) after anesthesia with xylazine (Miles Canada, 5 mg/kg im)
and ketamine (Rogar/STP, 40 mg/kg im). Subsequently, these guinea pigs
were treated with 5-propyl-2-thiouracil (Sigma) for 6-8 wk (0.05%
in drinking water). CaCl2 was
added to the drinking water of hypothyroid animals at a concentration of 1% to avoid hypocalcemia caused by parathyroid damage. Weight and
electrocardiograms (ECG) were obtained on a weekly basis. In the
hypothyroid group, the first ECG changes occurred after 4 wk, and the
ECG stabilized by the end of 8 wk. When ECG changes had stabilized,
animals were killed by cervical dislocation, and their hearts were
removed for cell isolation.
ECG recordings.
Six-lead ECG recordings were obtained after sedation with acepromazine
(0.1 mg/kg im) and ketamine (40 mg/kg im). The average of three
measurements was used to determine the R-R, P-R, QRS, and Q-T intervals
with ±2.5-ms precision. The corrected Q-T
(Q-Tc) interval was calculated
using Bazett's formula (2).
Cell isolation and solutions.
Left ventricular myocytes were isolated by enzymatic dissociation as
previously described (6). Guinea pigs were killed by cervical
dislocation, and the hearts were excised and mounted on a Langendorff
apparatus. The hearts were perfused with oxygenated (100%
O2, pH adjusted to 7.35 with NaOH)
Tyrode solution containing (mM) 136 NaCl, 5.4 KCl, 2.0 CaCl2, 1.0 MgCl, 0.33 NaH2PO4,
5 HEPES, and 10 glucose at 37°C. When clear of blood, the perfusate
was changed to nominally Ca2+-free
Tyrode solution until contraction ceased. Perfusion continued with the
same solution containing 0.03% collagenase (type II, Worthington
Biochemical) and 1% bovine serum albumin (Sigma) until left
ventricular tissue softened. Small pieces of subepicardial tissue were
removed and mechanically dissociated by trituration. The isolated cells
were kept at room temperature in a storage solution containing (mM) 20 KCl, 10 KH2PO4,
25 glucose, 40 mannitol, 70 L-glutamic acid, 10 
-hydroxybutyric acid, 20 taurine, and 10 EGTA, along with 1%
albumin (pH adjusted to 7.35 with KOH).
A small aliquot of cell-containing solution was placed in a 1-ml open
perfusion chamber. After a brief period for cell adhesion to the
chamber, the cells were perfused at 6 ml/min with (mM) 136 NaCl, 5.4 KCl, 2.0 CaCl2, 1.0 MgCl, 0.33 NaH2PO4,
5 HEPES, and 10 glucose (pH adjusted to 7.35 with NaOH) for the
recording of action potentials, inward rectifier current
(IK1), and
delayed rectifier current
(IK). To record
ICa, we used a
solution containing (mM) 136 choline chloride, 5.6 CsCl, 2.0 CaCl2, 1.0 MgCl2, 0.33 NaH2PO4,
5 HEPES, and 10 glucose (pH adjusted to 7.35 with CsOH). For
INa recording,
the solution contained (mM) 132.5 CsCl, 5.0 NaCl, 1.0 MgCl2, 1.0 CaCl2, 20 HEPES, and 11 glucose
(pH adjusted to 7.35 with CsOH). To record
IK1 and
IK,
ICa was blocked
with 5 µM nifedipine (Sigma). All experiments were performed at
36°C except for those studying fast
Na+ current
(INa), for
which the bath was held at 17°C with a Peltier-effect device. The
pipette solution contained (mM) 20 KCl, 110 K-aspartate, 1.0 MgCl, 10 HEPES, 5 EGTA, 5 Mg2ATP, 0.1 GTP,
and 5 phosphocreatine (pH adjusted to 7.2 with KOH) to record action
potentials, IK1, and IK. For
ICa recording,
the pipette contained (mM) 20 CsCl, 110 Cs-aspartate, 10 HEPES, 10 EGTA, 1.0 MgCl, 5 Mg2ATP, 0.1 GTP, and 5 phosphocreatine (pH adjusted to 7.2 with CsOH). To record INa, pipettes
were filled with a solution containing (mM) 135 CsF, 5.0 NaCl, 5.0 HEPES, 10 EGTA, and 5 Mg2ATP (pH
adjusted to 7.2 with CsOH).
Voltage-clamp technique and action potential recording.
Borosilicate glass electrodes (outer diameter 1.0 mm) with resistances
of 0.8-1.2 M
for
INa recording and
2.6-6 M for other experiments were connected to a patch-clamp
amplifier (Axopatch 200A, Axon Instruments). The sampling frequency was
10 kHz for rapidly changing currents (such as
INa or
ICa) and as low
as 0.4 kHz for long recordings of slowly changing currents like
IKs.
Membrane capacitance was larger in the hypothyroid group (167 ± 6 vs. 147 ± 6 pF in control, P < 0.05), so all mean current data are expressed as current densities.
Before compensation, series resistance
(Rs) averaged
13.7 ± 1.0 and 13.2 ± 1.1 M in control and hypothyroid
groups, respectively, and the capacitive time constants were 2,010 ± 192 and 2,081 ± 189 µs, respectively. After
compensation, Rs
values were 3.0 ± 0.2 and 3.3 ± 0.2 M, and capacitive time
constants were 424 ± 35 and 557 ± 42 µs. Cells with
significant leak current were rejected.
Action potentials were recorded in current-clamp mode, beginning 5 min
after membrane rupture. Stimulation with 2-ms pulses to 20 mV
was applied at 0.1-4 Hz, and action potential parameters were
recorded at steady state at each frequency. Recorded resting potentials
were corrected for the junction potential, which averaged 11.5 mV.
Data analysis.
Group data are expressed as means ± SE. Statistical comparisons
between groups were made by t-test,
with P < 0.05 considered statistically significant. A nonlinear least-square curve-fitting program in pCLAMP 6.0 or Sigma Plot was used for curve fitting.
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RESULTS |
In vivo effects of hypothyroidism.
Hypothyroidism was associated with typical electrocardiographic
changes, as illustrated by the representative ECG recordings in Fig.
1. The ECG recordings of control animals
were similar to the baseline recordings of animals in the hypothyroid
group (Table 1). At the time of euthanasia
for electrophysiological study, hypothyroid animals had significantly
longer R-R, Q-T, and Q-Tc
intervals, but hypothyroidism did not alter P-R and QRS intervals
(Table 1). During observation periods of 69 ± 5 days for controls
and 73 ± 6 days for hypothyroid guinea pigs, the hypothyroid
animals gained more weight than controls. On the day of experimental
study, the average weight of hypothyroid animals was 799 ± 22 g,
significantly larger than in the control group (697 ± 27 g,
P < 0.05). Serum concentrations of
Na+,
K+,
Ca2+, and
Cl
were unchanged in
hypothyroid guinea pigs.
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Fig. 1.
Representative electrocardiogram (ECG) recordings in control and
hypothyroid guinea pigs. Guinea pigs were sedated with acepromazine and
ketamine, and ECGs were recorded at a paper speed of 200 mm/s.
Decreased heart rate and marked prolongation in Q-T interval are
characteristic ECG changes in hypothyroidism. II, aVL, aVF, ECG
leads.
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Action potential characteristics.
In agreement with the Q-T prolongation observed in the ECG of
hypothyroid guinea pigs, APD was prolonged in single ventricular myocytes isolated from hypothyroid animals (Fig.
2). The degree of prolongation was greater
at slower rates, but the prolongation was statistically significant at
all frequencies. APD was prolonged to a similar degree at 20, 50, and
90% of repolarization (Table 2). In
contrast to APD, resting potential and action potential amplitude were
unaffected by hypothyroidism (Table 2). We did not observe early
afterdepolarizations (EADs) in myocytes from hypothyroid guinea pigs;
however, conditions were not designed to favor EADs, and we have not
seen EADs in control cells exposed to
IKr blockers
under the same conditions.
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Fig. 2.
Action potential changes in hypothyroidism.
A: representative action potentials in
control and hypothyroid ventricular myocytes stimulated at 1 (top) and 4 (bottom) Hz. Action potentials were
recorded at 37°C in single ventricular myocytes isolated from
control and hypothyroid guinea pig hearts. Repolarization is already
delayed at very positive potentials and throughout plateau phase of
action potential. Resting membrane potential is not corrected for
junction potential. B: frequency
dependence of action potential prolongation. Action potential duration
at 90% repolarization (APD90)
prolongation becomes less pronounced as stimulation frequency is
increased. *** P < 0.0001 for difference between control and hypothyroid animals
(n = 13 and 15 cells, respectively).
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Table 2.
Action potential parameters of control and hypothyroid ventricular
myocytes at 1and 4-Hz stimulation frequency
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Changes in IK density.
IK was recorded
with the use of a series of 3-s depolarizing pulses (0.1 Hz) from a
holding potential of 50 mV to test potentials from 40 to
+70 mV, followed by a 2-s repolarizing pulse to 40 mV to record
tail current
(IKtail).
Baseline measurements were performed 15 min after cell membrane
rupture, and the protocol was run at least three times for each cell in
10-min intervals to detect rundown of
IK. In cells with
a stable IK
(<10% rundown over 20 min), 1 µM dofetilide was added to the bath
solution to block
IKr. After an
equilibration period of 10 min the protocol was repeated. Washout of
dofetilide was obtained in five cells, and a mean reversal of 94% in
drug effect was observed. Cells with rundown >10% (4% of cells)
were rejected. To exclude differences between groups in the rate of
early IK rundown,
the current was recorded at 5 and 15 min after membrane rupture in 14 cells for each group from 10 control and 7 hypothyroid animals. In
control cells, IK
at +40 mV averaged 581 ± 91 pA at 5 min and 541 ± 93 pA at 15 min (7.3 ± 1.2% rundown). In hypothyroid cells,
IK averaged 191 ± 20 pA at 5 min and 176 ± 18 pA at 15 min (7.8 ± 0.7% rundown).
Representative IK
recordings in control and hypothyroid myocytes are illustrated in Fig.
3. Results are shown both before (Fig. 3,
A and
D) and after (Fig. 3,
B and
E) superfusion with 1 µM
dofetilide. Dofetilide-sensitive currents (corresponding to
IKr; Ref. 26)
obtained by digital subtraction are shown in Fig. 3,
C and
F. In cells isolated from hypothyroid
hearts, the amplitudes of the time-dependent activating and tail
currents were substantially smaller for total
IK and the slowly
activating, dofetilide-resistant component
IKs. In contrast,
the rapidly-activating, dofetilide-sensitive currents were not
obviously affected by hypothyroidism.
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Fig. 3.
Differences in delayed rectifier current in a control
(left) and a hypothyroid
(right) ventricular myocyte. From
holding potential of 50 mV, 3-s test pulses from 40 to
+70 mV (0.1 Hz) were applied to record step current, and tail currents
were measured on 2-s repolarization to 40 mV. Bath temperature
was 36°C, and 5 µM nifedipine was added to inhibit L-type
Ca2+ current
(ICa).
Recordings are presented before (A and
D) and after
(B and
E) addition of 1 µM dofetilide to
block dofetilide-sensitive current
(IKr).
IKr
(C and
F) were obtained by digital
subtraction. For reasons of clarity, in
A, B,
D, and
E, recordings at test voltages between
0 and +70 mV are shown (see inset),
whereas in C and
F recordings represent test voltages
between 20 and +20 mV. Arrows and 0 lines indicate holding and
zero-current level, respectively.
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Mean current densities are shown as a function of the voltage of the
test pulse (TP) in Fig. 4.
IKs step currents
had a linear IV relationship in both groups, with hypothyroid cells
showing a significant decrease in step current densities at all
voltages positive to +20 mV (Fig.
4A). For example, at +30 mV
IKs density was
1.23 ± 0.22 and 0.49 ± 0.11 pA/pF in control and
hypothyroid myocytes, respectively (P < 0.05). IKr
activated at more negative potentials, reached a maximum at +10 mV, and
decreased thereafter. There was no statistically significant difference
in IKr densities between control and hypothyroid cells, with current densities at 0 mV
of 0.31 ± 0.04 and 0.30 ± 0.02 pA/pF for cells from control and
hypothyroid myocytes, respectively
[P = not significant
(NS)]. IKs
tail currents also showed a smooth current-voltage relation, with
current densities from cells of hypothyroid animals significantly decreased at all test potentials positive to 20 mV (Fig.
4B). Mean values for
IKs tails at a TP
of +30 mV were 0.53 ± 0.10 pA/pF for controls and 0.10 ± 0.01 pA/pF for hypothyroid myocytes (P < 0.001). IKr tail
currents approached saturation at 0 mV and had similar current
densities for both groups of animals. For example, for an activating
pulse to 0 mV,
IKr tail current
densities were 0.27 ± 0.07 pA/pF in control and 0.25 ± 0.02 pA/pF in hypothyroid myocytes (P = NS). Mean IKr
step currents over the entire voltage range between 20 and +20
mV averaged 0.28 pA/pF for control cells and 0.24 pA/pF for hypothyroid
cells, and corresponding values for tail currents were 0.24 and 0.22 pA/pF, respectively.
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Fig. 4.
Reduced dofetilide-resistant current
(IKs) step and
tail current densities in hypothyroidism. Currents were elicited with
same pulse protocol as in Fig. 3. A:
IKs and
IKr step current
densities (means ± SE) in control
(n = 17) and hypothyroid
(n = 8) myocytes as function of test
potential (TP). Step current is defined as difference between current
immediately after decay of capacitive transient at onset of
depolarization and current level at end of 3-s test pulse.
Ba2+ (500 µM) was added to
decrease contamination by inward rectifier
K+ current
(IK1).
B: tail current densities for control
(n = 9) and hypothyroid
(n = 8) myocytes.
* P < 0.05, ** P < 0.01, *** P < 0.001 for difference
between control and hypothyroid results at same voltage.
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Kinetics and voltage dependence of
IK activation.
The reduction in the amplitude of whole cell
IKs could involve
a change in activation kinetics or a shift in the voltage dependence of
activation. The time course of activation of the dofetilide-insensitive step current was best fit with a biexponential function. Hypothyroidism did not alter the kinetic properties of dofetilide-insensitive IKs. For example,
at a test potential of +60 mV,
IKs had a fast time constant (
fast)
averaging 222 ± 22 ms for controls
(n = 5) and 248 ± 28 ms for
hypothyroid cells (n = 5, P = NS). The corresponding values for
the slow time constant (slow)
of IKs were
1,830 ± 214 and 1,439 ± 247 ms
(P = NS).
IKr activation
kinetics were similarly unaffected by hypothyroidism. At a test
potential of +10 mV, fast of
IKr was 68 ± 17 ms in controls (n = 5) and 81 ± 17 ms in hypothyroid myocytes (n = 6, P = NS), whereas
slow values were 1,785 ± 232 and 1,587 ± 253 ms for control and hypothyroid groups,
respectively (P = NS).
An analysis of the voltage-dependent activation of
IKr and
IKs (Fig.
5) was performed by normalizing tail
currents in each cell at each test potential to the current at the most
positive voltage. A Boltzmann function was used to fit the activation
curves of IKr and
IKs. Under
control conditions,
IKs
half-activation voltage
(Vh) was 21.3 ± 2.7 mV with a slope factor (k)
of 13.4 ± 1.1 mV, values equivalent to those previously reported in
guinea pig ventricle (26). In hypothyroid animals,
Vh of
IKs shifted to
more positive values and averaged 29.5 ± 2.2 mV
(P < 0.01 vs. control);
k was unchanged at 13.5 ± 1 mV
(P = NS vs. control). IKr activation
voltage dependence was not affected by hypothyroidism: Vh was
17.1 ± 3.1 mV in controls and 17.5 ± 3.3 mV in
hypothyroid myocytes, and k averaged
9.0 ± 1.2 and 7.5 ± 1.4 mV, respectively.
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Fig. 5.
Voltage-dependent activation of
IKr and
IKs in control
conditions and hypothyroidism. Isochronal activation curves of
IK,
IKr, and
IKs under control
(A) and hypothyroid
(B) conditions based on analysis of
tail currents. Continuous curves were obtained by fitting experimental
data by Boltzmann distribution function of the following form:
A = 1/{1 + exp
[(Vh Vm)/k]},
where A is activation variable
[tail current at TP
(Vm) divided by
tail current at +70 mV] and
Vh and
k are half-activation voltage and
slope factor, respectively. Results shown are from 17 control and 8 hypothyroid cells.
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Envelope of tails test.
To assess the composition of
IK under control
and hypothyroid conditions, we applied an envelope of tails analysis.
IK step currents
(IKstep) were
elicited by depolarization from 60 to +50 mV with test pulses
ranging from 60 to 4,200 ms, and tail currents were recorded on
repolarization to 40 mV. Mean values for the ratio
IKtail/IKstep
are shown in Fig. 6. Before blockade of
IKr (Fig.
6A), the envelope of tails test was
not satisfied in either the control or the hypothyroid group,
indicating that IK results from
the activation of more than one component. In hypothyroid myocytes,
IKtail/IKstep
was significantly larger at all intervals than in control cells,
reflecting the larger contribution of
IKr to total
IK because of the
much smaller IKs.
After dofetilide was added to the superfusate (Fig.
6B), the envelope of tails test was
satisfied. Furthermore,
IKtail/IKstep
was no longer different for hypothyroid compared with euthyroid cells,
reflecting the fact that the same single component
(IKs) remained
under each condition.
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Fig. 6.
Ratio of IK tail
current
(IKtail) to
IK step current
(IKstep) under
control conditions and in hypothyroidism. Envelope of tails were
recorded with pulse protocol shown in
inset at 0.1 Hz, and
IKtail/IKstep
was plotted as a function of pulse duration in 6 control and 6 hypothyroid ventricular myocytes before
(A) and after
(B) exposure to 1 µM dofetilide.
Before block of
IKr envelope of
tails test was not satisfied in either group. Note that
IKtail/IKstep
is larger in myocytes from hypothyroid animals for all pulse durations
(* P < 0.05). Where error bars
are absent, they fell within symbol for mean.  t, pulse
duration.
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Inward rectifier
K+ current.
Figure 7A
shows typical examples of
IK1 in control
and hypothyroid myocytes, and Fig. 7B
shows mean IK1
densities elicited with 200-ms pulses to test potentials between
90 and +30 mV from a holding potential of 40 mV from 12 control and 20 hypothyroid myocytes. Hypothyroidism did not alter
IK1. The reversal
potential for IK1
was 71.0 ± 1.4 mV in hypothyroid cells, compared with 69.6 ± 1.2 mV in controls.
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Fig. 7.
Inward rectifier K+ current
(IK1) is not
affected by hypothyroidism. A:
original recordings from representative control
(top) and hypothyroid
(bottom) ventricular myocyte. Pulse
protocol shown in inset was used to
elicit currents at frequency of 0.5 Hz. Steady-state currents were
measured at end of 200-ms test pulses; 0 indicates zero current levels.
B: mean current densities in control
(n = 12) and hypothyroid
(n = 20) cells at TP between 90
and +30 mV. Where error bars are absent, they fell within symbol for
mean.
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Ca2+ current.
ICa is the major
inward current during the plateau and is therefore crucial in the
determination of APD and refractoriness under physiological conditions
(17). The ICa
current-voltage relation was studied at 36°C, with 400-ms steps
from a holding potential of 80 mV at a frequency of 0.1 Hz. The
magnitude of ICa
was measured as the difference between the peak inward current and the
steady-state current at the end of the depolarizing step. ICa densities
were similar in control and hypothyroid myocytes at all voltages
tested, as illustrated in Fig. 8. Peak
current densities occurred at +10 mV in both groups and averaged 5.1 ± 0.3 pA/pF in controls and 5.9 ± 0.5 pA/pF in hypothyroid
guinea pigs (P = NS). In both groups a
T-type Ca2+ current
(ICa,T) was
noted as a shoulder on the total
Ca2+ current-voltage relation. The
density of ICa,T
was not significantly affected by hypothyroidism.
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Fig. 8.
Ca2+ currents in control and
hypothyroid ventricular myocytes. A:
family of ICa in
control (top) and hypothyroid
(bottom) conditions, recorded by
depolarizing voltage steps to voltages indicated in
inset from holding potential of
80 mV. Current amplitudes were measured as difference between
peak transient inward current and steady-state current at end of 400-ms
test pulse. To show clearly the transient component of each current
recording, only first 200 ms of each recording are shown.
B: mean current densities from 7 control and 9 hypothyroid cells. Pulse protocol was as in
A with TP between 70 and +60
mV. Note second peak at 40 mV, which represents T-type current.
Where error bars are absent, they fell within symbol for mean.
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Figure 9,
A and
B, shows
ICa activation
and inactivation voltage dependence, respectively, in 7 control and 10 hypothyroid cells.
Vh averaged
11.2 ± 0.6 mV in the controls and 11.9 ± 0.8 mV
in the hypothyroid group (P = NS), and
mean values for k were 7.4 ± 0.5 and 7.5 ± 0.8 mV, respectively (P = NS). Voltage-dependent inactivation was also unaffected by
hypothyroidism, with a mean Vh of 40.8 ± 2.3 mV in control and 37.8 ± 1.1 mV in hypothyroid myocytes and k values of 9.8 ± 0.8 and 8.6 ± 0.5 mV, respectively (P = NS).
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Fig. 9.
Voltage-dependent activation (A) and
inactivation (B) of
ICa in control
and hypothyroid myocytes (n = 7 and 10 cells, respectively). Activation voltage dependence was determined by
dividing current at each TP by driving force. Inactivation voltage
dependence was determined with 1,000-ms prepulses followed by 400-ms
test pulse to +10 mV. Data points represent mean values; smooth curves
were obtained by fitting data with a Boltzmann equation.
C: recovery of
ICa from
inactivation. Recovery of control (n = 5) and hypothyroid (n = 6)
cells is shown as function of interpulse (P1-P2) interval. Data were
best fit by a biexponential function (smooth curves). I1 and I2,
current amplitudes during P1 and P2. For details, see text. Where error
bars are absent, they fell within symbol for mean.
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These results indicate that differences in
ICa density and
voltage dependence cannot account for the APD differences between control and hypothyroid cells but do not exclude kinetic differences in
ICa that could
have major effects on APD. We therefore analyzed the time dependence of
ICa inactivation
development and recovery. The inactivation of
ICa at +10 mV was
best fit by a biexponential function with time constants of 5.1 ± 0.6 and 70.8 ± 4.8 ms (n = 5) in
cells from control guinea pigs. In the hypothyroid group, no alteration
was observed, with values of 6.1 ± 1.3 ms for
fast and 89.7 ± 8.4 ms for
slow
(n = 7, P = NS vs. control for each). The
recovery of ICa
from inactivation was studied with a two-pulse protocol (Fig.
9C) . Two identical 300-ms pulses to +10 mV (P1 and P2)
were delivered from a holding potential of 80 mV every 10 s at
increasing P1-P2 intervals. Recovery kinetics were analyzed on the
basis of the current amplitude during P2 relative to the amplitude
during P1 as a function of the P1-P2 interval. Recovery was rapid under
control and hypothyroid conditions and was best fitted with a
biexponential function. Mean
fast was 96.8 ± 11.4 ms in
controls and 99.5 ± 14.5 ms in hypothyroid cells
(n = 5 and 6 cells, respectively,
P = NS), whereas
slow averaged 779 ± 75 and
1,267 ± 230 ms, respectively (P = NS).
Na+ current.
INa is the other
major inward current in cardiac myocytes and is a particularly
important determinant of conduction and cellular excitability. We
studied INa at
17°C with 40-ms steps at 0.1 Hz to test potentials between
80 and 5 mV (with 5-mV increments) from a holding
potential of 120 mV. The groups had similar current densities,
voltages of peak current density (35 mV), and mean peak current
densities (48 ± 5 and 49 ± 4 pA/pF in 10 and 11 myocytes from
control and hypothyroid animals, respectively). Inactivation kinetics
of INa at
35 mV were best fit by biexponential functions and were not
altered by hypothyroidism, with mean
fast values of 3.5 ± 0.1 and 3.4 ± 0.2 ms and slow
values of 39.3 ± 7.8 and 38.4 ± 6.8 ms for 10 control and 11 hypothyroid cells, respectively.
| |
DISCUSSION |
In the present study, we have demonstrated that hypothyroidism leads to
important delays in guinea pig ventricular repolarization. This effect
was associated with large reductions in
IK, the primary repolarizing K+ current in the
guinea pig, which were exclusively caused by decreases in
IKs. No other
changes in K+,
Ca2+, or
Na+ currents were seen.
Comparison of ECG and action potential changes with previous reports
in the literature.
The ECG changes we observed in hypothyroid guinea pigs (a decrease in
heart rate and a prolonged Q-T interval) are typical of clinical
hypothyroidism (32) and are quantitatively similar to those described
previously in guinea pigs rendered hypothyroid by
131I (33). At 4 Hz, which
corresponds to the physiological heart rate in guinea pigs, APD at 90%
repolarization was 61% longer in hypothyroid guinea pigs than under
control conditions, whereas at slower frequencies the prolongation was
more pronounced. As in previous standard microelectrode studies, all
phases of repolarization were prolonged to a similar extent (11, 28).
Comparison with previous studies of ionic current changes in altered
thyroid state.
The voltage-dependent properties of
IKs and
IKr in myocytes
from control guinea pigs were similar to those reported previously for
this species (26) and comparable to delayed rectifier current in cells
from human atria (37) and ventricles (16). Under hypothyroid
conditions, Vh of
IKs was shifted
by 8.2 mV to more positive voltages, leading to a decrease in current
amplitude at a given voltage. However, this relatively small shift is
not sufficient to account for the total reduction of
IKs, because maximal IKs
conductance (obtained by dividing the current amplitude by the driving
force for K+) is decreased. For
example at +50 mV, maximum conductance was 3.21 µS in control cells
and 1.07 µS in cells from hypothyroid guinea pigs. Our results
regarding changes in
IK in hypothyroid guinea pigs differ from those of two previous publications by Binah et
al. (4) and Rubinstein and Binah (23), in which no difference in
IK was noted
between control and hypothyroid guinea pigs. The discrepancies between
our results and those of Binah and co-workers may be caused by a
variety of technical factors such as experimental temperature,
isolation technique, and method of
IK measurement.
Temperature can have a marked effect on
IK currents and
their response to interventions (10, 35), and IK is
particularly sensitive to isolation technique (39). The analysis of
current density is important in studies of hypothyroidism, because as
we found and others have reported previously (19, 30), hypothyroidism
can alter cell size.
IK1 contributes
to the late phase of repolarization at near-diastolic potentials.
Hypothyroidism did not influence
IK1 in our study.
To our knowledge, there are no published studies of the effects of
hypothyroidism on
IK1. Shimoni and
Banno (29) did not detect differences in
IK1 between
hyperthyroid and euthyroid rabbits.
A previous study by Rubinstein and Binah (23) reported a decrease in
ICa amplitude in
hypothyroid guinea pigs, whereas we found
ICa to be
unchanged by hypothyroidism. A variety of technical differences may
explain the discrepancy. Kosinski et al. (15) found no change in L-type
Ca2+ channel concentration with
hypothyroidism. Studies of hyperthyroid effects on
ICa have also
provided varying results, suggesting a decrease (9, 15), little change
(29), or an increase (14).
Potential mechanism of hypothyroid effect on
IKs.
Reduced thyroid hormone activity could decrease
IKs density by
several mechanisms. Thyroid hormone is known to control cardiac gene
expression (31). The channel that carries
IKs appears to result from the coassembly of KvLQT1 and minK proteins (1, 24, 38). We
reported preliminary findings suggesting that KvLQT1 mRNA
concentrations were reduced in hypothyroid animals (5); however,
because of problems with RNA stability, we have been unable to confirm
or refute these initial data. Posttranscriptional changes, such as
differences in protein kinetics, insertion in the membrane, or
regulation by neurohormones (3, 36) may also be involved in the
decrease in IKs.
Potential significance.
Thyroid hormone is known to be an important regulator of cardiac
repolarization. Our study is the first to provide a clear mechanism for
this phenomenon in a mammalian heart. The importance of
IKs in
repolarizing guinea pig ventricular myocytes has been suggested by
recent modeling work (40). The degree of prolongation in APD associated
with IKs
reduction in our guinea pigs (65% at 4 Hz) is of the same order as
predicted by the mathematical model (between 38 and 70% increase for
IKs reductions of
65-80%; Y. Rudy, personal communication), supporting the
relevance of the latter and the role it suggests for
IKs in
repolarization. On the other hand, removing
IKs from the
action potential model strongly promotes the generation of EADs (40),
which we did not see in the present study.
Because of the potential proarrhythmic effects associated with
currently available class III drug therapy (21), there has been
particular interest in the development of substances with novel ionic
targets. Jurkiewicz and Sanguinetti (12) demonstrated that
IKs contributes
to rate-dependent action potential abbreviation and suggested that
IKs may be an
interesting target for new antiarrhythmic drugs. Of note, although APD
was greater in hypothyroid guinea pigs at all frequencies, the
difference was greatest at low frequencies, i.e., some reverse use
dependence for APD prolongation was present. In hypothyroid patients,
Q-T prolongation is a typical electrocardiographic feature (32), but
torsades de pointes arrhythmias are rarely observed (22). Furthermore,
hypothyroidism is known to have significant antiarrhythmic actions (22,
34). Hypothyroidism may therefore be an interesting natural paradigm
for delayed repolarization with low associated risk of torsades de
pointes. The extent to which these electrophysiological actions of
hypothyroidism are caused by a depression in
IKs without
alteration in
IKr, as opposed to or in combination with other actions of hypothyroidism [e.g., reduced sympathetic activation (3)], remains to be determined. It
should be pointed out in this context that KvLQT1 mutations (and
presumably IKs
dysfunction) cause a common and life-threatening form of the long-Q-T
syndrome (1, 24, 38).
Potential limitations.
We studied left ventricular cells from guinea pigs, which have
important ionic differences from human ventricular myocytes. Transient
outward current, which has been shown to be an important repolarizing
current in human ventricle (20), is absent in the guinea pig.
Ito was found to
be markedly reduced in hypothyroid rat ventricle (30), whereas in
rabbit ventricle it was unchanged (29). It is therefore possible that
alterations in
Ito play a role
in the changes in repolarization in human ventricle under hypothyroid
conditions. On the other hand, both components of IK are present
and likely to play a significant role in human ventricular
repolarization (16). Furthermore, it is important to note that
Ito inhibition
reduces, rather than increasing, APD in dog ventricle (18). Other ionic
mechanisms, such as exchangers and pumps (27), may also be affected by
hypothyroidism and were not assessed in the present study. Thyroid
hormone has particularly important effects on the
Na+-K+-ATPase,
which is significantly downregulated by hypothyroidism (7). Thus
effects of hypothyroidism on cardiac pumps and exchangers may
contribute significantly to cardiac electrophysiological properties in
the presence of hypothyroidism, an issue that remains to be resolved.
IK is very
sensitive to isolation procedures (39), and therefore changes in
isolation technique could contribute to differences in
IK when several
groups are compared. To minimize possible effects of time-dependent
changes in enzymes, isolation success, etc., animals from each group
were studied concurrently in an alternating fashion. Rundown can be a
problem when studying
IK and
ICa. All currents
were therefore studied with serial measurements to screen for rundown.
All cells with significant rundown were rejected for analysis. Mean
values for IKr
step and tail currents on steps to between 20 and +20 mV were
slightly smaller (by 14 and 8%, P = NS for each) in hypothyroid compared with control cells. We cannot
exclude the possibility of a very small change in
IKr (of uncertain
physiological significance) in hypothyroid hearts.
We studied action potentials from isolated cells in whole cell mode.
These results may be different from those obtained with traditional
voltage followers and fine-tipped microelectrodes in multicellular
preparations. IKs
activation kinetics were determined during 3-s pulses, which were only
about twice the estimated time constant. This introduces an element of
imprecision, and the time constants should not be considered as
absolutely precise but rather as a quantitative tool to compare the
relative activation rates in control versus hypothyroid cells.
In conclusion, we have shown that hypothyroidism in guinea pigs is
associated with a pronounced delay in ventricular repolarization both
in vivo and in isolated ventricular myocytes. The only ionic current
change noted was an important reduction of the slow component of the
delayed rectifier K+ current, of a
magnitude potentially sufficient to account for the repolarization
changes observed. These results point to an important role of
IKs in
controlling repolarization in ventricular myocytes and in mediating the
regulation of cardiac repolarization by thyroid hormone.
| |
ACKNOWLEDGEMENTS |
The authors thank Johanne Doucet, Emma de Blasio, Dalie St-Georges,
and Mirie Levi for expert technical assistance and Luce Bégin and
Caroll Boyer for secretarial help with the manuscript.
| |
FOOTNOTES |
This work was supported by grants from the Medical Research Council of
Canada, the Quebec Heart Foundation, and the Fonds de Recherche de
l'Institut de Cardiologie de Montréal. R. F. Bosch was a fellow
of the Deutsche Forschungsgemeinschaft. Z. Wang is a scholar of the
Heart and Stroke Foundation of Canada.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Nattel,
Research Center, Montreal Heart Institute, 5000 Bélanger St.
East, Montreal, Quebec H1T 1C8 Canada (E-mail:
nattel{at}icm.umontreal.ca).
Received 11 September 1998; accepted in final form 11 March 1999.
| |
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ABSTRACT
INTRODUCTION
