(Circulation. 1999;99:2942-2950.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Troglitazone Inhibits Voltage-Dependent Calcium Currents in Guinea Pig Cardiac Myocytes
From the Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo (T.N., K.I., H.O., H.I., H.H., M.A., T.M., F.N., J.S., M.O.), and the Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba (S.S., Y.K., Y.O.), Ibaraki, Japan.
Correspondence to T. Nakajima, MD, Second Department of Internal Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
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Abstract |
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Background—It has been suggested that intracellular Ca2+ overload in cardiac myocytes leads to the development of diabetic cardiomyopathy. Troglitazone, an insulin-sensitizing agent, is a promising therapeutic agent for diabetes and has been shown to prevent diabetes-induced myocardial changes. To elucidate the underlying mechanism of troglitazone action on cardiac myocytes, the effects of troglitazone on voltage-dependent Ca2+ currents were examined and compared with classic Ca2+ antagonists (verapamil and nifedipine).
Methods and Results—Whole-cell voltage-clamp techniques were applied in single guinea pig atrial myocytes. Under control conditions with CsCl internal solution, the voltage-dependent Ca2+ currents consisted of both T-type (ICa,T) and L-type (ICa,L) Ca2+ currents. Troglitazone effectively reduced the amplitude of ICa,L in a concentration-dependent manner. Troglitazone also suppressed ICa,T, but the effect of troglitazone on ICa,T was less potent than that on ICa,L. The current-voltage relationships for ICa,L and the reversal potential for ICa,L were not altered by troglitazone. The half-maximal inhibitory concentration of troglitazone on ICa,L measured at a holding potential of -40 mV was 6.3 µmol/L, and 30 µmol/L troglitazone almost completely inhibited ICa,L. Troglitazone 10 µmol/L did not affect the time courses for inactivation of ICa,L and inhibited ICa,L mainly in a use-independent fashion, without shifting the voltage-dependency of inactivation. This effect was different from those of verapamil and nifedipine. Troglitazone also reduced isoproterenol- or cAMP-enhanced ICa,L.
Conclusions—These results demonstrate that troglitazone inhibits voltage-dependent Ca2+ currents (T-type and L-type) and then antagonizes the effects of isoproterenol in cardiac myocytes, thus possibly playing a role in preventing diabetes-induced intracellular Ca2+ overload and subsequent myocardial changes.
Key Words: troglitazone • myocytes • calcium • isoproterenol • diabetes • cardiomyopathy
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Introduction |
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Myocardial contractile dysfunction is a major complication of diabetes, known as diabetic cardiomyopathy.1 2 3 4 The subcellular mechanisms responsible for cardiomyopathy are unknown. However, several cellular defects, including depressions in sarcoplasmic reticular Ca2+ uptake,5 Na+-K+ pump,6 sarcolemmal Ca2+ pump, Na+-Ca2+ exchanger activities,7 and the alteration of mitochondrial functions,8 have been suggested to be contributors to the development of this disease. The net result of these changes in Ca2+ homeostasis causes an intracellular Ca2+ overload, thereby resulting in cellular damage and, ultimately, diabetic cardiomyopathy. Moreover, diabetes prolongs the action potential duration9 10 11 12 and increases the number of myocardial voltage-dependent Ca2+ channels,13 14 something that may also play a role in causing diabetic cardiomyopathy. In fact, it has been reported that in chronically diabetic rats, elevated tissue Ca2+ levels are present15 and treatment with verapamil or diltiazem, a voltage-dependent L-type Ca2+ channel blocker, lessens cardiac dysfunction.16 17 18 Thus, it is likely that excess Ca2+ influx through the voltage-dependent Ca2+ channels contributes to induce intracellular Ca2+ overload and consequently diabetic cardiomyopathy.
Troglitazone, a novel member of the insulin-sensitizing thiazolidinediones, has been widely used to treat patients with non–insulin-dependent diabetes mellitus and other insulin- resistant diseases. Treatment with troglitazone reduced hyperglycemia, plasma triglycerides, and blood pressure.19 20 21 22 Recent studies show that troglitazone attenuates high- glucose–induced abnormalities in relaxation and intracellular calcium in rat ventricular myocytes23 and may improve cardiac function in diabetic patients.24 Until now, the mechanisms underlying the beneficial effects of troglitazone on hearts have not been clearly established, but several articles have shown that troglitazone inhibits the voltage-dependent L-type Ca2+ currents (ICa,L) in vascular smooth muscle cells.25 26
Therefore, the purpose of the present study was to clarify the effects of troglitazone on the voltage-dependent Ca2+ currents (T-type [ICa,T] and L-type) in cardiac myocytes. We have also made comparisons with the classic Ca2+ antagonists verapamil and nifedipine.
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Methods |
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Cell Preparation
Single atrial myocytes were obtained from guinea pig hearts by an enzymatic dissociation procedure described elsewhere.27 Briefly, the animals were anesthetized with sodium pentobarbital, and their hearts were rapidly removed and retrogradely Langendorff-perfused at 35°C to 37°C with an oxygenated Tyrode's solution. The hearts were then perfused with Ca2+-free Tyrode's solution for
10 minutes
and subsequently with the same solution containing
collagenase (0.04% wt/vol type 1, Sigma Chemical Co) for
17 to 20 minutes. The digested hearts were stored in a
high-K+/low-Cl-
solution27 at 5°C for later experimentation. The atria
were then removed, and cells were obtained by gentle mechanical
agitation. This procedure consistently yielded an acceptable
number of quiescent and relaxed atrial cells.
Solution and Drugs
The composition of the normal Tyrode's solution was (in
mmol/L) NaCl 136.5, KCl 5.4, CaCl2 1.8,
MgCl2 0.53, glucose 5.5, and HEPES-NaOH buffer 5,
pH 7.4. The Ca2+-free Tyrode's solution was
identical to normal Tyrode's solution except that
CaCl2 was omitted. To record
voltage-dependent Ca2+ currents,
K+ currents were eliminated by the internal Cs
and external Ba (5 mmol/L), and
Ca2+-activated currents were blocked by
10 mmol/L EGTA and 2 mmol/L BAPTA in the internal solution.
The composition of the internal solution was (in mmol/L) CsCl 140,
EGTA 10, BAPTA 2, Na2-ATP 3, GTP (sodium salt,
Sigma) 0.1, MgCl2 1, and HEPES-CsOH buffer 5, pH
7.3. In the experiments in which the cells were held at -80 mV, the
bath was perfused with the following solution (in mmol/L) to block
the voltage-dependent Na+ current:
tetraethylammonium chloride (TEA-Cl) 140,
BaCl2 5, MgCl2 0.53,
glucose 5.5, tetrodotoxin (TTX) 0.01, and HEPES-CsOH buffer 5, pH 7.4.
Troglitazone was obtained from Sankyo Co Ltd. Troglitazone was
dissolved in DMSO to give a stock solution of 1 to 30 mmol/L, and
the final concentration of DMSO applied to the bathing solution was
0.1%. Nifedipine and verapamil were dissolved
in ethanol to give a stock solution of 10 mmol/L. In several
experiments, cAMP was added to the pipette solution. (±)Isoproterenol,
cAMP, verapamil, and nifedipine were purchased
from Sigma.
Recording Technique and Data Analysis
Membrane currents were recorded with patch electrodes in a
whole-cell clamp configuration27 28 and a patch-clamp
amplifier (EPC-7, List Electronics). The heat-polished patch electrodes
had a tip resistance of 3 to 6 M
. The membrane currents were
monitored with a high-gain storage oscilloscope (COS 5020-ST, Kikusui
Electronics). At the start of each experiment, the series resistance
was compensated. The data were stored on video cassettes with a PCM
converter system (RP-880, NF electronic circuit design). Later, the
data were reproduced, low-pass–filtered at 2 kHz (-3 dB) with a
Bessel filter (FV-665, NF, 48-dB/octave slope attenuation), sampled at
5 kHz, and analyzed off-line on a computer with p-Clamp
software (Axon Instruments). In general, we used a holding potential of
-40 mV at a frequency of 0.2 Hz to inactivate the
voltage-dependent Na+ current. In experiments to
evaluate the contribution of ICa,T
or voltage-dependence of the drug, a holding potential of -80 mV was
used in combination with the high-TEA solution containing
Ba2+ 5 mmol/L in place of
Ca2+ (see Methods). Statistical results are
expressed as mean±SD. Student's t tests were performed,
with a value of P<0.05 considered significant.
The first data were usually taken after the current amplitude of
Ca2+ currents had been stabilized (2 to 3 minutes
after the rupture of the membrane). After that, we could investigate
the effects of drugs on the voltage-dependent
Ca2+ currents for 15 to 20 minutes. In
experiments with cAMP, data were taken immediately after the rupture of
the membrane. To measure the amplitude of the voltage-dependent
Ca2+ currents, we subtracted from the peak
amplitude of Ca2+ currents in the original trace
to the current level in the presence of Cd2+
(1 mmol/L). In preliminary experiments, we confirmed that 0.1%
DMSO did not affect the voltage-dependent Ca2+
currents significantly. Furthermore, to exclude the effects of DMSO,
0.1% DMSO was always added to the bathing solution. The steady-state
inactivation parameters of the voltage-dependent
Ca2+ currents were analyzed with
double-pulse protocols. Conditioning voltage pulses (3 seconds in
duration) for various membrane potentials between -70 and +0 mV were
applied from a holding potential of -80 mV. Ten milliseconds after the
end of each conditioning pulse, a test pulse of +10 mV (0.2 seconds in
duration) was applied to elicit Ca2+ currents.
The ratio between the amplitude of the Ca2+
currents with conditioning pulse and that without conditioning pulse
was plotted for the membrane potential of each conditioning pulse. The
interval between sets of double pulses was 20 seconds.
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Effects of Troglitazone on Voltage-Dependent ICa,L
The effects of troglitazone on the voltage-dependent ICa,L were examined in single atrial myocytes (Figure 1
). The membrane
potential was held at -40 mV, and command voltage pulses (320 ms in
duration) to +0 mV were applied at 0.2 Hz. In control cells, a
transient inward current was elicited during each voltage pulse (Figure 1A, a). The inward current was blocked by nifedipine
1 µmol/L (see Figures 9 and 10),
verapamil 1 µmol/L (Figure 9), and
Cd2+ 1 mmol/L (Figure 1A, f),
indicating that it consisted of ICa,L.
Troglitazone 10 µmol/L reduced the amplitude of
ICa,L from -500 to -140 pA in this cell
within 2 minutes (Figure 1A, b). After washout,
ICa,L gradually returned to near control
level. The time courses of changes in the peak
ICa,L measured from the zero current level
are shown in Figure 1B. Troglitazone 30 µmol/L almost
completely blocked ICa,L (Figure 1A, d). After washout, the depressed current gradually increased, but the
inhibitory effects of troglitazone 30 µmol/L were
not easily reversible. Figure 2 shows the
effects of troglitazone on current-voltage relationships of
ICa,L. The cells were held at -40 mV, and
command voltage steps to various membrane potentials were applied at
0.2 Hz. The current-voltage relationships of
ICa,L measured at the peak inward current
are shown in Figure 2B. Troglitazone reduced the current
amplitude of ICa,L at any command voltage
without affecting the voltage dependence of
ICa,L. On average, troglitazone 5
µmol/L decreased peak ICa,L at +0 mV by
42±6% (n=5). Troglitazone 30 µmol/L almost completely blocked
ICa,L. The reversal potential for
ICa,L was not altered significantly by
troglitazone. These results suggest that troglitazone inhibited
ICa,L in cardiac myocytes. The effects of
various concentrations of troglitazone on the amplitude of
ICa,L are shown in Figure 3. The cells were held at -40 mV, and
the command pulses to +0 mV were applied at 0.2 Hz. Troglitazone at
concentrations >1 µmol/L decreased
ICa,L, and the half-maximal
inhibitory concentration (IC50) of
troglitazone on ICa,L was 6.3
µmol/L.
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Figures 4 and 5 illustrate the effects of troglitazone
on isoproterenol- and cAMP-enhanced ICa,L.
Isoproterenol 1 µmol/L increased the amplitude of
ICa,L (Figure 4, b). Immediately
after application of isoproterenol, there was a rapid small increase in
ICa,L, probably reflected by direct
activation of the GTP-binding proteins
(Gs),29 and then a large
increase in ICa,L was observed. The
additional application of troglitazone 30 µmol/L completely
abolished ICa,L (Figure 4, c).
Moreover, when cAMP was applied through the patch pipette,
ICa,L increased from -370 to -1080 pA in
this cell (Figure 5A and 5B). Troglitazone 30 µmol/L also
abolished ICa,L (Figure 5A, c and
5B, c). Figure 5D shows the current-voltage relationships of the
peak ICa,L in the presence of cAMP (Figure 5C, a) and with the additional application of troglitazone
30 µmol/L (Figure 5C, b). Troglitazone 30 µmol/L
decreased ICa,L at all command potentials.
These results suggest that troglitazone antagonizes the effects of
isoproterenol on ICa,L independently of
ß-adrenergic receptors.
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Effects of Troglitazone on the Voltage-Dependent
ICa,L and
ICa,T
The existence of 2 distinct Ca2+ currents
has been shown for cardiac myocytes in several kinds of mammalian
hearts.30 31 32 33 ICa,T
activates at low voltages and inactivates quickly;
ICa,L activates at high voltages
and inactivates slowly. In addition, the T-type
Ca2+ channel is about equally permeable to
Ca2+ and Ba2+ ions and has
the same inactivation kinetics in Ba2+ as in
Ca2+; the L-type Ca2+
channel is more permeable to Ba2+ and has a
dramatically slower inactivation time in Ba2+
than Ca2+. To clarify whether both types of
Ca2+ currents can be identified in guinea pig
atrial myocytes, we carried out tests under the conditions in which
extracellular Na+ ions were replaced by
impermeable TEA+ ions, and 5 mmol/L
BaCl2 was added in place of
Ca2+. Sodium removal induced cell contracture,
but under our conditions with EGTA 10 mmol/L and BAPTA 2
mmol/L in the patch pipette, the cell attached to the patch electrode
survives, probably owing to the diffusion of EGTA and BAPTA into the
cytosol. The cells were held at -40 or -80 mV (Figure 6A), and command voltage steps (320 ms in
duration) were applied to various membrane potentials. The
current-voltage relationships of the peak inward current are shown in
Figure 6C. At a holding potential of -40 mV, the inward current
was elicited at positive potentials to -30 mV (Figure 6A, right). A small fraction of current was inactivated at the
command pulses to -20 and -10 mV. Conversely, when the cell was held
at -80 mV, the transient inward current was recorded at a command
potential of -30 mV and was overlapped on the
noninactivated component at a command potential of -20 mV
(Figure 6A, left). The current traces subtracted from the
current of a holding potential of -80 mV to that of a holding
potential of -40 mV at command potentials of -30, -20, and +0 mV are
shown in Figure 6B. The transient inward current rapidly
inactivated within 50 ms and could be discriminated from
the sustained component. Cd2+ 1 mmol/L
abolished both types of inward current, but nifedipine
1 µmol/L (data not shown) failed to inhibit the transient
component. These findings suggest that both types of
Ca2+ currents exist in guinea pig atrial
myocytes. The fast inward current consisted of
ICa,T, and the slow component consisted
primarily of ICa,L. Figure 7 shows the effects of troglitazone on
both types of Ca2+ currents.
ICa,T and ICa,L
were elicited at a command voltage to -30 and +10 mV from a
holding potential of -80 mV, respectively. Troglitazone 10
µmol/L inhibited both types of Ca2+ currents
(Figure 7A and 7B) but inhibited
ICa,L more effectively than
ICa,T (Figure 7B).
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) and -80 mV (
). Note
that transient component (arrows) was observed at command steps (-40
and -20 mV) from a holding potential of -80 mV.
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Effects of Troglitazone on the Kinetic Parameters of
the Voltage-Dependent ICa,L
Figure 8 shows the effects of
troglitazone on the inactivation time courses of
ICa,L. Under conditions in which the cell
was perfused with normal Tyrode's solution, the inactivation time
courses of ICa,L were well fitted by the
sum of 2 exponentials (Figure 8A and
Table) as previously
described.34 Troglitazone 10 µmol/L did not affect
the time courses of inactivation of ICa,L
significantly (Figure 8 and Table). The differences
between the values of 
1 and
2 in the control and those in the presence of
troglitazone were not statistically significant.
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The use-dependent block of troglitazone was also examined and compared
with the classic Ca2+ antagonists
verapamil and nifedipine as shown in Figure 9
. The changes in the amplitude of
ICa,L elicited by successively applied
command pulses were measured in the absence or presence of each drug
with a test depolarizing pulse to +0 mV from a holding potential of
-40 mV at 0.2 Hz. The amplitude of ICa,L
recorded by the last pulse of a train stimulation before
application of the agents (Figure 9A through D) was
normalized to 1.0. In control conditions (Figure 9A), the
amplitude of ICa,L elicited by the first
command pulse (b) was not inhibited and remained nearly constant during
the successive repetitive pulses (c). The small decrease of the current
(8±3% of the first pulse, n=5) during 1-minute application of
repetitive stimulation was thought to be induced simply by
Ca2+ channel rundown. Verapamil
(1 µmol/L, Figure 9B) produced very little inhibition of
Ca2+ current in the absence of test pulses (b),
but blockade increased with repeated depolarizations (c). Conversely,
in studies with the same pulse protocol, nifedipine
blockade was different (Figure 9C). The first current after the
quiescent period gave a good estimate of the final level of blockade (b
and c). Figure 9D shows the use-dependent effects of
troglitazone 10 µmol/L. As in the case of
nifedipine, the inward current elicited by the first
command pulse after a 2-minute quiescent period was
consistently inhibited by 63±10% (n=5, P<0.01),
which was different from that recorded with verapamil.
With the repetitive stimulations, the inward current decreased
slightly, by 13±7% (n=5) from the first pulse during repetitive
stimulation (Figure 9D, b), but could not discriminate the
simple rundown of the Ca2+ channel. These results
suggest that troglitazone inhibited ICa,L
mainly in a use-independent manner.
The influence of the holding potential on the inhibitory
effects of nifedipine and troglitazone was compared as
shown in Figure 10. In these
experiments, the command voltage steps (320 ms in duration) to +10 mV
from a holding potential of -40 or -80 mV, where the
voltage-dependent Ca2+ currents consisted
primarily of ICa,L, were applied. The peak
amplitude of ICa,L in the absence of drugs
was normalized to 100. The percentage inhibition induced by
nifedipine and troglitazone is shown in Figure 10B.
Nifedipine 1 µmol/L reduced the amplitude of
ICa,L by 96±4% at a holding potential of
-40 mV, whereas it inhibited it by only 34±8% at a holding potential
of -80 mV. Conversely, troglitazone 10 µmol/L inhibited
ICa,L by 70% at each membrane
potential. Furthermore, the effects of troglitazone and
nifedipine on the voltage-dependent availability of L-type
Ca2+ channels were examined by means of
double-pulse protocols (Figure 11). The
test pulse to +10 mV from a holding potential of -80 mV was preceded
by a 3-second conditioning pulse to various membrane potentials. The
relationships between membrane potentials and the
f
value in the absence and presence of the
drug were fitted by the following Boltzmann equation using the
least-squares method:
f(V)=fmax/{1+exp[(V-a)/b]},
where fmax is the maximal value of
f (in control conditions, the value of
fmax=1), V is membrane potential in mV, a is
membrane potential at 1/2 fmax, and b is slope
factor. In the absence of the drug, fmax=1,
a=-21.2 mV, and b=5.13 mV. In the presence of nifedipine
1 µmol/L, fmax=0.68, a=-37.0 mV, and
b=6.6 mV (Figure 11A). Thus, nifedipine decreased
the maximal Ca2+ channel availability (0.69±0.05
of the control, n=5), with a significant shift of the curve toward the
negative (-18±4 mV, n=5). Conversely, in the absence of troglitazone
10 µmol/L, fmax=1, a=-21.3 mV, and
b=5.33 mV. In the presence of troglitazone,
fmax=0.39, a=-22.3 mV, and b=6.0 mV (Figure 11B). Thus, troglitazone reduced the maximal
Ca2+ channel availability (0.36±0.1 of control,
n=5) but did not show any significant shift of the voltage-dependent
inactivation curve (-23.4±3.4 mV in the control versus -25.9±5.0 mV
in the presence of troglitazone, n=5, P=NS).
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Discussion |
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We have demonstrated here that troglitazone had inhibitory effects on voltage-dependent Ca2+ currents in single atrial myocytes from the guinea pig. The inward Ba2+ current in place of Ca2+ could easily be divided into 2 components with distinct physiological properties, as described in mammalian cardiac myocytes.30 31 32 33 One component had characteristics identical to the ICa,L, including slow inactivation (Figure 6
) and sensitivity to dihydropyridine
(Figures 9 and 10) and isoproterenol (Figure 4).
The second component, ICa,T, had a fast
inactivation (Figures 6 and 7) and small amplitude
(-100 pA) even in the presence of Ba2+ and
insensitivity to dihydropyridine
(nifedipine 1 µmol/L) and isoproterenol (data not
shown). We found that troglitazone inhibited both types of
voltage-dependent Ca2+ currents in atrial
myocytes, although it inhibited ICa,L more
effectively than ICa,T. Thus, the effects
of troglitazone on Ca2+ currents might not be
restricted to L-type Ca2+ channels, in comparison
with the classic Ca2+ channel
antagonists nifedipine and
verapamil, because nifedipine and
verapamil 1 µmol/L did not inhibit
ICa,T significantly (data not shown).
Troglitazone 1 µmol/L reduced ICa,L
by 10% to 20% within 2 minutes of application, and 10 µmol/L
troglitazone reduced it by 60% to 80%. Troglitazone 30 µmol/L
almost completely abolished ICa,L, and the
IC50 value was estimated at 6.3 µmol/L.
The inhibitory potency of troglitazone on
ICa,L was less than that of
nifedipine and verapamil. However, because the
therapeutic plasma concentration of troglitazone was estimated to be
0.6 to 2.7 µmol/L,35 these concentrations are
nearly the same as those required for the inhibition of
ICa,L in this study. Thus, troglitazone may
affect cardiac function by inhibiting the channel. The direct evidence
showing that troglitazone inhibits ICa,L
has been shown in vascular smooth muscle cells.25 26
The IC50 of troglitazone on
ICa,L of vascular smooth muscle cells was
3 µmol/L,26 which was relatively lower than
that in the present study. However, we conclude that troglitazone
inhibited the L-type Ca2+ channels in cardiac
myocytes as well as vascular smooth muscle cells in therapeutic
concentrations.
It has been reported that voltage-dependent L-type
Ca2+ channel blockers such as
verapamil and diltiazem prevent the development of diabetic
cardiomyopathy.16 17 18 These
cardioprotective effects of Ca2+-blocking drugs
have also been reported in Syrian cardiomyopathic
hamsters36 and in patients with hypertrophic
cardiomyopathy.37 Therefore, the mode
of action of troglitazone on ICa,L was
compared with that of the classic Ca2+
antagonists verapamil and
nifedipine. As shown in Figures 10 and 11,
troglitazone reduced ICa,L but did not
cause a significant shift in the steady-state inactivation curve.
Conversely, nifedipine, a
dihydropyridine Ca2+
antagonist, which has a high affinity for the
inactivated state of the channel but much less affinity for
other states (eg, closed, open), showed strong voltage-dependent
effects and caused a distinct negative shift of the steady-state
inactivation curve. Thus, it is unlikely that troglitazone inhibits
ICa,L by preferentially binding the
inactivated states of the channels. Also, troglitazone did
not exhibit significant use-dependent characteristics, which was
different from verapamil (Figure 9), as previously
described.38 Potencies of the use-dependent
inhibition might be closely related to the ionization constants of the
drug as shown by Sanguinetti and Kass.39 According to this
model, charged forms of the drug can reach their receptors inside the
channel by a hydrophilic pathway available only when the channel gates
are open and hence are characterized by a significant use-dependent
block. In contrast, an uncharged form of the drug easily reaches its
receptors via a hydrophobic region of the membrane without channel
opening and thereby does not show significant use-dependent effects.
Verapamil (pKa=8.7) is almost
entirely in the charged form at pH 7.4, whereas troglitazone
(pKa=6.1)40 exists almost entirely
in the neutral form at the same pH. Thus, opening of the channels may
not be necessary for troglitazone to affect
ICa,L, as shown in Figure 9.
Furthermore, the time courses of Ca2+ current
decay were little affected by troglitazone. From these observations,
troglitazone did not appear to inhibit the Ca2+
channels by binding to activated Ca2+
channels. Thus, the mechanisms by which troglitazone affects the
voltage-dependent Ca2+ channels are unknown at
present, but troglitazone may interact with L-type
Ca2+ channels in a manner distinct from the
classic Ca2+
antagonists.41
The present study indicates that troglitazone inhibited the voltage-dependent Ca2+ currents (ICa,L and ICa,T) in cardiac myocytes in therapeutic concentrations. Under normal circumstances, the current through the T-type Ca2+ channel is unlikely to be very important in atrial and ventricular myocytes, because in a well-polarized cell, such as atrial and ventricular cells, Na current is much larger and activates in a similar voltage range. Also, because L-type Ca2+ channels inactivate more slowly, they are likely to be more important than T-type channels. However, T-type Ca2+ channels may contribute to the generation of pacemaker activities in pacemaker cells42 and may make hypertrophied ventricular myocytes more prone to spontaneous action potentials and increase the likelihood of arrhythmia in partially depolarized hypertrophied myocardium.43 Troglitazone may affect the electrical activities under these conditions by inhibiting ICa,T. Conversely, troglitazone inhibited ICa,L more effectively than ICa,T. The inhibitory effects of troglitazone on ICa,L did not show significant voltage- and use-dependent properties as observed in classic Ca2+ antagonists.38 From these unique actions, troglitazone may inhibit cardiac Ca2+ channels in a similar way in well-polarized cells as well as in partially depolarized cells. Also, it may antagonize the effects of isoproterenol on ICa,L. Several studies have shown that in diabetic animals, the duration of action potential in cardiac myocytes is markedly longer, whereas the resting membrane potential is not altered.9 10 11 12 In addition, an augmented number of Ca2+ antagonist receptor binding sites and an increase of voltage-dependent L-type Ca2+ channels have been reported in diabetic hearts.13 14 The increased influx of Ca2+ through the voltage-dependent Ca2+ channels may cause Ca2+ overload, which appears to be linked to the cardiac pathology in diabetic cardiomyopathy.16 17 18 The present study shows that troglitazone inhibits voltage-dependent Ca2+ currents (ICa,T and ICa,L) and then antagonizes the effects of isoproterenol in cardiac myocytes, which may play a role in preventing diabetes-induced intracellular Ca2+ overload and then myocardial changes. In fact, recent studies have shown that troglitazone attenuates high-glucose–induced abnormalities in relaxation and intracellular calcium in rat ventricular myocytes23 and improves cardiac function in diabetes mellitus.24 From these observations, troglitazone may be a unique agent for diabetic cardiomyopathy, but further studies are needed to clarify this possibility in diabetic patients.
Received October 5, 1998; revision received February 23, 1999; accepted March 9, 1999.
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