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Introduction
Ibuprofen (Fenbid) is a non-steroid anti-inflammatory
drug (NSAID) often used to relieve fever and the symptoms
of arthritis. Low doses of ibuprofen (200_1200 mg/d) are
available over the counter in many countries as an analgesic.
Its mechanism of action is believed to non-selectively
inhibit both variants of cyclooxygenase (COX-1 and COX-2)
and thus block prostaglandin synthesis. However, unlike
aspirin, which also blocks both COX isoforms and prevents
blood clotting, ibuprofen does not have antiplatelet effects
and thus is not routinely used to prevent cardiac infarction.
The wide use of ibuprofen attributes to its lowest
incidence in causing gastrointestinal dysfunction, a side-effect
common to all non-selective COX inhibitors. Despite this
advantage, the recent withdrawal of selective COX-2
inhibitor Vioxx (Merck & Co, Inc, Whitehouse Station, New
Jersey, USA). from the US market due to its cardiac
toxicity brought all COX inhibitors under the spotlight and raised
the question as to whether they could lead to elevated
cardiovascular risk.
The initial study of potential cardiac toxicity in an
ibuprofen cohort identified significantly higher rates of
life-threatening ventricular arrhythmias and cardiac arrest than
those in the control naproxen cohort[1]. To date, with the
extensive use of ibuprofen, the number of frequent
ventricular premature beats, atrial
fibrillation[2,3], and other untoward cardiac reactions have been reported as increasing
steadily[4_6].
In spite of these clinical studies and case reports, very
little work has been done to characterize the
electrophysiological effects of ibuprofen on the animal heart. In the
present study, we used isolated guinea pig hearts to identify
the arrhythmiogenic effects of ibuprofen on the
electrocardiogram (ECG); we examined its effects
on the fast- and slow-response cardiac action potentials (AP), and analyzed its
dynamic process of combination and dissociation with the
Na+ channels. In conclusion, our results demonstrate for
the first time that ibuprofen, a non-selective COX inhibitor,
may induce cardiac arrhythmias by shortening the effective
refractory period (ERP) and decreasing the excitation
propagation.
Materials and methods
Solution preparation In total, 300 mg ibuprofen was
dissolved in 5 mL DMSO to make a stock solution of 60 mg/mL.
The stock solution would be further diluted to the designed
concentrations in the Tyrode's solution. The normal Tyrode's
solution for perfusing the cardiac ventricular cells of guinea
pigs consisted of the following (in mmol/L): 137 NaCl, 23
NaHCO3, 0.5 MgCl2, 5.4 KCl, 1.8
CaCl2, 0.4
NaH2PO4, and 10 glucose, pH 7.4±0.05. The Tyrode's solution for the rabbit
sinus node preparations consisted of the following
(mmol/L): 140 NaCl, 1.0 MgCl2, 5.4 KCl, 1.8
CaCl2, 5 HEPES, and 10 glucose, pH 7.4±0.05. Both solutions were gas-saturated by
95% O2+ 5% CO2 and 100%
O2, respectively.
ECG recording and measurement in vivo Guinea pigs of either sex (weighing approximately 500 g) were
anesthetized by an intraperitoneal injection (ip) of urethane (20%,
5 mL/kg). Needle electrodes, inserted subcutaneously, were
used to record the ECG of standard limb lead II. The RR
interval, QRS duration, QT interval, and heart rate variation
(HRV) were measured, respectively. The corrected QT
interval (QTc) was calculated with the Bazett formula
(QTc=QT×RR-1/2) to reduce the influence of the
fluctuation of the heart rate on the measurement of the QT interval.
Different concentrations of ibuprofen were injected into
the external jugular vein to evaluate its effects on the heart.
ECG recording in isolated heart The hearts of the
anesthetized guinea pigs were quickly taken out of the chest
and perfused with Langendorff perfusion set-up at a
recorded pressure of 80 cmH2O. The temperature of the
Tyrode's solution was maintained at 37±0.5 °C. Three
platinum electrodes placed on the cardiac apex, right atrium,
and aortic root were used to record the ECG. The signals
were documented on computer using the PowerLab system (PowerLab ML135, ML 785, ADInstruments Castle
Hill, NSW, Australia). The RR interval, QRS duration, QT
interval, and QTc were analyzed.
Preparation of guinea pig papillary muscles Guinea pigs of either sex (weighing approximately 250~300 g) were
sacrificed by venesection under deep anesthesia with an
injection of sodium pentobarbital (35 mg/kg, ip). The hearts were
rapidly removed into the dissection chamber filled with the
Tyrode's solution. The right ventricular papillary muscles
were excised and pinned to the bottom of a recording chamber.
The recording chamber was perfused with the Tyrode's
solution at a constant rate of 3 mL/min and maintained at a
temperature of 37±0.5 °C.
Recording of fast-response AP Bipolar platinum
electrodes were used to drive the preparations with rectangular
current pulses at a stimulation frequency of 1 Hz. Each pulse
lasted for 0.1 ms, with the amplitude approximately 1.5 times
the threshold value. After 30 min stimulation, transmembrane
AP were recorded by a conventional glass
microelectrode, which was filled with 3 mol/L KCl and had a tip resistances of
15_20 MΩ. The records were sampled and stored in the
computer through the amplifier (MEZ8201, Nihon Kohden,
Tokyo, Japan) and PowerLab interface (PowerLab ML785,
ADInstruments, Australia)[7].
The calculated parameters for the fast-response AP
included the resting potential (RP), AP amplitude (APA),
maximum upstroke velocity of phase 0
(Vmax), AP duration at 50% and 90% repolarization
(APD50 and APD90), and the ERP.
The preparations were driven by a series of 8 stimuli
pulses at a frequency of 1 Hz. Then an additional pulse was
added following the eighth one. Adjusting the time interval
between the eighth and the extra stimulation pulse, the
minimum interval for the evoked AP by the extra stimulus would
be calculated as the ERP.
Measurements of time constants for ibuprofen combined
with and dissociated from fast Na+ channels The preparations were stimulated at a frequency of 1, 2, and 3 Hz,
respectively. For each frequency, the stimulation would last
until the Vmax decreased gradually to a steady state, which
could indicate the dynamics of the combination of ibuprofen
with the fast Na+ channels after repetitive activation of the
channels. The first-order exponential decay equation was
employed to fit the relationship between the decreased Vmax values and the duration of the stimuli. The time constant
(τon) thus obtained is the index of the combination dynamics
of ibuprofen with the fast Na+ channels. Origin 6 software
(Microcal Software, Northampton, MA, USA) was used to
analyze the data.
With a similar protocol, the preparations were activated
for 30 s at a frequency of 1, 2, or 3 Hz, respectively. After the Vmax for each frequency decreased to a steady state, a
various delayed supra-threshold stimulus was added to invoke
an additional AP, and the Vmax of the first evoked AP with
various delays were measured. As the drug needs time to
dissociate from the channels, it was found that the longer
the retardation time for evocation, the greater the Vmax of the evoked AP. The relationship of the Vmax value and the retardation time was fitted by the first-order exponential decay
equation. The time constant τoff) would represent the
dissociation index of ibuprofen from the
Na+ channels.
Recording of slow-response AP of guinea pig papillary
muscles When the preparations were perfused with the
Tyrode's solution containing 27 mmol/L KCl, the resting
potential would gradually change to approximately -45 mV.
The Na+ channels would be inactivated at this membrane
level. After 0.4 mg/L isoprenaline was added into the Tyrode's
solution and a stronger stimulus was employed to the
preparation, the slow-response AP, which were evoked by
the slow inward current of the Ca2+ rather than the
Na+ channels, could be
initiated[8].
Spontaneous AP of rabbit sinus node cells Adult rabbits of either sex were anesthetized by injection of sodium
pentobarbital (1%, 3 mL/kg). The hearts were removed to a
chamber containing the Tyrode's solution. The sinus nodes were
excised from the heart and pinned to the bottom of the
recording chamber, which was perfused with the special
Tyrode's solution for sinus nodes. An intracellular
microelectrode (containing 3 mol/L KCl with a tip resistance
of 25 MΩ) was employed to record the spontaneous AP of the
sinus node cells. The parameters of AP were sampled with
the same method as described in fast-response AP. The
spontaneous beating rate, APA, APD, Vmax and spontaneous depolarization rate of phase 4 (SDR) were calculated,
respectively.
Statistical analysis All of the data were sampled by Chart
5 software (ADInstruments, Australia). Origin 6 was used
for the statistical analysis. The results were expressed as
mean±SD. Statistical significance was determined by
Student's t-test for paired data. P<0.05 was considered
statistically significant.
Results
ECG from anesthetized guinea pigs ECG of standard
limb lead II from urethane-anesthetized guinea pigs were
recorded. Every 10 min, ibuprofen was consecutively
injected into the external jugular vein to reach a concentration
of 2, 5, 10, 20, 25 mg/kg, respectively (Table 1; Figure 1A).
After 25 mg/kg ibuprofen, ECG exhibited a marked increase
of the QRS duration (from 13±1 ms to 20±1 ms vs control) and RR intervals (from 230±4 ms to 268±5 ms) with percentage
changes of 53.8% and 16.5%, respectively. However, the
QTc decreased from 271±4 ms to 230±4 ms by a significant
change of 15.1%. A HRV analysis showed that the main
parameters of HRV (including the standard deviation of the
RR intervals (SDNN), the low frequency power density
component (LF), the high frequency power density component
(HF), LF/HF ratio (LF/HF)] were not changed significantly
after an intravenous injection of ibuprofen. In order to exclude the possible effect from DMSO, we performed the 5
control experiments, with an intravenous injection of DMSO
(417 µL/kg) alone, 50 min after the injection of DMSO, QRS
(ms), RR (ms), QT (ms), and QTc were measured. These
did not change significantly on the ECG of the guinea
pigs: 13.7±0.7, 213±7, 119±11, and 259±14 to 13.8±0.6, 206±9,
123±13, and 272±18, respectively. The results indicate that
DMSO has no significant effect on the ECG of guinea pigs in vivo (P>0.05).
Premature contractions and ventricular fibrillations
occurred in some animal experiments. Ventricular
fibrillations occurred in 2 of 6 guinea pigs, which later recovered
with an injection of a low dosage of ibuprofen (5 or 10
mg/kg); 25 mg/kg ibuprofen induced fatal ventricular
fibrillations in another two guinea pigs (Figure 1B).
Effects of ibuprofen on ECG in isolated hearts The isolated guinea pig hearts were perfused with the Tyrode's
solution. Every 10 min, the heart was consecutively
perfused with ibuprofen at concentrations of 5, 10, 20, 40, and 80
mg/L respectively, while the ECG was recorded. The
results in Table 2 show that like the in
vivo experiments, ibuprofen could prolong the duration of the QRS wave and
the RR interval (Figure 2A). Although ibuprofen decreased
the heart rate, it shortened the QT interval, especially the
QTc. When the hearts were exposed to 20 mg/L ibuprofen,
some ECG showed arrhythmias (especially ventricular
premature contractions), but most disappeared within 30 min.
With 40 mg/L ibuprofen, ventricular fibrillations occurred
in 3 of 7 experiments and recovered later after washing out
with the control Tyrode's solution. In another experiment
with a higher concentration of ibuprofen (80 mg/L), 2 hearts
stopped beating after sustained ventricular fibrillations
(Figure 2B).
Effects of ibuprofen on fast-response AP of guinea pig
papillary muscles In order to get a stable recording of
fast-response AP, the papillary muscles of the guinea pigs were
stimulated at a frequency of 1 Hz for approximately 30 min.
Then the preparations were perfused consecutively with 10,
20, 40, and 80 mg/L Tyrode's solution containing ibuprofen.
Each performance lasted for 10 min. The results are shown in
Figure 3 and Table 3. Ibuprofen can dose dependently shorten
the APD50 by 30.9% (from 165.8±12.5 ms to 114.5±6.4
ms), APD90 by 25.6% (from 203.4±14.3 ms to 151.3±9.8 ms), Vmax by 14.7% (from 225.4±11.3 V/s to 192.2±9.6 V/s), and ERP by
24.3% (from 186.6±11.2 ms to 141.3±8.3 ms), respectively.
No obvious influence of ibuprofen on RP and APA were
observed. When the preparations were washed out with the
control Tyrode's solution for 30 min, all the changes could
obviously be reversed.
Dynamics of ibuprofen combined with and dissociated
from the fast Na+ channels Ibuprofen can inhibit the Na+ channels; the Vmax of the fast-response AP is dramatically
depressed by ibuprofen (Table 3). In a series of stimuli with
frequencies lasting approximately 30 s, the value of the Vmax will decrease from the first evoked response, then gradually
diminish into a steady state (Figure 4). With the different
concentrations of ibuprofen (20 and 40 mg/L for 15 min,
shown in the lower two rows of the Figure 4), the
stimulation rate was increased from 1 to 2 or 3 Hz for more than 30 s
for each frequency. The combination time constant
(ton) of ibuprofen with the
Na+ channels was calculated and shown in Table 4. Our results show that the higher the
concentration of ibuprofen used, the shorter the time constant of ton; the higher the stimulus frequency, the shorter the time
constant of ton (Figure 4; Table 4). These results
indicate that ibuprofen is much easier to combine with the
Na+ channels at a high stimulation frequency than that at a
lower one.
The dissociation time constant is longer than that of the
combination ones for each different stimulation frequency
(Table 4). As the ibuprofen concentration or the stimulation
frequency increased, the dissociation course became much
faster.
Effects of ibuprofen on slow-response AP After the slow-response AP of papillary muscles were recorded, the
preparation were consecutively perfused with 5, 10, 20, 40, and 80
mg/L ibuprofen contained in Tyrode's solution at an
interval of 10 min for each concentration, as shown in Figure 5.
The results in Table 5 show that ibuprofen (80
mg/L) could attenuate APD50 (from 118.6 ms to 85.3 ms),
APD90 (from 131.9 ms to 98.5 ms), Vmax (from 31.3 V/s to 10.2 V/s),
and ERP (from 226.2 ms to 154.6 ms), with percentage changes
of 28.1%, 25.3%, 67.4%, and 31.7%, respectively. No
substantial alterations were observed regarding RP and APA.
Effects of ibuprofen on spontaneous AP The
spontaneous AP were recorded in rabbit sinus node preparations.
The sinus node were exposed to 5, 10, 20, 40, and 80
mg/L ibuprofen consecutively. The results in Table 6 demonstrate
that 80 mg/L ibuprofen decreases the Vmax by 40.2%, the beating rate by 12.4%, and the SDR by 38.5%, respectively.
These effects of ibuprofen are dose dependent and
reversible after washing out with control Tyrode's solution.
Figure 6 shows the effects of ibuprofen on the spontaneous AP
of sinus nodes.
Discussion
NSAIDs are chemicals that present analgesic and
anti-inflammatory effects, and most of them, including aspirin
and ibuprofen, act as non-selective inhibitors of
prostaglandin synthesis enzymes COX-1 and COX-2. By contrast,
selective COX-2 inhibitors specifically block the production of
the type of prostaglandins responsible for pain and
inflammation. Hence, it was believed that selective COX-2
antagonists had more curative and fewer side-effects.
Ever since Merck withdrew its COX-2 antagonist Vioxx, a
$2.5 billion/year sales blockbuster, because of high
cardiovascular risk, the safety concerns over all COX inhibitors
have received wide attention across the entire
pharmaceutical industry. The cardiotoxicity of COX-2 inhibitors has been
considered to result from the suppression of prostaglandin
synthesis in endothelial cells[9,10]. Reduced prostaglandin in
blood vessels would prevent vasorelaxation and facilitate
clot formation, and thus predispose patients with existing
heart diseases higher chances of myocardial
infarction[11,12]. Although a similar association was obtained in other large
population studies, in which high doses of traditional
non-selective COX inhibitors were used to compare with
placebo controls[13_15], the underlying mechanisms remain
unknown.
In the present research, we studied the effects of ibuprofen
on fast- and slow-response AP in guinea pig papillary
muscles, on the spontaneous AP of rabbit sinus nodes, and
the ECG of guinea pigs in order to elucidate its cardiac
side-effects. During both the in vitro and in vivo ECG recording, ibuprofen prolonged the duration of the QRS complex wave
and inversely shortened the QTc. The duration of QRS
represents the propagating velocity of excitation in the heart.
We also found that at a low dosage of ibuprofen (10_20
mg/L), certain types of arrhythmias, such as premature ventricular
contraction and short duration ventricular fibrillation, could
occur, and these events were reversible after ibuprofen was
washed out with control solution. At a high dosage of
ibuprofen (40_80 mg/L), 2 of 8 hearts stopped beating
because of long-lasting ventricular fibrillations. These
observations suggest that ibuprofen exhibit detrimental effects
on heart function.
Next, we investigated the effect of ibuprofen at the
cellular level, that is, its influence on cardiac AP. Figures 3
and 5 demonstrate that ibuprofen could dose dependently
suppress Vmax in fast-response AP by 14.7%, and decrease
the Vmax of slow-response AP by 67.4%, suggesting that
ibuprofen is able to block the fast Na+ channel and the slow
Ca2+ channel. Tables 3 and 5 further show that ibuprofen
could not only decrease the fast and slow AP duration
(APD50) by 30.9% and 28.1%, but also shorten the
effective refractory periods by 24.3% and 31.7%, respectively.
In sinus node cells, we found that ibuprofen decreased the
SDR, slowing the heart rate in 6 experiments.
Interestingly, our data also imply that ibuprofen may
directly interact with the cardiac Na+ channel. The changes of
the association and dissociation time constants
(τon and τoff) in Figure 4 provide evidence that the drug molecule may
interact and inhibit the Na+ channel quicker as its
concentration increase or during fast heart rates. The results
presented in Tables 2, 3, 5, and 6 indicate that these alterations
could not be reversed completely to the baseline after wash
out. We believe that this effect may be induced by the
prolonged exposure to high concentrations of ibuprofen during
recording, lack of solvent DMSO in the wash solution, or a
higher affinity of ibuprofen to the Na+ channel
(τon<τoff ), as
illustrated in Table 4.
It might be speculated that ibuprofen could dose
dependently suppress the Na+ and
Ca2+ channels, and decrease the excitation spreading within the heart. The slower
conduction is displayed on the ECG by the prolongation of the
QRS wave. The decrease of the APD in AP recording could
explain the shortening of the QTc produced by ibuprofen on
ECG.
It is well known that slow conduction, shortening of the
refractory period, and unidirectional blocking are the 3
important preconditions for re-entry that results in fibrillations
in the heart. Since ibuprofen could slow conduction and
decrease the refractory period, it might therefore lead to
arrhythmias in clinical practice.
On the contrary, some reports indicate that NSAIDs,
such as salicylic acid (SA), can reduce ventricular
dysfunction and arrhythmias[16_18]. The investigators proposed that
ibuprofen could prevent the atrial fibrillation mediated by
inflammation, and SA could trap the hydroxyl radicals to
reduce postischemic ventricular arrhythmias. Yet, those
experiments were conducted on patients with heart diseases
or on isolated ischemic hearts. By contrast, our study was
performed on healthy animals and heart preparations.
Nevertheless, they found that only low concentration of SA
(0.5 and 1.0 mmol/L) prevented the risk of arrhythmias; at
higher concentrations, the drugs lost their protective
functions in the trials. It is known that anti-arrhythmia drugs are
2-edged swords; they can be used to treat one type of
arrhythmia, but at the same time become pro-arrhythmic to
another. It is also known that their arrhythmiogenic effects
depend on, to some extent, the pathophysiological
conditions of the diseased heart. Taken together, the results
presented in this paper suggest that patients with existing heart
diseases should cautiously take NSAIDs, unless they are
under close observation of the side-effects.
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