inhibition of delayed rectifier potassium channels and induction of arrhythmia

25 Pages·2007·2.8 MB·English

by  Satpal Singh

JBC Papers in Press. Published on November 5, 2007 as Manuscript M708100200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M708100200
INHIBITION OF DELAYED RECTIFIER POTASSIUM CHANNELS AND INDUCTION OF
ARRHYTHMIA - A NOVEL EFFECT OF CELECOXIB AND THE MECHANISM
UNDERLYING IT
Roman V. Frolov1, Ilya G. Berim2 and Satpal Singh1
From the Departments of 1Pharmacology and Toxicology, and 2Medicine
State University of New York at Buffalo, Buffalo, New York 14214, USA.
Running Title: K+ Channel Mediated Arrhythmia Induced by Celecoxib
Address correspondence to: Satpal Singh, Department of Pharmacology and Toxicology, 102 Farber Hall,
State University of New York at Buffalo, Buffalo, New York 14214-3000. Telephone: [phone],
Fax: [phone], Email: [email protected]

Selective inhibitors of cyclooxygenase-2 attributed to a reduction in antithrombotic, COX-
(COX-2), such as rofecoxib (Vioxx), celecoxib 2-derived, prostacyclin (PGI ) without a reduction
2
(Celebrex) and valdecoxib (Bextra), have been in prothrombotic, COX-1-derived, platelet
developed for treating arthritis and other thromboxane. This shifts the balance towards a
musculoskeletal complaints. Selective inhibition prothrombotic state resulting in clot formation (7).
of COX-2 over COX-1 results in preferential The adverse effects of drugs like celecoxib
decrease in prostacyclin production over arising out of their selective inhibition of COX-2
thromboxane A2 production, thus leading to is the topic of intense current discussion (7,8). We D
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less gastric effects than those seen with non- now show that celecoxib can have additional, n
lo
selective COX inhibitors such as acetylsalicylic heretofore-unanticipated, effects on Drosophila a
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acid (Aspirin). Here we show a novel effect of heart and rat heart cells via a pathway independent d
celecoxib via a mechanism that is independent of cyclooxygenase inhibition. At low micromolar from
of COX-2 inhibition. The drug inhibited the concentrations, celecoxib reduced heart rate and h
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delayed rectifier (Kv2) potassium channels induced pronounced arrhythmia in Drosophila ://w
from Drosophila, rats and humans, and led to heart. A similar effect was observed in rat w
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pronounced arrhythmia in Drosophila heart cardiomyocytes in culture where the drug reduced .jb
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and arrhythmic beating of rat heart cells in the beating rate in clusters of cells and made the .o
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culture. These effects occurred in spite of the beating arrhythmic. These effects were not b/
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genomic absence of cyclooxygenases in mediated via cyclooxygenase inhibition but g
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Drosophila and the failure of acetylsalicylic involved inhibition of the voltage-activated es
acid, a potent inhibitor of both COX-1 and delayed rectifier K+ channels (Kv2) by the drug. t on
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COX-2, to inhibit rat Kv2.1 channels. A These observations reveal a new molecular o
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genetically null mutant of Drosophila Shab mechanism underlying the effects of celecoxib that m
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(Kv2) channels reproduced the cardiac effect of may operate in addition to its prothrombotic er 2
celecoxib, and the drug was unable to further influence. The data also raise the possibility of 4, 2
enhance the effect of the mutation. These effects on other tissues that express Kv2 channels. 01
8
observations reveal an unanticipated effect of
EXPERIMENTAL PROCEDURES
celecoxib on Drosophila heart, and on heart
cells from rats, and the inhibition of Kv2 Materials – Quinidine, 4-AP, acetylsalicylic
channels as the mechanism underlying this acid and other chemicals were purchased from
effect. Sigma Chemical. Celebrex capsules were used as
the source of celecoxib so as to examine the effect
Selective COX-21 inhibitors, or coxibs, were of the actual formulation used in clinical settings
developed for use as non-steroidal anti- (9). Celecoxib (4-[5-(4-methylphenyl)-3-
inflammatory drugs (NSAIDs) without adverse (trifluoromethyl)-1H-pyrazol-1-
gastric effects (1,2). Gastric effects of non- yl]benzenesulfonamide) from Celebrex capsules
selective cyclooxygenase inhibitors, such as containing 200 mg of the drug was dissolved in
acetylsalicylic acid (Aspirin), arise in main part dimethyl sulfoxide (DMSO). Additional details are
from inhibition of cyclooxygenase-1. A selective given in Supplemental Experimental Procedures.
inhibition of COX-2 by coxibs helps in avoiding Drosophila heart preparation – Strains of
these effects (3,4). However, the use of coxibs has Drosophila melanogaster were maintained at
been complicated by adverse cardiovascular 21°C. Activity of the heart was monitored at 21˚C
effects (5-7). These effects of coxibs have been using a larval preparation described previously
Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
(10). Heart rate (HR) was counted for 6 min before Scientific, Inc.). Recordings from rat Kv2.1
drug application (the basal rate), for 10 min in the channels were made in flow of a drug solution
presence of the drug, and for 10 min after within 10 sec from the start of perfusion.
washing-out the drug. Video recordings of heart A clone for human Kv2.1 channels in
activity were made at a resolution of 640 x 480 pTracerSV40 vector was provided by Dr. T.
pixels at 30 frames per second. Analysis of video Zimmer of Friedrich Schiller University in Jena,
data is described in Supplemental Experimental Germany (14). HEK-293 cells expressing the
Procedures. channels were held at -80 mV and 500 ms test
Rat ventricular cardiomyocytes – Rat fetuses pulses were applied in 10 mV increments.
(G20 - one day before birth) from timed-pregnant Recordings in the presence of the drug were made
Holtzman rats were kindly donated by Dr. J. 5 minutes after the start of drug perfusion.
Olson. The protocols used were approved by the The experiments were performed at 21-23 °C.
Institutional Animal Care and Use Committee of Kv2.1 current was measured at the end of 500 ms
the University at Buffalo. Ventricular myocytes test-pulses as mentioned above in the case of I .
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were isolated and cultured under aseptic The currents obtained in the presence of the drug
conditions. The cardiomyocyte cultures were used were normalized to the currents obtained before
in experiments 24-48 hours after seeding the cells the drug exposure. For more details, see
when cells were beating rhythmically. Beating Supplemental Experimental Procedures.
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activity in cardiomyocytes was monitored at 37˚C. ow
RESULTS n
The activity was video-recorded for 2 min each lo
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before drug application (basal rate), at the end of We examined if celecoxib affected cardiac de
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45-min exposure to the drug, and 15 min after rhythm in Drosophila heart and rat fro
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wash-out. Additional details and analysis of video cardiomyocytes. We first examined this in h
data are given in Supplemental Experimental Drosophila, as Drosophila serves as an important ttp
Procedures. model for human cardiac pharmacology and ://w
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Recordings from Drosophila K+ channels – cardiomyopathies (10,15-18) as well as other w
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microelectrode voltage-clamping. Recordings beating within a minute of applying 30 µM and ue
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were made from muscle fiber 12 (11). Currents within seconds of exposure to 100 µM celecoxib. t o
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were elicited by 500-ms voltage steps from a Dose-response curve showed EC (50% reduction N
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holding potential of –80 mV. IKS was measured as in HR) of 11.2 µM (Fig. 1C). DMSO, used for vem
current at the end of the 500 ms testing pulse and dissolving the drug, did not affect HR at 0.5%, the be
IA was measured as the initial peak current. Dose- highest concentration used in these experiments r 24
response curve for IKS (Fig. 4E) was generated by (Fig. 1A, B). , 20
1
examining the current for voltage steps to +40 In addition to decreasing HR, celecoxib 8
mV. For more detailed current voltage affected cardiac rhythm. Each trace in Fig. 2A
relationships (Fig. 4A, C), membrane potential shows HR from one animal as it varied over time
was stepped to between –40 and +40 mV in 10- with drug application. While HR fluctuations
mV increments. The currents were divided by cell averaged out in a pool of samples (Fig. 1A), they
capacitance to obtain current densities. For more were observed in individual animals even at low
details, see Supplemental Experimental concentrations (Fig. 2A). While 1 µM celecoxib
Procedures. did not affect rhythm in all cases (Fig. 2B), it
Recordings from rat and human Kv2.1 induced intermittent arrhythmia in 5 out of 17
channels expressed in HEK-293 cells – For rat cases (Fig. 2C). Similarly, 3 µM celecoxib
channels, the pcDNA-Kv2.1 vector, provided by induced pronounced intermittent arrhythmia (Fig.
Dr. H. Y. Gaisano of the University of Toronto 2A, D), with several minutes of arrhythmic
(12,13), was expressed in HEK-293 cells. After activity followed by several minutes of relatively
establishing the whole-cell configuration, the cell regular heartbeat. Arrhythmia became so severe at
was held at -70 mV and 500 ms test-pulses were 10 µM that in 4 out of 16 animals heartbeat was
applied in 10 mV increments to potentials up to occasionally punctuated by temporary heart
+40 mV. Drugs were applied using a fast stoppages (Fig. 2E). Irregularity of HR was
perfusion system (ValveLink 8 - Automate
2
quantified for beat-to-beat interval and for in Drosophila. Thus the effects reported above in
amplitude of the movement of heart wall. Drosophila may not occur via inhibition of COX-
Coefficient of variation, a dimensionless ratio of 2.
standard deviation to mean, was used as a measure Since K+ channels play a significant role in
of variability in the rhythm and the amplitude maintaining the integrity of the cardiac rhythm
(Supplemental Fig. 1). Celecoxib significantly (29-34), we examined possible effects of celecoxib
increased variability in interbeat intervals as well on K+ currents in Drosophila muscles. The
as in amplitudes of contractions (Fig. 2F). delayed rectifier K+ current in Drosophila (I ),
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To examine the effect of celecoxib on carried by Kv2 (Shab) channels (35-37), was
mammalian heart, we used cultured blocked by celecoxib at micromolar
cardiomyocytes from rat fetuses at stage G20 (one concentrations (Fig. 4A, C). I elicited at +40 mV
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day before birth). These cells can establish a (Fig. 4D) showed an effect in a similar
spontaneous beating rhythm in culture and provide concentration range as the effect on heartbeat.
a model for cardiac function in humans (21-25). Celecoxib inhibited I with IC of 10.3 µM with
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Celecoxib reduced the rate of beating in significant inhibition starting at 1 µM (Fig. 4E).
cardiomyocytes with significant effect occurring at This compares with EC of 11.2 µM for reduction
50
3 µM and with EC of 7.7 µM (Fig. 3). This in HR. Furthermore, while I showed very little
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compares with an EC of 11.2 µM for HR inactivation in the absence of the drug, celecoxib
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decrease in Drosophila. The drug also induced increased the rate of inactivation significantly ow
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irregularity in the beat. As in the case of (Fig. 4D). lo
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Drosophila, this irregularity was more marked in Celecoxib did not inhibit the remaining two de
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individual samples (Fig. 3B) and was averaged-out voltage-activated K+ currents in the muscles. fro
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in a pool of samples (Fig. 3A). Celecoxib These are I , carried by the Shaker channels
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rat cardiomyocytes and Drosophila heart in a (Fig. 4B, C), a concentration 20-fold higher than w
similar way. the concentration that significantly affected IKS .jbc.o
While the prothrombotic effects of coxibs (Fig. 4E) and cardiac rhythm (Fig. 2F). Similarly, rg
have been attributed to selective cyclooxygenase-2 celecoxib did not inhibit IKF at 20 µM (data not by g/
inhibition, there is no report on the presence of shown). u
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cyclooxygenases in Drosophila. We probed the Mammalian Kv2.1 channels are ortholog of t o
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existence of relevant sequences in the genomic Drosophila Shab (Kv2) channels and play a N
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and the proteomic databases of Drosophila. COX significant role in cardiac function (40-45). We ve
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is composed of three units: an epithelial growth examined the effect of celecoxib on rat Kv2.1 b
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factor (EGF)-like domain, a membrane-binding channels expressed in HEK-293 cells (12). r 2
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motif, and a domain with two catalytic sites, one Celecoxib inhibited Kv2.1 current in a similar , 2
0
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for cyclooxygenase activity and one for range of concentrations as in the case of I (Fig. 8
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hydroperoxidase reaction (26,27). Nucleotide- 5A, D). IC of 10.3 µM for Kv2.1 (Fig. 5D)
50
nucleotide, protein-nucleotide and protein-protein compares with an IC of 10.3 µM for Drosophila
50
BLASTs in Drosophila melanogaster databases I and an EC of 7.7 µM for reduction of rate of
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using human COX-2 sequence elicited some beating in cardiomyocytes. As in the case of I ,
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similarity with genes and proteins characterized by celecoxib made inactivation faster for rat Kv2.1
peroxidase activity or an EGF-like domain but did channels (Fig. 5C). The current started recovering
not yield a similarity with respect to COX-2 from the effect of low drug concentrations within a
activity. Amino acids 510-535 of COX-1 and minute, and it showed less recovery during this
COX-2 sequences, constituting cyclooxygenase time frame with increasing concentrations of the
active site, are highly conserved among species drug (Fig. 5E).
(Supplemental Table 1) (27). However, BLAST To confirm if inhibition of rat Kv2.1 current
searches using human active site sequences found occurred independent of COX-2 inhibition by
no similarity in Drosophila. Also, Ser 530, a celecoxib, we used acetylsalicylic acid, which
residue irreversibly acetylated by acetylsalicylic inhibits both COX-1 and COX-2. The current was
acid, and the surrounding amino acids are virtually not reduced by 1 mM acetylsalicylic acid (Fig.
identical in all species for both isoforms of COX 5B), a concentration that is more than 400-fold
(27,28) but were not detected in BLAST searches higher than the IC of 2.4 µM for COX-2
50
3
inhibition by acetylsalicylic acid (46). Thus examined if celecoxib had any effect on human
inhibition of COX-2 could not be mediating the Kv2.1 channels. There is a strong overlap among
effect of celecoxib on rat Kv2.1 channels. Drosophila Shab, rat Kv2.1 and human Kv2.1
To further examine the link between channels from molecular, pharmacological and
celecoxib’s effect on channels and its effect on physiological points of view. For example, rat and
heartbeat, we used Drosophila, where we can human Kv2.1 channels have identical amino acid
combine the strength of pharmacology with that of sequence over the membrane-spanning region
available genetic tools. Quinidine blocks (50). At a concentration of 3 µM, celecoxib
Drosophila Shab channels in a highly selective strongly inhibited the current through human
manner (47-49). If celecoxib induced arrhythmia Kv2.1 channels expressed in HEK-293 cells (Fig.
by inhibiting these channels, then quinidine also 7). In addition, as in the case of Drosophila Shab
would induce arrhythmia. Previous studies have and rat Kv2.1 channels, the drug increased the rate
shown reduction of HR in Drosophila by of inactivation in human Kv2.1 channels.

quinidine, without examining its effect on the
DISCUSSION
rhythm (10). In these previous studies, quinidine
reduces HR with an EC50 of 79 µM. In the current Until now, the adverse effects of celecoxib
experiments, 50 µM quinidine induced severe have been attributed to a single mechanism -
irregularity in heartbeat (Fig. 6A, B), again reduction in COX-2-derived prostacyclin (PGI2) D
implicating Shab channels in this phenomenon. but not in COX-1-derived platelet thromboxane - ow
n
While quinidine shows high selectivity for resulting in clot formation in response to a lo
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Shab channels in Drosophila (47), its effect on perturbation (51-53) (Fig. 8). The data presented de
d
other channels cannot be completely ruled out. A here reveal an important new effect of celecoxib. fro
m
genetic approach, using a mutation in the Shab At low micromolar concentrations, the drug h
gene, was pursued to further corroborate the link reduced heart rate in Drosophila and induced ttp
between Shab channel disruption and arrhythmia. pronounced arrhythmia. In a similar fashion, it ://w
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The Shab3 mutation is predicted to eliminate the reduced the rate of beating in rat heart cells in w
Shab channels (36). Heartbeat in Shab3 larvae culture and made the beating arrhythmic. .jbc
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showed a severe effect with full contractions of The above effects were mediated via an rg
heart interspersed between partial contractile unanticipated mechanism, i.e., through inhibition by/
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events, similar to fibrillation-type activity. In of delayed rectifier potassium channels (Fig. 8) ue
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addition, rate and pattern of contractions varied rather than through inhibition of cyclooxygenases, t o
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from larva to larva as well as at different times as Drosophila lacks these enzymes. Failure of N
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within the same larva. Fig. 6A shows two such acetylsalicylic acid to inhibit rat Kv2.1 channels ve
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recordings from Shab3 mutants. While HR, in supports the COX-independence of the channel be
terms of full contractions, appeared to be inhibition by celecoxib. The role of the channels in r 2
4
substantially reduced in Shab3 (Fig. 6A), it was mediating the cardiac effect in Drosophila was , 2
0
1
difficult to quantify HR because of many partial further substantiated by the induction of the effect 8
contractions with different amplitudes. Variability by a mutation in the Shab channels, and by the
in heart rhythm in Shab3 was compared with that inability of the drug to further influence this effect.
in the wild-type. As shown in Fig. 6B, Shab3 Inhibition of human Kv2.1 channels by
animals exhibited a strongly arrhythmic heartbeat. celecoxib, expression of Kv2.1 channels in human
If Shab channels are involved in mediating the atrial and ventricular cells (41,54), and the fact
action of celecoxib on cardiac rhythm, then the that dysfunction of delayed rectifier K+ (Kv2.1 and
drug may not be able to further augment the effect Kv1.5) channels can induce cardiac arrhythmias
of the Shab3 mutation, as the mutation has already and reduction in HR in mammals (55) make it
disrupted these channels. 10 µM celecoxib, which tempting to speculate on the likely cardiac
produced significant arrhythmia in itself, was implications of celecoxib therapy in humans. In
unable to further augment the arrhythmic effect of addition, Kv2.1 channels play an important
the mutation (Fig. 6C). This strongly supports the physiological role in several tissues such as
idea that the arrhythmic effect of celecoxib pancreas, pulmonary arteries, placental vasculature
occurred, at least in major part, via the Shab and hippocampal and cortical pyramidal neurons
channels. (56-58). An effect of celecoxib on Kv2.1 channels
As a preliminary step towards exploring the in these tissues could likely have an impact on the
relevance of the above observations to humans, we
4
functioning of the tissues. However, any extrapolated to human heart or other tissues
speculation on likely human implications of the without further experiments to directly explore
observations reported here would be premature. such possibilities. The data presented here make a
The extent to which human cardiac and other case for pursuing such experiments and for
functions may be altered cannot be gauged at this examining if the mechanistic direction described
stage because of likely differences between here for the effects of celecoxib has physiological
Drosophila, rats and humans in profiles of and clinical relevance in humans.
expression of various ion channels and accessory The data presented here do not allow for an
subunits, and in the role of these channels in action extrapolation to other selective COX-2 inhibitors
potentials and contractility. In addition, any effect because the observed effect did not depend on
in a particular tissue will depend on the COX inhibition. Whereas the previously described
concentration of the drug in that tissue. Celecoxib cardiovascular complications arising from the
concentration that shows significant Kv2.1 prothrombotic effects of coxibs have been seen to
inhibition (1 µM) is in the range of plasma a varying degree in the case of most coxibs, the
concentration achieved at therapeutic dosages in effect described here may or may not translate to
humans. For example, the peak drug concentration other coxibs depending on the nature of the
in human plasma after the minimal adult interacting domains between the channel and the
therapeutic dose of 200 mg is generally between drug molecule.
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1.7 and 2.8 µM and may be as high as 6.2 µM in As a fortuitous outcome of the above ow
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women aged 65 or above (9). However, available observations, lack of cyclooxygenases in lo
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drug concentration in specific tissues is not Drosophila makes it valuable for investigating de
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known, and drug binding to plasma proteins on side effects of COX-2 inhibitors and other fro
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one hand and a large volume of distribution on the NSAIDs that occur via pathways possibly
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other (9) makes it difficult to assess drug independent of COX inhibition. ttp
concentration in specific tissues. For all these ://w
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reasons, the effects reported here cannot be w
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ACKNOWLEDGEMENTS .jbc
.o
We are thankful to Dr. H. Y. Gaisano (University of Toronto) and Dr. T. Zimmer (Friedrich Schiller rg
University) for providing the rat and the human Kv2.1 clones respectively. We are particularly thankful to by/
g
Dr. M. Slaughter (from the University at Buffalo) for providing experimental set up, other laboratory u
e
s
facilities and advise on recordings from Kv2.1 channels expressed in HEK cells, and to Dr. J. R. Olson t o
n
(from the University at Buffalo) for providing ventricular tissue from rats. This work would not have been N
o
possible without their support. Mr. Anil Neelakantan, Mr. Jason Myers and Mr. Kevin Kransler provided ve
m
help in conducting these experiments. We are thankful to Dr. A. Bhattacharjee, Dr. R. D. Shortridge and b
e
Dr. T.-C. Lee (from the University at Buffalo) for discussions on the project. The study was supported by r 2
4
grants MCB-0094477 and MCB-0322461 from NSF. , 2
0
1
8
FOOTNOTES
1. Abbreviations used are: COX, cyclooxygenase; DMSO, dimethyl sulfoxide; EGF, epithelial growth
factor; HEK cells, human embryonic kidney cells; HR, heart rate; NSAIDs, non-steroidal anti-
inflammatory drugs; PGI , prostacyclin.
2

FIGURE LEGENDS
Figure 1. Effects of celecoxib on Drosophila heart rate (HR). A, Average HR is shown for 5 to 16
larvae as specified, before drug application, in the presence of celecoxib (horizontal bars) and after the
washout. B, A bar plot summarizing the effect of celecoxib on heart rate. Treatment with 0.5% DMSO,
the maximum concentration of DMSO used with celecoxib, did not change the HR significantly. A
significant effect was seen with celecoxib at 3 µM (p<0.05, ANOVA) and higher concentrations
(p<0.001); the inhibition of HR was partially reversed upon washing out the drug. (1): HR before drug
application; (2): HR in the presence of celecoxib; (3): HR after washout. C, A dose-response curve for the
effect of celecoxib on heart rate. The curve represents a fit to Hill equation. Drop lines show EC . In this
50
and the following figures, error bars show ± s.e.m., (n): number of experiments; *: P < 0.05; **: P <
7
0.001.

Figure 2. Effects of celecoxib on Drosophila cardiac rhythm. A, Celecoxib introduced a
pronounced variability in HR, shown here for individual animals as percentage of the HR before drug
application. B-E, Traces represent the relative displacement of an edge of the larval heart. Video
recordings of heart wall movements were obtained immediately before application of celecoxib (basal), at
8-10 min after wash-in of celecoxib, and at 8-10 min after washing the drug out. B, 1 µM celecoxib in
most cases slightly reduced HR, but in 5 out of 17 cases it also induced a severe transient arrhythmia (C).
D, 3 µM celecoxib reduced HR and induced arrhythmia. E, In addition to triggering severe arrhythmia in
most cases, 10 µM celecoxib induced transient heart arrest in 4 out of 16 larvae (data shown on a
compressed scale). F, To assess drug-induced irregularity in HR, coefficient of variation (CV) was used
as explained in Data Supplement, Fig. 1. Celecoxib significantly increased irregularity of HR at 1 µM and
higher concentrations (ANOVA).

Figure 3. Effects of celecoxib on beating rate and rhythm in cultured rat ventricular myocytes.
A, A change in beating rate (BR) on the application of celecoxib. B, Celecoxib induced a large variability
in beating rate, shown here for individual cell clusters as percentage of the beating rate before drug
application. C, A bar plot summarizing the effects of celecoxib on beating rate. Treatment with 0.1%
DMSO, the maximum concentration of DMSO used with celecoxib, did not change the beating rate
D
significantly. A significant effect was seen with celecoxib at 3 µM (P<0.05, ANOVA), and higher ow
n
concentrations (p<0.001). Inhibition of the beating rate was partially reversed upon washing out the drug. lo
a
D, Celecoxib increased irregularity in beating rate as assessed using coefficient of variation for the de
d
beating rate rhythmicity; 10 µM, P<0.05, paired Student’s t-test. E, A dose-response curve for the effect fro
m
of celecoxib on beating of ventricular myocytes. The curve represents a fit to the Hill equation. Drop lines
h
s how EC50. ttp://w
Figure 4. Celecoxib inhibited Shab-encoded voltage-activated delayed rectifier K+ channels w
w
(Kv2) in Drosophila. Averaged currents obtained from Drosophila larval body-wall muscles (A) in .jb
c
control and in the presence of 10 µM celecoxib. Voltage pulses were given from -80 mV to +40 mV in .o
rg
increments of 10 mV. As described in Methods, the currents are shown as current density. B, Celecoxib b/
y
showed no effect on IA at 20 µM concentration. C, Current-voltage relationships for IA and IKS. D, gu
Average currents are shown for a voltage step from a holding potential of -80 mV to +40 mV at various es
concentrations of celecoxib. E, Dose-response curve for the effect of celecoxib on I at +40 mV, as t on
KS N
measured at the end of the 500 msec pulse. Inset bar plot: celecoxib significantly affected I at 1 µM and o
KS v
e
higher concentrations. m
b
e
Figure 5. Celecoxib inhibited rat Kv2.1 channels expressed in HEK-293 cells. Averaged currents r 2
4
obtained from HEK-293 cells expressing Kv2.1 channels (A) in control and in the presence of 10 µM , 2
0
1
celecoxib. Voltage pulses were given from -70 mV to +40 mV in increments of 10 mV. As described in 8
Methods, the currents are shown as normalized to the currents obtained in the absence of the drug. B,
Current-voltage relationships in the presence of 10 µM celecoxib (Cel.) and 1 mM acetylsalicylic acid
(ASA). C, Average currents are shown for a voltage step from a holding potential of -70 mV to +40 mV
at various concentrations of celecoxib. D, Dose-response curve for the effect of celecoxib on Kv2.1
current at +40 mV, as measured at the end of the 500 msec pulse. Inset bar plot: celecoxib significantly
affected Kv2.1 current at 1 µM and higher concentrations. E, recovery of Kv2.1 currents within one
minute after wash-out of celecoxib. Designation of the plain and the hatched bars is the same as in Fig. 2.

Figure 6. Genetic or pharmacological knockdown of Shab-encoded channels induced cardiac
dysfunction in Drosophila. A, Quinidine, a selective blocker of I current in Drosophila over
KS
micromolar range, reduced HR and induced severe arrhythmia in wild-type larvae; these effects were
reversed upon wash-out. Basal HR in Shab larvae was severely irregular. The character of arrhythmia in
Shab mutants ranged from severe rhythm disorder (i) to fibrillation-like heart contractions (ii) that are
similar to those obtained with 10 µM celecoxib. B, Bar plot summarizes the effects of quinidine and the
Shab3 mutation on HR regularity. Quinidine makes the HR arrhythmic. Similarly, HR in Shab larvae is
significantly more irregular than in CS animals (ANOVA). C, Celecoxib fails to further enhance the
arrhythmia induced by the Shab3 mutation.

8
Figure 7. Effects of celecoxib on human Kv2.1 channels expressed in HEK-293 cells. Averaged
currents obtained from HEK-293 cells expressing human Kv2.1 channels (A) in control and in the
continuous presence of 3 µM celecoxib. Voltage pulses were given from -80 mV to +40 mV in
increments of 10 mV. As described in Methods, the currents are shown as normalized to the currents
obtained in the absence of the drug. B, Current-voltage relationships under control conditions and in the
presence of 3 µM celecoxib at the end of 500-ms voltage steps.

Figure 8. Cardiac effects of celecoxib mediated via delayed rectifier potassium channels. Until
now the only pathway that has been considered to be mediating the cardiovascular effects of selective
COX -2 inhibitors has been the prothrombotic state induced by the drugs. In contrast to non-selective
inhibitors, which inhibit both the pro-thrombotic and the anti-thrombotic branches of the pathway shown,
the selective COX-2 inhibitors block only the anti-thrombotic branch. The data presented in this paper
suggest that celecoxib may induce cardiac dysfunction by a completely independent pathway, that of
cardiac arrhythmia induced by inhibition of delayed rectifier potassium channels. Effects of celecoxib are
highlighted by thick lines.

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