(Circulation. 2000;101:1179.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
Dose-Dependence of 4-Aminopyridine Plasma Concentrations and Electrophysiological Effects in Dogs
Potential Relevance to Ionic Mechanisms In Vivo
From the Research Center and Department of Medicine, Montreal Heart Institute (S.N., E.D.B., W.H., D.L., L.Y.); Department of Medicine, University of Montreal (S.N.); and Department of Pharmacology, McGill University (S.N., C.M., L.Y.), Montreal, Quebec, Canada.
Correspondence to Dr Stanley Nattel, Research Center, Montreal Heart Institute, 5000 Belanger St E, Montreal, Quebec, Canada H1T 1C8. E-mail nattel{at}icm.umontreal.ca
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Abstract |
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Background—Previous investigators have administered 4-aminopyridine (4AP) to dogs to evaluate the role of transient outward current (Ito) in vivo; however, plasma concentrations of 4AP were not measured, and it is therefore uncertain which cardiac ion channels were blocked at the concentrations achieved.
Methods and Results—We applied high-performance liquid chromatography to measure 4AP concentrations produced by intravenous 4AP administration to dogs. A previously described dose regimen produced plasma concentrations that increased during the maintenance infusion but never exceeded 250 µmol/L and caused significant mortality. Whole-cell patch-clamp experiments on isolated canine myocytes showed that even the maximum 4AP concentrations achieved in vivo failed to alter ventricular Ito and had very small effects on atrial Ito; however, concentrations achieved in vivo had a strong inhibitory effect on the dog ultrarapid delayed rectifier (IKur.d), present only in atrial cells. We designed a loading and maintenance infusion regimen to produce stable 4AP plasma concentrations. At concentrations in the range of 25 and 50 µmol/L, 4AP had no effect on ventricular refractory period but increased atrial refractoriness significantly, consistent with the results of voltage clamp studies.
Conclusions—The interpretation of previous studies using intravenous 4AP administration to inhibit Ito in dogs in vivo needs to be reevaluated in light of the fact that the infusion regimens used produce plasma concentrations that are inadequate to affect ventricular Ito. Our findings also support the concept that selective inhibition of ultrarapid delayed rectifier current can prolong atrial refractory periods without affecting ventricular refractoriness.
Key Words: ion channels • antiarrhythmic agents • electrophysiology • action potentials • heart diseases
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Introduction |
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Over the past 10 years, there has been an explosion of knowledge regarding the ionic currents controlling repolarization in various species, as well as their molecular determinants and pharmacological properties.1 One of the challenges in cardiac electrophysiology has been to link these fundamental mechanisms identified in studies with isolated cells to in vivo electrical phenomena. A potential approach to determining the ionic mechanisms governing electrophysiological processes in the intact heart is to administer selective channel blocking drugs in vivo and to measure the resulting electrophysiological alterations. This approach was used by del Balzo and Rosen, who infused 4-aminopyridine (4AP) into morphine/chloralose-anesthetized, open-chest dogs to evaluate effects on T-wave memory.2 They found that ventricular pacing caused substantial T-wave alterations during subsequent sinus rhythm, alterations that were prevented by the infusion of 4AP. The results were interpreted as indicating an important role of transient outward current (Ito) in T-wave memory. Similarly, Tachibana et al3 used 4AP infusion in dogs to evaluate the possible role of Ito in the ventricular proarrhythmic effects of flecainide. Because 4AP appeared to abolish flecainide proarrhythmia, Ito was considered to play a central role in mediating the proarrhythmic action of flecainide-induced INa inhibition. The interpretation of these experiments assumes that 4AP infusion produced sufficient plasma concentrations in vivo to inhibit Ito; however, the plasma concentrations achieved were unknown.
The approach of administering K+ channel blockers in vivo to study ionic mechanisms requires knowledge of the resulting plasma drug concentrations in relationship to blocking concentrations for specific ionic currents. The present experiments were designed to (1) determine the plasma concentrations achieved by infusing 4AP in vivo to dogs, (2) design a loading and maintenance infusion regimen of 4AP that maintains stable plasma concentrations over time, (3) determine the effects of 4AP infusion on canine atrial and ventricular effective refractory period (ERP), and (4) relate any changes observed to 4AP-induced changes in ionic currents.
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Methods |
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Protocols
We performed preliminary experiments with methods and 4AP infusion techniques similar to those described by del Balzo and Rosen.2 Tachibana et al3 used a similar approach with doses that were
25% lower. We chose the del
Balzo-Rosen infusion rates because the Tachibana study had yet not been
published at the time that we designed our experiments. Because del
Balzo and Rosen used a larger 4AP dose than Tachibana et al, our
results reflect the maximum 4AP concentrations achieved in vivo with
previously described dose regimens. In initial experiments, dogs were
anesthetized with morphine (2 mg/kg SC) and 
-chloralose (100
mg/kg IV), and 4AP was infused as an initial dose of 1.6 mg/kg followed
by an infusion of 0.25 mg · kg-1 ·
min-1. All dogs handled in this fashion
developed clonic muscle contractions during 4AP infusion that made
experimentation difficult. We therefore elected to paralyze the dogs
with pancuronium 0.1 mg/kg IV initially, followed by subsequent doses
of 0.1 mg/kg each hour. The use of the paralytic agent necessitated
(for humane purposes) both loading (30 mg/kg) and maintenance
(10 mg · kg-1 ·
h-1) doses of intravenous
pentobarbital to ensure continuous and adequate general
anesthesia.4 This form of
anesthesia was therefore used instead of
morphine/chloralose in all subsequent in vivo studies. All experiments
described in the present article were performed with
continuous-infusion pentobarbital anesthesia. In the first series of in vivo experiments, 4AP was infused intravenously at a dose of 1.6 mg/kg followed by 0.25 mg · kg-1 · min-1. Blood samples were collected at the end of the initial loading dose (given over 2 minutes) and at 5, 10, 15, 20, 30, 40, 50, and 60 minutes thereafter for subsequent high-performance liquid chromatography (HPLC) assay of plasma 4AP concentration. On the basis of the results of these initial studies, we designed a loading and maintenance infusion of 4AP to produce and maintain stable plasma drug concentrations and tested it in a series of 5 dogs. We then used this approach to produce 2 plasma concentration levels of 4AP in dogs and evaluated the effects of 4AP infusion on atrial and ventricular ERP in 6 additional dogs. Finally, we applied patch-clamp techniques to determine the effects of 4AP on drug-sensitive ionic currents of isolated canine atrial and ventricular myocytes to relate the plasma drug concentrations in vivo to possible ionic mechanisms of the electrophysiological changes observed.
Methods for In Vivo Electrophysiological Study
General surgical methods (with cardiac access provided via a
right thoracotomy) and instrumentation were as described in detail
previously.5 6 To prevent interference from autonomic
reflexes and direct neural release of neurotransmitters, ß-adrenergic
receptor blockade was produced by administration of
intravenous nadolol (0.5 mg/kg initially, followed by 0.25
mg/kg every 2 hours) and atropine (1 mg every 2 hours). ERP was
measured in duplicate with the extrastimulus technique. Square-wave,
2-ms twice-threshold pulses were applied via bipolar hook electrodes in
the right atrial appendage or right ventricular free wall.
A single extrastimulus was applied after every 15 basic stimuli, with
the coupling interval reduced by 10-ms decrements until failure to
capture indicated the ERP.
HPLC Assay
HPLC assay was performed with general methods similar to ones we
have described in detail previously.5 6 7 8 In brief,
aliquots (0.5 mL) of plasma, with 0.1 mL of 1N NaOH added, were placed
in 15-mL tubes containing 10 µg of procainamide as the
internal standard. Ethyl acetate (2.5 mL) was then added to the tubes,
which were agitated vigorously for 30 seconds. After 10 minutes of
centrifugation at 3000 rpm for 10 minutes, 2 mL of the
upper organic phase was transferred to Reactivials and
evaporated to dryness with a stream of pure nitrogen gas. The residue
was dissolved in 500 µL of mobile phase, and 25 µL was introduced
into the injection loop. The mobile phase consisted of equal quantities
(by volume) of acetonitrile and 0.03 mmol/L potassium dihydrogen
phosphate aqueous solution, with the addition of 2 mL/L glacial acetic
acid and 0.006 mmol/L L-octane sulfonic acid
(Na+ salt). Separation was carried out on a
Spherisorb ODS 10-µm reverse-phase column from
Chromatography Sciences coupled to a Waters 501 HPLC
solvent delivery module and a Waters 481 UV visible detector. The
compounds (4AP and the internal standard, procainamide) were
detected at a wavelength of 263 nm, and the retention times for 4AP and
procainamide were 3.5 and 4.5 minutes, respectively, with
complete baseline separation. A standard curve was performed for each
set of sample determinations, with concentrations obtained from the
linear standard curve without extrapolation. The coefficient of
variation ranged from 1.7% to 2.7%.
Cell Isolation and Patch Clamp Methods
Canine atrial9 10 and
ventricular11 myocytes were isolated with
methods previously described in detail.9 10 11 Whole-cell
patch clamp was used to record the 4AP-sensitive currents
Ito and IKur.d
by methods previously described in detail.9 10 11 The
extracellular solution contained (mmol/L) NaCl 126, KCl 5.4,
MgCl2 1.0, CaCl2 1.0,
NaH2PO4 0.33, HEPES 5.0,
and dextrose 10 (pH set to 7.4 with NaOH).
Ito was studied at 37°C, whereas
IKur.d was evaluated at room temperature to
resolve its very rapid activation kinetics.10 In
addition, 1 µmol/L dofetilide (to suppress
IKr), 200 µmol/L
CdCl2 (to block ICa
and ICl.Ca), and 200 nmol/L atropine (to
inhibit any basal acetylcholine-dependent current) were added to the
perfusate. The intracellular (pipette) solution contained
(mmol/L) potassium aspartate 110, KCl 20, Mg2ATP
5, HEPES 10, sodium phosphocreatine 5, GTP 0.1, and EGTA 5 (pH set to
7.3 with KOH). For studies of atrial Ito,
IKur.d was suppressed with the use of
10 mmol/L tetraethylammonium, which
fully inhibits IKur.d without affecting
Ito.10 Ventricular cells lacked
IKur.d. Cell capacitance averaged 74.1±4.5 and
113±6 pF for atrial and ventricular cells, respectively,
and compensated series resistance averaged 2.1±0.2 M
.
Data Analysis
Because ERP values are not distributed according to a normal
distribution, comparisons between ERPs were performed with
Wilcoxon’s distribution-independent paired rank test. Group
data are presented as the mean±SEM. Two-tailed probabilities
were used for all comparisons, with P<0.05 considered
significant. Nonlinear curve-fitting of concentration-response data was
performed with commercially available software (Chebyshev
algorithm).
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Results |
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Plasma Concentrations Resulting From 4AP Infusion In Vivo
The previously described 4AP infusion regimen resulted in mean concentrations that averaged
80 µmol/L at the end of the
1.6-mg/kg loading dose, decreased to 50 µmol/L 5 minutes into
the maintenance infusion, and increased slowly thereafter to
reach a maximum in the range of 225 µmol/L after 60 minutes
(Figure 1A
). Because of the instability
of the plasma concentrations achieved by this dose regimen and
substantial lethality (3 of 6 dogs died before receiving the full
dose), we designed a dose regimen to achieve and maintain stable drug
concentrations and administered it to 5 additional dogs. This regimen,
consisting of an initial dose of 1 mg/kg, followed by 0.1 mg ·
kg-1 · min-1 for
20 minutes and then 0.05 mg · kg-1
· min-1 for 40 minutes, resulted in stable
plasma concentrations in the range of 20 to 30 µmol/L from 5
minutes after the end of the loading dose until the end of the
observation period at 60 minutes (Figure 1B).
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Electrophysiological Effects of 4AP
Infusion
We applied the loading and maintenance approach to study
the effects on atrial and ventricular ERP of stable 4AP
concentrations in the range of 25 (dose indicated above) and 50
(additional 1 mg/kg load, doubled maintenance doses)
µmol/L. After baseline values were obtained, we infused 4AP according
to the regimen shown in Figure 1B
(dose 1) and repeated the ERP
measurements. We then gave an additional 1-mg/kg loading dose, followed
by 0.2 mg · kg-1 ·
min-1 for 20 minutes and then 0.1 mg ·
kg-1 · min-1 (dose
2). The resulting electrophysiological
effects are shown in the Table.
Neither dose significantly altered ventricular ERP. Dose 1
increased atrial ERP slightly but not significantly, whereas dose 2
produced significant increases on the order of 25% to 30% in atrial
ERP.
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Concentration-Response Relations for 4AP Inhibition of Ionic
Currents
To assess the potential ionic mechanisms of the
electrophysiological effects of 4AP in
vivo, we studied the effects of 4AP on the ionic currents known to be
sensitive to the compound: Ito and
IKur.d in canine atrial
myocytes9 10 and Ito in
canine ventricular cells.11 Figure 2 (top) shows examples of dog atrial and
ventricular Ito before and
after the addition of 4AP at various concentrations. Corresponding
concentration-response curves, shown at the bottom, indicate that
relatively large concentrations of 4AP are needed to inhibit
Ito. The mean 50% inhibitory
concentration (IC50) averaged 1486±261
µmol/L for ventricular cells (n=4) and 471±97
µmol/L for atrial cells (n=3). Concentrations of 4AP in the range of
the largest 4AP concentrations achieved by intravenous
infusion in vivo, 200 µmol/L, reduced ventricular
Ito by an average of 10±4%
(P=NS versus control) and atrial
Ito by 25±4% (P=0.02 versus
control). Figure 3 shows the effect of
several concentrations of 4AP on IKur.d in
a representative myocyte (left), as well as the 4AP
concentration-response curve for IKur.d
inhibition (right). IKur.d was much more
sensitive to 4AP than was Ito, with an
IC50 of 5.3±0.7 µmol/L (n=8). At a
concentration of 50 µmol/L, in the range of concentrations
produced by dose 2 for the in vivo studies, 4AP reduced
IKur.d in atrial myocytes by an average of
99±1% at +40 mV. At the same concentration (50 µmol/L),
ventricular Ito was reduced by
1±1% (P=NS) and atrial Ito by
3±6% (P=NS).
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Discussion |
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We measured the plasma concentrations produced by a previously described 4AP infusion protocol in dogs. The plasma concentrations changed over time and reached a maximum just over 200 µmol/L, a concentration that had no significant effect on Ito in canine ventricular myocytes and a very small effect on Ito in canine atrial myocytes. In contrast, 4AP concentrations as low as 50 µmol/L completely inhibited IKur.d in atrial myocytes. A 4AP dose regimen was developed to produce and maintain stable plasma concentrations. With this dose regimen, 4AP significantly altered canine atrial but not ventricular ERP in vivo, in general agreement with effects on corresponding drug concentrations on ionic currents in vitro.
Relationship to Previous Studies of 4AP Pharmacokinetics
Relatively few published studies have evaluated the effects of 4AP
on cardiac electrophysiology in vivo. Del Balzo and Rosen used 4AP
infusion to evaluate the potential role of
Ito in T-wave memory in the dog. They
estimated that their infusion regimen would result in blood
concentrations of 3 mmol/L, assuming no loss from the
intravascular compartment.2 Our direct measurements
of plasma concentrations are not consistent with this
assumption and indicate that the infusion regimen reported by del Balzo
and Rosen produces much lower concentrations, averaging 80
µmol/L at the end of the loading dose, 50 µmol/L at 5 minutes,
and 225 µmol/L after 60 minutes of maintenance infusion.
In fact, the concentrations achieved were well below the threshold for
Ito inhibition in ventricular
myocytes.
Uges et al12 studied the pharmacokinetics of intravenous 4AP in humans. Their pharmacokinetic analysis indicated a serum drug clearance of 0.61 L · h-1 · kg-1. On the basis of the well-known pharmacokinetic relationship IR=Css · Cl,13 where IR is drug infusion rate, Css is steady-state drug concentration in the blood, and Cl is drug clearance from the blood, the pharmacokinetic parameters calculated by Uges et al would predict a steady-state concentration of 265 µmol/L with a 4AP maintenance infusion rate of 0.25 mg · kg-1 · min-1, as used by del Balzo and Rosen. This concentration is very similar to the concentrations (in the range of 225 µmol/L) that we measured after 60 minutes of maintenance infusion with the del Balzo-Rosen infusion protocol. Uges et al found that 4AP concentration-time data were best fit by a triexponential model in 5 patients and a biexponential model in 4 others. Mean central compartment volume estimates ranged from 0.0708 to 0.78 L/kg, which would predict a drug concentration between 240 and 22 µmol/L, respectively, after a loading dose of 1.6 mg/kg 4AP, as used by del Balzo and Rosen. The concentrations at the end of a 1.6-mg/kg 4AP load in our dogs varied from 31 to 111 µmol/L, with a mean of 80 µmol/L, quite consistent with predictions based on the pharmacokinetic analysis of Uges et al.12
Novelty and Potential Significance
The present study is the first of which we are aware to
measure the plasma 4AP concentrations achieved in dogs by
intravenous infusion regimens that have been applied to
study the role of Ito in cardiac
electrophysiological phenomena in intact
animals. Our results indicate that such infusion regimens do not result
in plasma drug concentrations that are sufficient to inhibit
ventricular Ito to any
significant extent. The work of del Balzo and Rosen2
has been widely interpreted as reflecting the role of
Ito in ventricular
repolarization, and particularly T-wave memory. Tachibana et
al3 recently described an effect of
intracoronary flecainide to induce ST alternans and
ventricular tachyarrhythmias in
anesthetized dogs. In some experiments, they administered
intravenous 4AP (1.2 mg/kg load followed by 0.17 mg
· kg-1 · min-1)
and found that the effects of flecainide were attenuated. On the basis
of the response to 4AP, they concluded that their results pointed to a
central role for a 4AP-sensitive current such as
Ito in flecainide proarrhythmia.
The doses of 4AP administered by Tachibana et al were 75% of those
used by del Balzo and Rosen, and according to our data would have been
most unlikely to directly inhibit outward currents in canine
ventricular myocytes. In fact, assuming coronary
flow rates in the range of 100 mL/min,14 the
proarrhythmic flecainide infusion rate (100 µg ·
kg-1 · min-1) that
they administered into the left anterior descending coronary
artery would have been expected to produce concentrations on the order
of 20 mg/L (100 µmol/L) in the coronary blood flow.
Flecainide reduces Ito effectively at
concentrations in the range of 10 µmol/L15 ;
thus, the flecainide concentrations achieved by Tachibana et al
(100 µmol/L) would in themselves have been expected to have a
very strong inhibitory effect on
Ito.
Other mechanisms that may account for the effects of 4AP noted by del Balzo and Rosen2 and Tachibana et al3 include interactions with cardiac autonomic innervation. Furukawa et al16 observed cardiac effects of 4AP that they interpreted to be a consequence of activation of parasympathetic ganglionic neurotransmission. Neuronal K+ channels may be very sensitive to 4AP,17 18 and altered autonomic nervous system tone is well known to have important effects on cardiac repolarization.19 4AP appears to increase sympathetic and parasympathetic outflow to the heart.20 Acetylcholine can have profound transmurally heterogeneous effects on ventricular repolarization that depend on the magnitude of Ito and are enhanced in the presence of sympathetic agonists.21 Thus, 4AP could perhaps act indirectly via the autonomic nervous system to suppress T-wave changes related to alterations in Ito. At the moment, there is no direct proof for this notion, and it remains speculative. In any case, our data suggest that direct inhibition of cardiac Ito is unlikely to have mediated the effects of 4AP infusion previously observed.
Ultrarapid delayed rectifier currents
(IKur) have been observed in cardiac
tissues from a wide variety of species.22 They have
been noted to play a role in repolarizing human23 and
canine10 atrial myocytes and mouse ventricular
myocytes.24 The human IKur is
absent in human ventricular cells,25 26
and it has been suggested that IKur may be
an interesting target for atrial-selective antiarrhythmic drugs. In the
present study, we observed IKur.d in
canine atrial myocytes and found no evidence of its presence in dog
ventricle. As shown in the Table, we found that 4AP is capable
of increasing atrial ERP in the dog in vivo without affecting
ventricular ERP. Because IKur.d
is the main outward K+ current affected by 4AP at
the concentrations achieved by the modified loading and infusion
regimen, these observations are consistent with the notion that
selective inhibition of ultrarapid delayed rectifiers can produce
atrial-selective refractoriness prolongation in vivo. To the best of
our knowledge, this is the first such demonstration in the
literature.
Our findings make it unlikely that the observations of del Balzo and Rosen regarding the effects of 4AP on T-wave memory in vivo2 were due to actions on Ito; however, this does not exclude a potentially important role for Ito in T-wave memory. Subsequent studies have shown that 3 mmol/L 4AP suppresses changes in action potential difference signals induced by altered activation pattern in canine ventricular slabs studied in vitro.27 Epicardial cells from the left ventricular base of dogs subjected to pacing at the left ventricular apex to induce T-wave alterations in sinus rhythm (T-wave memory) showed a decreased phase 1 notch, along with a decreased Ito density, altered Ito voltage dependence, slowed Ito recovery, and decreased Kv4.3 mRNA levels.28 Thus, strong suggestive evidence remains for a role of Ito in T-wave memory. Further work would be useful to establish more precisely the role of Ito, as well as that of other ionic currents and factors, such as changes in cell coupling and myocardial structural remodeling, in T-wave memory.
We noted quite different 4AP potencies for inhibition of canine atrial Ito (IC50 of 0.47 mmol/L) compared with ventricular Ito (1.49 mmol/L). Canine atrial and ventricular Ito are both believed to be composed of Kv4.3 subunits.29 30 The molecular basis for the difference in 4AP sensitivity between canine atrial and ventricular Ito is an interesting issue that merits further study.
Potential Limitations
Cell isolation can produce artifacts by affecting cardiac ion
channels; however, we have found that Ito
is relatively unaffected by the cell isolation
method.9 It could be argued that myocardial tissue
concentrations of a drug like 4AP may not be the same as plasma
concentrations. Although this may be true, the concentration of a drug
in the extracellular solution space is equivalent to plasma
concentrations of free (ie, non–protein-bound) drug. Because 4AP shows
negligible protein binding,12 plasma concentrations are
probably equivalent to the concentration in the extracellular space
with which the cell membrane is in equilibrium. Therefore, the effects
of a given 4AP concentration applied to single cells via the
extracellular (bath) solution should be equivalent to the effects of a
corresponding plasma concentration in vivo.
Conclusions
Our study shows that previously described 4AP infusion
regimens produce inadequate plasma drug concentrations to significantly
inhibit ventricular Ito. Given
the toxicity of these infusion regimens, it is unlikely that sufficient
4AP can be given intravenously to block cardiac
Ito in vivo. Thus, the interpretation of
the results of previous studies using intravenous 4AP in
vivo needs to be reevaluated, and caution should be used in designing
subsequent studies. Our data also suggest that selective inhibition of
atrial ultrarapid delayed rectifier currents can produce
atrial-specific ERP prolongation.
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Acknowledgments |
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Funding for this work was obtained from the Medical Research Council of Canada and the Quebec Heart Foundation. Dr Yue was supported by a Canadian Heart Foundation research studentship. The authors would like to thank Diane Campeau for secretarial help with the manuscript.
Received June 14, 1999; revision received September 2, 1999; accepted September 23, 1999.
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