© 1996 American Heart Association, Inc.
Articles |
Cellular Basis for the Negative Dromotropic Effect of Adenosine on Rabbit Single Atrioventricular Nodal Cells
From the Departments of Pharmacology (D.W., L.B.) and Medicine (J.C.S., L.B.), University of Florida, Gainesville.
Correspondence to Luiz Belardinelli, MD, University of Florida, Department of Medicine, PO Box 100277, Gainesville, FL 32610.
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Abstract |
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Abstract The effects of adenosine on action potentials, rate-dependent activation failure (the cellular basis for second-degree atrioventricular [AV] block), and the recovery of excitability in rabbit isolated single AV nodal cells were studied using the whole-cell patch-clamp technique. Adenosine (1 µmol/L) shortened the duration, depressed the amplitude, and reduced the rate of rise of the AV nodal cell action potential. Adenosine (10 µmol/L) caused a significant hyperpolarization (7±1 mV) of AV nodal cells. Adenosine increased the occurrence and the rate dependence of activation failure (Wenckebach periodicity) of AV nodal cells; this effect was concentration dependent and mediated by A1 adenosine receptors. The rate-dependent activation failure caused by adenosine was associated with a prolongation of the effective refractory period by 18±2 ms (P<.05), an increase in the duration of activation delay, and an elevation (from 0.22±0.04 to 0.30±0.03 nA, P<.05) of the threshold current amplitude required to activate AV nodal cells. The results suggest that the slowed recovery of excitability of AV nodal cells caused by adenosine forms the cellular basis for adenosine-induced second-degree AV block. Adenosine decreased ICa,L and activated IK,ADO of AV nodal cells. These actions of adenosine on ion currents may contribute to the effect of this nucleoside to depress excitability of AV nodal cells. The enhancement by adenosine of rate-dependent activation failure of AV nodal cells implies that the negative dromotropic effect of adenosine should be more pronounced during an episode of supraventricular tachycardia than during normal rhythm.
Key Words: excitability • refractory period • activation delay • K+ current • L-type Ca2+ current
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Introduction |
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An important function of the AV node is to limit the number of impulses being conducted from the atria to the ventricles. At high atrial rates, only a fraction of impulses is conducted to the ventricles, and the remainder are "blocked" in the AV node.1 2 3 Thus, the AV node serves as a filter to protect the ventricles from life-threatening arrhythmias during episodes of atrial fibrillation and/or atrial flutter.4 5 The mechanisms underlying second-degree AV block are not clear. Rate-dependent second-degree AV block is thought to result from discontinuous impulse propagation across junctional nonhomogeneous tissues (eg, atrionodal, nodal, and nodal–His bundle zones6 7 8 9 10 ). The demonstration of Wenckebach-like periodicity of guinea pig single ventricular myocytes11 and rabbit AV nodal cells12 indicates that rate dependence of activation is an intrinsic property of the individual cells. In a study of rabbit single AV nodal cells, Hoshino et al12 demonstrated that rate-dependent activation failure (Wenckebach periodicity) is due to postrepolarization refractoriness, which corresponds to the slow recovery of membrane excitability after a preceding action potential. A slow deactivation of IK appeared to be the cause of the delay in recovery of excitability of ventricular myocytes.13 Hoshino et al12 proposed that at least IK, IK1, and ICa are involved in the rate-dependent recovery of excitability of AV nodal cells.
Adenosine activates specific cell-surface
A1 receptors to slow AV node impulse conduction and has
been shown to cause second-degree AV block in both
animals14 and humans.15 This negative
dromotropic effect of adenosine forms the basis of its clinical
application in treatment of supraventricular
tachycardias in which the AV node is part of a reentrant
pathway.15 16 17 Adenosine shortens the
duration and
depresses the amplitude and rate of rise of the action potential of AV
nodal cells.14 Results of a preliminary study indicated
that in AV nodal cells, as in atrial cells, adenosine (
10
µmol/L) activated IK,ADO, but in addition,
it also caused a small reduction in basal
ICa,L.18 These effects of adenosine on
membrane currents of AV nodal cells may play a role in the negative
dromotropic effect of this nucleoside by decreasing the
excitability and/or increasing the effective refractory period of AV
nodal cells. To test this hypothesis, we studied the effects of
adenosine on the effective refractory period and recovery of
excitability of rabbit single isolated AV nodal cells. In addition,
because results of recent studies of guinea pig isolated hearts and
human patients revealed that the negative dromotropic effects of
adenosine and A1 adenosine receptor
agonists are frequency
dependent,19 20 21 22 23 24
we also studied the
effect of adenosine on the patterns of rate-dependent
activation failure (Wenckebach periodicities) as well as on the
threshold current amplitude required to excite AV nodal cells. The
results of our experiments showed that adenosine-induced
activation failure of AV nodal cells was concentration and rate
dependent. Adenosine also caused an increase in the duration of
activation delay and a prolongation of the refractory period of AV
nodal cells. The findings of the present study provide evidence
that the negative dromotropic effect of adenosine is dependent
on its depression of the excitability of individual AV nodal cells.
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Materials and Methods |
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Chemicals
Collagenase type II was purchased from Worthington Biochemical. CPX was purchased from Research Biochemicals International. Adenosine, carbachol, CsCl, CdCl2, and TEACl were purchased from Sigma Chemical Co.
Isolation of Single AV Nodal Cells
Single AV nodal cells were
isolated by enzymatic digestion of
hearts of adult New Zealand White rabbits (weighing 1.5 to 2 kg) by
using methods similar to those described by Hoshino et
al.12 Rabbits were heparinized (500 U IV) and then
anesthetized by intramuscular injection of a combination of
acepromazine maleate (1.0 mg/kg), ketamine (30 mg/kg), and
xylazine (6 mg/kg) to produce unconsciousness. The heart was quickly
excised, rinsed, and retrogradely perfused through the aorta for 5 to
10 minutes at a flow rate of 16 mL/min with modified
oxygenated (100% O2) 35°C K-H solution
containing (mmol/L) NaCl 127, KCl 4.6, CaCl2 2,
MgSO4 1.1, sodium pyruvate 2, glucose 10, creatine 10,
taurine 20, ribose 5, adenine 0.01, allopurinol 0.1, and sodium HEPES 5
(pH 7.4). The heart was then perfused for 5 to 10 minutes with a
Ca2+-free K-H solution and for 20 minutes with 0.6 mg/mL of
collagenase type II (286 U/mg) and 1.5 mg/mL of
albumin in Ca2+-free K-H solution. At the end of
this initial enzymatic digestion, a small 2x4-mm piece of the AV nodal
region was removed by dissection, on the basis of the anatomic
landmarks described by Anderson et al.25 The small piece
of tissue containing the AV node was minced and incubated at 35°C for
an additional 10 to 20 minutes with enzyme solution in a shaker bath.
The dissociated cells were collected, washed, and stored at room
temperature in low Ca2+ (0.1 mmol/L) K-H solution. Atrial
and ventricular myocytes were similarly isolated for use in
experiments comparing responses of these cells with responses of AV
nodal cells to adenosine.
Electrophysiological
Measurements
Cells were transferred into a recording chamber on the
stage of an inverted microscope (Axiovert 10, Zeiss). After the cells
settled on the bottom of the chamber (
10 to 15 minutes), superfusion
with Tyrode's solution containing (mmol/L) NaCl 140, KCl 4.6,
CaCl2 1.8, MgSO2 1.1, glucose 10, and sodium
HEPES 5 (pH 7.4) was started and maintained at a rate of 2 to 3 mL/min
(35°C). Both membrane potentials and currents were recorded in a
whole-cell patch-clamp configuration26 using an
Axopatch-200 amplifier (Axon Instruments). The patch electrodes
(Kimax-51, Kimble Glass Inc) had resistances of 2 to 4 M
when filled
with an internal pipette solution containing (mmol/L) KCl 10, potassium
aspartate 130, Na2ATP 4, MgCl2 1,
Na3GTP 0.1, KH2PO4 10, glucose 10,
and sodium HEPES 10 (pH 7.2). Under voltage-clamp mode, junction
potentials between pipette and bath medium were nulled to zero before
seal formation. Compensation of the electrode capacitance transient was
performed. The whole-cell clamp configuration was established by
applying a small negative hydrostatic pressure through the pipette
solution to the membrane patch. After breakthrough of the membrane, the
electrode resistance in series to the cell membrane was increased to 5
to 8 M, which was compensated (75% to 85%) to minimize the
distortion of the whole-cell current-voltage relationship. The
whole-cell capacitance was obtained from the direct readings of the
potentiometer of whole-cell capacitance compensation (Axopatch
200). Data acquisition and analysis were carried out using the
pClamp system (version 5.6, Axon Instruments) installed on an
IBM-compatible 386 personal computer (Northgate Computer Systems, Inc)
and an interface board (TL-1 DMA, Axon Instruments). Signals were also
displayed on a storage oscilloscope (Tektronix 5113) and recorded
using a chart recorder (Gould Instrument).
Experimental Protocols
Current Stimulation Protocols
Protocol to determine the effect of adenosine on
rate-dependent activation failure (Wenckebach periodicity).
The rate dependence of activation of single AV nodal cells was studied
with a repetitive stimulation protocol similar to that described by
Hoshino et al.12 A cell was driven by repetitive
depolarizing current pulses of fixed amplitude and duration (10 to 40
ms). The amplitude of the depolarizing current was adjusted to slightly
above threshold (0.2 to 0.3 nA) for stimulation at a BCL of 1500 ms.
After a stable 1:1 stimulus-response coupling was achieved, the BCL
was progressively shortened in steps of 10 to 100 ms, and the
activation ratio (number of responses divided by number of stimuli) was
determined at each BCL. A cell was stimulated 50 times at each BCL
before changing to the next BCL. Each cell was subjected to this
stimulation protocol at various BCLs in the absence and presence of
adenosine (1 µmol/L). The effect of adenosine was
reversed by either washout or addition of the adenosine
receptor antagonist CPX (0.1 µmol/L).
Protocols to determine the effect of adenosine on the recovery of excitability. To evaluate the effects of adenosine on excitability and refractoriness of single AV nodal cells, two different stimulation protocols (A and B) were used.
Protocol A. A standard S1-S2
premature stimulation protocol was used to determine the effects of
adenosine on the postrepolarization RP and on activation delay.
Briefly, a cell was driven by conditioning stimulus trains of 10 to 12
suprathreshold depolarizing S1 pulses (10 ms in duration)
applied at a BCL of 800 ms. After the last pulse of the S1
train, a single premature (test) stimulus S2 (10 ms in
duration) was delivered. The coupling interval between the last
S1 and the test stimulus S2 was
progressively shortened in steps of from 100 ms to as small as 5 ms.
The amplitude of S2 was selected to be just threshold for
coupling intervals (ie, BCL) of 800 ms. The protocol was repeated in
the absence (control) and in the presence of adenosine (1
µmol/L). The RP was defined as the shortest interval between a basal
(S1) and a single premature stimulus (S2) that
failed to elicit a near fully developed action potential (ie, an action
potential with an amplitude 80% of the amplitude of the preceding
action potential elicited by S127 ). Activation
delay was defined as the elapsed time from the beginning of the test
stimulus S2 to the peak of the action potential response
(R2).
Protocol B. To determine the effect of adenosine on the relationship between the S1-S2 coupling interval and the threshold current required for activation (the strength-interval relationship), the following protocol was used: the S1-S2 coupling interval was progressively decreased in steps of 10 to 100 ms, but rather than determining RP and activation delay, the threshold current amplitude (strength of stimulation) of the test pulse S2 that could elicit a near fully developed action potential (R2) was determined at each S1-S2 coupling interval in the absence (control) and in the presence of adenosine (1 µmol/L).
Voltage-Clamp Protocols
Cell input resistance
(Table
) was measured as the
inverse slope of the ramp current voltage relationship within the
linear range (usually from -100 to -75 mV). The
depolarizing ramp clamp pulse was applied from a holding potential of
-100 mV to a potential of +50 mV at a rate of 15 mV/s.
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Maximal inward current (Table
) of isolated myocytes was
measured using
a square pulse-clamp protocol. Depolarizing clamp pulses of 50-ms
duration were applied from a holding potential of -90 mV to
potentials of -85 to +5 mV in 10-mV steps. The maximal transient
inward current elicited by the fourth or fifth pulse was determined by
use of the CLAMPFIT program (Axon Instruments, Inc).
To evaluate the effect of adenosine on ICa,L, the cell membrane potential was held at -40 mV to inactivate fast Na+ current, and depolarizing clamp pulses of 250 ms were applied to potentials from -40 to +40 mV in 10-mV steps. ICa,L was defined as the difference between peak inward current and late outward current (at the end of the 250-ms-long clamp pulse). To reduce outward K+ currents in these experiments, CsCl (4.6 mmol/L) was substituted for KCl (4.6 mmol/L) in the external Tyrode's solution, and 50 mmol/L KCl of the internal pipette solution was replaced by CsCl (20 mmol/L) and TEACl (30 mmol/L).
To evaluate the effect of adenosine on IK,ADO, the holding current required to maintain the membrane potential at -40 mV was measured in the absence and presence of adenosine.
Data Analysis
Statistical analyses were performed using a
two-tailed t test or ANOVA (for multiple-comparison
data). All values are expressed as mean±SEM. Differences between group
means and control versus interventions were considered significant at
P<.05.
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Results |
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The AV nodal cells used in the present study were quiescent. The electrophysiological characteristics of the cells are presented in the Table
, in
which properties of atrial and ventricular myocytes are
included for comparison. As illustrated in Fig 1 and
summarized in the Table, the action potential configuration of
AV nodal
cells was that of a slow action potential (slow response), with a
maximum diastolic potential of -58±2 mV, a maximum
rate of rise of <15 V/s, and an amplitude of 85±1 mV (n=23).
The
capacitance of AV nodal cells was 42.3±0.4 pF (n=33). The
effects of
adenosine on action potentials of AV nodal, atrial, and
ventricular cells are shown in Fig 1. Adenosine (10
µmol/L) had no effect on the amplitude and the rate of rise but
shortened the duration of the atrial action potential.
Adenosine (10 µmol/L) also caused a relatively small
hyperpolarization of the atrial cell membrane
potential (<3 mV). In depolarized atrial myocytes, in which the action
potential resembled a "slow response" (not shown), 10 µmol/L
adenosine caused as much as 10 mV of
hyperpolarization, and concomitantly, the action
potential configuration was converted from a "slow" to a typical
"fast" action potential as shown in Fig 1A. In
comparison,
adenosine (1 µmol/L) not only hyperpolarized the membrane
potential and shortened the duration of the AV nodal cell action
potential but also depressed the amplitude and the rate of rise of the
action potential (Fig 1B). In contrast, adenosine neither
altered the action potential configuration nor caused
hyperpolarization of ventricular
myocytes (Fig 1C). Likewise, the effects of adenosine on
rate-dependent activation failure, on postrepolarization
refractoriness, and on membrane ion currents of AV nodal, atrial, and
ventricular cells were distinctly different (see below).
The relatively high-input resistance (Table), the small
IK1, and the relatively small peak maximum inward
current (<6 nA) in Tyrode's solution were other important features of
AV nodal cells.
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Effect of Adenosine on Rate-Dependent Activation of AV
Nodal Cells
Adenosine increased the occurrence of activation failure
of AV nodal cells (n=6). Figs 2 and 3
depict typical responses of two AV nodal cells to adenosine.
Rate-dependent activation failure (Wenckebach periodicity) was
enhanced by adenosine (1 µmol/L) and occurred at slower
pacing rates (longer BCLs) in the presence than in the absence of
adenosine (Fig 2A). At slow rates of pacing by critical current
stimuli (BCLs of 1500 to 800 ms) in the absence of adenosine,
the ratio of stimuli to responses was 1:1 (Fig 2). Unstable 5:4
and 3:2
stimulus-to-response ratios typical of rate-dependent
Wenckebach activation failure occurred when the BCL was shortened from
800 to 700 ms (Fig 2A). In the presence of adenosine (1
µmol/L), a stable 3:2 Wenckebach pattern occurred at a longer BCL
of 1000 ms. When BCL was shortened to 800 and further to 700 ms, a 2:1
pattern and advanced activation failure patterns (3:1) were achieved
(Fig 2A, right). A plot of the activation ratio (number of
responses
divided by number of stimuli) as a function of BCL (Fig 2B)
revealed
that rate-dependent activation failure of single AV nodal cells was
increased by adenosine in the range of BCL from 650 to 1200 ms.
The enhancement of activation failure by adenosine was
concentration dependent (Fig 3) and was reversed by the
selective
A1 adenosine receptor antagonist, CPX
(0.1 µmol/L, Figs 2B and 3). Although the
plot of activation ratio as
a function of BCL was different for each cell, similar patterns of
activation failure and responses to adenosine were obtained in
four other cells from a total of six cells isolated from three rabbit
hearts.
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) and presence of adenosine (1
µmol/L) (
) and presence of adenosine (1 µM) plus CPX
(0.1 µmol/L) (
) .
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Effects of Adenosine on the Recovery of
Excitability
The effects of adenosine on the refractory period,
activation delay, and the strength-interval relationship of single
AV nodal cells were examined.
Effects of Adenosine on Refractory
Period and Activation
Delay
Adenosine prolonged the refractory period and increased
the activation delay of single isolated AV nodal cells. Results shown
in Figs 4 and 5 illustrate these actions
of adenosine. In the example shown in Fig 4, the refractory
period of the AV nodal cell was 300 ms, and the maximal activation
delay was 80 ms under control conditions (measured immediately before
activation failure occurred). After the cell was exposed to
adenosine (1 µmol/L) for 2 minutes (Fig 4, middle), the
refractory period was prolonged from 300 to 330 ms, and the activation
delay was increased (eg, from 80 to 130 ms at
S1-S2 intervals of 310 and 340 ms in the
absence and presence of adenosine, respectively). On average,
adenosine (1 µmol/L) increased the AV nodal cell refractory
period by 18±2 ms (P<.05, n=7). These effects were
reversed upon washout of adenosine (Fig 4, right). The data of
activation delay are summarized in Fig 5. Abbreviation of the
S1-S2 interval was associated with an increase
in activation delay (S2-R2 interval).
Adenosine (1 µmol/L) increased the activation delay at all
stimulus intervals tested. Adenosine (1 µmol/L) increased the
activation delay by 14±3 ms (P<.05, n=7) at the
S1-S2 coupling interval of 350
ms.
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) and presence (
Effects of Adenosine on Strength-Interval
Relationship
To determine if adenosine depresses the excitability of
AV
nodal cells, the minimum amplitude (threshold) of current needed to
elicit an action potential was measured. The amplitude of threshold
stimulation was increased by adenosine (1 µmol/L) from 0.22
nA (control) to 0.28 nA (Fig 6; compare panels A and B).
After washout of adenosine, the amplitude of threshold
stimulation decreased to 0.24 nA (Fig 6C). In five experiments
similar
to that shown in Fig 6, adenosine (1 µmol/L) increased the
threshold current amplitude for activation from 0.22±0.04 to
0.30±0.03 nA (P<.05, n=5). To further examine the
effect
of adenosine on the amplitude of threshold stimulation for
activation of AV nodal cells, an alternative premature stimulation
protocol was used. The S1-S2 coupling interval
was fixed at 270 ms. The amplitude of the premature S2
pulse was increased from subthreshold to suprathreshold to determine
the threshold current amplitude needed to activate the AV nodal
cell. The threshold current amplitude of the premature pulse was 0.25
nA in the absence of adenosine, increased to 0.28 nA in the
presence of 1 µmol/L adenosine, and returned to 0.25 nA upon
washout of adenosine (Fig 7). To determine the
effect of adenosine on the strength-interval relationship,
the following experimental protocol was used. An AV nodal cell was
stimulated by a premature S1-S2 stimulation
protocol. As the coupling interval of S1-S2 was
progressively shortened, the amplitude of S2 stimuli was
increased from subthreshold to suprathreshold to determine the
threshold current amplitude of S2 stimuli at each
S1-S2 interval in the absence and presence of
adenosine. As shown in Fig 8A, adenosine
(1 µmol/L) increased the threshold amplitude of S2
stimuli at each S1-S2 interval tested. The plot
of the amplitude of current threshold as a function of
S1-S2 interval illustrates that
adenosine shifted the strength-interval curve upward and to
the right (Fig 8B). Qualitatively identical results were
obtained in
another AV nodal cell. The total number of cells studied was
three.
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Adenosine Activates IK,ADO,
Decreases ICa,L, and Hyperpolarizes the Membrane
Potential of AV Nodal Cells
Hyperpolarization of AV nodal cells (Figs
1 and 6) caused by increase of outward membrane
current could be a
mechanism underlying the depression of excitability by
adenosine. Adenosine (10 µmol/L) hyperpolarized
current-clamped AV nodal cells by 7±1 mV (n=6). Adenosine
(1 and 100 µmol/L) increased outward current by 32±3 pA
(n=5) and
86±4 pA (n=4), respectively, at a membrane potential of
-40 mV
(Fig 9A). The increase by adenosine of outward
current was antagonized by CPX (0.1 µmol/L, Fig 9A). The
magnitude of
the outward current activated by 0.5 µmol/L carbachol was
2.3-fold greater than that activated by 100 µmol/L
adenosine (Fig 9A). The outward current was inwardly
rectifying, blocked by Cs+ and TEA ions, and had an
apparent reversal potential of -64±3 mV (n=6;
[K+]o, 4.6 mmol/L). When
[K+]o was increased by 5.3-fold from 4.6
to
24.6 mmol/L, the apparent reversal potential of the current was shifted
in a positive direction by 48 mV. This evidence suggests that the
outward current is carried by K+ ions.
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Adenosine (100
µmol/L) decreased inward current in the
presence of Cs+ and TEA ions (Fig 9B). The
magnitude of
inward current activated by depolarizing clamp pulses was
reduced in the presence of adenosine, as shown in the
current-voltage curve for activation of inward current from
-40 to +40 mV (Fig 9B). Peak inward current was
decreased 29±3%
(n=4) by adenosine (100 µmol/L). Adenosine (1
µmol/L) caused a small but reversible decrease (<10%) of inward
current (not shown). The inward current was abolished by 100 µmol/L
Cd2+ (not shown), suggesting that this current was carried
by Ca2+ ions.
Effects of Adenosine on Rate-Dependent Activation Failure
and Refractoriness of Atrial, Ventricular, and AV
Nodal Myocytes
When Wenckebach activation failure of atrial myocytes
was elicited
by critical current stimuli at a fixed BCL, application of
adenosine (10 µmol/L) converted the Wenckebach periodicity
into a 1:1 stimulus-response pattern (Fig 10).
Consistent with this finding, adenosine shortened the
refractory period of atrial myocytes from 210 (control) to 150 ms
(Figure 11A), which was accompanied by a significant
reduction in action potential duration. In ventricular
myocytes, the activation failure pattern observed was an
all-or-none phenomenon (data not shown), and adenosine
at concentrations as high as 100 µmol/L had no effect on the
refractory period (Fig 11B). In comparison, adenosine (1
µmol/L) significantly prolonged the refractory period of AV nodal
cells (eg, from 300 to 340 ms; Fig 4). The effects of adenosine
on the refractory periods of atrial, AV nodal, and
ventricular cells are summarized in Fig 12.
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As many as six different types of cells in the AV nodal region may contribute to the rate-dependent delay of AV nodal conduction.10 Because we studied only myocytes that had electrophysiological properties typical of cells of the N region of the AV node, the effects of adenosine that we observed may not be similar to the effects of adenosine on cells from other regions of the AV node (eg, AN and NH regions).14 To our knowledge, there is no reliable method to isolate and to distinguish single cells from the various regions of the AV node.
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Adenosine Enhanced Wenckebach Periodicity of Single AV Nodal Cells
The Wenckebach phenomenon is manifested in the electrocardiogram as a progressive beat-to-beat prolongation of the PR interval that leads to a failed ventricular activation (dropped beat), followed by restoration of AV nodal transmission and another cycle of progressive increases in AV nodal conduction delay. Wenckebach periodicity of electrical activity of rabbit single AV nodal cells has been observed.12 Adenosine has been shown to cause Wenckebach AV block in multicellular cardiac preparations,14 in perfused isolated hearts,31 and in human patients.15 The negative dromotropic effect of adenosine was found to be associated with a depression by adenosine of the action potentials of AV nodal cells.14 The demonstration that adenosine and A1 adenosine receptor agonists cause greater slowing of AV conduction during fast than during slow pacing in guinea pig and rat isolated hearts19 20 23 and in human hearts22 24 led us to investigate the cellular basis of this phenomenon. Adenosine decreased action potential amplitude (Fig 1B
) and enhanced rate-dependent
activation
failure of single AV nodal cells (Figs 2 and 3).
The action of
adenosine was concentration dependent, reversible, and mediated
by A1 adenosine receptors. The findings provide the
cellular basis for the observation that the negative dromotropic effect
of adenosine is greater during supraventricular
tachycardia than during normal sinus
rhythm.23
Adenosine Decreased Excitability of AV Nodal
Cells
The adenosine-induced (or enhanced) activation failure
(Wenckebach periodicity) may be due to alteration of the excitability
of AV nodal cells. Adenosine decreased the excitability of
single AV nodal cells (Figs 4 to 8). In multicellular preparations, AV
nodal cells are characterized by action potentials with low amplitude,
slow upstroke velocity (Vmax), and refractory
periods longer than the action potential duration (ie,
postrepolarization refractoriness32 33 ). The rabbit
single
AV nodal cells used in the present study had
electrophysiological characteristics
similar to those of AV nodal cells in multicellular preparations from
rabbit and guinea pig hearts.14 34 35
Our findings show that adenosine prolongs the refractory period, increases the duration of activation delay, increases the current amplitude required for eliciting action potentials, and depresses action potential amplitude and the rate of rise of phase 0 of the action potential. These effects of adenosine on the action potentials of single AV nodal cells are consistent with the observations that adenosine and A1 receptor agonists increase AV nodal refractory period and cause Wenckebach AV block in laboratory animals and in human patients by slowing impulse conduction in the proximal portion of the AV node, ie, by prolonging the atrial–His bundle interval.17 31
Identification of Single AV Nodal Cells
Single viable
Ca2+-tolerant AV nodal cells from
collagenase-digested rabbit hearts were first described
by Nakayama et al30 as spherical or oval-shaped
"balls" with a relatively smooth and shiny surface. Hoshino et
al12 used a series of
electrophysiological parameters
to distinguish ellipsoid-shaped AV nodal cells from
ventricular cells, whereas Hancox et al36
described morphologically normal isolated AV nodal cells as rod- or
spindle-shaped. Because contamination of single AV nodal cell
preparations with atrial and ventricular myocytes is
inevitable, it is essential to establish criteria to distinguish AV
nodal cells from cardiomyocytes in adjacent regions of the
heart. The absent or small IK1, the high input
resistance, the action potential configuration (low amplitude and slow
rate of rise), the small cell capacitance, and the responses to
carbachol and adenosine are distinguishing characteristics of
AV nodal cells (Table). In our experience, rod-shaped cells
usually
had electrophysiological features typical
of atrial myocytes, whereas ellipsoid-shaped cells had
electrophysiological characteristics of AV
nodal cells. However, it was difficult, if not impossible, to
distinguish AV nodal from other cell types (eg, atrial) solely on the
basis of morphology. To identify AV nodal cells, the
electrophysiological and pharmacological
criteria summarized in the Table were found to be more reliable
than
cell shape alone. In particular, it was noted that adenosine
increased activation failure of AV nodal cells, whereas it decreased or
had no effect on activation failure of atrial and
ventricular cells, respectively. This differential effect
of adenosine on activation failure of isolated cells observed
in the present study is consistent with previously reported
effects of adenosine on cells in intact cardiac tissues. For
instance, in guinea pig isolated hearts, open-chest
anesthetized dogs, and human patients, adenosine is
well known to cause Wenckebach-type AV block and to induce atrial
flutter and/or
fibrillation.14 15 37 38 39
Ion Currents Mediating the Effects of Adenosine on
Excitability of AV Nodal Cells
The inward current responsible for the
depolarization of AV nodal
cells is carried by Ca2+ and Na+ ions through
kinetically "slow" channels similar to those found in sinoatrial
nodal cells.40 The relatively small inward depolarizing
current of AV nodal cells can be easily shunted by modest increases in
membrane K+ permeability.34 Changes in outward
and inward transmembrane currents are expected to affect excitability.
In AV nodal cells, adenosine causes an increase in outward
current and a decrease in inward current (Fig 9). Either change
could
account for the small (4- to 10-mV)
hyperpolarization of membrane potential and the
decrease of action potential duration (Figs 1B and
6). The
hyperpolarization will shift the membrane resting
potential away from the threshold potential and should be sufficiently
large to account for the decrease in excitability of AV nodal cells.
Unexpectedly, adenosine did not increase the rate of
repolarization of the AV nodal cell action potential (Fig 1B).
This
result may indicate that adenosine acts to alter other currents
in addition to those that we measured. It is clear that the ionic
mechanisms by which adenosine alters the shape of the AV nodal
cell action potential need to be investigated further.
Adenosine also
caused a decrease in inward current (Fig 9).
This current was blocked by Cd2+ (100 µmol/L) and thus
appears to be carried by Ca2+ ions. Even a small effect on
inward Ca2+ current may be important, because there is no
fast Na+ current in AV nodal cells to provide a robust
inward current for depolarization. Thus, we propose that the combined
activation of IK,ADO and decrease in ICa,L is a
potential basis of the decrease in excitability and, hence, may explain
the increased rate-dependent activation failure caused by
adenosine and A1 receptor agonists.
However, the ionic mechanism(s) of adenosine's action to decrease AV nodal cell excitability may be complex. The time-and frequency-dependent changes in excitability of AV nodal cells in the absence of drugs have been proposed to be related to the slow kinetics of deactivation of IK, IK1, and ICa,L12 A full explanation of the inhibitory effect of adenosine on recovery of excitability of AV nodal cells will require quantification of the magnitude and kinetics of IK,ADO and ICa,L, as well as the consideration of other current systems.
Recently, Liu et al41 suggested that inhibition of excitability secondary to a transient decrease in ICa,T plays an important role in concealed AV nodal conduction. Adenosine has no effect on ICa,T of guinea pig atrial myocytes.42 However, because of the potential importance of this current to the depolarization process of AV nodal cells, the effect of adenosine on ICa,T of rabbit AV nodal cells needs to be investigated. Likewise, because adenosine was found to depress an If-like current in AV nodal cells,43 the potential contribution of If to the effect of adenosine on recovery of excitability of AV nodal cells needs to be studied. Another ion current that could contribute to the rate-dependent activation of AV nodal cells is the Ito. Adenosine does not affect Ito of atrial and ventricular myocytes (authors' unpublished data, 1995). However, because this current mediates frequency-dependent modulation of action potential duration29 and has been identified in the rabbit AV node44 and in crista terminalis cells,45 its potential role in the rate-dependent negative dromotropic effect of adenosine needs to be investigated.
Comparisons Among Atrial, AV Nodal, and
Ventricular Cells
The shorter refractory period and decreased
activation failure of
atrial myocytes in the presence of adenosine (Fig 10) are
likely to be the cellular basis of the action of adenosine to
induce atrial flutter and/or fibrillation in laboratory animals and
humans.38 39 Adenosine was reported to shorten the
duration of the atrial action potential, an effect attributed to
activation of IK,ADO,46 but had little
or no effect on action potential amplitude (Fig 1A). In AV
nodal cells,
by contrast, adenosine shortened the duration of the action
potential but prolonged rather than shortened the duration of the
refractory period and decreased the amplitude of the action potential.
The differential effects of adenosine on atrial and AV nodal
cells may be due to the following: (1) The contribution of
ICa,L to the inward current in atrial cells is relatively
small, whereas it is a major portion of the inward current in AV nodal
cells. Consequently, a small decrease in ICa,L will have a
greater impact on the depolarization of AV nodal cells than of atrial
myocytes. (2) The input resistance of atrial cells is smaller than that
of AV nodal cells; hence, increases of outward current of equal
amplitude will cause less hyperpolarization and
less decrease of action potential amplitude and excitability of atrial
than of AV nodal cells.
The lack of effects of adenosine on action potential configuration and transmembrane currents of ventricular myocytes may be due to the absence or low density of IK,ADO channels in ventricular myocardium.47 48 Although adenosine can attenuate ß-adrenergic–stimulated ICa,L of ventricular myocytes, it does not affect basal ICa,L of guinea pig, bovine, and rabbit ventricular myocytes.49 The lack of effects of adenosine on resting membrane potential, phase-3 repolarization, IK1, IK, and the refractory period of ventricular myocytes is consistent with the insensitivity of major ionic currents in ventricular myocardium to adenosine, in the absence of other drugs.49
In summary, the present study provides evidence that the mechanism of adenosine-induced Wenckebach AV block is the depression of the excitability of AV nodal cells. This effect of adenosine can at least in part be attributed to the activation of IK,ADO and to a reduction of ICa,L. However, the involvement of other membrane currents in the action of adenosine on AV nodal cells cannot be ruled out. Regardless, the rate dependence of adenosine's effect on the activation of AV nodal cells explains why the negative dromotropic effect of adenosine is dependent on the rate of atrial pacing and is more pronounced during an episode of supraventricular tachycardia than during normal rhythm.19 20 21 22 23 24 25 Investigations of the relative contributions of IK,ADO and ICa,L, as well as the potential role of other membrane ion current systems, are needed before the cellular basis for the negative dromotropic action of adenosine is fully understood. In addition, because the electrophysiological actions of adenosine are cell type and species dependent, extrapolations of our findings to other species should be made cautiously.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grant HL-50488. The authors thank Dr Justus Anumonwo and Dr Mario Delmar at the State University of New York for teaching us the technique of isolation of single atrioventricular nodal cells from rabbit hearts and for valuable criticism and suggestions to improve the manuscript.
Received July 7, 1995; accepted December 22, 1995.
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References |
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|
TopAbstractIntroductionMaterials and Methods Results Discussion References |
|---|
1. Lewis T, Master AM. Observations upon conduction in the mammalian heart: AV conduction. Heart (Lond). 1925;12:209-269.
2. Wenckebach KF. Zur analyse des unregelmässigen pulses. Z Klin Med. 1899;37:475-488.
3. Simson MB, Spear JF, Moore EN. Electrophysiologic studies on atrioventricular nodal Wenckebach cycles. Am J Cardiol. 1978;41:244-258. [Medline] [Order article via Infotrieve]
4.
Slama R, LeClercq JF, Rosengarten M, Coumel PH,
Bouvrain Y. Multilevel block in the
atrioventricular node during atrial
tachycardia and flutter alternating with Wenckebach
phenomenon. Br Heart J. 1979;42:463-470.
5. Stanton MS, Miles WM, Zipes DP. Atrial fibrillation and flutter. In: Zipes DP, Jalife J, eds. Cardiac Electrophysiology: From Cell to Bedside. Philadelphia, Pa: WB Saunders; 1990:735-742.
6. Rosenblueth A. Mechanism of the Wenckebach-Luciani cycles. Am J Physiol. 1958;194:491-494.
7.
Merideth J, Mendez C, Mueller WJ, Moe GK.
Electrical excitability of atrioventricular nodal
cells. Circ Res. 1968;23:69-85.
8.
Jalife J, Antzelevitch C, Lamanna V, Moe GK.
Rate-dependent changes in excitability of depressed cardiac
Purkinje fibers as a mechanism of intermittent bundle branch
block. Circulation. 1983;67:912-922.
9.
Shrier A, Dubarski H, Rosengarten M, Guevara MR,
Nattel S, Glass L. Prediction of complex
atrioventricular conduction rhythms in humans with use
of the atrioventricular nodal recovery curve.
Circulation. 1987;76:1196-1205.
10.
Billette J. Atrioventricular
nodal activation during periodic premature stimulation of the
atrium. Am J Physiol. 1987;252:H163-H177.
11.
Delmar M, Michaels DC, Jalife J. Slow recovery
of excitability and the Wenckebach phenomenon in the single guinea pig
ventricular myocyte. Circ Res. 1989;65:761-774.
12.
Hoshino K, Anumonwo J, Delmar M, Jalife J.
Wenckebach periodicity in single atrioventricular nodal
cells from the rabbit heart. Circulation. 1990;82:2201-2216.
13.
Delmar M, Glass L, Michaels DC, Jalife J. Ionic
basis and analytical solution of the Wenckebach phenomenon in guinea
pig ventricular myocytes. Circ
Res. 1989;65:775-788.
14.
Clemo SHF, Belardinelli L. Effect of
adenosine on atrioventricular conduction, I:
site and characterization of adenosine action in the guinea pig
atrioventricular node. Circ
Res. 1988;59:427-436.
15.
Di Marco JP, Seller TS, Berne RM, West GA, Belardinelli
L. Adenosine:
electrophysiological effects and
therapeutic use for terminating paroxysmal
supraventricular tachycardia.
Circulation. 1983;68:1254-1263.
16. Belardinelli L, Lerman BB. Adenosine: cardiac electrophysiology. Pacing Clin Electrophysiol. 1991;14:1672-1680. [Medline] [Order article via Infotrieve]
17. Rankin AC, Brooks R, Ruskin JN, McGovern BA. Adenosine and the treatment of supraventricular tachycardia. Am J Med. 1992;92:655-664. [Medline] [Order article via Infotrieve]
18. Martynyuk AE, Kane KA, Cobbe SM, Rankin AC. Adenosine increases potassium conductance in isolated rabbit atrioventricular nodal myocytes. Cardiovasc Res. 1995;30:668-675. [Medline] [Order article via Infotrieve]
19.
Belardinelli L, Shryock JC. Does
adenosine function as a retaliatory metabolite in the
heart? News Physiol Sci. 1992;7:52-56.
20. Stark G, Sterz F, Stark U, Bachernegg M, Decrinis M, Tritthart HA. Frequency-dependent effects of adenosine and verapamil on atrioventricular conduction of isolated guinea pig hearts. J Cardiovasc Pharmacol. 1993;21:955-959. [Medline] [Order article via Infotrieve]
21.
Nayebpour M, Billette J, Amellal F, Nattel
S. Effects of adenosine on rate-dependent
atrioventricular nodal function: potential roles in
tachycardia termination and
physiological regulation.
Circulation. 1993;88:2632-2645.
22. Radzik D, Talajic M, Roy D, Lemery R, Hii JTY, Thibault B, Lavalle E, Nattel S. Rate-dependent actions of adenosine on the human atrioventricular node. Pacing Clin Electrophysiol.. 1993;16:891. Abstract.
23.
Belardinelli L, Lu J, Dennis D, Martens J, Shryock
JC. The cardiac effects of a novel
A1-adenosine receptor agonist in guinea pig
isolated heart. J Pharmacol Exp Ther. 1994;271:1-12.
24.
Lai WT, Lee CS, Wu SN. Rate-dependent
properties of adenosine-induced negative dromotropism in
humans. Circulation. 1994;90:1832-1839.
25.
Anderson RH, Janse MJ, van Capelle FJL, Billette J,
Becker AE, Durrer D. A combined morphological and
electrophysiological study of the
atrioventricular node of the rabbit heart.
Circ Res. 1974;35:909-922.
26. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391:85-100.[Medline] [Order article via Infotrieve]
27. Kao CY, Hoffman BF. Graded and decremental response in heart muscle fibers. Am J Physiol. 1958;194:187-196.
28. Thuringer D, Larribe P, Escande D. A hyperpolarization-activated inward current in human myocardial cells. J Mol Cell Cardiol. 1992;24:451-455. [Medline] [Order article via Infotrieve]
29.
Giles WR, Imaizumi Y. Comparison of potassium
currents in rabbit atrial and ventricular cells.
J Physiol (Lond). 1988;405:123-145.
30. Nakayama T, Kurachi Y, Noma A, Irisawa H. Action potential and membrane currents of single pacemaker cells of the rabbit heart. Pflugers Arch. 1984;402:248-257. [Medline] [Order article via Infotrieve]
31.
Belardinelli L, Fenton RA, West G, Linden J, Althaus
JS, Berne RM. Extracellular action of adenosine and the
antagonism by aminophylline on the atrioventricular
conduction of isolated perfused guinea pig and rat hearts.
Circ Res. 1982;51:569-579.
32. Hoffman BF, Cranefield PF. Electrophysiology of the Heart. Mt Kisco, NY: Futura Publishing Co; 1960:211-256.
33. Cranefield PF. The Conduction of the Cardiac Impulse. Mt Kisco, NY: Futura Publishing Co; 1975:1-151.
34.
Akiyama T, Fozzard HA. Ca and Na selectivity of
the active membrane of AV nodal cells. Am J Physiol. 1979;236:C1-C8.
35.
Zipes DP, Mendez C. Action of manganese ions and
tetrodotoxin on atrioventricular nodal transmembrane
potentials in isolated rabbit hearts. Circ
Res. 1973;32:447-454.
36.
Hancox HC, Levi AJ, Lee CO, Heap P. A method for
isolating rabbit atrioventricular node myocytes which
retain normal morphology and function. Am J Physiol. 1993;265:H755-H766.
37. Belardinelli L, Mattos EC, Berne RM. Evidence for adenosine mediation of atrioventricular block in the ischemic canine myocardium. J Clin Invest. 1981;68:195-205.
38.
Kabell G, Buchanan LV, Gibson JK, Belardinelli
L. Effects of adenosine on atrial refractoriness and
arrhythmias. Cardiovasc Res. 1994;28:1385-1389.
39. Di Marco JP, Miles W, Akhtar M, Adenosine for PSVT Study Group. Adenosine for paroxysmal supraventricular tachycardia: dose ranging and comparison with verapamil. Ann Intern Med. 1990;113:104-110.
40. Kokubun S, Nishimura M, Noma A, Irisawa H. The spontaneous action potential of rabbit atrioventricular node cells. Jpn J Physiol. 1980;30:529-540. [Medline] [Order article via Infotrieve]
41.
Liu YN, Zeng WZ, Delmar M, Jalife J. Ionic
mechanism of electrotonic inhibition and concealed conduction in rabbit
atrioventricular nodal myocytes.
Circulation. 1993;88:1634-1646.
42.
Cerbai E, Klockner U, Isenberg G.
Ca-antagonistic effects of adenosine in guinea
pig atrial cells. Am J Physiol. 1988;255:H872-H878.
43. Wang D, Belardinelli L. Effects of adenosine on phase 4 depolarization and pacemaker current (If) in single rabbit atrioventricular nodal myocytes. FASEB J. 1994;8:A611. Abstract.
44.
Nakayama T, Irisawa H. Transient outward current
carried by K+ and Na+ in quiescent
atrio-ventricular node cells of rabbit.
Circ Res. 1985;57:65-73.
45.
Giles WR, Van Ginneken A. A transient outward
current in isolated cells from the crista terminalis of rabbit
heart. J Physiol (Lond). 1985;368:243-264.
46.
Visentin S, Wu SN, Belardinelli L.
Adenosine-induced changes in atrial action potential:
contribution of Ca and K currents. Am J Physiol. 1990;258:H1070-H1078.
47.
Dascal N, Schreibmayer W, Lim NF, Wang WZ, Chavkin C,
Dimagno L, Labarca C, Kieffer BL, Gaveriaux-Ruff C, Trollinger D,
Lester HA, Davidson N. Atrial G protein activated
K+ channel: expression cloning and molecular
properties. Proc Natl Acad Sci U S A. 1993;90:10235-10239.
48. Kubo Y, Reuveny E, Slesinger PA, Jan YN, Jan LY. Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel. Nature. 1993;364:802-806. [Medline] [Order article via Infotrieve]
49.
Belardinelli L, Shryock JC, Song Y, Wang D, Srinivas
M. Ionic basis of the
electrophysiological action of
adenosine in cardiomyocytes. FASEB
J. 1995;9:359-365.
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