Cellular Basis for the Negative Dromotropic Effect of Adenosine on Rabbit Single Atrioventricular Nodal Cells
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.
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.
Materials and Methods
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 2×4-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.
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 MΩ 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 MΩ, 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).
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).
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.
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.
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.
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.
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.
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.
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.
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.
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⇓.
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.
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 β-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.
Selected Abbreviations and Acronyms
|BCL||=||basic cycle length|
|ICa,L||=||L-type Ca2+ current|
|ICa,T||=||T-type Ca2+ current|
|IK||=||delayed rectifier K+ current|
|IK,ADO||=||inwardly rectifying outward K+ current activated by adenosine (ADO)|
|IK1||=||inward rectifier K+ current|
|Ito||=||transient outward K+ current|
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.
- © 1996 American Heart Association, Inc.
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