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(Circulation. 1997;96:1275-1281.)
© 1997 American Heart Association, Inc.

Articles

Withdrawal of Acetylcholine Elicits Ca²⁺-Induced Delayed Afterdepolarizations in Cat Atrial Myocytes

Y.G. Wang, MD; J. Hüser, PhD; L.A. Blatter, MD, Dr med; ; S.L. Lipsius, PhD

From Loyola University of Chicago, Stritch School of Medicine, Department of Physiology, Maywood, Ill.

Correspondence to Stephen L. Lipsius, PhD, Department of Physiology, Loyola University Medical Center, 2160 S First Ave, Maywood, IL 60153. E-mail slipsiu{at}wpo.it.luc.edu

	Abstract

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Background Recent experiments in atrial myocytes indicate that withdrawal of cholinergic agonist can directly increase Ca²⁺ influx via L-type Ca²⁺ current and stimulate Ca²⁺ uptake into the sarcoplasmic reticulum (SR), thereby increasing intracellular Ca²⁺. Overload of cellular Ca²⁺ within the SR can initiate various types of atrial dysrhythmias. The present study was designed to determine whether withdrawal of acetylcholine (ACh) can elicit Ca²⁺-induced delayed afterdepolarizations (DADs) in atrial myocytes.

Methods and Results A nystatin perforated-patch whole-cell method and fluorescence microscopy (indo 1) were used to measure electrical activities and intracellular free Ca²⁺ ([Ca²⁺]_i), respectively. Withdrawal of ACh (1 µmol/L) increased action potential duration, shifted plateau voltage toward positive, and generated DADs that initiated spontaneous action potentials. Voltage-clamp analysis revealed that withdrawal of ACh elicited a rebound stimulation of L-type Ca²⁺ current (I_Ca,L) (+45%) and Na/Ca exchange current (I_NaCa) (+16%) and the appearance of transient inward current (I_ti) and spontaneous [Ca²⁺]_i transients. Each of these changes induced by withdrawal of ACh was abolished by Rp-cAMPs (50 to 100 µmol/L) or H-89 (2 µmol/L), inhibitors of cAMP-dependent protein kinase A. Ryanodine (1 µmol/L) abolished I_NaCa and the appearance of I_ti without decreasing the rebound stimulation of I_Ca,L elicited by withdrawal of ACh.

Conclusions Withdrawal of ACh can elicit cAMP-mediated stimulation of Ca²⁺ influx via I_Ca,L and uptake of SR Ca²⁺. As a result, cellular Ca²⁺ overload causes enhanced SR Ca²⁺ release and the initiation of DADs. These mechanisms may generate triggered and/or spontaneous atrial depolarizations elicited by withdrawal of vagal nerve activity.

Key Words: arrhythmia • electrophysiology • action potentials • calcium • adenosine

	Introduction

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Autonomic tone plays an important role in the initiation and modulation of cardiac dysrhythmias.^{1 2} Stimulation of vagal nerve activity can initiate atrial fibrillation by shortening atrial refractoriness or terminate paroxysmal supraventricular tachycardia by depressing AV nodal conduction. Vagal nerve–mediated inhibition of heart rate can also initiate or terminate certain types of ventricular dysrhythmias. However, the effects of withdrawal of vagal nerve activity have received less attention. In general, vagal withdrawal is considered arrhythmogenic,¹ although the mechanisms are less well understood. The most obvious effect of vagal withdrawal is an increased sinus rate, which can directly initiate dysrhythmias, particularly in the setting of myocardial ischemia. Because sympathetic and vagal nerve activities usually act reciprocally, autonomically induced dysrhythmias are generally attributed to enhanced sympathetic effects unleashed by withdrawal of vagal nerve activity. Thus, vagal nerve activity inhibits sympathetic release of norepinephrine and suppresses second messenger pathways activated by ß-adrenergic receptor stimulation.

Recent findings, however, indicate that in isolated atrial myocytes, withdrawal of ACh can directly stimulate atrial function by eliciting a rebound stimulation of I_Ca,L and contraction.³ Moreover, in atrial muscle preparations, withdrawal of cholinergic agonist elicits rebound stimulation of intracellular Ca²⁺ release and contraction.⁴ These findings indicate that after exposure to ACh, intracellular Ca²⁺ concentrations increase to levels above control. Elevation or overload of Ca²⁺ within the SR is known to underlie the development of various cardiac dysrhythmias, especially those caused by DADs.^{5 6 7} Therefore, rebound increases in intracellular Ca²⁺ may underlie the initiation of premature atrial beats elicited by withdrawal of vagal nerve activity.⁸ The purpose of the present study, therefore, was to determine whether withdrawal of ACh from isolated atrial myocytes can elicit the development of DADs that are mediated by overload of SR Ca²⁺.

	Methods

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Details of the isolation and recording methods have been published previously.⁹ Atrial myocytes were isolated from cat heart. Adult cats of either sex were anesthetized with sodium pentobarbital (70 mg/kg IP). Hearts were perfused via a Langendorff apparatus with a bicarbonate-buffered Tyrode's solution for 5 minutes followed by perfusion with a nominally Ca²⁺-free Tyrode's solution. After 5 minutes, the perfusion was switched to a low-Ca²⁺ (36 µmol/L) Tyrode's solution containing 0.06% collagenase (Worthington Biochemical, type II) for 30 to 40 minutes. After collagenase perfusion, both atria were cut into small pieces and agitated in fresh collagenase and 0.01% protease. Experiments were performed on either right or left atrial cells, with no discernable differences in their responses to withdrawal of ACh.

Cells used for study were transferred to a small tissue bath on the stage of an inverted microscope (Nikon Diaphot) and superfused with a modified Tyrode's solution containing (in mmol/L) NaCl 137, KCl 5.4, MgCl₂ 1.0, CaCl₂ 2.5, HEPES 5, and glucose 11 and titrated with NaOH to a pH of 7.4. Solution was perfused through a small (0.3-mL) chamber by gravity at 5 mL/min. The system required 20 seconds to completely exchange the bath contents. All experiments were performed at 35±1°C. Cells selected for study were elongated and quiescent. Membrane potential and ionic currents were recorded by a nystatin perforated-patch¹⁰ whole-cell recording method.¹¹ This method was used because it minimizes dialysis of intracellular constituents with the internal pipette solution and thereby maintains physiological buffering of intracellular Ca²⁺ and second-messenger signaling pathways.¹² Nystatin was dissolved in DMSO at a concentration of 50 mg/mL and then added to the internal pipette solution to yield a final nystatin concentration of 150 µg/mL. The pipette solution containing nystatin is strongly sonicated before use. The internal pipette solution contained (in mmol/L) cesium glutamate 100, CsCl 40, MgCl₂ 1.0, Na₂ATP 4, EGTA 0.5, and HEPES 5 and was titrated with CsOH to a pH of 7.2. To record I_Ca,L, ACh-activated K⁺ currents were blocked by Cs⁺ in the internal pipette solution and addition of 20 mmol/L CsCl to the external solution. If ACh elicited changes in background K⁺ conductance, the cell was discarded. In experiments designed to measure action potentials, the pipette solution contained K⁺ instead of Cs⁺.

A single suction pipette was used to record voltage (bridge mode) or ionic currents (discontinuous voltage-clamp mode) with an Axoclamp 2A amplifier (Axon Instruments, Inc). The switch (discontinuous) clamp avoids the potential effects of series resistance. Action potentials were elicited by stimulation (1 Hz) through the recording pipette (bridge mode) with 2- to 3-ms voltage pulses at twice diastolic threshold. Computer software (pCLAMP; Axon Instruments, Inc) was used to deliver voltage protocols and acquire and analyze data. In addition, all signals were digitally recorded on VCR tape.

[Ca²⁺]_i was measured by fluorescence microscopy with the cell-permeant Ca²⁺-sensitive fluorescent dye indo 1 acetoxymethyl ester (indo 1-AM; Molecular Probes, Inc). Atrial myocytes were loaded with indo 1-AM at room temperature for 20 minutes. The cell loading solution consisted of 2 mL Tyrode's solution containing 5 µmol/L indo 1-AM, 2.5 µL 25% wt/wt Pluronic F-127 (Molecular Probes; solubilized in DMSO), and 75 µL newborn calf serum (GIBCO). [Ca²⁺]_i was measured at 35±1°C by exciting indo 1 fluorescence with light of 360-nm wavelength and measuring emitted fluorescence signals simultaneously at 405 nm (F₄₀₅) and 485 nm (F₄₈₅). Single-cell fluorescence signals were recorded with photomultiplier tubes (model R2693; Hamamatsu Corp) by masking off individual cells with a pinhole positioned in the emission pathway. [Ca²⁺]_i transients were elicited by field stimulation (0.4 Hz) with 2-ms voltage pulses of suprathreshold amplitude through platinum wires. Relative changes in [Ca²⁺]_i are reported as changes in the fluorescence ratio F₄₀₅/F₄₈₅.

Drugs included acetylcholine chloride (Sigma Chemical Co), Rp-cAMPs (LC Laboratories), and H-89 (Seikagaku America, Inc). The present experiments as well as previous studies³ have shown that H-89 and Rp-cAMPs both effectively abolish the rebound stimulatory effects of ACh withdrawal. Because H-89 is much less expensive than Rp-cAMPs, H-89 was used in some experiments requiring larger volumes of superfusate. Cells studied were isolated on the same morning that the experiment was performed. In general, I_Ca,L was activated by clamping cells from a holding potential of -40 mV to inactivate fast Na⁺ channels to 0 mV for 200 ms every 10 seconds. Peak I_Ca,L was measured with respect to zero current and was not compensated for leak currents. I_NaCa was measured at 50% time to recovery, between the end of the clamp pulse and the point at which the current returned to baseline. The DAD coupling interval was measured from the action potential upstroke to the initial change in membrane potential of the DAD. Statistical significance of paired and unpaired data was determined by Student's t test at values of P<.05. Data are expressed as mean±SEM. The animal procedures followed in this study were in accordance with the guidelines of the Animal Care and Use Committee of Loyola University Medical Center.

	Results

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Fig 1 shows the effect of 1 µmol/L ACh on stimulated (1 Hz) action potentials recorded from a single atrial myocyte. Under control conditions, APD₉₀ was 152 ms and APP₅₀ was 2 mV. Exposure to ACh shortened APD markedly and hyperpolarized the resting membrane potential. Within 30 seconds of withdrawal of ACh, APD₉₀ and APP₅₀ exhibited a rebound increase to 182 ms and 15 mV, respectively. These action potential parameters recovered to control levels 4 minutes after withdrawal of ACh. In the 4 cells studied, withdrawal of ACh elicited a rebound increase in APD₉₀ from 159±8 to 182±8 ms (+15±2%; P<.01) and a positive shift in APP₅₀ of +7 mV (+9±2%; P<.05).

View larger version (6K): [in this window] [in a new window]   Figure 1. Withdrawal of ACh elicits a rebound increase in APD. Control: Action potential before ACh exposure exhibited an APD90 of 152 ms. ACh: Exposure to 1 µmol/L ACh for 2 minutes hyperpolarized resting membrane potential and dramatically shortened APD. Rebound: Within 30 seconds of withdrawal of ACh, APD rebound increased to 182 ms and plateau voltage shifted more positively. Recovery: Action potential configuration recovered to control 4 minutes after withdrawal of ACh.

Fig 2A shows typical effects of 1 µmol/L ACh on stimulated (1 Hz) action potentials recorded from another atrial myocyte. Exposure to ACh hyperpolarized the resting membrane potential, increased action potential amplitude, and shortened APD. In most experiments, stimulation failed to elicit action potentials during exposure to ACh because of membrane hyperpolarization. Action potentials reappeared immediately upon withdrawal of ACh. The inability to elicit action potentials during exposure to ACh had no discernable effect on the rebound responses elicited by withdrawal of ACh. Approximately 30 seconds after withdrawal of ACh (Fig 2Ab), action potential amplitude progressively increased, diastolic slope was more pronounced, and DADs appeared after each action potential. DADs reached threshold and generated spontaneous activity that gradually decayed and ended with damped oscillations in membrane voltage. About 4 minutes after withdrawal of ACh, stimulated action potentials recovered to control configuration (Fig 2Ac). DADs were elicited in 25 of 29 atrial cells studied (86%) and appeared within 41±1 seconds of withdrawal of ACh. The DAD coupling interval, measured in relation to the action potential upstroke, was 589±57 ms (n=8).

View larger version (30K): [in this window] [in a new window]   Figure 2. Withdrawal of ACh elicits DADs and spontaneous activity via stimulation of cAMP. A, Control responses to 1 µmol/L ACh for 2 minutes (solid line) followed by withdrawal of ACh. Aa, Exposure to ACh hyperpolarized resting membrane potential, shortened APD, and increased action potential amplitude. Ab, Within 30 seconds of withdrawal of ACh, action potential amplitude gradually increased, diastolic slope increased, and DADs developed and resulted in spontaneous activity. Ac, Action potentials recovered 4 minutes after withdrawal of ACh. B, Effect of 2 µmol/L H-89. Ba, Exposure to ACh elicited inhibitory effects similar to those under control conditions. Bb, Withdrawal of ACh failed to elicit changes in diastolic slope, appearance of DADs, or initiation of spontaneous activity. Bc, Recovery 4 minutes after withdrawal of ACh. Cells were stimulated at 1 Hz. All records obtained from same atrial cell.

The development of DADs and spontaneous voltage oscillations suggests that withdrawal of ACh induced cellular Ca²⁺ overload.⁷ Moreover, withdrawal of ACh has been shown to elicit a rebound increase in cAMP concentration.¹³ To determine whether a rebound stimulation of the cAMP signaling pathway underlies the development of DADs elicited by withdrawal of ACh, we repeated the experiment in the presence of 2 µmol/L H-89, an inhibitor of cAMP-dependent PKA.¹⁴ Previous experiments¹⁵ have shown that 2 µmol/L H-89 abolishes the stimulatory effects of 1 µmol/L isoproterenol on I_Ca,L. Fig 2B shows electrically stimulated action potentials recorded from the same myocyte in the continuous presence of H-89. Under these conditions, exposure to ACh elicited inhibitory effects similar to those obtained under control conditions. Withdrawal of ACh, however, failed to increase action potential amplitude, increase diastolic slope, elicit DADs, or initiate spontaneous oscillations in membrane potential (Fig 2Bb). Similar results were obtained in all 6 cells studied. In 3 additional cells, similar results were obtained with 50 µmol/L Rp-cAMPs, a selective cAMP-dependent PKA inhibitor.¹⁶ These results support the idea that stimulation of the cAMP-dependent PKA pathway is responsible for the dysrhythmic changes in action potential configuration elicited by withdrawal of ACh.

I_Ca,L triggers SR Ca²⁺ release, which in turn stimulates sarcolemmal I_NaCa. Because I_NaCa contributes to the action potential plateau voltage and duration¹⁷ and to the development of I_ti,^{18 19} we determined whether the withdrawal of ACh elicits a rebound stimulation of I_NaCa. As shown in Fig 3A, activation of I_Ca,L and stimulation of I_NaCa were elicited by short (30-ms) depolarizing clamp steps from -50 to 0 mV and then back to -70 mV. Depolarization elicited basal I_Ca,L (c), followed upon repolarization by a slowly declining inward tail current that is identified as I_NaCa.^{20 21} Exposure to 1 µmol/L ACh inhibited basal I_Ca,L (-23±9%; P<.05) and I_NaCa (-16±8%; P<.05) (n=5). Presumably, the inhibition of I_NaCa is secondary to the ACh-induced decrease in I_Ca,L, which would reduce loading and release of SR Ca²⁺. Within 30 seconds of withdrawal of ACh, there was a concomitant rebound stimulation of I_Ca,L and I_NaCa. I_Ca,L and I_NaCa recovered to control levels 4 minutes after withdrawal of ACh (r). In all 5 cells studied, withdrawal of ACh stimulated I_Ca,L and I_NaCa by 45±17% (P<.01) and 16±4% (P<.01) above control values, respectively. These results indicate that the rebound stimulation of I_Ca,L elicited by withdrawal of ACh is accompanied by an increase in SR Ca²⁺ load and/or SR Ca²⁺ release.

View larger version (12K): [in this window] [in a new window]   Figure 3. Withdrawal of ACh elicits rebound stimulation of ICa,L and INaCa via stimulation of cAMP. A, Control responses to a 2-minute exposure to 1 µmol/L ACh followed by withdrawal of ACh. Exposure to ACh decreased both ICa,L and INaCa, and withdrawal of ACh elicited rebound stimulation of ICa,L and INaCa. B, Effects of 50 µmol/L Rp-cAMPs. Exposure to ACh elicited a small decrease in ICa,L and INaCa, and rebound stimulations of ICa,L and INaCa were both abolished. C, Effects of 1 µmol/L ryanodine. Ryanodine abolished INaCa without decreasing rebound stimulation of ICa,L. Records in A and B were recorded from same atrial cell and those in C from another atrial cell. c indicates control recordings before ACh; r, recovery, several minutes after ACh withdrawal.

The role of cAMP in the rebound stimulation of I_NaCa was examined by repeating the short clamp protocol in the presence of 50 µmol/L Rp-cAMPs. As shown in Fig 3B, Rp-cAMPs elicited a small decrease in basal I_Ca,L and I_NaCa (c), consistent with inhibition of the effects of endogenous cAMP.³ In the presence of Rp-cAMPs, ACh still elicited a small inhibition of I_Ca,L and I_NaCa. Although not the focus of this study, one interpretation of these findings is that ACh may be acting to inhibit I_Ca,L, in part via a cAMP-independent mechanism. A similar response to ACh has been reported in sinoatrial node pacemaker cells.¹⁵ Withdrawal of ACh, however, failed to elicit a rebound stimulation of I_Ca,L or I_NaCa. Similar results were obtained in all 6 cells studied and in 2 additional cells with 2 µmol/L H-89 (not shown). Taken together, these findings indicate that withdrawal of ACh acts via stimulation of cAMP-dependent PKA to load SR Ca²⁺ content and enhance I_Ca,L-induced SR Ca²⁺ release.

To confirm that ACh-induced stimulation of I_NaCa is due to release of Ca²⁺ from SR, ACh was tested in the presence of ryanodine, an alkaloid that depletes SR Ca²⁺ content by opening SR Ca²⁺ release channels.²² As shown in Fig 3C, exposure to 1 µmol/L ryanodine for 10 minutes had little effect on the basal I_Ca,L, but it abolished I_NaCa. In the presence of ryanodine, 1 µmol/L ACh elicited inhibition and rebound stimulation of I_Ca,L without the appearance of I_NaCa. Similar results were obtained in all 4 cells studied. These findings indicate that I_NaCa and its modulation by ACh withdrawal are due to SR Ca²⁺ release.

Ca²⁺ overload is known to elicit spontaneous SR Ca²⁺ release and stimulation of I_ti. We therefore determined whether withdrawal of ACh elicits I_ti and whether this effect was mediated by cAMP. In Fig 4, atrial myocytes were clamped from -40 to +30 mV for 2 seconds and then repolarized to different voltages, before (A) and then shortly (35 seconds) after (B) a 2-minute exposure to 1 µmol/L ACh. Under control conditions, clamps to more negative voltages elicited decaying tail currents that appeared to reverse near -80 mV. Within 35 seconds of withdrawal of ACh, the same clamp steps revealed spontaneous oscillations of inward current recognized as I_ti. I_ti appeared larger at more negative voltages. About 4 minutes after withdrawal of ACh, I_ti diminished and currents returned to control levels (C). Exposure of the same cell to 100 µmol/L Rp-cAMPs had no obvious effect on control tail currents (D). In the presence of Rp-cAMPs, however, the withdrawal of ACh failed to elicit the development of I_ti (E). Similar results were obtained in all 5 cells studied. In 4 additional atrial myocytes, exposure to 1 µmol/L ryanodine abolished the development of I_ti elicited by withdrawal of ACh (not shown). These results indicate that the development of I_ti elicited by withdrawal of ACh is mediated via cAMP-dependent PKA activity and due to overload of SR Ca²⁺, similar to the changes in I_NaCa induced by withdrawal of ACh.

View larger version (23K): [in this window] [in a new window]   Figure 4. Withdrawal of ACh elicits Iti via stimulation of cAMP. Ionic current elicited by repolarization to different voltages after a 2-second depolarization from -40 to +30 mV. A, Currents elicited between -20 and -80 mV before exposure to 1 µmol/L ACh. B, Appearance of Iti 35 seconds after withdrawal of ACh. C, Recovery 4 minutes after withdrawal of ACh. D, Exposure to 100 µmol/L Rp-cAMPs. E, Rp-cAMPs abolished development of Iti typically elicited by withdrawal of ACh. All records were obtained from same atrial cell.

Fig 5 shows direct measurements of [Ca²⁺]_i recorded from a stimulated (0.4 Hz) atrial myocyte before (a), during (b), and within 30 seconds of withdrawal of (c) ACh. Fig 5B shows an expanded time scale of each phase of the recording shown in A. Exposure to 1 µmol/L ACh for 2 minutes slightly decreased basal [Ca²⁺]_i and decreased [Ca²⁺]_i transient amplitude to 50% of control (b). Upon withdrawal of ACh, basal [Ca²⁺]_i increased slightly, in part as a result of fusion of spontaneous and stimulated [Ca²⁺]_i transients. In addition, [Ca²⁺]_i transient amplitude steadily increased and irregular spontaneous [Ca²⁺]_i transients appeared, indicative of cellular Ca²⁺ overload (c). [Ca²⁺]_i transients returned to control values within 4 minutes after withdrawal of ACh (not shown). Similar results were obtained in all 7 cells studied.

View larger version (21K): [in this window] [in a new window]   Figure 5. Effects of ACh on [Ca2+]i measured in an electrically stimulated (0.4 Hz) atrial myocyte. A, Exposure to 1 µmol/L ACh for 2 minutes (solid bar) decreased basal [Ca2+]i and [Ca2+]i transient amplitude. Withdrawal of ACh resulted in a return of [Ca2+]i transient amplitude and irregular spontaneous [Ca2+]i transients, indicative of cellular Ca2+ overload. B, Selected traces recorded in A before (a), during (b), and after (c) withdrawal of ACh shown at an expanded time scale.

Fig 6 shows the effects of ACh on [Ca²⁺]_i transients recorded from another atrial myocyte in the continuous presence of 2 µmol/L H-89. H-89 slightly reduced control [Ca²⁺]_i transient amplitude compared with those recorded in the absence of H-89 (not shown). This is consistent with the observation that H-89 or Rp-cAMPs decreased basal I_Ca,L amplitude (Fig 3). Exposure to 1 µmol/L ACh for 2 minutes rapidly decreased [Ca²⁺]_i transient amplitude (b). During exposure to ACh, [Ca²⁺]_i transient amplitude appeared to recover slightly, probably due to desensitization. Withdrawal of ACh elicited a partial recovery of [Ca²⁺]_i transient amplitude toward control but failed to elicit the development of spontaneous [Ca²⁺]_i transients. In other words, inhibition of cAMP-dependent PKA activity prevented cellular Ca²⁺ overload typically induced by withdrawal of ACh. Similar results were obtained in all 3 cells studied.

View larger version (21K): [in this window] [in a new window]   Figure 6. Effect of H-89 on ACh-induced changes in [Ca2+]i in an electrically stimulated (0.4 Hz) atrial myocyte. A, Exposure to 1 µmol/L ACh for 2 minutes (solid bar) decreased [Ca2+]i transient amplitude. In the presence of 2 µmol/L H-89, however, withdrawal of ACh failed to elicit appearance of spontaneous [Ca2+]i transients. B, Selected traces recorded in A before (a), during (b), and after (c) withdrawal of ACh shown at an expanded time scale.

	Discussion

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The present study shows that in cat atrial myocytes, withdrawal of ACh can elicit changes in membrane voltage, ionic currents, and intracellular [Ca²⁺] that are consistent with cellular Ca²⁺ overload. These effects of ACh withdrawal are mediated by a rebound stimulation of the cAMP-dependent PKA pathway. This is the first demonstration that ACh withdrawal can directly elicit the development of Ca²⁺-induced DADs in atrial myocytes.

Exposure to cholinergic agonists directly inhibits atrial function, primarily through muscarinic receptor–mediated increases in ACh-activated K⁺ current,^{23 24} resulting in hyperpolarization and shortening of APD (Fig 1). In addition, inhibition of the adenylate cyclase–cAMP-PKA signaling pathway²⁵ via G_i protein is the primary mechanism by which ACh is thought to inhibit Ca²⁺ influx via I_Ca,L. The present results, however, indicate that withdrawal of ACh elicits a rebound stimulation of the cAMP-dependent PKA pathway to stimulate Ca²⁺ influx via I_Ca,L and stimulates SR Ca²⁺ uptake. The role of cAMP in the rebound phenomenon is supported by several studies. Thus, direct measurement of cAMP in chick heart cells shows that withdrawal of cholinergic agonist elicits an increase in cAMP concentration above control levels.¹³ Moreover, withdrawal of cholinergic agonist elicits a rebound stimulation of I_Ca,L in ventricular Purkinje fibers²⁶ and a rebound stimulation of chloride current in guinea pig ventricular muscle.²⁷ In both of these studies, the rebound response could not be elicited without continuous ß-adrenergic stimulation, presumably required to raise basal cAMP levels. In contrast to ventricular muscle, cat atrial cells exhibit significant endogenous or basal adenylate cyclase/cAMP activity and therefore do not require exogenous ß-adrenergic stimulation to elicit ACh-induced rebound responses.^{3 15} Basal adenylate cyclase/cAMP activity is evident in that exposure to inhibitors of cAMP-dependent PKA activity significantly decreased basal I_Ca,L amplitude. Moreover, exposure to 50 µmol/L isobutylmethylxanthine, a nonspecific inhibitor of phosphodiesterase activity, elicits maximum stimulation of I_Ca,L (unpublished observations, 1996). The mechanism of the rebound stimulation of I_Ca,L appears to be due, in large part, to ACh-induced inhibition of cAMP-dependent phosphodiesterase type III activity.³ Thus, after withdrawal of ACh, phosphodiesterase activity presumably recovers from inhibition more slowly than the inhibition of adenylate cyclase activity, resulting in transient elevation of cAMP concentration.

In the present study, we hypothesized that the rebound stimulation in cAMP concentration elicited by withdrawal of ACh stimulates Ca²⁺ influx via I_Ca,L and stimulates SR Ca²⁺ uptake and thereby overloads SR Ca²⁺ stores. Ca²⁺ overload is a primary condition predisposing for the development of DADs.^{5 6 7} Several of the present findings support our hypothesis that withdrawal of ACh induced overload of SR Ca²⁺. Thus, withdrawal of ACh elicited (1) an increase in APD, the development of DADs, and spontaneous voltage oscillations; (2) an increase in I_Ca,L and I_NaCa; (3) the appearance of I_ti; and (4) development of spontaneous [Ca²⁺]_i transients. Moreover, the finding that each of these responses was blocked by inhibition of cAMP-dependent PKA activity indicates that cAMP mediated the overload of SR Ca²⁺ induced by ACh withdrawal. This is consistent with the known actions of cAMP to stimulate both Ca²⁺ influx via I_Ca,L and SR Ca²⁺ uptake via Ca²⁺ ATPase activity.²⁸ Phosphorylation of SR Ca²⁺ release channels by cAMP/PKA activity may also promote SR Ca²⁺ release.^{29 30 31} A contributing factor that increases [Ca²⁺]_i is the effect of ACh to induce activation of a nonselective cation current carried primarily by Na⁺.^{32 33} The resulting increase in intracellular Na⁺ stimulates Ca²⁺ influx via Na/Ca exchange,^{34 35} and this Ca²⁺ would be expected to be transported into the SR.

The present findings indicate that the rebound stimulation in cAMP elicited by withdrawal of ACh mediates enhanced stimulation of I_NaCa. I_NaCa was elicited upon repolarization after relatively short depolarizing clamp steps, indicating that I_NaCa was stimulated by SR Ca²⁺ release. This is supported by the fact that ryanodine abolished I_NaCa. It seems likely that cAMP is acting indirectly to stimulate I_NaCa by stimulating SR Ca²⁺ uptake and subsequent Ca²⁺ release. That I_NaCa contributes to the atrial action potential configuration³⁶ would account for the rebound stimulation in APD and shift in plateau voltage to more positive voltages elicited by ACh withdrawal (Fig 1). Rebound stimulation of I_Ca,L also would be expected to contribute directly to these changes in action potential configuration. Several studies have reported that I_NaCa participates in the generation of I_ti.^{18 19 37 38} This is consistent with the present findings that ryanodine abolished both I_NaCa and I_ti. That ryanodine abolished the signs of Ca²⁺ overload without affecting rebound stimulation of I_Ca,L suggests that Ca²⁺ uptake into the SR is essential for the Ca²⁺ overload induced by withdrawal of ACh. I_ti is believed to underlie the development of DADs.^{5 6 7} In the present study, relatively long (2 seconds) depolarizing clamp steps were used to enhance intracellular Ca²⁺ and promote development of I_ti. During withdrawal of ACh, repolarization elicited spontaneous oscillatory inward currents that exhibited characteristics typical of I_ti recorded in cardiac myocytes under conditions of Ca²⁺ overload.^{19 38 39} The fact that inhibition of cAMP-dependent PKA activity abolished rebound stimulation of I_Ca,L, I_NaCa, spontaneous [Ca²⁺]_i transients, the appearance of I_ti, and DADs strongly supports our hypothesis that rebound stimulation of the cAMP-dependent PKA pathway elicited by withdrawal of ACh is the common underlying mechanism.

Implications
Generally, vagal stimulation to the heart is considered antiarrhythmic.¹ The present results indicate that withdrawal of cholinergic inhibition may be proarrhythmic as well. Clearly, withdrawal of vagal nerve activity does not normally provoke atrial dysrhythmias. In the present study, we used relatively prolonged exposure times and relatively high concentrations of ACh to illustrate possible effects of ACh. However, the rebound response and the development of DADs could be elicited by withdrawal from ACh exposures as short as 30 seconds. Moreover, previous work has shown that background levels of ß-adrenergic agonist that do not directly stimulate I_Ca,L potentiate cAMP-mediated rebound responses elicited by ACh withdrawal.³ This suggests that in the presence of background or elevated levels of catecholamines, withdrawal of vagal nerve activity in vivo may provoke marked increases in cellular [Ca²⁺]. This mechanism, therefore, may play an important role in generating triggered premature atrial depolarizations and the subsequent initiation of paroxysmal supraventricular tachycardia. Moreover, it may underlie, to some extent, the effectiveness of ß-adrenergic receptor antagonists to block certain types of atrial dysrhythmias. The present mechanisms also may be involved in vagally mediated cardiac dysrhythmias associated with REM sleep.¹ These dysrhythmias have been related to changes in autonomic tone that cause surges of cardiac sympathetic activity associated with withdrawal of vagal tone. Another important implication of the present findings may involve vagal maneuvers used clinically to break supraventricular tachyarrhythmias. Based on the present results, abrupt termination of carotid sinus massage or Valsalva maneuver may actually initiate spontaneous atrial activity. This may explain, in part, the relative ineffectiveness of these antiarrhythmic maneuvers. Finally, the present results suggest that dysrhythmias initiated by withdrawal of vagal nerve activity may be enhanced by conditions that elevate cellular [Ca²⁺], such as myocardial ischemia.⁴⁰

	Selected Abbreviations and Acronyms

 

ACh = acetylcholine APD = action potential duration APD90 = action potential duration at 90% repolarization APP50 = action potential plateau voltage at 50 ms [Ca2+]i = intracellular free Ca2+ concentration DAD = delayed afterdepolarization ICa,L = L-type Ca2+ current INaCa = Na/Ca exchange current Iti = transient inward current PKA = protein kinase A SR = sarcoplasmic reticulum

	Acknowledgments

 
Support was provided by grants from the National Institutes of Health (HL-27652 to Dr Lipsius and HL-51941 to Dr Blatter), the American Heart Association National Center (Dr Blatter), the Schweppe Foundation Chicago (Dr Blatter), and Loyola University Medical Center (Drs Lipsius and Blatter). Dr Blatter is an Established Investigator of the American Heart Association. We wish to thank C. Rechenmacher for her expert technical assistance with these experiments.

Received November 20, 1996; revision received February 4, 1997; accepted February 16, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 
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