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Am J Physiol Heart Circ Physiol 292: H1861-H1867, 2007. First published December 8, 2006; doi:10.1152/ajpheart.00826.2006
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Calcium-mediated triggered activity is an underlying cellular mechanism of ectopy originating from the pulmonary vein in dogs

Masamichi Hirose1 and Kenneth R. Laurita2

1Department of Molecular Pharmacology, Shinshu University School of Medicine, Nagano, Japan; and 2Heart and Vascular Research Center, MetroHealth Campus, Case Western Reserve University, Cleveland, Ohio

Submitted 1 August 2006 ; accepted in final form 4 December 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Paroxysmal atrial fibrillation associated with focal ectopy originating from the pulmonary vein (PV) can be preceded by variations in autonomic tone; however, the underlying cellular mechanisms are not clear. To determine the mechanisms of autonomically mediated PV ectopy, high-resolution optical mapping techniques were used to measure action potentials and Ca2+ transients from the PV and the ligament of Marshall area in the arterially perfused canine left atrium. Rapid pacing was used to initiate ectopic activity during pituitary adenylate cyclase-activating polypeptide (PACAP) injection (1 nmol), as a surrogate for autonomic imbalance, before (n = 9) and after (n = 6) verapamil (10 nmol) administration. In all preparations, spontaneous activity was absent before rapid pacing. During PACAP injection, rapid pacing induced ectopic activity in eight of nine preparations. In contrast, before PACAP injection, rapid pacing did not induce ectopic activity. Activation maps of each episode of ectopic activity indicated that the site of origin occurred more frequently in the PV (70%) than in the ligament of Marshall (30%) area. As rapid pacing cycle length increased, so did the ectopic beat coupling interval. In addition, PACAP-induced ectopic activity was associated with large Ca2+ transient amplitudes and was always suppressed by verapamil, a Ca2+ channel blocker (P < 0.05). Finally, during PACAP injection in the absence of an ectopic beat, spontaneous Ca2+ release and delayed afterdepolarizations were observed simultaneously after termination of rapid pacing. In conclusion, these data suggest that autonomically mediated PV ectopy may be due to Ca2+-mediated triggered activity arising from delayed afterdepolarizations.

delayed afterdepolarizations; pituitary adenylate cyclase-activating polypeptide; ligament of Marshall; high-resolution optical mapping

RECENT CLINICAL AND EXPERIMENTAL studies have shown that the pulmonary veins (PVs) are important sources of ectopic activity that can initiate paroxysmal and sustained atrial fibrillation (AF) (5, 7, 14). Autonomic tone may play an important role in the mechanism of ectopy originating from the PV (1, 5, 26). More specifically, paroxysmal AF (3) and paroxysmal AF associated with focal ectopy originating from the PV (37) have been linked to variations of autonomic tone, with a primary increase in adrenergic drive followed by vagal predominance. We previously showed that pituitary adenylate cyclase-activating polypeptide (PACAP) causes a similar variation in sympathovagal effects due to increased adenylate cyclase activity and activation of intracardiac vagal nerves (15). Interestingly, PACAP also causes AF spontaneously (16). Therefore, PACAP may help provide a better understanding of autonomically mediated ectopic activity originating from the PV.

Triggered activity and automaticity are well recognized as mechanisms of ectopic activity in the ventricle (32) and atrium (11, 36). Recently, Patterson et al. (25) demonstrated PV ectopy consistent with triggered activity caused by early afterdepolarizations (EADs) during combined adrenergic and cholinergic stimulation. They proposed that accelerated repolarization and elevated intracellular Ca2+ transients due to enhanced parasympathetic and sympathetic tone, respectively, may be required for arrhythmia. Ectopy caused by delayed afterdepolarization (DAD) can also occur under a variety of conditions when cellular Ca2+ increases and triggers a spontaneous (non-electrically driven) release of Ca2+ from the sarcoplasmic reticulum (13, 21). Similar to adrenergic stimulation of the heart, PACAP activates Ca2+ influx through voltage-dependent Ca2+ channels (10), which may promote triggered activity. PACAP also causes the activation of intracardiac vagal nerves, which contributes to rapid firing within the PV sleeve (25). Therefore, we hypothesized that PACAP can induce triggered activity originating from the PV. To test this hypothesis, we used optical mapping techniques to measure action potentials and Ca2+ transients from the endocardial surface of the PV in the isolated canine left atrium. Our results suggest that PACAP causes Ca2+-mediated triggered activity, originating primarily from the PV area.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
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Experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University.

Isolated canine left atrial preparation. A total of nine male dogs (20–25 kg body wt) were treated with heparin sodium (2 ml iv) and anesthetized with pentobarbital sodium (30 mg/kg iv). After right thoracotomy, hearts were quickly excised and placed in cold cardioplegic solution (in mM: 129 NaCl, 8.0 KCl, 1.8 CaCl2, 20.0 NaHCO3, 0.5 MgSO4, 0.9 NaH2PO4, and 5.5 dextrose). Then the left atrium was isolated from the heart, and the left coronary artery was cannulated and leaking arteries were ligated. The preparation was mounted to a custom-made frame (Fig. 1A). Each preparation was perfused under constant-flow conditions with oxygenated (95% O2-5% CO2) Tyrode solution [in mM: 129 NaCl, 4.0 KCl, 1.8 CaCl2, 20.0 NaHCO3, 0.5 MgSO4, 0.9 NaH2PO4, and 5.5 dextrose (pH 7.4 at 36 ± 1°C)]. The preparation was immersed in the coronary effluent, which was maintained at a constant temperature (equal to the perfusion temperature) with a heat exchanger, to avoid surface cooling. Perfusion pressure was measured with a pressure transducer (World Precision Instruments, Sarasota, FL), and flow was adjusted to maintain pressure at 50–60 mmHg. Atrial rhythm was monitored using three silver disk electrodes fixed to the chamber. The atrial electrograms were filtered (0.3–300 Hz), amplified (x1,000), and continuously displayed on a digital oscilloscope. Preparations were stained by direct coronary perfusion for 7–10 min with 100 ml of the voltage-sensitive dye di-4-ANEPPS (Molecular Probes, Eugene, OR) dissolved in 0.19 ml of ethanol at a final concentration of 15 µM and for ~40 min with the Ca2+-sensitive indicator indo 1-AM (Molecular Probes) dissolved in a 0.5-ml solution of DMSO and Pluronic (20% wt/vol) at a final concentration of 5 µM. Atrial rhythm, perfusion pressure, and flow were continuously monitored during each experiment. Preparations were maintained for 2–3 h without signs of deterioration, and our experimental protocol typically lasted 2 or 2.5 h. In addition, after each experiment, tissue viability was confirmed by staining with 10 ml of 2,3,5-triphenyltetrazolium chloride (14 mg/ml).


Figure 1
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  		Fig. 1. A: isolated left atrial preparation mounted on a custom-made frame. Optical action potentials and Ca2+ transients were recorded simultaneously from the mapping field over the pulmonary vein (PV) or the ligament of Marshal (LM) area. Arrows point to left PVs and LM region. LAA, left atrial appendage; LV, left ventricle; IAS, interatrial septum. B: representative action potential (red, AP) and Ca2+ transient (blue, CaF) signals recorded from the endocardium at a baseline pacing cycle length (BCL) of 600 and rapid pacing at 150 ms under control conditions and with pituitary adenylate cyclase-activating polypeptide (PACAP).

 
Optical mapping system. Perfused left atria were immersed in a coronary effluent-filled custom-built Lexan chamber specifically designed for optical recordings. In every experiment, measurements were made from the endocardial surface of the left atrium, with the mapping field positioned over the PV and/or ligament of Marshall (LM) region (Fig. 1A). A movable piston was used to apply gentle pressure to the surface of the atria opposite the mapping field, allowing the preparation to contract freely, except within the mapping field. The movable piston area extended ~1 cm beyond each side of the mapping field. No electromechanical uncouplers were used to reduce motion artifact. The dual Ca2+-voltage optical mapping system used in this study has been described in detail elsewhere (21, 22). Briefly, action potentials were measured using di-4-ANEPPS with filtered excitation light (514 nm) obtained from a 250-W quartz tungsten halogen lamp (Oriel, Stratford, CT) directed to the heart with a liquid light guide. Ca2+ transients were measured using indo 1-AM with filtered excitation light (365 nm) obtained from a 250-W mercury arc lamp (Oriel) directed to the atrium with a second liquid guide. Excitation light from both light guides was directed to the same location on the atrium. Fluoresced light from both dyes was collected by a tandem lens assembly, and a dichroic mirror (560 nm; Omega Optical) placed between the lenses passed longer-wavelength light to an emission filter (>690 nm; Shott Glass Technologies, Duryea, PA) and a 16 x 16 element photodiode array and reflected shorter-wavelength light to a second emission filter (485 nm; Chroma, Brattleboro, VT) and 16 x 16 element photodiode array. An optical magnification of x0.81 was used, corresponding to a mapping field of 2.1 x 2.1 cm and 0.13-cm spatial resolution between recording pixels. Ca2+ transients and action potentials were recorded simultaneously; however, sequential measurements were also performed under steady-state conditions when signal quality was reduced. Several factors could explain the reduction in signal quality. It is possible that the larger size of the preparation and mapping field reduced the amount of dye and excitation light, respectively, per recording site. In addition, we used no electrical mechanical uncouplers, so motion artifact may have lowered the apparent signal-to-noise ratio. Signals recorded from each photodiode and the ECG were multiplexed and digitized with 12-bit precision at a sampling rate of 1,000 Hz per channel (Microstar Laboratories, Bellevue, WA). To view, digitize, and store anatomic features, a mirror was temporarily inserted between the lenses of the tandem lens assembly to direct reflected light to a charge-coupled device video camera.

Experimental protocol. A polytetrafluoroethylene-coated silver bipolar electrode with 1-mm interelectrode spacing was used to stimulate the epicardial surface of the left atrial appendage at twice diastolic threshold current for 2 ms. Spontaneous activity was absent in all preparations, regardless of PACAP injection into the left coronary artery through the perfusion cannula, and baseline pacing was continuously performed, unless otherwise noted, at a cycle of 600 ms. A total of nine preparations were used in this study, and all were stained with both dyes. High-resolution optical mapping of action potentials and Ca2+ transients was performed from the PV in six of nine preparations and from the LM area in six of nine preparations (Fig. 1, Table 1). In three preparations, PV and LM mapping were performed sequentially. Representative action potentials (red) and Ca2+ transients (blue) recorded from the endocardial surface of the PV during steady-state baseline pacing (600-ms cycle length) under control conditions and during rapid pacing (150-ms cycle length) under control conditions and during PACAP injection are shown in Fig. 1B.


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  		Table 1. Electrophysiological characteristics during PACAP in each preparation

 
To compare the amplitude of Ca2+ transient fluorescence before and after PACAP injection, the Ca2+ transient fluorescence was recorded at a cycle length of 600 ms in four of nine preparations. To minimize the effect of fluorescence loss over time, recordings were made during and immediately before (<1 min) PACAP injection under control conditions without change in the intensity and position of exciting light. To initiate ectopic activity, rapid pacing for ~10 s from the left atrial appendage at four different cycle lengths was used during control and 1 min after PACAP bolus injection in all preparations and during PACAP treatment with verapamil (10 nmol) administration in six preparations. PACAP was injected 2 min after verapamil injection into the left coronary artery through the perfusion cannula. Because PACAP causes tachyphylaxis, we had to wait >30 min before a second administration and, thus, could not compare Ca2+ transient amplitudes with and without verapamil. Atrial tachyarrhythmia (AT) was defined as a rapid (<150-ms cycle length) rhythm lasting >10 beats.

Data analysis. In all experiments, automated algorithms were used to determine depolarization time relative to a single fiducial point (i.e., earliest activation time within the mapping field). Depolarization times were calculated for all action potential recordings and defined as the point of maximum positive derivative in the action potential upstroke (dV/dtmax). The method of Bayly et al. (2) was modified for optically recorded action potential maps to accurately quantify the direction and magnitude of conduction velocity (CV) and the local repolarization gradient at each recording site. Mean CV was calculated from the average of local CVs at 200 sites in the center of the mapping field. The ectopic beat coupling interval (S1A1) was defined as the difference between the depolarization time of the first ectopic beat (A1) and the last paced beat (S1) at the site where the A1 beat originated. The amplitude of Ca2+ transient fluorescence (CaAmp) was defined as the difference between maximum and minimum fluorescence during a single beat and was determined from an average of ≥40 equally spaced sites across the entire mapping field.

Values are means ± SE. Simple regression analysis was used to test the relation between S1S1 cycle length of rapid pacing and the ectopic beat coupling interval. Fisher's exact test was used for comparison of the incidence of ectopic activity before and after verapamil with PACAP. Student's t-test for paired or unpaired data was used for comparison between two groups, and P < 0.05 was considered statistically significant.

Drugs. Drugs were mixed fresh for each experiment. Human PACAP-27 (Peninsula Laboratories) was dissolved in distilled water before use. PACAP-27 at a dose of 1 nmol was injected into the left coronary artery through the perfusion cannula.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PACAP-induced ectopic activity. Spontaneous activity was absent before rapid pacing in all preparations. Under the control condition, ~10 s of rapid pacing from the epicardial surface of the left atrial appendage did not induce ectopic activity (i.e., an unstimulated beat) in any preparation. In contrast, after PACAP injection, ectopic activity consisting of at least one beat was initiated in eight of nine preparations after rapid pacing at a mean cycle length of 144 ± 4 ms (Table 1). Examples of ectopy induced by rapid pacing during PACAP in three separate preparations are shown in Fig. 2. Action potentials and Ca2+ transients recorded simultaneously are shown on termination of rapid pacing (S1). Multiple ectopic beats (preparations 2 and 3) were also observed at faster pacing rates, and AT occurred in preparations 2, 3, 5, and 8 (Table 1). Verapamil (a Ca2+ channel blocker) combined with PACAP completely suppressed ectopic activity during rapid pacing (P < 0.05) in all five preparations (Table 1).


Figure 2
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  		Fig. 2. PACAP-induced ectopic activity. Action potential (AP) and Ca2+ transient (CaF) signals were recorded simultaneously during 3 separate episodes (preparations 2, 3, and 9) of ectopic activity after PACAP injection (1 nmol). Multiple ectopic beats were observed in preparations 2 and 3.

 
The site of ectopic activity did not appear to be random. Ectopic activity from the PV occurred in five of six preparations when the PV area was mapped (Table 1). In contrast, ectopic activity from the LM area occurred in only two of six preparations when the LM area was mapped (Table 1). A representative example of ectopy from the PV after PACAP injection is shown in Fig. 3: activation maps of the last paced beat of rapid pacing at a cycle length of 160 ms (S1) and two ectopic beats (A1 and A2) originating near the PV. The S1 beat originated from the left atrial appendage; it entered at site g and propagated toward the upper and lower left (arrow), with no evidence of conduction block within the mapping field. Then a very stable focal pattern of activation was observed originating from the PV area (site b), with no evidence of impulse block. CVs of the two ectopic beats were 50 and 48 cm/s, respectively. Action potentials recorded from sites a–g demonstrate the site of origin of the S1 beat and ectopy originating from the PV area. Of the four episodes of AT initiated, two were initiated within the mapping field, one was associated with a focal pattern of activity, and the other was associated with spiral wave reentrant activity (data not shown).


Figure 3
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  		Fig. 3. Ectopic activity originating from the PV area during PACAP treatment. Contour maps show the activation pattern of the last paced beat (S1) from the LAA and 2 ectopic beats (A1 and A2). Activation maps are shown with 2-ms isochrones. Lighter contours indicate earlier time. Numerical data on each activation map indicate range of activation times (0–45, 281–316, and 495–535 ms) and mean conduction velocity (CV) within the mapping field. Action potentials recorded from sites a–g of the last paced beat (S1) demonstrate the sequence of activation in the mapping field. Ectopic beats were induced by rapid pacing at a BCL of 160 ms. Dotted lines on activation maps of the 2 ectopic beats (A1 and A2) outline the PV.

 
Ca2+-mediated triggered activity. To elucidate the mechanism of ectopic activity, the relation between ectopic beat coupling interval (S1A1) and rapid pacing cycle length (S1S1) was determined. The relation between S1A1 and S1S1 during PACAP treatment is shown in Fig. 4 for all episodes of ectopic activity that were initiated within the mapping field (10 episodes). As S1S1 cycle length increased, so did the S1A1 interval. This suggests that the mechanism for the first beat of PACAP-induced ectopic activity is triggered activity, rather than automaticity or reentry. The mechanism of ectopic activity also appears to depend on intracellular Ca2+. For example, CaAmp measured during PACAP was significantly larger than that measured during control (Fig. 5A). However, in one experiment CaAmp did not increase (Fig. 5B, preparation 6). Interestingly, this was the only preparation in which ectopic activity could not be induced (Table 1). Finally, as mentioned previously, ectopic activity could not be induced during PACAP with verapamil administration in the six preparations tested. Taken together, these data suggest that the mechanism of the first beat of ectopy during PACAP is Ca2+-mediated triggered activity.


Figure 4
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  		Fig. 4. Relation between the ectopic beat coupling interval (S1A1) and the cycle length of rapid pacing (S1S1) after PACAP injection. Each point represents a separate episode of ectopy initiated within the mapping field in 7 preparations. A positive correlation (r = 0.8, P < 0.01) suggests that the ectopic beats are triggered, rather than automatic or reentrant.

 

Figure 5
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  		Fig. 5. A: representative example of a Ca2+ transient recorded during control conditions and with PACAP. Amplitude of CaF (CaAmp) was larger with PACAP than under control conditions. B: percent change in CaAmp immediately before (control) and after PACAP injection in 4 preparations. ***P < 0.001 vs. control. In preparation 6, no change in CaAmp and no ectopic activity were observed (see Table 1).

 
PACAP-induced DAD. If the ectopic activity observed in this study is due to Ca2+-mediated triggered activity, then some DAD and spontaneous Ca2+ release (SCR) activity should be evident. A representative example of a DAD and an SCR during PACAP immediately after termination of rapid pacing in the absence of an ectopic beat is shown in Fig. 6. The contour map shows activation of the last S1 beat during rapid pacing from the left atrial appendage at a cycle length of 130 ms. Action potential and Ca2+ transient signals recorded simultaneously from the same site within the PV area demonstrate a subthreshold depolarization (DAD) and an SCR that occur simultaneously immediately after pacing is terminated. DAD and SCR activity occurred simultaneously at the origin of ectopic activity in three of five preparations.


Figure 6
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  		Fig. 6. PACAP-induced delayed afterdepolarization (DAD) and spontaneous Ca2+ release (SCR) in the absence of a triggered (ectopic) beat. Left: activation map of the last paced beat (S1) from the LAA with gray scale (2-ms isochrones) and corresponding numerical values. Right: action potential and Ca2+ transient signals recorded near (*) the PV (dashed line). Simultaneous DAD and SCR activity were induced by rapid pacing at a BCL of 130 ms.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study is the first to use dual Ca2+-voltage optical mapping techniques to investigate ectopy arising from the left atrium. We found that, during PACAP, a surrogate for sympathovagal imbalance, rapid pacing-induced ectopic activity originated primarily from the PV area. PACAP-induced ectopic activity was associated with large Ca2+ transient amplitudes and was suppressed by verapamil, a Ca2+ channel blocker. Moreover, in the absence of triggered action potentials, DAD and SCR activity were observed immediately on termination of rapid pacing during PACAP. Taken together, these data suggest that autonomically mediated PV ectopy may be due to Ca2+-mediated triggered activity arising from DADs.

Sympathovagal-mediated ectopy from the PV. Clinical and experimental studies have shown that AF and focal ectopy originating from the PVs are associated with variations in autonomic tone. For example, electrical stimulation at the PV and within the left pulmonary artery, which activates autonomic nerves, can evoke rapid ectopic beats from the PV and induce AF in canine hearts (26, 27). Patterson et al. (25) recently demonstrated the importance of a combined increase in sympathetic and parasympathetic stimulation in the formation of ectopic activity in the PV sleeve. Zimmermann and Kalusche (37) showed that, in patients with focal ectopy originating from the PV, sustained episodes of AF were associated with variations in autonomic tone, with a primary increase in adrenergic drive followed by vagal predominance. Similar changes in sympathovagal balance were observed in patients with paroxysmal AF (3). We previously showed that PACAP causes a similar variation in sympathovagal effects due to an increase in adenylate cyclase activity followed by activation of intracardiac vagal nerves (15, 16). Therefore, our demonstration that PACAP causes Ca2+-mediated triggered activity from the PV provides further evidence that variations in sympathovagal balance may have an important role in PV ectopy and AT.

Mechanism of PACAP-induced ectopy. Catecholamines or cAMP, which increase Ca2+ entry, are a mechanism of DAD and triggered activity in atrial and ventricular muscle (30, 36). PACAP increases adenylate cyclase activity, which enhances L-type Ca2+ current and, consequently, increases intracellular Ca2+ concentration. Our results demonstrate that ectopic beats were observed only when PACAP increased Ca2+ transient amplitude (Fig. 5). In addition, verapamil suppressed PACAP-induced ectopic activity, indicating that increased Ca2+ entry plays an important role. Finally, the presence of SCR and DAD activity provides further evidence that PACAP-induced PV ectopy is caused by Ca2+-mediated triggered activity (Fig. 6). This finding is very similar to the SCR and DAD activity we previously reported in the canine left ventricular wedge preparation under enhanced Ca2+ entry conditions (21). In contrast to catecholaminergic activation, the role of intracardiac vagal nerve activation by PACAP in PV ectopy is less clear. For example, it is well known that ACh suppresses pacemaker activity and decreases adenylate cyclase activity and, consequently, L-type Ca2+ current. However, recent studies have demonstrated that ACh withdrawal can directly elicit spontaneous premature beats in canine atrium (29) and the development of Ca2+-induced DADs in cat atrial myocytes (35). In addition, our previous study showed that atropine treatment abolished PACAP-induced spontaneous AF (15). Similarly, Burashnikov and Antzelevitch (4) showed that ACh promotes DAD activity in the pectinate muscle area of isolated arterially perfused canine right atrium.

Recently, Patterson et al. (25) proposed that the EADs and short coupled ectopy they observed in the PV sleeve during autonomic nerve stimulation was not caused by SCR activity but, rather, by activation of Na+/Ca2+ exchange current in the forward mode as a result of the decay of the Ca2+ transient far outlasting repolarization. In humans, the triggering of AF by short coupled beats is consistent with EADs as the underlying mechanism. However, Haissaguerre et al. (14) showed that repetitive focal discharges in the initiation of AF have irregular cycle lengths, ranging from 110 to 270 ms, which are similar to the range of S1A1 coupling intervals that we report (Fig. 4). In addition, Haissaguerre et al. showed that the coupling interval from the last sinus beat to the initial activation of AF was 212 ± 34 ms. So, triggering in the PV may not always occur at short coupling intervals. During rapid pacing under PACAP conditions, we did observe Ca2+ transients outlasting repolarization (Fig. 1B, bottom). However, ectopy (Fig. 2) and SCR activity (Fig. 6) occurred well after final repolarization in our experiments, suggesting that the underling mechanism is not mediated by EAD. Additional studies may be required to specifically address this issue.

PV and LM as the location of ectopy. Cells isolated from the PVs have been shown to exhibit pacemaker-type activity and isoproterenol-induced spontaneous activity (8). Moreover, recent preliminary data suggest that T-type Ca2+ current, which can trigger Ca2+ release from the sarcoplasmic reticulum (19), is higher in PVs (6). In addition to the PVs, it has been shown that the left atrial tract within the LM region may generate double potentials and may also serve as a source of left atrial ectopic activity during sympathetic stimulation (28). Hwang et al. (20) demonstrated that the LM could be a source of rapid activation in patients with paroxysmal AF. Their results also suggest abnormal automaticity from the muscle bundle within the LM. In addition, Doshi et al. (12) showed that the LM is a source of abnormal automaticity during isoproterenol infusion in isolated canine left atria that did not include the PV area. Therefore, the LM may be the origin of the ectopy induced by automatic activity in adrenergically mediated AF. Although the LM may be the origin of the ectopy in some clinical conditions (20), its importance relative to PV ectopy is not clear. Nevertheless, our results suggest that, with variations in autonomic tone (such as that associated with paroxysmal AF), the PV is more likely than the LM to be a source of the ectopy by Ca2+-mediated triggered activity.

In this study, we did not map the full length of the PV. However, during PV area mapping, most ectopy was initiated near the PV-left atrial junction. Recently, Tan et al. (31) reported the longitudinal distribution of autonomic nerves from the left atrium to the distal PV and demonstrated that autonomic nerve densities are highest in the left atrial region within 5 mm from the PV-left atrial junction. Therefore, in the induction of autonomically mediated AF, the trigger is more likely to occur around the PV-left atrial junction. Therefore, our results suggest that, in autonomic tone fluctuation, late coupled (e.g., DAD-induced) triggered activity is expected to originate near the PV-left atrial junction. However, this may not be true for all cases of AF.

Relation between PV ectopy and AF. In general, the exact mechanistic relation between PV ectopy and AF is unknown. Abnormal automaticity (7, 24), triggered activity (8, 9), and microreentry (18, 23) are possible mechanisms. Regardless of the mechanism, sympathovagal-mediated PV ectopy appears to play an important role in AF. One possibility that is consistent with our data is that fluctuations in autonomic tone provide a substrate for triggering AF. For example, enhanced Ca2+ entry associated with PACAP (as shown in the present study) or isoproterenol (1) may promote triggered beats originating from the PV area. Such beats may interact with the underlying electrophysiological substrate (1). In addition, shortening of atrial refractoriness associated with PACAP in vivo (17) or an increase in dispersion of refractoriness associated with vagal stimulation (34) may also play a role in the induction and maintenance of AF associated with PV ectopy. In the present study, however, we could not evaluate shortening and dispersion of refractoriness in the right atrium, where the effects of vagal activation are greatest. This may also explain why sustained AT was not observed in this study. Finally, even though PACAP is an endogenous substance known to be present in humans (33), whether or not PACAP plays a role in paroxysmal AF in humans is unclear. Nevertheless, PACAP may be a good model of the sympathovagal imbalance that often precedes paroxysmal AF.

Study limitations. In this study, the atrial pacing rates required for DAD-induced triggered activity during PACAP administration (3–5 min) were faster than that associated with PV firing in humans, when autonomic tone fluctuation continues for >20 min before the onset of paroxysmal AF. It is possible that, in our model, faster pacing was required to increase cellular Ca2+ loading because of the relatively short time of PACAP action. It is known that autonomic fluctuations cause paroxysmal AF in the clinical setting; however, a good model has yet to be developed. Although combined adrenergic and cholinergic nerve stimulation can be performed (25), it is difficult to stimulate these nerves selectively in the isolated atrium. Alternatively, adrenergic and cholinergic agonists could be used. However, the effect of autonomic tone fluctuation on atrial electrophysiological properties depends on the distribution of adrenergic and muscarinic receptors, rather than autonomic nerves, in the atrium. Given that PACAP activates intracardiac postganglinonic vagal nerves, it may be more appropriate than adrenergic and cholinergic agonist infusion.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported in part by Ministry of Education, Science, and Culture, Japan, Scientific Research Grant-in-Aid 13670083 (M. Hirose) and National Heart, Lung, and Blood Institute Grant HL-68877 (K. R. Laurita).


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. R. Laurita, Heart & Vascular Research Center, MetroHealth Campus of Case Western Reserve Univ., Cleveland, OH 44109-1998 (e-mail: klaurita{at}metrohealth.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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B. S. Stambler and K. R. Laurita
Atrial Fibrillation in Heart Failure: Steady Progress but Still a Long Way to Go
Circ Arrhythmia Electrophysiol, June 1, 2008; 1(2): 77 - 79.
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