BARIUM-INDUCED ABNORMAL AUTOMATICITY AND WITHDRAWAL OF ACETYLCHOLINE-INDUCED TRIGGERED ACTIVITY IN PULMONARY VEIN SLEEVES FROM NORMAL HEALTHY DOGS

Tsui-Min Wang¹, Hsiang-Ning Luk², Joen-Ron Sheu¹, Chern-En Chiang^3
1Graduate Institute of Medical Science, Taipei Medical University;
²Department of Anesthesiology, Chang-Gung Memorial Hospital;
³Division of Cardiology, Taipei Veterans General Hospital and National Yang-Ming University, Taiwan

Abstract: The presence of sleeves of atrial myocardium over pulmonary veins (PV) has been observed in many species. Cellular electrophysiological characterization of PV sleeves has been documented in guinea pigs, rabbits and dogs. Of particular interest, spontaneous activity and digitalis-induced triggered activity have been demonstrated in rodent PV sleeves. Recently, extremely high incidence of arrhythmogenic activities has also been shown in normal healthy dog and rabbit PV sleeves. In this study, we carefully re-characterized PV sleeves from 27 normal healthy dogs using conventional intracellular recording technique. Myocardial sleeves of PV were confirmed by histological staining. We found that in PV sleeves there were neither slow-response action potentials nor high-frequency irregular rhythm. All action potentials were fast response and resembled those of left atrium. Barium (1 mM) depolarized the cardiomyocytes and elicited low-frequency spontaneous activity in a reversible way. Isoproterenol (Iso) dose-dependently increased phase 4 slope and the effect could be antagonized by acetylcholine (ACh). During washout of ACh in the presence of Iso, spontaneous activity occurred and lasted for a few minutes. In conclusion, results of Ba²⁺-suppressed inwardly rectifying K⁺ currents and intracellular Ca²⁺ overload caused by rebound phenomenon during cholinergic withdrawal indicate pathological roles of canine PV sleeves.

INTRODUCTION

The presence of sleeves of atrial myocardium over pulmonary vein (PV) has been observed in many species. It is generally believed that these myocardial sleeves behave as throttle valves to prevent reflux of blood from the atrium into the veins during atrial systole. The active contraction of these myocardial sleeves can also facilitate the left atrial filling during atrial diastole.

Cellular electrophysiological characterization of PV sleeves has been documented in guinea pigs, rabbits and dogs. Ectopic foci in PV have recently been implicated in the pathogenesis in patients with paroxysmal atrial fibrillation. Of particular interest, spontaneous activity and digitalis-induced triggered activity have been demonstrated in rodent PV sleeves. Recently, extremely high incidence of arrhythmogenic activities has also been shown in normal healthy dog and rabbit PV sleeves. Its physiological significance remains to be determined. In this study, we re-characterized the electrophysiological features of canine PV and examined its arrhythmogenicity.

METHODS

Twenty-seven normal healthy dogs were anesthetized by pentobarbital (30 mg/kg, iv) and paralyzed by pancuronium (0.1 mg/kg). Mechanical ventilation was applied via an intubated endotracheal tube and respiration was controlled. Intravenous line was established via peripheral vein. Median sternotomy was performed and pericardium was opened. Right and left upper PVs were exposed and ligated. The heart and attached PV were quickly excised and stored in the warm oxygenated Tyrode solution. The PV were isolated under dissecting microscope and immediately mounted into oxygenated perfusion chamber. The whole procedure was completed in 15 minutes. The PV preparation consisted of part of ostium, whole part of cardiac sleeves and vessel part. The latter two regions could easily identified by a zig-zag line. The preparation was carefully fixed by silk threads and stimulated by bipolar electrode. The tissue was electrical driven by a 1-2 ms square pulse with a suprathreshold intensity by a Grass stimulator (S88, Grass Ins., W. Warwick, RI, USA). Stimulation frequency was set at 1 Hz and changed by need. The tissue was superfused with normal Tyrode solution bubbled with 97% O₂ and 3% CO₂. The composition of Tyrode solution was as follows (mM): NaCl 130, KCl 4, CaCl₂ 1.8, MgCl₂ 0.5, NaHCO₃ 12, glucose 5, and pH 7.4. All the experiments were performed at 37 ^oC. Electrophysiological recording was performed with conventional microelectrode technique. Micropipettes with tip resistance of 30 to 45 megaOhm were fabricated by vertical puller (David Kopf Ins., Tujunga, Ca, USA) and filled with 3 M KCl. The action potentials were recorded by Axonclamp 2-A (Axon Instrument, Foster City, Ca, USA) and stored in a VHS tape-recorder via a DA-AD interface (Digidata 1200, Axon), displayed in a digital oscilloscope and plotted in a plotter.

RESULTS

Action Potential Characteristics in Normal Health PV Sleeves

The transmembrane action potential parameters in 27 normal healthy dogs (10.9 ± 0.5 kg) were as follows: RMP –82.2 ± 1.0 mV, APA 122.7 ± 1.6 mV, Vmax 162.0 ± 6.9 V/s, APD₅₀ 105.2 ± 4.4 ms, APD₉₀ 119.1 ± 6.9 ms. In all preparations, we could not find any PV preparations occurred spontaneous activities and only fast response action potentials could be recorded in the cardiac sleeves region in the control conditions. There was neither automaticity nor delayed afterdepolarization (DAD), early afterdepolarization (EAD).

Barium-Induced Abnormal Automaticity

Figure 1.Barium superfusion induced depolarization and abnormal automaticity in PV sleeves.  The RMP of normal PV in panel A was –84 mV. Barium (1mM) superfusion resulted in depolarization (RMP –58mV) and inexcitability (Panel B).  Abnormal automaticity was elicited later with a cycle length of about 1500 ms (panel C). The spontaneous activity returned to a transient period of quiescence at the 23^rd min (panel D). About 1 to 2 minutes later, spontaneous activity reappeared and became stable at a cycle length of about 3000 ms (panel E) while the RMP was –41 mV. The abnormal automaticity disappeared and the normal action potential was recovered after 50-minute washout in normal Tyrode solution (Panel F).

Triggered Activity by the Withdrawal of Acetylcholine

Figure 2. Panel A shows the control action potential. Isoproterenol (Iso) 1 mM increased phase 4 depolarization (–84 to –88 mV) (Panel B) that could be antagonized by Acetylcholine (ACh) 10mM (Panel C). Withdrawal of ACh in the presence of Iso resulted in spontaneous activity (Panel D) that lasted for a few minutes and then stopped (Panel E). The increase in the phase 4 depolarization disappeared after washout of Iso (Panel F). It should be noted that PV strip displayed solely fast response action potentials in control condition. The scale is the same with Fig. 1.

DISCUSSION

In the present study, all the action potentials recorded from the control condition showed fast response activity. Neither slow response action potentials nor spontaneous activity could be observed. Our findings are different from others [1-3] (Table 1).  The discrepancy remains to be determined.

TABLE 1
Comparison of Arrhythmogenesis of Normal Healthy PV Sleeves

	Automaticity	DAD / EAD
Chen et al. Cardiovasc Res 48:265-73, 2000 (Dogs, tissues, N = 17)	71 %	0 % / 12 %
Chen et al. Circulation 104:2849-54, 2001  (Dogs, cells, N = 151)	40 %	7 % / 0 %
Chen et al. JACC 39:366-72,2002 (Rabbits, cells, N = 70)	51 %	6 % / 0 %
Wang et al. (Dogs, tissues, N = 27)	0 %	0 % / 0 %

Previous study demonstrated that low barium could induce abnormal automaticity in cat papillary muscle. In the present study we also found a similar finding in canine PV sleeves. It is suggested that this abnormal rhythm is due to barium-induced blockade of the inward rectifier potassium current.

Withdrawal of cholinergic agonist can directly increase Ca²⁺ influx and stimulate Ca²⁺ uptake into the sarcoplasmic reticulum (SR), thereby increasing intracellular Ca²⁺. Intracellular Ca²⁺ overload in the SR can initiate atrial arrhythmias. In the present study, we demonstrated that in PV sleeves this arrhythmogenic mechanism do exist. Our findings have provided a mechanistic rational for the arrhythmogenicity in PV.

REFERENCES

1.    Chen YJ, Chen SA, Chang MS, Lin CI. Arrhythmogenic activity of cardiac muscle in pulmonary veins of the dog: implication for the genesis of atrial fibrillation. Cardiovasc Res 2000;48:265-73.

2.    Chen YJ, Chen SA, Chen YC, et al. Effects of rapid atrial pacing on the arrhythmogenic activity of single cardiomyocytes from pulmonary veins: implication in initiation of atrial fibrillation. Circulation 2001;104:2849-54.

3.    Chen YC, Chen SA, Chen YJ, Chang MS, Chan P, Lin CI. Effects of thyroid hormone on the arrhythmogenic activity of pulmonary vein cardiomyocytes. J Am Coll Cardiol 2002;39:366-72.