Introduction

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Pacemaker activity is initiated from the pacemaker cells in the sinoatrial (SA) node in the normal mammalian heart (Lewis et al., 1910; Boineau et al., 1978, 1980; Schuessler et al., 1986). However, after excision of the SA node area, the pacemaker activity in anesthetized and conscious dogs is initiated from the dorsal cranial right atrium (in or near Bechmann's bundle) or the inferior right atrium along the sulcus terminalis (Randall et al., 1984; Ardell et al., 1991). In addition to the SA nodal region, in the anesthetized dog heart those areas become pacemaker sites when atrial rate is changed by sympathetic or parasympathetic interventions (Boineau et al., 1978, 1980; Jones et al., 1978; Schuessler et al., 1986).

Pacemaker activity of SA node cells in the mammalian heart is regulated by pacemaker potential, which consists of a slow inward Ca²⁺ current (ICa), a rapid type of delayed rectifier K⁺ current (IKr), a hyperpolarization-activated inward current (If), and others (Irisawa et al., 1993). Norepinephrine released from sympathetic varicocities activates -adrenoceptors followed by stimulatory G protein and adenylyl cyclase and then increases tissue cyclic AMP (cAMP). Activation of ICa, IKr, and If by cAMP increases the rate of the pacemaker potential and increases sinus rate. Subsidiary pacemaker activity is also increased by adrenergic interventions or exercises in dogs (Jones et al., 1978; Boineau et al., 1980; Ardell et al., 1991). Additionally, parameters of the pacemaker action potential are different between the central SA node cells and peripheral nodal cells of the isolated rabbit hearts (Hoffman and Cranefield, 1960; Bouman et al., 1968; Kodama and Boyett, 1985).

We hypothesized that the roles of ICa, IKr, and If on the pacemaker activity are different among the pacemaker cells of the SA node and the superior and inferior subsidiary pacemaker regions in the dog heart in situ. To test this hypothesis, we studied the effects of an ICa blocker verapamil, an If inhibitor zatebradine (Goethals et al., 1993), and an IKr blocker E-4031 (Sanguinetti and Jurkiewicz, 1990) on the atrial rate and pacemaker location and on the changes in atrial rate and pacemaker location in response to cardiac sympathetic nerve stimulation in the autonomically decentralized heart of the anesthetized dog. The pacemaker location was determined by the isochronal activation sequence map using the 48 unipolar electrodes, which covered a 15-mm × 35- to 40-mm area including the SA node region along the sulcus terminalis from the anterior to posterior right atrium.

	Materials and Methods

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The animal experiments were approved by the Shinshu University School of Medicine Animal Experimentation Committee, and animals were obtained through the Animal Laboratory for Research of the Shinshu University School of Medicine.

Preparation. Twenty-four mongrel dogs, weighing 11 to 20 kg, were anesthetized with pentobarbital sodium (35 mg/kg i.v.); supplemental doses of pentobarbital sodium were given as necessary to maintain stable anesthesia. A tracheal cannula was inserted, and intermittent positive ventilation (tidal volume, 20 ml/kg; frequency, 15 strokes/min) was started. The chest was opened transversely at the 4th intercostal space and covered with a plastic sheet to keep a constant temperature of the heart. Each cervical vagus nerve was ligated tightly and crushed at the neck, and each stellate ganglion was crushed with a tight ligature at its junction with the ansa subclavia. These maneuvers remove almost all tonic neural activity to the heart (Levy et al., 1966). The heart rate derived from the standard electrocardiogram lead II and the right femoral arterial blood pressure were recorded on an oscillograph (model RTA-1200; Nihon Kohden, Tokyo, Japan). The right femoral vein was also cannulated for drug injection and physiological saline infusion to adjust spontaneous fluid losses.

Epicardial Mapping. The epicardial activation sequence of the right atrium including the SA node region was obtained from 48 unipolar recording electrodes, which were fixed to 2 flexible templates made of soft plastic plates (Unique Medical, Tokyo, Japan). One template made of six unipolar electrodes (diameter, 1 mm; interelectrode distance, 3 mm) was fixed to the anterior, and another made of 42 unipolar electrodes (vertical interelectrode distance, 5 mm; horizontal interelectrode distance, 3 mm) was fixed to the posterior of the right atrium along the sulcus terminalis as shown in Fig. 1 (left panel). A bipolar reference electrode was attached to the right atrial appendage. Forty-eight unipolar electrograms, the reference electrogram, and a standard electrocardiogram lead II were recorded simultaneously and storaged on the disk for 4 s by using a computerized mapping system (HPM-7100; Fukuda Denshi, Tokyo, Japan) (Misaki et al., 1994). The bandwidth of the amplifiers was set under programmable control at 0.16 to 200 Hz and the sampling rate at 1000 Hz with 12-bit resolution. Each electrogram from a selected time window was automatically detected by the computer. The time of local activation for each electrogram was determined by the maximum negative rate of voltage change, dV/dt_max. They were indicated by vertical cursors on the screen and could be corrected on the halfway of the initial negativity by the operator, if necessary, when the initial successive negative waves were greater than 0.2 mV. The time reference was the time of earliest epicardial breakthrough occurring during spontaneous beats. Isochronal maps were automatically constructed from the activation times within selected time windows. Thus, we determined each isochronal sequence map from the digitized data of 48 electrodes as an average of three consecutive atrial electrograms, which showed the similar P-waves of the standard cardioelectrogram lead II (Fig. 1, right panel). In preliminary experiments, we determined the conduction velocity in spontaneously beating and paced hearts, and the range of the conduction velocity was 0.8 to 1.4 m/s as previously reported (Boineaue et al., 1980; Watanabe et al., 1985; Schuessler et al., 1986). Thus, we determined an isochronal activation map with a 3-ms interval. The earliest 3-ms activation area contains the earliest point, 0-ms point in each determination, and it covers the more than 5-mm-diameter area. We refer to this area as the 3-ms earliest activation region (EAR) (Fig. 1, right panel) because the area of the dominant pacemaker is 0.5 mm² (an approximate diameter, 0.8 mm) in dog hearts, although a very rapid conduction in the SA node region was suggested (Bromberg et al., 1995). Additionally, because we retrospectively found that the vertical line made of points N4, N11, N17, N23, N29, N35, N41, and N47 (Fig. 1) along the sulcus terminalis presented the earliest activation line, we analyzed these lines in the present study.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Schematic representation of two electrode templates positioned at the anterior right atrium and over the SA node region along the sulcus terminalis and an actual isochronal activation map obtained with 3-ms intervals (right panel). Left panel, one template at the anterior right atrium contains 6 unipolar electrodes (N1-N6; interelectrode distance, 3 mm), and another over the SA node region along the sulcus terminalis contains 42 electrodes (N7-N48; vertical and horizontal interelectrode distances are 5 and 3 mm, respectively). Dots show unipolar electrodes, and small open holes are used for fixing the template. SVC, superior vena cava; ST, sulcus terminalis; SAN area, sinoatrial node area; SAFP, sinoatrial fat pad; SANA, sinoatrial node artery; IVC, inferior vena cava; RA, right atrium; RV, right ventricle. Right panel, atrial isotemporal activation map obtained with a 3-ms interval when atrial rate was 122 beats/min in an autonomically decentralized heart of an anesthetized dog. Dotted line at the upper part of the activation map represents the border of the anterior and posterior right atrium.

Experimental Protocol. First, we investigated the effects of sympathetic nerve stimulation at 0.5, 1, 2, and 4 Hz on the atrial rate and the 3-ms EAR in 17 autonomically decentralized hearts of the anesthetized dogs. To stimulate the right and left ansa subclaviae, we placed two bipolar hook electrodes on the cardiac side of each stellate ganglion. These electrodes were connected to an electrical stimulator (SEN 7103; Nihon Kohden). We stimulated the sympathetic nerves with 10-V and 1-ms pulse duration at frequencies of 0.5, 1, 2, and 4 Hz for 30 s and determined the effects of sympathetic nerve stimulation on the EAR and atrial rate at the end of the stimulation.

Drugs. Drugs used in the present study were dl-verapamil hydrochloride (Eisai, Tokyo, Japan), zatebradine (1,3,4,5-tetrahydro-7,8dimethoxy-3[3-[[2-(3,4-dimethoxyphenyl)-ethyl]methylamino]propyl]-2H-3-benzazepin-2-one-hydrochloride), generously donated by Boehringer Ingelheim (Hyogo, Japan), and E-4031 {1-[2-(6-methyl-2-pyridyl)ethyl]-4-(4-methylsulfonyl-aminobenzoyl)piperidine)}, generously donated by Eisai.

Data Analysis. The data are shown as the mean ± S.E. Data were analyzed by ANOVA. When the F statistic was significant, we compared the data between two values with the Bonferroni t test. Sites of electrodes, levels of sympathetic nerve stimulation, and doses of each drug were considered to be fixed factors. P values of < 0.05 were considered to be statistically significant.

	Results

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The 3-ms EAR and Sympathetic Nerve Stimulation. In the autonomically decentralized heart of the open-chest anesthetized dog, the 3-ms EAR including the earliest activation point was usually observed in the SA node region (Figs. 1 and 2) as shown by Boineau et al. (1978, 1980). The EAR was stable during experiments for more than 4 h when the atrial rate and arterial blood pressure were stable.

View larger version (50K): [in this window] [in a new window]   Fig. 2.   Effects of sympathetic nerve stimulation at frequencies of 0.5 to 4 Hz on the isochronal activation sequence and mean activation times of the eight selected electrodes, N4, N11, N17, N23, N29, N35, N41, and N47, in 17 autonomically decentralized hearts of the anesthetized dog heart. The isochronal activation sequence maps were obtained with 3-ms intervals. The 3-ms earliest activation region is shaded, and a closed circle represents the earliest point, 0 ms. Numbers under the map show atrial rate (mean ± S.E.). Control, before sympathetic stimulation; SS, sympathetic stimulation.

Effects of Verapamil, an ICa Blocker on the Pacemaker Location and Atrial Rate. Before verapamil treatment, the EAR was in the upper part of the SA node region, and atrial rate was 153 beats/min in a representative dog heart (Fig. 3A). Sympathetic nerve stimulation at 2 Hz increased atrial rate to 241 beats/min, and it shifted the EAR to the superior pacemaker region (Fig. 3D). After treatment with verapamil at 0.1 µmol/kg i.v., the atrial rate decreased to 135 beats/min, but the EAR did not change (Fig. 3B); the responses to sympathetic stimulation did not change either (Fig. 3E). However, 1 µmol/kg (i.v.) of verapamil decreased atrial rate to 115 beats/min and shifted the EAR to the SA node region-low right atrium (Fig. 3C), and the EAR in the superior pacemaker region evoked by sympathetic stimulation shifted toward the SA node region-low right atrium with the attenuation of the increase in atrial rate from 88 to 27 beats/min (Fig. 3F).

View larger version (51K): [in this window] [in a new window]   Fig. 3.   Effects of verapamil at doses of 0.1 and 1 µmol/kg i.v. on the isochronal activation sequence and atrial rate (A-C) and these responses to sympathetic stimulation at 2 Hz for 30 s (D-F) in an autonomically decentralized heart of an anesthetized dog. The isochronal activation sequence maps were obtained with 3-ms intervals. The 3-ms earliest activation region is shaded, and a closed circle represents the earliest point, 0 ms. Numbers show atrial rate. SS, sympathetic stimulation.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effects of verapamil (0.1-1 µmol/kg i.v., n = 8), zatebradine (0.1-3 µmol/kg i.v., n = 7), and E-4031 (0.1-3 µmol/kg i.v., n = 9) on the atrial rate (A) and the effects of verapamil (n = 8), zatebradine (n = 7), and E-4031 (n = 8) on the positive chronotropic response to sympathetic stimulation at 2 Hz for 30 s (B) in the autonomically decentralized heart of the anesthetized dog. The effects of E-4031 on the positive responses to sympathetic stimulation were studied in eight of nine animals. , verapamil; , zatebradine; , E-4031. The increases in atrial rate in response to sympathetic stimulation in verapamil, zatebradine, and E-4031 treatment groups were 68 ± 5.0, 58 ± 5.3, and 74 ± 7.1 bpm, respectively.

View larger version (58K): [in this window] [in a new window]   Fig. 5.   Effects of verapamil at doses of 0.1, 0.3, and 1 µmol/kg i.v. on the isochronal activation sequence (A-D) and mean activation times (E) of the eight selected electrodes, N4, N11, N17, N23, N29, N35, N41, and N47, in eight autonomically decentralized hearts of the anesthetized dogs. The isochronal activation sequence maps were obtained with 3-ms intervals. The 3-ms earliest activation region is shaded, and a closed circle represents the earliest point, 0 ms. Numbers under the map show atrial rate (mean ± S.E.).

View larger version (49K): [in this window] [in a new window]   Fig. 6.   Effects of verapamil at doses of 0.1, 0.3, and 1 µmol/kg i.v. on the isochronal activation sequence (A-D) and mean activation times (E) in response to sympathetic nerve stimulation at 2 Hz in eight autonomically decentralized hearts of the anesthetized dogs. The isochronal activation sequence maps were obtained with 3-ms intervals. The 3-ms earliest activation region is shaded, and a closed circle represents the earliest point, 0 ms. Numbers under the map show atrial rate (mean ± S.E.). SS, sympathetic stimulation.

Effects of Zatebradine, an If Inhibitor. Zatebradine at doses of 0.1, 0.3, 1, and 3 µmol/kg i.v. decreased the atrial rate dose-dependently (P < .001; Fig. 4A) and inhibited the increase in the atrial rate in response to sympathetic stimulation to 47 ± 7.8% of the control-increased atrial rate (P < .001; Fig. 4B) in seven anesthetized dogs. Although the EAR was variable with the treatment with zatebradine (Fig. 7A-E). The mean activation times at the eight selected electrodes were affected (P = .01) by doses of zatebradine but were not dose-dependent (Fig. 7F). The EAR in the anterior right atrium evoked by sympathetic stimulation was shifted by zatebradine to the posterior-superior right atrium (Fig. 8A-E). The mean activation times at the eight selected electrodes were different (P = .001) as a function of a dose of zatebradine (Fig. 8F).

View larger version (60K): [in this window] [in a new window]   Fig. 7.   Effects of zatebradine at doses of 0.1 to 3 µmol/kg i.v. on the isochronal activation sequence (A-E) and mean activation times (F) of the eight selected electrodes, N4, N11, N17, N23, N29, N35, N41, and N47, in seven autonomically decentralized hearts of the anesthetized dogs. The isochronal activation sequence maps were obtained with 3-ms intervals. The 3-ms earliest activation region is shaded, and a closed circle represents the earliest point, 0 ms. Numbers under the map show atrial rate (mean ± S.E.).

View larger version (55K): [in this window] [in a new window]   Fig. 8.   Effects of zatebradine at doses of 0.1 to 3 µmol/kg i.v. on the isochronal activation sequence (A-E) and mean activation times (F) in response to sympathetic nerve stimulation at 2 Hz in seven autonomically decentralized hearts of the anesthetized dogs. The isochronal activation sequence maps were obtained with 3-ms intervals. The 3-ms earliest activation region is shaded, and a closed circle represents the earliest point, 0 ms. Numbers under the map show atrial rate (mean ± S.E.). SS, sympathetic stimulation.

Effects of E-4031, an IKr Blocker. E-4031 at 0.1 to 3 µmol/kg, i.v., decreased atrial rate (P < .001, n = 9; Fig. 4A), and it did not affect the increase in the atrial rate in response to sympathetic nerve stimulation in eight anesthetized dogs (Fig. 4B). On the other hand, E-4031 (0.1-3 µmol) tended to widen and to shift the EAR to the lower right atrium when the dose of E-4031 was raised (Fig. 9A-E). The mean activation times were not significantly changed by the dose of E-4031(Fig. 9F). The EAR in the superior pacemaker region induced by sympathetic stimulation was not changed by any dose of E-4031 (Fig. 10).

View larger version (66K): [in this window] [in a new window]   Fig. 9.   Effects of E-4031 at doses of 0.1 to 3 µmol/kg i.v. on the isochronal activation sequence (A-E) and mean activation times (F) of the eight selected electrodes, N4, N11, N17, N23, N29, N35, N41, and N47, in nine autonomically decentralized hearts of the anesthetized dogs. The isochronal activation sequence maps were obtained with 3-ms intervals. The 3-ms earliest activation region is shaded, and a closed circle represents the earliest point, 0 ms. Numbers under the map show atrial rate (mean ± S.E.).

View larger version (51K): [in this window] [in a new window]   Fig. 10.   Effects of E-4031 at doses of 0.1 to 3 µmol/kg i.v. on the isochronal activation sequence (A-E) and mean activation times (F) in response to sympathetic nerve stimulation at 2 Hz in eight autonomically decentralized hearts of the anesthetized dogs. The isochronal activation sequence maps were obtained with 3-ms intervals. The 3-ms earliest activation region is shaded, and a closed circle represents the earliest point, 0 ms. Numbers under the map show atrial rate (mean ± S.E.). SS, sympathetic stimulation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study, we first demonstrated that verapamil, an ICa blocker, zatebradine, an If blocker, and E-4031, an IKr blocker, differentially affected the EAR when the sympathetic tone was absent or present in the anesthetized dog heart. Our results suggest that the pacemaker activities among the atrial pacemaker complex are differentially regulated by ICa, If, and IKr in the dog heart in situ.

The EAR and Pacemaker Location. We obtained the EAR from the isochronal activation sequence map using the 48 unipolar electrodes that covered the area from the anterior to posterior right atrium, including the SA node region along the sulcus terminalis (Fig. 1). Horizontal and vertical interelectrode distances of the 48 electrodes are 3 and 5 mm, respectively, and the diameter of the electrode is 1 mm. In the present study, similar EARs were consistently and repeatedly observed more than several hours through experiments when the change in atrial rate was minimum. In the pacemaker site, a pacemaker cell would activate first and conduct radially surrounding atrial myocardial cells through the sinoatrial conduction delay (Hoffman and Cranefield, 1960; Sano and Yamagishi, 1965; James et al., 1966; Strauss et al., 1973). The area of the dominant pacemaker of the dog heart is 0.5 mm², an approximate diameter of 0.8 mm (Bromberg et al., 1995). In an individual autonomically decentralized heart of the anesthetized dog, the EAR was usually observed at the SA node region when the atrial rate was 126 ± 17.8 (S.D.) beats/min in 17 animals. Additionally, sympathetic nerve stimulation shifted the EAR from the SA node region to the anterior right atrium. That anterior right atrial region corresponds to a superior subsidiary pacemaker site in the SA node pacemaker region excised heart, as shown by Ardell et al. (1991). Boineau et al. (1978, 1980) referred to the SA node pacemaker region, superior pacemaker region, and others that control the atrial rate as the atrial pacemaker complex. Three sites of those sites, the anterior superior vena cava at its junction with the right atrium, the midpostero-lateral superior vena cava just above its junction with the right atrium (the position of the canine sinus node), and the superior vena caval-intercaval band junction, are main pacemaker regions when the rate is changed by autonomic interventions. It is, therefore, likely that the 3-ms EAR includes the functional pacemaker cell in the dog heart, although a very rapid conduction in the SA node region was suggested (Bromberg et al., 1995).

Effects of Verapamil, Zatebradine, and E-4031 on the Pacemaker Location and Rate. The pacemaker activity is regulated by ICa, IKr, If, transient Ca²⁺ current, Na⁺-Ca²⁺ exchanger current, and sustained inward current in mammalian SA node cells (Irisawa et al., 1993; Guo et al., 1995). In the central, transitional, and peripheral areas of the rabbit sinus node, the maximum diastolic potential, the action potential upstroke velocity, and the overshoot are greater in tissue from the peripheral nodal area (Kodama and Boyett, 1985). The electrophysiological characteristics of the subsidiary pacemaker activity present the longer spontaneous cycle length and lower maximum diastolic potential than those of the SA node, and the ultrastructural characteristics of P cells in the Eustachian ridge are similar to those of the SA node in the isolated cat cardiac tissue (Rubenstein et al., 1987). In the present study, verapamil, a Ca²⁺ channel blocker, shifted the EAR from the SA node region to the inferior pacemaker region of the right atrium in the autonomically decentralized heart of the anesthetized dog when the increasing dose of verapamil decreased the atrial rate in a dose-dependent manner (Figs. 3-5). These results suggest that the inhibition by Ca²⁺ channel blockers of ICa shifts the pacemaker location and decreases the atrial rate in the heart and that ICa regulates the pacemaker activity of the SA node region more dominantly than that of the inferior pacemaker region. Verapamil-used doses in the present study might not block -adrenoceptors because the same doses of verapamil do not attenuate the positive dromotropic response to sympathetic nerve stimulation (Furukawa et al., 1995).

Effects of Verapamil, Zatebradine, and E-4031 on the Pacemaker Location and Rate in Response to Sympathetic Stimulation. We confirmed that the EAR was shifted from the SA node region to the anterior-superior vena cava region in the autonomically decentralized dog heart when sympathetic nerve stimulation increased atrial rate (Fig. 2), as shown previously using the multielectrodes mapping system (Boineau et al., 1980; Schuessler et al., 1986). This anterior-superior pacemaker region of the right atrium includes the Bechmann's bundle area, and this site shows the pacemaker activity after excision of the SA node pacemaker area in the dog heart (Ardell et al., 1991). We also confirmed that verapamil and zatebradine but not E-4031 attenuated the increased atrial rate in response to sympathetic nerve stimulation in anesthetized dogs as previously reported (Furukawa et al., 1995; Imamura et al., 1996). The attenuation by zatebradine of the positive chronotropic responses was greater than that by verapamil.