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(Circulation. 1995;92:3061-3069.)
© 1995 American Heart Association, Inc.

Articles

Regional Differences in Transient Outward Current Density and Inhomogeneities of Repolarization in Rabbit Right Atrium

Takeshi Yamashita, MD; Toshiaki Nakajima, MD; Hisanori Hazama, MD; Eiji Hamada, MD; Yuji Murakawa, MD; Kouhei Sawada, PhD; Masao Omata, MD

From the Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Japan; and Tsukuba Research Laboratories (K.S.), Eisai Co, Ltd, Ibaragi-ken, Japan.

Correspondence to Takeshi Yamashita, MD, The Second Department of Internal Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.

	Abstract

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Background Recent experimental and clinical studies on atrial flutter have demonstrated that the crista terminalis (CT) plays an important role in the genesis of atrial reentry. To elucidate the underlying mechanism of its role, we characterized the electrophysiological repolarization properties of CT cells by comparing them with those of the pectinate muscles (PM).

Methods and Results After action potential properties of both regions were compared by conventional microelectrode technique in multicellular atrial tissues, the whole-cell clamp experiments were applied in atrial cells isolated from both regions. Action potential duration (APD) was more prolonged in CT than in PM in multicellular preparations (APD₉₀ 77±5 ms versus 52±8 ms at 1 Hz, P<.01), though the other properties did not differ significantly. Similarly, in isolated atrial cells, APD was more prolonged in CT cells than in PM cells (APD₉₀ 63±7 ms versus 41±6 ms at 0.1 Hz, P<.01). Isolated single cells were larger in CT than in PM. The whole-cell clamp recordings showed no definite distinctions in the density of the voltage-dependent L-type Ca²⁺ current and the inwardly rectifying K⁺ current between these cells but revealed a significant reduction of the density of the 4-aminopyridine–sensitive transient outward current (I_to) in CT cells compared with that in PM cells (6.3±0.7 pA/pF versus 10.3±0.8 pA/pF at +20 mV, P<.05). However, no differences in the kinetics or the voltage dependence of I_to were observed between the cells. The time course of recovery from inactivation of I_to was also similar in both types of cells.

Conclusions These results suggest that the preferential reduction in the density of I_to in the CT cells could contribute to prolong their APD, which may be related to the genesis of atrial reentry.

Key Words: action potentials • arrhythmias

	Introduction

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Recent progress in catheter ablation technique has made it possible to ablate reentrant arrhythmias.^{1 2 3} Because the target for catheter ablation has been focused on the slow conduction area in the reentrant circuit, the electrophysiological and anatomic basis for slow conduction is mandatory for successful elimination of the arrhythmias. Many experimental and clinical studies on atrial flutter have recently demonstrated that the anatomic architecture in the right atrium plays an important role in the genesis of atrial reentry.^{4 5 6 7 8 9 10} The CT in the right atrium, which is both a unique anatomic structure of the right atrium and physiologically a preferential conduction pathway from the sinus node to the right atrium,¹¹ provides an area for conduction block and delay, leading to initiation, maintenance, and termination of atrial flutter.^{4 5 6 7 8 9 10 11 12 13}

Conduction disturbances at initiation of reentrant arrhythmias are believed to result from alterations in cell electrophysiological properties and/or anisotropic conduction caused by alterations in cell-to-cell couplings.¹⁴ Whether the former or the latter alone can cause atrial reentry is not yet certain. Therefore, it is necessary to clarify electrophysiological characteristics of the CT from both viewpoints. In the present study, we focused on repolarization characteristics of the CT, known to be the area for unidirectional block at initiation of atrial flutter.^{6 7 8 12 13} To have an insight into the mechanism of the important role of the CT in the genesis of atrial flutter, we compared cellular electrophysiological properties between the CT and PM, using conventional glass microelectrode technique in multicellular atrial tissues and also whole-cell voltage-clamp technique in isolated atrial cells.

	Methods

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New Zealand White young rabbits weighing 1.0 to 1.2 kg (3 months of age) were used in the present study.

Multicellular Preparations
The rabbits were anesthetized with secobarbital 50 to 75 mg IV. The hearts were rapidly removed and perfused for 10 minutes with modified Tyrode solution aerated with 95% O₂+5% CO₂. Thereafter, the right atrial free wall was excised and pinned to the Sylgard-covered bottom of a 10 mL chamber with endocardial side facing upward. The preparation was then superfused with modified Tyrode solution at a rate of 15 mL/min. The superfusate temperature was maintained at 37°C. One hour was allowed for tissue equilibration before the experiments. APs were recorded from both the CT and PM in the mid right atrium by use of a conventional glass microelectrode technique. A glass microelectrode filled with 3 mol/L KCl and with tip resistance of 5 to 20 M was connected to a high-impedance microelectrode amplifier (MEZ-7200, Nihon Kohden).

Single Cell Preparation
Single atrial cells were enzymatically isolated as reported previously.^{15 16} Rabbits were anesthetized and the dissected heart was mounted on a Langendorff apparatus and perfused for 7 to 8 minutes with nominally Ca²⁺-free Tyrode solution containing 120 to 160 µg/mL collagenase (Yakult Ltd) at a perfusion pressure of 80 cm H₂O. Thereafter, the enzyme-containing solution was flushed out with storage solution. The right atrium was excised, and the endocardial tissues of the CT and PM regions were separated from other regions visually with a razor. Endocardial side was chosen to correlate data in single cells with data in multicellular preparations, because epicardial cells are known to have different electrophysiological properties from endocardial cells.¹⁷ Single cells in each region were obtained by gentle shaking, kept in the storage solution at 4°C for at least 1 hour before use, and dispersed in a recording chamber filled with normal Tyrode solution. Only rod-shaped and quiescent single cells were selected for the experiments. All experiments were performed at 33 to 35°C.

Electrophysiology With Single Cells
APs and ion currents were studied in the whole-cell configuration as described by Hamill et al and Kurachi et al^{18 19 20} with a patch-clamp amplifier (EPC 7, List) that was interfaced with a personal computer. The signals were filtered at a bandwidth of DC-1 kHz and stored on a videotape by a PCM converter system (RP-880, NF Electronic Circuit Design). Electrodes fabricated from 1.0 mm ID capillary tubes had a resistance of 0.5 to 2 M when filled with internal pipette solution. Pipette current was adjusted to zero just before it was placed on the cell. The input membrane resistance and capacitance of the cell were measured by applying either a small hyperpolarizing current in the current-clamp condition or a -10-mV step pulse in the voltage-clamp condition. The potential response in the former was fitted to a simple exponential curve, whereas in the latter, capacitive transfer and steady state current level were measured. The values of resistance and capacitance obtained from these different methods were very similar.

Solutions
The modified Tyrode solution used in the multicellular experiments consisted of (mM) NaCl 121, KCl 5.0, NaHCO₃ 24, MgCl₂ 1.0, Na₂HPO₄ 1.0, glucose 5.0, CaCl₂ 1.8, pH 7.4 after equilibration with 95% O₂+5% CO₂. The normal Tyrode solution used in single-cell experiments consisted of (mmol/L) NaCl 136.5, KCl 5.4, HEPES 5.5, Na₂HPO₄ 0.33, glucose 5.5, CaCl₂ 1.8, MgCl₂ 0.53, pH 7.4 adjusted with NaOH. The storage solution contained (mmol/L) glutamic acid 70, KCl 25, KH₂PO₄ 10, taurine 10, oxalic acid 10, glucose 11, EGTA 0.5, HEPES 10, pH 7.4 adjusted with KOH. The internal pipette solution contained (mmol/L) KCl 130, ATPK₂ 5, HEPES 5, MgCl₂ 1, EGTA 3, pH 7.2 adjusted with KOH.

Data Acquisition and Analysis
Membrane potentials and current signals replayed from videotapes were converted either onto a recorder or to the analog-digital board for digitization. The digitized data were analyzed with DSS4 software programs (Canopus) in multicellular data and with the P-CLAMP program (Axon Instruments) in single-cell data. To record I_to, 0.3 mmol/L CdCl₂ and TTX 10 µmol/L were added to the Tyrode solution in all the experiments. The amplitude of I_to was measured as the difference between the peak of I_to and the value of the current at 200 ms. In some experiments, the 4-AP–sensitive current was measured by subtracting the current in the presence of 2 mmol/L 4-AP from that in its absence (Fig 1). As indicated in Fig 1, 4-AP did not significantly change the current at 200 ms at any command voltage pulses and the 4-AP–sensitive current as shown in Fig 1B subsided to almost zero at 200 ms. From these observations, it is reasonable that we could determine I_to as the difference between the peak current and the value of the current at 200 ms. The difference between the peak amplitude of I_Ca in control and that after application of 0.3 mmol/L CdCl₂ was considered to be a measure of the amplitude of the voltage-dependent I_Ca. The amplitude of I_to, I_Ca, and I_K1 was normalized to the cell membrane capacitance. The voltage-dependent inactivation of I_to was studied with the use of double-pulse protocol. The conditioning voltage pulses (500 ms in duration) to various membrane potentials between -80 and +20 mV were applied from a holding potential of -80 mV. At 10 ms after the end of each conditioning pulse, a test pulse to +20 mV (500 ms in duration) was applied to evoke I_to. The ratio of the amplitude of I_to with or without the conditioning pulse was plotted for the membrane potential of each conditioning pulse. The interval between the sets of double pulses was 30 seconds. The time course of recovery of I_to from inactivation (reactivation) was also studied by a double-pulse protocol. The first pulse (PI, 500 ms in duration) was applied from a holding potential of -80 mV. Then, with a varying interpulse interval, the second pulse (PII, 500 ms in duration) was again applied. The reactivation time course was quantified by calculating the percent of decrease in I_to amplitude during the second pulse (percent of inactivation) and plotting this value on a semilogarithmic scale against the interpulse interval. The data were simply fitted by an exponential. Data are expressed as mean±SD. Student's t test was used for comparison between mean values. Statistical significance was set at a value of P<.05.

View larger version (17K): [in this window] [in a new window]   Figure 1. Graphs show 4-AP–sensitive Ito in rabbit atrial cells. A, Original current tracings before and after application of 4-AP 2 mmol/L. The cell was held at -80 mV, and various membrane potentials were applied at 0.1 Hz. B, 4-AP–sensitive Ito was obtained by subtraction between the currents in control and after application of 2 mmol/L 4-AP. Note that during each command voltage pulse, Ito subsided to zero within 200 ms.

	Results

Top Abstract Introduction Methods Results Discussion References

 
AP Properties
In multicellular preparations of atrial free wall, APs from the CT were found to be consistently prolonged in duration from those from the PM (Table 1, Fig 2A), as described in a previous report with canine right atrium.¹⁴ Other AP characteristics, including resting membrane potential, maximal upstroke velocity of APs, and AP amplitude, did not differ between the two regions. APD at a stimulation rate of 1 Hz was longer at both early and late repolarization phases in the CT [APD₅₀: 38±7 ms (n=10) versus 18±4 ms (n=10), P<.01; APD₉₀: 77±5 ms (n=10) versus 52±8 ms (n=10), P<.01]. Similar results were obtained from single cells isolated separately. A typical AP configuration recorded at a stimulation rate of 0.1 Hz was illustrated in a cell isolated from the PM (Fig 2Ba, left) and CT (Fig 2Bb, left). APD₅₀ was 15±6 ms (n=13) versus 35±7 ms (n=13) in PM cells and CT cells, respectively. APD₉₀ of the CT cells was also more prolonged in duration than that of the PM cells [41±6 ms (n=13) and 63±7 ms (n=13), P<.01]. Thus, AP recorded from the CT cells showed a relatively long plateau phase, while that from the PM had a triangle morphology, resulting in prolongation of APD of the CT cells. To investigate the ionic mechanisms underlying the difference of AP shapes between these cell types, the effects of 4-AP were examined in a cell isolated from the PM (Fig 2Ba) and the CT (Fig 2Bb). As indicated in Fig 2B, 4-AP 2 mmol/L markedly increased the plateau phase and prolonged the APD in both types of cells. 4-AP prolonged the APD in PM cells in particular, and the AP configuration of the PM cells approached that of the CT cells. These observations suggest that the different AP configuration in these types of cells may be attributable mainly to the 4-AP–sensitive current (I_to).

View this table: [in this window] [in a new window]   Table 1. AP Properties in the CT and PM in Multicellular Preparations

View larger version (12K): [in this window] [in a new window]   Figure 2. A, Representative recording of APs of the CT and PM in multicellular mid right atrial preparations. The atrium was stimulated at a frequency of 1 Hz. AP from CT showed a slight plateau phase, while that from PM had a triangular morphology, resulting in prolongation of APD of CT. The zero voltage level is indicated by the dotted line. B, AP configuration of single cells prepared from CT and PM. The typical recording of APs in two cell types (PM cell in a and CT cell in b) are indicated in control (left) and after application of 4-AP (2 mmol/L, right). APs were elicited at a stimulation rate of 0.1 Hz.

Cell Morphology
The single cells from the CT were significantly larger in size from those of the PM (Table 2), while the ratio of length and width were similar between the two types of cells. The capacitance of single cells and input resistance were measured under voltage-clamp condition. As indicated in Table 2, the CT cells showed a significant increase in cell capacitance compared with the PM [134±22 pF (n=20) versus 42±13 pF (n=20), P<.01]. Also, the CT had a lower input resistance. The characteristics of cell morphology in the CT cells were compatible with the previous report.²¹

View this table: [in this window] [in a new window]   Table 2. Cell Parameters in the CT and PM

L-type Calcium Current
To compare the I_Ca in the two cell types, we determined current-voltage relations in the CT and PM using voltage steps for 500 ms from the holding potential of -40 mV (Fig 3A).¹⁵ To minimize contamination of I_to, 3 mmol/L 4-AP was added to the bathing solution.^{15 21} Ca²⁺-sensitive I_to that was not blocked did not distort the peak of I_Ca.²² As indicated in Fig 3B, the current-voltage relations represented by the current density were almost superimposable in the CT cells (n=10) and PM cells (n=10). Thus, the current density of I_Ca measured from the zero current did not significantly differ between the two cells. Also, the amplitude of I_Ca was obtained by the difference between the peak amplitude of I_Ca in control and after application of Cd²⁺ 0.3 mmol/L, and the density of I_Ca was determined by dividing the current amplitude by each cell capacitance as indicated in Fig 4. The density of peak I_Ca was not significantly altered in the CT and PM cells [6.6±1.2 pA/pF (n=10) versus 7.4±1.0 pA/pF (n=10), P=NS].

View larger version (23K): [in this window] [in a new window]   Figure 3. A, Representative recording showing ICa in a CT cell and a PM cell. The holding potential was -40 mV, and the command voltage pulses for 500 ms were applied at 0.2 Hz. 4-AP 3 mmol/L was added to the bathing solution. When ICa was measured from the zero current level, ICa was larger in CT than PM because of the difference in cell size, but its density did not differ. The zero current level is shown as the triangle. B, Current-voltage relations of ICa and steady state current in the CT (C: n=10) and PM (P: n=10). The currents were divided by the cell capacitance at each membrane voltage. Open circles and squares represent the peak of ICa and steady state current in C, respectively. Closed circles and squares represent the peak of ICa and steady state current in P, respectively. The current-voltage relations showed no significant difference between the two cell types.

View larger version (29K): [in this window] [in a new window]   Figure 4. Bar graphs show comparison of density of Ito, ICa, and IK1 between the CT and PM cells. The density of Ito, ICa, and IK1 was estimated from the Ito at +20 mV, the peak ICa, and steady state current at -120 mV, respectively. The amplitude of Ito was measured as the difference between the initial outward peak current and the current at 200 ms of the pulse. ICa amplitude was obtained by the difference between the amplitude of peak currents in control and after application of Cd2+ 0.3 mmol/L. There was a significant difference only in the density of Ito (*P<.05).

Steady State Properties
The steady state membrane properties were determined with discontinuous voltage steps for 500 ms from the holding potential of -40 mV (Fig 3A). Currents measured at the end of pulses were consistently larger in the CT cells than in the PM cells. The voltage of the current being zero did not differ between the cells, which is compatible with the findings that the resting membrane potential in the two cells was similar as shown in Table 1. Steady state current density did not show any difference between the two types of cells at each voltage <+40 mV (Fig 3B). Because no time-dependent currents were observed at voltages <-50 mV, the result suggested no difference in the I_K1 density between the cells. Though the slight increase in steady state current at voltages >+30 mV in the CT was considered to be due to contamination of delayed rectifier K⁺ current, we did not recognize it as a major determinant of the APD because it was induced only by the pulses of high voltage and long duration.

Transient Outward Current
Rabbit atrial cells have a remarkable I_to that affects APs in a way similar to that in human atrial cells.^{15 21 23} We compared the I_to density between the two types of cells using voltage steps for 500 ms from the holding potential of -80 mV as shown in Fig 5A. To minimize overlapping of I_Ca and I_Na, 0.3 mmol/L Cd²⁺ and 10 µmol/L TTX were added to the bath solution. I_to has been known to consist of two components.^{24 25 26} Because the transient outward components were totally blocked by 5 mmol/L 4-AP in our experimental conditions with 0.3 mmol/L Cd²⁺ in the bathing solution and 5 mmol/L EGTA in the pipette as in the previous reports,^{22 23 24 25 26 27} we considered this component as a Ca²⁺-insensitive and 4-AP–sensitive current. The fact that Cd²⁺ changes the kinetics and voltage dependence of I_to modified the present results²⁸ but did not interfere in the purpose of the present study. A representative recording and current-voltage relations are shown in Fig 5. The CT cells showed a smaller initial peak I_to compared with that from the PM at any command voltages >0 mV. Therefore, I_to density estimated from the difference between the initial outward peak and the current at 200 ms of the pulses was more decreased in the CT cells (n=10) than in the PM cells (n=10) at command voltages >-10 mV (Fig 4C). The current-voltage relations of I_to showed linear relation in both types of cells, and the voltage dependence of I_to activation did not differ. The measured densities of I_K1, I_Ca, and I_to are summarized in Fig 4. The density of I_K1, I_Ca, and I_to was estimated from the current at -120 mV, the peak I_Ca, and the I_to at +20 mV, respectively. The voltage-clamp experiments in single cells revealed that there was a statistically significant difference (P<.05) of I_to density in single cells from the CT and the PM. The density of I_to was 6.3±0.7 pA/pF and 10.3±0.8 pA/pF in the cells isolated from the CT and PM, respectively. Next, we determined the kinetics and voltage dependence of I_to in both cells.

View larger version (21K): [in this window] [in a new window]   Figure 5. A, Representative recording showing Ito in a CT cell and PM cell. The holding potential was -80 mV, and the command voltage pulses (500 ms in duration) were applied at 0.1 Hz. Cd2+ 0.3 mmol/L and TTX 10 µmol/L were added to the bathing solution. Ito, measured as the difference between the initial outward peak current and the current at 200 ms of the pulses, was larger in CT than in PM. However, its density was more decreased in CT than in PM, because CT cell capacitance was larger than that of PM. This decrease could be shown also by the ratio of Ito and steady state current. The zero current level is shown as the triangle. B, Density of the initial outward current and current at 200 ms of the pulses. Open circles and squares represent the initial outward peak current and current at 200 ms of the pulses in the CT cells (C: n=10), respectively. Closed circles and squares represent the initial outward peak current and the current at 200 ms in the PM cells (P: n=10), respectively. The density of the initial outward peak current was more depressed in C than in P at voltages >0 mV. C, Density of Ito in C (open triangle, n=10) and P (closed triangle, n=10). Ito was measured as the difference between the initial outward peak current and the current at 200 ms of the pulses. Density of Ito was significantly more decreased in C than in P at voltages >-10 mV. *P<.05.

Kinetics and Voltage Dependence of I_to
I_to activation time course and its inactivation time course could be evaluated with TTP and the of current decay (Fig 6A). TTP was measured as time from the depolarizing step to the peak I_to. We calculated by fitting the decay of I_to with double exponential functions. TTP and showed no differences between the two cell types and were similar to those reported previously,²³ though TTP was much longer than that without Cd²⁺.²⁸

View larger version (25K): [in this window] [in a new window]   Figure 6. A, Graphs show TTP and of current decay of Ito in the CT (C) and PM cells (P). Open circles and squares represent TTP and in C. Closed circles and squares represent TTP and in P. TTP was measured as time from the depolarizing step to the peak Ito. was calculated by fitting the decay of Ito with double exponential functions (left: the calibrations are 100 pA and 10 ms). There were no differences in TTP and between the two types of cells, suggesting no differences in the kinetics of Ito activation and inactivation. B, Graph shows steady state inactivation of Ito in the CT and PM. Steady state inactivation was determined with double-pulse protocols (shown in text). The steady state inactivation curve of Ito did not show any differences between the CT and PM cells.

The voltage dependence of inactivation of I_to was determined by two-step voltage pulses: the first was a conditioning pulse to voltages from -80 to 20 mV for 500 ms from the holding potential of -80 mV, and the second was a test pulse to 20 mV with a delay of 10 ms after the first pulse. The interval between each test pulse was 30 seconds. The peak amplitude of I_to at each test pulse was normalized to the maximal amplitude of I_to. The normalized I_to was plotted against the conditioning voltages (Fig 6B). The normalized values were fitted to a Boltzmann distribution equation. The steady state voltage dependence of I_to inactivation was similar between the two cell types. The CT cells showed the mean voltage at half inactivation of -40.8±4.5 mV and the slope factor of 7.7±0.6 mV (n=5), while the PM ones showed -40.3±3.9 mV and 7.4±0.8 mV (n=5), respectively. The voltage of half inactivation was lower than that in the previous report²¹ for the rabbit CT probably because of the difference in concentration of Cd²⁺ used.

The time course of recovery of I_to from inactivation (reactivation) was also investigated by a double-pulse protocol. Two command pulses from a holding potential of -80 mV were applied with a varying interpulse interval. Fig 7A shows a typical example of a cell isolated from the PM. I_to was absent with an interpulse interval of 10 ms and increased gradually as interval was prolonged in both types of cells (Fig 7B). In Fig 7C, the reactivation time course was quantified by calculating the percent of decrease in I_to amplitude during the second pulse (percent of inactivation) and plotting this value on a semilogarithmic scale against the pulse interval. The reactivation time course could be approximately described by a single exponential function in both cell types. The reactivation time constant was 738±120 ms (n=6) in CT cells and 728±110 ms (n=6) in PM cells (P=NS).

View larger version (19K): [in this window] [in a new window]   Figure 7. Graphs and plots show time course of recovery from inactivation (reactivation) of Ito. A, Original current tracings at a holding potential of -80 mV. The data were obtained from a cell isolated from the PM. The protocols of pulses are indicated in text. B and C, Typical examples of reactivation of Ito in atrial cells isolated from the CT and PM. In B, the relative amplitude of Ito evoked by the second pulse with respect to that during the first pulse was plotted against the interpulse interval. The amplitude of Ito evoked by the first pulse was considered as 100%. In C, the reactivation time course was quantified by calculating the percent of decrease in Ito density during the second pulse (percent of inactivation) and plotting this value on a semilogarithmic scale against the interpulse intervals. The reactivation time course was fitted by a single exponential in both cell types, and was 626 ms vs 634 ms in these cells.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Major Findings
The major findings of the present study were (1) APD was significantly more prolonged in the CT than in the PM in both multicellular preparations and isolated single cells. (2) Cells in the CT were larger in size than those in the PM. (3) Whole-cell voltage-clamp recordings revealed a significant decrease in the density of I_to in the CT cells compared with that in the PM cells, though the kinetics and voltage dependence of I_to were not different between the two types of cells.

Differences in I_to
Using conventional microelectrode technique, Spach et al¹⁴ reported that inhomogeneities of repolarization exist in the canine right atrium. Also, the present study clearly illustrated that inhomogeneities of repolarization exist between the CT and PM in the rabbit right atrium. Moreover, the data of single cells using the whole-cell clamp technique suggested that the prolongation of the APD in the CT could be ascribed to intrinsic properties of single cells but not to electrotonic interactions of some specialized fibers. AP recorded from the CT showed a relatively long plateau phase, while that from the PM had a triangle morphology. While application of 4-AP (Fig 2) or the increase of stimulation rate from 1 Hz to 3 Hz (data not shown) increased the plateau phase and prolonged the AP in both types of cells, the APD in the PM cells was more prolonged than in the CT cells and the AP configuration in the former approached that in the latter. These observations strongly suggest that the difference in the APD between the CT and PM is mainly attributable to some differences in I_to in these cells. In the present study, using the whole-cell clamp technique, we have characterized I_to in these two types of cells. The measured density of I_to was decreased in the CT cells and not in the PM cells, although the voltage dependence of activation and steady state inactivation, and kinetics of activation, inactivation, and reactivation did not differ between the two types of cells. Therefore, the difference in the APD was possibly ascribed to a preferential reduction of I_to channel density in the CT cells, not to modified properties of the single channel. Similar results regarding the modification of I_to channel expression have been reported in other specimens. In both canine atrium and ventricle, epicardial cells have more density of I_to channel than endocardial ones.^{23 36 37 38} Ventricular cells from acromegalic rats are larger in size and have less density of I_to channel than normal cells.³⁹ These reports also have ascribed the change in the APD to a change in the density of I_to channel, not to any changes in the intrinsic channel properties.

Our results demonstrate a difference in the density of I_to in different regions of the rabbit right atrium (CT and PM). The density of I_to was 6.3±0.7 pA/pF in the CT cells and 10.3±0.8 pA/pF at +20 mV (33 to 35°C) in the PM cells. In rabbit ventricular cells, Fedida and Giles³⁸ reported that the density of I_to in epicardial, endocardial, and papillary muscle cells was 7.66 pA/pF, 6.45 pA/pF, and 3.69 pA/pF, respectively, at +20 mV at 35°C. Thus, the current density of I_to in the CT cells is somewhat similar to that reported in rabbit ventricular cells isolated from endothelium or papillary muscle. The density of I_to in the PM cells was much higher than that reported in rabbit ventricular cells,³⁸ which supports the observations of Giles and Imaizumi¹⁵ that the density of I_to is lower in ventricular than in atrial cells from the rabbit.

Role of Anatomic Architecture in the Right Atrium
A possible physiological role and cause of the decreased density of I_to in the CT should be discussed. In normal conditions, the CT is the earliest activation site next to the sinus node in the right atrium.^{11 30} If the atrial cells had almost the same APD irrespective of their locations, this different activation time would lead to different repolarization time, ie, early activation and repolarization in the CT and late activation and repolarization in the PM. However, prolongation of APs in the CT would shorten or cancel the time lag of repolarization time between the CT and PM caused by the different activation, resulting in the simultaneous repolarization of the whole atrium. This would lead to effective contraction of the atrium and at the same time to prevention of reentrant arrhythmias. However, in some pathological conditions that may damage rapid conduction through the CT as demonstrated in human atrial flutter³¹ and a canine or rabbit model,^{17 32} these regional inhomogeneities in repolarization may exert an arrhythmogenic effect by providing an area of conduction block from the PM to the CT. Without the early activation of the CT, its prolonged repolarization time, which would be caused by its prolonged APD and its late activation, would fascilitate the conduction block around the CT. This consideration is consistent with the fact that unidirectional block at initiation of atrial reentry always occurred around the CT in canine experimental atrial flutter including the sterile pericarditis model^{5 6} and our crista ligation model.^{4 7} Apart from the initiation of atrial reentry, however, the role of I_to should be limited in its maintenance, because I_to channel is almost inactivated rate dependently at a high frequency such as atrial flutter.

In regard to the cause, there are some circumstantial differences between the CT and PM. First, the CT has an embryological origin different from that of the PM.⁴⁰ CT originates in the right valve of the sinus venosus, while the PM originates in the right atrium. Second, the location of the CT that is fixed by the superior and inferior venae cavae at both ends might make it more difficult to weaken the wall stresses on it than those on the PM. The present results and the previous works might raise a hypothesis that the expression of I_to channel in a cell might be modified dynamically by the circumstances where it lives, by stretch, or by other regional factors including embryological origin. However, this is within the realm of speculation and remains to be determined by future studies.

Limitations
The present study has several limitations. First, cells that had spontaneous pacemaker activity, which was observed in approximately 10% of the cells, were excluded in the present study. This was more frequently observed in the cells isolated from the CT,²¹ and their roles remain unknown. Secondly, Ca²⁺-sensitive 4-AP–insensitive I_to (I_to2), which is known to be Cl^- current in rabbit hearts,^{23 28} was not determined in the present study. Its density was difficult to measure because it is affected by the rundown of I_Ca.²⁸ No differences in I_Ca density and relatively small amounts of I_to2 in the previous reports^{23 28} would make the role of I_to2 minimal in the differences in APD but not negligible, particularly at a high frequency rate. Though limited for these reasons, we believe that the present results provide understanding of the mechanisms underlying the regional inhomogeneities of repolarization in the right atrium, which may be related to the genesis of atrial reentry.

	Selected Abbreviations and Acronyms

 

4-AP = 4-aminopyridine AP = action potential APD = AP duration CT = crista terminalis ICa = inward L-type Ca2+ current IK1 = inwardly rectifying K+ current Ito = transient outward current PM = pectinate muscle = time constant TTP = time to peak current TTX = tetrodotoxin

	Acknowledgments

 
This work was partly supported by grants to N. Nakajima from the Ministry of Education, Science and Culture of Japan.

Received January 20, 1994; revision received May 17, 1995; accepted July 5, 1995.

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