Ionic Mechanisms of Regional Action Potential Heterogeneity in the Canine Right Atrium

From the Department of Medicine (Z.W., S.N.) and Research Center (J.F., L.Y., Z.W., S.N.), Montreal Heart Institute; the Department of Medicine (Z.W., S.N.), University of Montreal; and the Department of Pharmacology and Therapeutics (L.Y., S.N.), McGill University, Montreal, Quebec, Canada.

Correspondence to Dr Stanley Nattel, MD, Research Center, Montreal Heart Institute, 5000 Belanger St East, Montreal, Quebec H1T 1C8 Canada. E-mail nattel{at}icm.umontreal.ca

	Abstract

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Abstract—Atrial action potential heterogeneity is a major determinant of atrial reentrant arrhythmias, but the underlying ionic mechanisms are poorly understood. To evaluate the basis of spatial heterogeneity in canine right atrial repolarization, we isolated cells from 4 regions: the crista terminalis (CT), appendage (APG), atrioventricular ring (AVR) area, and pectinate muscles. Systematic action potential (AP) differences were noted: CT cells had a "spike-and-dome" morphology and the longest AP duration (APD; value to 95% repolarization at 1 Hz, 270±10 ms [mean±SEM]); APG and pectinate muscle cells had intermediate APDs (180±3 and 190±3 ms, respectively; P<0.001 versus CT for each), with APG cells having a small phase 1; and AVR cells had the shortest APD (160±4 ms, P<0.001 versus other regions). The inward rectifier and the slow and ultrarapid delayed rectifier currents were similar in all regions. The transient outward K⁺ current was significantly smaller in APG cells, explaining their small phase 1 and high plateau. L-type Ca²⁺ current was greatest in CT cells and least in AVR cells, contributing to their longer and shorter APD, respectively. The E-4031–sensitive rapid delayed rectifier K⁺ current was larger in AVR cells compared with other regions. Voltage- and time-dependent current properties were constant across regions. We conclude that myocytes from different right atrial regions of the dog show systematic variations in AP properties and ionic currents and that the spatial variation in ionic current density may explain AP differences. Regional variation in atrial ionic currents may play an important role in atrial arrhythmia generation and may present opportunities for improving antiarrhythmic drug therapy.

Key Words: ion channel • cardiac arrhythmia • atrial fibrillation • action potential duration • regional heterogeneity

	Introduction

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The potential importance of heterogeneity in atrial refractoriness for the maintenance of atrial fibrillation (AF) has long been recognized,¹ and increased refractoriness heterogeneity appears to play a significant role in a variety of experimental models^{2 3 4 5 6} and clinical populations^{7 8} with AF. Standard microelectrode experiments have demonstrated a potential substrate for atrial refractoriness heterogeneity, in terms of regional variability in action potential morphology.^{9 10 11} The most detailed findings have been reported in the dog, with consistent differences in action potential morphology and duration noted between the crista terminalis and several other right atrial regions.^{9 10} In the rabbit, action potential differences between the right and left atrial roofs¹¹ and between the crista terminalis and pectinate muscles¹² appear to involve differences in transient outward K⁺ current (I_to). Indirect evidence is compatible with regional variability in the importance of the rapid delayed rectifier K⁺ current (I_Kr) in the canine right atrium.¹³ Recent studies have pointed to variable regional expression of a variety of cloned ion channels in the ferret right atrium.¹⁴

Virtually no data are available regarding the ionic mechanisms underlying regional differences in canine atrial action potential morphology. Such information would be relevant because of the importance of refractoriness heterogeneity in canine AF models,^{2 3 6} because of evidence for a role of regional differences in sensitivity to the I_Kr blocker dofetilide in the response to the drug of experimental atrial flutter,¹³ and because of the similarity between the properties of a variety of ionic currents in the canine atrium and those in human atrial cells.^{15 16 17} We therefore performed the present study to establish in canine right atrial tissues (1) whether the regional action potential differences previously reported in multicellular preparations^{9 10} are also present in single cells isolated from corresponding regions, (2) whether cells from different regions have different ionic current properties, and (3) whether the ionic current differences are consistent with the regional differences observed in action potential properties.

	Materials and Methods

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Cell Isolation
Adult mongrel dogs of either sex weighing 24.9±3.7 kg (n=19) were anesthetized with sodium pentobarbital (30 mg/kg IV), and their hearts were quickly removed and immersed in Tyrode's solution at room temperature. All solutions used for dissection and perfusion were equilibrated with 100% O₂. Single atrial cells were isolated by arterial perfusion with enzyme-containing solutions as described previously.¹⁵ The right coronary artery was cannulated, and the right atrium was dissected free and perfused with Tyrode's solution at 37°C for 5 minutes until the effluent was clear of blood. Any leaks from arterial branches were stopped by ligation with silk thread to ensure adequate perfusion. The tissue was then perfused at a constant rate of 12 mL/min with Ca²⁺-free Tyrode's solution for 20 minutes, followed by 40 minutes of perfusion with the same solution containing collagenase (100 U/mL, type CLSII, Worthington Biochemical) and 0.1% BSA (Sigma Chemical Co). A small piece of tissue from each of 4 well-perfused regions (crista terminalis, appendage, pectinate muscles, and the lower free wall near the atrioventricular [AV] ring; see Figure 1) was removed with pincers at 5-minute intervals. The tissues were minced, and the cells were separated by gentle trituration with a Pasteur pipette. Cells were kept at room temperature in a high-K⁺ solution before being used.

View larger version (26K): [in this window] [in a new window]   Figure 1. Schematic diagram of canine right atrium. Areas from which cells were isolated are indicated: APG, appendage; PM, pectinate muscles; CT, crista terminalis; and AVR, AV ring. Anatomic landmarks are sinoatrial node (SAN), superior vena cava (SVC), inferior vena cava (IVC), and right ventricle (RV).

Only quiescent rod-shaped cells showing clear cross striations were used. A small aliquot of the solution containing the isolated cells was placed in a 1-mL chamber mounted on the stage of an inverted microscope. Five minutes was allowed for cell adhesion to the bottom of the chamber, and then the cells were superfused at 3 mL/min, initially with Ca²⁺-containing Tyrode's solution and then with the extracellular solution appropriate to each experiment as described below. Experiments to study action potentials and all ionic currents except for the ultrarapid delayed rectifier K⁺ current (I_Kur.d) were performed at 35°C with the use of a Peltier-effect device (N.B. Datyner). I_Kur.d was studied at room temperature (21°C to 23°C) to resolve more clearly its very rapid activation.

Solutions
The standard Tyrode's solution contained (mmol/L) NaCl 136, CaCl₂ 2, KCl 5.4, MgCl₂ 0.8, NaH₂PO₄ 0.33, dextrose 10, and HEPES 10 (pH 7.4 adjusted with NaOH). This solution was used for cell isolation and as the extracellular solution for action potential studies and was modified as indicated below when specific currents were studied. The high-K⁺ storage solution contained (mmol/L) KCl 20, KH₂PO₄ 10, dextrose 10, glutamic acid 70, ß-hydroxybutyric acid 10, taurine 10, and EGTA 10, along with 1% BSA (pH 7.4 adjusted with KOH). The pipette solution for action potential recording contained (mmol/L) GTP 0.1, potassium aspartate 110, KCl 20, MgCl₂ 1.0, ATP-Mg 5, HEPES 10, Na₂-phosphocreatine 5, and EGTA 0.05 (pH 7.4 adjusted with KOH). The pipette solution for K⁺ current studies contained (mmol/L) GTP 0.1, potassium aspartate 110, KCl 20, MgCl₂ 1.0, ATP-Mg 5, HEPES 10, Na₂-phosphocreatine 5, and EGTA 10 (pH 7.4 adjusted with KOH).

When K⁺ currents were recorded, Cd²⁺ (200 µmol/L) and atropine (200 nmol/L) were added to Tyrode's solution to inhibit L-type Ca²⁺ current (I_Ca), Ca²⁺-dependent Cl^- current (I_Cl.Ca), and cholinergic K⁺ currents, including acetylcholine-dependent K⁺ current (I_KACh) and choline-dependent currents. Classical delayed rectifier K⁺ current (I_K) was studied with the addition of 2 mmol/L 4-aminopyridine (Sigma) to block I_to and I_Kur.d (4-aminopyridine was not present when I_to or the inward rectifier K⁺ current [I_K1] was recorded). E-4031 (5 µmol/L, Essai Pharmaceuticals) was used to separate the rapid component (I_Kr) from the slower component (I_Ks). I_to was studied in the presence of 10 mmol/L tetraethylammonium (TEA) and 5 µmol/L E-4031 to inhibit I_K. I_Kur.d was recorded at room temperature (21°C to 23°C), in the presence of E-4031 (5 µmol/L) to suppress I_Kr, and with the use of a holding potential (HP) of -50 mV and an 80-ms prepulse to +40 mV at 10 ms before the test pulse to inactivate I_to, as previously described.^{16 17} When I_K1 was investigated, we recorded currents before and after the addition of Ba²⁺ (0.5 mmol/L). Ba²⁺-sensitive currents were taken to reflect I_K1. Contamination by the Na⁺ current (I_Na) was prevented by holding the cell at -50 mV or by isomolar substitution with Tris for Na⁺ when more negative HPs were necessary.

The extracellular solution for I_Ca studies contained (mmol/L) TEA-Cl 136, CsCl 5.4, MgCl₂ 1.0, CaCl₂ 2.0, NaH₂PO₄ 0.33, dextrose 10, and HEPES 10 (pH 7.4 adjusted with CsOH). In studies of I_Ca, 2 µmol/L ryanodine was added to suppress I_Cl.Ca, which could otherwise overlap with and contaminate I_Ca. The pipette solution for I_Ca recording contained (mmol/L) CsCl 20, cesium aspartate 110, MgCl₂ 5, EGTA 10, GTP 0.1, Mg₂ATP 5, and Na₂-phosphocreatine 5 (pH 7.4 adjusted with CsOH).

Data Acquisition
The whole-cell patch-clamp technique was used to record ionic currents in the voltage-clamp mode, and action potentials were recorded in current-clamp mode. Borosilicate glass electrodes (outer diameter, 1.0 mm) were filled with pipette solution and connected to a patch-clamp amplifier (Axopatch 1-D, Axon Instruments). Electrodes with tip resistances of 1 to 2 M were used to record whole-cell currents, and tip resistances were 3 to 5 M when action potentials were recorded. Action potentials were elicited by 2-ms twice-threshold pulses and were recorded at 0.1, 1, and 2 Hz. Action potential duration (APD) stabilized within 15 action potentials at each frequency, and steady-state APD was measured to 20% (APD₂₀), 50% (APD₅₀), and 95% (APD₉₅) of full repolarization. Only cells in which action potentials were stable for at least 20 minutes were used for analysis. Action potential measurements were begun 5 minutes after cell rupture. Voltage command pulses were generated by a 12-bit digital-to-analog converter controlled by pClamp6 software (Axon). Recordings were low pass–filtered at half the sampling frequency. Data were sampled at rates varying from 2 to 25 kHz (with sampling at 10 to 25 kHz used for the action potential and the rapidly activating currents, I_Ca, I_to, and I_Kur.d, and sampling at 2 kHz used for the slower currents, I_Kr and I_Ks) and then stored on the hard disk of an IBM-compatible computer.

Liquid junction potentials (2 to 8 mV) were zeroed before formation of the membrane-pipette seal in Tyrode's solution. Junction potentials between the bath and pipette solution averaged 10 mV and were corrected for action potentials only. Mean seal resistance for cells from each region is shown in Table 1, along with cell dimensions measured with a calibrated graticule in the microscope eyepiece. Several minutes after seal formation, the membrane was ruptured by gentle suction to establish the whole-cell configuration. The series resistance (R_s) was estimated by dividing the time constant obtained by fitting the decay of the capacitive transient by the calculated membrane capacitance (the time integral of the capacitive response to 5-mV hyperpolarizing steps from a HP of -60 mV, divided by the voltage drop) and was electronically compensated. Before R_s compensation, the decay of the capacitive surge was expressed by a single exponential having a time constant of 532±36 µs (cell capacitance, 73.2±4.9 pF; n=82). Precompensation R_s values averaged 7.5±0.4 M. After compensation, the time constant was reduced to 112±8 µs (cell capacitance, 71.2±2.7 pF), and R_s was reduced to 1.5±0.1 M. Currents rarely exceeded 2 nA, and the maximum voltage drop across the R_s did not exceed 3 mV. Input resistance was measured with the use of a series of 5-mV hyperpolarizations from -60 mV, as the slope of the line relating voltage change to resulting current flow. Cells from the crista terminalis were larger and had greater membrane capacitance compared with those from other regions (Table 1). Therefore, all current values are represented as current densities (ie, normalized to capacitance). Cells with significant leak currents were rejected, and leakage compensation was not applied.

To ensure representativity of voltage-clamp data, similar numbers of cells from each heart were studied with each protocol (ie, the cells were distributed evenly across dogs). Furthermore, to ensure that interanimal variability did not bias results from various regions, similar numbers of cells from each region were studied from each heart, and the same measurements were made for each region within a given heart.

Statistical Analysis
Group data are presented as mean±SEM unless otherwise stated. Nonlinear curve fitting was performed with the Clampfit routine in pCLAMP (Chebyshev algorithm). Statistical comparisons among groups were performed by ANOVA. If significant effects were indicated by ANOVA, a t test with the Bonferroni correction or a Dunnett test was used to evaluate the significance of differences between individual mean values. A 2-tailed value of P<0.05 was taken to indicate statistical significance.

	Results

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Regional Action Potential Heterogeneity
Figure 2 shows all action potentials recorded from the crista terminalis (panel A, 41 cells), appendage (panel B, 41 cells), pectinates (panel C, 68 cells), and AV ring region (panel D, 51 cells). We have chosen to show all cells, rather than just "representative" examples, in order to provide a full appreciation of the variation among and within regions. Action potentials within the crista terminalis were the longest in duration and commonly showed a "spike and dome" with a long plateau. Appendage cells showed the greatest variability in morphology but had a preponderance of cells with a small phase 1 and high plateau (ie, relatively positive plateau voltage). Both pectinate and AV ring cells had a large and prominent phase 1, with the major difference between the two being the shorter plateau and more triangular appearance of cells from the region of the AV ring. Figure 2E was created by digitally averaging all action potentials in each region. The "average" action potentials illustrate quantitatively the major overall differences among action potentials in different regions.

View larger version (25K): [in this window] [in a new window]   Figure 2. Action potentials in different regions. A to D, All action potentials recorded are shown for CT (n=41 cells) (A), APG (n=41 cells) (B), PM (n=68 cells) (C), and AVR (n=51 cells) (D). E, Action potentials obtained by digitally averaging all recordings from each respective region.

Resting membrane potential was not significantly different among the four regions, averaging -73.5±0.3 (n=41), -71.2±0.3 (n=41), -72.8±0.1 (n=68), and -70.9±0.2 (n=51) mV in the crista, appendage, pectinates, and AV ring region, respectively. Action potential amplitude also showed no significant differences, averaging 126±3, 120±3, 123±2, and 119±3 mV, respectively, at 1 Hz. Mean values of APD at various frequencies are shown for all groups of dogs in Table 2. APD was significantly longer in cells from the crista compared with cells from the other 3 areas. Appendage and pectinate muscle cell APDs were not significantly different, but APDs in both areas were substantially greater than in cells near the AV ring at 0.1 and 1 Hz.

Phase 1 amplitude was quantified in each cell as the voltage difference between the peak of the overshoot and the end of rapid phase 1 repolarization and averaged 49±2 mV in crista cells, 51±1 mV in pectinate muscle cells, 54±2 mV in AV ring cells, and 39±1 mV in appendage cells (P<0.001 versus each of the other regions). Plateau voltage was quantified as the most positive voltage after the end of phase 1 and averaged 6.1±1.3 mV in crista cells, 0.8±0.2 mV in pectinate muscle, -6.9±2.2 mV in AV ring cells, and 13.9±2.7 mV in appendage cells (P<0.001 versus other regions). To obtain a quantitative estimate of the presence and amplitude of spike-and-dome morphologies, we operationally defined the presence of such morphologies by a plateau voltage positive to the voltage at the end of phase 1, and we defined their amplitude by the voltage difference between the plateau and the end of phase 1. Spike-and-dome morphologies were seen in 35 of 41 crista cells (85%; mean amplitude, 7.8±1.1 mV), 9 of 41 appendage cells (22%; amplitude, 1.9±0.3 mV), 22 of 68 pectinate muscle cells (32%; amplitude, 3.3±0.4 mV), and 0 of 51 AV ring cells.

L-Type Ca²⁺ Current, I_Ca
Panels A to D of Figure 3 show typical I_Cas recorded from each of the regions studied. Possible contaminating effects of I_Ca rundown were minimized by beginning all studies 5 minutes after membrane rupture, by performing protocols in the same sequence in all cells, with the I_Ca density-voltage relation studied first, and by bracketing protocols with an I_Ca measurement at +10 mV. If I_Ca varied by >5% over the course of a protocol, the experiment was terminated, and the data were not used. I_Ca was largest in crista cells, intermediate in cells from the appendage and pectinate muscles, and smallest in AV ring cells. Mean±SEM I_Ca density-voltage relations (25 cells/group) are shown in Figure 3E. I_Ca density was significantly greater in crista cells than in other regions and was significantly smaller in AV ring cells than in appendage or pectinate regions.

View larger version (19K): [in this window] [in a new window]   Figure 3. A to D, Typical recordings of ICa from CT (A), APG (B), PM (C), and AVR (D). E, Mean±SEM ICa density-voltage relations for each region (n=25 cells/region). Voltage protocol (240-ms pulses to voltage indicated at 0.1 Hz) is shown in inset. ***P<0.001 vs APG and PM cells.

The voltage- and time-dependent properties of I_Ca in various regions were explored further, as illustrated in Figure 4. The voltage dependence of inactivation was explored with the use of 1-s prepulses to various voltages, followed by a 300-ms test pulse to +10 mV (HP, -80 mV; 0.1 Hz). The peak current elicited by the test pulse was normalized to current without a prepulse, providing the mean data in Figure 4A, which show no apparent regional differences in inactivation voltage dependence. The results between -90 and 0 mV were well fitted by a modified Boltzmann relation of the form (Iv-Imin)/(Imax-Imin)=1/{1+exp[(V-V_1/2)/s]}, where Iv is I_Ca at prepulse voltage V; Imax and Imin are I_Ca without a prepulse and at the prepulse producing maximum inactivation, respectively; V_1/2 is the voltage for half-maximal inactivation; and s is a slope factor. The V_1/2 values for I_Ca were -22.8±1.2, -23.1±1.3, -25.2±1.3, and -21.2±1.4 mV in crista, appendage, pectinate, and AV ring regions, respectively (n=10 cells/group, P=NS), and s values averaged 6.1±0.5, 6.9±0.7, 5.4±0.5, and 5.8±0.4 mV, respectively (P=NS). The voltage-dependent activation of I_Ca was determined by dividing peak current during depolarizing test pulses by the driving force (calculated as the difference between test potential and the I_Ca reversal potential from the current-voltage relation). The results (Figure 4A) were well fitted by a Boltzmann relation, with a half-maximal activation voltage of -11.5±0.8, -12.3±0.4, -10.3±0.7, and -10.9±0.8 mV in crista, appendage, pectinate, and AV ring region, respectively (n=10 cells/group, P=NS).

View larger version (26K): [in this window] [in a new window]   Figure 4. Voltage and time dependence of ICa in various regions. A, ICa inactivation was determined with the use of 1000-ms conditioning pulses (CPs) from an HP of -80 mV followed by a 300-ms test pulse to +10 mV. Activation voltage dependence was determined from the current-voltage relation, with the driving force corrected for by dividing by the difference between test potential (TP) and the reversal potential obtained from the intersection between the ascending limb of the current-voltage relation and the voltage axis. Results are mean±SEM of 10 cells/region. B, Inactivation time constants (1 and 2) were determined during 300-ms pulses to the voltages indicated at 0.1 Hz (10 cells/region). C, ICa recovery was studied with the use of paired 300-ms pulses delivered at 0.1 Hz. The current during pulse 2 (P2) was normalized to the current during pulse 1 (P1) and expressed as a function of the P1-P2 interval. Results are mean±SEM of 10 cells/region. D, Frequency dependence of ICa was determined from the current during the last pulse of a 15-pulse train (200 ms from -70 to +10 mV) at the frequencies indicated. Results are normalized to the current during the first pulse and represent the mean±SEM of 10 cells/frequency in each region.

The time course of I_Ca was fitted by biexponential functions, as previously reported.^{18 19} As shown in Figure 4B, the mean±SEM time constants of I_Ca inactivation were voltage dependent, but not different among groups (n=10 cells/group). The recovery kinetics of I_Ca at -80 mV were studied with a 2-pulse protocol, as shown in Figure 4C. Recovery proceeded at a similar rate in each region, with monoexponential time constants averaging 27.8±2.9, 33.2±3.1, 29.2±2.6, and 32.2±3.4 ms in crista, appendage, pectinate, and AV ring regions, respectively (n=10 cells/group, P=NS). The frequency dependence of I_Ca was tested with a train of fifteen 200-ms pulses from -70 to +10 mV, with current during the last pulse of the train normalized to first-pulse current. Mean data from 10 cells/group (Figure 4D) show that I_Ca frequency dependence had no regional differences.

Inward Rectifier K⁺ Current, I_K1
Figure 5 (panels A to D) shows representative I_K1 recordings from one cell in each region, as elicited by 300-ms pulses from an HP of -40 mV to voltages ranging from -120 to -20 mV. To separate I_K1 from potential contaminating currents, recordings were obtained before and after the addition of 0.5 mmol/L Ba²⁺ to the superfusate. Figure 5 shows Ba²⁺-sensitive currents obtained by digital subtraction of currents after Ba²⁺ application from those before Ba²⁺ application. Mean current-voltage relations are shown in panel E. No significant regional differences in I_K1 density were observed; eg, mean current densities at -120 mV averaged -5.6±0.3, -5.1±0.5, -5.3±0.5, and -5.0±0.4 pA/pF in crista, appendage, pectinate, and AV ring regions, respectively (n=25 cells in each group). The lack of regional variation in I_K1 is consistent with the lack of any regional differences in the resting membrane potential.

View larger version (29K): [in this window] [in a new window]   Figure 5. A to D, Typical recordings of IK1 from CT (A), APG (B), PM (C), and AVR (D). E, Mean±SEM IK1 density-voltage relations for each region (n=25 cells/region). Voltage protocol is shown in the inset of panel E, with 300-ms pulses applied at 0.1 Hz.

Regional Differences in I_toDensity
Figure 6 (panels A to D) shows typical recordings of I_to from cells in each region, as determined with the use of 100-ms pulses from an HP of -80 mV (0.1 Hz). Current amplitude was measured as the difference between peak and end-pulse steady-state current. There were no obvious differences in current time course, but currents tended to be smaller in cells from the appendage (panel B). Figure 6E shows mean I_to density as a function of test potential in each region (n=25 cells for each determination). There were no significant differences in I_to density among cells from crista, pectinates, and the AV ring region; however, I_to densities were approximately half as great in appendage cells compared with the other regions.

View larger version (30K): [in this window] [in a new window]   Figure 6. A to D, Typical recordings of Ito from CT (A), APG (B), PM (C), and AVR (D). E, Mean±SEM Ito density-voltage relations for each region (n=25 cells/region). Voltage protocol is shown in the inset of panel E, with 100-ms pulses applied at 0.1 Hz. ***P<0.001 vs other regions.

The voltage dependence and kinetics of I_to were analyzed in various atrial regions, as shown in Figure 7. Voltage-dependent inactivation was studied with a 1000-ms prepulse to various test potentials from an HP of -80 mV, followed by a 100-ms test pulse to +60 mV. The voltage dependence of activation was obtained from the current-voltage relation determined as shown in Figure 6, with changes in driving force corrected by dividing each current by the difference between test potential and the mean reversal potential of I_to tail currents recorded after a 3-ms pulse to +50 mV (in the presence of 5 mmol/L TEA to inhibit I_Kur.d). The I_to reversal potential averaged -74±4, -73±3, -74±3, and -73±3 mV in crista, appendage, pectinate, and AV ring cells, respectively (n=5 for each). Mean±SEM data for 10 cells/region are shown in Figure 7A, along with best-fit Boltzmann relations, and indicate no regional differences in I_to voltage dependence. The half-activation voltage averaged 12.9±1.0, 13.1±1.1, 12.3±1.2, and 12.7±1.0 mV in crista, appendage, pectinates, and AV ring regions, respectively, and the half-inactivation voltage averaged -19.9±1.8, -21.5±2.3, -22.6±2.1, and -18.5±2.0 mV.

View larger version (27K): [in this window] [in a new window]   Figure 7. Voltage and time dependence of Ito in various regions. A, Ito inactivation was determined with the use of 1000-ms conditioning pulses from an HP of -80 mV followed by a 100-ms test pulse to +60 mV. Activation voltage dependence was determined from the current-voltage relation with a correction for the driving force by dividing by the difference between TP and the reversal potential obtained from Ito tail current recordings in cells from each region. Results are mean±SEM of 10 cells/region. B, Inactivation (1,inact and 2,inact) and activation (act) time constants for Ito were measured during 100-ms pulses to the voltages indicated at 0.1 Hz (10 cells/region). Current activation was fitted by a function of the form A [1-exp(-t/)]3, and current inactivation was fitted by a function of the form A1 exp(-t/1)+A2 exp (-t/2). C, Ito recovery was studied with the use of paired 150-ms pulses delivered at 0.1 Hz. The current during P2 was normalized to the current during P1 and expressed as a function of the P1-P2 interval. Results are mean±SEM of 10 cells/region at -80 mV and 6 cells/region at -60 mV. D, Frequency dependence of Ito was determined from the current during the last pulse of a 15-pulse train (100 ms from -80 to +50 mV) at the frequencies indicated. Results are normalized to the current during P1 and represent the mean±SEM of 10 cells/frequency in each region.

The rapidity of I_to activation was assessed by fitting current activation by a third-order monoexponential relation, and inactivation kinetics were analyzed by fitting a biexponential relation to the time course of current decay during a depolarizing step. The time course of I_to activation and inactivation, as shown in Figure 7B by mean±SEM results obtained in 10 cells/group, did not show any significant interregional differences. I_to recovery kinetics were assessed with the use of paired 150-ms pulses from an HP to +50 mV with varying interpulse intervals (Figure 7C). The recovery of I_to was well fitted by monoexponential functions. In 6 cells in which recovery time constants were measured at -80, -70, and -60 mV for each cell, the time constants averaged 20±2, 23±3, 21±3, and 24±3 ms at -80 mV, 23±2, 25±1, 25±3, and 26±2 ms at -70 mV, and 34±3, 34±3, 34±4, and 35±3 ms at -60 mV, respectively, in crista, appendage, pectinate, and AV ring cells (P=NS for interregional differences). The frequency dependence of I_to was tested with a train of fifteen 100-ms pulses from -80 to +50 mV and showed no significant regional differences (Figure 7D, 10 cells/group).

View larger version (25K): [in this window] [in a new window]   Figure 10. A to D, Typical recordings of IKr (measured as E-4031– sensitive current) from CT (A), APG (B), PM (C), and AVR (D). E, Mean±SEM IKr density-voltage relations for each region (n=25 cells/region). F, Mean±SEM voltage-dependence of IKr activation based on tail current amplitudes after voltage steps indicated. Voltage protocol (3-s pulses to voltage indicated, followed by 1 s at -30 mV to record tail currents; protocol delivered at 0.1 Hz) is shown in the inset.

Delayed Rectifier K⁺ Currents
Three types of delayed rectifier currents are present in dog atrium: classical I_Kr and I_Ks¹⁵ and an ultrarapid delayed rectifier (I_Kur.d) with properties that resemble those of Kv3.1 channels.¹⁶ Figure 8 shows representative currents elicited by 140-ms pulses from an HP of -50 mV to various test potentials, followed by repolarization for 60 ms to -30 mV to record tail currents, under conditions (including a prepulse to inactivate I_to; see Materials and Methods for details) designed to isolate I_Kur.d. An HP of -50 and an 80-ms prepulse to +30 mV at 10 ms before the test pulse were used to suppress I_to and elicit selectively I_Kur.d as previously described. Typical original recordings from each region are shown in panels A to D and have the rapid activation and large tail currents characteristic of I_Kur.d, with no obvious regional differences. Mean current-voltage relations for 25 cells/region, shown in panel E, indicate a lack of significant regional variation in the current.

View larger version (27K): [in this window] [in a new window]   Figure 8. A to D, Typical recordings of IKur.d from CT (A), APG (B), PM (C), and AVR (D). E, Mean±SEM IKur.d density-voltage relations for each region (n=25 cells/region). Voltage protocol is shown in the inset of panel E, with 140-ms pulses applied at 0.1 Hz, preceded by an 80-ms prepulse to +40 mV to inactivate Ito.

Figure 9 shows an analysis of I_Ks in all groups. Panels A to D show recordings obtained with the use of 3-s depolarizing pulses to various voltages, followed by a 1-s repolarization to -30 mV in the presence of 5 µmol/L E-4031 (to inhibit I_Kr) and 2 mmol/L 4-aminopyridine to inhibit I_Kur.d and I_to. The overall form of original recordings was not different among the 4 groups, and mean time-dependent step current density-voltage relations (in 25 cells/group) showed no significant regional differences (Figure 9E). Similarly, there were no differences in tail current density (eg, after a step to +40 mV, values averaged 2.7±0.3, 2.5±0.3, 2.6±0.3, and 2.7±0.3 pA/pF in crista, appendage, pectinate, and AV ring regions, respectively).

View larger version (27K): [in this window] [in a new window]   Figure 9. A to D, Typical recordings of IKs from CT (A), APG (B), PM (C), and AVR (D). E, Mean±SEM IKs density-voltage relations for each region (n=25 cells/region). IKs was recorded in the presence of 5 µmol/L E-4031 to suppress IKr. Voltage protocol (3-s pulses to voltage indicated, followed by 1 s at -30 mV to record tail currents; protocol delivered at 0.1 Hz) is shown in the inset.

I_Kr was recorded with the voltage protocol shown in Figure 10, with results obtained before and after the addition of 5 µmol/L E-4031 and I_Kr expressed as the E-4031–sensitive current. Representative I_Kr recordings from each region are shown in panels A to D of Figure 10. Larger currents were consistently recorded in cells from the AV ring region. Mean step current density-voltage relations (from 25 cells/group) are shown in Figure 10E and indicate that I_Kr was significantly larger in the AV ring region compared with the other 3 regions studied. Similar differences were noted in I_Kr tail current density; eg, after a step to 0 mV, tail current densities averaged 0.93±0.09 pA/pF in crista cells, 0.89±0.08 pA/pF in appendage cells, 0.91±0.09 pA/pF in pectinate muscle cells, and 1.61±0.18 pA/pF (P<0.001 versus other regions) in AV ring cells. Shown in Figure 10F is the voltage dependence of I_Kr activation, based on E-4031– sensitive tail currents at -30 mV after 3-s steps to the voltages indicated. Half-activation voltages averaged -9±1, -11±1, -10±1, and -9±1 mV in crista, appendage, pectinate, and AV ring cells, respectively (n=15 cells/group). Activation kinetics were also similar among regions; eg, at +10 mV, the activation time constants averaged 115±14, 104±15, 119±14, and 109±13 ms in crista, appendage, pectinate, and AV ring cells, respectively (P=NS, n=25 cells/group).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present findings indicate discrete differences in action potential morphology and duration in cells isolated from different regions of the canine right atrium. These differences correspond to previously published observations obtained with standard microelectrode techniques and were associated with clear variations in the densities of specific ionic currents, notably, I_to, I_Ca, and I_Kr, whereas the densities of other currents did not show regional alterations.

Comparison With Previous Studies of Regional Action Potential Heterogeneity in the Atrium
Hogan and Davis⁹ described a variety of action potential morphologies recorded with standard microelectrodes in the canine right atrium. The action potentials they show from the crista terminalis, appendage, the "atrial roof" (tissue between pectinate muscles, corresponding to the region near the AV ring in our studies), and pectinate muscles are quite similar to our recordings from single cells isolated from corresponding regions. Spach et al¹⁰ showed a progressive decrease in plateau amplitude and APD from the crista terminalis to the pectinate muscles in multicellular canine right atrial preparations, with recordings quite similar to those we obtained in single cells from the crista and pectinate muscles, respectively. The similarity between our findings and previous reports in multicellular preparations points to the physiological relevance of our isolated cell preparation. The only other data regarding regional action potential variations in atrial tissue of which we are aware were obtained in the rabbit. Left atrial roof cells have a shorter duration and larger phase 1 than do right atrial cells,¹¹ and as in the dog, cells from the crista have a longer action potential than do cells from pectinate muscles.¹²

Ionic Mechanisms of Action Potential Heterogeneity
Over the past 10 years, there has been increasing awareness of the importance of action potential heterogeneity at the ventricular level.²⁰ Transmural heterogeneity has been well studied in canine ventricular preparations, in which epicardial action potentials are characterized by a prominent phase 1 and spike-and-dome appearance,²¹ and midmyocardial ("M cells") have a much longer APD.²² Similar observations have been made in guinea pigs,²³ rabbits,^{24 25} and humans.^{26 27} The limited phase 1 amplitude in epicardial myocytes is due to a reduced I_to density.^{21 28} The longer APD of M cells is due, at least in part, to a smaller I_Ks.^{29 30} These spatial gradients in repolarization underlie several clinically important phenomena, including differences in rate-dependent repolarization of epicardium versus endocardium,³¹ the electrocardiographic J wave,³² triggered activity caused by quinidine and digitalis,³³ and potentially arrhythmogenic responses to I_Na blockade,³⁴ ATP-sensitive K⁺ current activation,³⁵ myocardial ischemia,³⁶ and increased extracellular Ca²⁺ concentration.³⁷ Recent molecular work points to varying expression of Kv4.2 mRNA as the mechanism underlying transmural heterogeneity in I_to expression.³⁸

The ionic basis of atrial action potential heterogeneity is poorly understood, and no data are available in the literature regarding the ionic mechanisms of atrial action potential heterogeneity in the dog. In the rabbit, differences in I_to density appear to be particularly important,^{11 12} in keeping with the well recognized prominence of I_to as a repolarizing current in the rabbit.^{39 40} The ionic differences that we recorded can explain much of the interregional action potential variation we observed. The smaller I_to in appendage cells is consistent with their generally smaller phase 1 amplitude (Figure 1 of the present study and Figure 3 of Hogan and Davis⁹). The greater I_Ca density in crista cells accounts for their greater APD and, in conjunction with a sizable I_to, for their common spike-and-dome morphology (as shown in Figure 1 and in References 9 and 10^{9 10} ). Finally, the smaller I_Ca density and larger I_Kr can explain the smaller plateau and shorter duration of cells from the area near the AV ring.

Potential Significance of Our Findings
There is increasing evidence suggesting a role for repolarization heterogeneity in the genesis and perpetuation of AF.⁴¹ The present study provides information regarding the ionic substrate for refractoriness heterogeneity in the canine right atrium. Although further work will be necessary to ascertain the applicability of our findings to humans, the present study provides potentially valuable insight into the mechanisms of atrial repolarization heterogeneity.

Understanding the ionic basis for refractoriness heterogeneity may provide new insight into mechanisms of antiarrhythmic drug action and help in the development of new approaches to developing antiarrhythmic drug therapy. Cha et al⁴² have shown that dofetilide is more effective than quinidine in suppressing atrial flutter in a dog model and acts at least in part by reducing the dispersion in atrial refractoriness. Restivo et al¹³ have shown that dofetilide increases the refractory period more in the lower right atrium of dogs with sterile pericarditis than in other regions. Our findings may explain this result in terms of the significantly larger I_Kr that we found in lower right atrial cells (near the AV ring) compared with cells from other right atrial regions.

Potential Limitations
We found significant interregional differences in action potential properties and ionic currents among cells isolated from different right atrial regions. The differences in ionic current densities were potentially able to explain the differences observed in mean action potential properties. On the other hand, within each region there was considerable variability in action potential properties of individual cells (eg, see Figure 2A to 2D). Action potential variability was greatest in cells from the atrial appendage (Figure 2B), consistent with previous observations of variability in action potential morphology in atrial cells from individual right atrial appendage preparations.⁴³ Thus, although our findings explain overall interregional differences, we did not study the ionic mechanisms for the fine structure of action potential variability within atrial regions. It was not feasible within the context of the present study to assess the ionic mechanisms of intercell action potential variability within a given region, but this matter would be appropriate to pursue in future work.

The ionic current profiles of isolated cells are sensitive to variations in the nature and quality of the isolation. To minimize this potential source of variability, cells from each region were studied for each dog, action potentials were recorded from all isolates to ensure that ionic currents were recorded from preparations with comparable properties, and the same types of studies were performed on cells from all regions on each experimental day. The similarity between the action potential properties from cells in each region in our studies and previous observations with standard microelectrodes in multicellular preparations^{9 10} supports the validity of our observations.

Although we have described in detail the ionic substrate for spatial variability in canine right atrial action potential properties, the mechanisms responsible for creating regional differences and their physiological role remain to be determined. It has been suggested that the longer action potential of crista terminalis cells may help to direct impulses from the sinus node preferentially in the direction of the AV node.⁴⁴ This function may explain the consistent finding across species that action potentials are longer in the crista terminalis than in other right atrial regions.^{9 10 11} It remains to be determined whether other regional action potential properties have functional significance and whether they arise as a function of embryological origin, developmental factors, differential innervation, and hemodynamic factors, for example. Recently developed immunolocalization techniques may be very helpful in studying the distribution of ion channel mRNA and proteins.⁴⁵

We characterized the properties and density of I_Ca and several K⁺ currents, and the variations observed are consistent with regional differences in action potential properties. We cannot, however, exclude a role of currents and transport mechanisms that we did not measure (eg, I_KACh, Na⁺, K⁺-ATPase, Na⁺-Ca²⁺ exchanger, and Ca²⁺-ATPase). The outward component of I_K1 was small and variable in our studies (Figure 5), as in previous work with atrial myocytes. Although no significant regional differences in I_K1 were noted, we cannot include small differences in the outward component of I_K1 that were beyond the resolution of our methods but could contribute to differences in repolarization. The degree to which cell coupling in the multicellular in situ heart would attenuate action potential variability is an important issue that cannot be addressed by the present study. Acetylcholine decreases the space constant by increasing K⁺ conductance,⁴⁶ and vagal stimulation causes a substantial increase in atrial repolarization heterogeneity,^{47 48} which appears to be central in the clinically relevant ability of vagal stimulation to promote AF.⁵ It is conceivable that the ability of vagal stimulation to increase atrial repolarization heterogeneity and promote AF is related to its propensity to unmask spatial variations in action potential properties by reducing the influence of cell coupling on the spatial variability of repolarization.

Conclusions
We have shown that cells isolated from different regions of the canine right atrium have discrete action potential characteristics that are accompanied by different profiles of ionic current densities. The regional differences in ionic current expression account in large measure for regional action potential properties. These findings provide a basis for understanding regional heterogeneity in atrial repolarization, an important factor in the genesis and maintenance of atrial reentrant arrhythmias, and may lead to new insights into mechanisms of antiarrhythmic drug action and approaches to the development of novel antiarrhythmic therapies.

	Acknowledgments

 
This study was supported by the Medical Research Council of Canada, the Quebec Heart Foundation, and the Fonds de Recherche de l'Institut de Cardiologie de Montréal. Dr Wang is a Canadian Heart Foundation Research Scholar, and L. Yue is the recipient of a Canadian Heart Foundation Research Studentship. The authors thank Nathalie Talbot and Mirie Levy for technical assistance and France Thériault for secretarial assistance with the manuscript.

Received January 19, 1998; accepted June 16, 1998.

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				E. P. Anyukhovsky and L. V. Rosenshtraukh Electrophysiological responses of canine atrial endocardium and epicardium to acetylcholine and 4-aminopyridine Cardiovasc Res, August 1, 1999; 43(2): 364 - 370. [Abstract] [Full Text] [PDF]

				D. M. Roden and S. Kupershmidt From genes to channels: normal mechanisms Cardiovasc Res, May 1, 1999; 42(2): 318 - 326. [Abstract] [Full Text] [PDF]

				R. Gaspo, H. Sun, S. Fareh, M. Levi, L. Yue, B. G. Allen, T. E. Hebert, and S. Nattel Dihydropyridine and beta adrenergic receptor binding in dogs with tachycardia-induced atrial fibrillation Cardiovasc Res, May 1, 1999; 42(2): 434 - 442. [Abstract] [Full Text] [PDF]

				M. Courtemanche, R. J Ramirez, and S. Nattel Ionic targets for drug therapy and atrial fibrillation-induced electrical remodeling: insights from a mathematical model Cardiovasc Res, May 1, 1999; 42(2): 477 - 489. [Abstract] [Full Text] [PDF]

				S.-H. Lee, F.-Y. Lin, W.-C. Yu, J.-J. Cheng, P. Kuan, C.-R. Hung, M.-S. Chang, and S.-A. Chen Regional Differences in the Recovery Course of Tachycardia-Induced Changes of Atrial Electrophysiological Properties Circulation, March 9, 1999; 99(9): 1255 - 1264. [Abstract] [Full Text] [PDF]

				D. Li, L. Zhang, J. Kneller, and S. Nattel Potential Ionic Mechanism for Repolarization Differences Between Canine Right and Left Atrium Circ. Res., June 8, 2001; 88(11): 1168 - 1175. [Abstract] [Full Text] [PDF]

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