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(Circulation Research. 2001;88:1168.)
© 2001 American Heart Association, Inc.

Cellular Biology

Potential Ionic Mechanism for Repolarization Differences Between Canine Right and Left Atrium

Danshi Li, Liming Zhang, James Kneller, Stanley Nattel

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

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

Abstract

Abstract—Experimental and clinical evidence suggests a critical role for the left atrium (LA) in atrial fibrillation (AF). In animal models, repolarization is faster in the LA than in the right atrium (RA), leading to more stable reentry circuits with a shorter intrinsic period in the LA. The ionic mechanisms underlying LA-RA repolarization differences are unknown. Therefore, we evaluated ionic currents and action potentials (APs) with the whole-cell patch clamp in isolated canine atrial myocytes. The density of the rapid delayed rectifier current (I_Kr) was greater in the LA (eg, 1.83±0.10 pA/pF at +20 mV) than in the RA (1.15±0.07 pA/pF, P<0.01; n=16 cells per group). The slow and ultrarapid delayed rectifier, the inward rectifier, L-type Ca²⁺, and transient outward K⁺ currents were all comparable in the LA and RA. There were no differences in kinetic or voltage-dependent properties of currents in LA versus RA. Western blots of ether-a-go-go–related gene (ERG) protein in three RA and corresponding LA regions showed significantly greater ERG expression in LA. AP duration (APD) was shorter in the LA versus RA in both isolated cells and multicellular preparations, and the effective refractory period (ERP) was shorter in the LA compared with the RA in vivo. Dofetilide had significantly larger APD- and ERP-increasing effects in the LA compared with RA, and LA-RA repolarization differences were eliminated by exposure to dofetilide. We conclude that LA myocytes have larger I_Kr than do RA myocytes, contributing importantly to the shorter APD and ERP in LA. The larger LA I_Kr may participate in the ability of the LA to act as a "driver region" for AF, with potentially important implications for understanding AF mechanisms and antiarrhythmic therapy.

Key Words: channels • heterogeneity • electrophysiology

Several lines of evidence point to a particularly important role of the left atrium (LA) in maintaining atrial fibrillation (AF). Animal models^{1 2} and clinical studies³ suggest the presence of LA "driver regions" in AF. In an isolated sheep-heart model of AF, rotors maintaining the arrhythmia were always located in the LA.⁴ Linear LA lesions appear particularly important for the success of radiofrequency ablation procedures directed against AF.⁵ LA refractoriness is shorter than right atrial (RA) refractoriness,^{6 7 8} potentially explaining, at least in part, the preferential role of LA reentry in AF. Despite the evidence for differences between LA and RA refractoriness that might be important in the pathophysiology of AF, there are no published studies comparing the cellular ionic electrophysiology of LA with RA myocytes. The present work was designed to evaluate action potential (AP) properties and ionic currents governing repolarization in canine cells isolated from the LA free wall and to compare them with RA cells. Because our findings pointed to an important role for differences in rapid delayed rectifier current (I_Kr) between LA and RA, we also evaluated the effect of the I_Kr blocker dofetilide on differences between RA and LA electrophysiology in both multicellular atrial preparations and the in situ heart.

Materials and Methods

Cell Isolation and Patch-Clamp Methods
Hearts were excised from 33 dogs (25.6±3.2 kg) anesthetized with pentobarbital (30 mg/kg IV) and immersed in Tyrode’s solution containing 2 mmol/L Ca²⁺. All tissues for dissection and perfusion were equilibrated with 100% O₂. Atrial myocytes were isolated as in previous studies.^{9 10} The right coronary artery (to perfuse the RA free wall) and left circumflex coronary artery (to perfuse the LA free wall) were cannulated and removed along with the perfused segments of atrial tissue. The tissues were then perfused with Tyrode’s solution at 37°C until the effluent was clear of blood, followed by perfusion at 12 mL/min with Ca²⁺-free Tyrode’s solution for 20 minutes and then with the same solution containing collagenase (CLSII, 100 U/mL, Worthington) and 1% BSA. When tissues were softened, a section was minced, and cells were harvested by trituration. Cells were kept at room temperature in a high-K⁺ storage solution until they were used.

Cells in a 1-mL bath were superfused (3 mL/min, 35°C) with an extracellular Tyrode’s solution containing (mmol/L) NaCl 136, KCl 5.4, CaCl₂ 2, MgCl₂ 0.8, NaH₂PO₄ 0.33, dextrose 10, and HEPES 10, pH 7.4 (NaOH). For Ca²⁺ current (I_Ca) recording, tetraethylammonium chloride and CsCl replaced NaCl and KCl, respectively. For classic delayed rectifier current (I_K) studies, 2-mmol/L 4-aminopyridine was used to block transient outward current (I_to), after it was verified that 4-aminopyridine had no effect on I_K tail currents. For studies of the ultrarapid delayed rectifier (I_Kur.d), a holding potential (HP) of -50 mV and an 80-ms prepulse to +30 mV at 10 ms before a test pulse were used to inactivate I_to as previously described.¹⁰ I_to was studied in the presence of 10 mmol/L tetraethylammonium and 1 µmol/L dofetilide to inhibit I_Kur.d and I_K, after it was verified that neither drug affects I_to. For K⁺ current measurement, CdCl₂ (200 µmol/L) or (for studies of I_K) 5 µmol/L nifedipine was added to block I_Ca and the Ca²⁺-sensitive I_Cl. When HP negative to -50 mV was used in studies of K⁺ currents, Tris-HCl replaced NaCl. The standard pipette solution contained (mmol/L) potassium aspartate 110, KCl 20, MgCl₂ 1, GTP 0.1, Mg₂-ATP 5, Na₂-phosphocreatine 5, EGTA 10, and HEPES 10, pH 7.3 (KOH). For AP recording, the EGTA concentration was decreased to 50 µmol/L. For I_Ca recording, K⁺ was replaced by Cs⁺. The storage solution contained (mmol/L) KCl 20, KH₂PO₄ 10, glutamic acid 70, ß-hydroxybutyric acid 10, taurine 10, EGTA 10, and dextrose 10, along with 1% albumin (pH 7.4 with KOH or CsOH).

To minimize the potential effects of time-dependent changes in enzymes and isolation procedure over the course of the present study, LA and RA cells were studied concurrently in an alternating fashion. Cells from both LA and RA from each dog were evaluated with the same voltage-clamp protocols to study the same currents on each experimental day. To minimize any potential contaminating effect of current rundown (particularly for I_K and I_Ca), the current protocols were applied in the same order for LA and RA cells. Five minutes were allowed for stabilization after membrane rupture before recording, and if currents varied by >5% over the course of a protocol, the data were rejected.

Tight-seal patch clamp was used to record currents (voltage-clamp mode) and APs (current-clamp mode). Electrode resistances were 1 to 2 M for current recording and 3 to 5 M for AP recording. Cells with normal resting potentials (negative to -70 mV, 30% of all cells) were used for AP recording. APs (elicited by 2-ms twice-threshold pulses) were analyzed at steady state (15 to 20 APs) at each frequency. Recordings were low-pass–filtered at half the sampling frequency (2 kHz for I_Kr and the slow delayed rectifier current [I_Ks], 10 kHz for APs, I_Ca, and I_to).

Junction potentials (8 to 10 mV) were corrected for AP recordings only. Seal resistance averaged 8.8±1.3 G. Pipettes had resistances of 1 to 3 M when filled. Series resistances and capacitance compensation were applied. Leakage correction was not used; cells with significant leaks were rejected for study. Series resistances and capacitive time constants averaged 7.1±0.2 M and 512±12 µs, respectively, before compensation and 1.60±0.04 M and 129±6 µs, respectively, after compensation. Cell capacitance (the time integral of capacitive current divided by the voltage drop during 5-mV steps) was 81.7±2.3 pF in 164 LA cells and 72.0±1.7 pF in 164 RA cells (P<0.01), indicating that LA cells were larger. Therefore, current amplitudes are presented in terms of densities.

Multicellular-Preparation AP Recording
Hearts were excised from seven dogs (25.9±4.0 kg), and the right and left coronary arteries were cannulated and perfused with (mmol/L) NaCl 120, KCl 3.8, CaCl₂ 1.25, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25, and dextrose 5.5, 95%O₂/5%CO₂ at 37°C. RA and LA preparations (endocardial surface exposed) were fixed in a tissue bath, and the tissue was perfused at 12 to 14 mL/min. APs were recorded from the RA pectinate muscles (same region as for cell isolation) or the LA free wall at 1 Hz via 3 mol/L KCl–filled floating microelectrodes (10 to 30 M) and an Axoclamp-2B amplifier (Axon), before and after addition of 1 µmol/L dofetilide to the bath.

In Vivo Study
Dogs weighing 26.3±3.7 kg (n=8) were anesthetized with morphine (2 mg/kg SC) and -chloralose (120 mg/kg IV bolus, followed by 29.25 mg/kg per hour). A median sternotomy was performed, and bipolar polytetrafluoroethylene (Teflon)-coated stainless-steel electrodes were hooked into RA and LA free walls for recording and stimulation. The effective refractory period (ERP) was measured with 15 basic (S1) stimuli followed by a premature (S2) stimulus (all stimuli were 2-ms, twice threshold) with 5-ms S1S2 decrements (ERP is the longest S1S2 failing to capture, mean of three determinations). LA and RA ERPs were measured before and after 80 µg/kg of intravenous dofetilide (Pfizer).

Immunoblotting of ERG Protein
Ether-a-go-go–related gene (ERG) proteins were extracted from three regions (appendage, free wall, and posterior wall) of the RA and LA of eight dogs not used for other studies. Atrial tissues were removed from hearts excised under pentobarbital anesthesia, with tissue blocks excised rapidly in Tyrode’s solution, frozen in liquid nitrogen, and stored at -80°C for subsequent analysis. To extract membrane protein, 2 g of atrial tissue was pulverized in liquid nitrogen and suspended in 3 mL ice-cold RIPA buffer containing (mmol/L) dibasic sodium phosphate 9.1, monobasic sodium phosphate 1.7, dithiothreitol 5, NaCl 150, and sodium orthovanadate 100, along with 1% Igepal CA-630 (Sigma), 0.5% sodium deoxycholate, 0.1% SDS, 0.1 mg/mL benzamidine, 10 mg/mL phenylmethylsulfonyl fluoride, 5 µg/mL leupeptin, 5 µg/mL pepstatin A, and 5 µg/mL aprotinin. The suspension was homogenized and then centrifuged at 14 000g for 20 minutes at 4°C. The soluble fraction was retained and stored at -80°C. Protein concentration was determined by the Bradford method, with BSA used as the standard.

Protein extracts (60 µg) were denatured in Laemmli sample buffer and subjected to electrophoresis on 7.5% SDS-polyacrylamide gels. Proteins were transferred to Immobilon-P polyvinylidene difluoride membranes blocked with 5% nonfat dry milk in Tris-buffered saline solution (TBS) and incubated overnight with anti-ERG primary antibody (Alomone) at a 1:200 dilution. The primary antibody was a polyclonal preparation from rabbits hyperimmunized against the C-terminal domain of human ERG1 (residues 1118 to 1133). Protein samples probed with primary antibody that had been preincubated with the antigenic peptide were used as negative controls. After having been washed 5 times in 0.1% Tween 80–TBS (TTBS), the membranes were reblocked in 1% nonfat dry milk in TTBS for 10 minutes. They were then incubated in a 1:8000 dilution of horseradish peroxidase–conjugated anti-rabbit IgG in 1% nonfat dry milk in TTBS for 60 minutes, followed by four additional washes in TTBS. Antibody detection was performed with Western Blot Chemiluminescence Reagent Plus (NEN Life Sciences). A single band corresponding to ERG with the expected molecular mass of 160 kDa was detected. The density of bands was quantified by laser densitometry with Quality One software. Densitometric comparisons of different groups were performed on blots processed equally and exposed on the same x-ray film.

Statistical Analysis
Data are presented as mean±SEM. Nonlinear curve fitting was performed with pCLAMP6 (Clampfit, Chebyshev algorithm). Paired t tests and nonpaired t tests (2-tailed) were used to compare baseline and drug responses and LA and RA cells, respectively.

Results

AP Recordings and In Vivo ERP
Figures 1A and 1B show all APs recorded from isolated RA and LA myocytes, respectively. Overall AP morphology was similar in both regions, but LA AP duration (APD) was significantly shorter (Figure 1C). AP amplitude and resting potential were similar for RA and LA (Table). In agreement with AP recordings, LA ERP was significantly shorter in vivo than RA ERP (Figure 1D). Standard microelectrode recordings from multicellular RA and LA preparations are shown in Figures 1E and 1F, with LA APD being consistently shorter than RA (Figure 1G).

View larger version (30K): [in this window] [in a new window]   Figure 1. A and B, AP recordings from 50 RA (A) and 50 LA (B) myocytes. C and D, Mean values of APD90 in vitro (C) and ERP in vivo (D) (n=8). *P<0.05 vs RA at corresponding frequency. E and F, Representative APs from multicellular RA and LA free wall preparations recorded with floating microelectrode techniques before (control) and after dofetilide. G, Mean±SEM APD90 (n=15 cells per group) recorded in multicellular preparations and ERP at a cycle length of 400 ms (ERP400, n=8) measured in vivo before (control [CTL]) and after dofetilide (DOF). H, Percentage change at a BCL of 400 ms in LA and RA APD90 (in multicellular preparations) and ERP400 (in vivo) caused by DOF. *P<0.05, **P<0.01 vs RA.

View this table: [in this window] [in a new window]   Table 1. AP Characteristics of Canine RA and LA (1 Hz)

K⁺ Currents
Inward Rectifier Current, I_K1
The inward rectifier current (I_K1) was isolated by applying step pulses from HP of -40 mV before and after 0.5 mmol/L Ba²⁺ and by obtaining Ba²⁺-sensitive currents by digital subtraction. Figures 2A and 2B show Ba²⁺-sensitive I_K1 recordings in RA and LA cells, respectively. The I_K1 density-voltage relation (based on end-pulse current) showed no differences between RA and LA (Figure 2C).

View larger version (25K): [in this window] [in a new window]   Figure 2. A and B, IK1 recordings from RA cell (A) and LA cell (B), based on Ba2+-sensitive current. C, Mean±SEM IK1 density (n=25 cells per group, P=NS). Inset shows currents between -80 and +20 mV on an enlarged scale to show small outward component. There were no significant differences between RA and LA at any voltage. TP indicates test potential. D and E, IKur.d recordings from RA cell (D) and LA cell (E) obtained with the voltage protocol shown, delivered at 0.1 Hz and consisting of an 80-ms prepulse to +30 mV, followed 10 ms later by a 120-ms test pulse, and then a 70-ms repolarizing step to -30 mV to observe tail currents. F, Mean±SEM step and tail IKur.d densities in 12 LA and 12 RA cells (P=NS).

Ultrarapid Delayed Rectifier Current, I_Kur.d
Typical I_Kur.d recordings from RA and LA cells are shown in Figures 2D and 2E and show the rapid activation, inward rectification at positive voltages, and tail currents characteristic of the current. There were no differences in the form of currents in RA versus LA. As shown in Figure 2F, mean I_Kur.d step and tail densities in 12 LA and 12 RA cells showed no significant differences.

Transient Outward Current, I_to
Figures 3A and 3B show recordings of I_to from RA and LA, respectively. Current amplitude was measured as the difference between peak current on depolarization and end-pulse steady-state current. I_to density-voltage relations (Figure 3C) and normalized current-voltage relations (Figure 3D) were superimposable for LA and RA. Similarly, I_to voltage-dependent activation and inactivation showed no RA-LA differences (Figure 3E). Half-activation voltage averaged 10.2±0.9 mV in LA and 11.7±0.4 mV in RA; half-inactivation voltage averaged -25.1±1.9 mV in LA and -25.4±0.8 mV in RA. Figure 3F illustrates I_to recovery at -70 mV as determined with a paired-pulse protocol. Time constants averaged 35.5±3.0 (-70 mV), 56.5±6.5 (-60 mV), and 90.4±8.3 (-50 mV) ms in LA and 34.2±3.2 (-70 mV), 59.0±7.6 (-60 mV), and 92.7±8.8 (-50 mV) ms in RA, with no significant RA-LA differences (n=10 cells at each voltage per group.)

View larger version (34K): [in this window] [in a new window]   Figure 3. A and B, Ito recordings obtained with 100-ms depolarizing pulses (0.1 Hz) in RA myocyte (A) and LA myocyte (B). C, Mean±SEM Ito density vs TP (n=25 cells per group). D, Ito normalized to current at +50 mV for each cell. E, Voltage-dependent Ito inactivation (Inact.) and activation (Act.) (n=10 cells per group). Inactivation was assessed with 1-second prepulses to the TPs indicated, followed by 100-ms pulses to +60 mV. Activation voltage dependence was assessed from depolarization-induced currents, with driving-force changes corrected by TP-Erev, where Erev indicates reversal potential of Ito tail current. Curves are best-fit Boltzmann relations. F, Ito reactivation (currents I2 and I1) determined with the use of 150-ms paired pulses (P1 and P2) and the protocol shown, delivered at 0.1 Hz (n=10 cells per group).

Slow Delayed Rectifier Current, I_Ks
Figures 4A and 4B show I_Ks recorded from RA and LA myocytes in the presence of 1 µmol/L dofetilide to inhibit I_Kr. Mean step and tail-current density-voltage relations (Figures 4C and 4D, respectively) were the same for LA and RA. The voltage dependence of I_Ks activation was evaluated from the normalized tail-current/test-potential relation (Figure 4E). Half-activation voltages averaged 17.2±1.2 (LA) and 18.3±1.9 mV (RA, P=NS). I_Ks activation was well-fit by biexponential functions (correlation coefficients >0.99). Mean activation time constants did not differ between RA and LA (Figure 4F). Similarly, there were no significant differences in deactivation time constants: tail time constants averaged 87.7±4.6 and 370.4±35.8 ms in LA versus 84.4±3.1 ms and 344.2±31.4 ms in RA (P=NS).

View larger version (32K): [in this window] [in a new window]   Figure 4. A and B, IKs recordings obtained with 3-second depolarizing test pulses followed by 1-second repolarizations to -30 mV to observe tail currents (0.1 Hz) in RA myocyte (A) and LA myocyte (B). C and D, IKs density of step (C) and tail (D) current (n=31 cells per group). E, Voltage-dependent IKs activation based on normalized tail currents (n=25 cells per group). Boltzmann fits are shown. F, Activation time () constants based on biexponential fits of IKs step current (n=20 cells per group).

Rapid Delayed Rectifier Current, I_Kr
The protocol shown in Figure 5 was used to record I_K before and after 1 µmol/L dofetilide, with I_Kr obtained as the dofetilide-sensitive current. Recordings from RA and LA myocytes are shown in panels A and B, respectively. I_Kr was consistently and significantly larger in LA cells (Figures 5C and 5D). For example, at a test potential of +20 mV, step current density averaged 1.83±0.10 pA/pF in LA cells; this value was 60% larger than the value of 1.15±0.07 pA/pF in RA cells (P<0.01). Tail current density after a pulse to +20 mV averaged 0.99±0.10 pA/pF in LA versus 0.75±0.06 pA/pF in RA (P<0.05). I_Kr activation voltage dependence was similar for LA and RA (Figure 5E). Half-activation voltages were -10.1±1.6 mV (LA) and -9.4±1.3 mV (RA, P=NS). I_Kr activation was monoexponential, with a time constant at +10 mV averaging 138±16 ms in LA and 134±10 ms in RA (Figure 5F, P=NS). Deactivation was biexponential, with tail-current time constants at -30 mV (after a pulse to +10 mV) of 313±38 and 3851±259 ms in LA and 311±47 and 3719±323 ms in RA (P=NS).

View larger version (31K): [in this window] [in a new window]   Figure 5. A and B, IKr recordings obtained as 1 µmol/L dofetilide-sensitive current during 3-second steps followed by 1-second repolarizations to -30 mV (0.1 Hz) in RA myocyte (A) and LA myocyte (B). C and D, IKr step (C) and tail (D) current density (n=16 cells per group). *P<0.05 and **P<0.01 vs control. E, Voltage-dependent IKr activation based on tail currents. F, IKr step (monoexponential fit at +10 mV) and tail (biexponential fit at -30 mV) current time constants (Act/Deact , n=6 cells per group).

L-Type Ca²⁺ Current, I_Ca
Typical I_Ca recordings in RA and LA cells are shown in Figures 6A and 6B. There were no significant differences in I_Ca density between RA and LA (Figure 6C) or in the normalized current-voltage relationship (Figure 6D). I_Ca inactivation voltage dependence (Figure 6E) was explored with 1-second prepulses to various voltages, followed by 300-ms test pulses to +10 mV (HP -80 mV, 0.1 Hz). Half-inactivation voltage averaged -25.2±1.3 mV in LA cells and -25.5±1.9 mV in RA cells (n=10 per group, P=NS). The activation voltage dependence was assessed by dividing peak current during test pulses by the driving force (difference between test potential and I_Ca reversal potential). The half-activation voltage averaged -9.6±7.1 mV in LA and -9.5±1.7 mV in RA (P=NS). I_Ca recovery kinetics were studied with a 2-pulse protocol as shown in Figure 6F. Monoexponential recovery time constants averaged 32.7±2.3 ms in LA and 30.0±2.5 ms in RA (P=NS).

View larger version (31K): [in this window] [in a new window]   Figure 6. A and B, ICa recordings obtained with 240-ms depolarizing steps at 0.1 Hz in RA myocyte (A) and LA myocyte (B). C, ICa density (n=34 cells per group). D, ICa normalized (I/Imax) to current at +10 mV for each cell. E, ICa voltage-dependent inactivation (Inact.) and activation (Act.). Inactivation was assessed with 1-second prepulses from -80 mV, followed by 300-ms test pulses to +10 mV. Activation voltage dependence was assessed from depolarization-induced currents, with driving force corrected by dividing by TP-Erev, where Erev is the voltage axis intercept of the ascending limb of the current-voltage relation. Curves are best-fit Boltzmann relations to mean±SEM data (n=10 cells per group). F, ICa recovery studied with paired 300-ms pulses (0.1 Hz). Data are mean±SEM current during the test pulse (P2) normalized to current during the first pulse (P1). Best monoexponential fits are shown (n=10 cells per group).

Response to Dofetilide
If the differences between RA and LA repolarization were primarily due to differences in I_Kr, as suggested by the voltage-clamp results, blockade of I_Kr should equalize repolarization properties. Furthermore, if I_Kr is larger in LA, I_Kr block should increase APD and ERP to a greater extent in LA than in RA. Therefore, we studied the effects of dofetilide at a maximally effective I_Kr-blocking concentration, 1 µmol/L,¹¹ on APD in multicellular atrial preparations (to avoid artifacts due to cell isolation) in vitro and the effects of 80 µg/kg dofetilide IV¹² on ERP in vivo. Typical results from RA and LA preparations are shown in Figures 1E and 1F. Overall, mean APD at 90% repolarization (APD₉₀) was significantly shorter in LA preparations under control conditions (Figure 1G, left), but there were no significant differences after dofetilide. The percentage APD prolongation caused by dofetilide in LA preparations was significantly greater than in RA (Figure 1H, left). Similarly, ERP in vivo was significantly shorter in the LA before, but not after, dofetilide (Figure 1G, right), and dofetilide caused substantially greater ERP increases in LA than in RA (Figure 1H, right).

Atrial Expression of ERG Protein
To obtain an independent assessment of interatrial differences in expression of I_Kr-related protein, we used Western blot methods to evaluate the expression of ERG protein. Figure 7A shows ERG protein detected in three RA and three corresponding LA regions from one dog. The molecular weights were identical in all regions, and the bands appear denser in the LA samples. Negative controls probed with antibodies preincubated with ERG antigen show no signals, as expected (Figure 7B). Figure 7C shows mean ERG protein densities (eight hearts per measurement, with data for each region obtained in each heart). ERG expression was significantly stronger in LA compared with RA (P=0.007 for LA versus RA as a significant determinant by 1-way ANOVA).

View larger version (65K): [in this window] [in a new window]   Figure 7. A, Western blots of ERG protein from RA appendage (RAA), free wall (FW), and posterior wall (PW), as well as corresponding LA regions. B, Western blots from same regions as in panel A, with antibody preexposed to ERG antigenic peptide. Molecular mass markers (165 kDa) are shown to the left of each set of gels in panels A and B. C, Mean±SEM ERG protein density in three corresponding RA and LA regions.

Discussion

We have shown that APD and ERP are shorter in the LA free wall than in the RA free wall, that LA cells have a larger I_Kr density than do RA cells (with no differences in I_Kur.d, I_Ks, I_K1, I_to, and L-type I_Ca), and that the I_Kr blocker dofetilide eliminates LA-RA repolarization differences. These findings suggest that differences in I_Kr play an important role in repolarization differences between the LA and RA.

Functionally Important ERP Differences Between LA and RA
Several studies in animal^{1 2 4 13} and clinical models^{3 5} point to the importance of the LA in maintaining AF. Rensma et al¹⁴ showed that wavelength is a significant determinant of atrial reentry, particularly AF, in dogs, and that shorter refractory periods result in smaller wavelengths in the LA than in the RA. Shorter local ERP and wavelength values should translate into more rapid and stable local reentry. Subsequent experimental studies have consistently demonstrated shorter ERPs in the LA than in the RA.^{7 8 15 16} LA regions with shorter refractory periods generate more organized electrograms^{4 13 15} and, by virtue of more rapid intrinsic periods, appear to act as driver regions that maintain AF by activating other areas with slower periodicities.^{4 13} The available data suggest that the ERP differences between LA and RA allow for more stable and rapid reentry in the LA than in the RA, potentially contributing to the important role of the LA in AF.

Ionic Mechanisms Underlying Atrial AP and ERP Heterogeneity
There is growing awareness of the importance of electrophysiological heterogeneity at the atrial level. AP heterogeneity within the canine RA is well recognized,^{17 18 19} and we found that regional AP variability within the RA is associated with distinct patterns of ionic current density.¹⁹ In the present study, ERP differences between canine LA and RA were attributable to differences in APD. Furthermore, we found that LA free wall myocytes have shorter APDs and larger I_Kr density than do RA cells from the free wall (pectinate muscles). In our previous study, APD was similar in RA pectinate muscles and RA appendage and longer in RA crista terminalis, areas with comparable I_Kr densities.¹⁹ Taken together, the results of the present study and our previous work suggest that I_Kr density is greater and APD is shorter in the LA than in the RA appendage, pectinate muscles, and crista terminalis. We are not aware of any previous studies comparing ionic and/or AP properties between LA and RA. At the ventricular level, Volders et al²⁰ showed that midmyocardial cells from the canine right ventricle have larger I_to and I_Ks density than do cells from the left ventricle.

Potential Significance
The present study is the first of which we are aware that compares the ionic properties of atrial myocytes from the LA with those from the RA, relating ionic differences to AP properties at the single cell and multicellular level and also to ERP in vivo. Thus, our findings add potentially important information regarding ionic mechanisms of interatrial electrophysiological variation.

The treatment of AF remains suboptimal. An improved understanding of the fundamental basis of atrial electrophysiological function should help in the development of improved therapeutic approaches. We have recently shown that intravenous dofetilide increases ERP to a greater extent in the LA than in the RA in dogs with a substrate for AF caused by either congestive heart failure or sustained atrial tachycardia.²¹ Dofetilide was highly effective in terminating heart failure–related AF but was relatively ineffective in AF associated with atrial tachycardia–induced remodeling. In the latter group, dofetilide largely eliminated reentrant activity in the LA, but persistent reentry in the RA maintained AF. The findings of the present study may explain, at an ionic level, why dofetilide is more effective in prolonging canine atrial ERP and preventing atrial reentry in the LA than in the RA. Our results may also relate to recent studies of surgical therapy for AF that point to a role for the LA as a critical driver region for the arrhythmia.^{22 23}

Potential Limitations
Several ionic currents, including in particular I_K²⁴ and I_Kur.d, are quite sensitive to damage during cell isolation. We took precautions to ensure that the same currents were studied in both LA and RA myocytes isolated from the same dog on each experimental day. For this reason, we could not compare the data from our previous voltage-clamp study of RA ionic heterogeneity¹⁹ with the results obtained in the LA in the present experiments. Any differences noted could have simply been due to different qualities of cell isolation enzymes and of cells available during the previous experiments (performed 3 years before the present study) and the present studies. Therefore, concurrent voltage-clamp studies for RA had to be performed for direct comparison with LA myocytes. The greater density of I_Kr in LA was supported by the independent observation of greater ERG protein expression in the LA compared with the RA. Further evidence in support of the main findings in isolated cells is provided by the fact that key observations (including earlier repolarization in LA versus RA, a larger effect of dofetilide in the LA, and equalization of LA-RA repolarization in the presence of dofetilide) were reproducible in multicellular atrial preparations and in the in situ heart.

Regional heterogeneity within the RA necessitates the consistent use of cells from one RA region as a basis for comparison with the LA. We studied RA cells from the pectinate muscle region, which represents a large proportion of the tissue in the RA free wall. Our results cannot be directly extrapolated to other RA regions, such as the appendix or crista terminalis; however, because our previous studies have shown that these areas have mean I_Kr densities similar to those in pectinate muscles and mean APDs that are equal to or greater than those in the pectinate muscle,¹⁹ it is likely that LA I_Kr is greater compared with values in the RA appendix and crista terminalis. Our studies of ERG protein expression support this notion, although we cannot exclude the possibility that there may be different ERG splice variants in LA versus RA, with differing affinities for the antibody that we used. RA cells in the AV ring region had a significantly larger I_Kr density than did cells from other RA regions in our previous study,¹⁹ with mean values close to those of LA cells in the present study. Therefore, the differences in RA versus LA I_Kr density found in the present study would not extend to comparisons between LA free wall and RA AV ring cells. We have previously found differences in ionic currents other than I_Kr within the RA.¹⁹ For example, L-type I_Ca is larger in the crista terminalis than in the pectinate muscles or the appendage (where it is equivalent). In addition, I_to is smaller in the RA appendage. Thus, potential regional differences within each atrium need to be considered, and although greater I_Kr is likely an important feature contributing to the shorter LA APD and ERP, it would be wrong to think that either atrium can be considered to have a single homogeneous set of ionic properties. We would have liked to examine the ionic properties of cells from other LA regions, such as the appendage, AV ring region, and pulmonary vein areas. Unfortunately, we found these areas difficult to perfuse effectively via single coronary arteries, so that the cells obtained were of variable and often poor quality. Any data obtained from such cells would be of questionable value; therefore, we elected to focus on the well-perfused LA free wall and the analogous RA free wall pectinate muscle region. Sampling limitations are always present in studies of this type; eg, the only published studies of right ventricular versus left ventricular ionic properties limited themselves to single regions of well-defined location in each ventricle.^{20 25}

The present study examined repolarization properties of LA and RA, along with underlying ionic mechanisms. In no way do we wish to imply that repolarization differences are the only factor promoting LA arrhythmogenesis in AF. For example, the role of ectopic activity in the pulmonary vein region is now well recognized.^{26 27} Nonetheless, the ERP remains an important factor governing the rate and stability of reentry, and the differences noted in the present study are likely to be, at the very least, an important contributing factor to the role of the LA in maintaining AF.

Conclusions
We have shown that a larger I_Kr results in shorter APD and ERP in canine LA myocytes compared with RA myocytes. These results have potential significance for determining the interatrial differences in basic electrophysiology, the mechanisms underlying AF, and the treatment of the arrhythmia.>

View this table: [in this window] [in a new window]   Table 11. AP Characteristics of Canine RA and LA (1 Hz)

Acknowledgments

This study was supported by the Medical Research Council of Canada and the Quebec Heart Foundation. Dofetilide was kindly supplied by Pfizer Pharmaceuticals. Dr Li is a Heart and Stroke Scientific Research Corporation of Canada/AstraZeneca research fellow. J. Kneller is a Medical Research Council of Canada student. The authors thank Chantal Maltais and Chantal St-Cyr for technical assistance.

Footnotes

Original received January 4, 2001; revision received April 9, 2001; accepted April 9, 2001.

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