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1Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada; and 2Department of Bioengineering, University of California, San Diego, La Jolla, California 92093-0412
Submitted 25 April 2003 ; accepted in final form 13 July 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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action potential; atrial repolarization; mouse heart
In larger mammals, it is well known that regional intra-atrial heterogeneity of tissue refractoriness, which is a consequence of differing action potential durations (APDs), can contribute to the susceptibility of atrial tissue to reentrant arrhythmias and may lead to atrial fibrillation (27). Subsequent clinical observations and comprehensive research programs on several species have developed and added support to this concept (2, 5, 28). Interatrial differences in refractoriness have also been described. The effective refractory period (ERP) of the left atrium is shorter than that of the right atrium in dogs (20), rabbits (33, 34), pigs (47), and goats (46).
The general acceptance of the critical mass hypothesis (11) has led to the assumption that the volume and mass of mouse cardiac myocardium is too small to sustain reentrant arrhythmias or to fibrillate (27). Recently, however, reentrant arrhythmias in the ventricles and atria of mice have been described (41, 43). Thus the anatomic and electrophysiological substrates essential for induction of reentrant excitation are present in the mouse heart, and maneuvers that cause atrial fibrillation in larger mammals, such as intense vagal nerve stimulation, can result in arrhythmias in the mouse heart. These findings provide the basis for combining electrophysiological approaches with molecular genetic and transgenic methods to study the mechanisms of arrhythmias in the atria and ventricles of mice (18).
Previous electrophysiological studies have utilized transgenic animals and antisense oligodeoxynucleotide technology to identify the 
-subunits of voltage-gated K+ channels (Kv) that contribute repolarizing current in mouse atrium. It was found that Kv4 family channel -subunits encode the calcium-independent transient outward K+ current (Ito) (48) and that the Kv1.5 and Kv2.1 -subunits underlie the slowly inactivating K+ current in mouse atrium (4). However, it is not known whether the atria of rodents exhibit the heterogeneity of repolarization that is thought to facilitate the generation of reentrant arrhythmias in larger mammals. Accordingly, the goals of the present study on myocytes and atrial tissue from adult mice were 1) to compare the ERPs and APDs of the left and right atrium, and 2) to characterize the repolarizing K+ currents in myocytes isolated from the left and right atrium to determine whether intra- and interatrial heterogeneity of repolarization exists in mice.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Atrial ERP measurements. Beating hearts were removed and placed in ice-cold Krebs solution containing (in mM) 118 NaCl, 4.7 KCl, 1.25 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NAHCO3, and 11 D-glucose. This solution was bubbled continuously with a 95% O2-5% CO2 gas mixture, which yielded a stable pH of 7.4. The lungs, thymus, and ventricular tissue were dissected away, leaving both atria. The atrial preparations were then pinned by the aorta to the Sylgard-lined base of a 35-mm petri dish and continuously superfused with 10–12 ml/min Krebs solution maintained at 37°C.
Epicardial electrogram recordings were obtained by gently placing a Teflon-coated silver wire electrode that was chlorided at the tip (0.3 mm diameter) on the surface of either atrium. The unfiltered signals were amplified by a factor of 1,000 with the use of a custom-designed device (Anton Paar; Graz, Austria) with an AC-coupled input (cutoff frequency = 0.72 Hz). The ECG signals were digitized at a sampling rate of 4 kHz per channel, with 16-bit resolution by using an analog-to-digital converter (model PCIMIO16XE, Analog Instruments). Data were stored, monitored, and analyzed using custom-designed acquisition and analysis software (Cardiolyzer, Laback; Graz, Austria) on a personal computer (38). To pace the selected atrium, a bipolar (0.3 mm distance between electrodes) stimulating electrode was positioned at a fixed distance (
3 mm) from the recording electrode. After an equilibration period of 10 min, ERPs of each atrium were determined as follows. Atrial preparations were paced with stimuli of 2 ms duration and twice the threshold amplitude, using an S1 interval of 120 ms. After 10 S1 stimuli, an extrastimulus (S2) was applied. S2 intervals decreased in 1-ms increments from 40 to 0 ms. A 1-s recovery time followed each S1-S2 protocol. The ERP was defined as the longest S2 interval that failed to evoke an action potential. ERPs were determined 10 consecutive times for each atrium, and the average ERP for each atrium in each mouse was computed.
Myocyte isolation. Single atrial myocytes were isolated using very similar methods to those described by Mangoni and Nargeot (26) for the isolation of mouse sinoatrial node cells. The beating hearts were removed from the mice after methoxyflurane anesthesia and cervical dislocation. Left and right atria were then dissected away from the remaining supraventricular structures in a Tyrode's solution containing (in mM) 140 NaCl, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES, and 5.5 D-glucose that was warmed to 35°C. The pH of this solution was adjusted to 7.4 by the addition of NaOH. The left and right atria were then separated and cut into 10 strips for enzymatic treatment.
Atrial tissue strips were transferred to a solution containing (in mM) 140 NaCl, 5.4 KCl, 0.07 CaCl2, 0.5 MgCl2, 1.2 KH2PO4, 50 taurine, 5.5 D-glucose, 1 mg/ml bovine serum albumin (BSA), and 5 HEPES; pH was adjusted to 7.4. After three washes in this low Ca2+-containing solution, tissue strips were digested by the addition of collagenase type II (227 U/ml, Worthington Biochemical; Lakewood NJ), elastase (2.4 U/ml, Worthington), and protease type XIV (0.35 U/ml, Sigma; Oakville, Ontario, Canada) to the solution. Enzymatic digestion took place for 30–35 min at 35°C, with manual agitation every 5 min. Tissue strips were then washed five times in a modified Kraftbrühe solution containing (in mM): 100 K+ glutamate, 10 K+ aspartate, 25 KCl, 10 KH2PO4, 2 MgSO4, 20 taurine, 5 creatine base, 0.5 EGTA, 5 HEPES, 20 D-glucose, and 1 mg/ml BSA (pH adjusted to 7.2 with KOH). After this washing procedure, single atrial myocytes were isolated by gentle trituration at room temperature with the use of a fire-polished Pasteur pipette (3 mm inner diameter). Aliquots of this cell suspension were monitored by using a phase-contrast microscope (Zeiss ID 03) as trituration progressed. Trituration was continued until an acceptable yield of single atrial myocytes was achieved, usually within 10 min. The myocytes were then readapted to normal extracellular Ca2+ concentrations by the addition of solution containing 10 mM NaCl and 1.8 mM CaCl2, followed by normal Tyrode's solution containing 1 mg/ml BSA. Cells were then stored in a solution containing (in mM) 100 NaCl, 35 KCl, 1.3 CaCl2, 0.7 MgCl2, 14 K+ glutamate, 2 KH2PO4, 2 taurine, and 1 mg/ml BSA, until used in electrophysiology experiments.
Electrophysiology. For electrophysiological recordings of action potential and K+ currents, an aliquot of cell suspension from either left or right atrium was allowed to settle for 15 min in a 35-mm petri dish that was mounted on the stage of a Nikon Diaphot inverted microscope. Myocyte supensions were then continuously superfused at a flow rate of 1.5 ml/min at room temperature (22°C) with Tyrode's solution of the following composition (in mM): 140 NaCl, 5.4 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 5.5 D-glucose (pH adjusted to 7.4 with NaOH). Patch pipettes were made from borosilicate glass (World Precision Instruments; Sarasota, FL) by using a P-87 Flaming/Brown pipette puller (Sutter Instruments; Novato, CA). These pipettes had resistances between 1.5 and 4 M
when filled with a solution of the following composition (in mM): 130 KCl, 12 NaCl, 1 CaCl2, 1 MgCl2, 4 dipotassium salt of ATP, 10 EGTA, and 10 HEPES. pH was adjusted to 7.2 by the addition of KOH.
Whole cell voltage-clamp and current-clamp experiments were carried out at room temperature (22 ± 1°C) using an Axopatch 200 B patch clamp amplifier interfaced to a Digidata 1322A data-acquisition system that was driven by Clampex version 8.1 software (all from Axon Instruments; Foster City, CA). The acquired data were recorded to the hard drive of a personal computer and stored for post hoc analysis, by using pCLAMP software (Axon Instruments) and Origin 4.1 (Microcal Software; Northampton, MA). The capacitance of each myocyte was measured by integrating the capacitive current evoked during 5 mV depolarizing steps from a holding potential of –75 mV. The mean cell capacitance of left atrial myocytes, 44.2 ± 1.4 pF (n = 55) was significantly (P < 0.001) larger than that of right atrial myocytes, which was 37.5 ± 1.4 pF (n = 59). As a result, current amplitudes recorded in each myocyte were normalized to the cell capacitance and expressed as current density (pA/pF). The input resistance of each myocyte was measured using 5-mV depolarizing steps from –75 mV. Left atrial myocytes had input resistances of 385.7 ± 63 M (n = 10) and right atrial myocytes had input resistances of 412.5 ± 48 M (n = 10). Whole cell experiments proceeded only if the series resistance was <15 M, and data collected from myocytes whose series resistance changed by >20% over the course of an experiment were discarded. Series resistance was compensated electronically by 85%. Voltage errors resulting from uncompensated series resistance were small and no corrections were applied.
The whole cell current-voltage (I-V) relationship was determined by using a protocol of 500-ms voltage steps in 10-mV increments between –115 mV and +45 mV from a holding potential of –75 mV. The same protocol was also applied immediately after a 200-ms prepulse from –75 mV to –35 mV to inactivate Ito and voltage- and time-dependent Na+ current (INa).
A standard two-pulse protocol was used to quantify the voltage-dependence of steady-state inactivation of Ito; both pulses lasted 200 ms. The first (conditioning) pulse was applied from a holding potential of –75 mV to potentials between +35 mV and –105 mV in 10-mV increments. The second (test) pulse was to +35 mV. The amplitudes of the peak outward current and the steady-state current at +35 mV were recorded. The steady-state current was subtracted from the peak outward current to give an estimate of Ito amplitude, and this value was normalized to the amplitude of Ito in response to a step to +35 mV without a conditioning pulse. The mean ± SE normalized data were plotted as a function of the conditioning prepulse potential and were fitted with a Boltzman equation. Nonlinear curve fitting to mean data was carried out by using Clampfit 8.1 software (Axon Instruments) based on the Levenberg-Marquardt method. A two-pulse protocol was also used to quantify the rate of recovery from inactivation of Ito. This protocol consisted of two identical 500-ms voltage steps to +35 mV from a holding potential of –75 mV. The interval between the end of the first protocol and the start of the second (during which time the membrane potential was –80, –75, or –70 mV) varied from 0 to 510 ms in increments of 15 ms. Again, the amplitudes of the peak outward current and of the sustained outward current were measured and used to determine the amplitude of Ito. The amplitudes of Ito recorded in response to the second voltage step were normalized to the amplitude of Ito recorded during the first stimulus. Means ± SE data were plotted against the interval between stimuli and fitted by a single exponential function by using Clampfit version 8.1 software using the Chebyshev transform.
To measure time and voltage-dependent L-type calcium current (ICa), myocyte supensions from either atrium were superfused at a flow rate of 1.5 ml/min at room temperature with Tyrode's solution of the following composition (in mM): 140 tetraethylammonium-Cl, 10 HEPES, 5.5 glucose, 1 MgCl2, 2 CaCl2 (pH adjusted to 7.4 with CsOH). The pipette solution contained (in mM) 135 CsCl, 10 EGTA, 4 ATP-Mg, 6.6 sodium phosphocreatine, 10 HEPES, 0.01 GTP
s, and 1 MgCl2 (pH adjusted to 7.2 with tetraethylammonium-OH). ICa was measured using the following voltage clamp protocol: the membrane potential of myocytes was stepped for 250 ms between –60 and +60 mV in 10-mV increments immediately after a 500-ms prepulse to –45 mV to inactivate INa. ICa was measured as the difference between the current measured at the end of the 250-ms test pulse and the peak inward current.
Action potentials were recorded in response to 300–600 pA depolarizing current pulses lasting 4 ms using the Axopatch 200 B amplifier in current-clamp mode. These stimuli were applied at 1 Hz, and the durations from the peak of the action potential to the 50% and 90% repolarization level (APD50 and APD90) were measured. A total of 60 action potentials were recorded per myocyte, under control conditions and during superfusion of 100 µM 4-aminopyridine (4-AP), and the final 30 action potentials were averaged and analyzed to derive the APD50 and APD90 for that myocyte.
Statistical analysis. Data are presented as means ± SE. For comparison of electrophysiological parameters from left and right atria, unpaired two-tailed Student's t-tests were used. For analysis of the effects of pharmacological agents on K+ currents and APDs, paired two-tailed Student's t-tests were used. In each case, P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Measurements of the ERP at 37°C were also made from the left and right atria of nine hearts. In accordance with the single cell data, the average ERP of the left atrium was significantly (P < 0.05) shorter than that of the right atrium. When measured with the use of an S1 interval of 120 ms, the ERP of left atrium was 16.9 ± 0.9 ms, whereas that of the right atrium was 19.8 ± 1 ms (Fig. 1B).
The finding that the ERPs and APDs of myocytes from the left and right atria differ raises interesting questions regarding the expression levels of repolarizing K+ currents in myocytes isolated from each atrium. Myocytes from both the left and right atria exhibited a range of patterns of K+ current waveforms in response to voltage-clamp depolarizations. The currents shown in Fig. 2, A–F, were recorded in two different myocytes from the right atrium, and illustrate the range of K+ currents that were observed. The waveforms in Fig. 2A represent the interaction of several time- and voltage-dependent K+ currents, including Ito and rapidly activating and slowly inactivating K+ currents (IKUR) (29). However, in many myocytes from both atria, very little or no Ito could be identified (Fig. 2B; see also Fig. 4A). By applying a 200-ms depolarizing prepulse to –35 mV before the test voltage step, the time- and voltage-dependent Na+ current (INa) and Ito could be inactivated. This maneuver revealed the slowly inactivating K+ currents in atrial myocytes (Fig. 2, C and D). Digital subtraction of the currents in Fig. 2C from those in Fig. 2A yielded a measure of Ito in an atrial myocyte (Fig. 2E). As shown Fig. 2D; however, a prepulse to –35 mV caused no change in the K+ currents in this myocyte, i.e., the difference current (Fig. 2F) reveals very little Ito. Inward K+ currents, due to inwardly rectifying K+ channels (IK1; see Fig. 6) were recorded in all atrial myocytes in response to hyperpolarization of the membrane potential negative to –75 mV (Fig. 2, A, B, D, and E).
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Analysis of current waveforms, such as those in Fig. 2, of 55 myocytes from left atria and 59 myocytes from right atria yielded the I-V relations of means ± SE peak currents shown in Fig. 3. Figure 3 compares the I-V relations before (Fig. 3A) and after (Fig. 3B) a prepulse to inactivate Ito for left and right atrial myocytes. Note that the total outward current recorded in left atrial myocytes (including Ito) is significantly larger (P < 0.05) than in right atrial myocytes (Fig. 3A). This difference reached statistical significance at membrane potentials more positive than –15 mV. Importantly, after a prepulse that inactivates Ito, left atrial myocytes still had significantly (P < 0.05) larger outward currents than myocytes from the right atrium (Fig. 3B).
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Xu et al. (48) have reported previously that Ito contributes significantly to action potential repolarization in mouse atrial myocytes. We therefore investigated whether differences in the biophysical properties of Ito in myocytes from left and right atria can account for the observed differences in repolarization between the atria. The distribution of the amplitude of prepulse-sensitive Ito in myocytes from both atria is compared in Fig. 4A, in which peak amplitude of Ito recorded at +45 mV is presented. As shown, many myocytes from both atria have very little Ito. We then measured Ito in myocytes from left (n = 20) and right (n = 25) atria that had this K+ current to determine whether there were differences in the amplitude of this current between these two supraventricular tissues. Figure 4B shows an I-V plot of mean ± SE peak Ito (measured as the prepulse-sensitive difference current) and shows that the density of Ito was the same in left and right atrial myocytes at all membrane potentials in the physiological range. Figure 4C shows raw data from an experiment used to determine the voltage-dependence of steady-state inactivation of Ito. Details of the voltage-clamp protocol are given in MATERIALS AND METHODS. Summary data are presented in Fig. 4D. The mean ± SE data from 10 left atrial myocytes and 12 right atrial myocytes were fitted with a Boltzman relation. No differences in the voltage-dependence of steady-state inactivation of Ito between left and right atrium were observed. The half-inactivation voltage for Ito in myocytes from the left atrium was –60 ± 3.0 mV for left atrial myocytes and –60.5 ± 2.6 mV for right atrial myocytes.
Figure 5 shows experiments carried out to determine the time course of recovery from inactivation of Ito, and to measure its dependence on diastolic membrane potential. Figure 5A presents raw data from an experiment that measured the time course of recovery of Ito at –75 mV. The plots shown in Fig. 5B are single exponential fits for means ± SE. The data were obtained from four right and three left atrial myocytes at –80, –75, and –70 mV. For left atrial myocytes: 
= 32.4 ± 2.0 ms at –80 mV, 41.5 ± 0.8 ms at –75 mV and 63.9 ± 2 ms at –70 mV; and for right atrial myocytes: = 27.5 ± 0.6 ms at –80 mV, 38.7 ± 0.9 ms at –75 mV, and 58.8 ± 2 ms at –70 mV. No differences were detected between the time course of recovery of Ito in left and right atrial myocytes at any holding potential within the normal range of diastolic membrane potentials.
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It is evident from both I-V relations in Fig. 3 that there is a significant (P < 0.05) difference in the amplitude of IK1 recorded negative to –95 mV between the left and right atrium. To further examine the properties of IK1 in the left and right atrial myocytes, 100 µM BaCl2 was applied to block IK1. An example of such an experiment is shown in Fig. 6. Figure 6A shows current traces from a left atrial myocyte. This cell was stepped for 200 ms in 5-mV increments between –115 mV and +45 mV from a holding potential of –75 mV, immediately after a 200-ms step to –35 mV to inactivate Ito and INa. The protocol was then repeated after 5 min of superfusion with Tyrodes solution containing 100 µM BaCl2. As shown in Fig. 6B, the inward current was blocked by BaCl2, whereas the outward K+ current was not changed appreciably. Digital subtraction of the data in Fig. 6B from those in Fig. 6A yielded data shown in Fig. 6C, which are BaCl2-sensitive IK1. I-V relations for IK1 can then be constructed from analysis of such "difference" currents. The mean ± SE I-V data for 15 right and 17 left atrial myocytes in Fig. 6D show that the amplitude of IK1 was significantly (P < 0.05) larger not only in the negative to diastolic range of potentials but also in the region of negative slope on the I-V relation.
Because neither the peak amplitude nor the inactivation/reactivation properties of Ito contributed to the differences in outward current density between left and right atrial myocytes, pharmacological agents were used to examine whether a slowly inactivating K+ current might be responsible. E-4031 at a concentration of 5 µM is a potent inhibitor of the rapid delayed rectifier K+ current (IKr) in mouse heart (26). Seven myocytes from right atrium and six myocytes from left atrium were examined, and no effect of superfusion of E-4031 on K+ currents was observed (data not shown). Specifically, superfusion of myocytes with 5 µM E-4031 had no effect on the currents recorded in response to voltage steps from a holding potential of –60 mV to –20 and +50 mV, a voltage protocol that reveals IKr in adult mouse sinoatrial node cells (26).
Mouse ventricular myocytes have a rapidly activating sustained K+ current that is sensitive to low (micromolar) concentrations of 4-AP (10, 22). This current, whose -subunit is encoded by the Kv1.5 gene, has also been identified in the mouse atrium and is thought to contribute to the rapidly activating, slowly inactivating K+ current (IKUR) in this tissue (4). Accordingly, we examined whether there was a difference in the amplitude of sustained K+ current that could be inhibited by 100 µM 4-AP between the left and right atrium. Figure 7 shows K+ currents from a right atrial myocyte recorded before (Fig. 7A) and during (Fig. 7B) superfusion of 100 µM 4-AP, and illustrates the effect of 4-AP on the outward current. The voltage-clamp protocol used was the same as that illustrated in Fig. 2, C and D, where INa and Ito are inactivated by a prepulse to –35 mV. Note that 100 µM 4-AP caused a significant decrease in the peak amplitude of outward K+ current. Furthermore, the slowly inactivating component of outward K+ current was abolished by 100 µM 4-AP. Figure 7C shows the reduction of maximum outward current density at +45 mV during superfusion of a left atrial myocyte with 100 µM 4-AP, and demonstrates that the current density returns toward control levels after washout of 4-AP.
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Data from experiments on 13 myocytes from the left atrium and 13 from the right atrium are summarized in Fig. 7D. The mean ± SE maximum outward current densities of left and right atrial myocytes at +45 mV are presented, as are the current densities following superfusion with 4-AP, along with the "difference current," which corresponds to the density of the 4-AP-sensitive K+ current. Several features of these data are noteworthy. First, as described earlier, the mean outward current density of left atrial myocytes is significantly greater than that of right atrial myocytes. Second, superfusion of 100 µM 4-AP causes a significant decrease in current density in both left and right atrial myocytes. Third, the amplitude of the 4-AP-sensitive component is substantially (almost x3) larger in left atrial myocytes than right atrial myocytes. Finally, the amplitude of the 4-AP-resistant current is significantly larger in left than right atrial myocytes.
In the next series of experiments, current-clamp recordings were made in an attempt to determine the role of the K+ current, which was inhibited by 100 µM 4-AP in repolarization of mouse atrial action potentials. Figure 8, A and B, show examples of action potentials recorded at 1 Hz from single atrial myocytes from left (Fig. 8A) and right (Fig. 8B) atria at room temperature before and during superfusion with 100 µM 4-AP. It is evident that superfusion with 4-AP caused a larger action potential prolongation in the myocyte from left atrium. This experiment was repeated in a further five myocytes from each atria, and means ± SE summary data are presented in Fig. 8C. Superfusion with 4-AP significantly (P < 0.05) lengthened the APD50 and APD90 of myocytes from both left and right atria. However, the mean action potential lengthening was significantly larger in left than right atrial myocytes at both the 50% and 90% repolarization levels.
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Although a major goal of the present study was to determine whether a gradient of time- and voltage-dependent K+ current results in a gradient of repolarization across adult mouse atria, we also examined the possible contribution of ICa to differences in refractoriness between the left and right atrium. We recorded ICa from 11 left atrial myocytes and 10 right atrial myocytes. The means ± SE I-V relations of ICa of left and right atrial myocytes are presented in Fig. 9. There was no difference in ICa amplitude between left and right atrium, which suggests ICa plays no role in the differences in refractoriness between the left and right atrium of adult mice.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Li et al. (20) have described differences in ERP and APD between the left and right atrium of the dog. The ERP and APD were significantly shorter in canine left atrium, and a gradient of decreasing rapidly activating, inwardly rectifying delayed rectifier K+ current (IKr) from the left to right atrium was found to underlie this difference. In mammalian hearts, IKr is encoded by the combination of members of the ERG gene family with min K-related peptides (32). We never observed a measurable IKr in mouse atrium, as judged by the absence of K+ currents that were sensitive to 5 µM E-4031. Our findings are in agreement with previous developmental studies, which concluded that mouse ERG does not contribute repolarizing current in working myocardium in adult mice (23, 44). However, E-4031-sensitive IKr has recently been identified in adult mouse sino-atrial node (26).
K+ currents in adult mouse atrium. The most prominent difference in the expression of K+ current between left and right atria of adult mice was due to a rapidly activating K+ current that was blocked by 100 µM 4-AP (10). This current, sometimes denoted IKUR, was larger in left atrial myocytes, and superfusion of myocytes with 100 µM 4-AP caused a significantly larger action potential lengthening in left atrial myocytes, resulting in substantially reduced differences in APD between the left and right atria. It is likely that in mice, this 4-AP-sensitive current is generated by K+ channels, whose pore-forming -subunits are encoded by Kv1.5 (22, 51). 4-AP, at the concentration used in this study, also blocks Kv1.1 and Kv3.1 K+ channels (12). However, neither of these channel subunits contributes to repolarizing K+ current in mouse atrium (4).
Previous electrophysiological studies (19, 20, 33, 34, 46, 47) on other mammals, including dogs, rabbits, pigs, and goats, have revealed that the left atrium repolarizes more quickly than the right atrium. However, of the previous studies that have identified differences in refractoriness between left and right atrium, only Li et al. (20) addressed the ionic mechanism underlying the differences. As mentioned earlier, a gradient of IKr was identified as the cause of the repolarization difference in dog (20). We identified a different cause of the repolarization gradient in mice, but it remains to be determined whether the gradient of IKUR identified in the present work underlies the repolarization differences between left and right atrium observed in other mammals.
We also observed a significant difference between left and right atrium in the amplitude of a sustained (noninactivating) K+ current that was not inhibited by 100 µM 4-AP. Kv2.1 encodes a plausible candidate (K+ channel) for this current. Bou-Abboud et al. (4) used an antisense oligodeoxynucleotide strategy to downregulate Kv2.1 -subunits and thus implicate this transcript in generating the slowly inactivating K+ current in mouse atrium, because at present there are no selective pharmacological blockers for this current. Other families of K+ channels may also be functionally expressed in mouse atrium. Some recent studies (29, 39) have identified mRNA for TWIK-related K+ channel (TREK-1)-like K+ channels in mouse and rat atrium, and shown that TREK-1 encodes a time-independent K+ current in rat atrial myocytes. We did not study the possibility that there is functional TREK-1 K+ channel expression in mouse atrium.
Calcium-independent Ito has been recorded in working myocardium in all mammalian species examined. Transgenic mice that express a mutant Kv4 -subunit were examined to determine whether -subunits of the Kv4 family are responsible for the expression of Ito in the mouse atrium (48). Ito in the mouse atrium was abolished in mice expressing mutant Kv4 -subunits. Our findings on the biophysical properties of Ito are in agreement with those of Xu et al. (48) in terms of both current density and recovery kinetics. We recorded Ito currents that have a rapid (>200 ms) recovery from inactivation between –70 and –80 mV. This is characteristic of K+ currents encoded by members of the Kv4 family. However, our measurements of the voltage at which current inactivation reached 50% (V1/2) of steady-state inactivation of Ito are 20 mV more negative than previous reports of Ito in the mouse [e.g., (13)]. This large apparent discrepancy is most likely caused by the presence of 5 mM CoCl2 in the superfusate used by Guo et al. (13). At this concentration, CoCl2 can cause depolarizing shifts of 20 mV in the V1/2 of the steady-state inactivation curve of Ito (1).
A consistent feature of our K+ current measurements from atrial myocytes is that there was considerable cell-to-cell variability of current density of Ito within both atria. Previous descriptions of Ito in mouse atrium (48) did not draw attention to this. Regional heterogeneity of Ito amplitude within left and right atria has been demonstrated in guinea pig (45), rabbit (33, 49), and dog (9). We suggest that regional heterogeneity of Ito expression within adult mouse atria may account for the range of Ito amplitudes in our data. However, the small size and thin chamber wall of the atrial myocardium of adult mice precluded any systematic correlation of K+ current expression with location of single myocytes within atria.
IK1, which in mice is produced by channels encoded by the gene Kir2.1 and to a lesser extent Kir2.2 (50), contributes to the late phase of repolarization and is very important for maintenance of diastolic membrane potential in working cardiac myocardium (24, 37). In most mammalian species, IK1 is substantially higher in ventricle than in the atrium (36). The present study, when compared with the measurements of IK1 in mouse ventricle by Trepanier-Boulay et al. (40) suggest that there is little, if any, difference in current density between ventricle and atrium in adult mice. We measured the current density of IK1 as the current inhibited by 100 µM BaCl2, and consistently found that the current density of IK1 in substantially larger in myocytes from left than right atria. The gradient of IK1 amplitude between left and right atria of mice may be expected to have important consequences for late repolarization. However, as mentioned earlier, there was no significant difference in maximum diastolic potential between myocytes from left and right atrium. This finding can be explained by the distinct nonlinearity of the ion transfer characteristics of IK1 (Fig. 6), i.e., that the difference in current density of outward IK1 is very small.
Atrial repolarization and arrhythmogenesis. Our results show that the left atrium of the mouse has a shorter ERP than the right atrium. Previous electrophysiological studies (19, 20, 33, 34, 46, 47) on many other mammals, including dogs, rabbits, pigs, and goats, have revealed this same trend. Thus it appears that in several mammalian species the left atrium, which is depolarized after the right atrium, repolarizes more quickly than the right atrium. In this way, action potential repolarization can be more closely synchronized between atria and normal rightto left-atrial conduction can be maintained.
A classic theory on the generation and maintenance of reentrant arrhythmias in mammalian atria stems from the experimental work of Allessie and colleagues (2, 3, 35), who made use of the concept of the wavelength of reentry in describing and interpreting their findings. According to this hypothesis, for reentry to occur in a defined substrate, the time that it takes a conducted action potential to reach the site of its initiation must exceed the ERP of the initiating site. This implies that the wavelength of reentry will become shorter with decreasing ERP; thus, for two myocardial tissue masses of equal size and conduction velocities, the tissue with a shorter ERP will be more susceptible to reentry. Many clinical studies have been published within the past 5 years that suggest that the left atrium, in particular the myocardium surrounding the pulmonary veins, is capable of generating ectopic electrical activity (14, 31). The ectopic activity generated by pulmonary vein myocytes (6, 25), in addition to decreased refractoriness caused by the larger K+ currents in the left atrium, may render the left atrium more susceptible to reentry, which could then be converted into sustained reentrant arrhythmia (46) that may initiate the chain of events leading to atrial fibrillation (28). In this context, it is noteworthy that the expression level of Kv1.5 and the density of the corresponding K+ current IKUR are reduced in patients with chronic atrial fibrillation (42).
Over the past decade, there has been a resurgence of interest in mouse cardiovascular physiology. The single most important advantage that the mouse has over many species is the ease with which transgenic animals can be generated as models of human disease. One disadvantage of using mice is that the duration of atrial action potentials in mice is much shorter than in humans. Also, atrial anatomy plays a significant role in arrhythmogenesis, and the difference in the size and structure of the atria of mice versus humans is a drawback of using mouse models. On the other hand, the repolarizing currents in mouse and human are similar (4, 30, 48). Indeed, a recent study on the atria of pigs, which are thought to be the most suitable animal model of human atrial electrophysiology, reported significantly larger effects of blockade of IKUR on the ERP and vulnerability to reentrant arrhythmias of the left versus right atria (17). This is the same current found to underlie the left-right atrial repolarization gradient in the present study and indicates that studies of mice may yield valuable insight into atrial cellular electrophysiology.
In summary, the present study is the first to demonstrate regional electrical heterogeneity at the single cell level in the atria of rodents. These differences in repolarizing K+ currents within atria and between left and right atria of adult mice can explain the differences in APD and refractoriness, and may provide insight into the cellular electrophysiological substrate for reentrant arrhythmias in the mouse.
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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