Electrophysiological consequences of human I_Ks channel expression in adult murine heart

¹ Departments of Pharmacology and ² Medicine and ³ Center for Molecular Therapeutics, Columbia University, College of Physicians and Surgeons, New York, New York 10032

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We expressed human delayed rectifier K⁺ cardiac current (I_Ks) channels in the murine heart, which lacks native I_Ks, to determine their electrophysiological role. Mice expressing human I_Ks channels were anesthetized, and an electrocardiogram and monophasic action potentials (MAP) recorded from the left ventricle. Sinus rate was not different between wild-type mice (WT) and transgenic mice (TG). Infusion of isoproterenol accelerated WT heart rate but not TG. Lack of TG sinus rate responsiveness may have resulted from accumulated outward current in I_Ks channels in sinus node. Ventricular MAP duration of TG mice to 50% repolarization (APD₅₀) during ventricular pacing was shorter than WT, likely resulting from outward current through I_Ks channels. TG APD₅₀ showed enhanced responsiveness (shortening) to isoproterenol compared with WT. Ventricular tachyarrhythmias were initiated in TG mice by programmed stimulation but not in WT and were accelerated by isoproterenol. I_Ks channels impart -adrenergic sensitivity to the ventricles and may be responsible for ventricular tachyarrhythmias.

arrhythmia; -adrenergic; repolarization; heart rate; delayed rectifier K⁺ cardiac current

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE HUMAN delayed rectifier K⁺ cardiac current (I_Ks) channel hKCNQ1-hKCNE1 is the slowly activating and slowly deactivating component of the delayed rectifier K⁺ channel current (33, 40), which serves as the dominant repolarizing current during periods of increased heart rates (35, 43). Mutations mapped to the hKCNQ1 gene locus on chromosome 11 and the hKCNE1 locus on chromosome 21 have been implicated in causation of two variants of the long QT syndrome: LQT₁ and LQT₅ (3, 30, 34, 37). -Adrenergic regulation of the I_Ks channel is of particular importance because the fatal cardiac arrhythmias that occur in LQT₁ are precipitated by increased sympathetic nervous system activity (37, 38).

Transgenic (TG) mice offer a unique opportunity to study the physiological properties of ion channels. In mice, compared with humans, I_Ks channel expression peaks during the first week of life and then is downregulated rapidly, such that 7 days after birth, channel activity is recorded in only 10% of isolated cardiomyocytes (14). I_to emerges as the primary repolarizing (transient outward current) of the murine heart (31, 47). Therefore, insertion of the human I_Ks gene into the mouse genome provides a model in which the properties of this channel can be investigated without contamination by native current. Marx et al. (28) created such a TG mouse by expressing the human hKCNQ1-hKCNE1 channel in myocardial cells under control of the -myosin heavy chain promoter fragment for cardiac-specific expression. Although in this TG model the human I_Ks channel has been characterized in single myocytes with the use of patch-clamp techniques (28), its influence on the electrophysiological properties of the heart in vivo has not been previously determined.

Therefore, the purpose of our study was to use this mouse model to electrophysiologically characterize the effects of inserting the human I_Ks channel on ventricular repolarization, -adrenergic responsiveness, and arrhythmogenesis in vivo.

	MATERIALS AND METHODS

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TG Mouse Model

Electrophysiological studies were done in a TG mouse expressing an hKCNQ1-hKCNE1 fusion protein in the heart. As previously described by Marx et al. (28), HKCNQ1-hKCNE1 (2.6 kb) cDNA under the control of the -myosin heavy chain promoter for cardiac-specific expression was injected into pronuclei of fertilized mouse eggs. The TG mice genotyping used in this study was done by PCR to confirm gene expression. Functional insertion of the gene was also tested in some of the littermates of animals studied in this report, by patch-clamp analysis of membrane currents in isolated ventricular myocytes (see Fig. 3 in Ref. 28).

Electrophysiological Study

Animal preparation. Studies were performed in vivo on both male and female adult wild-type (WT) and TG mice (strain B6/cBAF1, 20-47 g wt). Surgical-depth anesthesia was achieved with a 50 mg/kg intraperitoneal injection of pentobarbital sodium, and supplemental doses were administered throughout the experiment when needed. A 24-gauge pediatric catheter with injection hub was inserted into the jugular vein for drug infusions. The trachea was intubated with a 20-gauge polyethylene catheter. Positive pressure ventilation was maintained at a tidal volume of 0.5 ml and a rate of 135 breaths/min (Harvard Apparatus Rodent Respirator, model 687). A rectal temperature probe was inserted, and a heating lamp was used to maintain core body temperature between 35° and 37°C.

Subcutaneous 22-gauge needles were placed in the four limbs to obtain ECG recordings. Before surgery, a six-lead ECG (I, II, III, aVR, aVL, and aVF) was observed on the screen of a recorder (model VR12, Electronics for Medicine; Pleasantville, NY). A midline sternotomy was then performed with fine scissors. Each chest flap was retracted and held with the use of a 4-0 silk tie secured to a metal post. With the heart fully exposed, the pericardium was removed with the use of fine forceps. An optimal ECG lead was selected by visual inspection (clear P and QRS deflections with minimal noise) for continuous recording during the experimental protocol.

Monophasic action potential recording. We used monophasic action potential (MAP) recordings to quantify ventricular repolarization in this study because the MAP has been shown to be a sensitive and accurate measurement (16). MAPs were recorded from the anterior surface of the left ventricle with a modified Franz MAP electrode (11, 16) composed of a silver-silver chloride wire. The tip of the wire in contact with the heart had a diameter of 0.25 mm, a size shown to accurately record the time course of repolarization in the murine heart (24). The electrode was attached to a micromanipulator (Stereotactic Instrument model 4, Lab-Tronics; Chicago, IL) that was used to position the electrode tip on the anterior left ventricular surface. The ground electrode, which was several millimeters above the MAP electrode tip, contacted a blood/tissue interface above the ventricle (usually the chest wall or the diaphragm). The MAP signals were led into a direct current-coupled preamplifier (model 111101, EP Technologies; Mountain View, CA). The MAP signals were digitized, and recorded along with the ECG, on a Windaq Waveform Browser (DATAQ Instruments; Akron, OH). A MAP signal was considered acceptable if the sequence of depolarization and repolarization remained uniform in shape and amplitude, with minimal baseline motion. Deterioration of the signal (progressive decrease in amplitude) necessitated slight repositioning of the electrode on the left ventricular surface and subsequent recalibration of the signal. Once in place, unless the signal deteriorated, the MAP electrode remained in position and continuously recorded throughout the duration of the experiment (generally 60 min).

Stimulation of ventricles. A second micromanipulator was used to position a bipolar stimulating electrode on the left ventricular surface, adjacent to the MAP recording electrode. The ventricular effective refractory period (VERP) was determined by pacing the ventricle at basic cycle lengths (BCL) of 100 and 70 ms and interpolating a single premature stimulus every 10 beats. Basic and premature stimulus parameters were 2× diastolic threshold and 1-ms duration. The premature impulse was decremented in 5-ms steps to a coupling interval of 55 ms and then by 2-ms steps. The VERP was defined as the longest coupling interval of a premature stimulus that did not elicit a propagated response, as seen on the MAP and ECG. To determine rate responsiveness of MAP repolarization, the ventricles were paced with 20-beat stimulus trains from 100- to 60-ms cycle lengths in 10-ms decrements; thereafter, the train was decremented by 5 ms until there was failure of 1:1 capture. Overall, for each animal, at least two pacing trials were done. Ventricular tachycardia (VT) sometimes occurred after ventricular stimulation. Reproducibly induced VT is defined in this study as two consecutive inductions with at least four consecutive beats of ventricular origin by the same stimulus protocol (i.e., either programmed stimulation or pacing at a constant cycle length).

Isoproterenol infusion. An isoproterenol solution (0.0002 mg/ml) was prepared in normal saline. Isoproterenol was administered via a jugular vein catheter at rates of 50, 125, and 200 ng · kg¹ · min¹ with the use of an intravenous infusion pump (model 341A, Orion Research Syringe Pump, Sage Instruments; Cambridge, MA). The maximum total volume of infusion was 0.75 ml. In preliminary experiments, we determined that a minimum infusion time of 8 min was required to achieve a steady-state effect of isoproterenol on the heart rate in WT animals. We therefore used an 8-min infusion of isoproterenol at each dose before conducting ventricular stimulation protocols.

Data Analysis

Sinus cycle length (the average of 10 R-R intervals on the ECG), VERP, the MAP duration to 50% repolarization (APD₅₀) and 90% repolarization (APD₉₀), and the inducibility of VT were determined for control and after an 8-min infusion of isoproterenol at each dose. The APD₅₀ was measured from the fastest part of the MAP upstroke to 50% repolarization. This represents 50% of the distance between the crest of the MAP (maximum level of depolarization after termination of the upstroke) and the diastolic baseline (16). The APD₉₀ was measured from the fastest part of the MAP upstroke to 90% repolarization. This represents 90% of the distance between the crest of the MAP and the diastolic baseline (16). The mean of five randomly selected APD₅₀ and APD₉₀ values was calculated at each BCL.

Statistical analysis was performed with Microsoft Office Excel and SYSTAT version 9 data-analysis software. Descriptive statistics generated means ± SD for measurements as noted. ANOVA with repeated measures was utilized for comparisons between WT and TG groups, and within each group.

	RESULTS

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Heart Rate

Both resting heart rate (sinus cycle length) and heart rate in response to increasing doses of isoproterenol were compared between WT and TG mice (Fig. 1). In the absence of isoproterenol there was no significant difference in the mean resting sinus cycle length between the two groups (P > 0.05). In the presence of isoproterenol there was a significant decrease in the sinus cycle length of WT mice (P < 0.01) but no significant change in the sinus cycle length of TG mice (P > 0.05) (Fig. 1). As a result, at isoproterenol doses of 125 and 200 ng · kg¹ · min¹, the sinus cycle length of WT mice was significantly less than that of TG mice (P < 0.01).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Effects of isoproterenol (Iso) on sinus cycle length (SCL) in five wild-type (WT) and five transgenic (TG) mice. *P < 0.01, statistically significant difference between the two groups. There was a significant decrease in SCL in WT mice after Iso (P < 0.01) but not TG mice (see text).

MAPs and VERP

A stable MAP was recorded in each experiment. MAP amplitudes ranged from 3.3 to 18.4 mV, with a mean of 7.7 ± 1.8 mV. There was no difference in amplitude between WT and TG, so all data for amplitude were pooled. Normalized MAP recordings from a WT and a TG mouse are shown in Fig. 2. The MAP recordings in both the WT and the TG mouse are characterized by a rapid depolarization spike (phase 0) and an initial rapid phase of repolarization (phase 1) that merges with a slow terminal repolarization phase (phase 3). There is little or no plateau phase (phase 2). The time course of repolarization (APD₅₀ and APD₉₀) for each MAP recording is marked, as described in MATERIALS AND METHODS. The APD₅₀ in the TG MAP recording is visually shorter than APD₅₀ in the WT MAP recording, while APD₉₀ for both TG and WT is nearly coincident.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Normalized monophasic action potential (MAP) recordings from WT and TG mice during ventricular pacing at basic pacing cycle length (BCL) = 100 ms. APD50 and APD90, action potential duration at 50% and 90% repolarization, respectively.

The time course of MAP repolarization (APD₅₀ and APD₉₀) during ventricular pacing in WT and TG mice is shown in Fig. 3. Rate-dependent shortening of the APD₅₀ occurred in both WT and TG animals over the range of stimulation cycle lengths from 100 to 60 ms (Fig. 3A) (P < 0.01) but is not demonstrated for APD₉₀ (Fig. 3B) (P > 0.05). By ANOVA with repeated measures, the APD₅₀ values for TG mice are significantly shorter than the corresponding APD₅₀ values in WT at all cycle lengths (Fig. 3; note asterisks). However, although APD₉₀ appears to be shorter in TG mice, the difference between TG and WT did not reach statistical significance at any cycle length (Fig. 3B) (P > 0.05).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Time course of repolarization in five WT and five TG mice. A: APD50, BCL of ventricular pacing from 100 to 60 ms. B: APD90, BCL of ventricular pacing from 100 to 60 ms. *P < 0.01, statistically significant difference between WT and TG mice.

VERP was determined at BCLs of 100 and 70 ms. An example of VERP determination is shown in Fig. 4. A comparison of VERP between WT and TG mice is shown in Fig. 5. The mean VERP of TG mice at a BCL of 100 ms (28.3 ± 4.2 ms) and at 70 ms (29.4 ± 4.7 ms) is significantly shorter than that of WT mice (48.5 ± 14.9 ms and 40.0 ± 7.2 ms, respectively) (P < 0.05). Neither WT nor TG mice demonstrate significant rate-dependent shortening of the VERP (P > 0.05 for WT and TG mice).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   ECG and MAP recordings from a TG mouse during ventricular effective refractory period (VERP) determination. A: programmed electrical stimulation (PES) with BCL = 70 ms (S1-S1) and premature coupling interval of 34 ms (S2) results in a propagated electrical response (arrow). B: PES with same BCL = 70 ms but premature coupling interval of 32 ms (S2) results in no propagated electrical response (arrow).

View larger version (12K): [in this window] [in a new window]   Fig. 5.   VERP of five WT and five TG mice. BCL = 100 and 70 ms. *P < 0.01, statistically significant difference between WT and TG mice.

Effects of isoproterenol on MAP and VERP. The time course of MAP repolarization (APD₅₀ and APD₉₀) for WT and TG mice was compared in the presence of increasing doses of isoproterenol at BCL of 80, 70, and 60 ms, respectively. Results for APD₅₀ and APD₉₀ at a BCL of 80 ms are shown in Fig. 6; similar results were obtained during ventricular pacing at 70 and 60 ms (data not shown). Isoproterenol at 50 ng · kg¹ · min¹ significantly shortened the APD₅₀ values measured for TG mice when compared with control (P = 0.05); increasing the dose to 125 or 200 ng · kg¹ · min¹ caused no further significant shortening of APD₅₀ (Fig. 6A) (P > 0.05). Isoproterenol had no effect on APD₅₀ of WT mice. At each dose of isoproterenol (50, 125, and 200 ng · kg¹ · min¹), APD₅₀ in TG mice was significantly shorter than the corresponding APD₅₀ in WT mice (Fig. 6A) (P < 0.01). There was no significant difference in APD₉₀ between TG and WT mice in control (Fig. 6B) (P > 0.05). Isoproterenol had no significant effect on APD₉₀ in WT or TG mice at any dose or cycle length (Fig. 6B) (P > 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effects of Iso on time course of repolarization in five WT and five TG mice during ventricular pacing at BCL = 80 ms. A: APD50; B: APD90. *P < 0.01, statistically significant difference between WT and TG mice. P = 0.05, statistically significant difference between control and Iso at 50 ng · kg1 · min1 in TG mice.

Figure 7 shows the comparison of VERP between WT and TG mice at a pacing cycle length of 70 ms. Isoproterenol at 125 ng · kg¹ · min¹ significantly shortened the VERP of TG mice (P < 0.05), with no further shortening of the VERP at 200 ng · kg¹ · min¹ (P > 0.05). In WT mice, shortening of VERP only reached significance at the highest dose of 200 ng · kg¹ · min¹ (P < 0.05). Although the VERP of TG mice was significantly less than WT mice in control, there were no statistically significant differences in VERP between WT and TG mice at any dose of isoproterenol (P > 0.05).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Effects of Iso on VERP of five WT and five TG mice. VERP is at a BCL of 70 ms. *P < 0.05, statistically significant difference between WT and TG mice in control only. P < 0.05, Statistically significant difference between control and Iso at 125 ng · kg1 · min1 in TG mice. **P < 0.05, statistically significant difference between control and Iso at 200 ng · kg1 · min1 in WT mice.

Ventricular Tachyarrhythmias

No episodes of spontaneous VT were seen in either WT or TG mice. VT was induced by rapid pacing or premature stimulation in four of the five TG mice, but was not induced in any of five WT mice. All induced arrhythmias self terminated. In two TG mice, VT was induced by premature stimuli with coupling intervals of 36 and 24 ms (BCL = 100 ms). Such short coupling intervals were not attained in the WT mice because they are less than the WT VERP. In two TG mice, tachycardia was induced during the first 10 impulses of pacing at cycle lengths of 28-36 ms when there was capture for each beat. In WT, 1:1 capture could not be established at cycle lengths <45 ms. Figure 8A demonstrates the inducibility of VT in a TG mouse under control conditions by a premature stimulus at a BCL of 100 ms. Isoproterenol increased the inducibility of VT in four of five TG mice (extended the range of premature or BCL at which VT was induced) but did not lead to inducible VT in WT. Increasing the dose of isoproterenol to 200 ng · kg¹ · min¹ in TG mice resulted in a significant shortening of the mean cycle length of induced VT (P < 0.01) (Fig. 8B). The duration of VT was not significantly affected (P > 0.05).

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Initiation of ventricular tachycardia in a TG mouse. A: ECG recording of ventricular tachycardia with a cycle length (CL) of 36 ms after PES. BCL of ventricular pacing (S1-S1) = 100 ms, with premature coupling interval (S2) = 36 ms. B: ECG of same mouse during infusion of Iso. Ventricular tachycardia CL = 26 ms, initiated with PES, S1-S1 = 100 ms, S2 = 26 ms.

	DISCUSSION

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TG Mouse Model Expressing Human I_Ks

The delayed rectifier K⁺ channels I_Kr and I_Ks play a significant role in repolarization of myocardial cells in several mammalian species (35, 49), including humans (10, 42). In the adult murine heart, I_Ks is of minor significance, being downregulated in the neonatal period to very low levels by 1 wk of age (31). The scarcity of native I_Ks facilitates the study of human I_Ks channel function by the insertion of genes controlling its expression into the murine genome. In our TG model, the introduction of human I_Ks channels into murine ventricular muscle cells results in an outward rectifying current having two components: a rapidly activating/inactivating early component (native I_to), followed by a second, slowly activating current that is prolonged, resulting in tail currents slow to extinguish (human I_Ks) (28). We determined how this electrical remodeling of ventricular cells influences the electrophysiological properties of the in situ ventricles, providing information on the functional role of the human I_Ks channel. We found that insertion of this channel into the murine heart had several notable effects including: 1) blunted sinus node responsiveness to -adrenergic stimulation, 2) accelerated early repolarization (decreased APD₅₀) and shortened VERP, 3) increased responsiveness of the ventricle to isoproterenol, and 4) increased propensity for induced ventricular arrhythmias. Although we propose that these effects are likely the results of I_Ks channel expression, the possibility of compensatory changes in other ion channels cannot be ruled out as contributory as discussed below (4, 21).

Blunted Sinus Node Responsiveness to -Adrenergic Stimulation

It is possible that the blunted response of the sinus node to isoproterenol (see Fig. 1) is a direct result of expression of I_Ks channels in sinus node pacemaker cells in this TG model, although nodal localization of I_Ks has not been looked for as yet. The I_f pacemaker current has been detected in mouse sinus node (27) and it has been suggested that the electrophysiological properties of the mouse sinus node is similar to other mammalian sinus nodes (32, 50). I_K has been consistently observed in isolated sinus pacemaker cells from many mammalian species (5, 8), with biexponential deactivation kinetics of I_K tail currents. Whether this behavior represents two distinct potassium conductance pathways is unclear (8). However, we are not aware of data on I_K in mouse sinus node. It appears that at rates native to the mouse heart (~600 beats/min, also the rate of the anesthetized mice in this study), insertion of the human I_Ks channel, if it indeed occurs in sinus node, does little to alter the ionic balance of the currents that control heart rate, thus no difference between WT and TG sinus cycle lengths. With the introduction of isoproterenol, sinus cycle length shortened in WT mice, likely the result of an increase in I_f current through stimulation of adenyl cyclase activity and cAMP production (13, 48). In the TG animals, -adrenergic stimulation did not decrease cycle length. A direct consequence of -adrenergic stimulation of I_Ks is increased I_Ks activation and slowing of channel deactivation during diastole (44, 45). If this occurs in the sinus node, this altered channel deactivation might contribute to a reduction in pacemaker slope that would offset the expected increase in heart rate.

APD₅₀ and VERP

Our study was focused on defining the effects of the human I_Ks channel in the ventricles by expressing it in an environment where native channels have only a minor physiological role. We found that early repolarization as reflected in the APD₅₀ of the MAP was significantly shorter in TG versus WT mice. Although the APD₉₀ was also shorter in TG mice, it did not reach statistical significance. These changes were small compared with the expected role of this channel in the human ventricle and may be a consequence of the already accelerated time course of repolarization in the mouse. Previous studies (49) have suggested that in mammalian ventricles that normally express I_Ks as a component of I_K, I_Ks provides the major repolarizing current during the plateau phase of the action potential. Thus addition of this channel to murine ventricular muscle cells increases the net flow of outward repolarizing current, resulting in a more rapid repolarization and shortening of APD₅₀. Although I_Ks might not be significantly activated at the short times for depolarization for each mouse ventricular action potential, owing to very slow deactivation, at the rapid heart rates activation would be expected to be cumulative. The kinetics of the I_Ks channel opening and inactivation would make this change more notable at APD₅₀, but might be diminished or lost at potentials closer to resting potential (near APD₉₀) where the driving force for I_Ks current is low and I_K1 may predominate (49). Additionally, given the triangulated nature of the mouse action potential, coupled to the rapid heart rate, changes that occur late in the repolarization process are difficult to resolve with great accuracy (2) and may have contributed to our failure to see any significant differences in APD₉₀ between the two groups. Because the VERP is primarily voltage dependent in the murine ventricles (11, 24), it is shorter as well in TG versus WT, reflecting a decrease in APD.

Despite studies showing little or no I_Ks in adult murine ventricle (31), others have shown prolonged QT interval with functional knockout of the KCNE1 gene encoding the minK portion of the I_Ks channel (14), functional suppression of the KCNQ1 gene (12), and targeted disruption of the KCNQ1 gene (9). The possibility exists that such changes may result from decrease in function of residual I_Ks channels or to compensatory changes in other membrane currents.

In our study, rate-dependent shortening of APD₅₀ was present in both the WT and TG mice. The mechanism for rate-dependent shortening of APD in many mammalian species may involve activation of I_Ks channels at rapid heart rates and slow deactivation leading to accumulation (43). Other ion channels also play a role (6, 17, 25, 49). The mechanism for the rate-dependent shortening of APD in murine ventricle may be a result of residual I_Ks and/or rate effects on other repolarizing currents. The addition of the I_Ks channel did not alter the rate responsiveness. It is possible that at the rapid control rate of the murine heart, these channels are already maximally activated, precluding an increase in I_Ks current when we increased the rate by pacing.

Effects of -Adrenergic Stimulation on Ventricles

The sympathetic nervous system has been shown to regulate delayed-rectifier channel activity (15). Previous studies (23, 36) in mammalian cells and tissues have shown that this regulation is most marked for the slow component I_Ks, rather than the fast-inactivating component I_Kr. Stimulation with a -agonist results in more rapid channel opening, and slowing of channel deactivation resulting in accumulation of open channels (28, 44, 45). Our study provides further evidence suggesting that -adrenergic effects on ventricular repolarization is dependent on I_Ks because insertion of these channels resulted in enhanced responsiveness to isoproterenol, shortening APD₅₀ at the lowest dose studied (50 ng · kg¹ · min¹). In this TG model, Marx et al. (28) have shown that the -adrenergic modulation of I_Ks requires targeting of cAMP-dependent protein kinase and protein phosphatase 1 to hKCNQ1 through the targeting protein yotaio. However, we cannot rule out the effects of isoproterenol on other membrane channels as contributing to the effects that we observed. For example, -adrenergic stimulation increases L-type calcium current (33), which in and of itself might prolong APD. However, the resultant increase in intracellular calcium might increase a background cAMP activated chloride current (19) or residual native I_Ks current (41). Future experiments using appropriate pharmacological blocking drugs might be able to provide further evidence for the role of the inserted I_Ks channels compared with other membrane channels. The VERP of WT mice was shortened only at the highest dose of isoproterenol that we studied (200 ng · kg¹ · min¹). This may have resulted from -adrenergic stimulation of residual I_Ks channels or other repolarizing currents that might be -adrenergic sensitive, as described above.

Arrhythmogenicity

We postulate that the enhanced susceptibility to ventricular arrhythmias in TG mice was related to the presence of the I_Ks channels. It is likely that these arrhythmias resulted from reentrant activity because they only occurred after electrical stimulation (22). In addition, increasing outward current through I_Ks would be expected to oppose automatic or triggered mechanisms of arrhythmias (46). It is speculative as to which arrhythrogenic mechanisms resulted from insertion of I_Ks into the ventricles in our experiments. It enabled impulses to be stimulated at shorter coupling intervals because of the shortened refractory period. Because there is dispersion in repolarization in the normal murine ventricle (24), shorter cycle lengths may have caused block in some regions and not in others because of the natural dispersion, leading to reentry. In addition, in mammalian ventricles in which I_Ks is naturally expressed, nonuniformity in expression has been identified as a cause of reentrant arrhythmias (39). The I_Ks channel by patch-clamp analysis is not present in uniform quantities in all ventricular myocytes in our TG mice (J. Kurokawa and R. S. Kass, unpublished data). By extending this observation to the in vivo ventricles, it is possible that the distribution of the I_Ks channels and its effects on repolarization were also nonuniform, establishing conditions for reentry (1, 18, 26, 29), which are exacerbated in the presence of -adrenergic stimulation that shortened VERP. Alterations in repolarizing currents in other TG murine models have also been associated with increased arrhythmogenicity attributed to nonuniform repolarization (2, 20). However, we did not determine whether increased dispersion occurred in our experiments, as was done in the study of Baker et al. (2). To further define the role of the I_Ks channel in this murine model, future experiments using the specific I_Ks blocker, chromanol-293B might be informative (7).

	ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institutes Grant HL-30557.

	FOOTNOTES

Address for reprint requests and other correspondence: A. L. Wit, Dept. of Pharmacology, College of Physicians and Surgeons of Columbia Univ., 630 W. 168th St., New York, NY 10032 (E-mail: alw4{at}columbia.edu).

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.

First published September 19, 2002;10.1152/ajpheart.00661.2002

Received 26 June 2002; accepted in final form 12 September 2002.

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