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Cardiovascular Research 2001 49(4):779-789; doi:10.1016/S0008-6363(00)00300-X
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Copyright © 2001, European Society of Cardiology

Transient outward current modulates discontinuous conduction in rabbit ventricular cell pairs

Delilah J. Huelsinga,*, Andrew E. Pollarda and Kenneth W. Spitzerb

aCardiac Rhythm Management Lab and Department of Biomedical Engineering, University of Alabama–Birmingham, Volker Hall B140, 1670 University Blvd., Birmingham, AL 35294, USA
bNora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah School of Medicine, Salt Lake City, UT 84112, USA

* Corresponding author. Tel.: +1-205-975-4718; fax: +1-205-975-4720 djh{at}crml.uab.edu

Received 2 August 2000; accepted 8 November 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Objective: While several studies have demonstrated that the L-type calcium current maintains discontinuous conduction, the contribution of the transient outward current (Ito) to conduction remains unclear. This study evaluated the effects of Ito inhibition on conduction between ventricular myocytes. Methods: An electronic circuit with a variable resistance (Rj) was used to electrically couple single epicardial myocytes isolated from rabbit right ventricle. We inhibited Ito with 4-aminopyridine superfusion, rate–acceleration, or premature stimulation to evaluate the subsequent effects on conduction delay and the critical Rj, which was quantified as the highest Rj that could be imposed before conduction failed. Results: Ito inhibition significantly enhanced conduction in all cell pairs (n = 23). Pharmacologic inhibition of Ito resulted in a 32±5% decrease in conduction delay and a 36±7% increase in critical Rj. Similarly, reduction of the basic cycle length from 2 to 0.5 s resulted in a 31±3% decrease in conduction delay and a 31±3% increase in critical Rj. Finally, premature action potentials conducted with a 41±4% shorter conduction delay and a 73±24% higher critical Rj than basic action potentials. Conclusions: Ito inhibition significantly enhanced conduction across high Rj. These results suggest Ito may contribute to rate-dependent conduction abnormalities.

KEYWORDS Arrhythmia (mechanisms); Cell communication; Conduction (block); Ion channels; K-channels; Membrane potential; Ventricular arrhythmias


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Slow conduction of the cardiac action potential occurs both physiologically at transitional regions [1–3] and pathologically after infarction [4–6]. During long conduction delays associated with increased junctional resistance (Rj), the balance of inward and outward ionic currents during the action potential plateau becomes increasingly important for the maintenance of conduction. The L-type calcium current (ICa,L) enhances conduction by providing most of the inward source current to maintain the action potential plateau. Indeed, several theoretical [7,8] and experimental studies [9–11] have demonstrated that during discontinuous conduction, ICa,L enhancement reduces conduction delay and increases safety factor for propagation, while ICa,L inhibition increases conduction delay and can result in conduction failure.

By comparison, the transient outward current (Ito) has been largely overlooked with respect to discontinuous conduction. In electronically coupled Purkinje and ventricular cell pairs, we concluded that asymmetries in Purkinje–ventricular conduction were importantly modulated by intrinsic differences in Ito and the plateau potential [12]. Ito has also been linked to supernormal conduction of premature beats [13]. Most recently, frequency dependence of discontinuous conduction was shown in rabbit atrial cell pairs [14]. As the major repolarizing current during the early phases of the action potential, Ito is responsible for the rapid phase 1 repolarization and the ‘spike-and-dome’ configuration observed in many cell types [15,16]. By setting the plateau potential, Ito can importantly modulate action potential configuration through voltage-dependent effects on other ionic currents. In particular, the slow recovery kinetics of Ito result in higher plateaus at faster pacing rates, which leads to frequency-dependence of action potential duration [17]. Additionally, the marked transmural heterogeneity in Ito density [18–20] results in an inherent dispersion of repolarization across the ventricular wall that can be arrhythmogenic [21–24].

While Ito clearly contributes to repolarization disparities across the ventricular wall, its early activation and interaction with the sodium and calcium currents suggest a potential role in conduction abnormalities as well, especially in regions with an increased Rj and long conduction delay. The purpose of this study was to test the hypothesis that Ito inhibition enhances conduction across high Rj in ventricular cells. To test this hypothesis, we used an electronic circuit with a variable Rj to couple single cells isolated from rabbit epicardium. We quantified the critical Rj as the highest Rj that could be imposed before conduction failed, and we compared the critical Rj under control conditions to that measured during Ito inhibition. During superfusion with 2 mM 4-aminopyridine, conduction delay decreased and critical Rj increased relative to control. Furthermore, conduction at shorter cycle lengths or during premature stimuli was similarly enhanced, despite a predicted inhibitory effect on ICa,L. These results suggest a novel, rate-dependent mechanism for conduction block or enhancement in regions with increased Rj.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 Cell isolation
Single myocytes were isolated from the right ventricular epicardial surface of adult rabbit hearts. Animal care and treatment conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Briefly, isolated hearts were perfused via the aorta with nominally Ca2+-free Tyrode's solution for 8–10 min, enzyme solution containing 0.1 mM Ca2+ for 18–20 min, and 0.1 mM Ca2+ Tyrode's solution for 5 min, as previously described [12,25]. Thin (<1 mm) slices were cut from the epicardial surface of the right ventricle and gently agitated in 0.1 mM Ca2+ for 10 min. Isolated cells were stored in 1.0 mM Ca2+ until use later that day.

2.2 Solutions
Nominally Ca2+-free Tyrode's solution contained (in mM): NaCl 126, KCl 4.4, MgCl2 5.0, glucose 22, NaH2PO4 1.0, taurine 20, creatine 5, sodium pyruvate 5, HEPES 24, with pH adjusted to 7.4 with NaOH. The enzyme solution had the same composition, except it also contained 1 mg/ml collagenase (Type II, Worthington Biochemical Corp., Freehold, NJ), 0.1 mg/ml protease (Type XIV, Sigma Chemical Co., St. Louis, MO), and 0.1 mM CaCl2.

The normal bathing solution during the experiments contained (in mM): NaCl 126, KCl 4.4, MgCl2 1.0, CaCl2 1.0, glucose 11, and HEPES 24, titrated with 13.0 mM NaOH (pH 7.4). In some experiments, the cells were superfused with 2 mM 4-aminopyridine (4-AP) (Sigma Chemical Co.) to inhibit Ito [17]. The pipette solution contained (in mM): NaCl 10, KCl 113, MgCl2 0.5, HEPES 10, K2ATP 5.0, dextrose 5.5, pH adjusted to 7.1 with 11 mM KOH.

2.3 Electrical recordings
Myocytes were placed in a glass-bottom, temperature-controlled bath (36°C) and continuously bathed with normal solution at 1–2 ml/min. Transmembrane potentials (Vm) were recorded with an Axoclamp 2B amplifier system (Axon Instruments, Inc., Foster City, CA). Suction pipettes were made from borosilicate glass (#7052, O.D. 1.65 mm, I.D. 1.20 mm, A-M Systems, Inc., Everett, WA). Pipette series resistance was compensated before cell attachment, and pipette capacitance was minimized by maintaining a low level (1 mm) of solution in the bath. Myocytes were stimulated with intracellular current injection at a basic cycle length (BCL) of 2 s unless noted otherwise. The stimulus duration was 3 ms, and the stimulus magnitude was approximately 1.1 times the current threshold. Vm was digitized at a minimum sampling rate of 10 kHz with a 12-bit A/D converter (Digidata 1200A, Axon Instruments) and recorded with a computer using pClamp 6 software (Axon Instruments).

We used an electronic circuit to couple myocytes. As previously described [12,25], this circuit included two amplifiers with variable gain to compute the voltage differences ±(Vm,1–Vm,2). That output was sent to voltage-to-current convertors with fixed gain to simultaneously supply equal and opposite coupling currents of ±(Vm,1–Vm,2)/Rj to the myocytes. Rj could be varied from 0 to 2000 M{Omega}. After establishing pipette attachments to both myocytes, we simultaneously stimulated both cells and recorded their intrinsic action potentials during normal solution bathing. We initiated conduction from the upstream cell to the downstream cell by pacing only the upstream cell and coupling the cells with a nominal Rj that ensured conduction.

2.4 Data analysis
We characterized conduction in terms of conduction delay, the extent of early partial repolarization, and the critical Rj [12]. Conduction delay was measured as the difference in activation times between the upstream and downstream cells, where activation time was the time of the maximum upstroke velocity. Early partial repolarization of the upstream cell reflected both intrinsic phase 1 repolarization and the electrical load imposed by the downstream cell. We measured early partial repolarization of the upstream cell as the voltage difference between the action potential peak and the local minimum established during conduction [12]. Conduction delay and early partial repolarization increased monotonically with Rj, as shown in Fig. 1. Thus, when cells were relatively well-coupled at Rj=40 M{Omega}, conduction delay and early partial repolarization were small and measured 2.9 ms and 14.2 mV, respectively. Successive increases in Rj resulted in increased conduction delay and early partial repolarization until conduction failed at Rj=130 M{Omega} (bottom right panel). Conduction failure was marked by a rapid repolarization of the upstream cell, while the downstream cell only demonstrated a passive response. The critical Rj, defined as the highest Rj that could be imposed before conduction failed, measured 125 M{Omega} in this cell pair (not shown).


Figure 1
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  		Fig. 1 Conduction delay and early partial repolarization increase monotonically with Rj. In the top panel, conduction delay (circles, solid line) and early partial repolarization (triangles, dashed line) are plotted as functions of Rj. Smooth fits were drawn between data points. All data were acquired from one cell pair. In the bottom panels, the initial portions of the action potentials are shown during conduction at the five Rj values plotted in the top graph and additionally at an Rj < the critical Rj, resulting in conduction failure. Vertical bar at Rj=40 M{Omega} indicates early partial repolarization of the upstream cell as the voltage difference between the action potential peak and the crossover point for the membrane voltages (see text).

 
To determine how Ito contributed to discontinuous conduction, we compared the critical Rj, conduction delay, and early partial repolarization measured under control conditions to those values measured during three interventions known to inhibit Ito: (i) 4-AP superfusion [17], (ii) increased pacing rate [18], and (iii) premature stimulation [17]. Summary statistics reflect mean±S.E.M., and statistical significance was established using a paired Student's t-test, where P<0.05 was considered statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 Response of uncoupled myocytes to Ito inhibition
Consistent with previous reports [17,18], inhibition of Ito increased the action potential amplitude, reduced the degree of phase 1 repolarization, and raised the action potential plateau. Fig. 2 shows uncoupled action potentials recorded under control conditions and during (A) 4-AP superfusion, (B) pacing at a reduced cycle length of 0.5 s, and (C) premature stimulation. The left portion of each panel depicts the first 50 ms of the action potentials, while the right inset shows the complete action potentials. As shown in the insets, action potentials were prolonged during 4-AP superfusion or premature stimulation, while a four-fold reduction in the steady-state cycle length shortened the action potential. More pertinent to these conduction studies were the changes in the early phases of the action potential. 4-AP increased the action potential amplitude by 5.6 mV and reduced phase 1 repolarization by 59% (Fig. 2A). Similarly, a reduction of BCL resulted in a modest increase in the action potential amplitude and a 61% reduction of phase 1 (Fig. 2B). Finally, the overlay of the basic and premature beats (Fig. 2C) demonstrates that a slight increase in amplitude and a 71% decrease in phase 1 repolarization occurred during the premature action potential.


Figure 2
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  		Fig. 2 4-AP superfusion, rate–acceleration, and premature stimulation reduce phase 1 repolarization. Initial phases (left panels) and full action potentials (insets) before and after the interventions: (A) 2 mM 4-AP superfusion, (B) rate–acceleration to BCL=0.5 s, (C) premature stimulation. Intrinsic phase 1 repolarization, indicated at the left of each panel, was reduced with each intervention.

 
3.2 4-Aminopyridine superfusion enhances conduction
Because an increase in action potential amplitude and reduction of phase 1 repolarization are thought to increase the current source for discontinuous conduction, we hypothesized that 4-AP superfusion would enhance conduction at high Rj. Fig. 3 shows the initial portion of the action potentials during conduction from the upstream cell (solid trace) to the downstream cell (dashed trace). Under control conditions (Fig. 3A), conduction at the critical Rj of 126 M{Omega} required 14.4 ms. Early partial repolarization of the upstream cell measured 48.7 mV. During 4-AP superfusion (Fig. 3B), early partial repolarization and conduction delay were both reduced during conduction at Rj=126 M{Omega}. Additionally, we found that when Ito was inhibited by 4-AP, the critical Rj increased to 168 M{Omega}, and conduction was sustained over a longer delay of 43.0 ms in this cell pair (Fig. 3C).


Figure 3
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  		Fig. 3 4-AP superfusion enhances conduction. Initial phases of the action potentials during conduction (A) at the critical Rj measured under control conditions, (B) during 4-AP superfusion at same Rj, and (C) at the critical Rj measured during 4-AP superfusion. Conduction delay is indicated above and early partial repolarization is indicated to the left of each set of action potentials.

 
Washout of 4-AP reversed these effects. Fig. 4 shows a train of action potentials before, during, and after 4-AP superfusion in another cell pair. When the cells were coupled at Rj=230 M{Omega}, conduction failed during normal solution superfusion (Fig. 4A). Three seconds after the switch to 4-AP, action potentials conducted 1:1. This enhanced conduction by Ito inhibition was fully reversible as every action potential failed to conduct 47 s after washout. Fig. 4B and C show action potentials at six different times during the train (labeled a–f in Fig. 4A) on an expanded scale. Before 4-AP superfusion (a), the upstream action potential could not provide sufficient source charge to elicit an action potential in the downstream cell because phase 1 repolarization was too large and rapid. Two seconds after 4-AP superfusion began (b), the voltage responses were slightly longer and phase 1 repolarization was slower. The very next action potential during 4-AP superfusion (c) conducted with 20.4 ms delay and 28.8 mV of early partial repolarization. Action potentials continued to conduct 1:1 in 4-AP, and conduction delay and early partial repolarization measured 17.2 ms and 21.4 mV, respectively, just before washout (d). During 4-AP washout, Ito gradually recovered from block, and early partial repolarization and conduction delay grew (e) until conduction failed again with every stimulus (f).


Figure 4
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  		Fig. 4 4-AP induces conduction across high Rj. (A) Stimuli were constantly applied to cell 1 at a BCL of 2 s while Rj was held constant at 230 M{Omega}. The first seven action potentials were recorded while cells were bathed in normal solution; the next 14 were recorded while cells were bathed in 2 mM 4-AP; the final 53 were recorded during washout. Small letters a–f below the voltage trace for cell 2 designate the action potentials shown in the panels below. (B) Left panel shows overlay of action potentials from both cells before (a) and immediately after (b) application of 4-AP. Right panel shows overlay of the last blocked action potential (b) and the first conducted action potential (c) during 4-AP superfusion. (C) Left panel shows overlay of the last conducted action potential in 4-AP and the last conducted action potential during washout (e). Right panel shows conduction failure after washout (f).

 
3.3 Rate–acceleration enhances conduction
Increased pacing rate reduced phase 1 repolarization, consistent with incomplete recovery of Ito [18]. As a result, conduction was enhanced when the pacing rate was increased, as shown in Fig. 5. At the BCL of 2 s (Fig. 5A), the critical Rj was 116 M{Omega}, with a conduction delay of 14.6 ms and early partial repolarization of 44.7 mV. Reducing the BCL to 0.5 s reduced the early partial repolarization by 76% and conduction delay by 29% (Fig. 5B). Additionally, the critical Rj increased by 45% at this faster rate (Fig. 5C). These effects were reversible when BCL was readjusted to the slower rate (not shown).


Figure 5
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  		Fig. 5 Rate–acceleration enhances conduction. Initial phases of the action potentials during conduction (A) at the critical Rj measured during pacing at BCL=2 s, (B) during pacing at BCL=0.5 s at same Rj, and (C) at the critical Rj measured during pacing at BCL=0.5 s. Same format as Fig. 2.

 
3.4 Conduction is enhanced during premature stimulation
Although premature stimulation is typically associated with conduction block [9], we found that conduction of premature action potentials was enhanced. Fig. 6 demonstrates that premature action potentials conducted with shorter delay and less early partial repolarization than action potentials elicited during basic stimuli. When the cells were bathed in normal solution (Fig. 6A), the basic action potential conducted with a delay of 9.2 ms and early partial repolarization of 30.6 mV, while conduction delay and early partial repolarization were reduced during the premature action potential. While this was consistent with reduced Ito availability during premature stimuli [26], we further tested this hypothesis by additionally superfusing the cells with 2 mM 4-AP. When these cells were bathed in 4-AP (Fig. 6B), the basic action potential conducted with shorter delay and less early partial repolarization, consistent with results shown in Fig. 3. However, premature stimulation did not further enhance conduction because Ito was already inhibited by 4-AP. In fact, the greater conduction delay and early partial repolarization observed during premature stimulation and 4-AP superfusion (Fig. 6B) relative to premature stimulation alone (Fig. 6A) suggest that the primary effect of the premature stimulus when Ito was already blocked was likely a reduction of ICa,L, which would tend to slow conduction [9].


Figure 6
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  		Fig. 6 Conduction of basic action potentials stimulated at BCL=2 s and premature action potentials elicited at short (<100 ms) diastolic intervals. (A) Top panel shows full action potentials. Bottom panel shows the initial portions of the basic and premature action potentials on an expanded time scale to highlight differences in conduction delay and early partial repolarization. (B) Full (top) and initial portions (bottom) of the action potentials recorded in the same cells during 2 mM 4-AP superfusion and premature stimulation. Rj was held constrant at 100 M{Omega}.

 
Additionally, we evaluated the change in critical Rj during premature stimuli. Fig. 7 illustrates the protocol and results in another cell pair. The critical Rj evaluated during the basic stimulus measured 200 M{Omega} in this cell pair (Fig. 7A). Premature stimuli were then introduced after every fifth basic stimulus, and the diastolic interval preceding the premature stimulus was 30–60 ms (Fig. 7B, inset). At higher Rj values, conduction failed during basic stimuli and succeeded during premature stimuli (not shown). Because conduction failure during the basic stimulus resulted in rapid repolarization of the upstream cell, the diastolic interval and time course of ionic currents would have presented very different initial conditions for the premature stimulus than if the basic action potential had conducted. As a result, we triggered a step increase in Rj during diastole between the basic and premature stimuli to ensure conduction at lower Rj values during basic stimulation while measuring higher critical Rj values during premature stimulation. Thirty ms before each premature stimulus, a step increase in Rj from 100 M{Omega} was instituted to identify the change in critical Rj with premature stimulation. In this cell pair, conduction succeeded at values as high as 248 M{Omega} during premature stimulation.


Figure 7
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  		Fig. 7 Conduction during basic and premature stimulation. Insets show pacing and coupling protocol used. (A) Rj was held constant at the critical Rj of 200 M{Omega} during basic pacing at BCL of 2 s. (B) Rj was set to 100 M{Omega} during basic stimuli and temporarily increased just before the premature stimulus to identify the critical Rj during premature stimulation.

 
Because enhanced conduction of premature action potentials was consistent with the slow recovery kinetics of Ito [26], the timing of the premature stimulus affected conduction delay by altering the availability of Ito. Fig. 8 shows basic and premature action potentials from one cell pair over a wide range of diastolic intervals. For clarity, only one basic action potential is shown, and consistent with the protocol described in Fig. 7, premature stimuli were introduced after every fifth basic stimulus. While the basic action potential conducted with a delay of 10.4 ms, a premature action potential with a diastolic interval of 142 ms conducted with a much shorter delay of 6.9 ms. Conduction delay increased monotonically as diastolic interval increased. Early partial repolarization was similarly modulated as longer diastolic intervals allowed greater reactivation of Ito and, therefore, greater phase 1 repolarization.


Figure 8
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  		Fig. 8 Conduction during basic and premature stimuli at different diastolic intervals. Premature stimuli were introduced at different diastolic intervals after every fifth basic action potential. Diastolic intervals are indicated in ms below and to the left of each set of premature action potentials. Conduction delay and early partial repolarization are indicated above and to the left, respectively, of each set of action potentials.

 
3.5 Summary of experimental data
Table 1 summarizes data from 4-AP, rate–acceleration, and premature stimulation experiments from which the critical Rj was quantified both before and after the intervention. Pharmacologic inhibition of Ito significantly enhanced conduction by increasing the critical Rj, while decreasing conduction delay and early partial repolarization in six cell pairs. Similarly, the critical Rj for conduction was significantly higher during pacing at BCL=0.5 s than at BCL=2 s in six cell pairs, while conduction delay and early partial repolarization were significantly reduced at the faster rate. Finally, premature action potentials demonstrated significantly higher critical Rj, shorter conduction delay, and less early partial repolarization than did basic action potentials in five cell pairs. Additionally, in twelve other cell pairs in which differences in the conduction of basic and premature action potentials were examined at an Rj that was approximately 80% of the critical Rj, mean conduction delay was reduced from 19.8±1.7 to 11.7±1.2 ms and mean early partial repolarization was reduced from 44.3±2.7 to 14.2±1.8 mV during premature conduction. These changes were statistically significant (P<0.0001). Taken together, these results strongly suggest that inhibiting Ito enhances conduction across high Rj.


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  		Table 1 Conduction delay, early partial repolarization, and critical Rj before and after interventionsa

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
Previous studies have shown that ICa,L significantly modulates action potential conduction across regions of increased intracellular resistance. Results from the present study indicate a major functional role for Ito as well during discontinuous conduction. Specifically, we showed that Ito inhibition by 4-AP superfusion, increased pacing rate, or premature stimulation enhanced conduction between ventricular cells. These findings suggest that an assessment of discontinuous conduction based solely on ICa,L is incomplete and that the interplay between Ito and ICa,L must be considered when long conduction delays are present.

This interplay exists because Ito and ICa,L share similar activation kinetics. In isolated rabbit ventricular myocytes, Ito and ICa,L have activation voltages between –30 and –20 mV [17,27]. Ito achieves a peak density of 7.6 pA/pF at +20 mV [18], while the peak density of ICa,L reaches 6.2 pA/pF at +10 mV [27]. The time courses of activation for Ito and ICa,L are also similar, as both activate rapidly upon depolarization [28,29]. Ito inactivates within 100–150 ms [26], while ICa,L inactivates within 200 ms [29]. Importantly, these currents peak as the downstream cell exerts the greatest electrical load on the upstream cell during discontinuous conduction. Because discontinuous conduction is defined as slow conduction across increased Rj, the downstream cell often remains unexcited while the upstream cell enters its plateau phase. As a result, the driving force for conduction is the difference between the plateau potential of the upstream cell and the (near) threshold potential of the downstream cell. Thus, ICa,L becomes the major current source maintaining conduction in the presence of a large conduction delay, while Ito works as a ‘conduction antagonist’ by moving the plateau to more negative potentials and thereby decreasing the driving force for conduction. We examined this interplay between Ito and ICa,L in our previous study on conduction between electrically coupled rabbit Purkinje and ventricular myocytes [12]. In that study, simulation results showed that the critical Rj for Purkinje-to-ventricular conduction increased with Ito inhibition or ICa,L enhancement and decreased with Ito enhancement or ICa,L inhibition.

Previous studies of discontinuous conduction that were performed with guinea pig cells [9] or a model of the guinea pig ventricular myocyte [7] examined only the contribution of ICa,L to discontinuous conduction. Notably, Ito is absent or very small in guinea pig ventricular myocytes [15], but is widespread in many other cardiac preparations including atrial [28], atrioventricular nodal [30], ventricular [26], and Purkinje myocytes [31] from dog [32], rabbit [18], rat [33], sheep [31], and human preparations [34]. Thus, possible modulation of discontinuous conduction by Ito cannot be overlooked.

In particular, our finding that conduction was enhanced during faster pacing is in direct contrast to results from a similar cell pair preparation utilizing guinea pig ventricular myocytes. In that study, single myocytes electrically connected by a variable resistance demonstrated longer conduction delays and conduction failure during more rapid pacing [9]. Because Ito is not a major component of the total ionic current in guinea pig ventricular cells, a shorter cycle length primarily reduced ICa,L, which in turn lowered the plateau potential and increased early partial repolarization. This reduced the driving force for conduction, resulting in slower or failed conduction. In contrast, shorter cycle lengths elevate the plateau in rabbit ventricular cells [18] because Ito is present and highly rate-dependent in these cells. Because the shorter cycle length increased the driving force for conduction in our study, conduction delays were shorter and conduction succeeded at higher Rj values. Our results were consistent with results from rabbit atrial cells coupled by a variable resistance [14]. In that study, discontinuous conduction was also enhanced at faster pacing rates because rabbit atrial cells demonstrate a large, rate-dependent Ito.

Similarly, we demonstrated that the higher plateau of premature action potentials provided a greater driving force for conduction. Again, this is in direct contrast to results from the guinea pig preparation [9]. In that study, premature action potentials conducted with greater conduction delay and early partial repolarization than action potentials initiated during basic stimuli because ICa,L was reduced during premature stimuli. In rabbit ventricular cells, both ICa,L and Ito are reduced during premature stimuli because slow recovery kinetics dictate their reduced availability [17]. However, because Ito was the primary determinant of phase 1 repolarization and the plateau height, we observed higher plateaus and enhanced conduction of premature action potentials in rabbit cell pairs.

Antzelevitch et al. [13] reported a similar observation in canine ventricular epicardial strips. These strips were mounted in a sucrose gap, and the shunt resistance was increased until action potential conduction across the gap failed during basic stimulation. Under those conditions, premature action potentials elicited at short diastolic intervals conducted while basic stimuli were blocked. We have extended those results by showing that conduction delay and early partial repolarization were always smaller during premature action potentials than basic action potentials. We additionally superfused the cells with 4-AP to ‘pre-inhibit’ Ito during premature stimuli. As expected, premature stimulation did not further enhance conduction under these conditions. Finally, conduction delay and early partial repolarization increased as the diastolic interval preceding the premature action potential increased, consistent with recovery of Ito. This is in contrast to decreases in conduction delay with longer diastolic intervals that would be expected with return to activation of ICa,L, suggesting a more prominent role for Ito in modulating discontinuous conduction of premature impulses.

We demonstrated that 2 mM 4-AP, a potent inhibitor of Ito [17], enhanced conduction. It is important to note that 4-AP may not be selective for Ito. For example, 4-AP blocks the delayed rectifier current in neurons [35] and smooth muscle [36], the ultrarapid delayed rectifier in atrial myocytes [37], and the inward rectifier current in sheep cardiac Purkinje fibers [38]. Though we could not find evidence in the literature for 4-AP block of the delayed or inward rectifier currents in rabbit ventricular myocytes, we cannot rule out the possibility that these currents may have been inhibited in our cells. However, the inhibition of these currents would not be expected to affect our results since the delayed and inward rectifier currents are minimal during phase 1 [28]. Additionally, 4-AP does not exert direct effects on ICa,L, suggesting that shorter conduction delays and higher critical Rj values during 4-AP superfusion are directly attributable to Ito inhibition.

We did not attempt to distinguish between the two components of the transient outward component: the Ca2+-independent, 4-AP-sensitive component [18,26] and the calcium-activated chloride current, ICl(Ca) [26,39]. Rather, we lumped their rate-dependent effects on conduction together. ICl(Ca) is insensitive to 4-AP but is abolished by Ca2+ channel blockers or agents that block Ca2+-induced Ca2+ release from the sarcoplasmic reticulum. It is likely that superfusion of these agents in addition to 4-AP would have exerted an even larger effect on conduction, though this was not explored in the present study.

Species-dependent differences in the reactivation kinetics of Ito may attenuate the rate-dependent effects on discontinuous conduction for other cell types. For example, recovery of Ito from inactivation is relatively slow in rabbit ventricular myocytes, which have fast and slow time constants of 105 and 1570 ms, respectively [26]. Human ventricular myocytes, however, have a faster recovery of Ito from inactivation with fast and slow time constants of 12 and 229 ms, respectively [19]. These data suggest a greater availability of Ito during premature stimulation of human ventricular myocytes, which would likely necessitate shorter diastolic intervals (<200 ms, compared to <1500 ms in rabbit) for enhanced conduction of premature action potentials.

Because Ito is an important determinant of action potential duration, arrhythmias linked to Ito have typically been related to repolarization abnormalities [22,40,41]. Our results suggest that Ito may also contribute to arrhythmias due to conduction abnormalities. For example, heterogeneous remodeling of Ito and gap junction resistance following cardiac hypertrophy [42,43] or myocardial infarction [4,23] might yield a substrate for reentry if unidirectional conduction block occurred from regions with high Ito density to regions with low Ito density. Additionally, because transitional areas typically demonstrate heterogeneity of membrane properties and an increased gap junctional resistance, our results suggest multiple scenarios in which Ito may affect conduction in these areas. For example, the high gap junctional resistances [44] and gradient of Ito from center to peripheral sinoatrial nodal cells [45] may serve as a protective mechanism to help prevent retrograde conduction and reentry since less source charge may be available to initiate peripheral-to-center conduction across high Rj. In the atrioventricular node, slow conduction [3] in combination with a heterogenous distribution of Ito [46] may contribute to rate-dependent differences in antegrade and retrograde atrioventricular nodal conduction [47] and reentrant atrial arrhythmias [3]. Finally, a high Rj [1] and dispersion of Ito density [48] are present at the Purkinje–ventricular junction. Simulation studies suggested that greater Ito density in Purkinje cells contributed to directional differences in conduction delay and critical Rj in electrically coupled Purkinje and ventricular cells [12]. Heterogeneous distribution of Itoin the Purkinje system [49] may contribute to reentry in a network of Purkinje–ventricular junctions if Purkinje-to-ventricular conduction succeeded at some sites (with low Ito) and failed at others (with high Ito).

Time for primary review 22 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
We gratefully acknowledge Bruce Steadman and Kent Moore for the design and construction of the electronic coupling system. This work was supported by grants from the American Heart Association to D.J. Huelsing; the National Science Foundation (BES-9457212 and BES-9903466), the National Heart, Lung and Blood Institute (HL52003), and the Southeast Affiliate of the American Heart Association to A.E. Pollard; and the National Heart, Lung and Blood Institute (HL52388) and the Nora Eccles Treadwell Foundation to K.W. Spitzer.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 

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