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Am J Physiol Heart Circ Physiol 291: H631-H637, 2006. First published March 24, 2006; doi:10.1152/ajpheart.00084.2006
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Effect of simulated Ito on guinea pig and canine ventricular action potential morphology

Min Dong,1 Xiaoyin Sun,1 Astrid A. Prinz,2 and Hong-Sheng Wang1

1Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio; and 2Department of Biology, Emory University, Atlanta, Georgia

Submitted 20 January 2006 ; accepted in final form 14 March 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transient outward current (Ito) is a major repolarizing current in the heart. Marked reduction of Ito density occurs in heart failure and is accompanied by significant action potential duration (APD) prolongation. To understand the species-dependent role of Ito in regulating the ventricular action potential morphology and duration, we introduced simulated Ito conductance in guinea pig and canine endocardial ventricular myocytes using the dynamic clamp technique and perforated patch-clamp recordings. The effects of simulated Ito in both types of cells were complex and biphasic, separated by a clear density threshold of ~40 pA/pF. Below this threshold, simulated Ito resulted in a distinct phase 1 notch and had little effect on or moderately prolonged the APD. Ito above the threshold resulted in all-or-none repolarization and precipitously reduced the APD. Qualitatively, these results agreed with our previous studies in canine ventricular cells using whole cell recordings. We conclude that 1) contrary to previous gene transfer studies involving the Kv4.3 current, the response of guinea pig ventricular myocytes to a fully inactivating Ito is similar to that of canine ventricular cells and 2) in animals such as dogs that have a broad cardiac action potential, Ito does not play a major role in setting the APD.

dynamic clamp; transient outward current; ventricular myocytes

THE TRANSIENT OUTWARD POTASSIUM current (Ito) is a key repolarizing current in the heart. The influence of Ito on cardiac action potential waveform and duration is remarkably dependent on species. In smaller animals such as mouse and rat, Ito is the dominant repolarizing current, and the high levels of Ito in these animals are mostly responsible for the short duration and triangular shape of the action potential (2, 23, 27). In large animals, including humans, it is well known that Ito is responsible for phase 1 repolarization of the action potential. However, the influence of Ito on action potential duration (APD) is less clear. The use of a pharmacological approach in studying the problem is hampered by the lack of specific Ito blockers. An alternative method, mathematical simulation, has produced somewhat inconsistent results, predicting that physiological levels of Ito do not affect (5, 34) or significantly prolong (11) the APD.

In an earlier study, we used the dynamic clamp technique to study the role of Ito in shaping the action potential in canine left ventricle (27). The dynamic clamp combines computer simulation with experimental electrophysiology and allows the introduction of programmable artificial conductances in living cells. We have shown that Ito, while being a key regulator of phase 1 repolarization, does not play a major role in regulating the APD over a wide current density range that encompasses the Ito densities found in canine ventricular cells. At densities above a threshold (a value that is much higher than the physiological Ito levels in canine ventricle), Ito precipitously shortens the APD and results in all-or-none repolarization.

Our study also suggests that the difference in the effect of Ito on action potential morphology in small and large animals is due, at least partially, to the dramatic difference in Ito density in these species (27). Interestingly, our results in canine ventricular cells contrast with previous studies (9, 10) in guinea pig ventricular myocytes. Guinea pigs are unusual in their lack of native Ito in the heart (4, 36), and in the studies by Hoppe et al., exogenous Kv4.3 current was introduced into guinea pig ventricular myocytes by either gene transfer or cell fusion techniques. Introduction of such an Ito-type current did not generate a phase 1 notch but progressively suppressed the action potential plateau and shortened the APD over the same Ito density range as what we have tested in canine ventricular cells. The strikingly different observations in canine and guinea pig ventricular cells raise the possibility that guinea pig cells are unique not only in their total lack of native Ito but also in their electrophysiological response to an exogenous Ito. It is plausible that due to the peculiar electrophysiological profile of the guinea pig cells, the threshold for all-or-none repolarization in these cells is much lower. As a result, the presence of even low to moderate densities of Ito suppresses the development of the action potential plateau and shortens the APD. To test this possibility and to further understand the species-dependent role of Ito in shaping the action potential, we performed in this study dynamic clamp simulations of Ito in guinea pig ventricular cells.

A notable limitation of our earlier study (27) is the use of the whole cell patch clamp and intracellular Ca2+ buffer, which can alter Ca2+ intracellular handling and affect the action potential properties. In the present study, to preserve physiological intracellular Ca2+ handling, we used the perforated patch clamp for all action potential recordings. We carried out simulation studies of Ito in canine endocardial myocytes using the perforated patch clamp to verify our earlier results and to allow a side-by-side comparison of canine and guinea pig ventricular cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of isolated guinea pig and canine ventricular myocytes. Handling and usage of animals were in accordance with protocols approved by the University of Cincinnati Institutional Animal Care and Use Committee. Adult guinea pigs, weighing 200–250 mg, of either sex were anesthetized by intraperitioneal injection of pentobarbital sodium (150 mg/kg body wt). Hearts were then quickly excised and mounted on a Langendorff perfusion apparatus and perfused with oxygenated Ca2+-free and then Ca2+-containing (1.5 mM) solution for 5 min each. The perfusion solution contained (in mM) 112 NaCl, 5.4 KCl, 1.7 NaH2PO4, 1.63 MgCl2, 4.2 NaHCO3, 20 HEPES, 5.4 glucose, and 10 taurine (pH = 7.6). This was followed by perfusion with the same solution containing zero Ca2+ and 85 U/ml collagenase (type II, Worthington) at 37°C until the hearts became flaccid. The ventricles were removed, minced, and pipette-triturated in oxygenated Kraft-Brühe (KB) solution containing (in mM) 83 KCl, 30 K2HPO4, 5 MgSO4, 5 Na-pyruvate, 5 beta-OH butyric acid (sodium salt), 20 taurine, 5 creatine, 10 glucose, 0.5 EGTA, 5 HEPES, and 5 Na2ATP (pH = 7.4). Cells were stored in the KB solution at room temperature and used on the day of isolation.

Adult dogs of either sex were euthanized with an intravenous injection of pentobarbital sodium at a concentration of 80 mg/kg body wt. The heart was excised, and a wedge-shaped left ventricular free wall was dissected. The tissue was then cannulated via a descending branch of the left circumflex artery and perfused with an oxygenated solution containing (in mM) 135 NaCl, 5.4 KCl, 1.0 MgCl2, 0.33 NaH2PO4, 10 HEPES, and 10 glucose (pH = 7.4) and then with the same solution containing 140 U/ml collagenase (type II, Worthington), 25 µM leupeptin, and 0.32 U/ml protease (type XIV, Sigma) at 37°C for 10–15 min. Thin slices of tissue (<2 mm in thickness) were then removed from the endocardial surface, minced, and gently shaken in the presence of a lower concentration of collagenase (110 U/ml). Isolated myocytes were harvested and stored in a standard Tyrode solution containing 0.1 mM Ca2+ at room temperature or 4°C for recordings on the same or the following day.

Electrophysiological recordings. Isolated cells were perfused with Tyrode solution containing (in mM) 140 NaCl, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES, and 10 glucose (pH = 7.4). Perforated patch-clamp recordings were used for action potential recordings and the dynamic clamp studies. Glass pipettes were back-filled with a pipette solution containing (in mM) 110 K-aspartate, 20 KCl, 8 NaCl, 10 HEPES, 2.5 MgCl2, 0.01 CaCl2, and 240 mg/ml amphotericin B (pH adjusted to 7.2 with KOH) and have a resistance of 1.5–2.0 M{Omega}. Cells were studied once stable series resistance of <7 M{Omega} was achieved; cells with unstable or a higher series resistance were rejected. Series resistance was fully compensated under current clamp. For K+-current recordings, whole cell patch-clamp recordings were used. CdCl2 (0.2 mM) was added to the external solution to block the Ca2+ current. Pipette solution contained (in mM) 110 K-aspartate, 20 KCl, 10 EGTA, 10 HEPES, 2.5 MgCl2, 4 NaCl, 1 CaCl2, 2 Na2-ATP, and 0.1 Na-GTP (pH adjusted to 7.2 with KOH). Both perforated and whole cell patch clamp recordings were performed with an Axopatch-1B amplifier. Data were collected using pCLAMP9 software through an Axon Digidata 1322A data acquisition system. All experiments were performed at 34°C with the exception of delayed rectifier K+ current (IK) recordings, which were carried out at room temperature (24°C). All chemical and drugs were from Sigma unless otherwise stated.

Implementation of the dynamic clamp. Dynamic clamp experiments were performed as previously described (27). A modified version of the Windows-based DynClamp software was used in the dynamic clamp studies (21). Voltage sampling of the dynamic clamp software and output of the current injection command were through an Axon Digidata 2100 board.

Ito was defined as a rapidly and fully inactivating outward current and was formulated based on our previous canine epicardial Ito model (27). Modifications were made to account for shifts of the voltage-dependence of Ito gating properties by external Cd2+ (26, 33). The Ito conductance was given by the following:

Formula 1(1)

Formula 2(2)

Formula 3(3)

Formula 4(4)

Formula 5(5)

Formula 6(6)

Formula 7(7)

Formula 8(8)
where m is the activation variable, h is the inactivation variable, Vm is the membrane potential, and R is the rectification factor. Figure 1 shows the waveform, true peak conductance-voltage relationship, and voltage-dependencies of steady-state and time constants of the m and h gates of the model Ito. EK, the reversal potential for K+, was set to –85 mV. The electrode junction potential was 12 mV and was corrected online in the computation. Action potentials were triggered with just-threshold 2-ms current steps and recorded at steady state. Action potentials were recontrolled after each simulation. Because gto in Eq. 1 does not equal the true simulated Ito conductance, the true peak current density in response to a depolarizing step from –80 to +40 mV is given to indicate the amplitude of the simulated Ito unless otherwise stated.


Figure 1
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  		Fig. 1. Mathematical modeling of canine epicardial transient outward current (Ito). A: waveform of the model Ito in response to voltage steps ranging from –60 to 50 mV from a holding voltage of –80 mV. B: peak conductance and time constant of activation (m) gate of the model Ito at various voltages (left) and steady-state and time constant of inactivation (h) gate at various voltages (right). gto, peak conductance of simulated Ito; Vm, membrane potential, au, arbitrary units.

 
Data analysis. Group data are presented as means ± SE. Statistical tests of the effects of the dynamic clamp simulation were performed using paired, two-tail Student’s t-tests. Other statistical tests used unpaired Student’s t-tests. A t value giving P < 0.01 was considered as significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Electrophysiological properties of guinea pig ventricular myocytes. We first examined the action potentials of guinea pig ventricular cells using the perforated current clamp. Figure 2A shows representative action potentials triggered at 1 and 3 Hz. Notably, the action potentials lacked phase 1 repolarization and had a waveform similar to that in canine endocardial ventricular cells (27). The average APD90 at 1 Hz was 196.4 ± 12.1 ms (n = 18 cells) and was abbreviated to 137.4 ± 9.9 ms at 3 Hz (n = 6 cells). Consistent with their lack of phase 1 notch, whole cell voltage clamp recordings showed no detectable Ito in guinea pig ventricular cells (Fig. 2B). By comparison, in response to the same voltage clamp protocol and under the same recording conditions, a robust Ito was evident in canine epicardial cells (Fig. 2B, inset). Repolarizing currents in guinea pig ventricular cells were dominated by a large IK (Fig. 2C).


Figure 2
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  		Fig. 2. Electrical properties of guinea pig ventricular myocytes. A: representative action potential traces recorded from guinea pig ventricular myocyte at 1 and 3 Hz. B: Ito is not present in guinea pig ventricular cells but was readily detectable in canine epicardial cells (inset). Cells were depolarized to voltages ranging from –30 to +50 mV in 10-mV increments from a holding potential of –70 mV at a frequency of 0.1 Hz. Notice the difference in current scales. C: delayed rectifier K+ current recorded from guinea pig ventricular myocyte at room temperature (24°C). Currents were in response to depolarizing steps ranging from –30 to +50 mV in 10-mV increments from a holding potential of –40 mV. Tail currents were recorded at –40 mV.

 
Dynamic clamp simulation of Ito in guinea pig ventricular cells. To understand the role of Ito in shaping the action potential, we introduced a simulated canine ventricular Ito conductance in guinea pig ventricular cells using the dynamic clamp. Action potentials were triggered at 1 Hz and recorded at steady state. Simulation of a typical canine epicardial-sized Ito in guinea pig ventricular cells produced a distinct phase 1 notch and a "spike-and-dome" action potential configuration but did not significantly affect the APD (Fig. 3A). The use of the perforated patch clamp presented a challenge for the implementation of the dynamic clamp. We only studied cells with stable series resistance of <7 M{Omega}, which was carefully monitored and fully compensated. The dynamic clamp output (Fig. 3A, inset) and the voltage traces were without oscillation or distortion.


Figure 3
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  		Fig. 3. Dynamic clamp simulation of Ito in guinea pig ventricular myocytes. A: action potentials recorded at 1 Hz from a guinea pig ventricular myocyte under control conditions (dashed line) and with the simulation of a canine epicardial Ito (25 pA/pF) using dynamic clamp (solid line). Bottom trace shows current output of dynamic clamp. A, inset: current output on a larger scale. B: action potentials recorded from a guinea pig ventricular myocyte with simulation of incremental densities of Ito. The "threshold" phenomenon with Ito = 48 (right) is indicated by bold lines. C: action potential duration (APD90, expressed as the ratio over control) versus Ito density relationships collected from 18 guinea pig ventricular myocytes. The average data are shown in D. Individual traces were aligned such that the last points before all-or-none repolarization fell into one group. Vertical and horizontal error bars are ± SE of the APD90 ratio and Ito density, respectively. *P < 0.01, statistical significance in a paired Student’s t-test.

 
We examined the density-dependent effect of simulated Ito on the action potential. Over a wide density range, simulated Ito produced a distinct phase 1 notch and a spike-and-dome action potential waveform (Fig. 3B). Low densities of Ito did not significantly affect the APD (Fig. 3B, left); as the Ito density was further increased, the APD was moderately prolonged (Fig. 3B, left, bold line) before reaching a threshold at which point the action potential alternated between one with a deep notch and a markedly prolonged APD and all-or-none repolarization (Fig. 3B, right, bold lines). Any simulated Ito larger than the threshold produced a brief, spike-like action potential (Fig. 3B, right). Figure 3C shows the Ito density-APD90 relationships for the 18 guinea pig cells we studied, and the average data are shown in Fig. 3D. When the simulated Ito was 21.9 ± 5.3 and 27.3 ± 6.2 pA/pF, APD90 was not significantly changed (0.98 ± 0.05 and 1.00 ± 0.04 times the control value, n = 16 and 14 cells, respectively; P > 0.2). Prolongation of APD was observed with 33.2 ± 5.5 and 39.4 ± 5.6 pA/pF of Ito, which resulted in APD90 values of 1.06 ± 0.05 and 1.18 ± 0.06 times control, respectively (n = 18 cells for both, P < 0.01). Larger Ito produced dramatic shortening of the APD: APD90 ratio was reduced to 0.21 ± 0.11 with a simulated Ito of 43.9 ± 5.9 pA/pF (n = 11 cells, P < 0.001).

Dynamic clamp simulation of Ito in canine endocardial myocytes. To allow comparison between guinea pig and canine ventricular cells and to verify our earlier Ito simulation results in canine ventricular cells, we performed dynamic clamp simulations of Ito in canine endocardial cells using the perforated patch clamp. The overall effect of simulated Ito on canine endocardial APD was similar to that in guinea pig cells (Figs. 3 and 4) and qualitatively similar to our earlier results (27). Low levels of Ito had little effect on the APD; further increases in Ito density progressively and moderately prolonged the APD before collapsing the plateau and markedly shortened the APD (Fig. 4). When the simulated Ito was 22.8 ± 1.7 and 28.6 ± 2.0 pA/pF, the APD90 ratios over control were 1.02 ± 0.01 and 1.05 ± 0.01, respectively (n = 8 and 5 cells, P > 0.02 and P < 0.01, respectively). APD90 was further prolonged to 1.12 ± 0.02 and 1.19 ± 0.03 times control with a simulated Ito of 33.2 ± 1.5 and 40.2 ± 1.9 pA/pF but was shortened to 19 ± 0.3% of control with an Ito of 44 ± 2 pA/pF (n = 7 cells, P < 0.01 for all groups).


Figure 4
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  		Fig. 4. Dynamic clamp simulation of Ito in canine endocardial ventricular myocytes. A: action potentials recorded from a canine endocardial myocyte with various densities of simulated Ito. A, left: Ito density ranged from 5.6 to 33 pA/pF in increments of ~5.6 pA/pF. B: average APD90 ratio vs. Ito density relationship for canine endocardial cells. Data are averaged and plotted as described in Fig. 3 from 7 cells. *P < 0.01, statistical significance in a paired Student’s t-test.

 
Effect of model formulation and stimulation rate. When compared with pure mathematical modeling, the dynamic clamp has the advantage of working with real living cells but still involves simulation of artificial conductances. To determine whether our Ito simulation results are unique to the particular formulations of our canine epicardial Ito model or instead represent a true Ito effect, we performed simulations of an atrial Ito model (24) in guinea pig ventricular cells and compared its effects with those of our ventricular Ito model. An example of such simulation is shown in Fig. 5. The two models used in the study are entirely unrelated and described by different formulations. The Ito conductances described by the two models have markedly different activation rates, conductance-voltage relationships, and steady-state inactivation properties. These differences resulted in noticeably different waveforms of dynamic clamp current output when simulated in the same guinea pig ventricular cell (Fig. 5, A and C, insets). Nevertheless, the effects of the two Ito models on action potential morphology and duration were remarkably similar (Fig. 5, A and C), and so were the shapes of the Ito conductance-APD90 relationship curves (Fig. 5, B and D). Similar experiments were performed in a total of five guinea pig cells and in three canine endocardial myocytes (data not shown). These findings argue that to the extent that these models describe a generalized Ito conductance, our simulation results are a representation of the true Ito functional property.


Figure 5
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  		Fig. 5. Simulations of different mathematical Ito models in guinea pig ventricular cells and at different pacing rates. Action potentials recorded from a guinea pig ventricular myocyte with various levels of Ito simulated based on canine ventricular Ito model (A) and canine atrial Ito model (C; see Ref. 24) (Ref. 14). A and C, insets: current output of the dynamic clamp. The APD90 ratio vs. Ito conductance relationships for A and C are shown in B and D. E: action potential traces from a guinea pig ventricular cell with incremental densities of simulated canine epicardial Ito at pacing rates of 3 Hz. F: APD90 vs. Ito density relationships at 1 and 3 Hz for the same cell as in E.

 
Guinea pigs have a heart rate >200 beats/min. At a higher and more physiological rate, cumulative activation of IK would favor repolarization of the action potential. We therefore performed simulation studies of Ito at pacing rates of 1 and 3 Hz. Simulation of Ito at 3 Hz had generally similar effects on guinea pig action potential as observed at 1 Hz (Fig. 5E), although a higher density of Ito was needed at 3 Hz to achieve all-or-none repolarization (Fig. 5F). This shift of the Ito density-APD90 curve at 3 Hz toward a higher Ito density probably reflects a higher degree of cumulative Ito inactivation at a higher pacing frequency.

Effect of sustained outward current on APD. Our dynamic clamp simulation results in guinea pig ventricular cells differ markedly from those using the gene transfer or cell fusion approaches to introduce Kv4.3 currents in the cells (9, 10). In these earlier studies, a sustained outward current was generated in the ventricular cells along with the Ito-like current. A previous computer modeling study suggested that the introduction of such a noninactivating current contributed to the shortening of the APD by the exogenous Kv4.3 current (6). To test this prediction in real guinea pig ventricular cells, we performed simulations of a modified canine epicardial Ito model that had a noninactivating component. Again, simulation of a fully inactivating Ito had the typical biphasic effect on action potential waveform and duration in guinea pig cells (Fig. 6A). In the same cell, simulation of an Ito with a 10 to 16% sustained component (a fraction similar to those in the studies of Hoppe et al.) progressively and significantly suppressed the plateau and shortened the APD over the entire density range we tested (Fig. 6B). With the presence of the sustained current, Ito density had a monotonic inverse relationship with APD90, instead of the biphasic relationship when the simulated current fully inactivated (Fig. 6C). Similar results were obtained in a total of four guinea pig cells. Results shown in Fig. 6, B and C, closely reproduced the observations of Hoppe et al., suggesting that the presence of a sustained outward current accounted for the monotonic shortening of APD by Ito in these earlier studies.


Figure 6
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  		Fig. 6. Effect of a sustained component in Ito (Isustained or Is) on action potential shape and duration. A: action potentials from a guinea pig ventricular cell with simulation of incremental densities of Ito from 8.8 to 60 pA/pF in increments of ~8.8 pA/pF. B: action potentials from the same cell with various densities of simulated Ito that had a noninactivating (sustained) component. Total simulated Ito density ranged from 8.9 to 44 pA/pF in 8.9-pA/pF increments. Sustained component was 16% of total peak current when elicited by a voltage step to +40 mV from a holding potential of –80 mV. C: APD90 ratio vs. Ito density relationships from the same cell with simulations of Ito that had various fractions of sustained component.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the action potential waveform in guinea pig ventricular cells shares similarities with those of endocardial ventricular myocytes in large animals, such as dog, the underlying ionic current distribution profile in guinea pig and large animals is significantly different. IK is more prominent in guinea pig ventricular cells than in canine ventricle (15, 17) and is balanced by a significantly larger L-type Ca2+ current (ICa-L) (8, 30). Indeed, it was shown that the effect of exogenous Kv4.3 current on action potential in guinea pig ventricular cells (9, 10) is distinct to that of Ito in canine ventricular cells (27). In the present study, we used the dynamic clamp technique to simulate Ito conductances in guinea pig and canine ventricular myocytes and have shown that, despite the significant differences in cellular electrophysiological background of the two myocytes, the effects of a fully inactivating Ito on the action potential morphology and duration of these two types of myocytes were remarkably similar. Importantly, a distinctive threshold (~40 pA/pF) existed that separated the effect of Ito into two phases. Below this threshold, increasing amounts of simulated Ito 1) generated an increasingly deep phase 1 notch and 2) did not significantly affect the APD at low densities and moderately prolonged the APD at higher densities. Ito above the density threshold led to all-or-none repolarization, resulting in spike-like action potentials with dramatically reduced APD. These effects of Ito are not linked to the particular mathematical formulations of the Ito model or a specific stimulation rate. Our experimental results are in agreement with the predictions of a modeling study of canine and guinea pig ventricular cells (6) and probably reflect a general role of the Ito conductance in regulating the morphology of broad, plateau-possessing cardiac action potentials.

The underlying mechanism of the biphasic APD-Ito relationship probably lies in the interplay between Ito and ICa-L, as suggested by the modeling study (6). It is clear from our simulation studies that the voltage trajectory at the end of phase 1 repolarization depends on the potential of the notch. Ito, via its regulation of phase 1 notch potential, either allows reactivation of ICa-L at the end of phase 1 and the development of the plateau (or the "dome") or turns off ICa-L by moving the notch potential below the activation voltage range of ICa-L and completely suppresses the plateau. Prolongation of the APD occurs when Ito causes a delay in the reactivation of ICa-L and a shift in ICa-L time course. In our experiments, the average notch potential for such transition between reactivation and deactivation of ICa-L was ~–20 mV for guinea pig ventricular cells and –24 mV for canine endocardial myocytes.

In our present study, we used perforated patch-clamp recording to preserve physiological intracellular Ca2+ handling and reexamined the effect of Ito in canine endocardial cells. Under these conditions, simulation of Ito in canine endocardial cells generated action potential waveforms that were remarkably similar to those recorded from canine epicardial tissue or myocyte using microelectrode recordings (13, 14, 16, 18). The APD-Ito density relationship is qualitatively similar to our previous findings using whole cell recordings (Fig. 4 of Ref. 27) but with a few noticeable differences. The small depression of APD when Ito was ~30 pA/pF was not found in the current study and was probably an artifact associated with intracellular Ca2+ buffering. Instead, progressive APD prolongation was observed over the entire Ito density range from ~28 pA/pF to the threshold for all-or-none repolarization. Importantly, in our present study, we found that simulation of canine epicardial level of Ito (~20 pA/pF) did not significantly affect the endocardial APD, supporting our previous conclusion that physiological levels of Ito do not play a significant role in regulating the APD in canine left ventricle. The same, however, cannot be said about Ito in canine right ventricular cells. Ito density is significantly higher in canine right epicardium than in the left (3, 25). With the assumption that the APD-Ito density relationship shown in Fig. 4 is shared in right ventricular cells, Ito probably moderately prolongs the APD in the right ventricular cells.

Ito is conventionally defined in modeling studies as the fully and rapidly inactivating component of the total outward current. Such definition is supported by experimental evidence. It is shown that in mouse ventricular cells, Ito inactivates fully and is separate from the noninactivating outward currents (35). In human atrial and mouse ventricular cells, antisense suppression or functional knockout of the Kv4-generated Ito leaves the noninactivating current intact (1, 7, 31), arguing that in these native systems, Ito and the noninactivating component are carried by channels with different molecular identifies. For these reasons, we defined and modeled Ito as a fully inactivating conductance in our simulation studies. With the simulation of even a small sustained conductance, the biphasic APD-Ito relationship in guinea pig cells was dramatically changed to a monotonic inverse relationship, strikingly similar to those reported earlier (9, 10). Simulation of a sustained conductance also eliminated the notch and suppressed the plateau, closely reproducing the results of Hoppe et al. These results point to the importance of the ultrarapid IK-type sustained current in regulating the action potential morphology. The marked effects of the sustained current are not surprising considering the small net current during the plateau phase. In canine ventricular cells, Ito inactivation is rapid and near complete (Fig. 2B). We argued in an earlier study that the large Ito size in smaller animals such as mouse is responsible for their brief action potentials (27). In addition to a large Ito, prominent slowly inactivating and noninactivating currents are present in mouse ventricle (35). These currents, when combined, can make up over half of the total outward current amplitude (35). It is likely that the presence of such slow and sustained currents also contributes importantly to the brief, spike-like action potential in mouse ventricle. In this case, species difference in cardiac action potential morphology is achieved by changes in both channel expression level and function.

Sudden cardiac death, mostly caused by ventricular arrhythmias, is responsible for about half of the mortalities in heart failure patients (20). One of the most characteristic electrophysiological changes in heart failure is the prolongation of the APD, which is believed to predispose the heart to afterdepolarization and reentrant arrhythmias (29). Accompanying the prolongation of APD, downregulation of Ito is consistently observed in heart failure patients as well as animal models (20, 28, 32). Based on their close correlation, it was proposed that Ito downregulation is an important contributor to APD prolongation in failure (12, 32). This is likely to be true in smaller animals, where Ito is much beyond the density threshold for all-or-none repolarization (27) and is the major determinant of APD. The same conclusion may not apply to large animals, such as humans (22), because the role of Ito in regulating action potential morphology differs significantly between small and large animals. Ito in canine (27) and human (19) left ventricle is well below the density threshold for all-or-none repolarization, and our APD-Ito density curves show that within this density range, reduction of Ito, regardless of the magnitude, will not prolong the APD under physiological conditions. In heart failure, a concerted change in ion channel expression occurs (33), and the APD-Ito density relationship reported in our study may be altered as a result of the remodelings of the cellular electrical background. Also, it is possible that downregulation of the Kv4 channel causes a concomitant decrease in the sustained component of the outward current, thereby resulting in APD changes. It would be important to expand our simulation studies to hypertrophy and failure settings.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H.-S. Wang, Dept. of Pharmacology and Cell Biophysics, Univ. of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575 (e-mail: wanghs{at}uc.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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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