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1 Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH, USA
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Abstract |
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(Received 11 October 2004;
accepted after revision 12 January 2005;
first published online 13 January 2005)
Corresponding author H.-S. Wang: Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, OH 45267-0575, USA. Email: wanghs{at}uc.edu
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Introduction |
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Transmural electrical heterogeneity is the result of differential expression of several ionic and exchange conductances across the ventricular wall. One notable and extensively studied transmural conductance gradient is that for the transient outward current, or Ito. In mammals such as the human and dog, Ito density is several-fold larger in epicardial myocytes than in the endocardium (Liu et al. 1993; Wettwer et al. 1994). It is well accepted that this transmural Ito gradient is responsible for the prominent phase 1 repolarization in epicardial but not endocardial cells. However, the role of Ito in regulating the APD and, consequently, the contribution of the Ito gradient to the transmural APD dispersion are less clear in large animals. The use of a pharmacological approach to study this problem is hindered by the lack of specificity of available Ito blockers, such as 4-aminopyridine (4-AP). An alternative approach, mathematical modelling, has yielded somewhat inconsistent results. It has been shown that the Ito conductance does not significantly affect the APD in the Luo–Rudy ventricular model (Gima & Rudy, 2002), and in human (Priebe & Beuckelmann, 1998) and canine (Winslow et al. 1999) ventricular cells. A separate study reported that Ito prolongs the APD at low levels, but dramatically shortens the APD at higher levels (Greenstein et al. 2000). Introduction of Ito using cell fusion into guinea-pig ventricular myocytes, where a native Ito is lacking, shortened the APD in a density-dependent manner (Hoppe et al. 1999). Interpretation of this result, however, is complicated by the introduction of a sustained outward current along with Ito into the myocytes (Greenstein et al. 2000).
In this paper, we use the real-time dynamic clamp, or dynamic clamp technique to study the role of Ito in shaping the action potential of canine epicardial and endocardial cells. This technique allows the simulation of membrane conductances in real, living cells to study the effect of these conductances on cellular electrical behaviour (Prinz et al. 2004). To do so, cells are recorded using standard electrophysiological techniques under current-clamp mode. The value of the simulated conductance is calculated by the dynamic clamp software based on the instantaneous membrane voltage and the algorithms describing the conductance, and a corresponding current is injected into the cell. Effectively, programmable artificial conductances can be ‘inserted’ in cells using the dynamic clamp. The power of the technique lies in its combination of computer simulation with real, biological cells. In this study, we used the dynamic clamp to ‘insert’ Ito in endocardial cells, and ‘block’ the native Ito in epicardial cells. The effects of these simulations on the action potential waveform and duration were examined.
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Methods |
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Handling and usage of animals were in accordance with protocols approved by the University of Cincinnati Institutional Animal Care and Use Committee. Six adult dogs of either sex were used in our study, and were killed with an intravenous injection of sodium pentobarbital at a concentration of 80 mg (kg body weight)–1. The heart was excised, and myocytes from the epicardium and endocardium of the left ventricle were dissociated using a trituration method as previously described (Wang & Cohen, 2003). Briefly, epicardial or endocardial ventricular chunks of 2 mm thickness were subjected to successive digestion and trituration at 37°C in a K+-reversed solution containing (mM): KCl 140, KH2CO3 8, KH2PO4 0.4, MgCl2 2, glucose 10, taurine 25, ß-OH-butyric acid 5, Na-pyruvate 5. The K+-reversed solution was bubbled with 95% O2 and 5% CO2 prior to use, and contained 40–80 µg ml–1 Liberase Blendzyme 4 (Roche Applied Science). Isolated myocytes were stored in oxygenated KB solution containing (mM): KCl 83, K2HPO4 30, MgSO4 5, Na-pyruvate 5, ß-OH butyric acid (sodium salt) 5, taurine 20, creatine 5, glucose 10, EGTA 0.5, Hepes 5, and Na2ATP 5 (pH = 7.2) at room temperature or 4°C, for recordings on the same day or the following day.
Hearts were excised from anaesthetized (sodium pentobarbital 70 mg kg–1 I.P.), 12-week-old mice of either sex, and mounted on a Langendorff perfusion apparatus. Hearts were perfused first with oxygenated Ca2+-free Tyrode's solution containing (mM): NaCl 112, KCl 5.4, NaH2PO4 1.7, MgCl2 1.63, NaHCO3 4.2, Hepes 20, Glucose 5.4, L-glutamine 4.1, taurine 10, MEM vitamins (Gibco) 1X, and MEM amino acids solution (Gibco) 1X, and then with the same Ca2+-free Tyrode's solution containing 40 µg ml–1 Liberase Blendzyme 4 at 37°C for 7–10 min. The ventricles were minced and pipette-triturated to disperse the myocytes. Isolated myocytes were stored in 1.8 mM Ca2+ Tyrode's solution at room temperature, and studied on the same day.
Electrophysiological recordings
Isolated cells were perfused with Tyrode's solution containing (mM): NaCl 140, KCl 5.4, MgCl2 1, CaCl2 1.8, Hepes 5, and glucose 10 (pH = 7.4). Whole-cell patch clamp recordings were performed with an Axopatch-1B amplifier. For Ito recordings, 0.2 mM CdCl2 was added to the external solution to block the Ca2+ currents. Action potentials were recorded in the current clamp mode, and were triggered with just-threshold 4 ms current steps at a stimulation rate of 1 Hz until a steady-state was reached. For both Ito and action potential recordings, glass pipettes were filled with solution containing (mM): K-aspartate 110, KCl 20, EGTA 10, Hepes 10, MgCl2 2.5, NaCl 4, CaCl2 1, Na2-ATP 2, and Na-GTP 0.1 (pH adjusted to 7.2 with KOH), and had a resistance of 1.5–2 M
. All recordings were performed at 34°C with the exception of Ito recordings from mouse ventricular cells, which were performed at room temperature (24°C).
Implementation of the dynamic clamp
A modified version of the Windows-based DynClamp software written by Dr Reynaldo Pinto was used in the dynamic clamp studies (Pinto et al. 2001). The software was installed on a Dell PC with a 1.6 GHz processor and 256 MB of memory. The software uses an exponential Euler integration method, and had an update rate of about 5 kHz in our Ito simulation. Membrane voltage was filtered at 3 kHz before being fed into the dynamic clamp software. Voltage sampling of the dynamic clamp software and output of the current injection command were through an Axon Digitdata 1200 board.
Ito is defined in our study as the rapidly, and fully inactivating component of the total outward current. Formulations of Ito were based on our voltage-clamp data on canine epicardial cells and a published canine ventricular Ito model (Dumaine et al. 1999). The Ito conductance was given by:
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were studied. The series resistance was fully compensated under current clamp. Action potentials were triggered at 1 Hz, and were recontrolled after each simulation. All drugs were purchased from Sigma (St Louis, MO, USA) unless otherwise stated. Data collection and analysis were performed using pCLAMP software (Axon Instruments, Foster City, CA).
Data analysis
Group data are presented as means ± S.E.M. Statistical tests of the effects of the dynamic clamp simulation were performed using paired, two-tailed Student's t tests. Other statistical tests used unpaired Student's t tests. A t-value giving P < 0.01 was considered to be significant.
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Results |
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We first compared the action potential and Ito in canine epicardial and endocardial ventricular myocytes using the whole-cell current- and voltage clamp. As previously reported (Lukas & Antzelevitch, 1993), the action potential duration (APD) is longer in endocardial cells than in epicardium (Fig. 1A and B). The average APD at 90% repolarization (APD90) for endocardial cells was 225.3 ± 5.7 ms (n = 28), and 192.7 ± 7.1 ms for epicardial cells (n = 17, P < 0.001). In addition, the action potential of epicardial cells, but not endocardial cells, had a prominent phase 1 repolarization followed by a ‘notch’, resulting in a ‘spike-and-dome’ configuration (Fig. 1A). Concordant with this transmural difference in phase 1 repolarization, there was a marked difference in Ito densities between the epi- and endocardial myocytes (Fig. 1C and D). The peak Ito density in epicardial cells at +40 mV was 20.0 ± 1.6 pA pF–1 (n = 12), and was 6.5-fold higher than that in endocardium (3.1 ± 0.3 pA pF–1, n = 13, P < 0.001).
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The steep gradient in Ito density between epi- and endocardial cells presents a suitable system for the application of the dynamic clamp technique. We first developed formulations of Ito based on voltage-clamp data from our laboratory on canine epicardial ventricular cells and a published canine ventricular Ito model (Dumaine et al. 1999). We followed the convention of existing modelling studies, and defined and modelled Ito as the rapidly and fully inactivating component of the total outward current. A small, non-inactivating current was also present in our recordings (Fig. 1C). We found no difference in the size of this sustained current between epicardial and endocardial cells. At +30 mV, the average sustained currents measured at the end of the 100 ms depolarizing steps in epicardial and endocardial cells were 88 ± 10 and 94 ± 14 pA, respectively. At +50 mV, the numbers were 147 ± 15 and 132 ± 20 pA (n = 10 for all groups, P > 0.5 at both voltages). These results suggest that the sustained current does not play a role in generating the differences between epi- and endocardial cells, and that this current is likely to be a separate current distinct from Ito. Therefore, the sustained current is not included in our Ito model.
Figure 2A shows the waveform of simulated Ito in response to a voltage-clamp protocol. The waveform closely approximated the native current. Figure 2B shows the steady-state values of the inactivation gate and the true peak conductance of the model and our voltage-clamp data. Time constants of the inactivation and activation gates and our experimental data are shown in Fig. 2C. Ito is given by eqn (1). Because of the introduction of the outward rectification factor R in the formulation, and the rapid inactivation rate of the channel, gto in the equation does not equal the maximum simulated Ito conductance. For this reason, the true peak current density at +40 mV is given hereafter for the simulated Ito.
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The native Ito is small in endocardial cells, particularly when compared to the epicardial cells (Fig. 1). The slow recovery kinetics of the endocardial Ito (Yu et al. 2000) should further reduce the current under repetitive stimulation. The absence of any significant Ito contribution to the endocardial action potential is reflected by the lack of phase 1 notch in the action potential (Fig. 1A). This allowed us to ‘insert’ a simulated Ito using the dynamic clamp in endocardial cells, on a near-‘blank’ background. Action potentials were recorded from isolated endocardial cells using the whole-cell current clamp at a firing rate of 1 Hz. ‘Insertion’ of a simulated, epicardial-sized Ito in endocardial cells endowed a prominent phase 1 notch, resulting in an action potential waveform characteristic of the epicardial cells (Fig. 3A). Interestingly, this did not significantly alter the endocardial APD. Engagement of the dynamic clamp simulation did not cause any noticeable distortion of the voltage. The dynamic clamp current output was smooth and without oscillation (Fig. 3A, inset). The simulation update rate of the software was about 5 kHz in our experiments.
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Such dose-dependent effects of simulated Ito were determined in 14 endocardial cells, and the APD90–Ito curves for these cells are shown in Fig. 4A (Ito levels that produced alternating APDs, such as that shown in Fig. 3D, were not included in the plot.) We averaged the data points such that the last points before the sudden APD drop fell into one group, and the rest of the points were aligned and grouped based on their positions relative to this point (Fig. 4B). When 20.3 ± 1.3 pA pF–1 of simulated Ito was introduced in endocardial cells, the average APD90 was 0.98 ± 0.01 times that of control (n = 13, P > 0.5). We did observe a small but significant dip in APD when the simulated Ito was increased to 28.4 ± 1 pA pF–1, which resulted in an APD90 ratio of 0.95 ± 0.03 over control (n = 7, P < 0.01). Further increase of the simulated Ito density to 35.0 ± 1.3 pA pF–1 produced a moderate prolongation of the APD (APD90 ratio = 1.07 ± 0.02, n = 14, P < 0.001).
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‘Blockade’ of the native Ito in epicardial myocytes using a dynamic clamp
Current Ito blockers such as 4-AP lack specificity. Instead, we used a dynamic clamp to ‘block’ the native Ito in epicardial cells by ‘inserting’ a simulated Ito that was of inward polarity (Fig. 5A). We adjusted the amplitude of the simulated inward Ito such that the notch of the epicardial action potential was eliminated. The resulting action potential waveform resembled that of the endocardial cells. Such ‘blockade’ of the native Ito in epicardial cells did not significantly change the APD. For the 11 epicardial cells we studied, the APD90 before and after the ‘blockade’ of Ito was 179.5 ± 7.3 and 181.3 ± 8.2 ms, respectively (Figs 5B, P > 0.5), with an average of 15.6 ± 2.3 pA pF–1 of simulated inward Ito ‘inserted’ to subtract the native current.
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Discussion |
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Using the dynamic clamp, we were able to examine the effect of a wide density range of Ito on the behaviour of ventricular cells. We did observe that a narrow range of simulated Ito produced a slight shortening of the endocardial APD (Fig. 4B). Although this range (around 28 pA pF–1) is outside the average epicardial Ito density, the Ito density reached this level in 2 out of 12 epicardial cells we examined, and therefore may have an effect on the APD. This potential effect, however, is small, with an average of 5% shortening of the APD. Further, in our Ito subtraction studies in epicardial cells, out of the 11 cells we examined, we did not observe any prolongation of the APD of more than 2% upon Ito ‘blockade’. These results indicate that even with a dispersed epicardial Ito density, the effect of Ito on epicardial APD, if any, is small, and not a common occurrence. Interestingly, further increase in the simulated Ito density in endocardial cells resulted in a noticeable prolongation of the APD (Figs 3B and 4B). Similarly, ‘enhancement’ of the native Ito in epicardial cells by introducing a simulated Ito also moderately prolonged the APD (Fig. 5C). These results are in partial agreement with the findings of a mathematical simulation study (Greenstein et al. 2000), which shows that canine Ito, at low to moderate levels and over a wide range, has a density-dependent prolongation effect on the APD. Our result suggests that although Ito is indeed capable of lengthening the action potential, the APD is more stable than this model study predicted, and the prolongation effect does not occur with physiological levels of Ito.
The role of Ito in shaping the cardiac action potential differs greatly among species, particularly between small and large animals. In small animals such as the mouse and rat, Ito is the dominant repolarizing current in the heart. The large Ito is responsible for the spike-like morphology of the action potential observed in these animals, and reductions of Ito result in prolongation of the APD (Clark et al. 1993; Wickenden et al. 1998; Xu et al. 1999b; Nerbonne, 2000). In our studies in canine endocardial cells, we have shown that the simulation of a large Ito eliminated the plateau phase and dramatically shortened the APD, producing an action potential reminiscent of the mouse ventricular action potential (Fig. 4). Although we were not able to simulate a mouse-level Ito in canine myocytes due to the large current size, our results agree, at least qualitatively, with a major role of Ito in shortening the APD in small animals. Other factors, such as the presence of a prominent slowly inactivating outward current in mouse myocytes (Xu et al. 1999a; Brunet et al. 2004) are also likely to contribute to the distinct action potential morphology.
The transmural Ito gradient is a near-ubiquitous feature of mammalian hearts. Instead of contributing to the transmural electrical dispersion, the primary physiological function of the Ito gradient in the hearts of large animals may be to regulate the mechanical properties of the ventricle via its influence on the L-type Ca2+ current (ICaL). Action potential clamp and modelling studies (Greenstein et al. 2000; Cordeiro et al. 2004) have shown that the Ito-induced phase 1 notch changes the activation time course of ICaL and results in a second ICaL peak. The notch also increases the driving force for the L-type Ca2+ current (ICaL) by moving the membrane potential away from ECa. Both effects probably contribute to the APD prolongation observed in our study. More importantly, they can significantly influence Ca2+ influx and sarcoplasmic reticulum Ca2+ load and release, and therefore the contractile properties of the myocytes (Sah et al. 2003). As such, the transmural Ito gradient may be an important determinant of the mechanics of the ventricle by producing transmural differences in the timing and/or strength of myocyte contraction.
In this study, we focused on epicardial cells and endocardial cells because of their marked difference in Ito density and otherwise similar cellular electrical properties, which provide a near-ideal system for the application of the dynamic clamp technique. In addition to epicardial and endocardial cells, a third electrophysiologically distinct cell type, the M cell, has been identified in the mid-myocardium (Antzelevitch et al. 1991; Sicouri & Antzelevitch, 1991; Drouin et al. 1995). The longer APD in the M cells is mainly due to a smaller slow delayed rectifier current (IKs, Liu & Antzelevitch, 1995). A larger late Na+ current and a larger Na2+–Ca2+ exchange current in these cells may also be contributing factors (Zygmunt et al. 2000, 2001). In an earlier study we also described a significant Ca2+ current heterogeneity across the canine left ventricular wall (Wang & Cohen, 2003). It would be of interest to expand our dynamic clamp study to include the M cells, and the role of these other conductances in the transmural electrical heterogeneity.
The dynamic clamp was first developed as a neurobiological tool over a decade ago (Robinson & Kawai, 1993; Sharp et al. 1993). It combines the strengths of in silico mathematical modelling and experimental electrophysiology, and has been widely used to study the electrical behaviour of neurones and neural circuits. Our study has demonstrated that the dynamic clamp is an equally effective and useful tool in the study of cardiac cells. In particular, the ionic basis for the electrical heterogeneity within the ventricle is not yet fully understood, and the dynamic clamp technique has great potential for studying the contribution of various membrane conductances to the transmural electrical heterogeneity.
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) and average peak conductance (•) of epicardial Ito. C, time constants of the inactivation (h) and activation (m) gates of the model Ito (solid lines), inactivation and recovery from inactivation time constants (
, 
