The transient outward current (Ito) plays an important role in shaping the early phase of the cardiac action potential in most mammalian species. As such, Ito may influence the balance of the currents during the plateau phase and thus modulate the duration of the action potential (APD; 1,2). Age-dependent differences in action-potential characteristics in different mammalian species have been explained by differences in current density and kinetics of Ito(3-5). In adult rabbit ventricular muscle, APD is lengthened when stimulation frequency is increased from 0.1 to 1.0 Hz. In neonatal rabbit ventricular preparations, a similar change in stimulation frequency produces no significant changes in APD (4,6). Differences in kinetic behavior of Ito in adult and neonatal rabbit ventricular cells can explain age-related effects of frequency on APD. The rates of apparent inactivation and recovery from inactivation of Ito are faster in neonatal than in adult rabbit ventricular cells (4).
APD is determined by a fine balance between inward and outward currents. Most class III antiarrhythmic drugs block cardiac K+ channels and cause prolongation of the action potential. The degree of blockade depends on drug concentration, activation frequency, and the voltage-dependent interactions. Furthermore, inhibition of one current can modify the action potential to limit or enhance activation of other currents (7). The age-dependent differences in action-potential characteristics make it likely that cardiac tissues from animals of different ages would not respond homogeneously to either pathologic processes or to cardioactive drugs (8). We hypothesized that the kinetic differences in Ito between neonatal and adult cells could affect the frequency-dependent effect of K+ channel blockers on APD. Therefore we studied the effects of the classic K+ channel blocker, 4-aminopyridine (4-AP) and the IKr blocker, almokalant, on APD at different stimulation frequencies and membrane currents of neonatal and adult rabbit ventricular muscle. Both 4-AP and almokalant increase APD (9,10). 4-AP has been shown to be an Ito blocker (9); almokalant inhibits the rapid component (IKr) of the delayed rectifying K+ current (11,12).
Neonatal (1- to 4-day-old) and adult (1.5-2.5 kg) White rabbits (New Zealand) were anticoagulated with heparin (1,000 U/kg, i.p.) and anesthetized with pentobarbital sodium (35 mg/kg, i.p.).
Right papillary muscles were dissected free and pinned to the bottom of a tissue chamber. The preparations were allowed to equilibrate for 1 h while superfused with an oxygenated (95% O2 and 5% CO2) solution of the following composition (in mM): NaCl, 125; KCl, 4.0; CaCl2, 1.8; MgCl2, 1.0; NaH2PO4, 0.42; NaHCO2, 24; and glucose, 11 (35 ± 0.2°C, pH 7.4).
Electrophysiologic recording in papillary muscles
The papillary muscles were stimulated at distinct frequencies (0.1-2.0 Hz) by means of rectangular stimuli (3-ms duration, 1.5 times diastolic threshold intensity) delivered by insulated (except at the tips) silver bipolar electrodes. Action potentials were recorded from the apical region of muscles, by using glass microelectrodes filled with 3 M KCl (15 to 20 MΩ resistance) coupled to the input of a high-impedance preamplifier (World Precision Instruments, New Haven, CT, U.S.A.). Action potential (AP) signals were digitized at a sampling rate of 5 kHz by use of an analog-to-digital board (Scientific Solutions, Solon, OH, U.S.A.) and stored on a hard disk, Axotape data-acquisition software (Axon Instruments, Burlingame, CA, U.S.A.), and a 486DX2 computer. The AP parameters were determined from the digitized records.
Ventricular myocytes were isolated from hearts by using an enzymatic perfusion method as previously described (4). Papillary muscles of the right ventricle were dissected free, and single cells were obtained by mechanical agitation with a pipette. The cells were stored in a high-K+, low-Na+, low-Cl− medium at 4°C until used for electrophysiologic experiments. The solutions had the following composition (mM): Tyrode's solution: NaCl, 125; KCl, 4.0; CaCl2, 1.8; MgCl2, 1.0; NaH2PO4, 0.42; NaHCO3, 24; glucose, 11; and taurine, 10; pH 7.4. Nominally Ca2+-free Tyrode's solution was prepared by omitting CaCl2. High-K+, low-Na+, low-Cl− solution: K-glutamate, 60; KCl, 50; taurine, 20; KH2PO4, 3; glucose, 11; HEPES, 10; and glycolbis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) 0.2; pH 7.4 was adjusted with KOH.
Membrane current recordings in single cells
All experiments were performed at 35°C. The cells were placed in a small-volume (0.2 ml) recording chamber on the stage of an inverted microscope (TMD; Nikon, Tokyo, Japan). Macroscopic current recordings were obtained by the whole-cell voltage-clamp method with an Axopatch 1C (Axon Instruments). Glass pipettes had a tip resistance of 1-3 MΩ when filled with internal solution. The inclusion of potassium aspartate caused a liquid junction potential of about −9 mV which was compensated electronically at the start of each recording. Series resistance was compensated (80%) to provide the fastest possible capacity transient without ringing. Currents were filtered at 2 kHz, digitized at 4 kHz, and stored on a 486DX2 computer (pClamp software; Axon Instruments). The capacitive transient in response to a −10 mV hyperpolarizing voltage step was integrated and divided by the voltage step to estimate cell capacitance. Currents were normalized to capacitance of individual cells to construct current-voltage relations.
The normal solution had the following composition (in mM): NaCl, 140; KCl, 4; CaCl2, 2.2; MgCl2, 1.0; HEPES, 10; and glucose, 11; pH was adjusted to 7.4 with NaOH. To record the membrane currents, we used a Ca2+/Co2+ solution of the following composition (mM): NaCl, 140; KCl, 4; CaCl2, 0.5; CoCl2, 2; MgCl2, 1; HEPES, 10; and glucose, 11; pH was adjusted to 7.4 with NaOH. The pipette solution had the following composition (mM): K-aspartate, 80; KCl, 40; KH2PO4, 10; MgSO4, 1; Na2ATP, 5; HEPES, 5; and EGTA, 5; pH was adjusted to 7.3 with KOH.
The drugs used were 4-AP (Sigma Chemical Co., St. Louis, MO, U.S.A.) and almokalant (a gift from Astra Hässle, Göteborg, Sweden).
Data are expressed as mean ± SD. Statistical significance was evaluated by the Student's paired t test where appropriate. Comparisons between multiple means was made with one-way analysis of variance. To compensate for simultaneous multiple comparisons, a method analogous to Bonferroni's method was used (13). A significance level of p < 0.05 was used.
In the first series of experiments, we studied the effects of 4-AP and almokalant on action-potential parameters in right ventricular papillary muscle from both neonatal and adult rabbits. 4-AP (1 mM) lengthened APD in papillary muscles from both adult (Fig. 1A and B; Tables 1 and 2) and neonatal (Fig. 1C and D; Tables 1 and 2) animals. 4-AP did not modify resting membrane potential, but significantly increased action potential amplitude in both age groups (Tables 1 and 2). In papillary muscle from adult rabbits, the effect on APD was more pronounced than in papillary muscles from neonatal animals. Moreover, the effect of 4-AP on adult preparations was frequency dependent, having a greater effect at lower stimulation frequencies (Fig. 2A). In papillary muscle from neonatal animals, the effect of 4-AP was not frequency dependent (Fig. 2C).
Almokalant significantly increased APD of papillary muscles in both neonatal and adult rabbits but did not modify resting membrane potential or action potential amplitude (Fig. 3; Tables 1 and 2). The effect of almokalant on APD was frequency dependent (Fig. 2B). In preparations from adult animals stimulated at 0.1 Hz, the effect of the drug was significant only on APD90. At frequencies between 0.5 and 2.0 Hz, the increase in the APD measured at 30, 50, and 90% of repolarization was significant (Fig. 2B). In neonatal preparations, the effect of almokalant was frequency dependent at all stimulation frequencies and measure of APD (Fig. 2D).
We next studied the effects of 4-AP and almokalant on membrane currents in ventricular myocytes from adult and neonatal rabbits. Figure 4A shows the effect of 4-AP (1 mM) on membrane currents in an adult ventricular cell. Currents were induced by application of depolarizing pulses (300 ms of duration) from a holding potential (HP) of −80 mV to membrane potentials between −30 and +50 mV, at a frequency of 0.1 Hz. 4-AP decreased the peak current amplitude and the current measured at 300 ms during depolarizing pulse (51 ± 6% and 53 ± 5% decrease in peak and steady-state current measured at +30 mV, respectively). In neonatal ventricular cells (Fig. 4B), 4-AP also decreased peak outward current amplitude measured at both the peak (−53 ± 7%) and steady-state current (−21 ± 4%) at a test potential of +30 mV.
Almokalant (1 μM) did not affect peak current amplitude (Fig. 5A and B). However, almokalant significantly decreased current measured at 300 ms during the depolarizing pulse, in neonatal and adult myocytes (Fig. 5A and B). Almokalant decreased current amplitude measured at 300 ms to +30 mV by 35 ± 5% in adult myocytes and by 51 ± 7% in neonatal myocytes. The decrease in steady-state current suggests that both drugs might block the delayed rectifier current (IK), as previously reported for almokalant (11). IK was activated by 3-s depolarizing pulses to membrane potentials ranging between −30 and +50 mV from a HP of −40 mV. Tail-current amplitude was measured on return to the HP used to quantitate IK. 4-AP decreased tail-current amplitude after the depolarizing pulse to +50 mV by 49 ± 6% in adult myocytes, and by 51 ± 5% in neonatal myocytes (Fig. 6). Almokalant decreased tail-current amplitude after a depolarizing pulse to +50 mV by 65 ± 8% in myocytes from adult and by 68 ± 6% in myocytes from neonatal rabbits (Fig. 7).
Frequency-dependent block of K+ currents
In the last series of experiments, we studied the effects of both drugs on membrane currents recorded under steady-state conditions at different stimulation frequencies ranging from 0.1 to 2 Hz. From an HP of −80 mV, 200-ms depolarizing pulses were applied to +30 mV. In Fig. 8A, control currents obtained at frequencies of 0.1 and 1.0 Hz from the myocyte of an adult animal are shown. As previously reported (4), peak current amplitude at 1.0 Hz was ∼40% of the peak current amplitude measured at 0.1 Hz. 4-AP decreased peak current amplitude by 54 ± 6% at 0.1 Hz (Fig. 8B). At higher stimulation frequencies, the blocking effect of 4-AP on peak outward current amplitude progressively decreased (Fig. 8E). The current measured at the end of the pulses was also decreased by 4-AP. This effect was not frequency dependent (Fig. 8F). In cells from neonatal rabbits under control conditions, peak outward current was not different at the stimulation frequencies used (Fig. 8C). 4-AP decreased peak outward current amplitude by 56 ± 7% at 0.1 Hz. The blocking effect was progressively relieved at stimulation frequencies from 0.5 to 2.0 Hz (Fig. 8E). 4-AP decreased amplitude of steady-state current. This effect was not significantly frequency dependent (Fig. 8F).
Almokalant (Fig. 9) did not modify the peak outward current in myocytes from either adult or neonatal rabbits, at any of the frequencies studied (Fig. 9E). The drug decreased current measured at the end of the pulses in myocytes from both adult and neonatal animals. The blocking effect of almokalant on steady-state current was not significantly frequency dependent (Fig. 9F).
Differences in the kinetics of Ito, inactivation, and recovery from inactivation can account for most of the age- and rate-dependent modulation of AP configuration in rabbit ventricular muscle (4). In this study, we extended these observations to demonstrate that kinetic differences in Ito also underlie age-dependent responses to K+ channel-blocker drugs.
Class III antiarrhythmic drugs have been recognized as promising candidates for antifibrillatory activity. Class III agents act by a specific prolongation of APD (14). To be effective against tachyarrhythmias, a drug should preferentially increase APD at high frequencies with no effect at low frequencies. However, many recently developed class III agents (i.e., E-4031, dofetilide, and almokalant) lengthen action potentials in a "reverse" rate-dependent manner; the agents have a greater effect at low versus high rate of stimulation. However, in experiments under voltage-clamp condition, these drugs produce a more pronounced block of IK at higher frequencies (10,11,15). Therefore, "reverse" use-dependent block of the current can be eliminated as responsible for the "reverse" rate-dependent effects of the drugs on APD. An alternative explanation for this phenomenon would be that these class III antiarrhythmic drugs affect (an)other repolarizing current(s) in a rate-dependent manner. However, these agents have no apparent effect on other currents, including IKs, ICa, Ito, IK1, Na-K pump, and Na-Ca exchanger (11,15). To understand the physiologic consequences of drug-induced inhibition of the different outward potassium currents (i.e., Ito, IK), we should realize that the APD is a complex phenomenon and the result of a fine balance between inward and outward currents and the relative contribution of these currents to the plateau change with frequency. It is clear that more work should be done to understand how the ionic mechanisms of drug action can be directly linked to changes in action potential. Because the preclinical studies of drug action are done in cardiac preparations from different species, it is important to have information about possible species- and age-dependent differences. Most of the preclinical studies on the electrophysiologic effects of antiarrhythmic drugs on isolated tissues are performed in guinea pig, which lacks Ito(12), and rabbit ventricular muscle, for which Ito is characterized by slow inactivation and recovery from inactivation kinetics (16).
In this study, we found that the rate-dependent effects of 4-AP and almokalant on APD are different in ventricular muscle from adult and neonatal rabbits.
One possible explanation for the differential effects of 4-AP and almokalant on neonatal and adult preparations could be that the blocking effects of both drugs on outward K+ currents were quantitatively different in both age groups. However, we found in experiments on isolated myocytes under voltage-clamp conditions that the effects of 4-AP and almokalant on Ito (peak transient outward current amplitude) and IK (tail current amplitude measured at −40 mV after a depolarizing pulse to +50 mV) were quantitatively similar in both age groups. 4-AP blocked both Ito and IK. Almokalant blocked only IK. Both Ito and IK current densities were significantly higher in adults than in neonates.
An alternative explanation for the differential effects of 4-AP and almokalant would be that the participation of outward currents (i.e., Ito, IK1, and IK) to repolarization of the action potential is different in adult and neonatal preparations. In addition, the relative contributions of the currents would depend on the stimulation frequency (4,16,17). In cells from adult rabbits stimulated at low frequencies (0.1 Hz), the action potential is short because Ito is prominent at this stimulation frequency, because the long interpulse interval permits a complete recovery from inactivation of Ito(4,16).
The slow recovery from inactivation of Ito (τ of 1-2 s) in cells from adult rabbits explains the diminished contribution of Ito to AP repolarization and the resulting increase in APD at higher stimulation frequencies (4,16,17). The rate-dependent changes in APD modulate the contribution of other outward currents like IK, with activation kinetics slower than Ito in action-potential repolarization. The relative contribution of IK to repolarization would be greater if the action potentials were of longer duration. In neonatal rabbit ventricular cells, Ito inactivation (τfast 3.7 ms, τslow 14 ms) and recovery from inactivation (τ of 113 ms) rates are much faster than in adult myocytes (4), because inactivation in fast Ito participates only during initial repolarization. The rapid recovery from inactivation results in a, Ito of similar magnitude at stimulation frequencies between 0.1 and 2.0 Hz. Therefore, in neonatal preparations, the participation of IK in action-potential repolarization would be significant at all stimulation frequencies.
From our results and previous work, it was found that almokalant at a concentration of 1 μM blocks IK (10,11; Figs. 5 and 7) without significantly affecting Ito(Fig. 5) and IK1 (J. Sánchez-Chapula, unpublished data). The lack of a significant effect of almokalant on APD30 and APD50 and its small effect on APD90 in adult papillary muscles stimulated at 0.1 Hz suggest that IK participation in repolarization at this frequency is small. As the frequency is increased, Ito is partially inactivated, and the duration of the plateau is increased. The increase in APD by almokalant at frequencies of 0.5-2.0 Hz was significantly increased as compared with that at 0.1 Hz, suggesting a greater participation of IK in repolarization at these frequencies. In neonatal preparations stimulated at 0.1 Hz, the effects of almokalant on APD were already significant, suggesting that at this stimulation frequency, IK participation is also significant. However, it is also clear that the effect of almokalant was increased at a stimulation frequency of 0.5 Hz in comparison with 0.1 Hz. At frequencies between 0.5 and 2.0 Hz, the increase in APD by almokalant was not frequency dependent.
The "reverse" rate-dependent effects of the Ito blocker 4-AP on APD of adult rabbit ventricular preparations also suggest that the participation of Ito on action-potential repolarization is rate dependent. In neonatal preparations, the lack of rate dependence on the increases in APD by 4-AP suggests that participation of Ito is similar at the different frequencies studied. However, the results with 4-AP are more difficult to interpret, because this compound also blocks IK (our results). In addition, the "reverse" use-dependent block of Ito(18,19) can partially explain the frequency-dependent effects of 4-AP on APD. It is clear that a voltage- and use-independent blocker of Ito is needed more rigorously to test our hypothesis.
Ito inactivation and recovery from inactivation rates are similar in atrial and ventricular myocytes from human (7,20-22), dog (23), cat (24), rat (25), and neonatal rabbits (4). Developmental changes in Ito of human atrial myocytes have been reported (21). However, in contrast to our results in rabbit ventricle, Ito recovery from inactivation rate constant was slower in young (τ = 293 ms) than in adult (τ = 138 ms) human myocytes. The effect of 4-AP on action potential and membrane currents was studied in human atrial muscle (6,26). At a concentration of 50 μM, 4-AP increased APD (27); at this concentration, 4-AP mainly blocks IKur(27). At a concentration of 500 μM, the drug produces a shortening in APD (26). This APD decrease was explained by arguing that the block of Ito increases the height and duration of the plateau, increasing the participation of IK, which in turn produces a shortening of APD during terminal repolarization (26).
In summary, the K+ channel blockers 4-AP and almokalant produce different rate-dependent increase in APD in neonatal and adult rabbit ventricular muscle. We propose that the kinetics differences in Ito between adult and neonatal preparations, slower inactivation, and recovery from inactivation rates in adult may determine differences in the participation of Ito and other outward currents like IK during repolarization at different rates of stimulation. Thus differences in the participation of Ito and IK would determine the rate-dependent changes induced by 4-AP and the IKr blocker almokalant on APD.
Acknowledgment: We thank Drs. M. Sanguinetti and M. Tristani-Forouzi for critical reading of the manuscript, Gousti Gould for editorial assistance, Juan Fernando Fernandez for technical assistance, and Juan Carlos Muñoz for preparing the figures. This work was supported by a grant from CONACyT (México) 2004-M9301 and from FOMES (México).
1. Kukushkin NI, Gaiullin RZ, Sosunov EA. Transient outward current and rate-dependence of action potential duration in rabbit cardiac ventricular muscle. Pflugers Arch
2. Campbell DL, Rasmusson RL, Comer MB, Strauss HC. The cardiac calcium-independent transient outward potassium current: kinetics. molecular properties, and role in ventricular repolarization. In: Zipes DP, Jalife J, eds. Cardiac physiology: from cell to bedside.
Philadelphia: WB Saunders, 1995:83-96.
3. Jeck CD, Boyden PA. Age-related appearance of outward currents may contribute to developmental differences in ventricular repolarization. Circ Res
4. Sanchez-Chapula J, Elizalde A, Navarro-Polanco R, Barajas H. Differences in outward currents between neonatal and adult rabbit ventricular cells. Am J Physiol
5. Kilborn MJ, Fedida D. A study of the developmental changes in outward currents of rat ventricular myocytes. J Physiol (Lond)
6. Saxon ME, Safranova VG. The rest-dependent depression of action potential duration in rabbit myocardium and the possible role of the transient outward current: a pharmacological analysis. J Physiol (Paris)
7. Wang Z, Fermini B, Nattel S. Effects of flecainide, quinidine and 4-aminopyridine on transient outward and ultrarapid delayed rectifier currents in human atrial myocytes. J Pharmacol Exp Ther
8. Jeck CD, Rosen MR. Use-dependent effects of lidocaine in neonatal and adult ventricular myocardium. J Pharmacol Exp Ther
9. Kenyon JL, Gibbons WR. 4-Aminopyridine and the early outward current of sheep cardiac Purkinje fibers. J Gen Physiol
10. Ohloer A, Amos GJ, Wettwer E, Ravens U. Frequency-dependent effects of E-4031, almokalant
, dofetilide and tedisamil on action potential duration: no evidence for "reverse-use dependent" block. Naunyn Schmiedebergs Arch Pharmacol
11. Carmeliet E. Use-dependent block and use-dependent unblock of the delayed rectifier K+
current by almokalant
in rabbit ventricular myocytes. Circ Res
12. Wettwer E, Grundke M, Ravens U. Differential effects of the new class III antiarrhythmic agent almokalant
, E-4031, D-sotalol, and quinidine, on delayed rectifier currents in guinea pig ventricular myocytes. Cardiovasc Res
13. Wallenstein S, Zucker CL, Fleiss JL. Some statistical methods useful in circulation research. Circ Res
14. Vaughan-Williams EM. A classification of antiarrhythmic action reassessed after a decade of new drugs. J Clin Pharmacol
15. Sanguinetti M, Jurkiewicz N. Two components of cardiac delayed rectifier K+
current: different sensitivity to block by class III antiarrhythmic agents. J Gen Physiol
16. Giles WR, Imaizumi Y. Comparison of potassium currents in rabbit atrial and ventricular cells. J Physiol (Lond)
17. Hiraoka M, Kawano S. Calcium-sensitive and insensitive transient outward current in rabbit ventricular myocytes. J Physiol (Lond)
18. Campbell DL, Qu Y, Rasmusson RL, Stauss HC. The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. II. Closed state reverse use-dependent block by 4-aminopyridine. J Gen Physiol
19. Castle NA, Slawsky MT. Characterization of 4-aminopyridine block of the transient outward K+
current in adult ventricular myocytes. J Pharmacol Exp Ther
20. Wettwer E, Amos GJ, Posival H, Ravens U. Transient outward current in human ventricular myocytes of subepicardial and subendocardial origin. Circ Res
21. Näbauer M, Beuckelmann DJ, Erdmann E. Characteristics of transient outward current in human ventricular myocytes from patients with terminal heart failure. Circ Res
22. Crumb Jr WJ, Pigott JD, Clarkson CW. Comparison of Ito
in young and adult human atrial myocytes: evidence for developmental changes. Am J Physiol
23. Liu DW, Gintant GA, Antazelevitch C. Ionic basis for electrophysiological distinctions among epicardial, midmyocardial and endocardial myocytes from the free wall of the canine left ventricle. Circ Res
24. Furukawa T, Myerbug RJ, Furukawua N, Basset AL, Kimura S. Differences in transient outward current of feline endocardial and epicardial myocytes. Circ Res
25. Josephson IR, Sanchez-Chapula J, Brown AM. Early outward current in rat single ventricular cells. Circ Res
26. Shibata EF, Drury T, Refsum H, Aldrete V, Giles W. Contributions of a transient outward current to repolarization in human atrium. Am J Physiol
27. Wang Z, Fermini B, Nattel S. Sustained depolarization-induced outward current in human atrial myocytes: evidence for a novel delayed rectifier K+
current similar to Kv 1.5 cloned channel current. Circ Res