© 2003 by European Society of Cardiology
Copyright © 2003, European Society of Cardiology
Calcium-activated transient outward chloride current and phase 1 repolarization of swine ventricular action potential
aInstitute of Cardiovascular Science and Medicine/Department of Medicine, The University of Hong Kong, Hong Kong SAR, China
bDepartment of Pharmacology, Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
* Corresponding author. Department of Physiology L04-55, Laboratory Block Faculty of Medicine Building, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong SAR, China. Tel.: +852-2819-2830; fax: +852-2855-9730. grli{at}hkucc.hku.hk
Received 17 July 2002; accepted 17 December 2002
 |
Abstract |
|---|
 Top Abstract 1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
Objective: It is unknown whether 4-aminopyridine- (4-AP-) sensitive transient outward K+ current (Ito1) and/or Ca2+-activated transient outward Cl– current (ICa.Cl or Ito2) contribute(s) to phase 1 repolarization of pig ventricular action potential (AP). The purpose of the present study was to determine ionic contribution of the phase 1 repolarization of AP in pig ventricle. Methods: We used whole-cell patch techniques to record APs and membrane currents, and Western immunoblotting analysis to detect expression of Ito1 protein (Kv4.2 or Kv4.3) in pig ventricular myocytes. Results: A transient outward current (Ito) was activated upon depolarization voltage steps to between –10 and +60 mV from –50 mV in pig ventricular cells, and the Ito was resistant to 4-AP application, but sensitive to the inhibition by ryanodine (10 µmol/l) and the Ca2+ channel blockade, and the Cl– channel blocker 4,4'-diisothiocyanostilben-2,2'disulfonic acid (DIDS, 150 µmol/l). The current was diminished by external Cl– (Cl–o) replacement and showed a ‘bell-shaped’ I–V relationship at room temperature, typical of Ito2. No difference in Ito2 was observed in the regional cells from epicardium, midmyocardium, and endocardium of left ventricle. APs showed significant phase 1 and ‘spike and dome’ in pig ventricular myocytes. The phase 1 and ‘spike and dome’ of APs were not affected by 4-AP (3 mmol/l), but abolished by replacing Cl–o and by application of 100 µmol/l DIDS, suggesting Ito2 contribution. Western immunoblotting analysis showed no evidence for the expression of 4-AP-sensitive Ito1 channel protein (Kv4.2 or Kv4.3) in pig ventricular cells. Conclusion: The results indicate that 4-AP-sensitive Ito1 is not expressed, and only Ca2+-activated Ito2 is present in pig cardiac cells, which contributes importantly to the phase 1 repolarization of ventricular APs in this species.
KEYWORDS Cl-channel; Ion channels; K-channel
|
1 Introduction |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
Depolarization-activated transient outward current (Ito) has been recognized in cardiac Purkinje fibers since the 1960s [1]. Ito was initially believed to be a Cl– current, and subsequently demonstrated in sheep cardiac Purkinje fibers to be predominantly due to an increase in K+ conductance. With 4-aminopyridine (4-AP), Kenyon and Gibbons firstly identified a 4-AP-resistant component in sheep cardiac Purkinje fibers that was decreased by external Cl– substitution [2]. Subsequently, distinctive 4-AP-sensitive component (transient outward K+ current) and 4-AP-resistant component (Ca2+-activated transient outward chloride current, ICl.Ca) were described in sheep Purkinje fibers [3], and in rabbit and dog cardiac myocytes [4–6]. The 4-AP-sensitive and 4-AP-resistant components are often termed ‘Ito1 and Ito2’ respectively, after Tseng and Hoffman [7]. Ito1 and Ito2 play important roles in the phase 1 repolarization of cardiac action potential (AP) [8–10].
Experimental studies demonstrated that the distribution of Ito1 and Ito2 was species-dependent. Both Ito1 and Ito2 were found in cardiac myocytes from rabbit atrium [4,10], ventricle [6], and Purkinje fibers [11], and canine cardiac cells [5,7,12], and also in sheep cardiac Purkinje fibers [2,3]. Only Ito1 was detected in myocardial cells in species including rat [13], ferret [14], and humans [9]. Earlier studies demonstrated Ito2 in calf Purkinje fibers [15], and elephant seal atrial fibers [16]. However, neither Ito1 nor Ito2 was present in guinea pig ventricular myocytes [17].
Pig is a species commonly used for the study of cardiovascular diseases [18]. However, cellular electrophysiology of the pig heart has not been well documented. Although a recent report described a significant phase 1 repolarization of ventricular APs in pig heart [19], ionic mechanisms are unknown. The present study was designed to study ionic contribution to the phase 1 repolarization of ventricular APs in pig heart. The results demonstrated that Ito2 contributed importantly to the phase 1 and ‘spike and dome’ of ventricular APs in pig heart, different from those observed in dog and humans [8,9,12].
|
2 Methods |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
2.1 Cardiac cell preparation
Left ventricular tissues from isolated hearts were obtained via a left thoracotomy after pigs (45–80 kg, with the Guide for Care and Use of Laboratory Animals of NIH publication no 85-23) were anesthetized with phentobarbital (IV, 30 mg/kg). The heart was initially placed in oxygenated Tyrode solution, the left anterior descending coronary artery was cannulated. Ventricular cells were enzymatically isolated with a procedure described previously [20]. Briefly, a free wall of the left anterior ventricle (
30x50 mm) was removed along with the coronary artery branch irrigating it. The free wall was perfused with oxygenated, nominally Ca2+-free Tyrode solution for 20–30 min, and the solution was then changed to one containing 200–300 U/ml collagenase (CLS II, Worthington Biochemical, Freehold, NJ) for 90–150 min. Ventricular cells were separated from the softened tissue. The rat ventricular myocytes were prepared with a Langendorff method as previously described [21]. All the cells were placed in a high-K+ storage solution (see Solutions) and gently triturated with a Pasteur pipette. The isolated cells were kept in the medium at least 1 h (at room temperature) before use. A small aliquot of the solution containing the isolated cells was placed in an open perfusion chamber (1 ml) mounted on the stage of an inverted microscope. Cells were allowed to adhere to the bottom of the dish for 5 to 10 min, and were then superfused at 2 to 3 ml/min with Tyrode solution. Only quiescent rod-shaped cells showing clear cross-striations were used.
2.2 Solutions
The Tyrode solution contained (mmol/l): NaCl 136, KCl 5.4, MgCl2 1.0, CaCl2 1.8, NaH2PO4 0.33, glucose 10 and HEPES 5; pH adjusted to 7.4 with NaOH. The high-K+ storage medium contained (mmol/l): KCl 20, KH2PO4 10, glucose 10, K-glutamate 70, taurine 20, EGTA 0.5, mannitol 20, albumin 0.1%; pH adjusted to 7.2 with KOH. The pipette solution contained (mmol/l): KCl 20, K-aspartate 110, MgCl2 1.0, HEPES 10, EGTA 0.05, GTP 0.1, Na2-phosphocreatine 5.0, Mg2-ATP 5.0; pH adjusted to 7.2 with KOH. K+ in the pipette and bath solution was replaced by equimolar Cs+ when K+-free conditions were used (specified). For determination of ICl.Ca (or Ito2), BaCl2 (0.5 mmol/l) was added or external K+ was omitted to inhibit inward rectifier K+ current (IK1). The experiments were conducted at room temperature (22°C for Ito2 recording) or 36°C (for AP recording, and Q10 determination of Ito2).
2.3 Data acquisition and analysis
The whole cell patch-clamp technique was used. Borosilicate glass electrodes (1.2 mm O.D.) were pulled with a Brown–Flaming puller (model P-97), and had tip resistances of 2 to 3 M
when filled with pipette solution. The tip potentials were compensated before the pipette touched the cell. A 3 M KCl–agar bridge was used as reference electrode. After a giga-seal was obtained, the cell membrane was ruptured by gentle suction to establish the whole-cell configuration. Liquid junction potentials after membrane rupture between the external and pipette solutions (10.4±0.3 mV) were not corrected except for the recording of AP. Data were acquired by the use of an EPC-9 amplifier (Heka Elektronik, Germany). Command pulses were generated by a digital-to-analog converter controlled by Pulse software (Heka Elektronik). Membrane capacity and series resistance was electronically compensated by the software. Recordings were stored on the hard disk of an IBM compatible computer.
2.4 Western immunoblotting analysis
Membrane fractions were isolated from dissociated single rat and pig ventricular myocytes using standard laboratory procedure. The Kv channel protein was determined by Western immunoblotting analysis. Briefly, 40 µg of total membrane proteins from the pig and rat cardiomyocytes were separated by electrophoresis on an 8% SDS polyacrylamide gel. Proteins in the gel were then transferred to a nitrocellulose membrane, which was subsequently incubated with anti-Kv4.2 or anti-Kv4.3 antibody (Alomone Labs, Israel). Alkaline phosphatase-conjugated secondary antibody and the appropriate substrate were used to develop the membranes. Bands corresponding to Kv proteins were identified according to their relative mobility on the gel. The specific Kv protein bands were analyzed with a gel documentation system (Bio-Rad Gel Doc 1000 and Multi-Analysis® version 1.1).
Nonlinear curve-fitting programs (SigmaPlot, SPSS Science, Chicago, IL) and/or Pulsefit software (Heka) were used to perform curve-fitting procedures. Paired and unpaired Student's t tests were used as appropriate to evaluate the statistical significance of differences between two group means, and analysis of variance (ANOVA) was used for multiple groups. Values of P<0.05 were considered to indicate statistical significance. Results are presented as the mean±S.E.
|
3 Results |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
3.1 Ito in pig ventricular cells
Fig. 1 displays Ito tracings recorded at room temperature (22°C) in different pig ventricular cells with low pipette EGTA (0.05 mmol/l) and normal bath Tyrode solution containing 0.5 mmol/l Ba2+ (to block IK1). An Ito was slowly activated after an initial inward ICa when voltage steps were positive to –10 mV under control conditions (Fig. 1A, D). The Ito was not affected by the application of 3 mmol/l 4-AP (Fig. 1B), and no 4-AP-sensitive Ito1 was observed (Fig. 1C). However, the Ito was decreased by the application of 10 µmol/l ryanodine to inhibit Ca2+ release from sarcoplasmic reticulum (SR) (Fig. 1E), and ryanodine-sensitive Ito was significant (Fig. 1F). Similar results were obtained in a total of 5 cells for each group. These observations suggest that the Ito is not 4-AP-sensitive Ito1, and may be Ca2+-activated Ito2.
|
3.2 Effect of blocking L-type Ca2+ current on Ito
If the Ito is Ca2+-activated Ito2, it would be inhibited by the blockade of L-type Ca2+ current (ICa), and the current–voltage (I–V) relationship would be ‘bell-shaped’. To study if the Ito would be decreased by blocking ICa, Cd2+ was applied in the bath solution. Fig. 2A illustrates Ito current elicited by the voltage protocol shown in the inset of Fig. 2B in control. The Ito was fully inhibited by the application of 200 µmol/l Cd2+ (Fig. 2B). Similar results were obtained in a total of 10 cells. Fig. 2C shows the I–V relationship of mean values of time-dependent peak Ito under control conditions, and in the presence of Cd2+ (n = 12). The I–V relation curve showed ‘bell-shaped’ under control conditions, typical of Ito2, indicating that 4-AP-resistant, ryanodine- and ICa-sensitive current is Ca2+-activated Ito2. The linear I–V relation curve in the presence of Cd2+ indicated a non-specific cation or leakage current.
|
), and linear I–V relation in the presence of Cd2+ measured from the zero (arrow), indicating the nonspecific cation or leakage current (
) (n = 12).3.3 Effect of lowering Cl–o or DIDS on Ito2
Previous studies have demonstrated that Ito2 is charged by Cl– ion [4]. To further determine if Ca2+-activated Ito2 was carried by Cl– in pig ventricular cells, Cl–o was reduced to 11 from 147 mmol/l by substituting bath NaCl with equimolar Na-aspartate. Fig. 3 displays membrane currents elicited by 200 ms voltage steps to +0, +20, and +40 mV from –50 mV before and after Cl–o replacement. Ito2 was significant upon the depolarization potentials under control conditions (Fig. 3A), and disappeared with Cl–o substitution (Fig. 3B). Fig. 3C shows Cl–o-sensitive Ito2 at corresponding voltages obtained by digital subtraction of currents before and after Cl–o replacement. A similar result was obtained in a total of 9 cells. The change of liquid junction potential was very limited with the reference electrode of 3 M KCl–agar bridge, and did not significantly affect experimental data. The result indicates that the Ito2 is carried by Cl– in pig ventricular cells. The density of Cl–o-sensitive Ito2 was 1.6±0.4, 2.5±0.6, and 2.9±0.7 pA/pF at 0, +20, and +40 mV, respectively (P<0.01 among potentials).
|
Ito2 is sensitive to the anion channel blocker DIDS [22]. We therefore observed the effect of DIDS on Ito2 in pig ventricular cells. Fig. 3D illustrates Ito2 elicited by 200 ms voltage steps to +10, +30 and +50 mV from –50 mV, and Fig. 3E shows the Ito2 inhibited by the addition of 150 µM DIDS in bath solution. Fig. 3F displays DIDS-sensitive Ito2 current tracings obtained at corresponding voltages by digital subtraction of current before and after DIDS application. Similar results were obtained in a total of 10 cells. DIDS-sensitive Ito2 was 2.1±0.5, 2.7±0.7, and 2.5±0.8 pA/pF at +10, +30, and +50 mV, respectively (P<0.01 among potentials).
3.4 Temperature-dependence of Ito2
It is well known that temperature may significantly affect the amplitude and kinetics of membrane currents [9,23]. Temperature-dependence of Ito2 was therefore determined under K+-free conditions at room temperature (22°C) and 36°C with 200 ms steps to between –40 and +60 mV from –50 mV. To directly analyze the temperature effect, we studied Ito2 at 22°C and then repeated the measurements at 36°C in the same cells to determine the temperature dependence of Ito2 amplitude in pig ventricular cells. The temperature coefficient (Q10) [9,23] for Ito2 density was calculated by using the equation Q10=1+10(A2–A1)/[A1(T2–T1)], where A1 and A2 are Ito2 density at different temperatures T1 and T2.
Fig. 4 displays the values of amplitude and density of Ito2 at 22°C and 36°C. Representative recordings are shown in Fig. 4A (at 22°C) and Fig. 4B (at 36°C) in the same cell. Ito2 amplitude substantially increased at 36°C. I–V relationships of mean values of Ito2 density at 22 and 36°C are shown in Fig. 4C. Interestingly, the typical ‘bell-shaped’ I–V relation of Ito2 at 22°C became linear as bath temperature was increased to 36°C, and density of Ito2 significantly augmented at 36°C. At +40 mV, Ito2 was increased from 2.1±0.3 to 5.3±0.9 pA/pF (increased by 152%, n = 7, P<0.01). The calculated Q10 was 1.45±0.05.
|
3.5 Contribution of Ito2 to phase 1 repolarization of cardiac APs
The previous study from Stamkovicova et al. [19] demonstrated that ventricular APs of pig hearts showed a significant fast repolarization phase (phase 1). To study the contribution of Ito2 to the phase 1 repolarization of pig ventricular APs, we recorded APs in current clamp mode. Fig. 5 illustrates transmembrane APs recorded at 1 Hz in cells from pig left ventricular epicardium under conditions of normal Tyrode solution at 36°C, showing a significant phase 1 and ‘spike and dome’ configuration in 3 different cells (left panels of Fig. 5A, 5B, and 5C). Fig. 5A displays that the phase 1 and ‘spike and dome’ were not affected by the application of 3 mmol/l 4-AP (right panel), which further suggests the lack of expression of Ito1 in pig ventricle. However, the prominent phase 1 and significant ‘spike-and-dome’ were diminished by lowering Cl–o (right panel of Fig. 5B), or by the application of the Cl– channel blocker DIDS (100 µmol/l, right panel of Fig. 5C), indicating that the Ito2 contributes to the phase 1 repolarization of ventricular APs. Similar results were obtained in 5–8 cells for the different experimental groups.
|
3.6 Ito2 and phase 1 of the AP in regional myocytes
Ito1 and the phase 1 of AP show a significant region-dependent distribution in myocytes from dog and human cardiac ventricles [8,9]. To study if Ito2 and the phase 1 of AP are region-dependent in pig cardiac ventricle, epicardial, midmyocardial, and endocardial myocytes were dissociated from pig ventricular free wall with a procedure as previously described [9]. Voltage-dependent Ito2 was recorded at 36°C with the protocol as in Fig. 4, while APs were recorded with protocol as in Fig. 5. Ito2 showed linear I–V relationship, and no significant regional difference was observed in the current density (Fig. 6A). At +40 mV, Ito2 was 4.1±0.6, 3.9±0.8, and 3.4±0.6 pA/pF in epicardial (n = 12) midmyocardial (n = 10), and endocardial (n = 11) cells (P = NS).
|
, n = 11) of pig left ventricular wall. B. Action potentials recorded with the protocol as in The three regional myocytes showed similar AP morphology with a prominent phase 1. Most of the cells had significant ‘spike and dome’. The phase 1 and/or ‘spike and dome’ of APs were insensitive to 3 mmol/l 4-AP (data not shown), but diminished by the application of 100 µmol/l DIDS in epicardial, midmyocardial, and endocardial cells. Representative recordings are shown in Fig. 6B. Similar results were obtained in 6 to 8 cells in each group.
3.7 Western immunoblotting analysis of Kv4.2 and Kv4.3
It is believed that Ito1 is molecularly ensemble to cloned Kv4.2 and Kv4.3 channels [24]. To further study whether Ito1 is expressed in pig ventricular myocytes, we determined the presence of the channel proteins, i.e. Kv4.2 and Kv4.3 with Western immunoblotting technique. We simultaneously determined Ito1, and Kv4.2 and Kv4.3 in rat ventricular myocytes as positive control under identical conditions. No Ito was observed in pig ventricular cells under conditions of 5 mmol/l EGTA in pipette solution (to buffer intracellular Ca2+), and 200 µmol/l Cd2+ (to block ICa) and 0.5 mmol/l Ba2+ (to block IK1) in bath solution (Fig. 7A). However, significant 4-AP sensitive Ito1 was observed in rat ventricular cells under the identical conditions (Fig. 7B). Similar results were obtained in 12 pig and 6 rat ventricular myocytes. Fig. 7C shows that the K+ channel proteins detected by specific antibodies. It was significant for the K+ channel proteins specific against Kv4.2 and Kv4.3 in the rat heart. In contrast, these K+ channel proteins were either minimal or undetectable by the antibody against Kv4.2 or Kv4.3 in the pig heart. The result is consistent with electrophysiological measurement, showing that pig ventricle does not express Ito1 channel.
|
|
4 Discussion |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
In the present study we have found a significant Ito in pig ventricular myocytes. This Ito is insensitive to the application of 4-AP, but sensitive to the blockade of ICa or Ca2+ release from SR, inhibited by DIDS or Cl–o replacement, and shows a ‘bell-shaped’ I–V relationship at room temperature, typical of Ca2+-activated Ito2. The Ito2 plays an important role in the phase 1 repolarization of pig ventricular APs.
4.1 Comparison with previous reports of pig cardiac electrophysiology
Pig is a commonly used species in studies on cardiac diseases, e.g. ischemia, heart failure and/or arrhythmia, etc. [18]. In addition, the pig heart is being studied for xenotransplantation [25]. However, studies on cardiac electrophysiology were limited to AP recording in isolated hearts [26] or papillary muscles [27], and to study changes in APs and Ca2+ current during transition to heart failure in isolated cells [28]. Characterization of pig ventricular AP properties was only studied with transmural ventricular slices [29] before Stankovicova et al. [19]. Stankovicova and colleagues [19] characterized in detail AP properties in isolated ventricular myocytes of pig heart, and demonstrated that M cells are present in pig ventricles. Also, the data from this group revealed that significant phase 1 repolarization of APs correlated significantly with the J wave of ECG, while the M cells with longest AP duration are related to ‘U’ wave formation of ECG in this species [19]. To date, there is limited information regarding ionic current characterization and distribution of pig cardiac myocytes. A recent report described the rapid and slow components (IKr and IKs) of delayed rectifier K+ current in pig sino-atrial cells [30]. In the present study, we have demonstrated for the first time the non-expression of 4-AP-sensitive Ito1, and the presence of Ca2+-activated Ito2 in pig ventricular cells. The Ito2 contributes importantly to the phase 1 repolarization of ventricular APs in this species.
4.2 Comparison with previous report of Ito2 in other species
4-AP-resistant, Ca2+-activated Ito2 was initially assumed to be charged by K+[3,7] and subsequently was demonstrated to be carried by Cl– ion [4,22]. The current shows the characteristics with ‘bell shaped’ I–V relationship at room temperature, sensitivity to the replacement of Cl–o, to the application of anion channel blockers, and to the blockade of Ca2+ release from SR or ICa in rabbit and dog cardiac cells [4,5]. The present observation has demonstrated that pig ventricular Ito2 is insensitive to 4-AP (Fig. 1), inhibited by the application of DIDS, ryanodine or Cd2+, and abolished by replacing Cl–o, and exhibited the characteristic of ‘bell-shaped’ I-V relationship at 22°C, consistent with those seen in other species [4,5]. We also observed that the ‘bell-shaped’ I–V relation curve became linear at 36°C, similar to the observation in rabbit ventricular cells [31]. The present study showed that the Q10 of Ito2 was 1.45, indicating that Ito2 is very sensitive to the temperature alteration (Fig. 4). It is well known that Ito2 is ligand (i.e. intracellular Ca2+) gated. The increase of Ito2 reflects an elevation of intracellular Ca2+ content, while the intracellular Ca2+ is involved in multiple mechanisms including inward ICa, reverse mode of Na+–Ca2+ exchanger, Ca2+-induced Ca2+ release [11,31], and a voltage-sensitive Ca2+ release mechanism [32]. The increased Ito2 (by 152%) and the linear I–V relationship at 36°C may be related to the enhanced activity of these mechanisms.
4.3 Distribution of cardiac Ito1 and Ito2
Ito1 and Ito2 play important roles in the phase 1 repolarization of the cardiac APs [8–10]. However, the distribution of Ito1 and Ito2 is species-dependent. The species with expression of both Ito1 and Ito2 in their cardiac myocytes are rabbit [4,6], canine [5,7,12], and sheep [1]. Only Ito1 is detected in myocardial cells in species including rat [13], ferret [14], and humans [9]. Ito2 is demonstrated in calf Purkinje fibers [15] and elephant seal atrial fibers [16]. The present observation has demonstrated that Ito1 is lack of expression, and only Ito2 is present in pig ventricular myocytes.
Ito1 plays an important role in phase 1 repolarization of cardiac cells in species including humans [9]. However, in cardiac myocytes with both Ito1 and Ito2, the contribution of either Ito to the phase 1 repolarization of cardiac APs is species-dependent. In dog, Ito1 contributes more to phase 1 and ‘spike and dome’ of ventricular AP than Ito2 [12], and is responsible for the formation of J wave of ECG [13]. Blockade of Ito1 significantly diminished the phase 1 and ‘spike and dome’, whereas inhibition of Ito2 slightly affected the shape of the AP [12]. It is believed that Ito1 shapes cardiac AP morphology and duration [10,12]. In rabbit, however, Ito2 seems to contribute more to the repolarization of cardiac AP due to the inactivation of Ito1 at normal heart rates [33,34]. The present observation has demonstrated that only Ito2 is present, and is responsible for the phase 1 and ‘spike and dome’ of APs in pig ventricular myocytes. No regional difference in Ito2 was observed in cells from specific regions: epicardium, midmyocardium, epicardium of left ventricular wall. Blockade of Ito2 significantly diminished phase 1 and ‘spike and dome’ of cardiac APs. Therefore, Ito2 would contribute to the J waves observed on the ECG in this species [19]. Neither Ito1 nor Ito2 is present in guinea pig cardiac myocytes, and so no significant phase 1 repolarization of cardiac AP is observed in this species [17].
4.4 Potential limitations
The present observation focused on determining Ito1 and/or Ito2 to study the ionic mechanisms of the phase 1 repolarization of pig ventricular APs. The transient outward K+ current with inward rectification (Ito.ir) observed in dog and guinea pig cardiac cells [20,21] was not determined. Although the contribution of Ito.ir to early repolarization of AP is limited in cardiac cells [20,21], further experimental studies are required to clarify cellular electrophysiology in this species. In addition, other repolarization currents, e.g. IKr and IKs, should be characterized in the future in detail in the ventricle of this species. On the other hand, Ca2+ transient was not studied in the present observation. However, correlation of Ca2+ transient to Ito2 was well studied in cardiac myocytes from other species, e.g. rabbit, dog, and calf [11,16,31].
In view of undetectable Ito1 in pig ventricular myocytes, one may reasonably question whether it might be a result of the experimental conditions (e.g. cell isolation). To exclude this possibility we have determined Kv4.2 and Kv4.3 expression in pig ventricular cells by Western immunoblotting analysis, in parallel with that in rat ventricular cells under identical conditions. Significant Ito1, and Kv4.2 and Kv4.3 were observed in rat ventricular cells, but no evidence for Kv4.2 or Kv4.3 was observed (Fig. 7), consistent with the electrophysiological observation.
4.5 Potential significance
As described above, pig is a commonly-used species in studies on cardiac diseases [18]. The size and coronary anatomy of pig heart are close to those of human heart; notwithstanding some differences [35], it is being studied for xenotransplantation [25], Although the ionic mechanisms for repolarization of ventricular APs of pig heart is not completely clarified from present study, our results demonstrate clearly the contribution of Ito2 to phase 1 repolarization of ventricular APs, and the non-expression of Ito1, which are different from those observed in human heart [9]. These important differences should be taken into consideration when extrapolating data from pig studies on electrical remodeling, assessing pharmacological efficacy for treatment study, and/or evaluating the pig heart for xenotransplantation.
In summary, we have demonstrated for the first time the lack of expression of Ito1, and only Ito2 is present in pig ventricular cells. Ito2 contributes importantly to the phase 1 and ‘spike and dome’ of APs in this species.
Time for primary review 23 days.
|
Acknowledgements |
|---|
The study was supported in part by the grants from Research Grant Council of Hong Kong (7338/01M), and from Institute of Cardiovascular Science and Medicine, HKU. The authors thank Professor TM Wong for his support throughout the study, and Ms Haiying Sun for the data analysis and excellent technical support.
|
References |
|---|
|
TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
|---|
- Dudel J., Peper K., Rudel R., Trautwein W. The dynamic chloride component of membrane current in Purkinje fibers. Pflugers Arch Gesamte Physiol Menschen Tiere (1967) 295:197–212.[CrossRef][Web of Science][Medline]
- Kenyon J.L., Gibbons W.R. 4-Aminopyridine and the early outward current of sheep cardiac Purkinje fibers. J Gen Physiol (1979) 73:139–157.
[Abstract/Free Full Text] - Coraboeuf E., Carmeliet E. Existence of two transient outward currents in sheep cardiac Purkinje fibers. Pflugers Arch (1982) 392:352–359.[CrossRef][Web of Science][Medline]
- Zygmunt A.C., Gibbons W.R. Calcium-activated chloride current in rabbit ventricular myocytes. Circ Res (1991) 68:424–437.
[Abstract/Free Full Text] - Zygmunt A.C. Intracellular calcium activates a chloride current in canine ventricular myocytes. Am J Physiol (1994) 267:H1984–H1995.[Web of Science][Medline]
- Hiraoka M., Kawano S. Calcium-sensitive and insensitive transient outward current in rabbit ventricular myocytes. J Physiol (1989) 410:187–212.
[Abstract/Free Full Text] - Tseng G.N., Hoffman B.F. Two components of transient outward current in canine ventricular myocytes. Circ Res (1989) 64:633–647.
[Abstract/Free Full Text] - Liu D.W., Gintant G.A., Antzelevitch C. Ionic bases for electrophysiological distinctions among epicardial, midmyocardial, and endocardial myocytes from the free wall of the canine left ventricle. Circ Res (1993) 72:671–687.
[Abstract/Free Full Text] - Li G.R., Feng J., Yue L., Carrier M. Transmural heterogeneity of action potentials and Ito1 in myocytes isolated from the human right ventricle. Am J Physiol (1998) 275:H369–H377.[Web of Science][Medline]
- Hiraoka M., Kawano S., Hirano Y., Furukawa T. Role of cardiac chloride currents in changes in action potential characteristics and arrhythmias. Cardiovasc Res (1998) 40:23–33.
[Abstract/Free Full Text] - Sipido K.R., Callewaert G., Carmeliet E. [Ca2+]i transients and [Ca2+]i-dependent chloride current in single Purkinje cells from rabbit heart. J Physiol (1993) 468:641–667.
[Abstract/Free Full Text] - Zygmunt A.C., Robitelle D.C., Eddlestone G.T. Ito1 dictates behavior of ICl(Ca) during early repolarization of canine ventricle. Am J Physiol (1997) 273:H1096–H1106.[Web of Science][Medline]
- Coraboeuf E., Coulombe A., Deroubaix E., Hatem S., Mercadier J.J. Transient outward potassium current and repolarization of cardiac cells. Bull Acad Natl Med (1998) 182:325–333.[Web of Science][Medline]
- Campbell D.L., Rasmusson R.L., Qu Y., Strauss H.C. The calcium-independent transient outward potassium current in isolated ferret right ventricular myocytes. I. Basic characterization and kinetic analysis. J Gen Physiol (1993) 101:571–601.
[Abstract/Free Full Text] - Siegelbaum S.A., Tsien R.W. Calcium-activated transient outward current in calf cardiac Purkinje fibres. J Physiol (1980) 299:485–506.
[Abstract/Free Full Text] - Maylie J., Morad M. A transient outward current related to calcium release and development of tension in elephant seal atrial fibres. J Physiol (1984) 357:267–292.
[Abstract/Free Full Text] - Sipido K.R., Callewaert G., Porciatti F., Vereecke J., Carmeliet E. [Ca2+]i-dependent membrane currents in guinea-pig ventricular cells in the absence of Na/Ca exchange. Pflugers Arch (1995) 430:871–878.[CrossRef][Web of Science][Medline]
- Verdouw P.D., Wolffenbuttel B.H. The effect of felodipine on ventricular fibrillation after coronary artery ligation in the anaesthetized pig. Br J Pharmacol (1983) 79:6–8.[Web of Science][Medline]
- Stankovicova T., Szilard M., De S I., Sipido K.R. M cells and transmural heterogeneity of action potential configuration in myocytes from the left ventricular wall of the pig heart. Cardiovasc Res (2000) 45:952–960.
[Abstract/Free Full Text] - Li G.R., Sun H., Nattel S. Characterization of a transient outward K+ current with inward rectification in canine ventricular myocytes. Am J Physiol (1998) 274:C577–C585.[Web of Science][Medline]
- Li G.R., Yang B., Sun H., Baumgarten C.M. Existence of a transient outward K+ current in guinea pig cardiac myocytes. Am J Physiol (2000) 279:H130–H138.[Web of Science]
- Sorota S. Insights into the structure, distribution and function of the cardiac chloride channels. Cardiovasc Res (1999) 42:361–376.
[Abstract/Free Full Text] - Kiyosue T., Arita M., Muramatsu H., Spindler A.J., Noble D. Ionic mechanisms of action potential prolongation at low temperature in guinea-pig ventricular myocytes. J Physiol (1993) 468:85–106.
[Abstract/Free Full Text] - Tseng G.N. Molecular structure of cardiac Ito channels: Kv4.2, Kv4.3, and other possibilities? Cardiovasc Res (1999) 41:16–18.
[Free Full Text] - Lambrigts D., Sachs D.H., Cooper D.K. Discordant organ xenotransplantation in primates: world experience and current status. Transplantation (1998) 66:547–561.[CrossRef][Web of Science][Medline]
- Janse M.J. Electrophysiological effects of myocardial ischaemia. Relationship with early ventricular arrhythmias. Eur Heart J (1986) 7 Suppl A:35–43.[CrossRef][Web of Science][Medline]
- Berman N.D., Wang L.Y., Ahmed A. The cellular electropharmacology of mexiletine combined with sotalol in porcine papillary muscle and Purkinje fibre. I. Alteration of mexiletine-induced Vmax depression. Can J Cardiol (1990) 6:416–422.[Web of Science][Medline]
- Mukherjee R., Hewett K.W., Walker J.D., Basler C.G., Spinale F.G. Changes in L-type calcium channel abundance and function during the transition to pacing-induced congestive heart failure. Cardiovasc Res (1998) 37:432–444.
[Abstract/Free Full Text] - Rodriguez-Sinovas A., Cinca J., Tapias A., Armadans L., Tresanchez M., Soler-Soler J. Lack of evidence of M-cells in porcine left ventricular myocardium. Cardiovasc Res (1997) 33:307–313.
[Abstract/Free Full Text] - Ono K., Shibata S., Iijima T. Properties of the delayed rectifier potassium current in porcine sino-atrial node cells. J Physiol (2000) 524:51–62.
[Abstract/Free Full Text] - Kawano S., Hirayama Y., Hiraoka M. Activation mechanism of Ca2+-sensitive transient outward current in rabbit ventricular myocytes. J Physiol (1995) 486:593–604.
[Abstract/Free Full Text] - Ferrier G.R., Howlett S.E. Cardiac excitation–contraction coupling: role of membrane potential in regulation of contraction. Am J Physiol (2001) 280:H1928–H1944.[Web of Science]
- Fermini B., Wang Z., Duan D., Nattel S. Differences in rate dependence of transient outward current in rabbit and human atrium. Am J Physiol (1992) 263:H1747–H1754.[Web of Science][Medline]
- Wang Z., Fermini B., Feng J., Nattel S. Role of chloride currents in repolarizing rabbit atrial myocytes. Am J Physiol (1995) 268:H1992–H2002.[Web of Science][Medline]
- Crick S.J., Sheppard M.N., Ho S.Y., Gebstein L., Anderson R.H. Anatomy of the pig heart: comparisons with normal human cardiac structure. J Anat (1998) 193:105–119.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. R. Ehrlich, C. Hoche, P. Coutu, C. Metz-Weidmann, W. Dittrich, S. H. Hohnloser, S. Nattel, and H. Gogelein Properties of a Time-Dependent Potassium Current in Pig Atrium: Evidence for a Role of Kv1.5 in Repolarization J. Pharmacol. Exp. Ther., November 1, 2006; 319(2): 898 - 906. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. O. Verkerk, A. C.G. van Ginneken, G. Berecki, H. M. den Ruijter, C. A. Schumacher, M. W. Veldkamp, A. Baartscheer, S. Casini, T. Opthof, R. Hovenier, et al. Incorporated sarcolemmal fish oil fatty acids shorten pig ventricular action potentials Cardiovasc Res, June 1, 2006; 70(3): 509 - 520. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Gwanyanya, B. Amuzescu, S. I. Zakharov, R. Macianskiene, K. R. Sipido, V. M. Bolotina, J. Vereecke, and K. Mubagwa Magnesium-inhibited, TRPM6/7-like channel in cardiac myocytes: permeation of divalent cations and pH-mediated regulation J. Physiol., September 15, 2004; 559(3): 761 - 776. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||