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Cardiovascular Research 1997 33(1):139-146; doi:10.1016/S0008-6363(96)00191-5
© 1997 by European Society of Cardiology
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Copyright © 1997, European Society of Cardiology

Differential effects of chronic membrane depolarization on the K+ channel activities in cultured rat ventricular cells

Weinong Guo*, Kaichiro Kamiya and Junji Toyama

Department of Circulation, Division of Regulation of Organ Function, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-01, Japan

Received 23 May 1996; accepted 12 August 1996


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Although there is widespread interest in the regulation of K+ channel gene expression by membrane depolarization, its effects on cardiac ion channel activity remain unclear. In the present study, we investigated the influences of chronic membrane depolarization on the functional expression of K+ channels in cultured rat cardiomyocytes. Methods: Single ventricular cells isolated from day-old rat hearts were cultured for nearly 10 days. From day 6, chronic depolarization induced by elevating the K+ concentration of growth medium to 20 mM was developed for 72 h. Whole-cell patch-clamp techniques were used to record action potentials and ion currents. Results: Compared with controls, longer action potential durations associated with relatively positive resting potentials were observed after 72-h high K+ incubation. Chronic membrane depolarization caused a significantly reduced density of transient outward current (Ito) without affecting the channel kinetics and voltage-dependence. Delayed rectifier K+ current (IK) in cultured cells could be inhibited by E-4031, showing the drug-sensitive and -resistant components with different kinetic properties. The E-4031-sensitive current activated rapidly, and the drug-resistant current was characterized by slow activation. Both the rapid (IKr) and slow (IKs) components constituted IK recorded from the control and depolarization-treated cells, while in the latter group the current density of IKr was slightly increased and that of IKs was enhanced by 80% with a small hyperpolarizing shift (5 mV) in the voltage-dependent activation curve. Conclusions: These observations suggest that the effects of chronic membrane depolarization differ depending on the phenotype of the cardiac K+ channels.

KEYWORDS Membrane depolarization, chronic; Potassium channel, cardiac; Cell culture; Patch clamp, rat


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Significant changes in voltage-gated ion channels have been recognized in both developing and diseased hearts, which contribute to the altered repolarization properties observed in these cardiomyocytes [1–4]. In rat ventricular cells, ion channel development is characterized by the prenatal enhancement of L-type calcium channel expression and postnatal increased density of transient outward current (Ito) [3, 5]. In contrast to this developmental increase in Ito, the majority of previous studies demonstrated reduced Ito density in hypertrophied heart failure and experimental myocardial infarction (see reviews [2, 6]). The underlying mechanisms of these contradictory changes remain to be elucidated.

One of the conspicuous changes in the transmembrane potential of cardiomyocytes is a dramatic hyperpolarization of resting potential paralleling heart development [4, 7], along with a depolarization of resting potential in heart failure [8, 9] and myocardial infarction (see review [10]). These results raise the possibility that cellular resting potential itself may play a role in the modulation of ion channel expression. Further evidence reported concerning the regulation of K+ channel gene transcription by membrane depolarization supports this interpretation. A depolarizing stimulus caused a substantial increase in Shaker-related potassium channel Kv1.4 mRNA levels in cultured neonatal rat heart cells [11], whereas it inhibited Kv1.5 gene and protein expression in pituitary cells [12]. It remains unclear whether the chronic membrane depolarization indeed affects cardiac K+ channel activity. In the present study, we investigated the effects of chronic membrane depolarization on the functional expressions of Ito and delayed rectifier potassium current (IK) in cultured neonatal rat ventricular myocytes, which had been widely used as an in-vitro model to study cardiac ionic functions during cell growth, differentiation and adaptation to stress[13, 14].


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
Neonatal Wistar rats were used for this study. The rats were handled in accordance with the Guidance on the Operation of the Animals (Scientific Procedure) Act 1986 published by HMSO, London.

2.1. Isolation and culture of neonatal rat ventricular myocytes
Single ventricular myocytes were isolated and cultured from day-old Wistar rats according to the protocols previously reported [15]. Briefly, animals were killed by cervical dislocation and their hearts were rapidly removed. Both ventricles were minced and enzymatically dissociated in Dulbecco's PBS solution at 37°C for 10 min, which contained 0.1 mg/ml of collagenase (Yakult, Japan). The cellular suspensions were pelleted by centrifugation at 1000 rpm for 5 min. Cells were then resuspended in normal growth medium at a density of 105 cell/ml and seeded on to 2 x 2 mm No. 1 coverslips (Matsunami Glass Ind., Ltd. Japan) in multiwell culture dishes (Falcon 3046, USA). The normal growth medium contained: 5% CO2/95% air bicarbonate-buffered Eagle's MEM ([K+] = 4.6 mM, Nissui Pharmaceutical Co., Ltd. Japan), 5% fetal bovine serum (Gibco, USA) and 0.3 mg/ml of glutamine. Cytosine arabinoside (Sigma) 10 µM was supplied continuously to the culture to inhibit overgrowth of non-muscle cells. The culture medium was maintained in a humidified incubator and renewed daily.

From day 6 after initial incubation, one set of cultures was grown in high-KCl medium for another 72 h. It was prepared as described for the normal growth medium except that additional K+ from a 3 M KCl stock solution was added to make a total K+ concentration of 20 mM. As a control, a second set of cultures was maintained in modified normal growth medium prepared by addition of the same volume of 3 M of NaCl. This protocol equalized the extracellular osmolarities between control and treatment groups [16].

2.2. Whole-cell patch-clamp procedures
The coverslips of cultures were transferred to a recording chamber mounted on an inverted microscope (Nikon, Japan). Cells were then perfused with Tyrode bath solution at 2 ml/min for at least 30 min before subsequent recordings. Whole-cell patch-clamp was performed at 34°C using a L/M EPC-7 patch-clamp system (D-6100 Darmstadt-13/w, Germany). Pipettes were pulled from borosilicate glass and, after firepolishing, had a resistance of 3–5 M{Omega} when filled with pipette solution. The liquid junction potential between the pipette and bath solutions was always corrected prior to the formation of a gigaohm seal. After breaking the membrane by applying suction to the pipette, action potentials were recorded in current-clamp mode. To further record K+ currents, Tyrode bath solution was then changed to various test solutions. The series resistance was electrically compensated by 70–80% and membrane capacitance (Cm) was calculated as the integrating area under capacitive transient evoked by an applied depolarizing pulse (5 mV) from a holding potential (HP) of – 80 mV. All records were digitized at 200 or 2K Hz for on-line storage in a NEC PC-9801 computer and subsequently analyzed using PC-based programs. The density of measured current was derived from normalization to Cm.

2.3. Solutions
Tyrode bath solution contained (in mM): NaCl 146.9, KCl 5.4, CaCl2 1.8, MgCl2 0.5, NaH2PO4 0.33, HEPES 5.0 and glucose 5.0 (pH = 7.4 adjusted by 1 M of NaOH). The composition of the pipette solution was (in mM): KCl 120, potassium glutamate 20, NaH2PO4 10, CaCl2 0.2, EGTA 10, MgATP 5.0 and HEPES 10 (pH = 7.2 adjusted by 1 M of KOH). While measuring Ito, TTX (10 µM) combined with nisoldipine (3 µM) was added to the Tyrode bath solution to form the test solution. When IK was recorded, a Na+-, Ca2+- and K+-free solution composed of (in mM) N-methyl-D-glucamine 149, MgCl2 5.0, HEPES 5.0 and nisoldipine 0.003 (pH = 7.4 adjusted by HCl) was used as the test solution. Under this recording condition, the inward rectifier K+ current, calcium current and the electrogenic Na+/Ca2+ exchange current were respectively blocked [17, 18].

2.4. Statistics
All data in the text are expressed as mean ± s.e.m. Error bars in the figures show the standard errors of the means. The means of different parameters in each group were compared by one-way analysis of variance (ANOVA). P-values < 0.05 were considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Primary culture of newborn rat cardiomyocytes always contains two types of cells: quiescent and spontaneously beating cells [19]. Each individual cell was observed 4 times per day and followed sequentially throughout the time of incubation. In 48 quiescent cells and 31 beating cells observed at day 5 from control group, 94% (45/48) and 90% (28/31), respectively, of the cells still maintained similar behavior after 72 h. However, in the depolarization-treated group, despite the unchanged activity for each initially quiescent cell, all of the 37 beating cells observed at day 5 became quiescent during the following 72-h high K+ incubation. Since these two types of cultured cells might have differences in expression of ion currents, we only used the constantly quiescent cells in control and treatment groups. Those myocytes showing spontaneous activity during any time in culture were excluded. There were no morphological differences in quiescent cells between control and treatment groups. In addition, these cells revealed no dramatic change in cell size or shape as compared with the age-matched cells maintained continuously in normal growth medium.

3.1. Changes in action potentials
Fig. GR1 shows representative recordings of action potentials from the control and depolarization-treated cells. Action potentials were elicited in current-clamp mode using 3–5 ms suprathreshold current pulses applied at 1.0 Hz. The action potential of the cell cultured in high K+ medium was characterized by long duration and a reduction in resting potential (RP). Compared with the controls, action potential durations at 75% repolarization (APD75, 192.4 ± 14.3 ms, n = 7) were prolonged by 24% (P < 0.05 versus 154.8 ± 12.9 ms, n = 9 in controls) and a depolarizing shift for nearly 8 mV in RP was observed (– 54.3 ± 2.7 versus – 61.9 ± 1.8 mV in controls, P < 0.05). In addition, although Cm in the depolarization-treated cells (97.1 ± 14.2 pF, n = 12) was slightly larger than that in controls (86.2 ± 11.4 pF, n = 7), no significance was found (P > 0.05).


Figure 1
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  		Fig. GR1 Representative action potentials recorded from control and chronic depolarization-treated cells. Action potentials were evoked in current-clamp mode using 3–5 ms suprathreshold current pulses. Cells were stimulated at 1.0 Hz. Measurements were made after reaching the steady state for 10 min. Records from a control (circle) and a depolarization-treated (square) cells are superimposed.

 
3.2. Changes in Ito
In rat heart cells, the Ca2+-independent 4-aminopyridine-sensitive Ito is abundant and predominantly contributes to action potentials [3, 20]. From a HP of – 60 mV, 300-ms depolarizing pulses to various test potentials were applied at an interval of 10 s. Outward current, which rose to a transient peak (Ito) followed by a rapid decay to plateau (Iss, Fig. GR2 A), was evoked when test potential was ≥ – 30 mV. These characteristics of brief depolarization-activated outward current in cultured neonatal rat ventricular myocytes were quite similar to those observed in freshly isolated adult cells with two distinct voltage-gated K+ channels, Ito and IK [21]. In the controls, Ito could be recorded in 8 of 11 cells tested. The percentage of cells expressing Ito (7 of 10) was unchanged in the depolarization group, but the peak current density measured at + 30 mV was significantly reduced (5.6 ± 1.6 versus 9.6 ± 0.5 pA/pF in controls, P < 0.01, Fig. GR2 C). In contrast, the Iss component at the end of a 300-ms voltage pulse was enhance in the depolarization-treated cells (4.4 ± 0.3 pA/pF at + 30 mV versus 3.4 ± 0.3 pA/pF in controls, P < 0.05), resulting in a significant decrease in the relative Ito/Iss amplitude (1.4 ± 0.3 at + 30 mV versus 2.6 ± 0.4 in controls, P < 0.01).


Figure 2
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  		Fig. GR2 Chronic membrane depolarization reduces peak Ito. Whole-cell Ito was evoked by the voltage protocol applied at 0.1 Hz (inset). Representative families of currents (at – 10, + 10, + 20 and + 30 mV) recorded from control and depolarization-treated cells are illustrated in panels A and B, respectively. Capacitive transients are blanked and the dotted lines indicate zero current level. (C) The peak current-voltage relation for cells from the control (n = 8, open circle) and depolarization-treated (n = 7, closed circle) preparations. Asterisks indicate a significant difference in current density (P < 0.05).

 
The current inactivation kinetics and voltage-dependence of Ito were then characterized in more detail. To minimize the influence of Iss, we analyzed the time course of the first 150 ms of Ito decay. Thus, Ito inactivation at test potentials ≥ + 10 mV could be precisely fitted by a double-exponential function: I(t) = a * exp(–t/Tf) + b * exp(–t/Ts) + c, where Tf and Ts are designated as the time constants of the fast (Ito,f) and slow (Ito,s) components of Ito inactivation, and a and b are values related to the respective amplitudes of two components. The fractional contribution of Ito,f is expressed as a percentage of total Ito (a/a + b). As shown in Fig. GR3(A,B), there was no obvious voltage-dependence of Tf and Ts in cultured cells. Although Tseng and Hoffman [22] reported voltage-dependence of Ito decay in canine ventricular cells, our observation is consistent with a previous study in adult rat ventricles showing similar no voltage-dependence of Tf and Ts[23]. At a test potential of + 30 mV, almost identical values regarding Tf (7.5 ± 1.3 versus 8.1 ± 0.9 ms in controls), Ts (78.0 ± 10 versus 72.5 ± 14 ms in controls) and fractional contribution of Ito,f (73.1 ± 8.7 versus 72.5 ± 11.8%) were obtained from control and depolarization-treated groups. In addition, the voltage-dependence of steady-state Ito activation and inactivation was determined by the conventional two-pulse protocols. To calculate the steady-state activation curve, tail current at – 20 mV was measured after a 15-ms prepulse at various potentials. The peak amplitude of tail current was then normalized to the maximal Ito tail to give I/Imax and plotted against the pre-pulse potentials (Fig. GR3 C). To assess the steady-state inactivation property, a conditioning pulse to various potentials for 500 ms was applied and followed by a test pulse at + 30 mV to activate Ito. The inactivation variable was the ratio of peak Ito to maximal current and plotted against the potentials of inactivating prepulse in Fig. GR3 D. Cells from the control and depolarization-treated groups had the same Ito steady-state activation (half-maximal activation potential, 6.9 mV; slope factor, 14.9 mV) and inactivation (half-maximal inactivation potential, – 60.5 mV; slope factor, 14.6 mV) curves which could be described by Boltzmann equations.


Figure 3
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  		Fig. GR3 Inactivation kinetics and voltage-dependence of Ito are not altered by chronic membrane depolarization. (A,B) Time course of macroscopic current decay of Ito as a function of membrane potentials. Fast (Tf) and slow (Ts) time constants were determined by a double-exponential fit of the first 150 ms of current inactivation. T vs voltage plots do not differ in cells from the control (open circles) and depolarization-treated (closed circles) groups (P = NS). (C) Voltage-dependence of steady-state Ito activation derived from tail current measurements in myocytes from control (open circles) and depolarization-treated (closed circles) preparations. From a HP of – 60 mV, Ito was activated by various voltage steps for 15 ms. The extent of activation was quantified by the peak amplitude of tail current measured at – 20 mV. Tail currents were normalized to maximal current to give I/Imax. Data obtained from the two groups fall on a similar sigmoidal curve and the solid line is a Boltzmann fit to the data. (D) Voltage-dependence of steady-state Ito inactivation. The peak Ito was measured at + 30 mV and current was normalized to that elicited after a conditioning pulse at – 120 mV for 500 ms. The solid line indicates a Boltzmann fit for the data derived from control (open circle) and depolarization (closed circle) cells.

 
In addition, the time course of recovery of Ito from inactivation was studied by a double-pulse protocol. Two 200-ms pulses, each to + 30 mV with a varying interpulse interval, were applied at 0.1 Hz from a HP of – 60 mV. The interpulse interval ranged from 0 to 2.0 s. The magnitude of peak Ito elicited by the second pulse was expressed as a fraction of Ito during the first pulse and plotted against the interpulse duration (not illustrated). Recovery of Ito in both the control (n = 6) and depolarization-treated (n = 5) cells could be fitted by a two-exponential function with similar values of time constants (fast time constants, 13.3 ms in control versus 12.8 ms in treatment group; slow time constants, 0.75 versus 0.81 s).

3.3. Changes in IK
It is well known that IK recorded from guinea pig [24] and canine ventricular cells [25] as well as human atrial and ventricular myocytes [26, 27] contains two distinct components, generally referred to as IKr (rapid IK) and IKs (slow IK). The elevated Iss observed during brief depolarizing pulses in chronic depolarization-treated cells (Fig. GR2 B) suggested an enhanced expression of IK. We focused on the functional expressions of two components of IK in cultured neonatal rat ventricular cells and studied the influences of chronic membrane depolarization.

Fig. GR4(A,B, left panels) illustrates the representative recordings of IK at test potentials of – 10 and + 20 mV, respectively. From a HP of – 40 mV, currents were evoked by 2000-ms depolarizing pulses applied at 0.1 Hz in the Na+-, Ca2+- and K+-free bath solution. After exposure to 5 µM E-4031, a specific IKr blocker, for 5 min, the currents activated during each depolarizing step were somewhat decreased by E-4031 while tail currents observed during repolarization at – 40 mV were reduced to a greater extent. E-4031-sensitive current, obtained by digitally subtracting the current in the presence of E-4031 from that in its absence, was characterized by several properties different from the E-4031-resistant component. First, the drug-sensitive time-dependent current showed a biphasic relation to voltage (Fig. GR4 C). Second, drug-sensitive current activated at more negative potentials (Fig. GR4 D) and more rapidly than the drug-resistant current. Finally, E-4031-sensitive current revealed a time-dependent decay during strong depolarization ≥ + 20 mV. These results are in agreement with the interpretation that IK in cultured neonatal rat ventricular cells represents two pharmacologically and kinetically distinct components, IKr and IKs.


Figure 4
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  		Fig. GR4 Effects of E-4031 on delayed rectifier potassium current recorded from a cultured neonatal rat ventricular myocyte. From a HP of –40 mV, currents were evoked by 2000-ms depolarizing pulse to potentials ranging from –40 to +40 mV at an interval of 10 s (insets). The representative currents recorded before (open circle) and after exposure to 5 µM E-4031 for 5 min (closed circle) are superimposed in the left panels of A (test voltage at – 10 mV) and B (test voltage at + 20 mV). E-4031-sensitive currents obtained by digital subtraction of currents in the presence of E-4031 from that in its absence are illustrated in the corresponding right panels. Capacitive transients are blanked, and the dotted lines indicate zero current level. Vertical calibrations are 0.5 nA and 0.2 nA referring to their respective column. (C) Current—voltage relation for the time-dependent current, defined as current at the end of 2000-ms voltage pulse. Results are shown for control conditions (open circles), after exposure to 5 µM E-4031 (E-4031-resistant current, closed circles) and the difference current suppressed by E-4031 (E-4031-sensitive current, triangles). (D) Activation curve of control (open circles), E-4031-resistant (closed circles) and E-4031-sensitive (triangles) currents based on analysis of tail currents at – 40 mV following 2000-ms activating pulses to various voltages shown.

 
The chronic effects of membrane depolarization on IKr and IKs were then investigated using different voltage protocols. Of the total 18 cells tested (8 control and 10 depolarization-treated cells), each of them expressed both IKr and IKs. The current density of IKr was determined as a normalized amplitude of tail current after 200-ms test pulses. This pulse duration was sufficient for nearly complete activation of IKr. As shown in Fig. GR5, chronic depolarization induced an increase in IKr of 18% (at + 10 mV, P < 0.05 versus controls). The IKs was analyzed by measuring the amplitude of E-4031-resistant tail current after 2000-ms pulses to various potentials. Such long pulses were required to ensure full activation of IKs (our unpublished observation). The current density of IKs recorded at + 10 mV was markedly enhanced by 80% in the depolarization-treated preparation, as compared with that of controls (P < 0.01, Fig. GR6). By using brief or long depolarizing pulses, the voltage-dependent activation of IKr and IKs based on tail current measurement was studied (Fig. GR7). Data derived from the control and depolarization-treated cells showed no difference in the voltage-dependent activation of IKr and fall on a similar sigmoidal curve which could be fitted by the Boltzmann equation (half-activation voltage, – 9.3 mV; slope factor, 10.2 mV). On the other hand, chronic membrane depolarization induced a small hyperpolarizing shift in the activation curve of IKs (half-activation potentials, 3.8 versus 8.6 mV in controls, P < 0.05; slope factors, 10.4 versus 11.3 mV in controls).


Figure 5
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  		Fig. GR5 Effects of chronic membrane depolarization on IKr. (A) Representative recordings of IKr from control (upper panel) and chronic depolarization-treated cells (lower panel). From a HP of –40 mV, myocytes were clamped by brief depolarizing pulse (200 ms) at + 10 mV and then repolarized at – 40 mV to observe tail current. Capacitive transients are blanked and the dotted lines indicate zero current level. (B) Tail current densities measured after 200-ms depolarizing pulse at + 10 mV. Data were obtained from 8 control cells (open column) and 10 depolarization-treated cells (hatched column). Asterisk indicates a significant difference in current density (P < 0.05).

 

Figure 6
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  		Fig. GR6 Effects of chronic membrane depolarization on IKs. (A) Representative recordings of IKs from control (upper panel) and chronic depolarization-treated cells (lower panel). From a HP of – 40 mV, myocytes were clamped by long depolarizing pulse (2000 ms) at + 10 mV and then repolarized at – 40 mV to observe tail current. All recordings were performed in the presence of 5 µM E-4031. Capacitive transients are blanked, and the dotted lines indicate zero current level. (B) Tail current densities measured after a 2000-ms depolarizing pulse at + 10 mV. Data were obtained from 8 control cells (open column) and 10 depolarization-treated cells (hatched column). Asterisk indicates a significant difference in current density (P < 0.01).

 

Figure 7
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  		Fig. GR7 Effects of chronic membrane depolarization on voltage-dependence of IKr and IKs. Voltage-dependent activation curves of IKr (A) and IKs (B) were determined by analysis of tail current at –40 mV after depolarizing pulses to various membrane potentials. Brief test pulses for 200 ms and long test pulses for 2000 ms were respectively applied during IKr and IKs measurements. IKs was recorded in the presence of 5 µM E-4031. Data obtained from 5–7 duplicate experiments in the control (open circles) and depolarization-treated (closed circles) preparations could be fitted by the Boltzmann equation. Note that the chronic membrane depolarization induced a small hyperpolarizing shift in the voltage-dependent activation curve of IKs.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Although there is widespread interest in the regulation of K+ channel gene expression by depolarizing stimulus[11, 12], this is the first study to report the differential effects of chronic membrane depolarization on cardiac K+ channel activities. It has important implications for understanding the regulation of K+ channel expression by electrical activity.

Maintaining cardiomyocytes in a depolarized state was developed by adding additional K+ to culture medium, as previously described [12, 16]. To equalize the extracellular osmolarities between control and treatment groups, control culture medium was supplemented with the same volume of NaCl. These manipulations would result in a slightly increased extracellular osmolarity as compared with that of normal growth medium. Therefore, the more appropriate experimental protocol would be to reduce [Na+] in culture medium by the amount corresponding to the increase in [K+]. In the present study, we observed no morphological differences between the cells under study and those age-matched cells incubated continuously in normal growth medium. This implies that a small increased osmolarity imposed by 20 mM K+ exerts a minor effect on cultured cells.

Ion channel development has been characterized in mammalian hearts [1, 5, 7]. During rat heart development, Ito could not be recorded in fetal cardiomyocytes [28] but demonstrated enhanced expression after birth [3]. Considering the age-related increase in resting potential around perinatal stage [4], there is a possibility that the membrane resting potential itself influences the developmental modulation of cardiac ion channel activity. By elevating the K+ concentration in growth medium for 72 h, a chronic depolarizing stimulus, we found reductions in Ito density and relative Ito/Iss amplitude associated with a significant decrease in RP. Although the Ito in depolarization-treated cells is likely to be inactivated at steady state due to the relatively positive RP, the suppressed Ito density may partially contribute to APD prolongation and a small increase in overshoot amplitude (Fig. GR1). In addition, these results are the opposite of the observed increases in Ito and Ito/Iss during heart development [1, 3, 14], suggesting a dedifferentiated change in Ito induced by chronic membrane depolarization. This may imply that the membrane potential level can affect Ito expression in developing cardiomyocytes. Similar results have been obtained from the regulation of A-current in cultured chick parasympathetic neurones [16]. On the other hand, the majority of previous experimental studies have shown a common decrease of Ito density in myocardial infarction and hypertrophied failing heart (see reviews [2, 6]). The suppression of Ito induced by membrane depolarization may be involved since reduced resting potential had been observed in these preparations [2, 8, 9]. Recently, Lee and co-authors [29] reported stage-dependent changes of Ito in rat ventricular cells subjected to monocrotaline-induced myocardial hypertrophy. Ito density increased at an early stage but decreased in the terminal stages associated with heart failure. In view of the unchanged resting potential in mild and moderate hypertrophied myocytes (see review[2]), modulation of Ito by chronic membrane depolarization may occur only during the severe heart failure.

The reduction in whole-cell Ito may result from a shift in the voltage-dependence of steady-state activation or inactivation. We observed that the voltage-dependence of Ito activation and inactivation curves did not differ in control and treatment groups. The kinetics of whole-cell current decay could be fitted with a double-exponential function, and the rates of decay did not change with the induction of chronic membrane depolarization. There were also no differences in the time course of Ito recovery from inactivation. The absence of changes in these characteristics would suggest that a reduction in channel number or single channel conductance is the reason for decreased Ito density in the cells treated with chronic membrane depolarization.

Numerous investigators have described two pharmacologically and kinetically distinct components of IK in guinea pig [24], canine [25] and mouse [7] ventricles as well as human atrial and ventricular myocytes [26, 27]. In rat ventricular myocytes, only Abrahamsson et al. [28] reported that IK recorded from fetal and adult rats could be blocked by almokalant, a IKr blocker, indicating the existence of IKr. In the present study, we first provided evidence suggesting that IK in neonatal rat ventricular myocytes is comprised of two distinct components, the properties of which correspond to those of IKr and IKs as described by Sanguinetti and Jurkiewicz [24] in guinea pig heart cells. In contrast to the changes in Ito, a chronic depolarizing stimulus induced a slight increase in IKr density, and a marked increase in IKs that could be partially attributed to a small hyperpolarizing shift in the voltage-dependent activation curve. Given the presence of 10 mM EGTA in the intracellular solution, it would be unlikely that the increase in IK is a secondary change resulting from a sustained rise in the cytoplasmic free Ca2+ concentration caused by high-KCl incubation [30], which may subsequently enhance IK via a calmodulin-dependent pathway [31].

Although we have not definitively distinguished whether the altered K+ channel activity induced by chronic membrane depolarization is the result of a change in the number of functional channels, or a change in structure and function of normally expressed channels, or a combination of both, previous reports have demonstrated that membrane depolarization and experimental myocardial infarction can affect K+ channel gene expression [11, 12, 32]. Therefore, it would be reasonable to consider that the observed differential effects of chronic membrane depolarization on macroscopic K+ currents may be caused by a change in the expression of functional channel proteins due to a change in gene transcription. However, our observations regarding a decrease in Ito and an increase in IK would appear to contradict the results of prior studies, which showed that the membrane depolarization enhanced the expression of rapidly inactivating Shaker K+ channel Kv1.4 mRNA related to Ito [11] and conversely inhibited Kv1.5 gene expression related to delayed rectifier current[12]. Thus, enhanced transcription of Kv1.4 or decreased expression of Kv1.5 caused by membrane depolarization may not necessarily be linked to a corresponding increase in Ito and a decrease in IK. Other mechanisms may be involved in chronic membrane depolarization-induced alterations in gene translation or post-translational modification.

This study clearly described the differential effects of chronic membrane depolarization on K+ channel activities in cultured newborn rat cardiomyocytes. The findings might be relevant to disease states (such as chronic heart failure and myocardial infarction) in which cardiac electrical activity is abnormal. However, it should be noted that these results obtained from an in vitro intervention are somewhat difficult to be extrapolated directly to clinical conditions. For example, substantial membrane depolarization and a high extracellular K+ concentration in ischemic areas are restricted to the acute phase of myocardial infarction for several hours [10]. Serum potassium up to 20 mM for 3 days is rarely present in patients. Although a decrease in RP had been reported in many experimental models of heart failure [8, 9], no significant difference in RP between cells isolated from normal hearts and from patients with terminal heart failure was observed [33]. Thus, further study is required to elucidate the effects of high-K+ culture at relatively lower concentrations and short-term administration of membrane depolarization.


   Acknowledgements
 
This work was supported by a Grant-in-aid for Cooperative Research from the Japanese Ministry of Education, Science, Sports and Culture (No. 07407073).


   Notes
 
* Corresponding author. Tel. +81 52 7893871; Fax +81 52 7893890; E-mail: g940015d@eds.ecip.nagoya-u.ac.jp Back


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

  1. Apkon M, Nerbonne J. Characterization of outward K+ currents in single ventricular myocytes from developing and adult rat heart. Biophys J (1986) 49:55a.
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