Sodium current in freshly isolated and in cultured single rat myocardial cells: Frequency and voltage-dependent block by mexiletine

J Mol

Cell

Cardiol

15, 431-444

Sodium Current Rat Myocardial

Steffen

(1983)

in Freshly Isolated and in Cultured Cells : Frequency and Voltage-Dependent Block by Mexiletine Hering,

Rolf

Bodewei

and

Albert

Single

Wollenberger

Division of Cellular and Molecular Cardiology, Central Institute of Heart and Circulation Research, Academy of Sciences of the GDR, 1115 Berlin-Buch, German Democratic Republic (Received 3 August 1982, accepted in revised form 12 January

1983)

S. HERING, R. BODEWEI AND A. WOLLENBERGER. Sodium Current in Freshly Isolated and in Cultered Single Rat Myocardial Cells: Frequency and Voltage-Dependent Block by Mexiletine. 3ournal of Molecular and Cellular Cardiolopy‘( 1983) 15, 431-444. Effects of mexiletine on the rapid inward sodium current (Zxa) were studied in freshly isolated single cells of the ventricular myocardium of adult rats and in single cultured ventricular muscle cells of newborn rats. The current was measured in internally perfused, voltageclamped cells by a single suction pipette technique. Mexiletine was applied extracellularly. INS was reduced by the drug in both preparations when the membrane was depolarized to -20 mV by short (8 ms) pulses delivered at a frequency of 0.1 Hz from a holding potential of - 100 mV. Mexiletine in a concentration of 50 p.~ diminished the Zxa under this condition by 70 5 8% (mean & s.D.) in the adult myomyocardial cells. A nearly equal reduction of the current (65 + 10%) was caused in the neonatal in the presence of 10 and cardial cells by 15 KM mexiletine. A use-dependent block of I xa was produced of 20 to 30 FM mexiletine, respectively, in the neonatal and the adult myocardial cells by repetitivse depolarizing test pulses applied at frequencies between 1 and 7 Hz. Prolongation of the pulse duration The frequency-dependent from 10 to 100 ms enhanced the use-dependent block of 2 xa in both preparations. action of mexiletine could be modulated by lOO-ms hyperpolarizing prepulscs from -80 to - 140 mV. The time course of the use-dependent block (prepulse off) and unblock (prepulse on) was monitored. The slope of the inactivation curve of Zxa, in the neonatal heart cells was reduced in the presence of mexiletine and the midpoint of the curve was shifted in the hyperpolarizing direction. These findings are interpreted as suggesting that binding of mexiletine to the sodium channel of the rat myocardial cells studied is enhanced when the cell membrane becomes depolarized. KEY

Suction

WORDS:

pipette;

Mexiletine; Intracellular

Isolated myocardial cells; Cultured perfusion; Frequency dependence;

Introduction Mexiletine, a compound structurally related to lidocaine, has in the past few years emerged as a valuable agent in the treatment of cardiac arrhythmias [30, 361. In order to arrive at an understanding of the mode of action of this drug a number of investigators have studied its effect on cardiac membrane currents with the aid of standard microelectrode technique [II, 12, 22, 23, 39, 411. It could be shown that mexiletine decreases the maximum rate of 0022-2828/83/070431+14

$03.00/O

cardiac Voltage

myocytes; Voltage clamping; dependence; Sodium current.

rise of the action potential [12, 23, 391. This effect was frequency-dependent [IO, 1?2]. On the other hand, conflicting findings were reported concerning the influence of the drug on the effective refractory period [II, 12, 23, 391. Some data strongly suggest that mexiletine may prolong the recovery of the fast sodium inward current [12, 391. The drug may also reduce the slow calcium inward current [ 121. In view of the prominence that mexiletine has acquired as an antiarrhythmic agent it 0

1983 Academic

Press Inc.

(London)

Limited

432

S. Hering

is perhaps surprising that its action on cardiac membrane currents has not yet been investigated under voltage clamp conditions. One of the reasons for this apparent reluctance may be the fact that multicellular preparations of heart muscle are not very satisfactory objects for voltage clamp experiments [Z, 241. Single heart muscle cells are more suitable in this respect. Such cells have indeed successfully been employed in recent voltage clamp studies of the rapid Naf inward current [3, 6, 34, 35, 44, 4.51, making use of adaptations of the suction pipette technique for internal perfusion that was introduced by Krishtal and Pidoplichko [33] and Lee et al. [33u] for the study of membrane currents in nerve cells. In continuation of work just mentioned [3] the action of mexiletine on some of the properties of the rapid Na+ current was examined in the present experiments both in freshly isolated and in cultured single muscle cells of the rat heart under the condition of internal perfusion and voltage clamping. Partly with the view at Hille’s [ZO] findings regarding the effect of antiarrhythmic drugs on the rapid Na+ current in myelinated nerve fibers special attention was paid to the frequency and voltage dependence of the action of mexiletine on the heart muscle cells. Methods cells were Single isolated myocardial employed. They were derived either from the ventricles of adult rats and used within a few hours after isolation or from the ventricles of newborn rats and used after several days of cultivation. Voltage clamp analysis of the rapid inward sodium current was performed at 22°C in internally perfused cells as detailed previously [3], with a few alterations indicated below. Isolation of adult rat myocardial cells Single ventricular myocytes were obtained according to a modification [3] of the method of Rais et al. [37] by perfusion of the excised hearts of heparinized adult male Wistar rats with a collagenase-containing solution, followed by disintegration of the minced ventricular tissue during gentle

et al. shaking in either ‘extracellular’ solution I (130 mM NaCl, 4 mM KCl, 0.5 mM MgCl,, 0.9 mM CaCI,, Il. 1 mM glucose, 10 mM Tris-HCl of pH 7.4) or ‘extracellular’ solution II (100 mM NaCl, 4 mM KCl, 0.5 mM MgCl,, 0.9 mM CaCl,, 11.1 mM glucose, 30 mM Tris-HCl of pH 7.4). Many of the isolated, not spontaneously contracting myocytes were capable of maintaining their cylindric shape, their smooth outline, and their cross-striations for as long as 5 to 7 h and could be used for voltage clamping. Culture of neonatal rat heart cells Cells of the heart ventricles of newborn rats were isolated and cultured by methods described elsewhere [13, 141. Calf serum (10%) and insulin (100 pU/ml) were added to culture medium SM 20-I. After 2 to 4 days ‘of cultivation as monolayers the cells were detached from the substratum by repeated washing of the monolayers with a stream of culture medium. The detached myocardial cells assumed a spherical shape (diameter 12 to 18 PM) and could be used for voltage clamping for as long as 24 h. The voltage clamp experiments with the cultured rat myocardial cells were carried out in ‘extracellular’ solution III (130 mM NaCl, 4 mM KCl, 0.5 mM MgCI,, 1.8 mM CaCl,, 11.1 mM glucose, 10 mM Tris-HCl of pH 7.4). Ionic current measurements The suction pipette technique developed in a voltage clamp study of neuronal cells and adapted to the measurement of ionic currents in isolated mammalian heart muscle cells [3, 44, 451 was used. A single isolated rat myocardial cell was placed in a test chamber filled with one of the extracellular solutions and was sucked under visual control onto the tip of the suction pipette. The suction pipette was perfused with ‘intracellular’ solution: 140 mM Tris phosphate of pH 7.3. The stimulation and reference electrodes (Ag-AgCl/S M KCl) were placed in the outilow section of the V-shaped pipette. The portion of the heart cell membrane protruding into the tip of the pipette was destroyed mechanically with a nylon fiber present inside the pipette.

Mexiletine

on Na

Current

For the voltage clamp experiments an electronic circuit was used that could compensate for series resistance (Rs) and leakage resistance (RL) [44].

of 1~~

Measurements

in fresh& isolated adult rat myocardial cells

In the experiments with adult myocardial cells the diameter of the suction pore was 10 to 16 pm and the wall thickness of the tip of the pipette was 20 to 30 pm. As described in our earlier work [3], the cells were largely drawn into the pipette and the membrane inside the pipette was destroyed as described above. By this procedure only a small portion of the total membrane area was left for voltage clamping, which may be the reason for the small current magnitudes in our experiments (see [3]) as compared to those in the study of Brown et al. [S, 61. The Rs values computed from the time constant of the capacitative current flow were 200 to 400 kQ; RI, approximated 10 MQ. Only preparations found to possess the following properties were selected for voltageclamp analysis: (1) Absence of ‘delayed activation kinetics’ of INa in the negative resistance range (see [3]). (2) An estimated Rs/RL-relationship that guaranteed a satisfactory temporal resolution for the measurements to be performed [3]. (3) A good

in

Single

Myocardial

Cells

433

voltage control of the I-V curves for peak 1~~ in the negative resistance range; the voltage for maximum current had to be :30 to 40 mV more positive than threshold (see Figure 4). Adequacy of voltage control during the measurements of 1~~ was asce:rtained by deactivating the current at its peak and observing the speed of its decay following the repolarization (Figure 1). 1~~ measurements in cultured neonatal rat myocardial cells In the experiments with cultured neonatal rat heart cells the type of suction pipette described in [32] was used. The diameter of the suction pore was 7 to 8 pm and tlhe wall thickness of the pipette was 5 to 8 ~.uI. The preparations were selected according to the criteria listed above for the experiments with adult cells. RL and Rs were determined to be 60 to 80 ML! and 400 to 800 kQ respectively. A rapid establishment of the membrane potential was the consequence of the resulting R~/RL relationship and of the small clamped membrane area. The ionic currents were displayed on a dual beam storage oscilloscope (Tektronix5113) and photographed with a Polaroid camera (C-5B-Tektronix). For command pulse generation a programmable 2-channel

\ \\

----

\

-\

--

f

\

\ \

a

L

-I

FIGURE 1. Deactivation of INa in (a) an adult and (b) - 100 mV; pulse potential = -40 mV. Calibration: vertical bars, 1 ms for (a) and 0.5 ms for (b). Note the rapid decay factory voltage control.

L

b a neonatal myocardial cell. Holding potential = bars, 1 nA for (a) and 0.3 nA for (b); horizontal of INa. following repolarization, indicating satis-

434

S. Hering

et al.

stimulator (Physiovar, Alvar-TR) was used. Simultaneously the currents were digitized every 10 ps using a universal wave form analyzer system (EMG-Typ TR-4910) and stored on tape recorder for off-line analysis. The slow inward current was blocked by 0.5 mM CdCl, added to the extracellular solutions. Mexiletine (1-methyl-2-(2,6-xylyloxy)ethylamine) hydrochloride was kindly supplied by the Vienna office of C. H. Boehringer Sohn, Ingelheim. It was added to the extracellular solutions.

Results

-__

&fleets of mexiletine on the I-V relationshi@ of 1~~ at low frequency of stimulation INa measurements in freshly isolated adult rat myocardial cells. Figure 2(a) shows a family of voltage clamp currents from a single internally perfused adult heart muscle cell surrounded by extracellular solution I. The test pulses applied to this cell gave rise to a sodium inward current that reached a maximum value at -30 mV after 1.5 ms. As shown in this figure, no outward currents could be detected in the investigated potential range after 10 min of internal perfusion with the intracellular solution. Following a 5 min exposure of the cell to 50 pM mexiletine the current was reduced by about 75% (Figure 2b). The average reduction of &, caused by 50 PM mexiletine in five cells was 70 f 8% (mean f s.D.). A 5 min wash restored the current to its control value (Figure 3). In these experiments short (8 ms) depolarizing pulses were applied at a low frequency (0.1 Hz) from a holding potential of - 100 mV. The current-voltage relations in the experiments in Figures 2(a) and (b) are shown in Figure 4. After treatment with mexiletine there was a shift of the half maximum activation in the I-V curve from -47 mV to -38 mV. The reversal potential of 1~~ could not be determined under present conditions, because the solution used for intracellular perfusion did not contain Na+ ions. The block of the sodium channels could be partly reversed by shifting the holding

- -- - --~ -

(b) FIGURE 2. Families of 1~~ curves in an adult rat myocardial cell (a) before and (b) after a 5-min exposure to 50 FM mexiletine in extracellular solution I. The cell was depolarized by 8-ms pulses applied at a frequency of 0.1 Hz. Holding potential VH = -100 mV. Calibration: vertical bar, 2 nA for (a) and 1 nA for (b), 20 mV; horizontal bar, 1 ms.

potential (- 100 mV) to more negative values (- 120 to - 140 mV) and also by applying hyperpolarizing prepulses 100 ms in duration (not shown). 1~~ measurements in cultured neonatal rat heart current records cells. Figure 5a shows obtained during voltage clamp of a neonatal rat heart cell cultured for 3 days. The threshold potential of the current was observed to be -65 mV and the current reached its maximum value at -20 mV. The rapid inward current was dependent on the extracellular sodium concentration and could be blocked by 5 PM tetrodotoxin. The currents registered following a 5-min exposure to 15 PM mexiletine are shown in Figure 5b. In five cells the amplitude of

Mexiletine

on Na

Current

the IN% was reduced by 40 f 10% (mean f SD.) when 10 PM mexiletine was added to the extracellular solution. The half maximum activation of the 1~~ (Figure 6) shifted from -48 mV under control conditions to -45 mV after mexiletine treatment. Figure 5(c) depicts currents measured in the same cell used for the experiment presented in Figures 5(a) and (b). In this case the holding potential was - 140 mV. As shown in the figure, the hyperpolarization partly removed the sodium channel block.

in

Single -60

Myocardial -60

-40

Cells -20

0

8

FIGURE 4. Current-voltage relationship for peak Zxa in the experiment shown in Figure 2. 0, before mexiletine; 0, after mexiletine.

(b)

FIGURE 3. Reversibility of the action of mexiletine on the rapid inward sodium current in an adult myocardial cell. (a) ZxB before (1) and after 5 min (2) of exposure to 60 w mexiletine. (b) Zxa of the mexiletine-treated cell after washing for 2 min (l), 3 min (Z), and 5 min _ (3). Holding potential = - 100 mV; pulse potential = -30 mV. Calibration: vertical bar, 2 nA; horizontal bar, 1 ms.

FIGURE 5. Families of Zxa curves of a neonatal rat myocardial cell cultured for 3 days; (a) before and (b) after a 5 min exposure to 15 WM mexiletine in extracellular solution III. The cell was held at a holding potential of -100 mV and depolarized by 8-ms test pulses applied at a frequency of 0.1 Hz in IO-mV steps from -65 to +45 mV (c): As in (b), but VH shifted to -140 mV. Calibration for (a.), (b) and (c): Vertical bar, 0.5 nA; horizontal bar, *1 Ins.

S. Hering

436

I&Al FIGURE peak INa and (b).

6. Current-voltage of the experiment l , before mexiletine;

relationship for the shown in Figures 5(a) 0, after mexiletine.

Frequency and voltage dependence of the action of mexiletine on 1~~ Freshb isolated myocardial cells. It is well established that in myelined nerve fibers a block of sodium channels by local anesthetics is enhanced by prolonged or repetitive depolarization [lo, 18, 19, 26, 27, 28, 421. It seemed therefore of interest to see, whether this might also be the case in isolated myocardial cells treated with mexiletine. In fact, this possibility was suggested by our finding (see Figure 5c) that the blockade by mexiletine of the Na channels of the isolated heart muscle cells could partly be removed by hyperpolarizing prepulses or by shifting the holding potential in the hyperpolarizing direction. The possibility that Na channel block by mexiletine may be frequency-dependent was examined in adult rat heart cells that were treated for 5 min with 25 FM mexiletine. The drug led on application of IO-ms depolarizing pulses from a holding potential of -80 mV to -20 mV at 0.1 Hz to a ‘tonic’ reduction [42] of IN, by 16 f 594 (mean & SD.). Trains of pulses of different duration and spaced at different intervals were then applied. After each train of pulses a rest period of 30 s was interposed during which the current amplitude returned to the initial value. Figure 7(a) presents the results of an experiment in which trains of IO-ms test

et al. pulses were applied in the presence of 25 PM mexiletine at frequencies ranging from 1 to 6 Hz. Peak INa is plotted here as a function of time. The figure shows that stimulation at high frequencies enhanced the blocking effect of mexiletine to a greater extent and sooner than did stimulation at low frequencies. At a stimulation frequency of 3 Hz, for example, INa was diminished by an additional 23q/, after 3 s of stimulation. As shown in Figure 8(a), the peak sodium current. was barely reduced in the absence of mexiletine even at frequencies higher than 5 Hz when IO-ms pulses were applied. The next experiment was concerned with the effect of pulse duration on the frequencydependent block by mexiletine of peak Na currents. After a 1 min rest period, during which the membrane was clamped to -80 mV and stimulated with lo-ms pulses at 0.1 Hz, the peak current had returned to the pre-train value. The test pulse duration was now extended to 100 ms. As seen in Figure 7 (b), the peak current was reduced by an additional 2094 when the 100 ms test pulses were applied at a frequency of 1 Hz. In Figures 8(a) and (b) the amplitude of INS at the end of each pulse train is plotted against the pulse frequency. In the absence of mexiletine a frequency-dependent inhibition of INa of 7 and 17:/,, respectively, was produced by 30-ms and IOO-ms depolarizing pulses applied at 5 Hz (Figure 8a). When 20 pM mexiletine was present in the extracellular solution, the block was enhanced, respectively, to 28 and 4294 (Figure 8b). Finally, the frequency-dependent inhibition by mexiletine of the Na+ channel in adult rat myocardial cells was studied by a procedure introduced by Courtney [IO]. In this pulse program the frequency-dependent effects of a given drug can be modulated by changes in the membrane potential during hyperpolarizing prepulses (see Figure illustrates the frequency9). This figure dependent inhibition and deinhibition of the sodium

channels

in

a

single

heart

muscle

cell. In these experiments the holding potential was -80 mV and continuous trains of IO-ms test pulses to -20 mV were applied at a frequency of 5 Hz. Prior to the first pulse train the cell was conditioned by a train of test pulses that was

Mexiletine

0

on Na

Current

2

3

I

in Single

5

4 Time

FIGURE 7a. Use-dependent ization intervals. The cell was The frequency of depolarization were then applied from VW of

0

FIGURE test pulses 12 nfL

7b. Use-dependent to -20 mV from

I

Myocardial

6

437

Cells

7

8

(s)

block of peak INa in a mexiletine-treated single heart cell at different depolarincubated for 5 min in extracellular solution II containing 25 FM mexiletine. during this period was 0.1 Hz. Trains of lo-ms test pulses of different frequencies -80 mV to -20 mV. Normalized current values, pre-train peak 1~~ = 1.

2

3

4

5

6

7

8

9

Time

(s)

IO

block of peak INa of the cell shown VH of -80 mV. Normalized values,

preceded by IOO-ms hyperpolarizing preMexiletine (30 PM) pulses to - 140 mv. diminished the peak sodium current to 80 * 7% (mean & SD.) of the control value. Upon subsequent removal of the prepulses 1~~ decreased progressively over the next 10 test pulses to 25 f lo:/, of the control value (upper part of Figure 9). The prepulse was then applied again and, as shown in the lower part of Figure 9, IN& returned gradually to the previous value. Figure 10 shows the time course of this ‘use-dependent’ block [IO] of the peak sodium current. With this procedure, which also has been employed by other authors in the investigation of the action of local anesthetics on myelined nerve fibers and skeletal muscle fibers [18, 19, 401, the time course of the voltage-dependent effects of

II

12

in Figure pre-train

I3

iti

I5

16 I7

5a, induced by applying lOO-ms peak INa = 1, corresponding to

mexiletine at a given frequency of stimulation could be followed (Figure 10). In some experiments (four cells) the blocking of sodium channels by mexiletine following a train of depolarizing pulses became so strong that no current could be registered after removal of the hyperpolarizing prep&e. Figure 11 depicts the gradual restoration of IN, in one of th,ese experiments. Cultered neonatal rat myocardial cells. Figure 12 shows the use-dependent block of INS by 10 PM mexiletine in a cultured myocardial cell from a newborn rat. In this experiment pulses of 100 ms duration were applied at a frequency of 5 Hz. As was the case in the experiments with adult myocardial cells (Figure 8a), a use-dependent inhibition was evident prior to mexiletine treatment. Again,

438

S. Hering

et al.

0.5C (a)

/ (b)

1

6 Fdquency

of stimulation

7

Test pulse

(Hz)

FIGURE 8. ZNa peak values at the end of pulse trains plotted as a function of pulse interval. The cell was depolarized to - 20 mV from VH of - 80 mV with test pulses of 10 ms (O), 30 ms (A), and (0) 100 ms (a) without mexiletine and (b) in the presence of 20 FM mexiletine in intracellular solution II.

-20 mV

-8OmV

Pulse

mexiletine accentuated the use-dependent effect (Figures 13a and b). Prolongation of the pulse duration enhanced the frequencydependent inhibition of INa (Figures 13a and b). The steady state inactivation curve of the INa of a representative cultured neonatal rat heart cell is shown in Figure 14. The prepulse duration was 500 ms. The midpoint potential (V,) of the curve was -87 mV and the curve fits the function 1

h, = 1 +

for k = 7.6 mV,

(ev-Vh,k)

where k is the slope of the curve. Mean values & S.D.S determined for V, and k in and 7.0& 6 experiments were -79h5 0.8 mV, respectively. Since the action of mexiletine on IN% is clearly voltage-dependent (Figure 5c), it is not justified to call the curve after mexiletine treatment a steady state inactivation curve. In agreement with Hille [18] this curve can simply be called ‘inactivation curve’. The midpoint voltage

-1f.O mV

on programme

1

FIGURE 9. Use-dependent block and unblock of peak ZN~ in a single adult heart muscle cell incubated in external solution II. Ten-ms test pulses to -20 mV were applied at a frequency of 5 Hz following tonic depression of ZN~ by 30 [LM mexiletine to 80% of the control amplitude. Then, as shown in the pulse programme, a lOO-ms hyperpolarizing prep&e was periodically turned off (block) and on (unblock); VH = -8OmV.

of the inactivation curve shown was - 102 mV and k was 11 mV. The corresponding average values (means f S.D.S from 4 experiments) were -93 + 10 and 10 f 1 mV. Discussion Changes in the rapid sodium inward current are believed to underlie the therapeutic action of antiarrhythmic drugs of the local anesthetic type on the heart [15, 20, 261. In the majority of previous studies the sodium channel blocking action of local

Mexiletine

on Na

Current

0 OO

0

I

Cells

439

000

2 Time

Myocardial

reduced in the presence of mexiletine over the entire potential range studied (Figures 4 and 6). The reversibility of the inhibitory effect of mexiletine on lx, is documented by the results shown in Figure 3 (see also [17]). The asymmetry of the I-V curve (Figure 4) in the adult cells which was noticed when the current magnitude was reduced by mexiletine to about 25% of the control value suggests that the voltage control

0

00

in Single

L

I

3

4

5

1.01J:::::‘..-

(5)

FIGURE 10. Time course of the use-dependent block and unblock of the peak ZN~ in the experiment shown in Figure 9. Normalized current peaks, the peak current after a train of prep&es of - 140 mV being given the value of 1; Vn = -80 mV. 0, extracellular solution II; 0, 30 FM mexiletine in solution II.

anesthetics on heart muscle preparations was studied only indirectly, using the maximum upstroke velocity (dl;Tldt),,, of the cardiac action potential .as a criterion [IO, 151. However, according to both theoretical considerations [?9, 431 and experimental data [7] V,,,,, is a nonlinear measure of sodium conductance in heart muscle preparations. In recent experiments with isolated myocardial cells of adult rat it was shown that the temporal resolution of voltage clamp measurements can decisively be improved with the aid of the technique of internal perfusion and attention was called to the potential usefulness of this method in the study of the influence of antiarrhythmic and other drugs on the myocardial Ixa [3, 341. In the present study, undertaken with internally perfused and voltage clamped single myocardial cells, it could be shown, first of all, that at a membrane potential of - 100 mV at which in both the freshly isolated adult and the cultured neonatal myocardial cells only a small minority of the Na channels are in inactivated state the h, curve passes through a value near 1.0 (Figure 14; see also [3]). IN& was severely

I . 0

I

OQQ.QiLQIMo 2

3

4

51

Time(s)

FIGURE 11. Effect of 40 FM mexiletine on the time course of the use-dependent block and unblock of ZN~ in a cell incubated in external solution II. Trains of depolarizing pulses of 10 ms duration to -20 mV were delivered at a frequency of 5 Hz from Vu -80 mV. In the presence of the drug the block became so strong that after turning the prepulse off the Zxa was completely blocked. The pulse program was the same as in the experiment of Figure 9. 0, external solution II; 0, 40 ~xM mexiletine in solution II.

FIGURE 12. Use-dependent block of Zxa in a neonatal rat myocardial cell cultured for 3 days and incubated in external solution III containing 10 ;FM mexiletine. The cell was depolarized from VH = -100 mV to -20 mV by a train of lOO-ms test pulses delivered at a frequency of 5 Hz. Calibration: vertical bar. 0.5 nA; horizontal bar, 1 ms.

S. Hering

05k----+-L. (a) Time

Time

2

t-s)

I (b)

et al.

2

I

I

-140

-120

-100

-80

-60

mV

(s1

FIGURE 13. Kinetics of block of Na channels with mexiletine. The cell was held at Vn = - 100 mV and depolarized to -2OmV (a) by lo-ms and (b) by IOO-ms test pulses. 0, extracellular solution III; 0, 10 v mexiletine in solution III.

FIGURE 14. Inactivation of Zm, at -2OmV in a neonatal myocardial cell on the third day of cultivation before (0) and after (0) addition of 10 FM mexiletine to external solution III. Vn = -80 mV. Pulse frequency 0.1 Hz; prep&e duration 500 ms.

during the current measurements in the adult myocardial cells may be affected by the influence of Rs to a greater extent than is suggested by the theoretical calculations of Bodwei et al. [3]. However, in our in which a single suction experiments, pipette was used, the voltage control was considerably better than that reported by Brown et al. [IO] (cf. Figure 6b in [IO] with Figure 3a in [3] and Figures 3a and 5a in the present study). We believe that in our experiments, performed with the type of suction pipette described in [3], a decisive factor for the satisfactory voltage control in the negative resistance range was a clamped membrane area (see Methods) sufficiently small to reduce the current amplitude to maximally 15 nA. It has been reported by several authors [lo, 18, 19, 38, 40, 421 the repetitive use of sodium channels caused by short depolarizing voltage pulses progressively reduces the currents amplitude of sodium during exposure to local anesthetics and similarly acting antiarrhythmic drugs. A similar

effect of the frequency of depolarization was presently found in freshly isolated adult and in cultured neonatal single myocardial cells treated with 20 to 30 and with 10 pM mexiletine, respectively, and depolarized by pulses of different length (Figs. 7a, b, and 13). In this context it is to be noticed that according to Khodorov et al. [28] and Hille [18, 191 local anesthetics promote the inactivation of the Na channel. The present observation of a use-dependent inhibition of I~J~ in the absence of the drug confirms the results of Lee et al. [34]. This inhibition was enhanced by prolongation of the pulse duration (Figures 7a, 8, and 13a, b) As a comparison of Figure 8a and b indicates, the use-dependent inhibition was enhanced by mexiletine and could be induced in the presence of the drug at lower frequencies and shorter depolarizing pulses than was the case in its absence. Additional experiments are required in order to establish which factors are responsible for the usedependent inhibition of Ixa. in the absence of mexiletine and whether the mexiletine-

Mexiletine

on Na

Current

induced use-dependent effect is an enhancement of this process or a separate phenomenon. Recently it was shown that the reactivation of IN& in myocardial cells is markedly slowed under the influence of mexiletine [17]. In view of the fact that the reactivation process of INa is voltage,dependent and slowed by depolarization [5, 61 the retardation of the reactivation process by mexiletine may be held partly responsible for the observed enhancement by the drug of the use dependence of INa. A more detailed study of the reactivation process of INa and of the frequency-dependent inhibition of this current may help to resolve the question whether the frequency-dependent inhibition might result from a prolongation of the reactivation time of INa. Our finding that a use-dependent inhibition of INa under the influence of mexiletine increases with an increase in pulse duration suggests that this drug, like the classical local anesthetics, interacts preferentially with inactivated Na channels. As could be seen in Figures 8(a) and (b), a rhythmic depolarization of the membrane by IO-ms trains of pulses applied either in the absence or presence of mexiletine at a frequency of 1 Hz failed to reduce INa, whereas with lOO-ms pulses applied at the same frequency in the presence of 20 PM mexiletine a usedependent depression of this current was clearly evident. This finding corroborates earlier reports about an interval-dependent inhibition of the upstroke of the cardiac action potential by mexiletine [II, 22, 391. With the aid of the pulse program presented in Figure 9 it could be shown that the frequency-dependent block of INa in mexiletine-treated heart muscle cells is modulated by changes in the membrane potential. The observed time course of the block (‘off reaction’) may be taken as an indication of a depolarization-induced increase in the binding of mexiletine to the sodium channel, while the time course of the ‘unblock’ (‘on reaction’) speaks for a facilitation of the dissociation of the drug from its binding site. Preliminary experiments with both the adult and the neonatal rat myocardial cells indicate. that the changes in the time to peak of IN% in Figure

in Single

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9 reflect an interference by the capacitive current in this particular experiment (compare with Figure 12). The experiments with the neonatal cultured heart cells have demonstrated that there are no fundamental differences between effects of mexiletine on these and on the freshly isolated adult myocardial cells. The fact that the cultured neonatal cells were more sensitive to mexiletine than were the freshly isolated adult cells might be ascribed to differences in the developmental state of the heart or, alternatively, to damage of the adult cells by the enzymic dispersion procedure. The concentration of mexiletine used in the experiments with the cultured neonatal rat myocardial cells (10 to 15 IAM) approach in magnitude the concentrations (2 to 10 FM) that have been reported to be present in the blood plasma of human patients [36] and of dogs [I] during treatment of cardiac arrhythmias by this drug. The results of the present study leave little doubt that the depressant effect of mexiletine on INa in adult and in cultured neonatal rat heart cells is dependent on membrane voltage and on the frequency of depolarization. As was shown for the adult cells, the inhibition of I~Q, by mexiletine in the neonatal cells was enhanced when the membrane was depolarized (Figures 13a and b) and the block could be removed by hyperpolarizing the membrane (Figure !jc) . According to results shown in Figures 5(b) and (c), 9, 10, 11, and 14 the dissociation of mexiletine from Na channels may be voltagedependent. In line with the view of Schwartz et al. [40] the changes in the INa inactivation curve depicted in Figure 14 may be taken to reflect changes in the inactivation mechanism in Na channels to which mexiletine molecules are bound. Similar changes in the steady state inactivation curve of INa have been found in the node of Ranvier under the influence of lidocaine and other local anesthetics [20]. These changes may represent instances of the drug-induced slow Na inactivation phenomenon described by Khodorov et al. [28]. From Figure 14 it may be gathered that the sodium channel block by mexiletine differs in its mode from that produced by compounds such as tetrodotoxin [7], quarternary derivatives of local

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anesthetics [28, 421, trimecaine [,!%I, and the antiarrhythmic agents ajmaline [46] and ethmozine [4, 261, all of which are believed to interact preferentially with Na channels in the open state. The question concerning a possible interaction of mexiletine with open sodium channels could not be resolved in the present study. In agreement with Hille’s [ZO] hypothesis of the mechanism of action of antiarrhythmic agents the present results may be interpreted as indicating that binding of mexiletine to the fast sodium channels of rat myocardial cells is facilitated when the cell membrane becomes depolarized. The present study is the first of its kind in which an ionic current was measured in cultured myocardial cells by the suction pipette method for internal perfusion and voltage clamping. These cells are easier to handle by this method than are freshly isolated heart cells. They are truly living cells and not merely surviving as are the isolated non-cultured myocardial cells. Out of 100 cultured neonatal rat myocardial cells detached from their substratum an average of 80 cells can be used for the voltage clamp experiments. The corresponding figure for the freshly isolated adult

et al. rat heart cells is 20. As stated above, no qualitative differences were found between the cultured neonatal and the freshly isolated adult myocardial cells with regard to the properties of the rapid inward sodium current and the action of mexiletine. The present voltage clamp study has dealt with the effect of externally administered mexiletine on the fast sodium inward current of internally perfused myocardial cells. The question arises what effect mexiletine might have on the IN, of the myocardial cells when it is added to the internal perfusing solution. Since local anesthetics have been found in the node of Ranvier [18, 421 and at the motor end plate [25] to be as effective on intracellular as they are on extracellular application, there is all the more reason to raise this question, for which an answer will be sought in experiments to come. Acknowledgements We thank Mr J. Ludecke for skilful electronic engineering, Dr V. I. Pidoplichko for technical guidance, Dr S. V. Revenko for helpful discussions, and Prof. B. Khodorov for constructive comments on the manuscript.

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