Pharmacological Research, Vol. 25, No. 1, 1992
75
ELECTROPHYSIOLOGICAL ACTIONS OF A NEW ANTIARRHYTHMIC DRUG, BISARAMIL, ON ISOLATED HEART PREPARATIONS MARGIT PAROCZAI*, EGON KARPATI*, RAISA MARKO? and VALERIA KECSKEMETIt
*Chemical Works of Gedeon Richter Ltd, Pharmacological Research Centre, Budapest; ?Semmelweis University of Medicine, Department of Pharmacology, Budapest, Hungary Received in final form 5 June 1991
SUMMARY Electrophysiological effects of bisaramil--a new antiarrhythmic drug under clinical trial--were investigated on isolated heart preparations, at a concentration range of 2.3-23x10 -6 M. Bisaramil dose dependently decreased the maximum rate of depolarization (Vmax), action potential amplitude (APA) and overshoot (OS) both in auricle and in papillary muscle of guinea-pig heart. There was no significant and obvious effect on the duration of the action potential and the resting membrane potential was also unchanged. Bisaramil slowed the spontaneous frequency of pacemaker cells in rabbit sinus node preparation due to its inhibitory effect on slow diastolic depolarization (SDD). Bisaramil was able to inhibit slow Ca2+-action potentials induced by isoprenaline on K+-depolarized papillary muscle. Results obtained with transmembrane current measurements revealed that bisaramil inhibited both fast Na+-current and slow Ca2+-current in frog sinoauricular fibres at the same concentration. Bisaramil with a mixed mode of the action seems to be a very promising drug. KEYWORDS:transmembrane action potential, pacemaker type action potential, 'slow Ca2+-action potential', transmembrane ionic currents.
INTRODUCTION Bisaramil is a new antiarrhythmic drug under clinical trials. It is chemically unrelated to any currently available antiarrhythmic drug. Its antiarrhythmic activity was investigated on different experimental arrhythmic models and animal species [1]. The antiarrhythmic properties of bisaramil were also studied [2] and Correspondence to: Dr Margit Paroczai, Chemical Works of Gedeon Richter Ltd, Pharmacological Research Centre, H-1475. Budapest, 10, P.O.B. 27, Hungary. 1043-6618/92/010075-11/$03.00/0
© 1992 The Italian Pharmacological Society
76
Pharmacological Research, Vol. 25, No. 1, 1992
these experiments indicated that bisaramil had a strong class I (local-anaesthetic type) activity accompanied by a significant Ca-antagonistic property. The aim of this study was to investigate the electrophysiological effects of bisaramil on various parts of myocardium (left auricle, papillary muscle, sinoauricular fibres) and furthermore, to obtain additional information about the mode of action of bisaramil.
METHODS
Guinea-pig left auricle or papillary muscle Guinea-pigs of either sex were killed, and the heart was placed in warm (34 °C) oxygenated Locke solution containing (mM): NaC1, 125; KC1, 5.6; NaHCO3, 25; glucose, 11; CaCI2, 2.16. Left auricle or papillary muscle with the free wall of the right ventricle were excised distinctly and pinned in a tissue chamber (3 ml) and superfused with oxygenated Locke solution. The preparation was stimulated through bipolar Pt-electrodes with 1 Hz frequency using square-wave pulses 0.5 ms in duration and twice threshold. Transmembrane action potentials were recorded by means of conventional glass microelectrodes filled with 3 M KC1, having a resistance of 8-20 Mr2. The output of the microelectrode was led to a high input impedance, capacitance-neutralizing amplifier and displayed on an oscilloscope. The maximum rate of depolarization was recorded on the second beam of the oscilloscope using an electronic differentiating network. Oscilloscope tracings were photographed, and evaluated using a microfilm reading instrument. Details of the technique were described previously by Kecskemeti et al. [3]. After measuring the control value of the parameters, the preparation was superfused with Locke solution containing bisaramil in various concentrations. After 30 rain equilibration measuring was repeated. The following parameters were recorded: APA (amplitude of the action potential), RP (resting potential), OS (overshoot), Vm,x (the maximum rate of depolarization), APD20, APDs0, APD90 (duration of the action potential until 20%, 50%, 90% of repolarization). Bisaramil was superfused in three concentrations: 2.3×10 -6 M, 1.2X10-5 M, 2.3X 10-5 M. Results are given as means_+sEM and per cent change compared to control. Data were analysed statistically using paired Student's t-test. Rabbit sinus-node preparation Rabbits weighing 1.5-2.0 kg were killed and the heart rapidly excised. The right auricle was separated from the other parts of the heart and placed in warm oxygenated Locke solution. We isolated a spontaneously beating piece of tissue 10 mm long and 3 mm wide and pinned it without stretching in a tissue chamber. Pacemaker type action potentials were recorded with conventional microelectrode techniques mentioned earlier. Tissue was allowed to equilibrate in Locke solution for at least 1 h. After measuring control parameters, the preparation was superfused with Locke solution containing bisaramil at a concentration of 2.3x 10 6 M. After 30 rain superfusion, the following parameters were recorded: APA
PharmacologicalResearch, Vol. 25, No. 1, 1992
77
(amplitude of the action potential), MDP (maximum diastolic potential), TP (threshold potential), SDD (slow diastolic depolarization), Td (duration of the action potential from the threshold potential to the maximum diastolic potential), CL (cycle length). Results were analysed using paired Student's t-test. Values are mean+_s~M.
K+-depolarized papillary muscle Papillary muscle from the right ventricle of guinea-pigs was isolated after killing animals and then placed in a tissue chamber containing normal Locke solution. Preparation was stimulated through bipolar Pt-electrodes with 1 Hz frequency using square-wave pulses 0.5 ms in duration and twice threshold. After a 1 h equilibration period, the preparation was placed in Locke solution containing 25 mM KCI, frequency was decreased to 0.3 Hz and the stimulus intensity was increased to 10 times higher than threshold potential. Details of the technique have been described previously [4]. Under such circumstances electrical activity ceased within a short time. After 30 rain isoprenaline at a concentration of 1×10 -6 M was given to Locke solution containing 25 mM K + in order to induce 'slow responses'. The effect of bisaramil at a concentration of 2.3×10 -6 M on slow responses was investigated after 15 and 30 min incubation with the drug. Data are given as mean_+szM. Paired Student's t-test was used for statistical analysis of results. Transmembrane ion currents using voltage clamp method Experiments were carried out on frog (Rana esculenta) sinoauricular fibre of 100 ~ m diameter and 3-4 mm length. A voltage clamp technique based on a double sucrose gap arrangement according to Rougier et al. [5] was used. Ringersolution of the following composition was applied: 111.1 mM NaC1, 1.8 mM CaC12, 1.0 mM KC1, 1.2 mM NaHCO3. Temperature was maintained at 18_+0.3 °C. Drug was dissolved in Ringer-solution, and perfused in central compartment. Details of the technique have been described previously [6, 7]. Holding potential was set to 65-75 mV, corresponding to the resting membrane potential of frog myocardium. Membrane potential was clamped between - 3 0 and +140 mV steps of 10 mV. Command pulses of 500 ms in duration were given at a frequency of 0.6 Hz. Double impulses were used to investigate the effect of bisaramil on the inactivation process of Na+-current. The first (conditioning) pulse was of 500 ms duration and its amplitude was continuously changed between - 2 0 and - 4 0 mV as compared to holding potential. The second pulse was of 100 ms duration and its amplitude was +50 mV compared to holding potential. The amplitude of the current (I) measured after the first conditioning pulse was related to the maximal amplitude of the current (/max) measured after the second pulse. All experiments were self-controlled. RESULTS
Guinea-pig left auricle and papillary muscle The effect of bisaramil (2.3-23.3×10 -6 M) on the characteristics of the auricular
Pharmacological Research, Vol. 25, No. 1, 1992
78
action potential are displayed in Fig. 1. The control values of these parameters are shown in Table I. The main effect of bisaramil was seen to be the decrease of Vmax, and OS in a concentration-dependent manner, with no change in RP. A small, but statistically significant decrease of APA was also measured. Bisaramil slightly prolonged the initial and terminal repolarization phase, but this effect was not dependent on the concentration, and was significant only in the presence of the highest concentration of bisaramil. Figure 2 summarizes the results obtained on papillary muscle. Control values of
%1
[~
1.16 x I0 -5 M [~
~
I0~ 0
2.3x IO-e M
APA
2.3 x lO-Sm
RP
OS
V,,,ox
--
o
~,
o.
-io
APD2o
.E
g c
-so
APDso
APDoo
-
--50 --70
---
--90
-X-~
--
Fig. 1. Electrophysiological effects of bisaramil on guinea-pig left auricle. Bars refer to mean per cent change in parameters measured +SEM. APA, action potential amplitude; RP, resting potential; OS, overshoot; Vma×,the maximum rate of depolarization; APD20,50,90,action potential duration to 20, 50, 90% repolarization; n=39.
Table I Pre-drug value of parameters of guinea-pig left atrium APA (mV)
RP (mV)
OS (mV)
99.0 1.25
78.5 0.74
20.5 1.22
APD (ms)
Vmax
APD2o
APDso
APD9o
23.7 2.5
43.6 3.14
64.2 3.66
(V/s) 141.8 5.00
All data are expressed as means of 39 preparations ±SEM. Abbreviations used are: APA, amplitude of the action potential; RP, resting potential; OS, overshoot; V.... maximum rate of depolarization; APD, duration of the action potential; APD20, APDso, APD9o, duration of the action potential until 20, 50, 90% repolarization.
79
Pharmacological Research, Vol. 25, No, 1, 1992
parameters are shown in Table II. Results of the experiments carried out on papillary muscle were similar to those obtained on left auricle, but the decrease of Vmax and OS in the presence of bisaramil was higher. Furthermore, besides the concentration-dependent inhibitory action of bisaramil on the Vmax and OS, its effect on the APA was also concentration dependent. A consistent effect on the duration of the action potential was not found. While significant prolongation of the initial repolarization phase could be observed, the considerable effect on APD90 was only seen in the presence of the highest concentration of the drug. Rabbit sinus-node preparations The effects of bisaramil at the concentration of 2.3×10-6 M on the pacemaker type action potential are summarized in Table III. Pre-drug values of parameters
% 50
30
[ ] ] •2 . 3 [~
x IO -6 M
1.16 x I 0 - s M
[~2.3
io APA ,., O ~' - I O * * * ~ .
x 10-eM
RP
®
*
.E
g g --30
ac: o
**
APD2o
APDso
APD,,o
*P
75
ELECTROPHYSIOLOGICAL ACTIONS OF A NEW ANTIARRHYTHMIC DRUG, BISARAMIL, ON ISOLATED HEART PREPARATIONS MARGIT PAROCZAI*, EGON KARPATI*, RAISA MARKO? and VALERIA KECSKEMETIt
*Chemical Works of Gedeon Richter Ltd, Pharmacological Research Centre, Budapest; ?Semmelweis University of Medicine, Department of Pharmacology, Budapest, Hungary Received in final form 5 June 1991
SUMMARY Electrophysiological effects of bisaramil--a new antiarrhythmic drug under clinical trial--were investigated on isolated heart preparations, at a concentration range of 2.3-23x10 -6 M. Bisaramil dose dependently decreased the maximum rate of depolarization (Vmax), action potential amplitude (APA) and overshoot (OS) both in auricle and in papillary muscle of guinea-pig heart. There was no significant and obvious effect on the duration of the action potential and the resting membrane potential was also unchanged. Bisaramil slowed the spontaneous frequency of pacemaker cells in rabbit sinus node preparation due to its inhibitory effect on slow diastolic depolarization (SDD). Bisaramil was able to inhibit slow Ca2+-action potentials induced by isoprenaline on K+-depolarized papillary muscle. Results obtained with transmembrane current measurements revealed that bisaramil inhibited both fast Na+-current and slow Ca2+-current in frog sinoauricular fibres at the same concentration. Bisaramil with a mixed mode of the action seems to be a very promising drug. KEYWORDS:transmembrane action potential, pacemaker type action potential, 'slow Ca2+-action potential', transmembrane ionic currents.
INTRODUCTION Bisaramil is a new antiarrhythmic drug under clinical trials. It is chemically unrelated to any currently available antiarrhythmic drug. Its antiarrhythmic activity was investigated on different experimental arrhythmic models and animal species [1]. The antiarrhythmic properties of bisaramil were also studied [2] and Correspondence to: Dr Margit Paroczai, Chemical Works of Gedeon Richter Ltd, Pharmacological Research Centre, H-1475. Budapest, 10, P.O.B. 27, Hungary. 1043-6618/92/010075-11/$03.00/0
© 1992 The Italian Pharmacological Society
76
Pharmacological Research, Vol. 25, No. 1, 1992
these experiments indicated that bisaramil had a strong class I (local-anaesthetic type) activity accompanied by a significant Ca-antagonistic property. The aim of this study was to investigate the electrophysiological effects of bisaramil on various parts of myocardium (left auricle, papillary muscle, sinoauricular fibres) and furthermore, to obtain additional information about the mode of action of bisaramil.
METHODS
Guinea-pig left auricle or papillary muscle Guinea-pigs of either sex were killed, and the heart was placed in warm (34 °C) oxygenated Locke solution containing (mM): NaC1, 125; KC1, 5.6; NaHCO3, 25; glucose, 11; CaCI2, 2.16. Left auricle or papillary muscle with the free wall of the right ventricle were excised distinctly and pinned in a tissue chamber (3 ml) and superfused with oxygenated Locke solution. The preparation was stimulated through bipolar Pt-electrodes with 1 Hz frequency using square-wave pulses 0.5 ms in duration and twice threshold. Transmembrane action potentials were recorded by means of conventional glass microelectrodes filled with 3 M KC1, having a resistance of 8-20 Mr2. The output of the microelectrode was led to a high input impedance, capacitance-neutralizing amplifier and displayed on an oscilloscope. The maximum rate of depolarization was recorded on the second beam of the oscilloscope using an electronic differentiating network. Oscilloscope tracings were photographed, and evaluated using a microfilm reading instrument. Details of the technique were described previously by Kecskemeti et al. [3]. After measuring the control value of the parameters, the preparation was superfused with Locke solution containing bisaramil in various concentrations. After 30 rain equilibration measuring was repeated. The following parameters were recorded: APA (amplitude of the action potential), RP (resting potential), OS (overshoot), Vm,x (the maximum rate of depolarization), APD20, APDs0, APD90 (duration of the action potential until 20%, 50%, 90% of repolarization). Bisaramil was superfused in three concentrations: 2.3×10 -6 M, 1.2X10-5 M, 2.3X 10-5 M. Results are given as means_+sEM and per cent change compared to control. Data were analysed statistically using paired Student's t-test. Rabbit sinus-node preparation Rabbits weighing 1.5-2.0 kg were killed and the heart rapidly excised. The right auricle was separated from the other parts of the heart and placed in warm oxygenated Locke solution. We isolated a spontaneously beating piece of tissue 10 mm long and 3 mm wide and pinned it without stretching in a tissue chamber. Pacemaker type action potentials were recorded with conventional microelectrode techniques mentioned earlier. Tissue was allowed to equilibrate in Locke solution for at least 1 h. After measuring control parameters, the preparation was superfused with Locke solution containing bisaramil at a concentration of 2.3x 10 6 M. After 30 rain superfusion, the following parameters were recorded: APA
PharmacologicalResearch, Vol. 25, No. 1, 1992
77
(amplitude of the action potential), MDP (maximum diastolic potential), TP (threshold potential), SDD (slow diastolic depolarization), Td (duration of the action potential from the threshold potential to the maximum diastolic potential), CL (cycle length). Results were analysed using paired Student's t-test. Values are mean+_s~M.
K+-depolarized papillary muscle Papillary muscle from the right ventricle of guinea-pigs was isolated after killing animals and then placed in a tissue chamber containing normal Locke solution. Preparation was stimulated through bipolar Pt-electrodes with 1 Hz frequency using square-wave pulses 0.5 ms in duration and twice threshold. After a 1 h equilibration period, the preparation was placed in Locke solution containing 25 mM KCI, frequency was decreased to 0.3 Hz and the stimulus intensity was increased to 10 times higher than threshold potential. Details of the technique have been described previously [4]. Under such circumstances electrical activity ceased within a short time. After 30 rain isoprenaline at a concentration of 1×10 -6 M was given to Locke solution containing 25 mM K + in order to induce 'slow responses'. The effect of bisaramil at a concentration of 2.3×10 -6 M on slow responses was investigated after 15 and 30 min incubation with the drug. Data are given as mean_+szM. Paired Student's t-test was used for statistical analysis of results. Transmembrane ion currents using voltage clamp method Experiments were carried out on frog (Rana esculenta) sinoauricular fibre of 100 ~ m diameter and 3-4 mm length. A voltage clamp technique based on a double sucrose gap arrangement according to Rougier et al. [5] was used. Ringersolution of the following composition was applied: 111.1 mM NaC1, 1.8 mM CaC12, 1.0 mM KC1, 1.2 mM NaHCO3. Temperature was maintained at 18_+0.3 °C. Drug was dissolved in Ringer-solution, and perfused in central compartment. Details of the technique have been described previously [6, 7]. Holding potential was set to 65-75 mV, corresponding to the resting membrane potential of frog myocardium. Membrane potential was clamped between - 3 0 and +140 mV steps of 10 mV. Command pulses of 500 ms in duration were given at a frequency of 0.6 Hz. Double impulses were used to investigate the effect of bisaramil on the inactivation process of Na+-current. The first (conditioning) pulse was of 500 ms duration and its amplitude was continuously changed between - 2 0 and - 4 0 mV as compared to holding potential. The second pulse was of 100 ms duration and its amplitude was +50 mV compared to holding potential. The amplitude of the current (I) measured after the first conditioning pulse was related to the maximal amplitude of the current (/max) measured after the second pulse. All experiments were self-controlled. RESULTS
Guinea-pig left auricle and papillary muscle The effect of bisaramil (2.3-23.3×10 -6 M) on the characteristics of the auricular
Pharmacological Research, Vol. 25, No. 1, 1992
78
action potential are displayed in Fig. 1. The control values of these parameters are shown in Table I. The main effect of bisaramil was seen to be the decrease of Vmax, and OS in a concentration-dependent manner, with no change in RP. A small, but statistically significant decrease of APA was also measured. Bisaramil slightly prolonged the initial and terminal repolarization phase, but this effect was not dependent on the concentration, and was significant only in the presence of the highest concentration of bisaramil. Figure 2 summarizes the results obtained on papillary muscle. Control values of
%1
[~
1.16 x I0 -5 M [~
~
I0~ 0
2.3x IO-e M
APA
2.3 x lO-Sm
RP
OS
V,,,ox
--
o
~,
o.
-io
APD2o
.E
g c
-so
APDso
APDoo
-
--50 --70
---
--90
-X-~
--
Fig. 1. Electrophysiological effects of bisaramil on guinea-pig left auricle. Bars refer to mean per cent change in parameters measured +SEM. APA, action potential amplitude; RP, resting potential; OS, overshoot; Vma×,the maximum rate of depolarization; APD20,50,90,action potential duration to 20, 50, 90% repolarization; n=39.
Table I Pre-drug value of parameters of guinea-pig left atrium APA (mV)
RP (mV)
OS (mV)
99.0 1.25
78.5 0.74
20.5 1.22
APD (ms)
Vmax
APD2o
APDso
APD9o
23.7 2.5
43.6 3.14
64.2 3.66
(V/s) 141.8 5.00
All data are expressed as means of 39 preparations ±SEM. Abbreviations used are: APA, amplitude of the action potential; RP, resting potential; OS, overshoot; V.... maximum rate of depolarization; APD, duration of the action potential; APD20, APDso, APD9o, duration of the action potential until 20, 50, 90% repolarization.
79
Pharmacological Research, Vol. 25, No, 1, 1992
parameters are shown in Table II. Results of the experiments carried out on papillary muscle were similar to those obtained on left auricle, but the decrease of Vmax and OS in the presence of bisaramil was higher. Furthermore, besides the concentration-dependent inhibitory action of bisaramil on the Vmax and OS, its effect on the APA was also concentration dependent. A consistent effect on the duration of the action potential was not found. While significant prolongation of the initial repolarization phase could be observed, the considerable effect on APD90 was only seen in the presence of the highest concentration of the drug. Rabbit sinus-node preparations The effects of bisaramil at the concentration of 2.3×10-6 M on the pacemaker type action potential are summarized in Table III. Pre-drug values of parameters
% 50
30
[ ] ] •2 . 3 [~
x IO -6 M
1.16 x I 0 - s M
[~2.3
io APA ,., O ~' - I O * * * ~ .
x 10-eM
RP
®
*
.E
g g --30
ac: o
**
APD2o
APDso
APD,,o
*P
