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Diastolic tension and contraction amplitude in calcium-loaded rat ventricular myocardium are differently affected by drugs
Gen. Pharmac. Vol. 22, No. 5, pp. 793-796, 1991 Printed in Great Britain. All rights reserved
0306-3623/91 $3.00+ 0.00 Copyright © 1991 PergamonPress plc
DIASTOLIC TENSION A N D CONTRACTION AMPLITUDE IN CALCIUM-LOADED RAT VENTRICULAR MYOCARDIUM ARE DIFFERENTLY AFFECTED BY DRUGS STEFAN HERZIG,* KLAUS MOHR, SONKE SCHMIDT and JOACHIM SPLIETH Department of Pharmacology, University of Kiel, Hospitalstrasse 4, 2300 Kiel, Fed. Rep. Germany [Tel. 597-3501]
(Received 5 February 1991) Abstraet--l. To obtain a measure of drug effects on myocardial function during diastole, the following experimental protocol was designed: rapid electrical stimulation (5 Hz) at high Ca2o+ caused an elevated diastolic tension, which could be subjected to drug-induced alterations. 2. Antiarrhythmic drugs (quinidine, propafenone, procainamide, mexiletine) were able to lower diastolic force without appreciably decreasing contraction amplitude. Calcium antagonists (nifedipine, verapamil) lowered both parameters in parallel. 3. Veratridine and Bay K 8644 both enhanced diastolic tension, but only Bay K 8644 concomitantly elevated contraction amplitude. 4. These findings may be explained when taking into account differential actions of sodium- and calcium channel modulating drugs, respectively, on cellular Ca 2+ movements. In quantitative terms, the non-linear dependence of myocardial force on Cai2+ also had to be considered.
Drug effects on myocardial force of contraction are often caused by changes in the intracellular calcium (Cai2+) transients. Force measurements in isolated cardiac preparations are easy experiments to screen for such effects. However, since Ca 2+ is subthreshold for the activation of the contractile proteins during diastole, these experiments can only give an indication about systolic, but not about diastolic Cai2+. We sought for a simple approach to monitor drug effects simultaneously both in systole and diastole. Therefore, we stimulated ventricular myocardial strips from rats at a high rate, in the presence of an elevated Cao2+ . In principle, such a procedure has been used before (Lakatta and Lappe, 1981; Yue et al., 1986). The rat heart seems particularly susceptible to calcium overload and a diastolic tension increment (Herzig and Mohr, 1984). This increment was well reproducible and could be modified by drugs in either direction. Here we describe the effects of negative inotropic drugs from two pharmacological groups, i.e. antiarrhythmic drugs (quinidine, propafenone, procainamide and mexiletine) and calcium antagonists (nifedipine and verapamil). Positive inotropic counterparts of these drugs, i.e. the sodium channel "activator" veratridine and the calcium channel "activator" Bay K 8644, were studied in comparison.
< 1 ram) were dissected from the free wall of the right ventricle. They were mounted in organ baths containing 20 ml of Tyrode's solution composed of (raM): NaCi 136.8; KC1 5.4; CaCI2 1.8; NaHCO 3 11.9; MgC12 1.05; NaH2PO4 0.21; glucose 5.5. The temperature was maintained at 32°C, and the solution was gassed with 95%O2/5%CO2, yielding a pH of 7.3. The muscles were preloaded with 5 mN, and tension was recorded isometrically (force transducer SG-45, SWEMA, Sweden, chart recorder Type 21807, Hellige, Fed. Rep. Germany). During equilibration (1 hr), the preparations were electrically driven (5 msec rectangular pulses, 50% above threshold) at l Hz (Stimulator T, Hugo Sachs, Fed. Rep. Germany), then Ca2o+ was elevated to usually 5.4 mM. 10 min later, stimulation was switched off. Separated by 15 min-periods of quiescence, 5 trains of stimulation (frequency 5 Hz, 6 min duration each) were successively applied. Drugs were added during the third train. In order to quantify the drug effects on diastolic tension and contraction amplitude, respectively, the ratios were calculated of the values at the end of the fifth train over the preceeding corresponding value at the end of the second train. These figures were close to 100% in the absence of drugs. The following drugs were kind gifts from manufacturers: nifedipine and ( + / - ) - B a y K 8644 (Bayer, Fed. Rep. Germany), (+/-)-verapamil and (+/-)-propafenone (Knoll, Fed. Rep. Germany), (+/-)-mexiletine (Boehringer Ingelheim, Fed. Rep. Germany), and procainamide (Hoechst, Fed. Rep. Germany). Quinidine was obtained from Merck (Fed. Rep. Germany), veratridine from Sigma (Fed. Rep. Germany). Experiments with dihydropyridines were carried out in sodium light.
MATERIAI~ AND METHODS
RESULTS
Sprague-Dawley rats of either sex (250-300 g body wt) were killed by a blow on the neck. The excised hearts were placed into Tyrode's solution and two to four strips (dia
Figure 1 shows original tracings from three experiments. In the upper row (control), it becomes apparent that repeated trains of rapid stimulation led to a sustained elevation of diastolic tension, which remained stable over the second half of the 6 min
INTRODUCTION
*To whom all correspondence should be addressed.
793
STEFAN HERZIGel al.
794 Control
Tension {%1 100
601
Ouinidine 10wM
4O 20
Nifedipine 31JM 10
.
.
.
.
.
.
0
10.6
~-t. [M]
10-5 Verapomil
6rain Fig. l. Original mechanograms demonstrating the effects of repetitive 5-Hz-stimulation trains on diastolic tension and contraction amplitude in rat right ventricular strips. In the upper row, a control is depicted. The addition of quinidine (10 #M, middle row), and of nifedipine (3 #M, lower row) are marked by arrows. period. Contraction amplitude, after reaching a transient maximum early during the train, was also stabilized at its end. Repeated stimulations of this kind gave reproducible results when applied at intervals of 15 min between the trains. The drugs tested revealed remarkable differences in their ability to affect diastolic tension: quinidine (10/~M) lowered diastolic tension by half without an effect on contraction amplitude (Fig. 1, middle trace), whereas nifedipine did so with a marked concomitant depression of contraction amplitude (Fig. 1, lower trace). Figure 2 shows that this differential influence is found not only between quinidine and nifedipine in particular, but between antiarrhythmic drugs and calcium antagonists in general: all drugs were given in concentrations sufficient to lower diastolic tension to c a 50% (open bars). With the antiarrhythmics, there was nearly no change in contraction amplitude (hatched bars), whereas for the calcium antagonists verapamil and nifedipine, contraction amplitude was reduced to less than 50%.
Tension 1%]
16o 60
201 ?-I
I
0
,
1D'-5
10L4
IMI
Mexiletine Fig. 3. Dose-response curves of verapamil (upper graph) and mexiletine (lower graph) to decrease contraction amplitude (O) and diastolic tension (O). Means + SEM are given for n = 4--8 muscles per point.
To check whether this difference in action between antiarrhythmics and calcium antagonists was just a matter of the particular condition chosen for comparison, dose response curves were evaluated. For each group one member was studied, i.e. mexiletine and verapamil (Fig. 3). Again, one drug concentration was investigated in each preparation only. The lowering of the contraction amplitude (solid
contraction amplitude 100%" -
-
-
-
O~
IO0OA
!
diastolic tension Control
Propofenone 1wM Ouinidine lOpM
Me~itetine 30pM I~inomide 300pM
V~mil 10pM Nif{Klipir~ 3pM
Fig. 2. Influence of different cardiodepressant drugs on contraction amplitude (hatched columns) and on diastolic tension (open columns). The drugs were administered as illustrated in Fig. 1. For evaluation, values recorded at the end of the fifth stimulation train were expressed as percentage of the respective values of the second train. Mean values + SEM of 4-6 experiments are given. Drug concentrations were chosen to lower diastolic tension to about 50% (dashed line).
Drug effects on diastolic tension 180 Tension [%]
0
10-7
10:6
[M]
10'-6
[M]
Boy K 8644 Tension [°/4 180
140
100
? O' "
10.-7 Verotridine
Fig. 4. Dose-response curves of Bay K 8644 (upper graph) and veratridine (lower graph) to affect contraction amplitude (@) and diastolic tension (O). In these experiments, Ca2o+ was elevated to only 3.6 raM. Means + SEM of 4-7 muscles per point are given. circles) was clearly shifted to the right in case of mexiletine, whereas it closely paralleled the lowering of the diastolic tension (open circles) for verapamil. It was intriguing to find out whether similar effects could be observed when instead of cardiodepressant drugs, positive inotropic agents were applied, i.e. the calcium channel "activator" Bay K 8644 and the sodium channel "activator" veratridine. In these experiments, Ca:o+ was elevated to only 3.6mM, which already caused diastolic tension to rise, but not to the maximum possible extent. Bay K 8644 (Fig. 4) enhanced both diastolic tension (open circles) and contraction amplitude (solid circles). In resemblance of the findings made with verapamil, both dose-response curves for Bay K 8644 nearly superimposed. In contrast, veratridine did not appreciably enhance contraction amplitude under the present conditions. The diastolic tension, however, was elevated in a concentration-dependent manner, comparable to the effect of Bay K 8644. DISCUSSION
The striking finding of the study is that an elevated diastolic tension can be almost selectively influenced by drugs acting on the cellular sodium homeostasis, i.e. by antiarrhythmic sodium channel blockers and the sodium channel toxin veratridine. In contrast, compounds acting on calcium channels, i.e. verapamil, nifedipine and Bay K 8644, always changed
795
diastolic tension and contraction amplitude in parallel. It is therefore tempting to speculate upon a link between the mode by which the drugs influence cardiac Cai2+ and the effect they have on both force parameters. It shall be assumed that the Ca~+-actomyosin interaction remained unaltered by the drugs, but this seems likely in the studied concentrations. At least for quinidine, 30-times higher concentrations than those studied would have been needed to lower the calcium sensitivity of actomyosin (Su and Libao, 1984). A confounding effect on actomyosin sensitivity possibly induced in the preparations by cellular acidosis (Blanchard and Solaro, 1984), or accumulation of inorganic phosphate (Kentish, 1986) cannot be ruled out either, but would not explain any of the qualitative differences between the observed drug effects (e.g. Fig. 3). With respect to the mode of action, calcium channel modulators alter the amount of Ca 2+ entering the cell during systole, and consequently to be extruded in diastole. Sodium channel modulators will alter the diastolic extrusion of Ca 2+ via sodium/calcium exchange, since they affect primarily Nai+; the systolic Ca 2+ influx should therefore remain unaltered. Thus, in qualitative terms, it seems trivial that the sodium channel modulators had less effects on contraction amplitude than the calcium channel modulators. A severe complexity has to be considered, however, when trying to interpret contractility data in terms of cellular Ca~ + changes, i.e. the non-linearity of the calcium-actomyosin interaction: already the force of skinned fibers steeply depends on Ca 2+ in a sigmoidal fashion [e.g. Hibberd and Jewell (1982)]. The sigmoidicity is even more pronounced in an intact muscle (Yue et al., 1986; Urthaler et al., 1990). This non-linearity has to be taken into account for a quantitative understanding of the observed drug effects, and of the differences between drugs. Yet, as illustrated in the Appendix, the simple assumption-compounds acting on sodium channels should affect diastolic Cai2+, whereas drugs influencing calcium channels should alter both diastolic Ca~ + and the size of the Ca 2+ transient--does not only qualitatively but also quantitatively predict the dose response curves found, when the non-linear relationship between Ca~ + and tension is incorporated. In conclusion, it is possible to simultaneously monitor drug effects on both diastolic and systolic force of contraction, when rat ventricular myocardium is Ca2+-loaded. There are qualitative differences observed according to the pharmacological groups to which these drugs belong. These differences hint that monitoring an elevated diastolic tension allows to differentiate between drugs which primarily interfere with either systolic Ca 2+ entry, or with Ca 2+ extrusion from the cytosol. It seems worthwhile to test these ideas by direct measurements of systolic and diastolic Cal2+ in the presence of drugs, for instance with Ca2+-sensitive fluorescent indicators. REFERENCES
Blanchard E. M. and Solaro R. J. (1984) Inhibition of the activation and troponin calcium binding of dog cardiac myofibrils by acidic pH. Circ. Res. 55, 382-391.
STEFANHERZIG et aL
796
Herzig S. and Mohr K. (1984) Action of ouabain on rat heart: comparison with its effect on guinea-pig heart. Br. J. Pharmac. 82, 135-142. Hibberd M. G. and Jewell B. R. (1982) Calcium and length-dependent force production in rat ventricular muscle. J. Physiol. 329, 527-540. Honerj/iger P. (1982) Cardioactive substances that prolong the open state of the sodium channel. Rev. Physiol. Biochem. Pharmac. 92, 1-74. Kentish J. C. (1986) The effects of inorganic phosphate and creatine phosphate on force production in skinned muscles from rat ventricle. J. Physiol. 370, 585-604. Lakatta E. G. and Lappe D. L. (1981) Diastolic scattered light fluctuation, resting force and twitch force in mammalian cardiac muscle. J. Physiol. 312, 369-394. Su J. Y. and Libao R. G. (1984) Intracellular mechanism of quinidine action on muscle contraction. A comparison between rabbit cardiac and skeletal muscle. Naunyn-Schmiedeberg's Arch. Pharmac. 326, 375-381. Urthaler F., Walker A. A., Reeves R. C. and Hefner L. L. (1990) Excitation-contraction coupling model to estimate the recirculating fraction of activator calcium in intact cardiac muscle. Can. J. Physiol. Pharmac. 68, 1041-1048. Yue D. T., Marban E. and Wier W. G. (1986) Relationship between force and intracellular (Ca 2+) in tetanized mammalian heart muscle. J. Gen. Physiol. 87, 223-242. APPENDIX It is conceivable that the different types of drug effects observed are a reflection of the non-linearity between Ca~ + and force. This idea is illustrated in Fig. 5. It depicts a model calculation intended to match the actual observations found with Bay K 8644 and with veratridine. A steep interrelationship between cytosolic Ca 2+ and tension [Hill function with a Hill coefficient of 6, half-maximal activation at 400 nM Ca 2+, Yue et al. (1986)] is assumed. In the absence of drugs, Ca :+ is taken to oscillate between 300 nM in diastole and 350 nM during systole. Bay K 8644, an agent promoting systolic Ca 2+ influx, is assumed to increase both the size of the Ca~2+ transient and the level of diastolic Ca 2+. As a result, both contraction amplitude and diastolic tension are enhanced. In contrast, veratridine is believed to impair Ca~ + extrusion rather than to promote Ca 2+ entry during systole, since by elevating Nai+, it should primarily reduce the ability ofNa+/Ca 2+ exchange to extrude Ca 2+ from the cytosol [see Honerj~iger (1982)]. Therefore, veratridine has been modelled to solely affect the diastolic level of Ca 2+, to which then adds systolically an unchanged Ca 2+ transient of 50 nM. This behaviour would lead to a positive inotropy and a normally shaped dose-response curve for contraction amplitude, when diastolic Ca 2+ starts at a lower level (e.g. at 150 nM, not shown). At an elevated diastolic Ca 2+, however, only a very small effect on contraction amplitude is visible, and the dose-response curve is bell-shaped. Again, however, diastolic tension rises concentration-dependently.
Simulohon Tension [%]
, °°...°..-'"°'°'°°" 40 ,.% 2C
t
I
10-7
10-6 [M] Boy K 8644
Tension [%1
°°°o°......°°'" .... 4C
° °°°'°
3(: 2(
,°°.°°
°°.-"
..'°
./"
o°,"
,/
10'-7
10'-6i M] Vemtridine
Fig. 5. Model to illustrate how drugs (abscissa) which cause differential elevations in cellular Ca 2+ induce changes in muscle tension (ordinate) during diastole (broken lines) and systolic contraction amplitude (solid lines). The following assumptions are made: tension is a Hill function of Ca 2+ (Hill = 6, half maximal effect at Ca 2+= 400riM). In the absence of drugs, Ca 2+ is 300 nM in diastole and increases by 50 nM in systole. Bay K 8644 (upper graph) enhances these two Ca2+-terms concentration-dependently (Hill = 1, maximal effects are elevations by 100 nM Ca2+; for half maximum effect, the Bay K 8644 concentration is 10-~ M). As the only difference to Bay K 8644, veratridine (lower graph) does not change the size of the Ca 2+ transient, but only elevates the diastolic Ca 2+.
A rise in diastolic tension would be virtually absent when calculating with a lower starting baseline for diastolic Ca~2+. The differential behaviour observed with calcium channel blockers versus sodium channel blockers can be modelled in a similar fashion (not shown). One has to assume that the former drugs lower diastolic Ca~ + and the systolic Ca~ + transient, whereas the latter compounds act solely by lowering the level of diastolic Ca~ +.
0306-3623/91 $3.00+ 0.00 Copyright © 1991 PergamonPress plc
DIASTOLIC TENSION A N D CONTRACTION AMPLITUDE IN CALCIUM-LOADED RAT VENTRICULAR MYOCARDIUM ARE DIFFERENTLY AFFECTED BY DRUGS STEFAN HERZIG,* KLAUS MOHR, SONKE SCHMIDT and JOACHIM SPLIETH Department of Pharmacology, University of Kiel, Hospitalstrasse 4, 2300 Kiel, Fed. Rep. Germany [Tel. 597-3501]
(Received 5 February 1991) Abstraet--l. To obtain a measure of drug effects on myocardial function during diastole, the following experimental protocol was designed: rapid electrical stimulation (5 Hz) at high Ca2o+ caused an elevated diastolic tension, which could be subjected to drug-induced alterations. 2. Antiarrhythmic drugs (quinidine, propafenone, procainamide, mexiletine) were able to lower diastolic force without appreciably decreasing contraction amplitude. Calcium antagonists (nifedipine, verapamil) lowered both parameters in parallel. 3. Veratridine and Bay K 8644 both enhanced diastolic tension, but only Bay K 8644 concomitantly elevated contraction amplitude. 4. These findings may be explained when taking into account differential actions of sodium- and calcium channel modulating drugs, respectively, on cellular Ca 2+ movements. In quantitative terms, the non-linear dependence of myocardial force on Cai2+ also had to be considered.
Drug effects on myocardial force of contraction are often caused by changes in the intracellular calcium (Cai2+) transients. Force measurements in isolated cardiac preparations are easy experiments to screen for such effects. However, since Ca 2+ is subthreshold for the activation of the contractile proteins during diastole, these experiments can only give an indication about systolic, but not about diastolic Cai2+. We sought for a simple approach to monitor drug effects simultaneously both in systole and diastole. Therefore, we stimulated ventricular myocardial strips from rats at a high rate, in the presence of an elevated Cao2+ . In principle, such a procedure has been used before (Lakatta and Lappe, 1981; Yue et al., 1986). The rat heart seems particularly susceptible to calcium overload and a diastolic tension increment (Herzig and Mohr, 1984). This increment was well reproducible and could be modified by drugs in either direction. Here we describe the effects of negative inotropic drugs from two pharmacological groups, i.e. antiarrhythmic drugs (quinidine, propafenone, procainamide and mexiletine) and calcium antagonists (nifedipine and verapamil). Positive inotropic counterparts of these drugs, i.e. the sodium channel "activator" veratridine and the calcium channel "activator" Bay K 8644, were studied in comparison.
< 1 ram) were dissected from the free wall of the right ventricle. They were mounted in organ baths containing 20 ml of Tyrode's solution composed of (raM): NaCi 136.8; KC1 5.4; CaCI2 1.8; NaHCO 3 11.9; MgC12 1.05; NaH2PO4 0.21; glucose 5.5. The temperature was maintained at 32°C, and the solution was gassed with 95%O2/5%CO2, yielding a pH of 7.3. The muscles were preloaded with 5 mN, and tension was recorded isometrically (force transducer SG-45, SWEMA, Sweden, chart recorder Type 21807, Hellige, Fed. Rep. Germany). During equilibration (1 hr), the preparations were electrically driven (5 msec rectangular pulses, 50% above threshold) at l Hz (Stimulator T, Hugo Sachs, Fed. Rep. Germany), then Ca2o+ was elevated to usually 5.4 mM. 10 min later, stimulation was switched off. Separated by 15 min-periods of quiescence, 5 trains of stimulation (frequency 5 Hz, 6 min duration each) were successively applied. Drugs were added during the third train. In order to quantify the drug effects on diastolic tension and contraction amplitude, respectively, the ratios were calculated of the values at the end of the fifth train over the preceeding corresponding value at the end of the second train. These figures were close to 100% in the absence of drugs. The following drugs were kind gifts from manufacturers: nifedipine and ( + / - ) - B a y K 8644 (Bayer, Fed. Rep. Germany), (+/-)-verapamil and (+/-)-propafenone (Knoll, Fed. Rep. Germany), (+/-)-mexiletine (Boehringer Ingelheim, Fed. Rep. Germany), and procainamide (Hoechst, Fed. Rep. Germany). Quinidine was obtained from Merck (Fed. Rep. Germany), veratridine from Sigma (Fed. Rep. Germany). Experiments with dihydropyridines were carried out in sodium light.
MATERIAI~ AND METHODS
RESULTS
Sprague-Dawley rats of either sex (250-300 g body wt) were killed by a blow on the neck. The excised hearts were placed into Tyrode's solution and two to four strips (dia
Figure 1 shows original tracings from three experiments. In the upper row (control), it becomes apparent that repeated trains of rapid stimulation led to a sustained elevation of diastolic tension, which remained stable over the second half of the 6 min
INTRODUCTION
*To whom all correspondence should be addressed.
793
STEFAN HERZIGel al.
794 Control
Tension {%1 100
601
Ouinidine 10wM
4O 20
Nifedipine 31JM 10
.
.
.
.
.
.
0
10.6
~-t. [M]
10-5 Verapomil
6rain Fig. l. Original mechanograms demonstrating the effects of repetitive 5-Hz-stimulation trains on diastolic tension and contraction amplitude in rat right ventricular strips. In the upper row, a control is depicted. The addition of quinidine (10 #M, middle row), and of nifedipine (3 #M, lower row) are marked by arrows. period. Contraction amplitude, after reaching a transient maximum early during the train, was also stabilized at its end. Repeated stimulations of this kind gave reproducible results when applied at intervals of 15 min between the trains. The drugs tested revealed remarkable differences in their ability to affect diastolic tension: quinidine (10/~M) lowered diastolic tension by half without an effect on contraction amplitude (Fig. 1, middle trace), whereas nifedipine did so with a marked concomitant depression of contraction amplitude (Fig. 1, lower trace). Figure 2 shows that this differential influence is found not only between quinidine and nifedipine in particular, but between antiarrhythmic drugs and calcium antagonists in general: all drugs were given in concentrations sufficient to lower diastolic tension to c a 50% (open bars). With the antiarrhythmics, there was nearly no change in contraction amplitude (hatched bars), whereas for the calcium antagonists verapamil and nifedipine, contraction amplitude was reduced to less than 50%.
Tension 1%]
16o 60
201 ?-I
I
0
,
1D'-5
10L4
IMI
Mexiletine Fig. 3. Dose-response curves of verapamil (upper graph) and mexiletine (lower graph) to decrease contraction amplitude (O) and diastolic tension (O). Means + SEM are given for n = 4--8 muscles per point.
To check whether this difference in action between antiarrhythmics and calcium antagonists was just a matter of the particular condition chosen for comparison, dose response curves were evaluated. For each group one member was studied, i.e. mexiletine and verapamil (Fig. 3). Again, one drug concentration was investigated in each preparation only. The lowering of the contraction amplitude (solid
contraction amplitude 100%" -
-
-
-
O~
IO0OA
!
diastolic tension Control
Propofenone 1wM Ouinidine lOpM
Me~itetine 30pM I~inomide 300pM
V~mil 10pM Nif{Klipir~ 3pM
Fig. 2. Influence of different cardiodepressant drugs on contraction amplitude (hatched columns) and on diastolic tension (open columns). The drugs were administered as illustrated in Fig. 1. For evaluation, values recorded at the end of the fifth stimulation train were expressed as percentage of the respective values of the second train. Mean values + SEM of 4-6 experiments are given. Drug concentrations were chosen to lower diastolic tension to about 50% (dashed line).
Drug effects on diastolic tension 180 Tension [%]
0
10-7
10:6
[M]
10'-6
[M]
Boy K 8644 Tension [°/4 180
140
100
? O' "
10.-7 Verotridine
Fig. 4. Dose-response curves of Bay K 8644 (upper graph) and veratridine (lower graph) to affect contraction amplitude (@) and diastolic tension (O). In these experiments, Ca2o+ was elevated to only 3.6 raM. Means + SEM of 4-7 muscles per point are given. circles) was clearly shifted to the right in case of mexiletine, whereas it closely paralleled the lowering of the diastolic tension (open circles) for verapamil. It was intriguing to find out whether similar effects could be observed when instead of cardiodepressant drugs, positive inotropic agents were applied, i.e. the calcium channel "activator" Bay K 8644 and the sodium channel "activator" veratridine. In these experiments, Ca:o+ was elevated to only 3.6mM, which already caused diastolic tension to rise, but not to the maximum possible extent. Bay K 8644 (Fig. 4) enhanced both diastolic tension (open circles) and contraction amplitude (solid circles). In resemblance of the findings made with verapamil, both dose-response curves for Bay K 8644 nearly superimposed. In contrast, veratridine did not appreciably enhance contraction amplitude under the present conditions. The diastolic tension, however, was elevated in a concentration-dependent manner, comparable to the effect of Bay K 8644. DISCUSSION
The striking finding of the study is that an elevated diastolic tension can be almost selectively influenced by drugs acting on the cellular sodium homeostasis, i.e. by antiarrhythmic sodium channel blockers and the sodium channel toxin veratridine. In contrast, compounds acting on calcium channels, i.e. verapamil, nifedipine and Bay K 8644, always changed
795
diastolic tension and contraction amplitude in parallel. It is therefore tempting to speculate upon a link between the mode by which the drugs influence cardiac Cai2+ and the effect they have on both force parameters. It shall be assumed that the Ca~+-actomyosin interaction remained unaltered by the drugs, but this seems likely in the studied concentrations. At least for quinidine, 30-times higher concentrations than those studied would have been needed to lower the calcium sensitivity of actomyosin (Su and Libao, 1984). A confounding effect on actomyosin sensitivity possibly induced in the preparations by cellular acidosis (Blanchard and Solaro, 1984), or accumulation of inorganic phosphate (Kentish, 1986) cannot be ruled out either, but would not explain any of the qualitative differences between the observed drug effects (e.g. Fig. 3). With respect to the mode of action, calcium channel modulators alter the amount of Ca 2+ entering the cell during systole, and consequently to be extruded in diastole. Sodium channel modulators will alter the diastolic extrusion of Ca 2+ via sodium/calcium exchange, since they affect primarily Nai+; the systolic Ca 2+ influx should therefore remain unaltered. Thus, in qualitative terms, it seems trivial that the sodium channel modulators had less effects on contraction amplitude than the calcium channel modulators. A severe complexity has to be considered, however, when trying to interpret contractility data in terms of cellular Ca~ + changes, i.e. the non-linearity of the calcium-actomyosin interaction: already the force of skinned fibers steeply depends on Ca 2+ in a sigmoidal fashion [e.g. Hibberd and Jewell (1982)]. The sigmoidicity is even more pronounced in an intact muscle (Yue et al., 1986; Urthaler et al., 1990). This non-linearity has to be taken into account for a quantitative understanding of the observed drug effects, and of the differences between drugs. Yet, as illustrated in the Appendix, the simple assumption-compounds acting on sodium channels should affect diastolic Cai2+, whereas drugs influencing calcium channels should alter both diastolic Ca~ + and the size of the Ca 2+ transient--does not only qualitatively but also quantitatively predict the dose response curves found, when the non-linear relationship between Ca~ + and tension is incorporated. In conclusion, it is possible to simultaneously monitor drug effects on both diastolic and systolic force of contraction, when rat ventricular myocardium is Ca2+-loaded. There are qualitative differences observed according to the pharmacological groups to which these drugs belong. These differences hint that monitoring an elevated diastolic tension allows to differentiate between drugs which primarily interfere with either systolic Ca 2+ entry, or with Ca 2+ extrusion from the cytosol. It seems worthwhile to test these ideas by direct measurements of systolic and diastolic Cal2+ in the presence of drugs, for instance with Ca2+-sensitive fluorescent indicators. REFERENCES
Blanchard E. M. and Solaro R. J. (1984) Inhibition of the activation and troponin calcium binding of dog cardiac myofibrils by acidic pH. Circ. Res. 55, 382-391.
STEFANHERZIG et aL
796
Herzig S. and Mohr K. (1984) Action of ouabain on rat heart: comparison with its effect on guinea-pig heart. Br. J. Pharmac. 82, 135-142. Hibberd M. G. and Jewell B. R. (1982) Calcium and length-dependent force production in rat ventricular muscle. J. Physiol. 329, 527-540. Honerj/iger P. (1982) Cardioactive substances that prolong the open state of the sodium channel. Rev. Physiol. Biochem. Pharmac. 92, 1-74. Kentish J. C. (1986) The effects of inorganic phosphate and creatine phosphate on force production in skinned muscles from rat ventricle. J. Physiol. 370, 585-604. Lakatta E. G. and Lappe D. L. (1981) Diastolic scattered light fluctuation, resting force and twitch force in mammalian cardiac muscle. J. Physiol. 312, 369-394. Su J. Y. and Libao R. G. (1984) Intracellular mechanism of quinidine action on muscle contraction. A comparison between rabbit cardiac and skeletal muscle. Naunyn-Schmiedeberg's Arch. Pharmac. 326, 375-381. Urthaler F., Walker A. A., Reeves R. C. and Hefner L. L. (1990) Excitation-contraction coupling model to estimate the recirculating fraction of activator calcium in intact cardiac muscle. Can. J. Physiol. Pharmac. 68, 1041-1048. Yue D. T., Marban E. and Wier W. G. (1986) Relationship between force and intracellular (Ca 2+) in tetanized mammalian heart muscle. J. Gen. Physiol. 87, 223-242. APPENDIX It is conceivable that the different types of drug effects observed are a reflection of the non-linearity between Ca~ + and force. This idea is illustrated in Fig. 5. It depicts a model calculation intended to match the actual observations found with Bay K 8644 and with veratridine. A steep interrelationship between cytosolic Ca 2+ and tension [Hill function with a Hill coefficient of 6, half-maximal activation at 400 nM Ca 2+, Yue et al. (1986)] is assumed. In the absence of drugs, Ca :+ is taken to oscillate between 300 nM in diastole and 350 nM during systole. Bay K 8644, an agent promoting systolic Ca 2+ influx, is assumed to increase both the size of the Ca~2+ transient and the level of diastolic Ca 2+. As a result, both contraction amplitude and diastolic tension are enhanced. In contrast, veratridine is believed to impair Ca~ + extrusion rather than to promote Ca 2+ entry during systole, since by elevating Nai+, it should primarily reduce the ability ofNa+/Ca 2+ exchange to extrude Ca 2+ from the cytosol [see Honerj~iger (1982)]. Therefore, veratridine has been modelled to solely affect the diastolic level of Ca 2+, to which then adds systolically an unchanged Ca 2+ transient of 50 nM. This behaviour would lead to a positive inotropy and a normally shaped dose-response curve for contraction amplitude, when diastolic Ca 2+ starts at a lower level (e.g. at 150 nM, not shown). At an elevated diastolic Ca 2+, however, only a very small effect on contraction amplitude is visible, and the dose-response curve is bell-shaped. Again, however, diastolic tension rises concentration-dependently.
Simulohon Tension [%]
, °°...°..-'"°'°'°°" 40 ,.% 2C
t
I
10-7
10-6 [M] Boy K 8644
Tension [%1
°°°o°......°°'" .... 4C
° °°°'°
3(: 2(
,°°.°°
°°.-"
..'°
./"
o°,"
,/
10'-7
10'-6i M] Vemtridine
Fig. 5. Model to illustrate how drugs (abscissa) which cause differential elevations in cellular Ca 2+ induce changes in muscle tension (ordinate) during diastole (broken lines) and systolic contraction amplitude (solid lines). The following assumptions are made: tension is a Hill function of Ca 2+ (Hill = 6, half maximal effect at Ca 2+= 400riM). In the absence of drugs, Ca 2+ is 300 nM in diastole and increases by 50 nM in systole. Bay K 8644 (upper graph) enhances these two Ca2+-terms concentration-dependently (Hill = 1, maximal effects are elevations by 100 nM Ca2+; for half maximum effect, the Bay K 8644 concentration is 10-~ M). As the only difference to Bay K 8644, veratridine (lower graph) does not change the size of the Ca 2+ transient, but only elevates the diastolic Ca 2+.
A rise in diastolic tension would be virtually absent when calculating with a lower starting baseline for diastolic Ca~2+. The differential behaviour observed with calcium channel blockers versus sodium channel blockers can be modelled in a similar fashion (not shown). One has to assume that the former drugs lower diastolic Ca~ + and the systolic Ca~ + transient, whereas the latter compounds act solely by lowering the level of diastolic Ca~ +.