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Effects of Ca+ and K+ on oscillatory afterpotentials in dog ventricular muscle fibers
Journalof
Molecular
andCellular
Effects
Cardiology
(1979)
11,999-1015
of Ca+ and K+ on Oscillatory in Dog Ventricular Muscle
Afterpotentials Fibers
MASAYASU HIRAOKA, YASUTAKA OKAMOTO for Cardiovascular Diseass, Tokyo Medical and Dental Tokyo, Japan
AND
Institute
TOYOMI
University,
SAN0
Tiishima,
Bunkyo-ku,
M. HIRAOKA, Y. OKAMOTO AND T. SANO. Effects of Ca+ and K+ on Oscillatory Afterpotentials in Dog Ventricular Muscle Fibers. Journal of Molecular and Cellular Cardiology (1979) 11,999-1015. Oscillatory afterpotentials were observed in dog ventricular muscle fibers at normal resting potential levels. These oscillatory afterpotentials appeared in the the train of K+-free, high-Gas+ solutions containing 3.5 nu4 Ca2+ or higher and followed applied impulses. The amplitude of the oscillatory afterpotentials was dependent upon the external Casf concentration. The oscillatory afterpotentials became larger by the application of isoproterenol (4 x 10-s M) and a low concentration of caffeine (1 mM) but a high concentration of 5 and 10 mM, stopped the oscillatory afterpotentials. The application of verapamil (1 or 4 x 10-s M) suppressed the oscillatory afterpotentials but took more than 20 min to achieve a complete abolition of these activities. The oscillatory afterpotentials were associated with tension development. Mild reduction of the external Na+ to 75% of the control increased the oscillatory afterpotentials, but further reduction suppressed them. The addition of 2.7 mM K+ or Rbf in the perfusing medium easily stopped the oscillatory after-potentials. In the presence of a lower concentration than 2.7 mM, both the K+ and the Rb+ showed concentration-dependent suppression of the oscillatory afterpotentials and the effects were similarly observed at the same concentration of each ion. The threshold to stop the oscillatory afterpotentials was found to be at a concentration between 1.08 and 1.35 mu using both K+ and Rb+. These results suggest that increased internal Gas+ and/or reduced K+ conductance have an important role in the appearance of the oscillatory afterpotentials. KEY
WORDS:
Internal
Gas+;
Oscillatory afterpotentials; K+-free, high-Gas+ solution.
Ventricular
muscle
fibers;
Triggered-activity;
1. Introduction Automatic normal abnormal latory
activity pacemaker impulse afterpotentials
important factors This is because
of cardiac mechanism formations
cells exhibits
of several
(OAPs) amongst the OAPs
muscle which have
repetitive
002%2828/79/100999+
excitations;
be slow
sorts
recently
brought
about
diastolic
of mechanisms been
thus
[19,
recognized
Dedicated to Richard 17$02.00/O
sustained
J. Bing M.D. 0
by
The
rhythmic
the or by
431.
as one
evoking another excitation not show automaticity. forming
either
depolarization,
those classified as abnormal impulse can bring the membrane to its threshold
their increased amplitudes ; thereby in the cells which, previously, do cause
can
The of
oscil-
the
most
formations potential
[6-j. from
(triggered-activity) OAPs can activity
or
also
runs
on his 70th birthday. 1979 Academic
Press Inc.
(London)
Limited
of
1000
WI. HIRAOKA
ET AL.
tachycardia. The role of the OAPs in arrhythmogenesis has been stressed in digitalis-induced arrhythmias [II] and those observed in depolarized fibers [S]. Experimental demonstrations of the OAPs and the triggered-activity, were limited to specialized tissues of particular regions of the heart or some fibers, when they were depolarized to around -60 mV or less [7, 8, 13, 16,35,36, 41, 421. Recently, we have shown that ventricular myocardium at normal resting potential levels between -90 and - 70 mV can also develop these activities uniformly when it is exposed to K+-free, high-Gas+ solutions [18]. The basic mechanism of the OSPs has not been clarified yet, although the involvement of a transient inward current of an undetermined ion specificity has been suggested in digitalis-induced OAPs of Purkinje fibers [17, 21, 22, 261. To understand the mechanism of the OAPs in ventricular muscle fibers further, the present experiments were done to examine the effects of various external ions and of agents known to affect the Casf and the K+ movement of cardiac muscles, because the OAPs and the tirggered-activity might be an important factor in abnormal automaticity developed in the working myocardium. 2. Materials
and
Methods
Mongrel dogs, weighing 8 to 15 kg, were anesthetized with sodium pentobarbital (30 to 35 mg/kg of body weight, i.v.) and the hearts rapidly removed through a thoracotomy. Fine papillary or trabecular muscles, which appeared not to contain Purkinje fibers, were dissected from the right ventricle. The usual size of these preparations was 5 to 8 mm in length and 1 to 2.5 mm in diameter. Preparations were placed in the tissue chamber, where a normal Tyrode solution was perfused. The normal Tyrode solution had the following millimolar composition : NaCl 137, KC1 2.7, CaCls 1.8, MgCls 1.0, NaHsP04 0.42, NaHCOs 11.9 and glucose 5.5. pH of the solution was adjusted to 7.2 to 7.4. Kf-free and K+-free, Cazf-free solutions were made up by omitting KC1 and CaC12 from the normal Tyrode solution. The K+-free, high-Gas+ solutions were prepared by adding extra-CaCls (3.6 to 9.0 mM) to the K+-free solution. Solutions containing K+ or Rbf were made up by adding either KC1 or RbCl to the K*-free, high-Ca2+ solution, as indicated in the text. To adjust the osmolarity constant in each experiment, sucrose in amounts equimolar to the extra CaCls in the K+-free, high-Caa+ solutions, was isoproterenol added to the K+-free and K+-free, Ca 2+-free solutions. Verapamil, (1-isoproterenoLHC1) and caffeine (CsHroN40s.HsO) were added to the bathing media in appropriate amounts. All the solutions were bubbled with 95% 02 + 5% COs throughout the experiments. Temperature of the perfusate was maintained at 36 f 1°C. Conventional glass microelectrodes, filled with 3 molar KC1 and having resistances to 10 to 20 MQ and tip potentials less than 5 mV, were used to record membrane potentials. Records of membrane potentials, obtained through conven-
Cit+
AND
K+
ON
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OAPS
IN
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1001
tional techniques, were displayed on an oscilloscope (VC-SA, Nihon Kohden Co., Tokyo) and photographed with a camera (PC-2B, Nihon Kohden Co., Tokyo) or recorded using a direct writing recorder (Rectigraph SS, San-ei Sokki Co., Tokyo). The maximum upstroke velocity (max. dV/dt) of action potentials was obtained using the R-C differentiator and having a time constant of 1.5 ms. The isometric tension was measured using electro-mechanical transducer (ME402 1, Medical Electronics Commercial Co., Tokyo). Preparations were driven by rectangular pulses (2 or 5 ms in duration and twice threshold voltage) obtained from a pulse generator (MSE-40, Nihon Kohden Co., Tokyo). Stimuli, passed through an isolation transformer, were delivered to one end of the preparations through bipolar silver electrodes. Particular care was paid to exclude the possible inclusion of Purkinje fibers, in the preparations as described in the previous report [18], and the same care was also applied in the present experiments. The exact method of inducing the OAPs, in ventricular muscle fibers, appeared in the previous report [18]. Following the same method as described, we could provoke the OAPs in almost all the preparations. 3. Results
Effects of high [Caz+], uponthe OAPs After a train of impulses was applied to the muscle preparations and suddenly stopped in the K+-free, high-Ca2+ solution, the OAPs appeared following the last driven action potential. They were, also, seen during the diastolic periods between the train of action potentials, while there was a slow frequency of stimulation. From our 40 preparations, we were able to record the appearances of the OAPs, in all the impalements that included from six to 20 different cells in each preparation. This indicated to us that the OAPs originated from the impaled individual cells, thus excluding the possibility that all the impaled cells represented Purkinje fibers. Also, the appearance times, of the OAPs, amongst the different cells, separated by the surface distance of 5 mm or more, were almost identical to each other while the difference of the peak time, of the OAPs, did not exceed more than 50 ms. There was no evidence of an electrotonic spread from action potentials, in the limited region, nor from a re-entrant action potential. The OAPs easily developed in the K+-free, high-Ca2+ solutions containing 3.6 mM or the higher Ca2f concentration, but they were not seen in the 1.8 mM and 0 mM CaZ+. Further, it was easier to induce the OAPs in the fiber, with an initial exposure to the K+free, Caaf-free solution before the high-Ca 2+ than without such an exposure. The amplitude of the OAPs showed a dependence upon the external Gas+ concentrations, once they appeared, and the typical examples are presented in Figures 1 and 2. Figure 1 shows the relationship of both the amplitude (a) and the time to peak (b) of the OAPs to the basic cycle length (BCL) of the train of impulses, examined
1002
M. HIFtAOK.4
B-AL.
(0-O) (A..4
7.2 mt4 Ca”+ 3.6 rnM Co*+
/% /..,......A’ yy-j#" (0-O) (&..A) I 0
250 Basic
FIGURE 1. Effects shows the amplitude in cycle length of the train 7.2 mM Casf solution, amplitude of the OAPs length of the train (a). Gas f (b) .
I
I
500 cycle
7.2 rnM Co’+ 3.6 rnt.i Ca2+ 750
length
I
1000
(ms)
of different (Cas+)s upon the oscillatory afterpotentials (DAPs). The ordinate (a) and the time to peak of the OAPs in (b). The abscissa represents the basic of applied impulses. The circles represent values obtained in the Kc-free, and the triangles are those in the Kf-free, 3.6 mM Gas+ solution. Note that the was always larger in the 7.2 mu Ca 2. than in the 3.6 rnM Gas+, at every cycle Time to peak of the OAPs was shorter in the 3.6 rnM than in the 7.2 rnx
in two different Ca2+ concentrations (7.2 and 3.6 1nM). The amplitude was measured from the maximum negative membrane potential immediately preceding the OAP to the peak of the OAP. The time to peak was measured as the interval from the point of the maximum negative membrane potential immediately preceding to OAP to that of the peak of the OAP. The amplitude of the OAPs was dependent upon the RCL of the train of impulses [18]. When this characteristic was examined in the 7.2 mM and in the 3.6 mM CaZ+ solutions, it was also seen in both solutions and the amplitude was always larger in the 7.2 EIM Gas+ than in the 3.6 mM Caaj- at every cycle length of the applied impulses [Figure 1(a)]. The time to peak of the OAPs became shorter in the 3.6 mM Ca2+ than in the 7.2 mM CaZ+ [Figure 1(b)]. The amplitude of the OAPs was also dependent upon the number of the preceding beats or applied impulses[18]. The dependence was again demonstrated in the present experiments and the amplitude of the OAPs always became larger in the 7.2 mM Caa+ than in the 3.6 IILM Caa+ of each number of preceding beats (Figure 2). The characteristics asshown in Figures 1 and 2 were always observed in six preparations, soakedin the K+-free, high-Caa+ solutions containing 3 6 to 9.0 mM Caaf.
Cit+
AND
K+
ON THE
OAPS
IN
Is
Is FIGURE impulses potential indicate K+-free, Note that of applied
1003
MYOCARDIUM
2. Dependence of the oscillatory afterpotentials (OAPs) upon the preceding applied in the different [Cas+]s. In each picture, the upper trace represents the membrane and the lower is the max. dV/dt of action potentials. Numbers in the left of the pictures the number of preceding applied impulses. Records in the left row were taken in the 3.6 mM Gas+ solution, and those in the right, were in the K+-free, 7.2 rnM Gas+ solution. the OAPs were always larger in the 7.2 rnM Ca sf than in the 3.6 rnM Ca*+, at each number impulses. The basic cycle length of the train was 320 ms.
Effects
of isojmoterenol
upon the OAPs
Catecholamine has been shown to induce or augment the OAPs of other preparations [41, 421. Therefore, the effect of isoproterenol on the OAPs of the ventricular muscle fibers was examined. The application of isoproterenol (4 x lo-7 M) always increased the amplitude of the OAPs and, frequently, brought about a triggeredactivity in the fiber which did not show it before the addition of the drug. The effect was easily stopped using the wash-out of isoproterenol (Figure 3). These changes were always observed in five preparations. Further the addition of isoproterenol (4 x lo-7 M), to the Kf-free, 1.8 mM Gas+ solution, sometimesinduced the OAPs in the fiber, which never showed them with the sameperfusate before the isoproterenol application (two preparations). E$ects of ca$eine upon the OAPs
Caffeine has been shown to affect, mainly, the Gas+ uptake and release from the
1004
M. HIRAOKA
ET
AL.
Gas+ storage sites as well as the Gas+ current in cardiac muscles [23, 29, 401. Therefore, the effect of caffeine upon the OAPs in our preparations was studied. The addition of 1 mM caffeine in the K+-free, high-Gas+ solution did not cause any marked changes in action potentials induced by the train of impulses but produced a change of the OAPs. The effect usually appeared within 5 min after the addition of caffeine [Figure 4(a)]. However, when the higher concentrations of 5 or 10 rnM of caffeine were applied to the preparation, soaked in the K+-free, high-Gas+ solution, therepolarization phase of action potentials was slowed with an appearance of the prominent negative afterpotential. Further, it was noted that the change of the OAPs was never developed and the pre-existing OAPs frequently disappeared after the application of 5 or 10 mM of caffeine. A wash-out of caffeine easily recovered the OAPs [Figure 4(b)]. Similar effects as described above were confirmed in four preparations.
0
-100 mV
FIGURE 3. Effects of isoproterenol upon the oscillatory afterpotentials (OAPs). (a) shows the record taken before the addition of the Isoproterenol. (b) represents the record taken during the perfusion of the 4 x IO-7 M isoproterenol. The amplitude of the OAPs became markedly increased and the triggered-activities developed as indicated by white arrows. (c) is the record taken after the wash-out of the drug. The OAPs became as small as in (a) and no triggered-activity was seen. The Gas+ concentration of the perfusate was constant at the 7.2 rnhs, throughout the experiment. The basic cycle length of the train was 640 ms.
Ca+
AND
K+
ON
THE
OAPS
IN
1005
MYOCARDIUM
Efects of verapamil upon the OAPs The effect of verapamil on the OAPs, in the ventricular muscle fibers, was examined, since verapamil is known to block the transmembrane Casf current [25]. When 1 or 4 x 10-s M verapamil was added to the K+-free, high-Gas+ solution, after the preparation developed the OAPs, and, sometimes, triggeredactivity, the amplitude of the OAPs decreased within 10 min after the applied drug to easily stop the triggered-activity. It, usually, was more than 30 min, however, before the complete suppression of the OAPs were achieved in 1 x 10V6 M
50 mV
-0
.
0
i - 100 mV Is
FIGURE 4. Effects of caffeine upon the oscillatory afterpotentials (OAPs). (a) shows the records taken before (1) and during (2) the application of 1 rn~ of caffeine. Note that the augmentation of the OAPs was seen in (2). The basic cycle length of the tram was 640 ms. (b) represents the records taken before (l), during (2) and after (3) the application of 10 rnM of caffeine. Note that the prominent negative afterpotentials, without the OAPs, were observed in 10 rnM of caffeine (2). The wash-out of caffeine recovered the OAPs and also the fiber developed triggered-activity (white arrow) (3). The basic cycle length of the train was 400 ms. (a) and (b) were obtained in different preparations.
1006
M. HIRAOKA
( b) IO min
verapamil
(c)
vcrapamil
40
min
ET AL.
IS
FIGURE 5. Effects of verapamil upon the oscillatory afterpotentiais (OAPs). In each picture, the upper trace represents the membrane potential and the lower shows the max. dV/dt of action potentials. (a) is the record taken before the application of verapamil. Well-developed OAPs and triggered-activities (last two action potentials) were observed after ten driven action potentials. The record in (b) was recorded 10 min after the start of the 1 x 10-s M verapamil. The amplitude, of the OAPs, was decreased and triggered-activity was not elicited any more. (c) shows the record obtained after 40 min of the perfusion by the 1 x 10-s M verapamil. The OAPs were almost completely stopped. The basic cycle length of the tram was 320 ms. The Gas+ concentration in the perfusing medium was constant at 7.2 rnM throughout the experiment.
verapamil. A typical example is shown in Figure 5. The effect of 4 x 10-s M verapamil was more marked than that in the 1 x 10-s M verapamil, and the complete elimination of the OAPs was usually obtained after 20 min of the application. A wash-out of the drug slowly recovered the OAPs and it was a longer time than the suppressive effect by the verapamil application. Similar results as those described above were obtained in six other preparations.
Cat
AND
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Tension development associated with OAPs
To examine whether an appearance of the OAPs was closely associated with a development of tension or not, six preparations were studied with simultaneous measurementsof both membrane potential and tension. Small aftercontractions, following twitch tension and closely associatingwith the timing of the OAPs were observed in all six preparations after the preparation developed the OAPs (Figure 6) Upon changing the mode of the applied impulses, the amplitude of the twitch tension induced by the train of action potentials differed with the BCL of the train. The aftercontractions developed, following the termination of the train or every twitch contraction, similar to the OAPs, following the tram of action potentials. The amplitude of the aftercontractions were grossly in parallel with that of the OAPs. The larger the amplitude of the OAPs, the greater the strength of the aftercontractions (Figure 6). Although Figure 6 showsa marked positive staircaseof the twitch contractions, in the BCL of 250 ms, and the largest of the aftercontractions, (a ) XL-250
ms
-0
50
w
IS
FIGURE 6. Tension development accompanying the oscillatory afterpotentials (OAPs). In each picture, the upper trace shows the membrane potential and the lower is the tension record. The train of ten impulses, with three different basic cycle lengths, was employed. The basic cycle length (BCL) of the train was indicated by the number in ms, at the left of each picture. Note that the development of the OAPs was associated with the aftercontraction of the small amplitude. The shorter the basic cycle length, the larger the amplitudes of both the OAPs and associated tension.
1008
M. HIRAOKA
ET AL.
the amplitude of the twitch contractions, induced by the train, did have a direct correlation with that of the aftercontractions. Thii is the records of the BCL of 500 ms and 1000 ms, respectively, since the twitch contractions was a little different between the two but contractions was quite different. Efect of reduced [.Na+].
not appear to apparent from the strength of that of after-
upon the OAPs
The effect of reduced external Na+ concentrations upon the OAPs was also examined, because Na+ ions are known to affect Caa+ movements of cardiac muscles [27, 321. When the Naf concentration was reduced to 75% of the normal Tyrode solution, in the K+-free, high-Caaf condition, the OAPs became larger than those in the normal Ida+ solution, and, frequently, reached the threshold to develop a triggered-activity (Figure 7, middle record). The augmentation of the OAPs was seen during the first 10 to 15 min from the 75% Naf perfusion. Wowever, the perfusion of the 75% Na+ solution with the constant Gas+ concentration of more than 30 min or further reduction of the external Na+ to 50% of the control condition, markedly decreased the amplitude of the OAPs (Figure 7, bottom record). Essentially the same results were obtained in five other preparations.
IS
FIGURE 7. Effects of the Na+ reduction upon the oscillatory afterpotentials (OAPs). The external Na+ concentration, relative to the control, was indicated at the left of each picture. When the Na+ concentration was reduced to 75% of the control, the OAPs became larger than those in the control solution and reached the threshold to develop a triggered-activity (middle record). Further reduction of the Na+ to 50% of the control. decreased the OAPs (bottom record). The Ca2+ concentration was constant at 4.5 rnM from the top to the bottom record. The basic cycle length of the train was 320 ms.
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E$ects of [K+], and [Rb+], upon the OAPs So far, the results indicated that the K+-free condition, in addition to the high-Caz+ concentration, was necessary in the development of the OAPs [18]. This may imply that the decreased K+ permeability and/or the block of the active Naf-K+ pump is required to reveal the OAPS, because the Kf-free condition is known to cause a decrease in both K+ permeability [4] and the activity of the Naf-K+ pump [30]. Therefore, the effects of external Kf and the Rbf, on the OAPs, were examined to clarify the exact purpose of the K+-free conditions in their appearance, because K+ and Rbf are known to activate the Naf-K+ pump but the membrane permeability of two ions is assumed to be different [I, 281. After the OAPs were induced in the K+-free, high-Casf solution, either K+ or Rbf contents of the perfusates were varied with constant [Ca2+lo. An addition of either 2.7 ells K+ or 2.7 mu Rb+, in the perfusates, easily stopped the OAPs, within 5 min of the start of the K+ or Rb+ solution. A removal of K+ or Rbf ions from the perfusate quickly recovered the OAPs. However, when much lower concentrations than 2.7 mM of either ions were used, the OAPs could be observed with a decreased amplitude as compared to that in the K+-free solution. The amplitude of the OAPs decreased from increasing either the external K+ or Rb+ concentration. The dependence of the amplitude of the OAPs was examined in solutions containing intermediate concentrations of either K+ or Rbf between 0 and 2.7 mM. Results of six such experiments are summarized in Figure 8. Most of the preparations showed the OAPs in 0.54 mM and 1.08 InM K+ or Rb+ solutions. However, the OAPs could not be induced in 1.35 InM K+ or Rb+ solutions except for one fiber in 1.35 mM K+
[K+&or
[Rb+]ohM)
FIGURE 8. Effects of the presence of external K+ and Rbf on the aplitude of the oscillatory afterpotentials (OAPs). The ordinate represents the amplitude of the OAPs. The abscissa indicates the c&entrati& of either the K+ or thekb+, in the perfusing medium containing 7.2 rnM Gas+, and this is plotted bv the semi-logarithmic scale. The OAPs were induced bv the train of ten imuulses with the basic cycle length of 320 ms. The values at 0 mM K+ and Rb+ represent the mean of, at least, two measurements, obtained in the K+-free and the Rb+-free solution, before and after the changes of the K+ or of the Rb+ concentrations. Circles (0 ) show the values obtained in the K+containing solutions. Crosses (X) are those in the Rb+-containing solutions.
1010
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ETAL.
Rb+=O K+=O
(cl K+=O Rb+=O
(d) 50
mV
FIGURE 9. Experimental records demonstrating the suppression of the oscillatory a&r-potentials (OAPs) by the external K+ and Rb+. In each record, the upper trace shows the membrane potential and the lower is the max. dV/dt of action potentials. The concentrations of either K+ or Rb+ are indicated at the left of each picture in mM. All the perfusing medium from (a) to (d) also contained 7.2 mM Gas+. The basic cycle length of the train was 320 ms. All the records were obtained with the same preparation and were 10 min soaked in each solution from (a) to (d) in that order.
solution. Therefore, the threshold concentration, blocking the development of the OAPs, lay between 1.08 and 1.35 mM in both K+ and Rb+. There were no apparent differences in the threshold concentrations between the two ions (Figure 9).
4. Discussion Our previous afterpotentials
[18] and the present experiments have shown that the oscillatory (OAPs) can be observed in ventricular muscle fibers, as found
Ca+
AND
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1011
similarly in Purkinje and other specialized tissues [7, 8, 13, IS, 35,36, 4.2, 421. This indicates that the OAPs are not unique features of the specialized tissues of the heart but represent a general characteristic, inherent to all the cardiac muscles. Further, the OAPs have appeared at membrane potentials more negative than -60 mV; thus, excluding a view that the activities represent limited properties of depolarized fibers [S]. These results confirmed the observations made by a few reports, which described a phenomenon like the OAPs of the present paper in the working myocardium [see 6, II], but the characteristics including the exact origination of the OAPs from the working myocardium or the actual development of the OAPs into the triggered-activity were not demonstrated. From the previous [18] and the present experiments, it was shown that the distribution of two ion species, Ca2f and K+, in the external fluid, is a major factor in the development of the OAPs in the working myocardium, because they appeared in the Kf-free, high-Caa+ condition. As to the Ca2+ function, evidence suggested that the OAPs were caused from an event closely coupled with the Ca2+ movement across the cell membrane or in the cytoplasma. This idea is supported from the following experimental findings: The OAPs were observed in the condition of a sudden increase of external Gas+, following the Caa+-free perfusion, which would cause a great increase in the Gas+ gradient across the cell membrane. The amplitude of OAPs showed the dependence on external Caf concentrations. The OAPs became larger from the application of isoproterenol and caffeine. Catecholamine is known to increase the Caa+ current across the sarcolemma and to enhance the accumulation of Gas+ by the sarcoplasmic reticulum [9, 24, 33, 38, 391. Caffeine has been shown to act, mainly, on the inhibiting Gas+ uptake of the sarcoplasmic reticulum; thus increasing the internal Gas+, although there is some evidence of the increase in the slow inward current [23, 29, 401. The OAPs were suppressex from the application of verapamil which was shown to block the Gas+ current [251. Further, the OAPs were associated with tension development, indicating a transient increase in the cytoplasmic Gas+ concentration. The slow inward current is assumed to be one of the most important factors in increasing the internal Caa+ in the heart muscle [32]. The current was once suggested as a likely candidate for the mechanism of the OAPs induced by digitalis [12] (the authors used the term “transient depolarization” of the OAPs in the present paper). Although the OAPs in our preparations showed dependence upon the external Casf concentration, and were suppressed by verapamil, as in the digitalis-induced OAPs [12, 24, these results could not be used as crucial proofs that the OAPs were brought about by the slow inward current. The slow inward current is activated upon depolarization to -40 mV or more positive potentials [31, 371, but the OAPs in our preparations, appear following the driven action potentials upon the repolarization to -60 mV or more negative potentials. These findings are strongly suggestive against the idea of considering the slow inward current as a mechanism of these OAPs. Lederer and Tsien [26] have shown the
1012
M. HIRAOKA
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presence of a transient inward current upon the repolarization from the preceding depolarizing pulses when Purkinje fibers developed the OAPs, using the treatment of digitalis and suggested the current forming the basis of the OAPs (transient depolarization). Further, this current was shown to have different properties from those of the slow inward current [17, 21, 221. We cannot rule out, however, the possibility of the OAPs being carried by the Ca2+ current, which has different characteristics from those of the slow inward current. Increased internal Gas+ may be important in developing the OAPs because of their relation to the conductance change. This view may depend upon the assumption that the aftercontractions reflect a temporal increase in the internal Ca”+, which regulate the membrane permeability to other ions. There have been presented some evidences in heart muscles that the internal Cast regulate the membrane permeability [Z, 201. As suggested by Kass et al. [21], the key step in this assumption is a transient or oscillatory release of the Casf from an intracellular store. In cardiac muscle preparations, with disrupted sarcolemma, tension oscillations, brought about by the phenomenon of “the Gas+-induced Gas+-release”, from the sarcoplasmic reticulum, have been shown in the Gas+ overloaded state [3, JO]. Glitch and Pott [14] have reported similar tension oscillations in Caoverloaded, intact cardiac cells. Although the present experiments did not give any direct evidence proving the assumption that increased internal Gas+, caused by the Gas+ release from the store, can bring the membrane conductance change; results, such as the effects of the high-Gas+, isoproterenol and caffeine on the OAPs, supported the theory that the increased internal Ca2f might be involved in the genesis of the OAPs. Verapamil suppression of the OAPs was probably due to a decrease in the internal Gas+ from blocking the Gas+ influx. Effects of the reduction of external Na+ upon the OAPs may be explained as a result of the increased internal Ca s+. Low Na+ solutions reduce the Naf gradient across the cell membrane and, thereby, decrease the Casf extrusion via the Na-Ca exchange mechanism [15, 341. Low external Na+ also cause an increase in the Gas+ influx through the action of the Na-Ca antagonism at the membrane [27]. Therefore, muscle cells were Ca s+-loaded in the low Naf perfusion. The suppression of the OAPs, after long exposure to the mildly reduced Na+ or in the moderate reduction of external Naf, may indidate that the optimum increase of the internal Casf is necessary to the appearance of the OAPs or Naf ions carry some fraction of charges forming the OAPs [22]. The result that high concentrations of caffeine did stop the OAPs, may support the explanation of the optimum Casf concentration in the cell in the appearance of the OAPs. Another important condition, to the appearance of the OAPs, was removal of the external K+. Two conditions may be induced from the K+ reduction, namely, the decreased membrane conductance and the inhibition of the Na+-K+ pump activity. The present results showed that externally applied K+ and Rb+ equally suppressed the OAPs and that there were no apparent differences in the threshold
Ca’
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1013
to suppress the OAPs. From the above results, one cannot draw a definite conclusion that either the decreasedK+ conductance or the inhibition of the Na+-K+ pump activity, or both, reveal a causative role of the OAPs. If the concentration
decrease in the Kf conductance contributes to the genesis of the OAPs, the fluctuating nature of the OAPs cannot be explained solely by changes in the K+ current since the kinetics of the K+ conductance, reported so far, have not shown such oscillatory behaviour [37]. Therefore, the contribution of other current systems must be essential, in addition to the decreased K+ conductance in the development of OAPs. On the other hand, the inhibition of the Na+-Kf pump activity will cause an increase in the internal Na+, thereby, reducing the Naf gradient, which ultimately leads to the accumulation of the internal Caaf.
REFERENCES 1. ADRIAN, R. H. & SLAYMAN, C. L. Membrane 2. 3.
transport
of sodium, potassium and rubidium
970-1014
(1966).
potential and conductance during in frog muscle. Journal of Physiology 184,
BASSINGTHWAIGHTE, J. B., FRY, C. H. & MCGUIGAN, J. A. S. Relationship between internal calcium and outward current in mammalian ventricular muscle; a mechanism for the control of the action potential duration?Journal of Physiolo~ 262, 15-37 (1976). BLOOM, S., BRADY, A. J. & LANGER, G. A. Calcium metabolism and active tension in mechanically disagregated heart muscle. Journal of Molecular and Cellular Cardiology 6, 137-147
(1974).
CARMELIET, E. E. Chloride and Potassium Permeability in Cardiac Purkinje Fibres. Bruxelles : Press Acad. European (1961). CRANEFIELD, P. F. The Conduction of the Cardiac Imjndse: The Slow Resfionse and Cardiac Arrhythmias. Mount Kisco, New York : Futura ( 1975). CRANEFIELD, P. F. Action potentials, afterpotentials, and arrhythmias. Circulation Research 41,415-423 (1977). CRANEFIELD, P. F. & ARONSON, R. S. Initiation of sustained rhythmic activity by 8. 9. 10. 11. 12. 13.
single propagated action potentials in canine cardiac Purkinje fibers exposed to sodium-free solution or to ouabain. Circulation Research 34,477-481 (1974). DAVIS, L. D. Effects of changes in cycle length on diastolic depolarization produced by ouabain in cardiac Purkinje fibers, Circulation Research 32,206-Z 14 (1973). ENTMAN, M. L., LEVEY, G. S. & EPSTEIN, S. E. Mechanism of action of epinephrine and glucagon on the canine heart: evidence for increase in sarcotubular calcium stores mediated by cyclic 3’, 5’-AMP. Circulation Research 25,429-438 (1969). FABIATO, A. & FABIATO, F. Excitation-contraction coupling of isolated cardiac fibers with disrupted or closed sarcolemmas. Circulation Research 31,293-307 (1972). FERRIER, G. R. Digitalis arrhythmias: role of oscillatory afterpotentials. Progress in Cardiouascular Diseases 19,459-474 (1977). FERRIER, G. R. & MOE, G. K. Effect of calcium on acetylstrophanthidin-induced transient depolarizations in canine Purkinje tissues. Circulation Research 33, 508-515 (1973). FERRIER, G. R., SAUNDERS,J. H. & MENDEZ, C. Cellular mechanism for the generation of ventricular arrhythmias by acetylstrophanthidin. Circulation Research 32, 600-609 (1973).
1014 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
25.
26. 27. 28. 29. 30. 31. 32. 33.
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ET AL.
GLITSCH, H. G. & POTT, L. Spontaneous tension oscillations in guinea-pig atria1 trabeculae. Pfliigers Archiu fir die gesamte Physiologie des Men&en und der Tiere 358, 1 l-25 (1975). GLITSCH, H. G., RJXUTER, H. & SCHOLZ, H. The effect ofthe internal sodium concentration on calcium fluxes in isolated guinea-pig auricles. Journal of Physiblogy 209, 25-43 (1970). HA~HIMOTO, K. & MOE, G. K. Transient depolarizations induced by acetylstrophanthidin in specialized tissues of dog atrium and ventricle. Circulation Research 32,618--624 (1973). HIRAOKA, M. Membrane current changes induced by acetylstrophanthidin in cardiac Purkinje fibers. Japanese Heart Journal 18,851-859 (1977). HIRAOKA, M., OKAMOTO, Y. & SANO, T. Oscillatory afterpotentials and triggeredautomaticity in mammalian ventricular muscle fibers at high resting potentials. Experientia 35,500-501 (1979). HOFFMAN, B. F. & CRANEFIELD, P. F. The physiological basis of cardiac arrhythmias. American Journal of Medicine 37,670-684 (1964). ISENBERC, G. Is potassium conductance of cardiac Purkinje fibers controlled by [Ca2+]i? .Nature, London 253,273-274 (1975). KAss, R. S., LEDERER, W. J., TSIEN, R. W. & WEINGART, R. Role of calcium ions in transient inward currents and aftercontractions induced by strophanthidin in cardiac Purkinje fibres. Journal of PhysioloQ 281, 187-208 (1978). KASS, R. S., TSIEN, R. W. & WEINGART, R. Ionic basis of transient inward current induced by strophanthidin in cardiac Purkinje fibres. Journal of Physiology 281,209-226 (1978). KIMOTO, Y., SAITO, M. & GOTO, M. Effects of caffeine on the membrane potentials, membrane currents and contractility of the bullfrog atrium. Japanese Journal of Physiology 24,53 1-542 ( 1974). KIRCHBERGER, M. A., TADA, M., REPKE, D. & KATZ, A. M. Cyclic adenosine 3’, 5’-monophosphate-dependent protein kinase stimulation of calcium uptake by canine cardiac microsomes. Journal of Molecular and Cellular Cardiology 4, 673680 (1972). KOHLHARDT, M., BAUER, B., KRAUSE, H. & FLECKENSTEIN, A. Differentiation of the transmembrane Na and Ca channels in mammalian cardiac fibres by the use of specific inhibitors. PjSigers Archiv.fiir die gesamte Physiologie des Menschen und der Tiere 335, 309-322 ( 1972). LEDERER, W. J. & TSIEN, R. W. Transient inward current underlying arrhythmogenic effects of cardiotonic steroids in Purkinje fibres. Journal of Physiolopy 263,73-100 (1976). L~~TTGAU, H. C. & NIEDERGERKE, R. The antagonism between Ca and Na ions on the frog’s heart. Journal of Physiology 143,486-505 (1958). MILLER, P. Potassium and rubidium exchange across the surface membrane of cardiac Purkinje fibres. Journal of PhysioloD 177,453%462 (1965). NAYLER, W. G. Effect of inotropic agents on canine trabecular muscle rendered highly permeable to calcium. American Journal of Physiology 225,918-924 (1973). PAGE, E. & STORM, S. R. Cat heart muscle in vitro. VIII. Active transport of sodium in papillary muscles. Journal of General Physiology 48,957-972 (1965). REUTER, H. Divalent cations as charge carriers in excitable membranes. Progress in Biophysics and Molscular Biology 26, 3-43 (1973). REUTER, H. Exchange of calcium ions in the mammalian myocardium. Mechanisms and physiological significance. Circulation Research 34,599-605 (1974). REUTER, H. Localization of beta adrenergic receptors, and effects of noradrenaline
Cit+AND
K+ONTHE
OAPSINMYOCARDIUM
1015
37.
and cyclic nucleotides on action potentials, ionic currents and tension in mammalian cardiac muscle. Journal of Physiology 242,42945 1 ( 1974). REUTER, H. & SEITZ, N. Dependence of calcium efflux from cardiac muscle on temperature and external ion composition. Journal of Physiology 195,451-470 (1968). ROSEN, M. R., GELBAND, H. & HOFFMAN, B. F. Correlation between effects of ouabain on the canine electrocardiogram and transmembrane potentials of isolated Purkinje fibers. Circulation 47,65-72 (1973). SAITO, T., OTOGURO, M. & MATSUBARA, T. Electrophysiological studies on the mechanism of electrically induced sustained rhythmic activity in the rabbit right atrium. Circulation Research 42, 199-206 (1978). TRAUTWEIN, W. Membrane currents in cardiac muscle fibers. Physiological Review 53,
38.
793-835 TSIEN,
34. 35. 36.
39.
40. 41. 42. 43.
(1973). R. W.,
GILES, W. & GREENGARD, P. Cyclic AMP mediates the effects of adrenaline on cardiac Purkinje fibres. Nature, .New Biology 240, 181-183 (1972). VASSORT,G.,ROUGIER,O.,GARNIER, D., SAUVIAT, M.P., CORABOEUP, E. & GARGOUIL, Y. M. Effects of adrenaline on membrane inward currents during the cardiac action potential. Pjiigers Archiv ftir die gesamte Physiologie des Menschen und der Tiere 309, 70-81 (1969). WEBER, A. The mechanism of the action of caffeine on sarcoplasmic reticulum. Journal of General Physiology 52,760-772 (1968). WIT, A. L. & CRANEFIELD, P. F. Triggered activity in cardiac muscle fibers of the simian mitral valve. Circulation Research 38,85-98 (1976). WIT, A. L. & CRANEFIELD, P. F. Triggered and automatic activity in the canine coronary sinus. Circulation Research 4L435-445 (1977). WIT, A. L., ROSEN, M. R. & HOFFMAN, B. F. Electrophysiology and pharmacology of cardiac arrhythmias. II. Relationship of normal and abnormal electrical activity of cardiac fibers to the genesis of arrhythmias. A. Automaticity. American HeartJournal 88, 515-524 (1974).
Molecular
andCellular
Effects
Cardiology
(1979)
11,999-1015
of Ca+ and K+ on Oscillatory in Dog Ventricular Muscle
Afterpotentials Fibers
MASAYASU HIRAOKA, YASUTAKA OKAMOTO for Cardiovascular Diseass, Tokyo Medical and Dental Tokyo, Japan
AND
Institute
TOYOMI
University,
SAN0
Tiishima,
Bunkyo-ku,
M. HIRAOKA, Y. OKAMOTO AND T. SANO. Effects of Ca+ and K+ on Oscillatory Afterpotentials in Dog Ventricular Muscle Fibers. Journal of Molecular and Cellular Cardiology (1979) 11,999-1015. Oscillatory afterpotentials were observed in dog ventricular muscle fibers at normal resting potential levels. These oscillatory afterpotentials appeared in the the train of K+-free, high-Gas+ solutions containing 3.5 nu4 Ca2+ or higher and followed applied impulses. The amplitude of the oscillatory afterpotentials was dependent upon the external Casf concentration. The oscillatory afterpotentials became larger by the application of isoproterenol (4 x 10-s M) and a low concentration of caffeine (1 mM) but a high concentration of 5 and 10 mM, stopped the oscillatory afterpotentials. The application of verapamil (1 or 4 x 10-s M) suppressed the oscillatory afterpotentials but took more than 20 min to achieve a complete abolition of these activities. The oscillatory afterpotentials were associated with tension development. Mild reduction of the external Na+ to 75% of the control increased the oscillatory afterpotentials, but further reduction suppressed them. The addition of 2.7 mM K+ or Rbf in the perfusing medium easily stopped the oscillatory after-potentials. In the presence of a lower concentration than 2.7 mM, both the K+ and the Rb+ showed concentration-dependent suppression of the oscillatory afterpotentials and the effects were similarly observed at the same concentration of each ion. The threshold to stop the oscillatory afterpotentials was found to be at a concentration between 1.08 and 1.35 mu using both K+ and Rb+. These results suggest that increased internal Gas+ and/or reduced K+ conductance have an important role in the appearance of the oscillatory afterpotentials. KEY
WORDS:
Internal
Gas+;
Oscillatory afterpotentials; K+-free, high-Gas+ solution.
Ventricular
muscle
fibers;
Triggered-activity;
1. Introduction Automatic normal abnormal latory
activity pacemaker impulse afterpotentials
important factors This is because
of cardiac mechanism formations
cells exhibits
of several
(OAPs) amongst the OAPs
muscle which have
repetitive
002%2828/79/100999+
excitations;
be slow
sorts
recently
brought
about
diastolic
of mechanisms been
thus
[19,
recognized
Dedicated to Richard 17$02.00/O
sustained
J. Bing M.D. 0
by
The
rhythmic
the or by
431.
as one
evoking another excitation not show automaticity. forming
either
depolarization,
those classified as abnormal impulse can bring the membrane to its threshold
their increased amplitudes ; thereby in the cells which, previously, do cause
can
The of
oscil-
the
most
formations potential
[6-j. from
(triggered-activity) OAPs can activity
or
also
runs
on his 70th birthday. 1979 Academic
Press Inc.
(London)
Limited
of
1000
WI. HIRAOKA
ET AL.
tachycardia. The role of the OAPs in arrhythmogenesis has been stressed in digitalis-induced arrhythmias [II] and those observed in depolarized fibers [S]. Experimental demonstrations of the OAPs and the triggered-activity, were limited to specialized tissues of particular regions of the heart or some fibers, when they were depolarized to around -60 mV or less [7, 8, 13, 16,35,36, 41, 421. Recently, we have shown that ventricular myocardium at normal resting potential levels between -90 and - 70 mV can also develop these activities uniformly when it is exposed to K+-free, high-Gas+ solutions [18]. The basic mechanism of the OSPs has not been clarified yet, although the involvement of a transient inward current of an undetermined ion specificity has been suggested in digitalis-induced OAPs of Purkinje fibers [17, 21, 22, 261. To understand the mechanism of the OAPs in ventricular muscle fibers further, the present experiments were done to examine the effects of various external ions and of agents known to affect the Casf and the K+ movement of cardiac muscles, because the OAPs and the tirggered-activity might be an important factor in abnormal automaticity developed in the working myocardium. 2. Materials
and
Methods
Mongrel dogs, weighing 8 to 15 kg, were anesthetized with sodium pentobarbital (30 to 35 mg/kg of body weight, i.v.) and the hearts rapidly removed through a thoracotomy. Fine papillary or trabecular muscles, which appeared not to contain Purkinje fibers, were dissected from the right ventricle. The usual size of these preparations was 5 to 8 mm in length and 1 to 2.5 mm in diameter. Preparations were placed in the tissue chamber, where a normal Tyrode solution was perfused. The normal Tyrode solution had the following millimolar composition : NaCl 137, KC1 2.7, CaCls 1.8, MgCls 1.0, NaHsP04 0.42, NaHCOs 11.9 and glucose 5.5. pH of the solution was adjusted to 7.2 to 7.4. Kf-free and K+-free, Cazf-free solutions were made up by omitting KC1 and CaC12 from the normal Tyrode solution. The K+-free, high-Gas+ solutions were prepared by adding extra-CaCls (3.6 to 9.0 mM) to the K+-free solution. Solutions containing K+ or Rbf were made up by adding either KC1 or RbCl to the K*-free, high-Ca2+ solution, as indicated in the text. To adjust the osmolarity constant in each experiment, sucrose in amounts equimolar to the extra CaCls in the K+-free, high-Caa+ solutions, was isoproterenol added to the K+-free and K+-free, Ca 2+-free solutions. Verapamil, (1-isoproterenoLHC1) and caffeine (CsHroN40s.HsO) were added to the bathing media in appropriate amounts. All the solutions were bubbled with 95% 02 + 5% COs throughout the experiments. Temperature of the perfusate was maintained at 36 f 1°C. Conventional glass microelectrodes, filled with 3 molar KC1 and having resistances to 10 to 20 MQ and tip potentials less than 5 mV, were used to record membrane potentials. Records of membrane potentials, obtained through conven-
Cit+
AND
K+
ON
THE
OAPS
IN
MYOCARDIUM
1001
tional techniques, were displayed on an oscilloscope (VC-SA, Nihon Kohden Co., Tokyo) and photographed with a camera (PC-2B, Nihon Kohden Co., Tokyo) or recorded using a direct writing recorder (Rectigraph SS, San-ei Sokki Co., Tokyo). The maximum upstroke velocity (max. dV/dt) of action potentials was obtained using the R-C differentiator and having a time constant of 1.5 ms. The isometric tension was measured using electro-mechanical transducer (ME402 1, Medical Electronics Commercial Co., Tokyo). Preparations were driven by rectangular pulses (2 or 5 ms in duration and twice threshold voltage) obtained from a pulse generator (MSE-40, Nihon Kohden Co., Tokyo). Stimuli, passed through an isolation transformer, were delivered to one end of the preparations through bipolar silver electrodes. Particular care was paid to exclude the possible inclusion of Purkinje fibers, in the preparations as described in the previous report [18], and the same care was also applied in the present experiments. The exact method of inducing the OAPs, in ventricular muscle fibers, appeared in the previous report [18]. Following the same method as described, we could provoke the OAPs in almost all the preparations. 3. Results
Effects of high [Caz+], uponthe OAPs After a train of impulses was applied to the muscle preparations and suddenly stopped in the K+-free, high-Ca2+ solution, the OAPs appeared following the last driven action potential. They were, also, seen during the diastolic periods between the train of action potentials, while there was a slow frequency of stimulation. From our 40 preparations, we were able to record the appearances of the OAPs, in all the impalements that included from six to 20 different cells in each preparation. This indicated to us that the OAPs originated from the impaled individual cells, thus excluding the possibility that all the impaled cells represented Purkinje fibers. Also, the appearance times, of the OAPs, amongst the different cells, separated by the surface distance of 5 mm or more, were almost identical to each other while the difference of the peak time, of the OAPs, did not exceed more than 50 ms. There was no evidence of an electrotonic spread from action potentials, in the limited region, nor from a re-entrant action potential. The OAPs easily developed in the K+-free, high-Ca2+ solutions containing 3.6 mM or the higher Ca2f concentration, but they were not seen in the 1.8 mM and 0 mM CaZ+. Further, it was easier to induce the OAPs in the fiber, with an initial exposure to the K+free, Caaf-free solution before the high-Ca 2+ than without such an exposure. The amplitude of the OAPs showed a dependence upon the external Gas+ concentrations, once they appeared, and the typical examples are presented in Figures 1 and 2. Figure 1 shows the relationship of both the amplitude (a) and the time to peak (b) of the OAPs to the basic cycle length (BCL) of the train of impulses, examined
1002
M. HIFtAOK.4
B-AL.
(0-O) (A..4
7.2 mt4 Ca”+ 3.6 rnM Co*+
/% /..,......A’ yy-j#" (0-O) (&..A) I 0
250 Basic
FIGURE 1. Effects shows the amplitude in cycle length of the train 7.2 mM Casf solution, amplitude of the OAPs length of the train (a). Gas f (b) .
I
I
500 cycle
7.2 rnM Co’+ 3.6 rnt.i Ca2+ 750
length
I
1000
(ms)
of different (Cas+)s upon the oscillatory afterpotentials (DAPs). The ordinate (a) and the time to peak of the OAPs in (b). The abscissa represents the basic of applied impulses. The circles represent values obtained in the Kc-free, and the triangles are those in the Kf-free, 3.6 mM Gas+ solution. Note that the was always larger in the 7.2 mu Ca 2. than in the 3.6 rnM Gas+, at every cycle Time to peak of the OAPs was shorter in the 3.6 rnM than in the 7.2 rnx
in two different Ca2+ concentrations (7.2 and 3.6 1nM). The amplitude was measured from the maximum negative membrane potential immediately preceding the OAP to the peak of the OAP. The time to peak was measured as the interval from the point of the maximum negative membrane potential immediately preceding to OAP to that of the peak of the OAP. The amplitude of the OAPs was dependent upon the RCL of the train of impulses [18]. When this characteristic was examined in the 7.2 mM and in the 3.6 mM CaZ+ solutions, it was also seen in both solutions and the amplitude was always larger in the 7.2 EIM Gas+ than in the 3.6 mM Caaj- at every cycle length of the applied impulses [Figure 1(a)]. The time to peak of the OAPs became shorter in the 3.6 mM Ca2+ than in the 7.2 mM CaZ+ [Figure 1(b)]. The amplitude of the OAPs was also dependent upon the number of the preceding beats or applied impulses[18]. The dependence was again demonstrated in the present experiments and the amplitude of the OAPs always became larger in the 7.2 mM Caa+ than in the 3.6 IILM Caa+ of each number of preceding beats (Figure 2). The characteristics asshown in Figures 1 and 2 were always observed in six preparations, soakedin the K+-free, high-Caa+ solutions containing 3 6 to 9.0 mM Caaf.
Cit+
AND
K+
ON THE
OAPS
IN
Is
Is FIGURE impulses potential indicate K+-free, Note that of applied
1003
MYOCARDIUM
2. Dependence of the oscillatory afterpotentials (OAPs) upon the preceding applied in the different [Cas+]s. In each picture, the upper trace represents the membrane and the lower is the max. dV/dt of action potentials. Numbers in the left of the pictures the number of preceding applied impulses. Records in the left row were taken in the 3.6 mM Gas+ solution, and those in the right, were in the K+-free, 7.2 rnM Gas+ solution. the OAPs were always larger in the 7.2 rnM Ca sf than in the 3.6 rnM Ca*+, at each number impulses. The basic cycle length of the train was 320 ms.
Effects
of isojmoterenol
upon the OAPs
Catecholamine has been shown to induce or augment the OAPs of other preparations [41, 421. Therefore, the effect of isoproterenol on the OAPs of the ventricular muscle fibers was examined. The application of isoproterenol (4 x lo-7 M) always increased the amplitude of the OAPs and, frequently, brought about a triggeredactivity in the fiber which did not show it before the addition of the drug. The effect was easily stopped using the wash-out of isoproterenol (Figure 3). These changes were always observed in five preparations. Further the addition of isoproterenol (4 x lo-7 M), to the Kf-free, 1.8 mM Gas+ solution, sometimesinduced the OAPs in the fiber, which never showed them with the sameperfusate before the isoproterenol application (two preparations). E$ects of ca$eine upon the OAPs
Caffeine has been shown to affect, mainly, the Gas+ uptake and release from the
1004
M. HIRAOKA
ET
AL.
Gas+ storage sites as well as the Gas+ current in cardiac muscles [23, 29, 401. Therefore, the effect of caffeine upon the OAPs in our preparations was studied. The addition of 1 mM caffeine in the K+-free, high-Gas+ solution did not cause any marked changes in action potentials induced by the train of impulses but produced a change of the OAPs. The effect usually appeared within 5 min after the addition of caffeine [Figure 4(a)]. However, when the higher concentrations of 5 or 10 rnM of caffeine were applied to the preparation, soaked in the K+-free, high-Gas+ solution, therepolarization phase of action potentials was slowed with an appearance of the prominent negative afterpotential. Further, it was noted that the change of the OAPs was never developed and the pre-existing OAPs frequently disappeared after the application of 5 or 10 mM of caffeine. A wash-out of caffeine easily recovered the OAPs [Figure 4(b)]. Similar effects as described above were confirmed in four preparations.
0
-100 mV
FIGURE 3. Effects of isoproterenol upon the oscillatory afterpotentials (OAPs). (a) shows the record taken before the addition of the Isoproterenol. (b) represents the record taken during the perfusion of the 4 x IO-7 M isoproterenol. The amplitude of the OAPs became markedly increased and the triggered-activities developed as indicated by white arrows. (c) is the record taken after the wash-out of the drug. The OAPs became as small as in (a) and no triggered-activity was seen. The Gas+ concentration of the perfusate was constant at the 7.2 rnhs, throughout the experiment. The basic cycle length of the train was 640 ms.
Ca+
AND
K+
ON
THE
OAPS
IN
1005
MYOCARDIUM
Efects of verapamil upon the OAPs The effect of verapamil on the OAPs, in the ventricular muscle fibers, was examined, since verapamil is known to block the transmembrane Casf current [25]. When 1 or 4 x 10-s M verapamil was added to the K+-free, high-Gas+ solution, after the preparation developed the OAPs, and, sometimes, triggeredactivity, the amplitude of the OAPs decreased within 10 min after the applied drug to easily stop the triggered-activity. It, usually, was more than 30 min, however, before the complete suppression of the OAPs were achieved in 1 x 10V6 M
50 mV
-0
.
0
i - 100 mV Is
FIGURE 4. Effects of caffeine upon the oscillatory afterpotentials (OAPs). (a) shows the records taken before (1) and during (2) the application of 1 rn~ of caffeine. Note that the augmentation of the OAPs was seen in (2). The basic cycle length of the tram was 640 ms. (b) represents the records taken before (l), during (2) and after (3) the application of 10 rnM of caffeine. Note that the prominent negative afterpotentials, without the OAPs, were observed in 10 rnM of caffeine (2). The wash-out of caffeine recovered the OAPs and also the fiber developed triggered-activity (white arrow) (3). The basic cycle length of the train was 400 ms. (a) and (b) were obtained in different preparations.
1006
M. HIRAOKA
( b) IO min
verapamil
(c)
vcrapamil
40
min
ET AL.
IS
FIGURE 5. Effects of verapamil upon the oscillatory afterpotentiais (OAPs). In each picture, the upper trace represents the membrane potential and the lower shows the max. dV/dt of action potentials. (a) is the record taken before the application of verapamil. Well-developed OAPs and triggered-activities (last two action potentials) were observed after ten driven action potentials. The record in (b) was recorded 10 min after the start of the 1 x 10-s M verapamil. The amplitude, of the OAPs, was decreased and triggered-activity was not elicited any more. (c) shows the record obtained after 40 min of the perfusion by the 1 x 10-s M verapamil. The OAPs were almost completely stopped. The basic cycle length of the tram was 320 ms. The Gas+ concentration in the perfusing medium was constant at 7.2 rnM throughout the experiment.
verapamil. A typical example is shown in Figure 5. The effect of 4 x 10-s M verapamil was more marked than that in the 1 x 10-s M verapamil, and the complete elimination of the OAPs was usually obtained after 20 min of the application. A wash-out of the drug slowly recovered the OAPs and it was a longer time than the suppressive effect by the verapamil application. Similar results as those described above were obtained in six other preparations.
Cat
AND
K+
ON
THE
OAPS
IN
1007
MYOCARDIUM
Tension development associated with OAPs
To examine whether an appearance of the OAPs was closely associated with a development of tension or not, six preparations were studied with simultaneous measurementsof both membrane potential and tension. Small aftercontractions, following twitch tension and closely associatingwith the timing of the OAPs were observed in all six preparations after the preparation developed the OAPs (Figure 6) Upon changing the mode of the applied impulses, the amplitude of the twitch tension induced by the train of action potentials differed with the BCL of the train. The aftercontractions developed, following the termination of the train or every twitch contraction, similar to the OAPs, following the tram of action potentials. The amplitude of the aftercontractions were grossly in parallel with that of the OAPs. The larger the amplitude of the OAPs, the greater the strength of the aftercontractions (Figure 6). Although Figure 6 showsa marked positive staircaseof the twitch contractions, in the BCL of 250 ms, and the largest of the aftercontractions, (a ) XL-250
ms
-0
50
w
IS
FIGURE 6. Tension development accompanying the oscillatory afterpotentials (OAPs). In each picture, the upper trace shows the membrane potential and the lower is the tension record. The train of ten impulses, with three different basic cycle lengths, was employed. The basic cycle length (BCL) of the train was indicated by the number in ms, at the left of each picture. Note that the development of the OAPs was associated with the aftercontraction of the small amplitude. The shorter the basic cycle length, the larger the amplitudes of both the OAPs and associated tension.
1008
M. HIRAOKA
ET AL.
the amplitude of the twitch contractions, induced by the train, did have a direct correlation with that of the aftercontractions. Thii is the records of the BCL of 500 ms and 1000 ms, respectively, since the twitch contractions was a little different between the two but contractions was quite different. Efect of reduced [.Na+].
not appear to apparent from the strength of that of after-
upon the OAPs
The effect of reduced external Na+ concentrations upon the OAPs was also examined, because Na+ ions are known to affect Caa+ movements of cardiac muscles [27, 321. When the Naf concentration was reduced to 75% of the normal Tyrode solution, in the K+-free, high-Caaf condition, the OAPs became larger than those in the normal Ida+ solution, and, frequently, reached the threshold to develop a triggered-activity (Figure 7, middle record). The augmentation of the OAPs was seen during the first 10 to 15 min from the 75% Naf perfusion. Wowever, the perfusion of the 75% Na+ solution with the constant Gas+ concentration of more than 30 min or further reduction of the external Na+ to 50% of the control condition, markedly decreased the amplitude of the OAPs (Figure 7, bottom record). Essentially the same results were obtained in five other preparations.
IS
FIGURE 7. Effects of the Na+ reduction upon the oscillatory afterpotentials (OAPs). The external Na+ concentration, relative to the control, was indicated at the left of each picture. When the Na+ concentration was reduced to 75% of the control, the OAPs became larger than those in the control solution and reached the threshold to develop a triggered-activity (middle record). Further reduction of the Na+ to 50% of the control. decreased the OAPs (bottom record). The Ca2+ concentration was constant at 4.5 rnM from the top to the bottom record. The basic cycle length of the train was 320 ms.
Ca+
AND
K+
ON
THE
OAPS
IN
MYOCARDIUM
1009
E$ects of [K+], and [Rb+], upon the OAPs So far, the results indicated that the K+-free condition, in addition to the high-Caz+ concentration, was necessary in the development of the OAPs [18]. This may imply that the decreased K+ permeability and/or the block of the active Naf-K+ pump is required to reveal the OAPS, because the Kf-free condition is known to cause a decrease in both K+ permeability [4] and the activity of the Naf-K+ pump [30]. Therefore, the effects of external Kf and the Rbf, on the OAPs, were examined to clarify the exact purpose of the K+-free conditions in their appearance, because K+ and Rbf are known to activate the Naf-K+ pump but the membrane permeability of two ions is assumed to be different [I, 281. After the OAPs were induced in the K+-free, high-Casf solution, either K+ or Rbf contents of the perfusates were varied with constant [Ca2+lo. An addition of either 2.7 ells K+ or 2.7 mu Rb+, in the perfusates, easily stopped the OAPs, within 5 min of the start of the K+ or Rb+ solution. A removal of K+ or Rbf ions from the perfusate quickly recovered the OAPs. However, when much lower concentrations than 2.7 mM of either ions were used, the OAPs could be observed with a decreased amplitude as compared to that in the K+-free solution. The amplitude of the OAPs decreased from increasing either the external K+ or Rb+ concentration. The dependence of the amplitude of the OAPs was examined in solutions containing intermediate concentrations of either K+ or Rbf between 0 and 2.7 mM. Results of six such experiments are summarized in Figure 8. Most of the preparations showed the OAPs in 0.54 mM and 1.08 InM K+ or Rb+ solutions. However, the OAPs could not be induced in 1.35 InM K+ or Rb+ solutions except for one fiber in 1.35 mM K+
[K+&or
[Rb+]ohM)
FIGURE 8. Effects of the presence of external K+ and Rbf on the aplitude of the oscillatory afterpotentials (OAPs). The ordinate represents the amplitude of the OAPs. The abscissa indicates the c&entrati& of either the K+ or thekb+, in the perfusing medium containing 7.2 rnM Gas+, and this is plotted bv the semi-logarithmic scale. The OAPs were induced bv the train of ten imuulses with the basic cycle length of 320 ms. The values at 0 mM K+ and Rb+ represent the mean of, at least, two measurements, obtained in the K+-free and the Rb+-free solution, before and after the changes of the K+ or of the Rb+ concentrations. Circles (0 ) show the values obtained in the K+containing solutions. Crosses (X) are those in the Rb+-containing solutions.
1010
M. HIRAOKA
ETAL.
Rb+=O K+=O
(cl K+=O Rb+=O
(d) 50
mV
FIGURE 9. Experimental records demonstrating the suppression of the oscillatory a&r-potentials (OAPs) by the external K+ and Rb+. In each record, the upper trace shows the membrane potential and the lower is the max. dV/dt of action potentials. The concentrations of either K+ or Rb+ are indicated at the left of each picture in mM. All the perfusing medium from (a) to (d) also contained 7.2 mM Gas+. The basic cycle length of the train was 320 ms. All the records were obtained with the same preparation and were 10 min soaked in each solution from (a) to (d) in that order.
solution. Therefore, the threshold concentration, blocking the development of the OAPs, lay between 1.08 and 1.35 mM in both K+ and Rb+. There were no apparent differences in the threshold concentrations between the two ions (Figure 9).
4. Discussion Our previous afterpotentials
[18] and the present experiments have shown that the oscillatory (OAPs) can be observed in ventricular muscle fibers, as found
Ca+
AND
K+
ON
THE
OAPS
IN
MYOCARDIUM
1011
similarly in Purkinje and other specialized tissues [7, 8, 13, IS, 35,36, 4.2, 421. This indicates that the OAPs are not unique features of the specialized tissues of the heart but represent a general characteristic, inherent to all the cardiac muscles. Further, the OAPs have appeared at membrane potentials more negative than -60 mV; thus, excluding a view that the activities represent limited properties of depolarized fibers [S]. These results confirmed the observations made by a few reports, which described a phenomenon like the OAPs of the present paper in the working myocardium [see 6, II], but the characteristics including the exact origination of the OAPs from the working myocardium or the actual development of the OAPs into the triggered-activity were not demonstrated. From the previous [18] and the present experiments, it was shown that the distribution of two ion species, Ca2f and K+, in the external fluid, is a major factor in the development of the OAPs in the working myocardium, because they appeared in the Kf-free, high-Caa+ condition. As to the Ca2+ function, evidence suggested that the OAPs were caused from an event closely coupled with the Ca2+ movement across the cell membrane or in the cytoplasma. This idea is supported from the following experimental findings: The OAPs were observed in the condition of a sudden increase of external Gas+, following the Caa+-free perfusion, which would cause a great increase in the Gas+ gradient across the cell membrane. The amplitude of OAPs showed the dependence on external Caf concentrations. The OAPs became larger from the application of isoproterenol and caffeine. Catecholamine is known to increase the Caa+ current across the sarcolemma and to enhance the accumulation of Gas+ by the sarcoplasmic reticulum [9, 24, 33, 38, 391. Caffeine has been shown to act, mainly, on the inhibiting Gas+ uptake of the sarcoplasmic reticulum; thus increasing the internal Gas+, although there is some evidence of the increase in the slow inward current [23, 29, 401. The OAPs were suppressex from the application of verapamil which was shown to block the Gas+ current [251. Further, the OAPs were associated with tension development, indicating a transient increase in the cytoplasmic Gas+ concentration. The slow inward current is assumed to be one of the most important factors in increasing the internal Caa+ in the heart muscle [32]. The current was once suggested as a likely candidate for the mechanism of the OAPs induced by digitalis [12] (the authors used the term “transient depolarization” of the OAPs in the present paper). Although the OAPs in our preparations showed dependence upon the external Casf concentration, and were suppressed by verapamil, as in the digitalis-induced OAPs [12, 24, these results could not be used as crucial proofs that the OAPs were brought about by the slow inward current. The slow inward current is activated upon depolarization to -40 mV or more positive potentials [31, 371, but the OAPs in our preparations, appear following the driven action potentials upon the repolarization to -60 mV or more negative potentials. These findings are strongly suggestive against the idea of considering the slow inward current as a mechanism of these OAPs. Lederer and Tsien [26] have shown the
1012
M. HIRAOKA
ET AL.
presence of a transient inward current upon the repolarization from the preceding depolarizing pulses when Purkinje fibers developed the OAPs, using the treatment of digitalis and suggested the current forming the basis of the OAPs (transient depolarization). Further, this current was shown to have different properties from those of the slow inward current [17, 21, 221. We cannot rule out, however, the possibility of the OAPs being carried by the Ca2+ current, which has different characteristics from those of the slow inward current. Increased internal Gas+ may be important in developing the OAPs because of their relation to the conductance change. This view may depend upon the assumption that the aftercontractions reflect a temporal increase in the internal Ca”+, which regulate the membrane permeability to other ions. There have been presented some evidences in heart muscles that the internal Cast regulate the membrane permeability [Z, 201. As suggested by Kass et al. [21], the key step in this assumption is a transient or oscillatory release of the Casf from an intracellular store. In cardiac muscle preparations, with disrupted sarcolemma, tension oscillations, brought about by the phenomenon of “the Gas+-induced Gas+-release”, from the sarcoplasmic reticulum, have been shown in the Gas+ overloaded state [3, JO]. Glitch and Pott [14] have reported similar tension oscillations in Caoverloaded, intact cardiac cells. Although the present experiments did not give any direct evidence proving the assumption that increased internal Gas+, caused by the Gas+ release from the store, can bring the membrane conductance change; results, such as the effects of the high-Gas+, isoproterenol and caffeine on the OAPs, supported the theory that the increased internal Ca2f might be involved in the genesis of the OAPs. Verapamil suppression of the OAPs was probably due to a decrease in the internal Gas+ from blocking the Gas+ influx. Effects of the reduction of external Na+ upon the OAPs may be explained as a result of the increased internal Ca s+. Low Na+ solutions reduce the Naf gradient across the cell membrane and, thereby, decrease the Casf extrusion via the Na-Ca exchange mechanism [15, 341. Low external Na+ also cause an increase in the Gas+ influx through the action of the Na-Ca antagonism at the membrane [27]. Therefore, muscle cells were Ca s+-loaded in the low Naf perfusion. The suppression of the OAPs, after long exposure to the mildly reduced Na+ or in the moderate reduction of external Naf, may indidate that the optimum increase of the internal Casf is necessary to the appearance of the OAPs or Naf ions carry some fraction of charges forming the OAPs [22]. The result that high concentrations of caffeine did stop the OAPs, may support the explanation of the optimum Casf concentration in the cell in the appearance of the OAPs. Another important condition, to the appearance of the OAPs, was removal of the external K+. Two conditions may be induced from the K+ reduction, namely, the decreased membrane conductance and the inhibition of the Na+-K+ pump activity. The present results showed that externally applied K+ and Rb+ equally suppressed the OAPs and that there were no apparent differences in the threshold
Ca’
AND
K+
ON
THE
OAPS
IN MYOCARDIUM
1013
to suppress the OAPs. From the above results, one cannot draw a definite conclusion that either the decreasedK+ conductance or the inhibition of the Na+-K+ pump activity, or both, reveal a causative role of the OAPs. If the concentration
decrease in the Kf conductance contributes to the genesis of the OAPs, the fluctuating nature of the OAPs cannot be explained solely by changes in the K+ current since the kinetics of the K+ conductance, reported so far, have not shown such oscillatory behaviour [37]. Therefore, the contribution of other current systems must be essential, in addition to the decreased K+ conductance in the development of OAPs. On the other hand, the inhibition of the Na+-Kf pump activity will cause an increase in the internal Na+, thereby, reducing the Naf gradient, which ultimately leads to the accumulation of the internal Caaf.
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