College of Medicine, Salt Lake City, Utah, U.S.A.

Transcription

1 J. Phy8iol. (1968), 196, pp With 7 text-figurms Printed in Great Britain FACILITATION OF HEART MUSCLE CONTRACTION AND ITS DEPENDENCE ON EXTERNAL CALCIUM AND SODIUM By R. K. ORKAND From the Department of Physiology, University of Utah College of Medicine, Salt Lake City, Utah, U.S.A. (Received 19 September 1967) SUMMARY 1. The magnitude and time course of the facilitation of contraction following previous stimulation have been studied in strips of frog ventricle when the external concentrations of Na and Ca were varied. The maximum tension and the maximum rate of rise of tension have been used as indices of activation of the contractile element. Under the experimental conditions these two parameters changed similarly. 2. At low stimulus frequencies (3-12 beats/min), and moderate concentrations of external Ca (2-5 mm), the facilitation of contraction with repetitive stimulation can be predicted by assuming each contraction to produce an amount of facilitation which sums linearly with that remaining from previous responses. Thus, the staircase can be constructed from the decay of facilitation following a single contraction. 3. The decay of facilitation appears to consist of two components. The first has a half-time of about 3 sec; the second about 5 sec. The decay of the second component of facilitation is slowed and its magnitude increased by raising the external Ca from 2 to 5 mm. 4. Reducing the external Na concentration increases both the initial strength of contraction and the amount of facilitation. 5. When the ratio Ca/Na2 is kept constant while changing the Ca and Na concentrations, the first contraction in a series remains constant, but facilitation is less at low concentrations of Ca and Na. Facilitation, therefore, does not depend solely on the amount of initial activation of the contractile system. 6. The results are consistent with the hypothesis that facilitation depends on the amount of calcium retained in some cellular store.

2 312 R. K. ORKAND INTRODUCTION Repetitive stimulation of previously resting heart muscle leads to a progressive increase in contractile tension, the well-known staircase phenomenon. The increase in peak tension as well as velocity of contraction is obtained without a corresponding increase in the amplitude or duration of the action potential and appears to result from a facilitation of those processes linking membrane depolarization to contraction (for a review, see Koch-Weser & Blinks, 1963). The strength of contraction of the heart is increased when the external calcium concentration is raised or when the external sodium concentration is reduced (Liittgau & Niedergerke, 1958). In both of these cases the movement of calcium into the heart cells is enhanced (Niedergerke, 1963a, b). It was suggested, on the basis of the mechanical responses of the heart in different ionic environments, that the staircase results from an accumulation of calcium in the heart cell (Niedergerke, 1956). In addition, the depression of the overshoot of the cardiac action potential with repetitive stimulation at high external calcium concentrations could be explained by such an accumulation of calcium (Niedergerke & Orkand, 1966). The main purpose of the present work has been to study changes in facilitation of contraction produced by varying the external calcium and sodium concentrations, and to relate these changes to the known calcium fluxes and behaviour of the cardiac action potential. A preliminary report of this work has been presented (Orkand, 1967). METHODS Frogs (Rana pipien8) were maintained at room temperature C at least 1 week before use. Quiescent ventricle strips, 8-15 mm long and mm thick, were dissected as previously described (Dale, 1932) and observed for about 5 hr to make sure that the ventricular pace-maker tissue had been removed. The strips were mounted vertically in a 1 ml. polyethylene chamber. They were tied with fine silk thread at one end to a stainless-steel hook immersed in the fluid; the other end was attached by cm of thread to an isometric gauge (Micro-sensor) which was displaced about -5 mm during the strongest contractions recorded. During the course of an experiment the resting tension tended to decrease, probably due to stretching of the elastic elements in the preparation. In a control experiment the resting tension was varied from 75 to 36 mg. This increased the twitch tension of the ventricle strip from 145 to 25 mg, but did not affect the amount of facilitation produced by a three-shock train of stimuli at 1 sec intervals. Nevertheless, although the resting tension was occasionally adjusted in the course of an experiment, it was not altered between a series of runs in which the amount of facilitation was to be compared. The output of the strain gauge was recorded with a Tektronix 52 A oscilloscope and also differentiated with a passive network with a 1 msec time constant. The differentiated signal was recorded on the second channel of the oscilloscope. The outputs of both channels of the oscilloscope were displayed on a CEC rectilinear pen recorder which had a frequency response of -7 c/s (3 db down). The amplitudes of the responses were measured directly from the oscillograph

3 FACILITATION OF CONTRACTION 313 trace. The ventricle was stimulated with a shock 2 msec in duration and a strength of up to 2 times threshold applied to two platinum electrodes on either side of the strip. One electrode was connected to earth. Figure 1 shows the relation between the ventricular action potential, tension response and the derivative of the tension response. It can be seen that the rise of the contraction continues until the action potential terminates but that the rate of rise reaches a maximum before the end of the action potential. Thus, although the peak tension response Fig. 1. Relation between action potential, contraction and rate of change of tension. Top: intracellularly recorded action potential of frog ventricle. Solid line at zero membrane potential. Middle: tension response. Bottom: first derivative of tension response obtained by differentiation of middle trace with a passive RC network with a time constant of 1 msec. is sensitive to changes in the duration of the action potential, its derivative is very much less sensitive to such changes. The present study was carried out under conditions where the duration of the contraction varied less than 15 %. The observed changes, therefore, represent differences in the amount of activation of the contractile apparatus and do not result primarily from changes in the duration of the action potential (see also, Niedergerke, 1956). Limitations of the analy8si. The determination of the decay of facilitation was limited to six measurements in each experiment because the slowness of the process made necessary an interval of up to 15 min between each test and the preparation was often not indefinitely stable. In addition, the decay was not followed for more than 1 or 2 min because it was more reliable to work with relatively large amounts of facilitation. Thus, small errors in the

4 314 R. K. ORKAND measurement of the amplitudes (2-5 %) did not lead to large errors in the estimation of the facilitation. In most experiments, the facilitation did appear to decay exponentially after intervals greater than 5-1 sec. Therefore, results were assessed by comparing the slope of the calculated regression line relating facilitation (on a log scale) to interval between stimuli. However, the limited number of points obtained and the lack of accurate data at very low values of facilitation leave open the possibility that the slow phase of facilitation does not decay in a purely exponential fashion. Solutions. The solutions were identical to those previously employed (Niedergerke & Orkand, 1966). The normal Ringer solution contained in mm: NaCl, 116; NaHCO3, 2; KCl, 3; CaCl2, 2 or 3. Fluids with reduced Na concentrations were prepared with equal volume parts of 125 mm choline chloride (in the presence of 1-' M or 2 x 1-6 m atropine) or 21 mm sucrose. When sucrose was used as a substitute, the concentrations of KCI and CaCl, were modified so as to correct for the changes in their activities at the reduced ionic strength. Solutions were changed by twice emptying the chamber and refilling with new solution. At least i hr was allowed for the preparation to equilibrate with the new solution. It was then tested at 15 min intervals until the responses stabilized. Often this took considerably longer than 3 min when large changes in Ca or Ca/Na2 were involved. This long delay, up to 9 min, possibly represented the time necessary for the electrolyte composition of the intracellular compartment to equilibrate. In all experiments where changes in solutions were compared, the control responses represent the means of the before and after runs. The experimnents were done at room temperature, C. RESULTS The normal staircase. Quiescent strips of frog ventricle were equilibrated with the normal Ringer solution containing 2 mm-ca for at least 1 hr and not stimulated for 2 min before testing. Figure 2A and B shows the progressive increase in both tension and maximum rate of rise of tension when such a strip is electrically stimulated 5 times at 1 sec intervals. In Fig. 2B, it can be seen that at this low stimulus frequency the time to peak of the contraction is changed little. In this analysis the amount of facilitation (f) will be defined as the fractional change in amplitude of the test response (P.) compared with the initial response (P), and is given by the equations: f = (Pn-P)/P = Pn/P-l. In Fig. 2C the amount of facilitation of both tension and maximum rate of rise during successive responses has been graphed. Under the conditions of these experiments both parameters were found to change in a similar fashion. A possible explanation for the progressive increase in facilitation is that each contraction leaves behind an amount of facilitation which sums linearly with the facilitation remaining from previous contractions. If this were the case, one could accurately predict the rise of facilitation and, therefore, the successive responses, solely from knowledge of the decay of facilitation following a single contraction. This explanation has been found to be sufficient to explain the results. In Fig. 3 a conditioning

5 FACILITATION OF CONTRACTION 315 stimulus was given at time zero and the amount of facilitation remaining at different intervals was assessed by recording the contraction resulting from a second stimulus at various intervals. Ten minutes elapsed between each of the separate tests. Figure 3A shows some of the records obtained. The earliest test response is the largest and, as the interval between stimuli A 5 sec At It 1 <@ s B 5, I -5sec J -5 sec C 1* S:g~~~~~~w a4 1* - *5 - S S - I I I I Time (sec) Time (sec) Fig. 2. Growth of tension response and maximum rate of rise of tension with repetitive stimulation. Quiescent ventricle strip in 2 mm-ca stimulated 5 times at 1 sec intervals. A, top: tension responses; bottom: derivative of tension response (derivative of falling phase of contraction cut off in this and subsequent records). B: superimposed tracings of tension response (top) and rate of change of tension (bottom) from the same preparation. Numbers indicate first, third, and fifth responses. C: graphs of facilitation (f ) of tension and of maximum rate of rise for each response in the series. Lines connect points. Facilitation initially increases almost linearly but approaches a plateau later on. 4

6 316 R. K. ORKAND is lengthened, the contractions are found to decrease in amplitude and approach that of the initial conditioning response. The amount of facilitation of tension at each interval tested has been graphed on a semi-log scale in Fig. 3A. The facilitation appears to decay in two phases. In 2 mm-ca Ringer the early phase of facilitation of both tension and maximum rate A 5 sec e 8- -_ -6 a 4 B Interval (sec) o e 1- -, I I I Time (sec) Fig. 3. Relation between decay of facilitation following a single conditioning stimulus and growth of facilitation during a train of stimuli (2 mm-ca). A, top: records of control response and responses at intervals of 4, 1, 2, 4, and 6 sec (five separate runs); bottom: graph indicating facilitation of responses at various stimulus intervals (means + s.e. of points, where the S.E. is greater than the size of the point in this and subsequent graphs, determined before and after test series in B). Dashed line is calculated regression line through points at 1, 2, 4, 6 sec intervals with half-time for decay of 67 sec. B: facilitation of tension during five responses at 1 sec intervals. Dashed line is predicted rise of facilitation calculated by linearly summing the facilitation remaining from preceding stimuli using the graph in A.

7 FACILITATION OF CONTRACTION 317 of rise decayed to one half in 2-4 sec, whereas the slower phase of facilitation of tension decayed to one half in sec (mean S.E. in 4 strips) and that of maximum rate of rise in 26-7 sec (mean s.e.). It was not possible to make accurate estimates of the decay of the fast phase because the long duration of the cardiac action potential precluded testing at intervals of less than 2 sec. Knowing the decay of facilitation following a single contraction, one can then determine the sum of the residual facilitation at any time during a series of stimuli. For example, if the facilitation sums linearly, the fourth contraction in a series at 1 sec intervals should have 1 sec of facilitation remaining from the third contraction, 2 sec from the second and 3 sec from the first. The amount of facilitation at each of these times can be read off from the graph in Fig. 3A and the sum should equal the facilitation found for the fourth contraction of the series. In Fig. 3B this hypothesis has been tested by measuring the facilitation during a series of six contractions and comparing the observed values to those predicted by summing the facilitation observed at various intervals following a single shock. The excellent agreement obtained in Fig. 3B between the calculated dashed line and the observed increase in facilitation indicates that under the conditions of this experiment the facilitation of contraction during the staircase is simply the linear sum of the residual facilitation from the preceding contractions. To test this hypothesis further, the decay of facilitation was determined in another strip before and after a series of seven contractions at 1 sec intervals with 'extrasystoles' produced after the second and fourth stimuli. Figure 4A illustrates the decay of facilitation in this strip obtained by stimulating at various intervals after a conditioning stimulus. Figure 4B (top) shows the successive tension responses. The arrows mark the two 'extrasystoles.' In Fig. 4B (bottom) the observed irregular development of facilitation is compared with that predicted solely by summation of facilitation from the decay curve in Fig. 4A. The good agreement between the predicted and observed facilitation confirms that, even with irregular intervals between beats, the successive changes in contractile strength can be predicted from a knowledge of the decay of facilitation following a single contraction. Similar good agreement was found for the facilitation of the maximum rate of rise of tension. It should be stressed that these predictions are only accurate when the time to peak of the contraction does not change appreciably with repetitive stimulation. In addition, the hypothesis has only been carefully tested at 2 mm-ca and under conditions where the decay of facilitation curve was determined and found to be essentially identical before and after the test series. It follows from this analysis that one should be able to obtain the curve for the decay of facilitation by subtracting the total facilitation at any time

8 318 R. K. ORKAND from the sum of facilitation produced by previous beats in a series. In practice, however, the procedure is usually inaccurate because it involves taking the difference between two large amounts of facilitation to obtain a small amount. The effect of varying the external calcium concentration. When the external calcium concentration bathing the strip was altered, a marked change in -7 i 5 A. ' *1~ I I I I I Interval (sec) B 5 sec 2 I I O 2-~~~~~. // sp /~~~~~~~, /~~~~~~~~~~~~~~~ Tixne (sec) Fig. 4. A. Decay of facilitation in a ventricle strip in 2 mms-ca following a single conditioning stimulus with a test stixnulus at various intervals. Dashed line is calculated regression line for points at 1, 2, 4, 6 see with half-time for decay of 46 sec. B, top: records of tension responses from a ventricle strip stimulated 7 times at 1 sec intervals with 'extrasystoles' (indicated by arrows) produced after the second and fourth stimuli; bottom: facilitation of successive tension responses (open circles) compared with the predicted responses derived from the decay of facilitation curve and assuming linear summation of facilitation remaining from preceding stimuli. Dashed line dravii through predicted points.

9 FACILITATION OF CONTRACTION 319 the magnitude and time course of facilitation was observed. The facilitation was found to be directly proportional to the calcium concentration. Figure 5 illustrates the increased facilitation observed when the calcium 5- ID 4- e W 3- M *5 1.* - 5 mm-ca V X 2 mm-ca b " 5 mm-ca 2 mm-ca 5 sec _/ > 4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Time (sec) hk B, Iv t- t _ 5 mm-ca -5 N 2mM-Ca to N 'a I'll 11-1 o -5 ;I -1 - I -_ I I I -I Interval (sec) 4 11 r 11r - -,o s *1* Time (sec) _-.. 5 mm-ca N11 X 2 mm-ca NI I IN A1 _ u x ~~~~~~No I I II I I -- I Interval (sec) Fig. 5. Effect of increasing Ca on facilitation of contraction. A: records of successive contractions, A1, and maximum rates of rise, A2, of a ventricle strip stimulated 3 times in 5 mm-ca (top records) and 2 mm-ca (bottom records). Note change in calibration. Graphs of facilitation of tension and facilitation of maximum rate of rise in 5 and 2 mm-ca. Open circles indicate means of four trains of stimuli at 1 sec intervals in 2 mm-ca, two before and two after 5 mm-ca, and two trains in 5 mm-ca (filled circles). B: same experiment as shown in A. Graphs of decay of facilitation of tension, B1, and facilitation of maximum rate of rise of tension, B2, following a single conditioning shock in 2 and 5 mm-ca. Lines are calculated regression lines for intervals of 1, 25, 6 and 1 sec. 21 Physiol. Ir96 -

10 32 R. K. ORKAND concentration was raised from 2 to 5 mm. Figure 5A shows the tension responses to a train of stimuli at 1 sec intervals in the different calcium concentrations, and the changes in the rate of rise of tension in the same experiment. For both parameters the increase in calcium concentration resulted in a marked increase in the steepness of the staircase as well as the known increase in the initial response which occurs when the calcium concentration is raised. The decay of facilitation at different calcium concentrations is shown in Fig. 5B. It can be seen that at the higher Ca the facilitation following a single shock was increased and its decay slowed. In three experiments comparing the decay of the slow phase of facilitation in 2 mm and 5 mm-ca, the average time for one-half decay of tension in 2 mm-ca was S.E. sec and in 5 mm-ca S.E. sec. For the maximum rate of rise of tension the values were 2 mm-ca S.E. and 5 mm-ca s.e. A t-test was applied to compare the slopes in each experiment. In two of three experiments the slope of the calculated regression line of tension decay was significantly steeper in 2 mm-ca than in 5 mm-ca (P <.5). In two of the three the decay of the facilitation of the maximum rate of rise was significantly steeper in 2 mm-ca than in 5 mm- Ca. In the third experiment the two slopes did not differ significantly (P >.1). One difficulty in obtaining consistent results is that the responses must remain stable over the period of about 5 hr which is required to determine the curve in 2 mm-ca, 5 mm-ca and then again in 2 mm-ca. If the regression line for the slow phase is extrapolated to time, the average amount of facilitation at time in 2 mm-ca was S.E. for tension and S.E. for maximum rate of rise, and in 5 mm-ca s.e. for tension and s.e. for maximum rate of rise. These results indicate that raising the external calcium from 2 to 5 mm increases the slow phase of the facilitation and slows its decline. As indicated previously, under the conditions of these experiments, it was not possible to study carefully the fast phase of the facilitation. However, an attempt was made to assess the effect of Ca on this phase by determining facilitation at 2 sec in high and low Ca and subtracting that amount due to the slow phase (the difference between the 2 sec point and the calculated regression line in Fig. 5B). The difference in 2 mm-ca was *27 + *5 S.E. for tension and S.E. for the maximum rate of rise and in 5 mm-ca s.e. for tension and S.E. for the maximum rate of rise. Because of the large scatter in the data these values are not significantly different. When the external calcium concentration was reduced, the amount of facilitation produced by repetitive stimulation decreased. In six of seven experiments, when the Ca was reduced below 1 mm, the second and third contractions in a series at 5 or 1 sec intervals were actually smaller in

11 FACILITATION OF CONTRACTION 321 amplitude than the initial contraction. The maximum rate of rise of the contraction decreased similarly. I mm-ca b4 I mm-ca 5% Na 5 sec Fig. 6. Effect of reducing Na on successive tension responses in 1 mm-ca. 5 sec intervals between stimuli. Sucrose replacement of Na. (Note change in calibration.) Effect of decreasing the external sodium concentration. When the external calcium concentration was 1 mm or less, a reduction of external Na produced an increase in the amplitude of the initial contraction as well as an increase in the amount of facilitation. The tension responses in Fig. 6 show the marked effect of Na reduction on both the amplitude of contraction and facilitation when a ventricle strip was stimulated repetitively at 5 sec intervals. Such results raise the question of whether the increase in amplitude of the initial contraction is causally related to the increase in facilitation. That this is not the case has been demonstrated in the following experiment. When the ratio of Ca/Na2 in the external bathing solution is constant, the amplitude of the contraction remains constant (Liittgau & Niedergerke, 1958). It is therefore possible to compare the facilitation when the initial contraction is constant but the concentrations of Ca and Na are different. The results of two such experiments are illustrated in Fig. 7. Figure 7A (top) shows the first three tension responses in 3 mm-ca, -68 mm-ca 5% Na and -75 mm-ca. The ratio of activities Ca/Na2 is constant in the first two and the Ca activity the same in the latter two solutions. In Fig. 7A (bottom) the facilitation during a train of seven stimuli at 1 sec intervals has been plotted. As indicated above, when the Na concentration is reduced, the facilitation as well as the initial tension response increases. However, although the initial tension responses are practically the same in 3 mm-ca and X68 mm-ca 5% Na, the facilitation 21-2

12 322 R. K. ORKAND was less in the latter solution. Thus, the amount that the contractile apparatus is activated is not the sole factor determining the amount of facilitation for successive contractions. Similar results were obtained when the maximum rate of rise was studied. In order to rule out the possible effect of changes in ionic strength, a few experiments were performed with A1 t o~~~~~~b } I [tjjo J\J~~Jj 5 sec r- 3 mm-ca -68 mm-ca 5% Na -75 mm-ca A2 4 bo 5 ~~~~~,I 3 mm-ca C) S bo.a a * B1 A mm-ca9 x~~~~~~~~~~- 3 mm-ca Interval (sec) Interval (see) Fig. 7. Effect of reducing Na at constant ratio Ca/Na2 on facilitation of contraction. Two experiments, 1 and 2. A1, A2, top: First three tension responses of a series of stimuli at 1 sec intervals in 3 mm-ca, -68 mm-ca 5% Na and -75 mm-ca; bottom: graphs indicating progressive changes in facilitation of tension for eight stimuli at 1 sec intervals. Sequence: 2 trains in 3 mm-ca, 2 trains in -75 mm-ca (one train -75 in B1), 2 trains in -68 mm-ca 5% Na, 2 trains mm-ca. in 3 Points give mean facilitation S.E. B1, B2: Decay of facilitation following a single conditioning stimulus in 3 mm-ca and at -68 mm-ca 5 % Na, same experiments. Dashed line in B indicates that at 25 sec intervals in -68 mm-ca 5% Na no facilitation was observed.

13 FACILITATION OF CONTRACTION 323 choline chloride in the presence of atropine 1-5 M or 2 x 1 M. The atropine was used to block the shortening of the action potential produced by choline. Under these conditions the facilitation was also less at low Na and Ca when the Ca/Na2 ratio was kept constant. The experiments with choline were complicated by an effect of atropine on the time course of facilitation. With atropine in the normal solution the facilitation was greater 2-3 sec after a single test response than after 1 sec. Under these conditions the staircase became progressively steeper with time after the first two or three stimuli at 1 sec intervals. In Fig. 7B the decays of facilitation in 3 mm-ca and -68 mm-ca 5% Na have been compared. The results of these experiments illustrate the difficulty of trying to come to any conclusion about the relative rates of decay in the two solutions. It can only be concluded that for the first 2 min following a single contraction the facilitation in low Ca and low Na was less than in high Ca and high Na, although the initial conditioning contraction was the same. DISCUSSION The present results can be interpreted using the model proposed by Niedergerke (1963 a, b) for the initiation of contraction in the frog ventricle. In this model a decrease in membrane potential allows external Ca to move inward down its electrochemical gradient and release 'active Ca' from the inner surface of the membrane. The rise in concentration of 'active Ca' within the cell leads to contraction. The 'active Ca' is rapidly inactivated, possibly by being bound to the inner surface ofthe membrane. The strength of contraction is therefore a function of both the amount of Ca which enters during the depolarization and the amount of active Ca able to be released from the inner surface of the membrane. The Ca which enters during the depolarization presumably joins the pool of active Ca and the initial conditions are restored by a pump which moves Ca out of the cell. Such a scheme is consistent with the observation that contraction is accompanied by an increase in both the influx and efflux of Ca (Niedergerke, 1963 a, b). If a second influx of Ca occurred before the excess active Ca in the cell was pumped out, a larger amount of active Ca would be released and the second contraction would be greater. According to this hypothesis the decay of facilitation would reflect the time course of restoration of the bound Ca to its resting value. Thus, facilitation would result from the accumulation of Ca within the heart muscle cell. This scheme successfully predicts the major results of the present work. At low external Ca, the influx and efflux of Ca are about the same and consequently there would be no facilitation. When the Ca is raised, the influx increases and transiently exceeds the efflux. Ca accumulates and facilitation is observed. With prolonged stimulation the ventricle comes into a steady

14 324 R. K. ORKAND state where influx and efflux are balanced and the tension responses plateau. The slower decay of facilitation in high Ca might result either from a saturation of the pump or from the greater difficulty of pumping against an increased chemical gradient and high resting influx. Ca influx is also increased at constant external Ca when the Na concentration is reduced (Niedergerke, 1963b). Under these conditions in the present experiments, the facilitation also increased. This result suggests an accumulation of Ca rather than Na as the cause of facilitation because when the external Na is decreased, the inward Na current decreases (Brady & Woodbury, 196). When the ratio of Ca/Na2 was constant and the concentration of Ca and Na varied, the initial tension response was the same (Luttgau & Niedergerke, 1958), and consistent with Niedergerke's (1963b) finding that the influx of Ca is constant under these conditions. However, the facilitation was always less at low Ca and Na. This result strongly supports the present hypothesis because under these conditions Niedergerke (1963b) found the accumulation of tracer Ca to be less. It is difficult to make quantitative comparisons between the present results of tension studies and tracer results. In the latter experiments, it was necessary to stimulate the preparations 1 or 6 times in order to obtain sufficient tracer exchange to make the required measurements. These results are concerned only with the first 5-1 beats following a rest period. The present findings of an increase in the facilitation (staircase) on raising Ca or decreasing Na are at first view contrary to the findings of previous workers who found a decrease in the staircase under these conditions (Fiddes, 1929; Dale, 1932; Nayler, 1961). However, these authors did not appreciate the very slow decay of facilitation (half-time about 1 min) in these preparations. They stimulated at base-line frequencies where the preparations were nearly maximally facilitated and then looked for changes in facilitation by making relatively small changes in frequency of stimulation. Such changes as they might have observed would have been in the early component of facilitation which did not appear to be sensitive to ionic changes in the present experiments. Alternatively, because of the frequencies of stimulation employed, any results that they obtained on the activation of the contractile mechanism could have been obscured by primary changes in the duration of the contraction. In the present experiments, both the maximum tension and maximum rate of rise of tension were used as indices of activation of contraction and care was taken in the selection of ionic environments and stimulus frequencies to be sure that changes in the duration of the contraction were not primarily responsible for the observed changes. Finally, the present results can be compared with comparable studies of facilitation of transmitter release at synapses. Both processes have the

15 FACILITATION OF CONTRACTION 325 point in common that they are brought about by depolarization of the cell membrane and their intensities are related to the external Ca concentration. Mallart & Martin (1967) found that at the neuromuscular junction of the frog there were two components to facilitation and that, as in the present studies, the growth of synaptic potentials during a series of stimuli was the result of the linear summation of facilitation remaining from previous impulses. However, the time courses of the processes are markedly different. The slow component of facilitation of transmitter release decays with a half-time of about 25 msec, whereas in the present work the slow component decayed within a half-time of about 5 sec. I am grateful to Drs A. R. Martin and A. Mallart for helpful discussion and criticism and to Dr R. Niedergerke for his comments on the manuscript. This work was supported by U.S.P.H.S. Grant No. NB REFERENCES BRADY, A. J. & WOODBURY, J. W. (196). The sodium-potassium hypothesis as the basis of electrical activity in frog ventricle. J. Phy8iol. 154, DALE, A. S. (1932). The staircase phenomenon in ventricular muscle. J. Physiol. 75, FIDDES, J. (1929). Studies on the cardiac muscle of lower vertebrata. Q. Jn exp. Phy8iol. 19, KOCH-WESER, J. & BLINKS, J. R. (1963). The influence of the interval between beats on myocardial contractility. Pharmac. Rev. 15, LUTTGAU, H. C. & NIEDERGERKE, R. (1958). The antagonism between Ca and Na ions on the frog's heart. J. Physiol. 143, MALLART, A. & MARTIN, A. R. (1967). An analysis of facilitation of transmitter release at the neuromuscular junction of the frog. J. Phy8iol. 193, NAYLER, W. G. (1961). The importance of calcium in poststimulation potentiation. J. gen. Physiol. 44, NIEDERGERKE, R. (1956). The 'staircase' phenomenon and the action of calcium on the heart. J. Phy8iol. 134, NIEDERGERKE, R. (1963a). Movements of Ca in frog heart ventricles at rest and during contractures. J. Physiol. 167, NIEDERGERKE, R. (1963b). Movements of Ca in beating ventricles of the frog heart. J. Physiol. 167, NIEDERGERKE, R. & ORKAND, R. K. (1966). The dual effect of calcium on the action potential of the frog's heart. J. Phy8iol. 184, ORKAND, R. K. (1967). Facilitation of heart muscle contraction and its dependence on external calcium and sodium. Physiologi8t, Lond. 1, 268.