Absence of Bowditch phenomenon in the ventricular muscle of hamsters with hereditary cardiomyopathy

Experimental and laboratory

Absence muscle

reports

of Bowdiitch of hamsters

phenomenon with

in the ventricular

hereditary

cardiomyopathy

Stephen Hajdu Christian J. Posner Bethesda, &Id.

T

he contractility of most mammalian cardiac muscle in vitro is the result of two independent phen0mena.l Their contribution to the overall contractility, however, varies according to the frequency of stimulation. While in the middle frequency range the generation of contractions is shared by both phenomena, at high frequencies it is furnished almost entirely by the Bowditch phenomenon (BP), and at low frequencies by the Woodworth phenomenon (WP). Since the heart rate in vivo is in the range of stimulation called “high frequency” under in vitro conditions, the importance of the BP in support of life is overwhelming. When it was reported2 that a strain of hamsters spontaneously developed primary cardiac failure as a result of hereditary factors, an opportunity was presented to study the effect of the disease on these phenomena, for the hamsters have hearts small enough for in vitro studies and exhibit both phenomena. It was thought that if, as the result of the disease, both phenomena are equally depressed, it would have to be concluded that the defect in the contractile system is the common cause of the depression, since it would be highly unlikely that the disease would affect two independent functions equally (BP and WP). On the other hand, if the disease is From

the National Heart & Lung Institute, mental Cardiovascular Diseases, Bethesda, Received for publication July 24. 1970.

Vol. 81, No. 6, pp. 781-789

June, 1971

Laboratory Md.

restricted to only one of the phenomena, leaving the other intact, this would indicate a normal contractile system with the point of attack on the specific mechanism serving only this phenomenon. The present report demonstrates that the disease is restricted to the mechanism responsible for the BP in a manner that explains the primary weakness of the heart muscle. Methsds The hamsters used in these experiments were divided into three groups. Group I consisted of seven healthy golden hamsters (NIH strain) with a body weight of 101 + 14 grams and a wet weight of both ventricles of 216 & 27 mg. Group 2 consisted of five hamsters with cardiomyopathy (BIO 82.62) without symptoms of cardiac failure. The average age was 88 & 6 days; body weight, 84 ZIZ 12 grams, and ventricular weight, 219 f 48 mg. Group 3 consisted of seven animals with cardiomyopathy (BIO 14.6) and with severe congestive heart failure. The average age was 327 i 14 days; body weight, 111 f 9 grams; and ventricular weight, 428 f 82 mg. Since the animals in Groups 1 and 2 were considerably younger than those in Group 3, an additional group of ten healthy golden hamsters whose age range was 270 to 420 days, body weight, 135 & 18 grams, and ventricular of Kidney

and

Electrolyte

Metabolism,

Section

American Heart Journal

on Experi-

781

weight, 13.5 f 18 mg. was later added to serve as appropriate age controls for the BIO 14.6 hamsters with severe congestive heart failure. The weight of the ventricles given above does not include the weight of the triangular-shaped muscle strip used for the measurement of tension. Since the body and heart weights exhibited some variation among the different groups of animals and in order to obtain comparable values in the measurements of contractile tension, strips of about the same size and shape were dissected from the right ventricular wall. The average dimensions (width at the base X stretched length in millimeters) were as follows: 5.6 X 11.7 for the NIH strain; 6.5 X 11.8 for BIO 82.62*‘; 5.4 X 15.1 for BIO 14.6; and 5.5 X 15.0 for the age control animals. The animals were killed by exsanguination, the chest was opened and a triangularshaped strip of muscle was excised from the free wall of the right ventricle beginning at the pulmonary artery. The muscles were suspended in a lucite bath by attaching the pulmonary artery by means of a thin (26 gauge) stainless steel tubing to a Statham transducer (G7A-0.15-350). The narrow base of the muscles was tied to a lucite bar firmly attached through rack and pinions to the transducer. One electrode was attached to the base of the muscle, the other to the pulmonary artery. Ringer-Krebs solution3 was used throughout the experiment, vigorously gassed with a mixture of 9.5 per cent oxygen and 5 per cent carbon dioxide. The temperature of the bath was kept between 21 and 22” C. in order to avoid spontaneous rhythmicity, which usually occurs above 23’ C. The transducer was coupled through a Brush Electronics (Model BL-520) amplifier to a Brush (B2902A) oscillograph. A Grass (Model S4) stimulator was used with a SIU4 stimulus-isolation unit; at intervals longer than 10 seconds the stimulator was controlled by an external time clock.

The over-all state of contractility of the cardiac muscle is best studied by measuring %AnimaIs of the BIO 82.62 Experimental Laboratory Maine 04609.

strain

were supplied by the Trenton Animal Company, Bar Harbor,

isometric tension over the widest possible frequency range, for the contribution of the two basic constituents of contractility (BP and WI’) varies widely with the frequency of stimulation. Fig. 1 shows the tension at various frequencies (expressed as interval between stimuli) in three groups of hamsters: Group 1, normal golden hamsters (NIH strain); Group 2, young hamsters with cardiomyopathy (BIO 82.62) prior to the development of cardiac failure; and Group 3, hamsters with cardiomyopathy (BIO 14.6) in the final stages of congestive heart failure. The interval-tension curves of Groups 1 and 2 are similar. They show the characteristic curve of a species endowed with both the BP and WP, demonstrated by the rising tension at both ends of the curve. The wide separation of the two phenomena leaves the middle frequencies with scarcely any contractile tension. This is in contrast to rat cardiac muscle, in which the tension in the middle frequency range is only slightly depressed, due to a considerable overlap of the two phen0mena.r The interval-tension curve of the diseased hamster heart (Fig. 1, BIU 14.6) shows a parallel course with the curves obtained from the two control groups, but only over the range of the WP (2 to 60 second interval). During the stimulus interval, when the BP begins to develop in the normal animals (2 seconds and less), the curve of the diseased animals continues to decline, as though the BP were missing in these hearts. The interval-tension curve of those animals serving as age controls for the BIO 14.6 group (see methods) is parallel to that of Groups 1 and 2 with an average tension of 1.8 gram at 0.5 second and 0.4 gram at 2 second intervals, demonstrating that the BP is present in the old healthy hamsters and that the absence of BP in the diseased animals is not due to their advanced age. In order to determine whether the BP is absent in the diseased hearts, all three groups of animal hearts were treated with ryanodine. It has been shown that this drug irreversibly destroys the WP, but leaves the contractility produced by the BP intact.l In essence,contractility remaining after treatment with ryanodine can be regarded as the result of BP alone. All hearts of the three

Absence of Bowditch

fihenowenon

I

I

I

I

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23

5

IO

20

183

in hamsters

I.6

0

I 0.5

INTERVAL

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BETWEEN

STIMULI,

I 60

SEC.

Fig. 1. Interval-tension relationship of the right ventricles of normal hamsters (NIH strain), hamsters with cardiomyopathy without cardiac failure (2310 82.62), and those with cardiac failure in the terminal stage (BIO 14.6). Each point represents the average of 5 measurements on 5 different muscles.

2.0

I,6

0.4

0 0.5

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3

5

IO 20 INTERVAL BETWEEN STIMULI, SEC..

60

Fig. 2. Interval-tension relationship of the right ventricles of normal hamsters (NIH strain), hamsters with cardiomyopathy without cardiac failure (BIO 82.62), and hamsters with cardiac failure (2310 14.6) after treatment with 0.05 mcg. per milliliter of ryanodine. Each point represents the average of 5 measurements on the same hearts as those used in Fig. 1.

of animals were treated with 0.05 mcg. per milliliter of ryanodine in Krebs solution for one hour, after which the hearts were washed free of the drug and the interval-tension curves constructed. Fig. 2 shows the interval-tension curves

groups

of the heart muscles after treatment with ryanodine. No tension was recorded over the Woodworth range, but good contractility with undiminished tension was observed in the Bowditch range of the two control groups (NIH strain and BIO 82.62).

784

Hajdu

and Bosner

Fig. 3. Unretouched tracing of the isometric tension of the heart of the hamster with cardiomyopathy cardiac failure (BIO 82.62) (upper tracing) and the hamster with cardiac failure @IO 14.6) (lower At the beginning of the tracing 0.05 mcg. per milliliter of ryanodine was added to the bathing solution muscles. For a detailed description see text.

The hearts of the animals with congestive failure, on the other hand, developed no tension over the entire frequency range, showing that all tension exhibited by the hearts of these animals before ryanodine treatment was produced by the WP. The interval-tension curve of the diseased animals (Fig. 1) is thus the first record of a pure WP, undistorted by the presence of the BP, and its general course is in agreement with the one predicted,” The difference in contractile activity of the hearts before and after the development of cardiac failure is shown in Fig. 3 by a pair of original tracings. Two extreme frequencies of stimulation were used alternately. A very low frequency (60 second interval) shows up on the tracings as individual contractions and represents contractility due to the WP. The high frequency stimulation (0.5 second interval) interposed alternately for 30 seconds shows up on a tracing as a solid column. The individual contractions cannot be seen very well due to the s!ow speed of the chart paper. This represents the contractility produced by the BP. Since 0.05 mcg. per milliliter of ryanodine was added to both muscles at the beginning of the tracing, the selective effect of the drug is shown as it slowly comes to completion (end of tracing) ~ The first seven contractions, paced at 60 second intervals, show that both muscles give comparable tensions in the Woodworth range. At exactly the middle of the eighth 60 second period, 60 fast beats were interposed by stimulating the muscle every

without tracing). of both

0.5 second. The first of the fast beats is still high, since it comes after a 30 second rest; the second is very small, being the first beat with a 0.5 second interval before it. From then on, while the still healthy animal increases its tension stepwise in the following 58 beats, the heart of the animal in failure shows no increase in contractility. This part of the tracings also clearly demonstrates that the development of the new steady state after a change of frequency occurs instantaneously in the case of the WP (lower tracing), but takes many contractions in the case of the BP, which still has not reached its maximum in 60 beats (upper tracing). The first beat with a 60 second interval immediately after the fast frequency stimulation is the ‘istrongest possible contraction”4 or poststimulation potentiation seen only on the upper tracings. This is caused by the additive effect of the instantaneously reappearing W’P and the slowly disappearing BP. On the lower tracings in the same position only a normalsized contraction is seen, corresponding to a 60 second interval (WP) without any sign of the slowly disappearing BP. If the tracing is followed from this point, the continuous disappearance of the WE’ due to the effect of ryanodine is obvious. Meanwhile the height of the tension produced by the intermittent high frequency stimulation attests to the presence of an undiminished BP in the healthy animals (upper tracing). The tension produced by the heart of animals with congestive failure is completely abolished at both frequencies during ryanodine treatment, showing that all preryano-

Absence of Bowditch

fihenomenon in hamsters

785

c,, 0.8

ra

0.2

0 0.5

I

2

3

INTERVAL

5

IO 20 BETWEEN STIMULI ,SEC,

60

Fig. 4. Typical interval-tension relationship of the right ventricle of a normal hamster before treatment with ryanodine (Before I+.), after the addition of 0.05 mcg. per milliliter of ryanodine (0.05 pg Ry.), same relationship upon addition of 2.5 mcg.- _ per milliliter of scilliroside (2.5 pg SC&L), and after addition of 5.0 mcg. per miilili~er of sciiliroside (5.0 pg S&U.).

dine tension was the result of the WP only. These experiments leave little doubt of the presence of a normal BP in the heart of young animals with cardiomyopathy which are still free of symptoms of cardiac incompetence, but they reveal its complete absence after the cardiac failure has reached its final stage. Since the absence of BP in the heart of any species is without precedent, drugs known to influence the BP were tried first in order to gain some insight into the changes which led to the absence of this phenomenon. There is a long list of drugs and natural products known to do this,5 but none is more potent than the cardiac glycosides. Scilliroside (Schering) was selected, since it was found (unpublished observation) that this glycoside has a very powerful effect on rodent hearts, which, as is commonly known, are rather insensitive to the preparations generally used. In order to avoid the complicating effect of the WP, the hearts were first treated with

ryanodine. Fig. 4 shows an example of the interval-tension relationships of a control animal (NIH strain) before and after treatment with ryanodine, and the effect of 2.5 and 5 mcg. per milliliter of scilliroside at steady state. As shown by the intervaltension curves, the BP shifted toward the lower frequency range in a dose-dependent fashion as has already been demonstrated on frog hearts.5 Fig. 5 shows the results of similar experiments carried out on the hearts of the diseased animals. The intervaltension curve before and after treatment with ryanodine shows the absence of BP (see Figs. 1 and 2). The addition of 2.5 mcg. per milliliter of scilliroside showed no measurable effect on the tension at any frequency; 5 mcg. per milliliter of scilliroside, although it increases the tension of the ryanodine-treated muscle somewhat at every frequency, did not elevate the tension at the high frequencies, which would have indicated the return of the BP. Potassium-free Krebs solution, which

0.5

I

23 INTERVAL

Fig, 5. Typical 14.6) with severe per milliliter of liroside (5.0 fig change occurred the same curve

5 BETWEEN

10

20

60

STIMULI,SEG

interval-tension relationship of the right ventricle of a hamster with cardiomyopathy (BIO cardiac failure before treatment with ryanodine (Before Ry.), after the addition of 0.05 mcg. ryanodine (O.OS pg Ry.), and the same relationship after addition of 5.0 mcg. per milliliter of scilStill.). Since upon the administration of 2.5 mcg. per milliliter of scilliroside no measurable in the interval-tension relationship observed after treatment with ryanodine (0.05 pg Ry.), also describes the effect of 2.5 mcg. per milliliter of scilliroside.

has an effect on the BP very similar to that of the glycosides,5 was also tried with identical results. Unimportant changes in contractility were observed across the whole frequency range, even upon extreme potassium deprivation, which finally produced contracture, Discussion

The following hypothesis was advanced for the explanation of the WP, based on experiments made on isolated skeletal and heart musc1es.r Muscle fibers contain a large supply of calcium that is restricted in its movement. A fraction of this calcium is altered during the rest interval, so that it can enter and trigger the contractile proteins upon depolarization of the membrane. The amount of calcium thus taking part in the electromechanical coupling is dependent on the duration of the rest interval and on the nature of the muscle. The release of calcium yielding one full contraction requires a rest interval of about one second in the case of skeletal muscle,

while the time required to accomplish the same is about one minute for the heart musc1e.r Due to the sluggish release of calcium in cardiac muscle the role of the WP is very limited in vivo, because of the relatively short rest periods. This probably explains why animals without WP, that either naturally do not have it or were deprived of it artificially by injection of ryanodine,’ can keep up a normal circulation. The relative unimportance of the WP has also been demonstrated in the hamsters with cardiomyopathy which were in frank cardiac failure in spite of an undiminished WP. Thus at a physiological heart rate, the mechanism mainly responsible for producing the contraction is the BP. This is why the hearts of all animals exhibit this phenomenon and when it is absent the heart can no longer support life, as was observed in the case of the hamsters in the terminal stage of cardiac failure. The underlying mechanism by which the BP regulates the contractility of the heart is not fully understood. Some experimental

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Absence of Bowditch

facts collected over the years, however, have enabled us at least to formulate a theory giving a possible reason for the absence of BP in these hamsters. The most obvious quality of the heart of animals possessing only the BP is the dependence of contractility upon the concentration of the extracellular calcium to such a degree that a sudden change in its concentration during the course of a contraction influences the final outcome of the same beat.6 This shows a direct involvement of the extracellular calcium in initiating the contraction in the Bowditch range. Another obvious quality of the heart in this range is that the contractile tension increases as the frequency increases, in other words, every frequency is characterized by a certain amount of tension (Fig. 2). The development of this characteristic tension upon a change of frequency takes time : The greater the difference between the old and new frequency, the longer it takes to reach a new steady state. When changing from 60 to 0.5 second intervals (Fig. 3, upper tracing) the tension rises within 60 contractions from almost zero tension to a level which is still not that which would have been reached had more time been made available. During the development of the steady state of tension there is a parallel change in the intracellular potassium content of the muscle.7-s The adjustment of the intracellular potassium is thought to take place in the following way.lO During every contraction the muscle loses some potassium but regains the same amount by an active process from outside during the rest interval. Upon increase of frequency the loss of potassium occurs more often, and because o’f the shortened rest intervals the return transport of potassium also decreases, as a result there is a net loss of potassium. The intracellular potassium decreases in the following beats, until finally the potassium influx becomes equal to the efflux, so that a new steady state of intracellular potassium content is achieved. Thus there is a characteristic level of intracellular potassium for every frequency of stimulation. The higher the rate of stimulation is, the lower the steady state of intracellular potassium.. It is thus possible that the primary occurrence upon a change of frequency is a change in intracellular

phenomenon in hamsters

787

potassium and that the change in contractility is the consequence thereof. This would seem to be probable, since it was found that a decrease in intracellular potassium without any change in frequency, by reducing extracellular potassium or by the use of cardiac glycosides5J1 (Fig. 4)? increased contractility, and that a net uptake of potassium by the heart, again without any change of frequency, decreased the tension.g How a decrease in intracellular potassium can bring about an increased contraction upon depolarization raises further difficulties. It had been shown originally that as the tension increased upon increase of frequency, the amount of calcium per beat entering the fiber also increased.12 This would have furnished a simple explanation for the increased contractility. However, a more recent study13 shows that there is no increase in calcium flux upon increased frequency. If the latter is correct we must suppose that the decreased intracellular potassium itself renders the actomyosin more sensitive to the same amount of calcium. Whichever is the case, the important point for the purpose of this discussion is that the increase of tension produced by increased frequency is the result of two consecutive steps, namely, a decrease of intracellular potassium and the entry of calcium into the fiber. Consequently, the absence of the BP could be the result of a malfunction of either step. Due to the restricted number of animals available in the terminal stage of cardiac failure, we were able to test only the functional state of the potassium mechanism. Lowering the intracellular potassium with cardiac glycosides7,g (Fig. 4) or decreasing the extracellular potassium, which resulted in increased contractility in the control hearts at the same frequency, did not bring back the missing BP in the failing heart, although this was not due to lack of response upon change of intracellular potassium brought about by these interventions. Their effectiveness was demonstrated by the slowing of relaxation and, finally at the toxic stage, by the appearance of contractures. But the decrease of intracellular potassium caused by these interventions did not give rise to increased contractility in the Bowditch range, probably because of failure in the

next step in these events, namely lack of influx of extracellular calcium during depolarization. In support of this possibility it should be mentioned that these hearts are able to give full contractions at the very low frequencies at which all the calcium needed for the electromechanical coupling originates froim bound sources (Fig. 1). From whatever source it comes, once sufficient calcium enters the fiber it contracts well, which shows that all the following steps in the contraction cycle, namely the shortening of the contractile protein, energy transfer, sequestration of calcium, and the relaxation of actomyosin, proceed normally. Thus we see a heart with good contractile properties at low frequencies, but without a BP in the physiological range. A heart muscle with these characteristics cannot function adequately. It cannot increase the cardiac output to an appreciabfe degree by increasing the heart rate, because this leads to decreased contractility (Fig. 1). On the other hand, a slower heart rate, which would increase contractility, would diminish the output by decreasing the minute volume. The hypothesis that the cardiac glycosides exert their beneficial effect through a mechanism involved in BP5,7 was further supported by the finding that the glycosides have no therapeutic effect in the absence of BP. This means that at the terminal phase of cardiomyopathy, during which the need for the action of glycosides would be the greatest, their effectiveness seems to be Iowest.14 The reason is that the glycosides, working through the potassium mechanism, obviously do not have an effect on the permeability of the membrane toward the influx of extracellular calcium, which seems to be the primary cause of the cardiomyopathy in these animals. According to this hypothesis the change that takes place in these animals later in life, leading to a fatal cardiac failure, is a complete impermeability of the membrane to the entrance of free extracellular calcium during the depolarization of the membrane. This is not without precedent among the contractile tissues. In fact, this is the way the skeletal muscle functions under physiological conditions, since it does not

use extracellular calcium for electromechanical coupling at al1.l The skeletal muscle functions very competently for hours in a calcium-free medium, for it has a very efficient WP which can supply enough calcium from bound sources at any frequency, including tetanus. In contrast to this, as the membrane of the hamster with cardiomyopathy becomes impermeable to extracellular calcium, the animal fails to compensate for this by increasing the efficiency of the release of calcium from bound sources, as shown by the finding that even at the terminal stage of the disease the WP is not more efficient than that of the normal animal (Fig. 1).

Summary The interval-tension relationship of right ventricular muscles of normal hamsters was compared to that of hamsters with hereditary cardiomyopathy, with or without congestive heart failure. The contractility of the heart of animals with cardiomyopathy without circulatory insufficiency did not differ from that of the normal animals at any frequency. The cardiac muscle of animals with severe congestive heart failure showed normal contractility at a low frequency of stimulation (range of the Woodworth phenomenon) but did not show the increased contractility upon high frequency stimulation (Bowditch phenomenon) seen in all other animals. Ryanodine, which eliminates only the contractility caused by the Woodworth phenomenon, abolished the contractility of the hearts of animals with congestive failure over the whole frequency range. Cardiac glycosides, which are known to potentiate the Bowditch phenomenon, were ineffective on the cardiac muscle of animals without Bowditch phenomenon. The probable cause of cardiac failure in hamsters with hereditary cardiomyopathy is discussed. The authors are greatly indebted to Dr. Eijrs Bajusz of the Bio-Research Institute for his generous gift and careful selection of the hamsters with congestive heart failure (&IQ 14.6). REFERENCES Haidu, S. : Mechanism of the Woodworth case phenomenon in heart and skeletal Amer. J. Physiol. 216:206, 1969. 2. Bajusz, E.: Hereditary cardiomyopathy:

1.

stairmuscle, A new

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3.

4.

5.

6.

7.

8.

9.

disease model, AMER. HEART J. 77:686, 1969. Krebs, H. A., and Henseleit, K.: Untersuchung ueber die Harnstoffbildung im Tierkoerper, Hoppe Seyler Z. Physiol. Chem. 210:33, 1932. Woodworth, R. S.: Maximal contraction, “staircase” contraction, refractory period, and compensatory pause of the heart, Amer. J. Physiol. 8:213, i902. Haidu. S.: Bioassav for cardiac active principies based on the staircase phenomenonbf the frog heart, J. Pharmacol. Exp. Ther. 120:90, 1956. Weidman, S.: Effect of increasing the calcium concentration during a single heart-beat, Experientia 15:128, 1959. Hajdu, S.: Mechanism of staircase and contracture in ventricular muscle, Amer. J. Physiol. 174:371, 1953. Vick, R. L., and Kahn, J. B., Jr.: The effect of ouabain and veratridine on the potassium movement in the isolated guinea pig heart, J. Pharmacol. Exp. Ther. 1X:389, 1957. Sarnoff, S. J., Gilmore, J. P., McDonald, R. H.,

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Jr., and Mansfield, P. B.: Relationship between myocardial K+ balance, 02 consumption and contractility, Amer. J. Physiol. 211:361, 1966. 10. Leonard, E., and Hajdu, S.: Action of electrolytes and drugs on the contractile mechanism of the cardiac muscle cell, Handbook of Physiology, Section 2, Vol. 1, Baltimore, 1962, The Williams & Wilkins Company p. 174. 11. Niedergerke. R.. and Harris. E. T.: Accumulation of calcium (or strontium) under conditions of increasing contractility, Nature (London) 179:1068, 1957. 12. Winegard, S., and Shanes, A. M.: Calcium flux and contractility in guinea pig atria, J. Gen. Physiol. 45:371, 1962. 13. Haacke, H., Luellmann, H., and van Zwieten, P. A.: Calcium metabolism in atria1 tissue during frequency potentiation and paired stimulation, Arch. Ges. Physiol. 314:113, 1970. 14. Burch, G. E., and DePasquale, N. P.: Heart muscle disease, Disease-a-Month, Chicago, May, 1968, Year Book Medical Publishers, Inc., p. 59. -

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