(From the Physiological Laboratory, Cambridge.) relation of temperature to heart rate in the English frogs (Rana ternporaria

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1 6I THE RELATION OF TEMPERATURE TO THE HEART RATE OF THE SOUTH AFRICAN FROG (XENOPUS DACTYLETHRA). BY N. B. TAYLOR. (From the Physiological Laboratory, Cambridge.) THE experiments embodied in the present report were done as an extension of the work of Barcroft and Izquierdol [1931] upon the relation of temperature to heart rate in the English frogs (Rana ternporaria and R. pipiens). Some observers have found that the curve relating heart rate to temperature when the latter did not rise above or fall below a level detrimental to the cardiac tissue, was a straight line. Others have maintained that the curve was of the logarithmic type, as it should be were the temperature-frequency relationship of the heart governed by a simple application of the van 't Hoff rule. Know lton and Starling [1912] found a linear relationship to exist between the temperature and the heart rate in the heart-lung preparation of the dog between 260 and 400 C., and for this reason insist that it is misleading to speak of a temperature coefficient of the heart since it is not the same within different temperature ranges. Barcroft and Izquierdo found that the slowing or acceleration of the excised heart of the English summer frog, which occurred in response to cooling or heating between 4 and 200 C., bore, as a rule, a roughly linear relationship to the temperature. We have carried out a similar series of experiments upon the excised heart of the English frog in summer. Izquierdo's technique for the suspension of the heart in saline was followed with but a single modification, namely, instead of attaching a thread to the sinus itself, the pericardium was employed as a means of attachment and suspension of the organ. Izquierdo [1930] found that the heart increased in rate for a time after excision, and a steady rate was not attained until after the organ had been immersed in saline for 30 minutes or more. For this reason the temperature in our experiments was kept constant for a time 1 The reader is referred to their paper for a review of the literature upon the subject.

2 TEMPERATURE ON SOUTH AFRICAN FROG'S HEART. 157 at the level at which the frogs had been living for the previous 24 hours or more. Not until the rate had remained unchanged for 30 minutes or so was the temperature raised or lowered. At the end of this preliminary period, what may be taken as the experiment proper was commenced. The temperature was lowered gradually by the addition of ice to the water jacket, surrounding the heart in its saline bath. This period of cooling usually lasted for about 2 hours. The temperature was then raised gradually until a level was reached at which the heart ceased to beat. The frequency of beat was timed by a stop-watch, the beats being counted over a period of 1 minute at the higher, and for 2 minutes Temperature, 0C. Fig. 1. Temperature-frequency curves plotted from experiments upon excised heart of British summer frogs. at the lower rates. The time of heating extended over a period of about 3 hours. We have been able in these experiments to confirm the observations of Barcroft and Izquierdo with regard to the form of the temperaturefrequency curve. The points, as may be seen from Fig. 1, which shows several of these curves, fall along practically straight lines. The hearts of the frogs collected in England cannot withstand a temperature much higher than or lower than 4 C. Above or below this range the heart ceases to beat and, after exposure to the higher temperature, is usually permanently damaged and cannot be resuscitated. It is, obviously, most desirable that observations should be

3 158 N. B. TA YLOR. made if possible upon a heart that will function without deterioration when subjected to greater temperature changes, with the view of ascertaining whether or not the linear form of the temperature-frequency curve is maintained over the wider range. Prof. Hogben suggested that the hearts of South African frogs (Xenopus dactylethra) would be suitable lui * U 30 :u 10o Temperature, C. Fig. 2. Temperature-frequency curves from three experiments upon the excised heart of South African frog. +, cooling; *, heating. for the test, and kindly shipped some fifty frogs to the laboratory for this purpose. OBSERVATIONS UPON THE TEMPERATURE-FREQUENCY RELATIONSHIP OF THE EXCISED HEART OF THE SOUTH AFRICAN FROG. The heart was suspended in the saline in the manner already referred to and the beat of the sinus counted at short intervals, while the tem-

4 TEMPERATURE ON SOUTH AFRICAN FROG'S HEART. 159 perature of the bath was gradually lowered from that of the room to 10 C. or less, and then raised again to about 330 to 35 C. The height to which the temperature could be raised before the heart was permanently damaged varied with different hearts. In most of the animals, however, the temperature range over which the functions of the sinus were apparently unimpaired was greater by some 10 or 11 degrees than when the English frogs were employed. It was not difficult to obtain data over a range of 350C. or more, i.e. nearly double the range that was possible with the English species. It cannot be said, however, that the cardiac functions were normal toward the upper limits of this range. This series Fig w tb 50 S C0 10 I Temperature, 'C. Comparison of typical temperature-heart rate curves of British +, British; *, South African. and South African frogs respectively. of experiments was carried out over a period extending from the end of May to the beginning of July. Fig. 2 shows temperature-frequency curves drawn from data furnished by three of these experiments. In Fig. 3, the curves of the English and South African frogs are given for comparison. Between 10 or 20 and about 28.50C. the graphs, as may be seen, are practically straight. When the temperature had reached a certain level a slackening in the rate occurred. This was an invariable phenomenon. The point at which the slowing appeared varied within a comparatively narrow range in the different experiments. The lowest temperature at which it was observed was C. and the highest C. The effect of

5 160 N. B. TA YLOR. higher temperatures upon the heart's frequency is shown by the sharp fall in the curves in Fig. 2. In some instances there was a plateau extending over 3 or 4 degrees at this critical temperature. When the temperature is raised higher still the rate accelerates again and is found to attain a speed of from 95 to 106 beats per minute. The temperature at which the heart commences to race in this way covers a comparatively narrow range. The lowest temperature at which it appeared in the several experiments was C. and the highest C. Cessation of the heart almost immediately follows any rise above that temperature at which the phase of rapid beating made its appearance; indeed, death of the heart soon occurs even if this temperature is maintained for any length of time. When the heart has stopped as a result of the excessive temperature it can sometimes be revived by cooling again. During the phase of acceleration the electrocardiograms, obtained by placing a silver wire electrode above and below the sinus, strongly suggest the establishment of a circus movement. The rapid heating is heralded by double beats, and later by tripling or short runs of extra systoles. Whatever may be the explanation of these alterations in rate at higher temperatures, the events themselves are indisputable. For the sake of clearness only three of the curves have been reproduced, but in all the experiments of this series the curves are almost identical in form and practically superimposable. THE EFFECT OF TEMPERATURE UPON THE RATE OF THE HEART IN THE INTACT SOUTH AFRICAN FROG. Considerable interest has been directed to the problem concerning the extent to which the inherent property of the heart to respond to temperature changes is governed or modified in the intact animal by nervous influences or other body functions. A simple linear relationship between temperature and heart rate is not of course to be expected, and we have found that the curves are more complex. In this series of experiments the animal was immobilized by being bandaged to a small wooden frame. It was immersed in a bath, and the temperature of this was gradually lowered by the addition of ice or raised by heating with a gas flame one end of a copper plate, the other end of the copper being submersed in the water. The heart rate was recorded electrocardiographically. Two inches or so of the bulbous ends of two silver probes were used for electrodes. One of these was passed through the anus high up into the bowels, the

6 TEMPERATURE ON SOUTH AFRICAN FROG'S HEART. 161 other into the cesophagus. Since the heart of the South African frog is situated in the mid-line of the chest with its long axis coinciding practically with the long axis of the body, this position of the electrodes corresponds to lead II or axial lead, employed in mammals. In some of the animals there was, apparently, a region of very high resistance lying somewhere between the two electrodes, and it was found that when the lower electrode was inserted through a small incision beneath the skin at the lower margin of the chest, much better records were procured. In frogs so immobilized the heart rate does not remain constant at constant temperature, but falls off about one beat per hour, sometimes more. If, therefore, the frog is first cooled and then heated, the curves Fig Temperature in gut, 0C. Curves obtained from experiments upon intact heart of South African frog. Temperatures recorded by means of thermocouple in the bowel. relating frequency to temperature diverge at the higher temperatures. The body temperature was recorded by means of a thermocouple, sealed within a large serum needle, the pointed end of which was rounded off. This instrument was inserted through the anus and passed for a distance of about 5 or 6 cm. up the bowel so that its tip, and consequently the thermoelectric junction itself lay not far below the lower limit of the heart. It was thought that by such an arrangement the temperature of the heart could be determined with a negligible error. The temperature-frequency curves of the intact heart were found to present two eminences (Fig. 4). The first of these covers a temperature range of from 10 to 20 degrees, and has its highest point corresponding to a temperature of about 150 or 160 C. The second peak is usually more abrupt and, commencing at a temperature of from 220 to 260 C., rises

7 162 N. B. TAYLOR. rapidly to a maximum and then falls away as quickly to a low level. This abrupt decline in the curve occurs at nearly the same temperature as the pronounced slowing of the rate of the excised heart, already referred to, makes its appearance. The heart invariably ceased to beat at the end of the period of slowing. Further elevation of the temperature never caused acceleration, as it did in the case of the isolated heart. On the other hand, a paradoxical quickening of the beat was several times observed when, during this phase of reduced frequency, the bath temperature was lowered again. The general form of the temperature-frequency curves in all the experiments of the series was essentially the same, and seven animals were examined in this way. Nevertheless, the unusual characteristics of the curves constituted a challenge to the accuracy of the method employed for recording the temperatures. It was thought possible, for instance, that the blood returning to the heart from the skin, since it would be sooner cooled or warmed by corresponding alterations in the temperature of the bath, might cause the heart temperature to be appreciably different from that of the bowel wherein the thermocouple had been placed. It may be mentioned in this connection that the rectal temperature of the marmot is known to be from 10 to 2 C., and under certain circumstances, 150 C. lower than that of the heart. The possibility of the unevenness in the curves being due to causes of the nature just mentioned made it necessary that the bowel and heart temperatures should be recorded independently and compared with one another. For this purpose a thermocouple constituted of very fine copper and constantin wires (gauge 40) was placed unsheathed within the ventricle and registrations made simultaneously from it and a similar instrument (sheathed) placed high up within the bowel. The frog was anmesthetized and the chest and upper abdomen opened. The unsheathed thermocouple was attached to a fine curved needle by means of a cotton thread and drawn into the ventricle. After being pushed well up towards the sinus the needle was removed and the thermocouple tied in position by means of the attached thread. The chest and abdomen were again closed by sutures. Very little bleeding from the heart was occasioned by this operation and the heart beat apparently normally for several hours afterwards. The anal thermocouple was then inserted and the frog placed in the bath. The temperature of this was gradually lowered and then raised as in the previous experiments, while simultaneous readings were made from the two instruments at frequent intervals. Fig. 5 shows graphs constructed from data obtained in this way. The bowel tempera-

8 TEMPERATURE ON SOUTH AFRICAN FROG'S HEART. 163 ture always lagged by a half to two degrees, depending upon the rate of heating or cooling, behind the heart temperature. Though the bath temperature was altered very slowly a disparity always existed between the temperatures of the two regions. The reason for this seemed to be simply a matter of heat conduction; the heart is placed very superficially and as a consequence its temperature is more quickly influenced by changes in the temperature of the bath-water than the more deeply buried abdominal organs. The graphs, however, run approximately parallel. It was concluded, therefore, that though the actual temperature of the heart had not been il 35 l 1 S -4a p4 I4) 15~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ so 25 _ K 4 V Ili 3i''' r L[11[UITT1Tr Minutes Fig. 5. Comparison of bowel and heart temperatures. 9, bowel temperatures; +, heart temperatures. recorded in the previous experiments, nevertheless, the temperature as registered by the bowel thermocouple must have borne a fairly constant relationship to the heart temperature, and consequently the form of the curves as drawn from the data secured by these experiments must represent true events. It was considered advisable, notwithstanding, to repeat the experiments and to register the heart temperatures directly by means of a thermocouple within the ventricle. When the data obtained from these later experiments were charted, the curves were found to agree closely with those of the original series. These curves are shown in Fig. 6. The chief characteristics, namely, the two elevations-one in the middle and the other toward the end are clearly seen. The departure of the temperature-frequency curve of the intact heart

9 164 N. B. TA YLOR. from the linear form characteristic of the excised heart must arouse enquiry into its cause. Several possible explanations suggest themselves. It might be due, for instance, to a rather sudden alteration in vagal tone, or of augmentor tone, or of both together acting in a reciprocal fashion. Again, it might be the result of the liberation of adrenaline into the blood stream. The terminal decline in the curve is very probably due to causes similar in nature to those which caused slowing of the excised heart at about the same temperature. If variations in vagal tone be responsible for the unevenness of the curve, then the administration of atropine should abolish this effect and Fig Temperature, 0 C. Temperature-frequency curves of intact heart of South African frog. Temperatures recorded by means of thermocouple in the ventricle. level the curve to the simple linear form. Barcroft and Izquierdo found that atropine had no effect upon the heart rate of the English frog, and concluded as a result of this that the vagus of this species possessed no tonic action. Indeed, it is generally stated that the amphibian heart is not under tonic vagal control [see Clark, 1927]. It was therefore not expected that the administration of atropine to the South African frogs would alter the form of the curve. The possibility, nevertheless, was put to the test of experiment. Though the curves were found to be of the same general shape as in the unatropinized animal, the inclination is considerably steeper. The first elevation (Fig. 7) rather than being flattened out is on the contrary more pronounced than in most of the curves of the unatropinized animal. The other possibilities,

10 TEMPERATURE ON SOUTH AFRICAN FROG'S HEART. 165 namely, the liberation of adrenaline or alterations in augmentor tone have not been investigated. THE EFFECT OF ATROPINE UPON THE HEART RATE OF THE SOUTH AFRICAN FROG. Though the effect of atropinization, so far as the general contour of the temperature-frequency curves of the heart is concerned, proved negative, the drug, nevertheless, was found to cause a very decided acceleration of the hearts of these animals. Barcroft and Izquierdo found that the dose necessary to paralyse the vagus of the English frog, which weighs about g., was 0 5 mg. The South African frogs weigh about 75 g. Calculating the dosage upon a weight basis 1-5 mg. of (D Ca "~ k Temperature, 0C. Fig. 7. Temperature-frequency curves of intact heart of South African frog after atropinization. +, cooling; O, heating. atropine were given subcutaneously. The frog remained in air and the room temperature was kept practically constant throughout the experiment. The animal was bandaged upon a frame, and the drug was not injected until the heart had been beating at a steady rate for an hour or so. A thermometer was kept in contact with the skin of the abdomen and the temperature read at intervals. The temperature of the animal recorded in this way, and which was practically that of the room, varied in different experiments from 160 to 180 C. Within from 6 to 7 minutes a very pronounced acceleration of the rate occurred'. The acceleration appeared quite suddenly at this time, 1 Experiments shortly to be described by Ivanitzki and Vasselenko show that atropine in such small doses does not accelerate the excised heart in Xenopu.

11 166 N. B. TA YLOR. when the rate was usually more than doubled. A few minutes later the frequency was trebled or quadrupled. This high degree of acceleration was maintained, throughout the entire period of observation which in some experiments lasted for 9 hours or more. As little as 0 12 mg., which was the smallest dose employed, produced just as great an effect. Control experiments in which distilled water equal in quantity to the atropine solution was injected were performed, but no increase in heart rate resulted. Examination of the electrocardiograms taken in these experiments revealed the fact that the increase in rate was brought about chiefly, if not almost entirely, by the shortening of the diastolic period (T-R Al Fig. 8. Time in seconds Electrocardiogram showing effect of atropine upon the heart rate of South African frog. A. Immeciately before aijection of O-5 mg. atropine sulph. B. 6 minutes after injection. interval). The length of the ventricular complex (R-T interval) was scarcely at all curtailed (Fig. 8). To cite an example: Before the administration of atropine the rate of the heart was 9 per minute. The length of ventricular systole as measured by the R-T interval was 1*5 sec., while diastole was 6-5 sec.' Twelve minutes after the injection of the drug the rate had increased to 24 per minute. The length of 'systole was unchanged at 1*5 sec., whereas diastole was now 1 It should be pointed out that in several of these experiments the initial rate was lower than was encountered in previous experiments upon these frogs, and it was in those with the slowest rates to start with that the greatest response to atropine was observed. It is possible, therefore, that the long preliminary period of immobilization which was employed in these experiments in order to establish a steady rate of beat had the effect of increasing the tone of the vagus.

12 TEMPERATURE ON SOUTH AFRICAN FROG'S HEART. 167 only 1X3 sec. in duration. Predominant shortening of the diastolic period as the heart rate increases is generally accepted as a reliable criterion of the release of vagal tone (see Reid Hunt). When the rate of the heart is increased by a rise in temperature both the diastolic and systolic periods are shortened to about an equal extent. Several experiments were performed for the purpose of determining the effect of atropinization upon the heart rate after this had been reduced to about 3 per minute by cooling the animal to 10 C. Atropine under these circumstances caused no acceleration of the beat so long as the low temperature was maintained. But upon removing the animal from the bath and allowing it to reach the temperature of the room (170 C.) the drug exercised its usual effect. The heart beat at from two to three times the rate at which it had been beating prior to the period of cooling. The failure of atropine to accelerate the heart so long as the temperature was very low, suggested that the low temperature had itself abolished vagal tone and that this action then was probably a protective measure to prevent undue slowing of the beat by low temperatures. If this were true one would expect that at high temperatures maximal vagal tone would be established in an attempt to hold the heart in check. Consequently atropine should produce an exaggerated effect. It was found on the contrary that the drug was without any effect whatever when this had been increased by raising the temperature of the animal to 24 C. It would appear therefore that in these frogs vagal tone is exercised within a certain temperature range-the precise limits of this were not determined. Vagal tone is absent, evidently, at very low or at unusually high temperatures. CONCLUSIONS. 1. Observations on the excised heart of the common frog confirm the statement that the sinus rate bears a roughly linear relation to the temperature when the tests are carried out in summer. 2. In the South African frog the same is true up to about 270 C. Above that temperature there is a slight slowing, followed at about 330 C. by a sudden and great acceleration. The cause of the slowing is obscure. That of the acceleration appears to be a "circus movement." 3. In the intact South African frog there are usually two summits on the curve relating the heart rate to the temperature. The first is at about C., the second at about 250 C.

13 168 N. B. TA YLOR. 4. The heart of the South African frog is subject to vagal tone at temperatures which are neither very high nor very low-a fact which may in part account for the drop in pulse rate between 12 and 160 C. a phenomenon which is not noticed in the British frog in which vagus tone appears to be absent. REFERENCES. Barcroft and Izquierdo (1931). J. Phy8iol. 71, 145. Clark (1927). Comparative Phy8iology of the Heart, p. 55. Cambridge. Izquierdo (1930). J. Phy8iol. 68, 363. Knowlton and Starling (1912). J. Phy8iol. 44, 206.