Temperature and the Responsive Mechanism of Cardiac Muscle: I. Temperature and the Duration of Contraction

Despite this close relationship between the separate processes, it is significant to note that each exhibits its own characteristic temperature coefficient. It is, therefore, essential to determine the effect of temperature upon these processes with reference to the velocity of the process as a function of temperature, and also to determine whether this function for each process may be modified independently of the other processes.

This paper is the first of a series dealing with these two problems in the responsive mechanism of cardiac muscle. It presents an analysis of the velocity of contraction of the auricular muscle as a function of temperature and an analysis of the stability of the system under various conditions. Other phases of the responsive mechanism of auricular muscle will be dealt with in later papers of the series.

The tissue used throughout these experiments is prepared either from the heart of the turtle Pseudyms elegans or Chrysemys emarginate The heart upon excision is immediately immersed in a modified Ringer solution containing adrenalin i part in 100.000. The adrenalin serves to prevent the tonic smooth muscle contraction which otherwise hinders ready dissection of the tissue. The two auricles are severed from the ventricle by an incision passing through the auriculo-ventricular junction, and are then separated from each other by an incision around the auricles along the inter-auricular septum. The rhythmic beating of the auricles is stopped by removing the sinus tissue in which the excitatory impulse originates. To prevent the subsequent assumption of rhythmicity by the auricle 0· 01 per cent. MgCl₂ is added to the Ringer solution (Smith, 1926). The auricle to be used is then placed upon its posterior surface and opened to form a rectangular strip by two parallel incisions along either side from the auricular opening almost to the tip of the auricle. One end of this strip is spread upon a small block of ebonite or of paraffined wood and fastened with a silk thread. The other end is secured in the muscle clamp on the muscle holder.

The muscle holder consists of an ebonite disc which carries supported from its lower surface two glass rods; the ends of these pass through an ebonite bar which has on its upper surface a small ebonite clamp. One end of the muscle is held by this clamp, while the other end of the muscle strip is tied tightly to a small ebonite block attached to the muscle lever by a silk thread. Also suspended from the large ebonite disc are two electrodes consisting of glass tubes, in the ends of which are sealed wires. In the case of platinum electrodes the tubes are filled with mercury and appropriate contact maintained at the top. In the case of silver electrodes they are coated with silver chloride and copper-wire connections are made between the silver and the binding post. Holes in the ebonite disc allow the insertion of a thermometer graduated to 0·1° C. and a tube for oxygenating the chamber.

The ebonite disc with the accessory parts is fastened in position by a clamp in such a way that it can be raised or lowered, or the muscle chamber can be removed at any time without disturbing the muscle holder.

For recording the isometric contraction of the muscle, an isometric lever is used. The lever is of the torsion wire type. It consists of a piece of fine steel watch spring, 2·5 cm. in length, fastened at either end by clamps which can be adjusted so as to place the band under tension. At the centre of the strip of steel and at right angles to it is soldered the lever arm. A small mirror 5 mm. square is fastened rigidly to the spring at the point of attachment of the lever arm. The lever holder is rigidly mounted upon an adjustable post so constructed that it is possible to raise, lower or revolve the spring holder in a horizontal plane. The adjustable post itself can be turned to any desired position in a vertical plane. The lever used has a period of 200 per second which is quite sufficient for the purpose in hand. By means of a suitable lens the image of the single filament of a 4-volt electric bulb reflected from a mirror is brought to a focus upon the recording surface. The movement of the beam of light is recorded upon a moving sheet of bromide paper in an electro cardiographic camera. The recording system gives a magnification of 75 times.

All observations are made upon muscle immersed in 400 c.c. of a modified Ringer solution. This is made up from stock solutions of the salts in glass-distilled water. The salts are present in the following proportion: NaCl 0·59 per cent., KC1 0·029 Per cent., CaCl₂ 0·017 Per cent., and MgCl₂ 0·049 per cent.

The solution is buffered to a pH of 7·0–7·2 by the addition of Na₂HPO4. The amount necessary is determined by titration and rarely exceeds 0 01 per cent.

The temperature is controlled by the use of Dewar flasks. At the beginning of the experiment nine of these flasks are filled with modified Ringer solution which has been adjusted to the desired temperature, and tightly stoppered. These maintain the temperature constant to within 0·1° C. during the time of recording, and, when stoppered, within 2° C. at the lower and higher temperatures for from 4 to 5 hours.

In the analysis of the records, the duration of the contractile phase of the response is taken as the time from the onset of contraction to the peak of the contraction. The investigations of Wiggers, as well as unpublished data of the author concerning the genesis of the isometric myogram of this tissue, justify this procedure in that they have shown that the values so determined represent the duration of the contractile phase of the first region of muscle stimulated.

In the analytical treatment of the data, the velocity of the process as a function of temperature is expressed in terms of the Arrhenius equation. This procedure makes possible the comparison of the results with those of other investigators and facilitates the comparison of several processes occurring simultaneously in the same tissue. Although it would be more suitable to use a general expression of the same form to which the theoretical implications of the Arrhenius equation would not be attached, it seems advisable not to introduce any new equation to express the velocities of the processes as a function of temperature, but rather to emphasise the physiological factors inherent in the system under investigation, which limit the use of the Arrhenius equation¹.

In Fig. 1, B, are plotted the data given in Table I from a representative experiment. Each point represents the average value for the first three or four twitches except where otherwise indicated. The twitches were recorded after a 15-minute period for temperature equilibration; a stimulation interval of 15 seconds was employed at the time of recording.

The results show that the velocity of contraction as a function of temperature is adequately described by this equation from 0·7° to about 20°. Above 20 the results deviate from the linear relation obtaining below this temperature, and in a direction indicating a decrease in velocity (increase in the duration) of the contractile phase of the response. The data is such that either a smooth curve, with a sharp inflection at 20°, or two straight lines intersecting at a point corresponding to 20°, may pass through the points. For purposes of calculation, there are advantages in the latter method of treatment. For purposes of analysis, however, it is sufficient to assume that the Arrhenius equation adequately describes the relation between the temperature and the velocity of the underlying process, whatever it may be, and that the deviations shown to exist owe their origin either to factors associated with the cellular mechanism itself, or that they are inherent in the experimental methods employed.

In such an analysis, it is essential to determine whether the results express the velocity of the contractile process as a function of temperature alone, or as a function of temperature plus cellular changes likewise varying as a function of temperature. Perhaps the most significant concomitant cellular change which could influence the results is the progressive change in functional state, or “deterioration,” which an isolated tissue undergoes when immersed in a physiological solution. To determine if possible to what extent the deterioration of the tissue is modifying the velocity temperature relation, experiments are performed in the following way: A first record is obtained following a 10-minute exposure of the tissue at 20°. The tissue is then immersed immediately in Ringer solution at a temperature around °. Records are obtained at this temperature and successively at higher temperatures at intervals of 3° to 30°, and then at decreasing temperatures at 3° intervals till 10° is reached. In Fig. 1, A, are plotted the results of a typical experiment performed by this method.

These experiments show that a deterioration of the tissue, progressing as a function of the time and resulting in an increase in the duration of contraction, is occurring at all temperatures. Below 20° the rate of deterioration is so slow that in the time involved it is not appreciable. At temperatures above 20°the rate of deterioration increases rapidly with the temperature. The degree of deterioration at these temperatures is obviously sufficient to play an important part in determining the results.

Since this “deterioration” is a function of the time of exposure and of the temperature, it may be controlled to a considerable extent by experimental treatment in the following way. A first record is obtained, following a 10-minute exposure of the tissue at 20°. The tissue is then immersed immediately in Ringer solution at a temperature in the vicinity of 3°. Records of the simple twitch are then obtained at this temperature and successively at temperatures of 8°, 12°, 16° and 20°, after a 10-minute equilibration period in every case. Following the 20° record the tissue is immersed quickly in Ringer solution at about 36° to 39°. After an exposure of 2 to 3 minutes at this temperature, three contractions are recorded, and the tissue immersed in Ringer solution at 15°. A record is then obtained after a 5-minute equilibration period at this temperature.

The results of an experiment performed in this fashion are plotted in Fig. 2, A. They show above 20° a deviation from the linear relationship existing below that temperature. In the particular experiment of the figure the value obtained at 38° deviates 19 per cent, from the value expected on the assumption that the Arrhenius equation adequately describes the relation for the entire range of temperature. For purposes of comparison, the results of an experiment, involving a prolonged exposure to progressively higher temperatures, are plotted in Fig. 2, B. The deviation from the expected linear relation in this experiment is much greater than in Fig. 2, A. It cannot be considered, however, as being similar in origin as it is completely irreversible. This is shown in Fig. 1, A, where the deterioration which has occurred permanently alters the subsequent values for the lower temperatures from those found before the tissue was exposed to the higher temperatures. This result is directly opposite to the results shown in Fig. 2, A, where the deterioration which occurred at higher temperatures did not appreciably alter the subsequent values at low temperatures. These results make it seem very unlikely, therefore, that the deviation from the expected linear relation shown in Fig. 2, A, can be due to the deterioration of the tissue at high temperatures. This conclusion does not preclude the fact that the exposure to high temperatures results in some irreversible changes in the tissue; it may be considered, nevertheless, as showing that such changes as do occur cannot be recorded as changes in the duration of contraction by any methods ordinarily employed, but that changes in the system do occur is shown by an irreversible decrease in the total tension of the twitch (as opposed to the duration of contraction) subsequently recorded at temperatures below 20° following an exposure to a high temperature.

The results from experiments involving the use of an isotonic method of recording substantiate the foregoing results. They show that an irreversible progressive detonation occurs, as a function of time, at temperatures above 20°. The deterioration may be partially eliminated as a modifying factor in the results, in this case, also, by proper regulation of the time of exposure to the higher temperatures.

Under the foregoing experimental conditions, which tend to limit the progressive deterioration occurring at temperatures above 20°, the result may be satisfactorily described by the Arrhenius equation-over a temperature range from 0·7° C. to 20° C. A marked deviation is still shown to exist above this temperature. If the degree of deterioration under-these conditions be considered as negligible,. the relation between the velocity of contraction and the temperature obtained may be considered as being jointly determined by two factors: (a) the actual reactions involved in the contraction of the muscle, and (b) associated reversible changes in the physico-chemical organisation of the cell at different temperatures, which might modify the reactions included under (a).

Although there is little direct evidence bearing on the point, we may take it as exceedingly improbable that the fundamental changes underlying the contraction are characterised by discontinuity, e.g. we may be reasonably certain that the neutralisation of lactic acid by the buffer system in the muscles proceeds above 20° in essentially the same way as it does below 20°. An intracellular property, however, which has been suggested as accounting for these discontinuities is the property of viscosity, and the results of investigations upon protoplasmic viscosity certainly furnish evidence of a gross physico-chemical reorganisation in the cell progressing as a function of temperature. The viscosity of the protoplasm of the Arbacia egg and Amoeba dubia (Heilbrunn, 1925, 1929), for example, is high at low temperatures, and, as the temperature increases, rapidly falls to a minimum, rises to a maximum, decreases to a second minimum and increases again at very high temperatures. The maximum in Arbacia is at 15°, and in Amoeba dubia is at 25°. Snyder (1911), Pütter (1914), and others have suggested that changes in viscosity may be responsible for the deviations from the accepted linear relation between the velocity of biological processes and the temperature, and Heilbrunn (1925) has emphasised the possibility of such changes in the viscosity of protoplasm affecting the velocity of cellular reactions. In the case of muscle, it is possible that in the vicinity of 20° the response is modified by rapid physico-chemical changes in the system associated with the condition of maximum viscosity at that temperature, but for reasons which will be developed in a later paper, the viscosity per se is not considered as directly affecting the system, but rather as one expression of a physico-chemical reorganisation with which may be associated those changes in the system responsible for the deviation from the linear relation.

The determination of the nature of the changes grouped under (a) would be facilitated if a treatment of the kinetics of the contractile system were available. The use of the Arrhenius equation in describing the relation between the velocity and temperature, however, involves a very elementary treatment of the kinetics of the system. Thus, in applying this equation to the data, it is assumed that the reciprocal of the duration of contraction is proportional to the velocity constant of the reaction at each temperature; this assumption implies either that the concentration of material involved in the reaction is constant at all temperatures or that the vderity constant is independent of the concentration of the reacting substances. An adequate-consideration of the foregoing factors may account for the deviations from the expected linear relation; it should be emphasised, however, that the results plotted in Fig. 2, A, accurately describe the velocity of contraction as a function of temperature under the conditions established by the experimental methods employed, and, moreover, that any treatment of the kinetics of the system must be in accordance with the results of such an experiment. In fact, the nature of the intra-cellular changes associated with the discontinuities referred to are unknown, and must remain unknown until the kinetics of the system are solved.

The tissue deterioration which has been shown to increase the duration of contraction also results in a decrease in the total tension produced during contraction. This result might be brought about either by the deterioration resulting in a decrease in the quantity of material released at stimulation, or by the deterioration producing modification in the method by which the foregoing material is utilised in producing contraction of the muscle, or by both. In either of the above cases, it is possible that such alterations due to deterioration would result in a change in the velocity of the process as a function of temperature.

To determine, if possible, whether deterioration in the tissue results in an alteration in the velocity-temperature relation, experiments were performed upon the tissue in which various degrees of deterioration had been produced. The effects of such treatment are best shown by a consideration of an actual experiment. In Fig. 1, A, are plotted the results of an experiment performed on the same tissue immediately after removal from the body of the turtle, after an exposure to room temperature and after an exposure to high temperature of sufficient duration to produce a considerable effect. These results show that the velocity as a function of temperature was not affected by such treatment. However, the slope of the line is greatest between the points 1 and 2 recorded immediately after removal from the turtle; following an exposure to a temperature of 20° for 3 hours, the slope is less. As the exposure is made longer and longer, the decrease in slope diminishes and reaches a quite constant value. If the slope is expressed in terms of μ, the initial values would be about 14,800 ± 500; after an exposure of 2 hours to a temperature of 20° the value would be 14,000 ± 500, and after prolonged exposure it would reach a lower limit of about 13,500 ± 500.

The exposure of the tissue to anaesthetising concentration of alcohol similarly results in an irreversible deterioration of the tissue which causes an increase in the duration and a decrease in the total tension of contraction. In this case also the deterioration fails to modify the velocity-temperature relation although it causes a decrease in the slope which expressed in terms of μ gives a value of 13,500 ± 500.

The relation between the velocity of contraction and the temperature shown to exist in the auricular muscle is a special case determined by the stimulation interval employed. In these experiments only the first response following a 10-minute rest period was considered. The magnitude and duration of this response is determined by the cellular conditions existing during the “steady state” of the resting muscle at that particular temperature. A similar relationship might be obtained under conditions of repeated stimulation, either at a constant or a variable rate, provided the same relative cellular conditions existed at each temperature, as in the first response following a period of rest, but such a condition is not attained readily under conditions of repeated stimulation. If a constant rate of stimulation is maintained throughout the experiment, the acceleration of the recovery process with an increase in temperature serves to convert the stimulation interval, constant with respect to time, into a stimulation interval of variable length with reference to the cellular processes themselves. Thus, a stimulus occurring relatively early in the recovery phase at low temperatures would occur relatively late in the recovery phase at high temperatures. Since the magnitude and duration of the second response depends upon the time at which it occurs during the recovery period of the first response, the cellular conditions at the different temperatures are not identical. The situation is complicated further by the fact that these effects of regular stimulation of the tissue are associated with irregularities in the magnitude and duration of the successive responses during continuous stimulation. In the latter phenomenon, known as treppe, the first twitch following a period of rest is usually larger than the second, after which each succeeding twitch increases in size until a plateau is reached, the height of the plateau depending upon the rate of stimulation. The slope of the temperature-velocity curve, therefore, depends upon the particular stimulation interval employed and the duration of stimulation, and the proper choice of interval and duration can result, over a portion of the temperature range, in a slope practically identical to that obtained when the average duration of the first few twitches only is used in the calculation. When a variable stimulation interval is employed at different temperatures almost any variation in the curve may be obtained. This is clearly shown by the results of Clark (1920) on the effect of temperature upon the duration of contraction of the rhythmically beating auricular strip. It is evident, therefore, that in an investigation of the effects of temperature upon the contractile mechanism, only those results are adequate which are based upon the duration of contraction of the first two or three twitches following an adequate period of rest, and stimulated at an interval sufficient to allow adequate recovery at all temperatures.

The foregoing discussion should be adequate to show that little significance can be attached to the values of μ. obtained in the sense of their chemical significance. The μ values obtained for various portions of the curve furnish, however, a convenient means of. comparing the constancy of the function in various tissues under different conditions. In Table II are plotted some of the values of μ obtained in these experiments. It should be noted that the μ values for the temperature range 0·7° to 20° are highest when experimental methods tending to limit the heat deterioration of the tissue are employed. Similarly, for the temperature range from 20° to 38° the highest values of μ were obtained under the same conditions, and the difference in μ value for these two ranges of temperature is well outside the experimental error; the values for the upper range of temperature cannot, of course, be considered as accurate as those for the lower range where the deterioration of tissue is practically negligible. For this reason, in comparing the values for the velocity of contraction of the auricular muscle with similarly determined values for the velocity of contraction of other types of muscle, the most accurate temperature range over which to make a comparison is that from o° to 20°, or to that temperature at which the lower value of μ is approached.

The fact that the μ value depends on the experimental methods employed renders difficult any exact comparison of the values obtained in this investigation with those of other investigators. The μ value of 14,500 for the auricular muscle of the turtle as obtained in this investigation is higher than that found by Eckstein (1920) for frog heart. At the same time the results are more consistent. The value, however, compares favourably with the μ value of 14,000 for the velocity of contraction of the gastrocnemius muscle of the frog for the same temperature range. This is shown in Fig. 1, D, in which are plotted the values obtained by Fulton (1926) for the gastrocnemius muscle. The complete lines are those applied by the author on assumption that the values obtained at lower temperatures are the most accurate, and gives a μ value of 14,000. The μ value of 12,500 obtained by Fulton is the same as would be obtained for the auricular muscle if points only in the upper temperature range were considered; the difference between the results of this paper and those of Fulton disappear, however, if the lower temperature range is used. This procedure, for reasons already stated, is preferable. It should be pointed out that if the value 14,000 is selected as best for skeletal muscle on the grounds that the observations in the lower temperature range of Fulton’s data are the most accurate, the μ value is not in accord with that of 12,500 obtained over the same temperature range, for the heat production per gram tension in skeletal muscle (Hartree and Hill, 1921; Fulton, 1926).

1. The velocity of contraction of the auricular muscle strip as a function of temperature is described adequately from 0·7° C. to about 20° by the Arrhenius equation. Above 20^° the results deviate from the linear relationship obtaining below this temperature in a direction indicating a decrease in the velocity of contraction. The cause of the deviation is unknown.

2. The deviations occurring above 20° C. are still present when methods are employed to reduce to a minimum the progressive deterioration of the isolated tissue at high temperatures.

3. Deterioration of the tissue due to prolonged exposures to low temperatures and brief exposures to high temperatures, or due to exposure to alcohol, produces no appreciable alteration in the velocity of contraction as a function of temperature. The μ value between 0·7° and 20°, however, is slightly lower.

4. The rate of stimulation can profoundly alter the relation between the velocity of contraction and the temperature.