The heart of Palinurus lalandii and the ventricle of Octopus horridus maintain their normal rhythm for long periods in solutions containing only sodium and calcium chlorides.
The removal of calcium from the perfusion fluids causes systolic arrest in both hearts.
Both hearts are sensitive to very low concentration of potassium when perfused with potassium-free fluid.
Excess of magnesium and strontium produce the same effect as excess of calcium, but magnesium cannot be successfully substituted for calcium in the perfusing fluid.
The physiological range of H ion concentration is determined for the two preparations.
Both hearts have low temperature optima, and the high limit of their range is in the neighbourhood of 20° C.
Although numerous papers have appeared during the last 20 years dealing with the influence of the saline medium upon invertebrate heart muscle, it is only in recent years that the perfection of the technique of isolated perfusion and the recognition of the important rôle of hydrogen and hydroxyl ions have made possible the collection of any significant body of evidence with regard to the influence of specific ionic constituents of the medium upon the mechanism of the heart. And since the observations that have so far been made available have shown a considerable degree of variation among genera and even among species, it was thought that some additional data on the subject would be valuable from the point of view of comparative physiology.
The experiments hitherto recorded dealing with the hearts of marine invertebrates have been performed almost exclusively on species from English and Italian waters; consequently some information concerning two South African species, Palinurus lalandii and Octopus horridus, should be of interest.
It has been claimed for a large number of crustacean and molluscan hearts that their rhythm is satisfactorily maintained in sea water. L. Fredericq (1922) calls it “un excellent liquide” for Palinurus vulgaris. Our results, in this respect, are in striking disagreement with all preceding ones, the heart of neither Palinurus nor Octopus could be induced to beat in sea-water, and the results presented in this paper show that in more respects than one sea-water is definitely unsuitable.
The attempt was made at the outset to establish the simplest mixture of salts that would serve adequately to preserve the normal heart rhythm, on the grounds that the specific rôle of an ion is more readily determined when the number of possible combinations is reduced, to a minimum. The earlier investigators in this field erred on the side of excessive complexity. H. Fredericq (1914), for example, employed an artificial medium containing seven different salts. The advantages of a simple medium are emphasised by Hogben (1925). Starting with a fluid of simple composition, the effects of the common monovalent and divalent cations have been investigated.
All the experiments reported in this paper were performed upon the isolated heart preparation. The technique employed for the heart of Palinurus was in every respect similar to that described by Hogben (1925) for the hearts of Maia and Homarus, and the device for maintaining constant pressure described by him was adopted without modification.
REGULATION OF TEMPERATURE
These experiments were carried out during January and February, the two hottest months of the year in the Cape Peninsula. Room temperature at this time ranges from 21 to 26° C. It was found at the outset that the normal rhythm of neither the molluscan nor the crustacean heart could be maintained for more than ten minutes when removed from the body, and no modification in the composition of the perfusion fluid produced any improvement. We were driven to the conclusion that the temperature was unsuitable, which conclusion proved to be correct. The device adopted for cooling the perfusion fluid is illustrated in Fig. 1. The fluid flows through a spiral tube surrounded by an ice-water mixture and is delivered into the vessel where a constant head of pressure is maintained. Into this vessel a thermometer is inserted, so that the temperature of the perfusing fluid can be recorded immediately preceding its passage through the heart. A tube supplying air may also be inserted at this point, but it was found to be unnecessary; during its flow through the spiral cooling tube, the fluid is adequately oxygenated.
The temperature of the fluid depends, of course, on its rate of flow through the cooling system. By reducing the rate to a minimum a temperature of 8 ° C. could be obtained, the room being at about 24° C. But the extent to which the rate of flow can be reduced depends ultimately upon the rate at which the heart empties itself, for a constant head of pressure must be maintained in the cannula vessel. It was found with large hearts that the flow was too rapid to permit of adequate cooling, and in order to obviate this difficulty the stumps of the vessels through which the heart is emptied (the ophthalmic artery of Palinuros and the cephalic artery of Octopus) were carefully constricted by means of a cotton ligature. By this means the rate of flow through the cooling system was adjusted so as to establish the desired temperature (10−12° C.) in the cannula vessel.
The influence of temperature upon the heart beat of Octopus is shown in Fig. 2. The temperature was varied by the simple device of increasing the rate of flow through the cooling system, and then slowing it again. On being subjected for less than two minutes to a temperature of 18 ° C. the amplitude and regularity of the rhythm are very much impaired.
PART I. THE HEART OF PALINUROS LALANDII
Calcium and Magnesium
It was found that neither magnesium nor potassium were essential to the maintenance of the normal beat of the heart of Palinuros. Like the heart of Maia (Hogben 1925, Wells 1928) this heart will maintain a normal rhythm almost indefinitely on a suitable mixture of sodium and calcium chlorides. The optimum ratio was found to be 100 : 5, and no significant variation in different, individuals was observed in this respect.
Removal of calcium from a heart perfused with sodium and calcium chlorides produced complete systolic stoppage in less than a minute, preceded by a slight decrease in systolic tone (Fig. 3). Recovery was almost immediate but was followed by a permanent increase of diastolic tone.
A significant increase in the concentration of calcium ions produces immediate diastolic arrest (Fig. 4). Recovery is very much prolonged and may take as long as five minutes, but it is characteristic of this effect that with the first recovery stroke the normal amplitude is regained, although the frequency may be much reduced.
The influence of magnesium on the heart of Palinuros was found to be quite analogous to its action on Homarus, as described by Hogben (1925). Addition of magnesium to a solution containing the optimal concentration of calcium provoked a reduction in systolic tone, with, finally, complete diastolic stoppage when the molar concentration of magnesium was equivalent to twice the optimal concentration of calcium (Na 100 : Ca 5 : Mg 10). Conversely, when, from a heart being perfused with a solution containing both calcium and magnesium ions, magnesium was removed, there resulted an immediate and rapidly reversible increase in amplitude (Fig. 5). It must be remarked, however, that although five hearts were shown to beat regularly and permanently in solutions containing magnesium and calcium in the proportion Na 100 : Ca 5 : Mg 1−5, the same result was not obtained with perfusion fluids in which the magnesium was replaced by an equivalent concentration of calcium, i.e. Na 100 : Ca 5−10. It appears, therefore, that in the presence of the optimal concentration of calcium, the action of magnesium is not as pronounced as that of calcium. In the total absence of calcium, however, the reverse appears to be the case. In no case was magnesium found to be a satisfactory sub stitute for calcium. Replacing Ca 5 with Mg 5 produced in some hearts diastolic arrest, in others a progressive loss of systolic tone leading ultimately to stoppage. The nature of the response evoked by the removal of calcium from the perfusion fluid depends therefore upon the concentration of magnesium present. This is illustrated in Figs. 6 and 7. When calcium is removed from a heart perfused with Na 100 : Ca 5 : Mg 2 the characteristic contraction results, when calcium is removed from a solution containing Mg 4 the effect is reversed and the heart stops in diastole. An intermediate condition may sometimes be obtained by careful adjustment of the concentration of magnesium.
A striking feature of our experiments was the extreme sensitivity of the heart of Palinurus to potassium. All the hearts investigated in this respect could be brought to diastolic standstill by the addition of K 0·5 (Fig. 8) and with K1 the effect was immediate and was accompanied by considerable loss of diastolic tone (Fig. 9). In some cases even K0·2 stopped the heart and K0·05 evoked a reduction in frequency and a loss of systolic tone. This last is equivalent to a molar concentration of 0·00025. We are not aware that such extreme sensitivity to potassium has been recorded for any other contractile mechanism1.
Strontium and Barium
The effect of strontium upon the heart rhythm appears to be one of considerable complexity and different, not only in degree but also in kind, from that of calcium and magnesium. Large amounts of strontium had to be added to the perfusion fluid to evoke any noticeable response. When Sr 10 was added to the normal medium there was an increase in tone without any other apparent difference (Fig. 10). With double this amount of strontium the rise in tone was sometimes though not always obtained, but the amplitude was considerably increased and the frequency much reduced (Fig. 11). The delayed diastolic stoppage seen in Fig. 11 was not invariably associated with this effect.
The sensitivity of the heart of Palinurus was very much greater to barium than to strontium, but no such extreme sensitivity was observed as is recorded by Hogben for the heart of Maia. The action of barium appears to be quite sui generis in that its effects permanently damage the heart and no complete recovery is possible. The nature of the effect is illustrated in Fig. 12. Increased tone accompanied by reduced frequency leads ultimately to a condition of contracture, from which only partial recovery can be produced.
The hydrogen ion
The heart of Palinurus appears to be far more sensitive to hydroxyl ions than to hydrogen ions. The optimum pH lies in the neighbourhood of 5·0 (Fig. 13) which is even more acid than the optima recorded by Hogben for Maia and Homarus. Slight increments in acidity beyond this point become rapidly injurious, and the increase in tone characteristic of those two species was observed also for Palinurus. On the alkaline side of its range the heart loses tone in the neighbourhood of pH 8·0 (Fig. 14) and there is a reduction in frequency followed by stoppage in the relaxed condition after prolonged perfusion at this pH.
PART II. THE HEART OF OCTOPUS HORRIDUS
Calcium and Magnesium
Although the rhythm of the Octopus ventricle could be maintained in a solution containing the chlorides of sodium and calcium only, the addition of a small amount of magnesium was found to promote a greater amplitude and regularity of beat. Any given quantity of magnesium did not always produce the same effect on different hearts, the sensitivity to the magnesium ion being subject apparently to considerable individual variation. Every heart tested showed a marked improvement on the addition of Mg 1, and some even with Mg 2·5 (Fig. 15). With others the addition of this amount of magnesium resulted in lowering of the frequency and irregular stoppages. Further increase in the concentration of magnesium usually caused diastolic arrest, as with Palinurus, and Mg 10 always did.
The effect of calcium on the Octopus heart was quite similar to its action in the case of Palinunis. The optimal concentration was Na 100 : Ca 8. With suboptimal concentrations of calcium relaxation was much reduced, and in its total absence a condition approaching systolic arrest was established (Fig. 17). In the absence also of magnesium the systolic stoppage was complete. With excess of calcium the usual diastolic standstill resulted (Fig. 18). In its relation to calcium, therefore, the heart of Octopus resembles the hearts of Helix (Hogben) and Pecten (Mines, 1912).
In its relation to potassium the Octopus heart is unlike any molluscan hearts hitherto investigated, and is on the other hand very similar to the heart of Palinurus. The addition of K 0·5 (= 0·0025 M) to a heart perfused without potassium brings about diastolic arrest (Fig. 19). In this connection a puzzling unexplained phenomenon must be recorded. During the first ten minutes of perfusion the Octopus heart goes into systolic contracture for somewhat less than a minute, after which the normal rhythm is re-established. This event is shown in Fig. 20. The question has been raised whether this effect may not be associated with the withdrawal of potassium from the muscle tissue. Evidence at present is lacking to warrant a further discussion of this point. (See footnote on page 91.)
Strontium and Barium
The influence of strontium and barium on the mechanism of the Octopus heart does not appear to differ materially from its effect on the crustacean heart investigated by us. Typical effects are shown in Figs. 21 and 22. As recorded by Hogben for Homarus, by Mines (1911) for amphibian skeletal muscles and by ourselves in this paper for Palinuros, strontium falls physiologically into the calciummagnesium group, and of the three it seems to be the least active, since its presence in comparatively high concentration is necessary for the production of total diastolic arrest.
The view advanced by Mines (1911) that barium forms an insoluble compound with some cell constituent gains from our results a considerable measure of support. The Octopus heart could not be induced to return to a normal rhythm after its beat had been impaired by the addition of a small amount of barium.
The hydrogen ion
The pH range of the Octopus heart lies considerably on the alkaline side of the range of Palinuros. Even pH 8·4, although it caused a reduction in frequency and a certain irregularity of rhythm, did not seriously injure the heart (Fig. 23). On the acid side, 5·0 induced a pronounced reduction in amplitude (Fig. 24), which, in some cases, led to complete standstill in three minutes.
Previous investigations on the hearts of Palinuros and Octopus have been carried out by H. and L. Fredericq (1914, 1922). Fry (1909) worked on the closely allied genus, Eledone, but, being concerned with the innervation of the heart, he did not determine the effects of varying the ionic constituents of his medium. He noted, however, the injurious effects of high temperatures 23–25° C. upon the heart of this animal, although he was inclined to attribute them to “some other unknown physiological factor.” H. Fredericq (1914) perfused the isolated heart of Octopus vulgaris and made observations on effects of varying the saline constituents of his medium. He found that the presence of Na, Ca and K was essential, while Mg was unnecessary. We are led to conclude, therefore, that the South African Octopus horridas differs profoundly from the European species.
The remarkable sensitivity to potassium exhibited by the hearts of the two species studied by us is an observation entirely at variance with all data recorded for European species1. It is particularly surprising that two species belonging to separate phyla should manifest reactions to potassium that are almost quantitatively identical.
The nature of the mechanism involved in the modification of invertebrate cardiac rhythm by changes in the ionic constitution of the saline medium has been exhaustively discussed by Hogben (1925). Nothing that has since come to light would warrant a rediscussion of the whole question.
Two propositions, however, appear to be strengthened by our results. Calcium in excess, and probably also magnesium and strontium, paralyse the excitatory mechanism, causing stoppage in the relaxed condition. Recovery from this condition is prolonged, but when eventually it is achieved, the full amplitude is usually regained with the first stroke, indicating that the contractile mechanism is not impaired.
It seems highly probable that the specific rôle of calcium is to be accounted for by the Clowes phenomenon. The increased permeability that results in emulsoid systems, from the removal of calcium, may be assumed to occur also in the case of tissues. According to this view the penetration of sodium ions will be conditioned directly by the Na/Ca ratio. That sodium in the absence of calcium acts directly on the contractile mechanism seems to us to be strongly indicated by the fact that systolic stoppage occurs. The only other ion capable of producing an effect resembling that of sodium is the hydrogen ion, and its unusual mobility and powers of penetration are well known. The validity of this view will be determined by the investigation of the relation of this effect to the electrical variation. This we hope to accomplish in the near future.
The experiments here recorded were undertaken at the suggestion of Prof. Lancelot Hogben. We are indebted to him for much advice, criticism and encouragement.
DESCRIPTION OF PLATE III
Recent experiments have shown that this sensitivity to potassium only develops in hearts that have been perfused for a short time with potassium free medium. When potassium is included in the medium from the beginning of perfusion the heart will beat normally in Na 100 : Ca 5 : K I, and will tolerate a triple increase of potassium without injurious effects. The complete removal of potassium, however, invariably brings about a marked improvement in tone, frequency and regularity of beat.
In a private communication from Prof. Hogben we learn that in some unpublished experiments carried out at Plymouth on Portunus and Cancer he obtained an analogous sensitivity to the inhibitory action of potassium.