87

THE RELATION OF ELECTROLYTES TO THECARDIAC RHYTHM OF PALINURUS {JASUS)

LALANDII AND OCTOPUS HORRIDUS

BY ALEXANDER ZOOND AND DAVID SLOME.

(From the Department of Zoology, University of Cape Town.)

{Received 2gth May 1928.)

(With One Text-figure and One Plate.)

CONTENTS.PAGE

I n t r o d u c t i o n . . . . . 8 7T e c h n i q u e . . . . . . 8 8R e g u l a t i o n o f t e m p e r a t u r e . . . 8 9P a r t I . T h e h e a r t o f Palinurus lalandii 9 0

C a l c i u m a r i d M a g n e s i u m . . 9 0P o t a s s i u m . . . . . 9 1S t r o n t i u m a n d B a r i u m . . . 9 1T h e h y d r o g e n i o n . . . . 9 2

P a r t I I . T h e h e a r t o f Octopus horridus 9 2C a l c i u m a n d M a g n e s i u m . . 9 2P o t a s s i u m . . . . . 9 2S t r o n t i u m a n d B a r i u m 9 3T h e h y d r o g e n i o n . . . . 9 3

D i s c u s s i o n 9 3S u m m a r y 9 4

INTRODUCTION.

ALTHOUGH numerous papers have appeared during the last 20 years dealing withthe influence of the saline medium upon invertebrate heart muscle, it is only inrecent years that the perfection of the technique of isolated perfusion and therecognition of the important role of hydrogen and hydroxyl ions have made possiblethe collection of any significant body of evidence with regard to the influence ofspecific ionic constituents of the medium upon the mechanism of the heart. Andsince the observations that have so far been made available have shown a consider-able degree of variation among genera and even among species, it was thought thatsome additional data on the subject would be valuable from the point of view ofcomparative physiology.

The experiments hitherto recorded dealing with the hearts of marine inverte-brates have been performed almost exclusively on species from English and Italianwaters; consequently some information concerning two South African species,Palinurus lalandii and Octopus horridus, should be of interest.

ALEXANDER ZOOND and DAVID SLOME

It has been claimed for a large number of crustacean and molluscan heartsthat their rhythm is satisfactorily maintained in sea water. L. Fredericq (1922)calls it "un excellent liquide" for Palinunis vulgaris. Our results, in this respect,are in striking disagreement with all preceding ones, the heart of neither Palinurusnor Octopus could be induced to beat in sea-water, and the results presented in thispaper 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 saltsthat would serve adequately to preserve the normal heart rhythm, on the groundsthat the specific role of an ion is more readily determined when the number ofpossible combinations is reduced.to a minimum. The earlier investigators in thisfield erred on the side of excessive complexity. H. Fredericq (1914), for example,employed an artificial medium containing seven different salts. The advantages ofa simple medium are emphasised by Hogben (1925). Starting with a fluid of simplecomposition, the effects of the common monovalent and divalent cations have beeninvestigated.

TECHNIQUE.

All the experiments reported in this paper were performed upon the isolatedheart preparation. The technique employed for the heart of Palinunis was in everyrespect similar to that described by Hogben (1925) for the hearts of Maia andHomarus, and the device for maintaining constant pressure described by him wasadopted without modification.

The procedure for isolating the heart of Octopus was as follows: When the armsof the animal are cut off with one stroke of a sharp butcher's knife, the body canthen be dissected in comfort. After exposing the viscera by a median incision in thedorsal aspect of the mantle, the heart can easily be located by following down theprominent dorsal aorta. When the heart has been exposed a cannula is tied intothe left auricle and the heart is lifted out of the body, about an inch of the dorsalvessel being left attached to the heart, for a purpose which will presently be stated.It is not necessary to flush out the heart following cannulation, since molluscanblood does not coagulate on exposure to air. Fry's (1909) observations on theinjurious effects of exposing the heart of Eledone to air were not confirmed; whensuitably perfused the Octopus heart continued to beat normally in contact with airfor several hours. The perfusion fluids were made up volumetrically from solu-tions of chlorides (Merck). The molarity of the sodium chloride solution was o-6,that of all the others was 0-5. The concentration of each solution employed wascontrolled by titration with tenth normal silver nitrate. The sodium chloridesolution was buffered with 4 c.c. per 10 litres of saturated disodium hydrogenphosphate. After the addition of the other ingredients, the solution was at

pH = 6-9-7-2.

Electrolytes and Cardiac Rhythm

REGULATION OF TEMPERATURE.

These experiments were carried out during January and February, the twohottest months of the year in the Cape Peninsula. Room temperature at this timeranges from 21 to 260 C. It wasfound at the outset that the normal Experimental—>==:n (7==z<^~Perfusion

Fluid Fluid

Water icemixture

Thermometer-

Air ~

rhythm of neither the molluscan northe crustacean heart could be main-tained for more than ten minuteswhen removed from the body, and nomodification in the composition ofthe perfusion fluid produced any im-provement. We were driven to theconclusion that the temperature wasunsuitable, which conclusion provedto be correct. The device adopted forcooling the perfusion fluid is illus-trated in Fig. 1. The fluid flowsthrough a spiral tube surrounded byan ice-water mixture and is deliveredinto the vessel where a constant headof pressure is maintained. Into thisvessel a thermometer is inserted, sothat the temperature of the perfusingfluid can be recorded immediatelypreceding its passage through theheart. A tube supplying air may alsobe inserted at this point, but it wasfound to be unnecessary; during itsflow through the spiral cooling tube,the fluid is adequately oxygenated.

The temperature of the fluid de-pends, of course, on its rate of flowthrough the cooling system. By re-ducing the rate to a minimum atemperature of 8° C. could be ob-tained, the room being at about 240 C.But the extent to which the rate offlow can be reduced depends ulti-mately upon the rate at which theheart empties itself, for a constanthead of pressure must be maintained in the cannula vessel. It was found with largehearts that the flow was too rapid to permit of adequate cooling, and in orderto obviate this difficulty the stumps of the vessels through which the heart is

Jl

Fig. 1. Cooling system for perfusion of heart atconstant temperature.

90 ALEXANDER ZOOND and DAVID SLOME

emptied (the ophthalmic artery of Palinurus and the cephalic artery of Octopus)were carefully constricted by means of a cotton ligature. By this means the rate offlow through the cooling system was adjusted so as to establish the desired tem-perature (10-120 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 flowthrough the cooling system, and then slowing it again. On being subjected for lessthan two minutes to a temperature of 180 C. the amplitude and regularity of therhythm are very much impaired.

PART I. THE HEART OF PALINURUS LALANDII.

Calcium and Magnesium.

It was found that neither magnesium nor potassium were essential to the main-tenance of the normal beat of the heart of Palinurus. Like the heart of Maia(Hogben 1925, Wells 1928) this heart will maintain a normal rhythm almostindefinitely on a suitable mixture of sodium and calcium chlorides. The optimumratio was found to be 100 : 5, and no significant variation in different individualswas observed in this respect.

Removal of calcium from a heart perfused with sodium and calcium chloridesproduced complete systolic stoppage in less than a minute, preceded by a slightdecrease in systolic tone (Fig. 3). Recovery was almost immediate but was followedby a permanent increase of diastolic tone.

A significant increase in the concentration of calcium ions produces immediatediastolic arrest (Fig. 4). Recovery is very much prolonged and may take as longas five minutes, but it is characteristic of this effect that with the first recoverystroke the normal amplitude is regained, although the frequency may be muchreduced.

The influence of magnesium on the heart of Palinurus was found to be quiteanalogous to its action on Homarus, as described by Hogben (1925). Addition ofmagnesium to a solution containing the optimal concentration of calcium provokeda reduction in systolic tone, with, finally, complete diastolic stoppage when themolar concentration of magnesium was equivalent to twice the optimal concen-tration of calcium (Na 100 : Ca 5 : Mg 10). Conversely, when, from a heart beingperfused with a solution containing both calcium and magnesium ions, magnesiumwas removed, there resulted an immediate and rapidly reversible increase in ampli-tude (Fig. 5). It must be remarked, however, that although five hearts were shownto beat regularly and permanently in solutions containing magnesium and calciumin the proportion Na 100 : Ca 5 : Mg 1-5, the same result was not obtained withperfusion fluids in which the magnesium was replaced by an equivalent concen-tration of calcium, i.e. Na 100 : Ca 5-10. It appears, therefore, that in the presenceof the optimal concentration of calcium, the action of magnesium is not as pro-nounced as that of calcium. In the total absence of calcium, however, the reverseappears to be the case. In no case was magnesium found to be a satisfactory sub-

Electrolytes and Cardiac Rhythm 91

stitute for calcium. Replacing Ca 5 with Mg 5 produced in some hearts diastolicarrest, 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 perfusionfluid depends therefore upon the concentration of magnesium present. This isillustrated in Figs. 6 and 7. When calcium is removed from a heart perfused withNa 100 : Ca 5 : Mg 2 the characteristic contraction results, when calcium is re-moved from a solution containing Mg 4 the effect is reversed and the heart stopsin diastole. An intermediate condition may sometimes be obtained by carefuladjustment of the concentration of magnesium.

Potassium.

A striking feature of our experiments was the extreme sensitivity of the heart ofPalinurus to potassium. All the hearts investigated in this respect could be broughtto diastolic standstill by the addition of K 0*5 (Fig. 8) and with K 1 the effect wasimmediate and was accompanied by considerable loss of diastolic tone (Fig. 9).In some cases even K 0*2 stopped the heart and K 0-05 evoked a reduction infrequency and a loss of systolic tone. This last is equivalent to a molar concen-tration of 0*00025. We are not aware that such extreme sensitivity to potassium hasbeen recorded for any other contractile mechanism1.

Strontium and Barium.

The effect of strontium upon the heart rhythm appears to be one of considerablecomplexity and different, not only in degree but also in kind, from that of calciumand magnesium. Large amounts of strontium had to be added to the perfusionfluid to evoke any noticeable response. When Sr 10 was added to the normalmedium there was an increase in tone without any other apparent difference(Fig. 10). With double this amount of strontium the rise in tone was sometimesthough not always obtained, but the amplitude was considerably increased andthe frequency much reduced (Fig. 11). The delayed diastolic stoppage seen inFig. 11 was not invariably associated with this effect.

The sensitivity of the heart of Palinurus was very much greater to barium thanto strontium, but no such extreme sensitivity was observed as is recorded byHogben for the heart olMaia. The action of barium appears to be quite sui generisin that its effects permanently damage the heart and no complete recovery is pos-sible. The nature of the effect is illustrated in Fig. 12. Increased tone accompaniedby reduced frequency leads ultimately to a condition of contracture, from whichonly partial recovery can be produced.

1 Recent experiments have shown that this sensitivity to potassium only develops in hearts thathave been perfused for a short time with potassium free medium. When potassium is included inthe medium from the beginning of perfusion the heart will beat normally in Na ioo: Ca 5: K i, andwill tolerate a triple increase of potassium without injurious effects. The complete removal ofpotassium, however, invariably brings about a marked improvement in tone, frequency andregularity of beat.

92 ALEXANDER ZOOND and DAVID SLOME

The hydrogen ion.

The heart of Palinurus appears to be far more sensitive to hydroxyl ions thanto 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 andHomarus. Slight increments in acidity beyond this point become rapidly injurious,and the increase in tone characteristic of those two species was observed also forPalinurus. On the alkaline side of its range the heart loses tone in the neighbourhoodof pH 8-o (Fig. 14) and there is a reduction in frequency followed by stoppage inthe 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 solutioncontaining the chlorides of sodium and calcium only, the addition of a smallamount of magnesium was found to promote a greater amplitude and regularityof beat. Any given quantity of magnesium did not always produce the same effecton different hearts, the sensitivity to the magnesium ion being subject apparentlyto considerable individual variation. Every heart tested showed a marked improve-ment on the addition of Mg 1, and some even with Mg 2*5 (Fig. 15). With othersthe addition of this amount of magnesium resulted in lowering of the frequencyand irregular stoppages. Further increase in the concentration of magnesiumusually 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 thecase of Palinurus. The optimal concentration was Na 100 : Ca 8. With suboptimalconcentrations of calcium relaxation was much reduced, and in its total absencea condition approaching systolic arrest was established (Fig. 17). In the absencealso of magnesium the systolic stoppage was complete. With excess of calcium theusual 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).

Potassium.

In its relation to potassium the Octopus heart is unlike any molluscan heartshitherto 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 bringsabout diastolic arrest (Fig. 19). In this connection a puzzling unexplained pheno-menon must be recorded. During the first ten minutes of perfusion the Octopusheart goes into systolic contracture for somewhat less than a minute, after whichthe normal rhythm is re-established. This event is shown in Fig. 20. The questionhas been raised whether this effect may not be associated with the withdrawal ofpotassium from the muscle tissue. Evidence at present is lacking to warrant afurther discussion of this point. (See footnote on page 91.)

Electrolytes and Cardiac Rhythm 93

Strontium and Barium.

The influence of strontium and barium on the mechanism of the Octopusheart does not appear to differ materially from its effect on the crustacean heartinvestigated by us. Typical effects are shown in Figs. 21 and 22. As recorded byHogben for Homarus, by Mines (1911) for amphibian skeletal muscles and byourselves in this paper for Palinurus, strontium falls physiologically into the calcium-magnesium group, and of the three it seems to be the least active, since itspresence in comparatively high concentration is necessary for the production oftotal diastolic arrest.

The view advanced by Mines (1911) that barium forms an insoluble compoundwith 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 itsbeat 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 therange of Palinurus. Even pH. 8-4, although it caused a reduction in frequency anda certain irregularity of rhythm, did not seriously injure the heart (Fig. 23). Onthe acid side, pH 5-0 induced a pronounced reduction in amplitude (Fig. 24),which, in some cases, led to complete standstill in three minutes.

DISCUSSION.

Previous investigations on the hearts of Palinurus and Octopus have been carriedout by H. and L. Fredericq (1914, 1922). Fry (1909) worked on the closely alliedgenus, Eledone, but, being concerned with the innervation of the heart, he did notdetermine the effects of varying the ionic constituents of his medium. He noted,however, the injurious effects of high temperatures 23-250 C. upon the heart ofthis animal, although he was inclined to attribute them to "some other unknownphysiological factor." H. Fredericq (1914) perfused the isolated heart of Octopusvulgaris and made observations on effects of varying the saline constituents of hismedium. He found that the presence of Na, Ca and K was essential, while Mg wasunnecessary. We are led to conclude, therefore, that the South African Octopuskorridus differs profoundly from the European species.

The remarkable sensitivity to potassium exhibited by the hearts of the twospecies studied by us is an observation entirely at variance with all data recordedfor European species1. It is particularly surprising that two species belonging toseparate phyla should manifest reactions to potassium that are almost quantitativelyidentical.

The nature of the mechanism involved in the modification of invertebratecardiac rhythm by changes in the ionic constitution of the saline medium has been

1 In a private communication from Prof. Hogben we learn that in some unpublished experi-ments carried out at Plymouth on Portunus and Cancer he obtained an analogous sensitivity to theinhibitory action of potassium.

94 ALEXANDER ZOOND and DAVID SLOME

exhaustively discussed by Hogben (1925). Nothing that has since come to lightwould warrant a rediscussion of the whole question.

Two propositions, however, appear to be strengthened by our results. Calciumin excess, and probably also magnesium and strontium, paralyse the excitatorymechanism, causing stoppage in the relaxed condition. Recovery from this con-dition is prolonged, but when eventually it is achieved, the full amplitude is usuallyregained with the first stroke, indicating that the contractile mechanism is notimpaired.

It seems highly probable that the specific role of calcium is to be accounted forby the Clowes phenomenon. The increased permeability that results in emulsoidsystems, from the removal of calcium, may be assumed to occur also in the caseof tissues. According to this view the penetration of sodium ions will be conditioneddirectly by the Na/Ca ratio. That sodium in the absence of calcium acts directlyon the contractile mechanism seems to us to be strongly indicated by the fact thatsystolic stoppage occurs. The only other ion capable of producing an effect resem-bling that of sodium is the hydrogen ion, and its unusual mobility and powers ofpenetration are well known. The validity of this view will be determined by theinvestigation of the relation of this effect to the electrical variation. This we hopeto accomplish in the near future.

SUMMARY.

1. The heart of Palinunis lalandii and the ventricle of Octopus horridus main-tain their normal rhythm for long periods in solutions containing only sodium andcalcium chlorides.

2. The removal of calcium from the perfusion fluids causes systolic arrest inboth hearts.

3. Both hearts are sensitive to very low concentration of potassium whenperfused with potassium-free fluid.

4. Excess of magnesium and strontium produce the same effect as excess ofcalcium, but magnesium cannot be successfully substituted for calcium in theperfusing fluid.

5. The physiological range of H ion concentration is determined for the twopreparations.

6. Both hearts have low temperature optima, and the high limit of their rangeis in the neighbourhood of 200 C.

The experiments here recorded were undertaken at the suggestion of Prof.Lancelot Hogben. We are indebted to him for much advice, criticism and en-couragement.

DESCRIPTION OF PLATE III.FIG. 2. Heart of Octopus. Perfused 65 min. Effect of temperature. Perfused with Na 100: Ca 8 : Mg 1;pH. 7-3; io° C. Each signal represents change in temperature of i° C. Time signal—1 min. in allrecords.

FIG. 3. Heart of Palinurus. Perfused 15 min. Perfusion fluid—Naioo:Ca5; pR 6-9; xz° C.At signal calcium removed. Time signal—1 min.

JOURNAL OF EXPERIMENTAL BIOLOGY VOL. VI, PLATE III.

ZOOND AND SLOME—THE RELATION OF ELECTROLYTES TO THE CARDIACRHYTHM OF PALINURUS (JASUS) LALANDII AND OCTOPUS HORRIDUS (pp. 87-95).

Electrolytes and Cardiac Rhythm 95

FIG. 4. Heart of Palinurus. Perfused 45 min. Perfusion fluid—NaiooiCas; pH 6 9 ; io° C.At signal changed to Na 100 : Ca 15.

FIG. 5. Heart of Palinurus. Perfused 12 min. Perfusion fluid—Na 100 : Ca 5 : Mg 5; pH 6 9 ;150 C. At signal changed to Na 100 : Ca 5.

FIG. 6. Heart of Palinurus. Perfused 10 min. Perfusion fluid—Na 100 : Ca 5 : Mg 2; pH6g;150 C. At signal changed to Na 100 : Mg 2.

FIG. 7. Heart of Palinurus. Perfused 5 rnin. Perfusion fluid—Na 100 : Ca 5 : Mg4;pH 6 9 ; 150 C.At signal changed to Na 100 : Mg 4.

FIG. 8. Heart of Palinurus. Perfused 27 min. Perfusion fluid—Na 100 : Ca 5; pH. 6-9; 12° C. Atsignal same with K 0-5.

FIG. 9. Heart of Palinurus. Perfused 8 min. Perfusion fluid—Na 100 : Ca 5; pH 6-9; 120 C. Atsignal changed to same with K 1.

FIG. 10. Heart of Palinurus. Perfused 5 min. Perfusion fluid—Na 100 : Ca 5; pH 6-9; 150 C. Atsignal changed to same with Sr 10.

FIG. 11. Heart of Palinurus. Perfused 10 min. Perfusion fluid—Na 100 : Ca 5; pH 6-9; 120 C.At signal changed to same with Sr 20.

FIG. 12. Heart of Palinurus. Perfusion fluid—Na 100 : Ca 5; pH 6*9; io° C. At signal same withBai .

FIG. 13. Heart of Palinurus. Perfused 27 min. Perfusion fluid—Na 100 : Ca 5; pH yz; n ° C. Atsignal changed to pH. 5*0.

FIG. 14. Heart of Palinurus. Perfused 47 min. Perfusion fluid—Na 100 : Ca 5; pH 7-2; n ° C.At signal changed to pH 8-o.

FIG. 15. Heart of Octopus. Perfused 8 min. Perfusion fluid—Na 100 : Ca8; pH6-g; n ° C . Atsignal changed to same with Mg 2-5.

FIG. 16. Heart of Octopus. Perfused 7 min. Perfusion fluid—Na 100 : Ca 8; pH 6-9; n ° C. Atsignal changed to same with Mg 5.

FIG. 17. Heart of Octopus. Perfused 5 min. Perfusion fluid—Na 100 : Ca 8 : Mgz;pH. 6-9; 120 C.At signal changed to Na 100 : Mg 2.

FIG. 18. Heart of Octopus. Perfused 55 min. Perfusion fluid—Na 100 : Cay-s; pH 6-9; 10-5° C.At signal changed to Na 100 : Ca 20.

FIG. 19. Heart of Octopus. Perfused 27 min. Perfusion fluid—Na 100 : Ca8*5; pH 6-9; io° C.At signal changed to same with K 0-5.

FIG. 20. Heart of Octopus. Perfused 5 min. Temporary contracture occurring at beginning ofperfusion. Perfusion fluid—Na 100 : Ca 8 : Mg 1; pH 7-3; io° C.

FIG. 21. Heart of Octopus. Perfused 55 min. Perfusion fluid—Na 100 : Ca 8 : Mg 1; pH 7-3;io° C. At signal changed to same with Sr 15.

FIG. 22. Heart of Octopus. Perfused 10 min. Perfusion fluid—Na 100 : Ca 8 : Mg 1; pH 7-3;I I ° C. At signal changed to same with Ba 1.

FIG. 23. Heart of Octopus. Perfused 20 min. Perfusion fluid—Na 100 : Ca 8 : Mg 1; pH 7*3;io° C. At signal changed to pH 8-4.

FIG. 24. Heart of Octopus. Perfused 30 min. Perfusion fluid—Na 100 : Ca 8 : Mg 1; pH 7-3;io° C. At signal changed to pH 5-0.

REFERENCES.FREDERICQ, H. (1914). Arch. Int. Physiol. 14, 126.FREDERICQ, L. (1922). Ibid. 19, 309.FRY (1909). Journ. Physiol. 39, 184.HOGBEN, L. T. (1925). Quart. Journ. Exp. Physiol. 15, 263.MINES, G. (1911). Journ. Physiol. 42, 251.

(1912). Ibid. 42, 467.WELLS, G. P. (1928). Brit. Journ. Exp. Biol. 5, 258.