The Japanese Journal of Physiology

18,pp.570-575,1968

ELECTRICAL MANIFESTATION OF CALCIUM

RIGOR OF FROG'S HEART

Yasu-Ichiro FUKUDA

Department of Physiology,Chiba UniversitySchool of Medicine,Chiba

Since the time of Sydney Ringer it has been well established that the Caions in the RINGER'S solution are necessary for cardiac beats of the frog's

heart and their increase strengthens the contracting force and ultimately

induces a ventricular contracture.Today the significance of Ca ions hasbeen evaluated in the excitation-contraction coupling mechanism and the

entrance of Ca ions into the cardiac muscle following excitation(depolarization)

has been proved to be necessary for the initiation of contraction.The con-

tractile mechanism can be activated simply by increasing the intracellular Ca

ion concentration1).Therefore it would be expected that an extremely high

extracellular Ca concentration might allow sufficient Ca ions to diffuse into

the heart to induce a contracture.The designation of"Ca contracture"or"Ca rigor"would indicate a state of sustained contraction without electrical

activities.We have been unable to find any complete reports of the electrical

manifestation during the Ca rigor of the frog's heart other than a statement

by RODECK2)that an extreme increase in Ca ion concentration(Ca/K=40-50)in the bathing fluid induces an immediate shortening of QT duration and

monophasic deformation of the ventricular complex and that after the ap-

pearance of irreversible contracture no electrical activity can be induced evenby strong stimulation.His experiments,however,were done under the specialcondition of a constant rhythmical stimulation of the auricle in order to

minimize the effect of changes in the cardiac rate upon the QT duration.

The present report is an attempt to clarify the electrical manifestation in

the Ca rigor under more physiological conditions.Together with this actual

increase in the Ca content of the heart muscle in the Ca rigor was determined

and the mechanism by which K ions in the medium counteract the Ca rigor

was examined.

After finishing this manuscript the recent paper by HAUCK and JACOB8)

(1962)came to our attention.They showed that the Ca contracture consistedof a short-lived ventricular flutter and true contracture without electrical

Received for publication December 24,1967

福田康一郎

570

CALCIUM RIGOR 571

manifestation.However,their experiments were done in winter at low experi-

mental temperatures(18-20•Ž).Under these condition the Ca contracture was

difficult to induce unless extremely high Ca concentration were used.Thus

results cannot be compared with that herein obtained on summer frogs.

METHODS

Experiments reported here were done on hearts of Rana catesbiana and Bufo

vulgaris during the summer months at room temperatures(25-30•Ž).The Yagi-

Hartung's perfusion method was used and the circulating Ringer's solution was continu-

ously aerated in order to supply oxygen.The millimolar concentrations of materials in

the Ringer's solution were as follows:NaCl 111,KC1 2.0,CaCl2 1.0,NaHCO3 2.5,NaH2PO4

0.1 and glucose 6.0mM/L.The ECG was recorded with electrodes at the apex and

auricle.

Analysis of the ventricular Ca and K content were made flamephotometrically3).

Excised ventricular strips were vigorously washed in a cold isotonic saline solution for

30 seconds and weighed after blotting with filter paper.As the mineral contents

showed variations according to the state of frogs,comparisons were made on the same

animal groups captured simultaneously and kept similarly.With this precaution in-

dividual variations were sufficiently reduced.However,such procedures could be made

only with Rana catesbiana.Therefore,the chemical analysis were made exclusively

on them.

RESULTS

The wax and wane and the electrical manifestations of the Ca rigor.As

shown in FIG.1,the Ca rigor induced by adding isotonic CaCl2 solution tothe circulating RINGER'S solution was not an irreversible one.It showed

waxing and waning,interrupted regularly by a period of complete relaxation.T irin o- the extreme contracture no visible movement indicating flutter or

fibrillation was noticed on the surface of the ventricle.The auricle continued

to beat normally throughout the whole period.

FIG.1. Wax and wane of the calcium rigor.

The Ca concentration was raised up to about 4.0mM/L by

adding isotonic CaCl2 solution to the perf using Ringer's.

Further elevation of Ca concentration did not materially

influence the course.Summer frog:28•Ž.

572 Y.FUKUDA

The ECG showed fairly regular oscillating waves of large amplitude,with

no isoelectric interval,and with a rapid rate between 500 and 1000 beats per

minute(FIG.2.).The onset of the rigor was characterized by the appearance

of small electrical oscillations following the ventricular complex,then the

contraction height was enormously increased.The small electrical oscillations

grew rapidly into the large ones with which the rigor was set off(FIG.2A).After the rigor was started for the first time,often an incomplete relaxation

of short durations was repeated.Thereby the electrical oscillation suddenly

disappeared and the normal sequence of ECG resumed.However,after a few

cycles it reverted to oscillating waves again(FIG.2B),as if the conditions

necessary for the continuation of the rigor oscillation seemed to be not yetsufficiently provided.During the long continued waning of the rigor the

circumstances were quite different.This was conditioned by cessation of

conduction of auricular excitation.As shown in FIG.2 C only the auricular

waves were noticed.The reappearance of the rigor was assured by the recovery

of A-V conduction and the oscillation was started following the auricular

wave at the time of PQ interval.Thus it was clear that the rigor oscillationof the ventricle was not due to the ventricular automaticity,as is the case with

ventricular flutter or fibrillation.It was conditioned by the auricular excitations

conducted to the ventricle.

Bubbling with pure oxygen instead of air made the contracture period

longer and the interposed relaxation period shorter.The reversed effects

FIG.2. ECG changes in the calcium rigor.

A:At the onset of the rigor.B:During the temporary,

incomplete relaxation.C:Reappearance of the rigor after

long continued waning of the rigor.

CALCIUM RIGOR 573

TABLE 1.

Concentrations of Ca and K in ventricles in calcium rigor.

Each value is the mean of 6-7 measurements with+S.E.

Excess Ca:The normal concentration of Ca in Ringer's(1mM/L)was raised upto about 7.5mM/L by adding isotonic CaCl2 solution.Experiments in room temper-ature(25-30℃).*At 8℃.

appeared when oxygenation was stopped.The A-V block easily appeared

and the ventricle was relaxed in complete arrest.Thus it became clear that

for the maintenance of the Ca rigor the conduction of auricular excitation to

the ventricle is necessary and the waxing and waning of the rigor results

from insufficient oxygen supply during the rigor state to which the A-V con-

duction is most susceptible.

Calcium content of the ventricle in the rigor state.It is well established

that the inotropic effect of Ca ions can be antagonized by K ions.The in-

duction of the Ca rigor became difficult with increasing concentration of K

ions in the RINGER'S solution(here 4 times the normal).Moreover,the Ca

rigor became difficult to induce at low temperature(lower than 13•Ž).These

circumstances were examined in relation to alterations of the Ca and K con-

tent of the ventricle.In TABLE 1 the analytical data are presented which

were obtained from ventricles dissected from the preparations 3 minutes of ter

raising the Ca concentration of the perf using RINGER'S up to 7.5mM/L by drop-

ping isotonic CaCl2 solution in it for 12 minutes.In case of the normal RINGER'S

in which the Ca rigor was started the Ca content was raised to 2 times the

control value.This rise in Ca content was prevented,together with the rigor,

in cases of experiment at 8•Ž or with RINGER'S solution containing 4 times

the normal K.A marked rigor appeared only when the Ca content was

raised to above 10mM/Kg.

DISCUSSION

It was shown that an increase of ventricular Ca content beyond a certain

limit is a necessary condition.for the induction of the ventricular rigor ac-

companied by the characteristic electrical oscillations.The environmental

temperature and the K concentration of the medium were found to have

marked influences upon the entrance of Ca into the ventricle,It was re-

574 Y.FUKUDA

markable that high Ca content of the ventricle itself does not induce the rigor

mechanism,unless the auricular excitation is conducted regularly.The most

likely explanation is that the increased cellular Ca is stored in an inactive

form in some cellular structures,for example,in the endoplasmic reticulum.

Repeated releases of this stored Ca by the regular ventricular excitations

would keep the intracellular Ca ion concentration sufficiently high to induce

the persistent contraction.

It is beyond the scope of this paper to speculate the mechanism of the

rapid electrical oscillations designated as the"rigor oscillation".As was

noticed,it was not due to the appearance of ventricular automaticity.The

conduction of the auricular excitation to the ventricle was necessary for its

establishment.Thus it differed from the ventricular flutter or fibrillation.

Whether the " rigor oscillation"was the cause of the mechanical rigor or

merely the result of it was not clear.In this respect rapid successions of

membrane depolarization and repolarization would be difficult to reconcile with

the well established fact that a high external Ca ion concentration tends to

stabilize the cell membrane".The phase theory of bioelectricity5) should also

be considered.

It is well known that a lack of extracellular K,like an excess of Ca,

increases the force of contraction.THOMAS')has shown that amount of

labeled Ca,taken up by the ventricle perf used with K-free RINGER'S,increases

at a linear rate over a 60minutes period to twice the normal value coinciding

with the gradual development of contractures and coinciding with a cellular

K loss and Na gain.In our experiences the K-free contracture induced by

continuous perfusion of the heart preparation with K-free RINGER'S was a

temporary phenomena succeeded by permanent relaxation.However,during

the contracture the characteristic"rigor oscillation"was also observed,indi-

cating the similarity with the Ca rigor here mentioned.The so-called K-

contracture which is induced by a sudden increase in K ion concentration in

the medium,although for its induction the presence of Ca ions in the medium

is known to be necessary",was found to be of a quite different nature

from the Ca rigor.The ECG showed a mere A-V block.

The experiments reported here were performed during the summer on

freshly captured animals.On frogs or toads stocked outdoors in the autumn

and winter the inotropic effect of Ca became markedly reduced and Ca rigor

was not induced by raising the Ca concentration which does not markedly

reduce the Na concentration of perfusing fluid.Also the K-lack contracture

was not induced.The membrane permeability of winter frogs seemed to be

reduced even in high experimental temperatures.The mechanism of this

pronounced seasonal variation is now under investigations.

CALCIUM RIGOR 575

SUMMARY

The well documented Ca contracture or Ca rigor of the frog's ventricledue to excess Ca in the medium was found to be accompanied by rapid regu-

lar electrical oscillations("rigor oscillation").Not only an increase in the

Ca content of the ventricle beyond a certain limit,but also the excitationconducted from the auricle were found necessary for the induction of the

rigor.The rigor was interrupted by the A-V block due to hypoxia during

the state of rigor,and reappeared after recovery of the conduction.

The K in the medium antagonized the rigor by inhibiting the increase in

the cellular Ca content.

The auther wishes to express his cordial thanks to Prof.Tokuro FUKUDA for hisadvice and criticism in carrying out the present experiments and for preparing themanuscript.

REFERENCES

1) NIEDERGERKE, R.(1955). Local muscular shortening by intracellulary appliedcalcium. J. Physiol., 128:12.

2) RODECK, H.(1947). Uber die Wirkung des Calciums auf den Aktionsstrom desKaltbluterherzens. Pflugers Arch., 249:470.

3) GEYER, R. P. AND BOWIE, E. J.(1961). The direct microdetermination of tissuecalcium by flame photometry. Analytical Biochem., 2:360.

4) WEIDMANN, S.(1955). Effect of calcium ions and local anaesthesia on electrical

properties of Purkinje fibres. J. Physiol., 129:568.5) TROSHIN, A. S.(1966). Problems of Cell Permeability. Pergamon Press. Oxford.6) THOMAS, L. J.(1960). Increase of labeled calcium uptake in heart muscle during

potassium lack contracture. J. Gen. Physiol., 43:1193.7) NIEDERGERKE, R. (1956). The potassium chloride contracture of the heart and its

modification by calcium. J. Physiol., 134:584.8) HAUCK, G. AND JACOB, R.(1962). Der caiciumbedingte systol.ische Herzstillstand.

Z. f. Kreislaufforschg., 51: 1184.