Trypanosoma cruzi - BMJ

British Heart Journal, 19703 32, I89.

Effect of Peruvosid (CD4I2) on excitabilityand functional refractory period of atrialand ventricular tissues in cardiomyopathycaused by Trypanosoma cruzi

Frederico Moleiro, Alfonso Anselmi, Regulo Suarez, Jose Angel Suarez,and Alberto DrayerFrom Laboratory of Experimental Cardiology, Institute of Tropical Medicine,Box 8250, Caracas, Venezuela

Attempts were made to produce myocarditis by Trypanosoma cruzi inoculation in healthy dogpuppies 6 to 8 weeks old. Significant electrocardiographic abnormalities were produced, coincidingwith interstitial inflammatory processes in the cardiac tissue and with degenerative changes in themyocardial fibres. In puppies showing these changes, profound changes in the excitability and thefunctional refractory period of the atrial and ventricular muscular tissue were observed.

The administration of Peruvosid in doses of o00240 to o-o647 mg./kg. tended to diminish theexcitability, previously increased by the inflammatory process, at the same time increasing thefunctional refractory period duration which had previously been shortened. The fact that Peruvosidcorrects these fundamental factors in the genesis of cardiac arrhythmias suggests that the drugmay be useful in the treatment of cardiac insufficiency produced by Chagas' myocardiopathy, inwhich arrhythmias are one of the basic characteristics.

Chronic inflammatory processes in the myo-cardium, caused by natural or experimentalinfections induced by Trypanosoma cruzi, pro-duce important changes in cardiac rhythm;it is this that provoked from Carlos Chagas(I928) the statement that 'arrhythmia con-stitutes the most important symptom at thisstage of the illness'.

Chagas and Villela (I922) related arrhyth-mias to changes in the main functions of thecardiac muscle; they considered that extra-systoles were due to changes in the excita-bility, an opinion also held by Cossio (I943).

Recent studies (Pifano et al., I962; Rod-riguez, I963) have shown that the smallerinterval between two propagated responses,the functional refractory period (Rosenblueth,Alanis, and Mandoki, I949), is shortened andthe velocity of propagation of the impulse isdiminished in the atrial and ventricular tissuein Chagas' myocarditis. The change occurringin these properties of the myocardium isgreater during the chronic than during theacute phase of the illness, and explains atrialand ventricular extrasystole, atrial flutter andfibrillation, and sudden death from ventricular

Received 23 June 1969.

fibrillation, which were first reported byChagas and Villela (I922).Treatment of cardiac arrhythmias in

Chagas' chronic myocarditis is still a problem.Preparations available to date have not pro-duced encouraging results. This paper exam-ines the effect of a new glucoside preparation(Peruvosid) on some of the fundamentalelectrophysiological properties of the myo-cardium of the dog, in which an inflammatoryprocess had been induced previously by theinoculation of T. cruzi.

Materials and methodsForty-nine healthy puppies, between 6 and 8weeks of age, were subjected to clinical and elec-trocardiographic tests before experimental inocu-lation with T. cruzi. The inoculations were madesubcutaneously and intraperitoneally with the'Bertoldo strain' of T. cruzi, maintained in highlyhomozygotic Carworth FarmsWebster white mice.The mice were initially obtained from an inbredstock developed by Carworth farms from theWebster-Rockefeller Swiss mouse strain (Law,1948). Each puppy was inoculated with a I1/2 ml.solution consisting of i ml. mouse blood in 3 ml.sodium citrate 3-8 per cent. The blood used forpreparing the inoculation showed approximately20 parasites per field. Six puppies died, with a

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190 Moleiro, Anselmi, Sudrez, Sudrez, and Drayer

picture of paralysis of the hind-quarters; in IO,no electrocardiographic changes were observed ina series of tests made after the infection, andhistological examination showed no changes inthe myocardium. Eight puppies died immediatelyafter anaesthesia, owing to profound changes inthe myocardium.

Artificial respiration was applied through atracheal cannula with an aspiring-impelling pumpof the Harvard type, for recording the intensity-duration curves and the functional refractoryperiod of atrial and ventricular tissue. The heartwas exposed by mid-section of the sternum.Pentobarbitone sodium was used for the anaes-thesia in doses of 35 mg./kg. An electroencephalo-graph, Schwarzer model E-5o2 with six channels,was used as the recording apparatus. Rectangularpulses of variable intensity and duration, gener-ated by Grass stimulators model S4E, were usedfor stimulating the tissues. The drug was adminis-tered with a Harvard pump, model 6o0-goo,through one of the saphenous veins, in a solutionof o-6 mg. of Peruvosid dissolved in 50 ml. of 5per cent glucose solution, at an infusion speed of0382 ml./minute.The histological examination of the heart was

carried out by the usual technique: fixation informol IO per cent; embedding in paraffin; sectionsof 5-7 ,u. The pigments used were haematoxylin-eosin, PAS, Gomori's trichrome, and Wilder'sreticulin.

Volt100 l

50\

20

l0-

5 -

2

0-5

0 2- CONTROL

002OO5E062 0.5 2 S 10 20 0 10(msec.

FIG. i The average values (considered to benormal in this study) of the atrial and ventri-cular intensity-duration curves from I2 healthydogs.

ResultsOf the 25 dogs infected with T. cruzi, 7were used to study the ventricular functionalrefractory period and atrial functional refrac-tory period, 3 for atrial excitability, and 4 forventricular excitability. We were unable toelicit responses to strong stimulation in 6dogs; this was explained by the conspicuoushistological alterations in the atrial and ven-tricular myocardium.

Atrial and ventricular excitability In-tensity-duration curves were plotted using acartesian co-ordinates system in which fixedvoltages were represented on the ordinate,and the required time to obtain a response forthat given voltage was represented on theabscissa. The minimum voltage necessary toelicit a response with infinite time is therheobase.With the object of comparing the changes

produced by T. cruzi cardiomyopathy in atrialand ventricular excitability, the intensity-duration curves were studied in I2 healthydogs; the averages, which are normal values,are shown in Fig. i.

Fig. 2 shows the curve of a dog (LCH4D4)with Chagas' infection of eight weeks' dura-tion. Electrocardiograms during the workingperiod showed, in comparison with the con-

FIG. 2 Dog LCH4D4 with 8 weeks of Chagas'infection. The electrocardiogram after infectionshows a complete right bundle-branch block,conspicuous left axis deviation of the QRS,and primary changes in the T wave. In the leftatrium the control intensity-duration curveshows, on the ordinate, an important reductionof the voltage intensity to stimulation of shortduration. These values go up after administra-tion of CD412.

CONTROL ECG Bweeks after infection

_k_;pi P4 VPE

Volt100.

50120.10.

21

05

02

X " F P3Pl2S4

LCH4D4Atrium

-O0O2'0<0502 05 1 2 5 1020 0130Imsec.



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Effect of Peruvosid (CD412) in cardiomyopathy i9i

FIG. 3 (A) Dog LCH4D4: Infiltration with monocytes. (H. and E. x 176.) (B) Dog LCH4D4:Extensive infiltration with monocytes that dissociate the fans of cardiac muscular fibres. Someof the fibres show moderate degenerative changes. (H. and E. x 305.) (C) Dog LCH4D5:Cross-section of muscular fibres showing smallfoci of monocyte infiltration, slight oedema,and atrophy of some muscular fibres. (H. and E. x 305.) (D) Dog LCH4D5: Conspicuousinfiltration of monocytes with necrotic areas in some fibres. (H. and E. x 305.)

trol, a complete right bundle-branch blockwith an accentuated deviation of the electricalaxis towards the left and primary changes inthe T wave in the left praecordial leads. Thesechanges pointed to histological changes of thekind seen in Chagas' myocarditis (Anselmiet al., I964). Histological examination showed(Fig. 3A and B) a dense and diffuse infiltrationof monocytic cells at the atrial and ventricularlevel, and slight degenerative changes of myo-cardial fibres. The control curve of atrial in-tensity-duration shows variations, as com-pared to normal dogs. In fact, it was seen thatthough the rheobase remained normal, i.e.for long-duration stimuli the required voltagewas the same as in normal dogs (Fig. i),for short-duration stimuli the voltage requiredto elicit a response was less than that in nor-mal dogs (I5 V per oo2o msec.). An infusionof o-o96 and 0-0252 mg. Peruvosid increased

the values of the rheobase and of the voltagerequired to produce responses with shortduration stimuli.

Fig. 4 shows the atrial intensity-durationcurves of dog LCH4D5, obtained 8 weeksafter infection with T. cruzi. The electro-cardiographic abnormalities, suggesting myo-carditis, consisted of complete right bundle-branch block, with accentuated deviation ofthe electrical axis towards the left (Laranja,Pellegrino, and Dias, I949; Pellegrino, 1946);the morphology of the right ventricle in allthe praecordials suggested dilatation of thesecavities (Fig. 5). Histological examination(Fig. 3C and D) showed dense and diffuselymphoplasmohistiocytic infiltrations and de-generative processes in myocardial fibres.The control curve of the intensity-duration

shows important changes, consistent with theaccentuated decrease of the rheobase, as well

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192 Moleiro, Anselmi, Suarez, Sudrez, and Drayer

Volt100

50

20

l0

0502-

l

0-1-\0 05 LCH4D4

Atrium0-02-i ~~~~~~~~~~~CONTROL

0,60-2 0-02 05 2 5 10 20 50Omsec.

FIG. 4 The atrial intensity-duration curve ofdog LCH4D5. The control curve shows a dis-tortion and an important reduction of the rheo-base and the voltage intensity for stimulationof short duration. The administration of Peru-vosid resulted in a normal trace.

FIG. 5 The changes in dog LCH4D5. Theelectrocardiogram 8 weeks after inoculationwith T. cruzi shows complete right bundle-branch block with conspicuous left axis devia-tion of the QRS. There is a right ventricularpattern in all the chest leads. An importantreduction in voltage intensity is seen as a re-sponse to short duration stimulation in theventricle intensity-duration curve. An increasein this value is seen after the administration ofCD412.

)

CONTROL ECG 8w5e after infection

LCH4D5 0-144 mg.

VentricleNTO

0324mq.

001002 cCC6 0i 02 06 i 2 5 10 20 50 100msec.

as the reduction of the short stimulus inten-sity (io V for stimuli of 0-02 msec.). A doseof o-o96 mg. CD412 corrects the atrial excita-bility curve, increasing the values of the rheo-base and the voltage required for obtainingshort duration stimuli. A lethal dose of o-42omg. does not modify significantly the resultsobtained by the previously mentioned dose.

Fig. 5 shows the ventricular intensity-dura-tion curve after the appearance of the electro-cardiographic changes described above. Thereis an important reduction in the intensity ofthe short-term stimuli voltage (I5 V per sti-mulus of o0o2 msec.), a dose of oi44 mg.CD4I2 diminishing the ventricular excitabilityand a lethal dose of o0324 mg. producing nosignificant changes.

Atrial and ventricular functional refrac-tory period The functional refractoryperiod was obtained with the method de-scribed by Mendez and Mendez (I953).

Fig. 6 shows the changes in the absolutefunctional refractory period in atrial (con-tinuous line) and ventricular (dashed line)tissue with increased doses of Peruvosid.The study of atrial and ventricular func-

tional refractory period presented difficultiesbecause of the profound histological changesin the tissues, the dog having died after thefirst control curves were completed. Fig. 7shows the result of the study of the atrialfunctional refractory period in a dog, histo-logical examination of which showed smallchanges consisting of inflammatory infiltra-tions of slight density and diffusion. The con-

FIG. 6 Absolute functional refractory periodin the ventricular tissue of 7 dogs andabsolute functional refractory period in theatrial tissue of 5 dogs.

300250

240 ---- X230- / - - R

22--

1200 - 'p"' f

I90

160 -----Absolute FRP in ventriculartissue of 7 dogsISO -Absolute ~~~~FRP in atrial

140-tissue of 5 dogs

0.01 0-02 0-03 004 0-05 0.06 0-07

Peruvosid (mg./kg.)

VoltC00

50

20

10

5

2-

0.5

02



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Effect of Peruvosid (CD412) in cardiomyopathy I93

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240

200

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DiscussionInfection of puppies with a highly virulentstrain of T. cruzi produces violent reactionson a level with the heart, a fact that has beenfully confirmed by various investigators(Pifano et al., I962; Taquini, 194I; Anselmiet al., I967). Recent studies have shown that

O312mq. a considerable reduction in the atrial and ven-tricular functional refractory period in thesemuscles is caused by Chagas' myocarditis

0240mg. LCHlD(Pifano et al., I962; Rodriguez, I963; Rodri-

*084m. 1 guez et al., I964). Atrial and ventricular con-*120mq. duction decreases in its transmission velocityONTROL when the impulse passes through tissues60 240 280320..... 360 W 4S2 showing inflammatory processes and degener-

msec. ate myocardial tissues (Rodriguez andAnselmi, I963; Anselmi et al., I965). The

7 The changes in atrial functional re- change in these two fundamental propertiesry pertod curve after progressive ad- explains, according to Wiener and Rosen-ration of Peruvosid. There is a linear blueth (1946), the high frequency of atrialon between drug dosage and increase in fibrillation in this type of cardiomyopathyional refractory period. (Rosenbaum and Alvarez, I953; Tejada and

Castro, 1958).curve is superimposed on the normal Doses of 0o0470 mg./kg. of Peruvosid pro-s. An infusion of Peruvosid produced a duced considerable increases in the values of

*essive increase in tissue functional re- atrial functional refractory period. In the ven-ry period. tricle, the increase in the functional refractory

8 shows the functional refractory period was produced with doses of 00240d curves of the same animal, obtained mg./kg. The effect of the drug, in increasingventricular muscular tissue. The control the duration of the functional refractoryvcan be considered as being within nor- period, consists in correcting a most impor-mits, as it was recently observed that a tant factor which, according to the circulariening of the functional refractory movement theory, maintains atrial flutter andd coincided with the infusion of cD4t2o fibrillation. The effect ofCD4I2, in increasingd the functional refractory period of the atrial

and ventricular muscles in myocarditis in-8 The ventricular functional refractory duced by T. cruzi, is similar to the effect re-Iof dog LCH1Dj and the changes with ported by Mendez and Mendez (I953) inessive doses of Peruvosid. There is a dogs with cardiac insufficiency produced byrelation between the amount of drug and digitoxin and ouabain. In this connexion, weion of the functional refractory period. must note that of the 12 puppies with myo-

carditis in which the functional refractoryperiod was studied, i i showed global dilata-tion of all the cavities and clinical signs ofheart failure, all showed dense and extensive

0°P inflammatory infiltrations, and only in onecase (LCH1Dl) were the inflammatory pro-cesses mild and the cardiac dilatation slight.The atrial and ventricular intensity-dura-

,/4>O, /\NvU tion curve showed significant modifications inmyocarditis induced by T. cruzi, as much inthe rheobase values and in the intensity ofstimuli of short duration as in the distortionsof the shape of the curve itself. Comparing theintensity-duration curves of normal dogs with

LCH1 Di those of dogs with electrocardiographic andhistological changes compatible with Chagas'myocarditis, we found that in the latter the

200 240 280 320 3f, s,Is voltage and time required to elicit a responsemsec. was less than in the former. These changes



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194 Moleiro, Anselmi, Sudrez, Sudrez, and Drayer

show that the dogs with Chagas' myocarditishave an increased excitability in both atrial andventricular tissue. This fact might be the causeof the arrhythmias so often found in thisdisease. The 7 puppies in which excitabilitywas studied showed that there was a close con-nexion between the changes in the intensity-duration curves and the histological changesproduced by the inflammation. Doses ofO0OI47 mg./kg. of Peruvosid produced an in-crease in the values of the rheobase, and dosesof o-o647 mg. raised the threshold of short-duration stimuli, making the values of theintensity-duration curve approximate to thenormal.

ReferencesAnselmi, A., Gurdiel, 0., Suarez, J. A., and Anselmi,

G. (x967). Disturbances in the A-V conductionsystem in Chagas' myocarditis in the dog. Circula-tion Research, 20, 56.

-, Pifano, F., Suirez, J. A., and Dominguez, A.(I964). Experimental Chagas myocarditis. VII.Interamerican Congress of Cardiology, Montreal.Abstracts, p. io5.-,-, -, -, Diaz Vizquez, A., and An-selmi, G. (I965). Experimental Schizotrypanumcruzi myocarditis. American Heart3Journal, 70, 638.

Chagas, C. (1928). Sur les alterations du coeur dans latrypanosomiase americaine (maladie de Chagas).Archives des Maladies du Coeur et des Vaisseaux,21, 641.

-, and Villela, E. (1922). Forma cardiaca da Try-panosomiase Americana. Memdrias do InstitutoOswaldo Cruz, 14, 5.

Cossio, F. (1943). Estado actual de neustros conocimi-entos sobre la miocarditis chagasica. RevistaMedica del Norte (Tucumdn), 7, II.

Laranja, F. S., Pellegrino, J., and Dias, E. (I949).Experimental Chaps' heart disease. AmericanHeart_Journal, 37, 646.

Law, L. W. (I948). Mouse genetics news. Number 2.Journal of Heredity, 39, 300.

Mendez, R., and Mendez, C. (1953). The action ofcardiac glucosides on the refractory period of hearttissues. Journal of Pharmacology and ExperimentalTherapeutics, 107, 24.

Pellegrino, J. (1946). 0 eletrocardiograma na fasecr6nica da doenp de Chagas experimental no cao.Memorias do Instituto Oswaldo Cruz, 44, 6I5.

Pifano, F., Anselmi, A., Alemin, C., Suarez, J. A., andDiaz Vazquez, A. (1962). Miocardiopatia chagasicaexperimental. Archivos Venezolanos de MedicinaTropical y Parasitologia, 4, 37.

Rodriguez, M. I. (i963). Realizaciones experimentalesde los principales tipos de trastorno de la conduc-cion intracardiaca. In Memorias del IV CongresoMundial de Cardiologfa, Mexico, I962, Vol. 2, p.I72.

-, and Anselmi, A. (1963). Cardiac conduction dis-turbances in experimental Chagas myocarditis.Federation Proceedings, 22, 346.

- , -, Gutierrez, R., and Calder6n, R. (I964).The functional refractory period of cardiac tissuesin experimental Chagas myocarditis. VII. Inter-american Congress of Cardiology, Montreal.Abstracts, p. I85.

Rosenbaum, M. B., and Alvarez, A. J. (I953). Elbloqueo de rama derecha en la miocarditis cr6nicachagasica. I Conferencia Nacional sobre enferme-dad de Chaps, p. I05, Buenos Aires.

Rosenblueth, A., Alanis, J., and Mandoki, J. J. (i949).The functional refractory period of axons. journalof Cellular and Comparative Physiology, 33, 405.

Taquini, A. C. (i94i). El electrocardiograma en laenfermedad de Chagas experimental. RevistaArgentina de Cardiologia, 8, II5.

Tejada, C. V., and Castro, F. (1958). Miocarditiscr6nica en Guatemala. Revista del Colegio Midicode Gautemala, 9, 63.

Wiener, N., and Rosenblueth, A. (I946). The mathe-matical formulation of the problem of conductionof impulses in a network of connected excitableelements, specifically in cardiac muscle. Archivosdel Instituto de Cardiologia de Mbxico, x6, 205.



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