686

Conduction Defects and Arrhythmias in Chagas' Disease:Possible Role of Gap Junctions and Humoral Mechanisms

ANTONIO C. CAMPOS DE CARVALHO.*** MASAKI O. MASUDA,*HERBERT B. TANOWITZ/^ MURRAY WITTNER,^

REGINA CS. GOLDENBERG/i^ and DAVID C. SPRAY='= ^^-

From the *lnstituto de Biofisica Carlos Chagas Filho. Universidade Federal do Rio de Janeiro. Brazil: and the Departmentsof **Neuroscience. tMedicine. and ^Pathology. Albert Einstein College of Medicine, Bronx. New York

Gap Junctions and Humoral Factors in Chagas' Disease. The protozoan parasiteTrypanosoma critzi causes ChuKas' disease, a major cause of cardiac dysfunction in LatinAmericans. Chagas' disease exhibits both acute and chronic phases, and each may be charac-terized by cardiac conduction disturbances. In acutely infected cultures of rodent heart cells,synchronized spontancctus beating becomes less regular, and coupling between cells is reduced.The basis of tbis decreased conduction is apparently in localization of tbe gap junction protein(Cx43) inside infected cells. Altbough total Cx43 is normal in infected cells, little is recognizableat appositional membranes. Electrophysiological properties are also altered by tbis infection.Action potentials are shortened, resting Ca-* levels are elevated, and response to (i -adrenergicagonists was altered, compared to controls. Humoral factors may contribute to tbe conductiondefects in chronic Chagas* disease. Sera from chronically infected rabbits produced KC(; abnor-malities in I.angendortl-perfused rabbit hearts. Tbese findings indicate tbat chagasic infection maymodify ion channel function in the heart, and we suggest that these cbanges may be manifested inthe conduction disturbances that characterize this disease. fJ Cardiovasc Electrophysiol Vol. 5. pp.686-698. August J 994)

heart, parasite, connexin. Trypanosoma crtizi, immumJy

Introduction

Chagas" disease, caused by the hemoflagellateprotozoati parasite., Trypanosoimi cmzi, is otie ofthe leading causes of heart disease in South Amer-icans. Approximately 25% of the population is atrisk of contractitig the disease (altnost 90 millionpeople) and more than 4% are actually infected(16 million people) in Latin American countries.'In endemic areas the insect vectof, a reduviid or

The work presented was supported hy grams from the NationalResearch Council (CNPq-Brasil) and FINEP K) ACC and MOM:by NIH grants AI12770 and H1.38449 to DCS. HBT. and MW:and the Beatrice F. Parvin Grant-in-Aid Award rn>iTi the NewYork Chapter of the American Heart Association to DCS.

Address tor correspondence: Antonio C. Campos dc Carvalhii.Instituto de Biofi'sica Carlos Chagas Filho. Universidade Federaldo Rio de Janeiro. Cidade Universitaria, llha do Fundao, Rio deJaneiro, RJ 2I94()-9(H). Brazil. Fax: 55-21-280-8193.

Manuscripi received 14 December 1993: Accepted for publication6 June 1994.

triatomine bug called "barbeiro" (barber), due toits habit of biting the uncoveied faces of sleepingpeople (or the "kissing" or "assassin" bug), sharesthe poor iTiral dwellings, usually tnud htits. withthe populaiion. Migtation of infected individualsto metropolitan areas within and outside LatinAtnetica has spread the disease to the cities thtx)ughtransfusion with infected blood. In Bolivia, forexample. 63% of bkx)d donors are seropositive forT. cruzi.^ In the Utiited States, a recent study foutidChagas' .seropositivity in 1.1% of voluntiiry bkniddonors in Los Angeles county-; thus, it is notsuiprising that there have been several reports ofttansfusion-communicated Chagas' disease.''

T. cruzi has distinct developmental stages thatare characterized by residence of the parasite indiffetent hosts and are identified moiphologicallyby the relative position of the kinetoplast in rela-tion to the cell nucleus and the emergence of theflagellutn. Trypomastigotes are the nondividingfoniis of the patasite that circulate within tlic blixxl

npos de Carvalho. et at. Gap Junctions and Humoral Factors in Chagas' Disease 687

of the mammalian host; amastigotes are the intra-cellular replicative forms of the parasite. In themid-gut of the insect vectors, trypomastigotesingested with the bkxxi meal differentiate into epi-mastigotes. There are also different strains of T.cruzi. These strains vary in the extent of parasitemiaachieved in different mammalian hosts, as wellas their virulence, pathogenicity., antigenicity, andsensitivity to pharmacologic agents. Some strainsmay be predominiintly myotropic (e.g., Colombianand Brazilian strains), while others are predomi-nantly reticulotropic (e.g., Y and Tulahuen sanins).

The disease caused by T. cruzi exhibits bothacute and chronic phases. Acute infection is asso-ciated with parasitemia and parasitism of manycell types'* and usually results in appearance ofantibody. Cardiac and skeletal muscle cells, thenervous system (specifically autonomic ganglia),and reticuloendothelial systems iire preferentiallyparasitized.^ Amastigotes often exist in so-calledpseudocysts. Although most patients with acuteinfection are asymptomatic, clinical manifestationsmay include fever, hepato- and splenomegaly,lymph node enlargement, and subcutaneous edema.Cardiac symptoms, when present in acute infec-tion, are largely those of myociirditis, with sinustachycardia or bradycardia, low QRS voltage, andfirst-degree AV blockage.*' The signs and symp-toms of acute infection are generally present forapproximately 1 to 2 months and then graduallydisappear, even in the absence of targeted thera-peutic treatment.

The chronic phase of Chagas' disease is anintriguing pathophysiological process due to thelack of obvious parasitemia and virtual absence ofobvious tissue parasitism. Moreover, latency toexpression of clinical abnormalities may beextraordinarily long. Infected patients may remainasymptomatic for years, decades, or throughouttheir lives; alternatively, they may exhibit cardiacand/or gastrointestinal manifestations, such as themega-syndromes.

Chronic Chagas' disease may present clinicallywith arrhythmias, congestive heart failure, or throm-boembolic events. Dilated cardiomyopathy is animportant manifestation that usually occurs manyyears after infection and is often associated with aleft ventriculiir apical aneurysm. Histologic exam-ination of the heart reveiUs myonecrosis. myix:y-tolysis, and contraction band necrosis. In addition,there are focal and diffuse areas of myocellularhypertrophy that may be associated with inflamma-tion. There is little tissue parasitism. Conductionpathways are damaged with resultant conduction

disturbances. Microvascular injury has also beenreported and may underlie these pathologicalchanges.

The electrophysiologic and echocardiographicalterations accompanying chronic Chagas' disea.seare well described. Right bundle branch block andleft anterior hemiblock are common ECG findings.Seropositive asymptomatic individuals may havecineographic and radionucleotide evidence of leftventricular alterations such as apical dyskinesia.

The etiology of the dilated cardiomyopathy asst>-ciated with Chagas' disease has been the subjectof many investigations. Autoimmune, neurogenic,and microvascukir etiologies have been proposedto explain its pathogenests. Autonomic abnormal-ities of parasympathetic and sympathetic functionhave been described in patients with chronic Cha-gas' disease; in this regard, selective destructionof cardiac autonomic ganglia has been proposedas one of tbe causes of cardiomyopathy. Duringthe course of acute murine T cntz.i infection, focalmicrovascular spasm and microaneurysm forma-tion have been described. The focal myocardialchanges seen in chagasic cardiomyopathy are likelydue to these discrete alterations in the coronarymicrovascular that supply small volumes of mycx:ar-dial tissue. In support of a microvascular etiologyof Chagas' disease, it was recently reported thatverapamil administration ameliorated the patho-logical and biochemicaJ alterations associated withacute and chronic murine infection. Ihus main-taining coronary microvascular pertusion.''*' Thescarcity of parasites has led to the notion thatautoimmune mechanisms are responsible for thepathology of chronic disease. Numerous reportsimplicate cellular and humoral autoimmunity inthe pathogenesis of chronic chagasic cardiomy-opathy. Recent studies suggest the involvement ofautoimmune mechanisms as one of the causes forthe observed pathology.'' " However, the linkbetween these observations and the pathogenesisof Chagas' disease remains unclear.

Even though many clinical studies have beencarried out concerning the electrocardiographicalterations present during both the acute and chronicphases of Chagas' disease, few studies have con-centrated on the electrophysiologic disturbancescaused by the presence of the parasite in myocar-dial cells. Data are also scarce and controversialregarding whether sera from chagasic :mimals orpatients might confer deleterious effects on phys-iological properties of normal cardiac tissues, suchas tbe electrogenesis and conduction of tbe cardiacaction potential. In recent years, our laboratories

688 Journal of Cardiovascular Electrophysiology Vol. 5. No. 8. Augti.st 1994

have focused on some of these topics, and. inthis review, we summarize some of the findingsthat we think are pertinent to a better understand-ing of the cellular mechanisms involved in thepathogenesis of acute and chronic phases of Cha-gas' disease.

The Biology of 7". Cruzi Infection

The interaction of T. cmz.i with mammalian cellsinvolves three distinct stages: adhesion, internal-ization., and vacuole formation. The extracellularor trypomastigote form of T. cntzi adheres to mam-malian cells via glycoconjugatesj '-^ some of whichhave been identified.'^"* The adhesion and inter-nalization steps can be dis.sociated at low temper-ature or by using drugs that interfere with thephagocytic machinery of the cell. The molecularsignals involved in the internalization stage arelargely unknown, but cyclic nucleotides andpolyamine levels may influence the process.'^-"Intemalization occurs via endixytosis within a par-asitophorous vacuole. Within this vacuole parasitesmay be killed by cytocida! mechanisms, such asthe production of hydrogen peroxide, often asso-ciated with lysosomal fusion; moreover, the para-site must escape this vacuole in order to prolifer-ate freely in the cytoplasm. This process isaccomplished by the synthesis by irypanosomes ofa hemolytic pore-forming protein, which is opti-mally active at acid pH and is capable of lysingthe parasitophorous vacuole.-'-- The number ofamastigotes within a single cell, whether result-ing from replication within the host cell or frommultiple parasitic penetrations, can be quite high.The high degree of parasitism is illustrated in Fig-ure 1, which shows the intracellular kxralization ofamastigotes within a cardiac myocyte, using theDNA stain propidium iodide. In this reconstruc-tion from serial sections obtained with a scanningconfocal microscope, parasites appeal" as uncon-nected satellites surrounding the host cell nucleus.

An Experimental Model for the Acute Phaseof Chagas' Disease

We have used primary cultures of cardiacmyocytes obtained from neonatal rats and fromembryonic mice as model systems in which tostudy the acute phase of Chagas' disease follow-ing infection by vjirious strains of T. cfuzi. Becauserodent species may differ in regard to susceptibil-ity to as well as progression of the chronic diseasestate, and because the parasite strains differ in vir-

ulence and time course of parasitemia, we hopedto obtain data that could be applicable to both piir-asites and host regarding the basis of mycx-ardialdysfunction. Cardiac myocytes in control andinfected cultures were then compared with regiu'dto differences in resting potentials, action poten-tial amplitudes and durations, spontaneous beatrates, chronotropic responses to adrenergic ago-nists, degree of dye and electrical coupling,, expres-sion of the main ciirdiac gap junction protein (con-nexin43), propagation of the cardiac impulse andconti action waves, and intracellular Ca * levels andCa-" mobilization by adrenergic agonists.

Resting and Action Potentials

Resting and action potentials were examined in3"day-old primary cultures of mouse embryonicventricular cells infected with the Y strain of T.cnizi. Conventional electrophysiologic techniqueswere used to impale either isolated cells or cellswithin clusteT-s of about ten cells from infected andage-matched, noninfectcd cultures. Figure 2 showsaction potentials obtained from control (A) andinfected cultures (B): a significant shortening ofthe action potential and an increase in spontaneousfiring rate can be observed in the infected cul-ture. Results obtained from experiments satisfyingthe criterion that impalements be suible for at least5 minutes are shown in Table 1. We observed thatunder conditions in which the parasites residedwithin the infected myocytes, the action potentialduration was significantly shorter in the infectedcultures than in controls (P < 0.05). Thisdecrease in the action potential duration cannot beattributed solely to the increase in the spontaneousfiring rate seen in infected cells (see below),since in the infected cultures the linear relationbetween beat rate and action potential duration islost. It will be particularly interesting tt) investi-gate whether the activation of Ca^ -dependent potas-sium currents are enhanced in the infected cardiacmyocytes. since it has recently been reported thatbasal cytoplasmic Ca'^ activity in the infected cellsis higher than in eontrois^^ (see below). Maximumdiastolic potential and action potential amplitudewere not significantly different between the infectedand uninfected groups of caidiac mytxytes, as illus-trated in Figure 2. Additionally, it was observedthat during this stage of infection, the propaga-tion of the depolarizing wavefront within mostclusters containing infected cells was still presei"ved,and no apparent desynchronization in contractionwas observed; all cells within a cluster beat syn-

Campos de Carvatho. el ai. Gap .lunclions and Humoral Factors in Chagas' Disuasv (589

Figure 1. Three-dimensional reamslruclion often scanning confocal l-/jm serial sfclions from cultured embryonic mousecardiac myocytes infected with the Y strain ofT. cruzi for 48 hours. Cell nuclei (N) and intracellular forms of the parasite(unuistif-otes. a few of which are indicated hy arrowheads) were labeled hy propidium iodide. Note the extreme parasitemia ofthe cell that is located at the center of the field.

chronously, a characteristic feature of normal, non-infected cultures. This finding obtained fromcells infected for 48 hours differed strikingly fromthe asynchronous contractions seen at later times(see below).

Spontaneous Beat Rates

Spontaneous beat rates were examined incultured cardiac myocytes from both rat andmouse. In mouse myocytes infected with theY strain of T. cruzi., beat rate was significantlyincreased (by 40%) 48 hours after infection(Fig. 2 and Table 1). At 24 and 72 hourspostinfection. however, beat rates were almostidentical in control and infected cultures. Theincrease in heart rate observed at 48 hours ofinfection In mouse heart cells appears to beiciated to the endoeytie activity (or conse-quences of this process) involved in parasite

intemalization, since cultures of the same agepresented comparably accelerated beat rateafter internalizing ferritin particles.-•* In ratmyocytes infected with the Tulahuen strain ofthe parasite, beat rate was significantly reduced(by > 50%. see Fig. 6) at 72 hours after infec-tion, the only time point examined in thesestudies."-^^ The opposite effects of infectionon heat rates in myocytes cultured from thesetwo murine species is puzzling and remainsunexplained, but the difference is paralleledby differences in adrenergic responses dis-cussed below. Because ehronotropic andinotropic effects of adrenergic agonists onmurine cardiac myocytes reverse from posi-tive to negative at early developmental timepoints,-'' infection-related differences obtainedin the two species might reflect different timecourses of this developmental program in theseanimals.

690 Journal of Cardiovascular Electrophysiology Vol. 5. No. 8. August 1994

B

J JFigure 2. Spontaneous action potentials (upper traces) obtained from 3-day-old cultures of embryonic mouse ventricularmyocytes. Panel A is a record obtained from a control culture, and panel B is from an age-matched culture infected with the Y.strain ofT. cruzi for 24 hours. In A the action potential duration (APD) measured at 50% amplitude was about 200 msec, andthe spontaneous firing rate was 70 beats/min. Corresponding values for the action potentials in B are about 130 msec and 150beats/min. Action potential amplitudes are slightly reduced in the infected compared to control culture.^, reflecting incompleterepolarization. Arrows indicate 0 mV level. Calibration bar in A corresponds to 20 mV. Time marks above the action poten-tials correspond to 1-second intervals.

Dye and Electrical Coupling

We have examined dye coupling in both mouseand rat cardiac myocyte culUires infected by eitherthe Y or Tulahuen strains of T cruzi. In both setsof experiments, a significant decrease in dye cou-pling was observed 72 hours after infection, andthis effect was confined to the parasitized indi-vidual cells (Fig. 3). In contrast, af 24 and 48 hoursafter infection, no significant change in dye cou-pling was detected between eontrol and infectedcultures. Cardiac myocytes in control, noninfecteddishes remained well coupled with respect to dyepassage after 72 hours in culture, as did nonpara-sitized celfs in infected cultures (Fig. 3). This findingindicates that the reduction in coupling is not dueto factors .secreted into the medium or even intothe extracellular matrix by the parasites or by the

TABLE 1Spontaneous Action Potential Characteristics in Control

and Infected Culture

ControlMeanSE(N)

InfectedMeanSE(N)

FR(beats/min)

89*9

20

t27*1013

MDP(mV)

- 5 33

17

-462

12

APA(mV)

693

20

623

12

ADP1 msec)

217*2317

141*1411

FR = Spontaneous firing rate; MDP = maximum diastolicpotential; APA = action potential amplitude: APD = actionpotential duration measured at 50% amplitude; SE = stan-dard error of the mean; N = number of cells, ""representparameters ihtit were signiUcanily different from controlvalues (/*< 0.05).

parasitized cells, but is directly associated with thepresence of the parasite inside the cell.

Electrical coupling was determined by thedual whole cell, patch clamp technique in pairsof rat myocytes 72 hours after infection. A markeddecrease in junctional conductance was observed,which again was tbtind to be confined to only thosemyocyte pairs where one or both cells of the pairwere parasitized.-'

Expression of Connexin43

Expression of connexin43 was examinedusing Northern and Western blot analyses and alsothrough indirect immunofluorescence in age-matched control and infected cultures of both ratand mouse cardiac myocytes. No change in totalconnexin43 mRNA levels was detected by North-em blots 72 hours after infection-^ (Fig. 4A). Con-nexin43 protein levels were also unaffected byinfection at that time, as shown from immunoblotsin Figure 4B. Immunofluorescence staining for thelocalization of connexin43 in the cultures, how-ever, showed a marked decrease in the expres-sion of connexin43 at the surface membrane(Fig. 5), a finding that correlated well with theobserved reduction in dye and electrical cou-pling. The disappearjince of junctional protein fromthe surface membrane without a correspondingdecrease in the total amount of connexin43 detectedby Western blot might be due to antigenic sitesbecoming "cryptic" after parasitism.-** However,we consider this possibility unlikely due toexperiments in which similar changes were seenwith antibodies raised to different connexin43 deter-

Campos de Carvalho. et al. Gap .lunction.s and Iliimonil Factors in Chagas' l>isca.sc 691

Figure 3. Reduced dye couplinii in iiifatcd cultures is not due to the release of a soiuhiv factttr into the culture mrdiiim. Dvecoupling is extensive in ceil pairs fnmi uninfected di.'ihes (A. B) and for nonparasitized cell pairs identified within infecteddi.she.s (C, D). In E atui F. the middle cell in an infected culture di.sh was injected with dye. and there was no detectablespread of Lucifer Yellow to either of the parasitized neighboring cells. Five percent fwt/vot) Lucifer Yellow was injected usingoicilUiting current through a microelectrode into the upper cell in the pairs in B and D and into the middle cell in F; all pho-tographs were taken appro.ximately I minute after injection.

niinants (DC Spray. C Roy. AC Campos de Car-valho. and EL Hertzberg. unpublished observa-tions). Thus, in response to int'cction, connexin43appe£ired to be removed from the surface and toaccumulate intraccllularly. either through mecha-nisms involving incivased remt)val, decreased inser-tion, or both. Comparison of the number ofimmunoreactive sites on the surface membrane andin intracellular compartments fuiiher indicated thatthe infection process leads to Eiiarkedly increasedintemalization of the junctional prcUein from thecell surtace (R Dermietzel. AC Campos de Car-valho. and DC Spray, unpublished observations).However, total eonnc\in43 protein legalized withinrecognizable nienihrane structures decreased withtime after infection. Since total connexin43 pro-tein as detected by Western blot did not decreaseunder this situation, the balance between the pro-tein lost ftx)m immunocyttx:hemically distinct com-

partments and that detected by Western blot pre-sumably reflects protein free within the cytoplasm.

Propagation of the Impulse and Synchronyof Contraction

To examine whether the detected changes incoupling after parasite infection could affect thepropagatitm of cardiac action potentials through-out the monolayer cultures, we used the brightnessversus time utility software of an image analysissystem (Image I AT/FL. Univei"sal Imaging, Media.PA, USA) and measured changes in transmittedlight by identifying regions of interest corrcsi^mdingto appiu.ximately I- to 4-/jni- areas of tiie cell nn.)no-layer. When cells contracted, movements of thecells resulted in changes in measured brightnessof these areas of the image. Figure 6 illustratessample records obtained in sister cultures of con-

692 Journal of Cardiovascular Eiectrophysioiogy Vol. 5. No. 8. August 1994

A B

- O

o -Figure 4. Infection does not affect connexin43 (Cx43) mRNA or protein abundance in cardiac myocytes. (A) Northern bint ofcontrol and infected cultures using a 20-mer oligonucleotide probe reveals no difference between mRNA levels for Cx43 incontrol (C lane) and infected (I lane) cultures. Lower and upper arrows indicate respective positions of lHS and 28S rRNA.(B) We.stern blot of control and infected cultures using a peptide-specific anti-C.\43 antibody for residues 346-360 of the pro-tein. Total Cx43 content of infected cultures (I lane) is not noticeably different from control cultures (C lane).

troi (Fig. 6A) and infected (Fig. 6B) rat cardiacmyocytes at 72 hours alter infection. As mentionedabove, frequency of beating was reduced in infectedcultures. Furthermore, interbeat interval was muchmore variable in infected than in control cardiacmyocytes (Fig. 6), and asynchronous contractionswere often observed.-^ These findings demonstratedthat the reduction in heart cell-lo-cell couplingobserved in the infected cells was correlated withthe impairment of normal rhythmic contractionof the cultured cells.

Responses to Adrenergic Agonists

Cultured ventricular myocytes from mouseembryonic hearts infected by the Y strain for 48hours displayed rates of spontaneous beating thatwere higher than uninfected controls. This effectof parasitism was mimicked by the addition of fer-ritin to the culture medium, suggesting that the ele-vated beat rate in infected cells might be a non-specific effect of increased endocytotic activity.Moreover, infected myocytes displayed a patternof chronotropic response to norepinephrine thatdiffered from control cultures, in 3-day-old cul-tures. 65% of the tested cells from 48-hour infectedcultures presented positive cbronotropic responses.

10% showed negative chronotropy. ajid in 10% thebeat rate did not change. In the noninfected group,86% exhibited positive chronotropic responses, 8%decreased their beat rate, and 8% did not respond.Thus, the major changes following infectionappeared to be an increase in the number of non-responding cells and a decrease in the fraction ofthe cells showing positive chronotropic responses.Furthermore, the infected mouse cardiac myocytesshowed a smaller increase in spontaneous beat ratein response to norepinephrine (concentrations rang-ing from 10"'' to lO""* M), when compared toage-matched control cultures. The presence of par-asites inside the cells (rather than increased endo-cytosis per se) appeared necessary for this effect,since cultures that had taken up ferritin particlesby endocytosis did not display altered responsesto norepinephrine. "*

Somewhat dilTerent results were reported in sim-ilar studies of neonatal rat cardiac myocytes in cul-tures infected with the Tulahuen strain. Theseinfected myocytes displayed a larger increase inspontaneous beat rate in response to 10 ^ M nor-epinephrine when compared to age-matched con-trol cultures.-" In contrast, responses to the /?-ago-nist isoproterenol were unchanged by infection,and cAMP-dependent substrates were found to be

Campos de Carvalho, et al. Gap Junctions and Humorai Factors in Ciiagas* Disease 693

Figure 5. hwnunofiuore.scence of control and infected cultures using affinity-purified antibody again.st polypeptide 346-360ofCx43. A shows the bright field and B the fiuorescent labeling obtained in control cultures. C ami D show the correspondingrecords obtained in infected culture.';. Immunolabeling localized to gap junctions is present between uninfected cells in thefield (arrows in C imd D) and is much less extensive between infected celts (amastigotes in (me cell indicated hv Tc in C andD). In contrast to results obtained with nonaffinity-purified antibody (not shown}, the ama.stigotes are not labeled by theaffinity-purified antibody. E shows the immunofiuore.scence obtained during competition of the labeling with the peptide anti-gen {note the lack of staining of either amastigotes or junctional membranes in this panel compared to panel F). F indicatesthe immunofiuorescence obtained using the preimmune serum in infected cell cultures (note that staining of the intracellularparasites is evident). Scale bar in F corresponds to 50 fim.

unaltered by infection. However, consistent withheightened a-adrenergic responses, Ca * levels werefound to be elevated in infected cells,'- and theCa-* mobilization by norepinephrine was exag-gerated. As noted above, the striking differencesreported by the two groups of investigators, bothon chronotropic and on ff-adrencKeptor mediatedresponses, may result from the use of differentstrains of the parasite, different host cells, orfrom different combinations of host cells andparasites. For example, it is known that myo-genie differentiation is not altered by the Y strain

of T. cruzi in cardiac myoblasts,-'' whereas theBrazilian strain impairs differentiation of L,,Egmyoblasts.-^" Experiments involving a collabora-tion between the two laboratories are underway toidentify the mechanisms underlying the dilTerentresponses of the different murine models.

An Experimental Model for ChronicChagas' Disease

Infected cultures of cardiac myocytes areobviously not an appropriate model in which the

694 Journal of Cardiovascuiar Electrophysiology Vol. 5. No. S, August 1994

S 120c

I ^= 4H

e15

Seconds

Figure 6. Beating rate is reduced and interbeat interval is more variable in infected (72 hours) myocardiai celts from neona-tul rat compared to controt cultures. Contraction of myocytes in various areas of the visual field were monitored as pixelintensity within specified regions of interest, using the "brightness versus time" function contained in the Image I AT/FLimage analysis package. Each panel represents the brightness over time of three areas within a single microscope field: upperand lower panels are from sister cultures. Under control conditions (A), contractions of all celts wittiin the fietd occurred syn-chronou.sly and witti a rapid heat rate. In infected cuttures (B), interbeat interval was more variatite. sp<mtaneous beat ratewas significantly slower, and contractions corresponding to extrasystolic beats (seventh beat in B) were tnore frequent.

entire repertoire of pathological changes of chronicChagas' disease may be studied, since amastigotesare rarely found in cardiac fibers during thisstage of the disease. In view of the evidence thatcellular and humoral mechanisms may be involvedin the pathogenesis of the chronic phase, we havetested the effects of serum from chronically infectedrabbits on electrophysiologic properties of wholeheart preparations perfused by the Langendorfftechnique.

Sera from five chronically infected rabbits wereperfused through isolated whole rabbit hearts whilemonitoring the ECG. Sera from three of these rab-bits reduced sinus ntxie frequency by 10% to 30%and, in many of tbe experiments, induced or poten-tiated ventricular ectopic foci. One of these .seraproduced third-degree AV block in two differentexperiments (Fig. 7) and reduced sinus frequencyby 30% in another trial (Fig. 8. upper record). Thissame reduction in frequency was obtained when

Campos de Carvalho, et al. Gap Junctions and Humoral Factors in Chagas' Disease 695

A

a J,

B

a

Figure 7. ECG recordings obtained from isolated rabbit hearts perfused by the Langendorff technique. A represents anexperiment where whole .lerum. diluted 1:100 (v:v). from a rabbit chronically infected with T. cruzi was u.sed to perfuse theisolated heart. Record a is the control record obtained during perfusion with normal Tyrode 's solution. Record b was takenduring perfusion with the chagasic serum, and the 2:1 AV blockage, evident in this sample record, persisted throughout the20 minute perfusion period. Record c was obtained after washout with normal Tyrode s solution and illustrates the totalreversibility of the serum effect. The .same type of protocol was used in the e.xperiments shown in B where serum of a normalrabbit was used to perfuse the isolated heart (record b). No effect other than a small and progressive decrease in firing ratewas detected under these conditions.

only the IgG fraction of this serum was used topertuse the heart (Fig. 8. lower record). The tworemaining chagasic sera and four control rabbitsera did not alter ECG recordings. We are nowtesting effects of IgG fractions from these sera onisolated myocytes from rabbit hearts by the wholecell patch clamp method in order to evaluatechanges in monibrane currents. Preliminary resultsindicate that both the chagasic serum thatinduced AV bUxrkade and its IgG-enriched frac-tion increase peak slow inward current and alsoincrease tbe steady-state outward current in tbe iso-lated myiK'ytes (Masuda et al.. unpublished results).We are now beginning to use the rabbit Langen-dorff preparations to screen sera frxjm chronic cha-

gasic patients in whom arrhythmias either are orare not present. We are also examining whethersuch action may involve antibodies to /3-adrener-gic or musciirinic cholinergic receptors, as has beenreported previously."'-

In conclusion, tbe experiments presented herecombine cell biological and electrophysiologicapproaches to provide novel infonnation regard-ing potential mechanisms contributing to both acuteand chronic chagasic cardiomyopathy. It hasbecome increasingly clear that cardiac conduc-tion disturbances are, to a large extent, disturbancesin intercellular communication. Thus. decrea.ses ingap junction abundance have been recently detectedat infarct border zone both acutely'^ and chroni-

696 Journal of Cardiovascular Electrophysiology Vol. 5, No. 8, August 1994

SERUM

I g G

Figure 8. ECG recordings obtained from isolated rabbit hearts under Langendorff perfusion. Upper three records (SERUM)were obtained during heart perfusion with a different serum sample obtained from the same chagasic rabbit used in theexperiment illustrated in Figure 7. Record a corresponds to the control condition, b shows the record obtained during perfu-sion with the serum (diluted 1:100: v:v). and c is the record after washout with normal Tyrode's soluticm. In this experimentno AV blockage was detected during perfusion with the serum, but a significant reduction in firing rate was observed (approx-imately 30%), which was almost totally reversed on washout. The lower three records (IgG) were obtained from an experi-ment where only the IgG-enriched fraction from the serum was used. Record a corresponds to the control condition, b wasobtained during perfusion with the IgG fraction (diluted 1:1000 in Tyrode's). and c is the record after washout. Note that theIgG-enriched fraction induced a decrease in firing rate, which is similar to that obtained after perfu.non with whole serum.Horizontal bars represent 1 second.

Furthermore, resculpturing of ventricularbundles occurs during the aging process,whereby collagen deposition physically divides thetissue into meandering conduction pathways. ^ Thestudies reviewed in this article indicate that T. cruziinfection of cardiac myocytes results in profoundchanges in gap junction distribution between thecells. Infected cultures of neonatal rodent cardiacmyocytes exhibit changes in gap junction distrib-ution, which are consistent with decreased cou-pling between the cells and with asynchronous andtemporally variable contractions throughout the cellmonolayer. Moreover, signal transduction pathways

are selectively altered in infected cells, as are rest-ing Ca-+ levels and the potential for mobilizationof intracellular Ca'* by a-adrenergic agonists.Finally, exposure of adult rabbit hearts to sera fixjmchronic chagasic rabbits resulted in acute abnor-malities in conduction, pRwiding yet another mech-anism that may contribute to altered cardiacfunction associated with chronic Chagas' disease.Although these studies on rodent models provideinfonnation regarding the effects of infection oncardiac cells in vitro, it should be noted that tbedegree to which they can be extrapolated to thehuman disease state remains to be detennined.

Campos de Carvalho, et al. Gap Junctions and Humoral Factors in Chagas' Disease 697

References

1. World Health Organization: TDR News 1992:40:3-5.2. Kerndt PR. Waskin HA. Kirchhotf LV, et al: Preva-

lence of antibody to Trypanosoma cruzi among blooddonors in Los Angeles, California. Transfusion1991:31:814-818.

3. Kirchhoff LV: Perspectives on Chagas' disease inLatin America and United States. In Walker DH. ed:Global Medicine: Current Status and Directions forthe Twenty-First Century. Springer Verlag, New York.1992. pp. 219-226.

4. Torres M: Estudo do miocSrdio na mol6stia de Chagas(forma agtida). Altera^oes na fibra muscular cardfaca.Mem Inst Oswaldo Cruz 1917:9:114-135.

5. Vianna G: Contribuigao para o estudo da anatomiapatologica da "Molestia de Carlos Chagas." Mem InstOswaidoCruz 1911:3:276-293.

6. Rassi A: A clinica: Fase aguda. In Brener Z. AndradeZA, eds: Trypanosoma cruzi e doent,a de Chagas.Guanabara Koogan. Rio de Janeiro, 1979, pp. 249-264.

7. Factor SM. Cho S. Wittner M. et al: Ahnormalilies ofthe coronary microcirculatlon in acute murine Chagas'disease. Am J Trop Med 1985:34:246-253.

8. Morris SA. Tanowiiz HB. Wittner M. et a!: Pathophys-iological insights into the cardiomyopathy of Chagas'disease. Circulation 1990:82:1900-1909.

9. Ribeiro dos Santos R, Pirmez C, Savino W: Role ofautoreactive immunological mechanisms in chagasiccarditis. Res Immunol 1991:142:134.

10. Ribeiro dos Santos R, Rossi MA, Laus JL, et al: Anti-CD4 abrogates rejection and reestablishes long-termtolerance to syngeneic newborn hearts grafted in micechronically infected with Trypanosoma cruzi. J ExpMed 1992.175:29-39.

11. Minoprio P, ltohara S, Heusser C, et al: lmmunobiol-ogy of murine T. cruzi infection: The predominance ofparasite-nonspecific responses and Ihe activation ofTCRIT cells. Immunol Rev 1989:112:183-189.

12. Morris SA. Hatcher V, Bilezikian JP. et al: Alterationsin intracellular calcium following infection of humanendothelial cells with Trypanosoma cruzi. MolBiochem Parasitol 1988:29:213 221.

13. Zingales B. Coli W: Ttypanosoma cruzi. Interactionwiih host cells. Curr Top Microbiol Immunol1985:129-152.

14. Aratijo-Jorge TC: The biology of Trypanosoma cruzi-macrophage interaction. Mem Inst Oswaldo Cruz1989:84:441-462.

15. Andrews NW, Katzin AM, Colli W: Mapping of sur-face glycoproteins of Trypanosoma cruzi by two-dimcnsionai electrophoresis. A correlation with cellinvasion capacity- EurJ Biochem 1984:140:599-606.

16. Souto-Padron T, Campeteita OE, Cazzulo JJ, et al:Cysteine proteinase in Trypanosoma cruzi'- Immunocy-tochemical localization and involvement in parasite-host cell inieraction. J Cell Sci 1990:96:485-490.

17. Schenkman S, Man-Shiou J, Hart GW, et al: A novelcell surface trans-sialidase of Trypanosoma cruzi gen-

erates a stage specific epitope required for invasion ofmammalian cells. Cell 1991:65:1117-1125.

18. Ortega-Barria E. Pereira MEA: A novel T. cruziheparin-binding protein promotes fibroblast adhesionand penetration of engineered bacteria and try-panosomes into mammalian cells. Cell 1991:67:411-421.

19. Wirth J, Kierszenbaum F: Macrophage mediation ofthe inhibitory effects of elevated intracellular levels ofadenosine-3'5' cyclic monophosphate (cAMP) onmas^xo^Wdgt-Trypanosoma cruzi infection. J Immunol1984:131:3028-3031.

20. Kierszenbaum F, Wirth J, McCann P, et al: Argininedecarboxylase inhibitors reduce the capacity of 7Vv-panosoma cruzi to infect and multiply in mammalianhost cells. Proc Natl Acad Sci USA 1987;84:4278-4282.

21. Andrews NW, Schenkman S, Ley V, et al: Try-panosoma cruzi: Mechanism of cell-invasion and intra-cellular survival. Mem Inst Oswaldo Cruz1988:83:452-455.

22. Andrews NW, Abrams CK. Slatin SL, et al: A T. cruzi-secreted protein immunologically related to the com-plement component 09: Evidence for membrane pore-forming activity at low pH. Cell 1990:61:1277-1287.

23. Bergdoll B. Tanowitz H. Morris SM, ei al: Try-pano.soma cruzi: Effects of infection on mobilizationand chronotropy in cardiac myocytes. Exp Parasitol1994:78:149-160.

24. Aprigliano O, Masuda MO. Meirelles MNL. et al:Heart muscle cells acutely infected with Trypanosomacruzi: Characterization of electrophysiology and neuro-transmitter responses. J Mol Cell Cardiol1993:15:1265-1274.

25. Campos de Carvalho AC. Roy C. Dermietzel R, et al:Gap junction distribution is altered between cardiacmyocytes infected with Trypanosoma cruzi. Circ Res1992:70:733-742.

26. Bilezikian JP, Steinberg SF, Horn EM, el al: Protein-adrenergic interactions in the heart. Mol Cell Biochem1988:82:5-11.

27. Campos de Carvalho AC. Tanowil/ HB. Wittner M, etal: Trypanosome infection decreases junclional com-munication between cardiac myocytes. In Hail JE.Zampighi GA. Davis RM, eds: Gap Junctions:Progress in Celt Research. Volume 3. Elsevier. Ams-terdam, The Netherlands, 1993, pp. 193-197.

28. Hossain MZ, Sawchuk MA, Murphy LJ. et al: Kainicacid induced alterations in antibody recognition o! con-nexin43 and loss of astrocytic gap junctions in ratbrain. Glla I994:(In press).

29. Meirelles MNL. Araujo-Jorge TC. Miranda CF. et al:Interaction of Trypanosoma cruzi wiih hearl musclecells: Ultrastructural and cytochemicai analysis ofendoeytie vacuole formation and effect upon myogene-sis. Eur J Cell Bio! 1986:41:198-206.

30. Rowin KS, Tanowitz HB. Murray M. et al: Inhibitionof muscle differentiation by Trypanosoma cruzi. Prix;Natl Acad Sci USA 1983:80:6390-6394.

698 Journal of Cardiovascular Electrophysiology Vol. 5, No. 8, August 1994

31. Sterin-Borda L, Gorelik G, Borda ES: Chagasic IgGbinding with cardiac muscarinic chofinergic receptorsmodifies cholinergic-mediated cellular transmembranesignals. Clin Immunol Immunopathol i991;61:387-397.

32. Goin JC, Borda E, Segovia A, et al: Distribution ofantibodies against beta-adrenoreceptors in the courseof fiuman Trypanosoma cruzi infection. Proc Soc ExpBiolMed f991;f97:I86-192.

33. Luke RA, Saffitz JE: Remodeling of ventricular con-duction patfiways in healed canine infarct border

zones. J Clin fnvest 199I;87:1594-16U2.34. Smith JH, Green CR, Petres NS, et al: Altered patterns

of gap junction distribution in ischemic heart disease.An immunohistochemical study of human myocardiumusing laser scanning confocal microscopy. Am J Patho!1991;139:801-8I3.

35. Spach MS. Dobler PC: Relating extracellular potentialsand their derivatives to anisotropic propagation at amicroscopic level in human cardiac muscle: Evidencefor electrical uncoupling of side-to-side fiber connec-tions with increasing age. Circ Res 1986;58:356-362.