919

J. Parasitol., 88(5), 2002, pp. 919–925q American Society of Parasitologists 2002

CARDIAC MYOCYTE HYPERTROPHY AND PROLIFERATING CELL NUCLEAR ANTIGENEXPRESSION IN WISTAR RATS INFECTED WITH TRYPANOSOMA CRUZI

Marıa Rosa Arnaiz, Laura Edith Fichera, and Miriam Postan*Instituto Nacional de Parasitologıa ‘‘Dr. Mario Fatala Chaben’’/ANLIS and Consejo Nacional de Investigaciones Cientıficas y Tecnicas(CONICET), Buenos Aires, Argentina. e-mail: miriampostan@yahoo.com

ABSTRACT: Chagasic cardiomyopathy is a major life-threatening complication of Trypanosoma cruzi infection in human beings.This study focuses on the hypertrophic and hyperplastic mechanisms underlying the structural changes of the heart duringexperimental infection. Proliferating cell nuclear antigen (PCNA) expression, transversal diameter, nuclear area, and number ofnuclei per unit volume were determined in the ventricular myocytes of T. cruzi–infected Wistar rats. PCNA expression wasenhanced throughout the inflamed myocardium and in the spared areas of the left ventricular wall and the septum. Myocytewidth increased from 26 to 75% at the inflammation-free myocardium (P , 0.0001), whereas it decreased 25% at the inflamedleft ventricular wall areas (P , 0.001). Nuclear size increased in the inflammation-free myocardium of the left ventricle and theseptum (.10–36%, P , 0.01 and .0.2–32%, P , 0.03, respectively) and decreased at the inflamed areas of the left ventricularwall (10–22%, P , 0.02) with respect to the controls. The number of nuclei per unit volume decreased at the inflamed myocardiumregardless of topographical location (36–65%) with respect to the controls (P , 0.0001) and in the inflammation-free myocardiumof the right ventricle and the septum (,21–37%, P , 0.002 and ,8–39%, P , 0.002, respectively). These results show that theheart responds to T. cruzi infection with DNA repair and cell multiplication in the inflamed sites and with hypertrophy of theunaffected myocardium.

Chagas’ disease is a major life-threatening complication ofTrypanosoma cruzi infection in human beings (Morel, 2001).Longitudinal studies indicate that approximately 45% of theinfected individuals who become chronic develop progressiveheart or digestive disease (or both). Cardiac involvement ischaracterized by degeneration and loss of cardiac myocytes,mononuclear cell infiltration with cytokine secretion, and fibro-sis of the myocardium. The roles of both the parasite and thehost immune system in the outcome of chagasic myocarditishave been extensively studied, but the mechanisms underlyingthe structural changes of the cardiomyopathy remain unknown.

The normal adult heart responds to the loss of myocardialcells and altered hemodynamic conditions by increasing thevolume of the remaining myocytes (hypertrophy) or the totalnumber of cells (hyperplasia) (or both) (Olivetti et al., 1991;Anversa and Kajstura, 1998). Myocyte hypertrophy or hyper-plasia in the pathological heart appears to result from the mag-nitude of the hemodynamic stress (Anversa and Kajstura,1998). Proliferating cell nuclear antigen (PCNA), a nuclear pro-tein expressed in the late G1-phase and during the S-phase ofthe cell cycle, is used frequently to detect DNA replication andhyperplasia, as well as DNA repair in viable cells (Shivji et al.,1992; Kajstura et al., 1994; Beltrami et al., 1997; Anversa andKajstura, 1998). In the present study we address the issuewhether cardiac myocytes located in T. cruzi–induced inflam-matory lesions and in the spared myocardium undergo hyper-trophy or increase the synthesis of DNA during experimentalinfection (or both).

MATERIALS AND METHODS

Infection of animals

Four- to six-wk-old male and female Wistar rats (Comision de Ener-gıa Atomica, Buenos Aires, Argentina) and T. cruzi clone Sylvio-X10/4 were used. The rats were selected at random and inoculated intraper-itoneally with 106 bovine skin and skeletal muscle (BESM) cell culture–derived trypomastigotes, as described previously (Postan et al., 1983).

Received 11 January 2002; revised 24 April 2002; accepted 26 April2002.

* To whom correspondence should be addressed.

Sham-inoculated rats were used as controls. Infected and control ratswere housed separately, with food and water ad libitum.

Tissue sampling

Infected and control rats were ether anesthetized and bled by intra-cardiac puncture immediately before killing by cervical dislocation. Theheart was removed, and samples were obtained from halfway betweenthe base and the apex containing the free right ventricle (RV) and leftventricle (LV) walls and the septum. Tissues were fixed in 10% bufferedformalin, processed routinely, and embedded in paraffin. Five-mm sec-tions were cut and stained with hematoxylin and eosin, periodic-acid–Schiff (PAS) counterstained with hematoxylin, and Masson’s trichromefor routine histopathological analysis. Infected rats exhibiting myocar-ditis and controls with normal histology were selected for the study. Atotal of 39 rats that met the selection criteria were included as follows:8 infected and 7 controls rats were killed at 30 days postinfection (PI),8 rats per group were killed at 375 days PI, and 4 rats per group werekilled at 515 days PI.

Histomorphometric analysis

Precoded heart sections underwent double-blind examination underthe light microscope, and the code was broken at the end of the exper-iment. Myocardial areas exhibiting obvious inflammation or fibrosis (in-fected inflamed [II]) (or both), and unaffected (infected noninflamed[INI]) myocardium were evaluated separately. Each field was first iden-tified at low-power magnification to determine the presence or absenceof inflammation; then, they were selected as the initial condition forfurther study. Only cardiac myocytes with intact cell membrane iden-tified with PAS staining, and centrally located nuclei were analyzed.The morphometric parameters studied were myocyte transversal diam-eter (MTD), nuclear transectional area (NTA), and the number of myo-cyte nuclei per unit volume of myocardium (N(n)v) (Loud et al., 1978;Anversa et al., 1985). MTD and NTA were determined in longitudinallyoriented sections at 31,000, using an ocular micrometer disc. Measure-ments were performed in mononucleated myocytes taken at randomfrom inflamed and inflammation-free myocardium at the LV, RV, andseptum (50 cells per tissue status per wall). The distance between bothsides of the myocytes (MTD) was determined at the plane across thenucleus. Transversal nuclear diameter (TDn) was measured at the nar-rowest site of the nucleus. Nuclear area was calculated with the equationfor elliptic cross-section bodies: (longitudinal nuclear diameter/2) 3(TDn/2) 3 p (Liu et al., 1998). An ocular reticle delineating a squarearea of 0.062 mm2 in the microscopic field at a magnification of 3400was used to quantify the number of nuclear profiles per unit area(N(n)A) in transversally oriented myocardial tissue sections. Four ofsuch fields per inflamed and inflammation-free myocardium per wallwere analyzed in infected animals (1.488 mm2 per rat) and on randomlyselected areas per wall in the controls (0.744 mm2 per rat). The equation

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N(n)v 5 (N(n)A)/LDn, described by Anversa et al. (1985), was appliedto determine cell hypertrophy, keeping in mind that the relative pro-portions of mononucleated and multinucleated myocytes remain con-stant both in the aging and in the pathological heart (Olivetti et al.,1996).

Immunohistochemistry

A direct avidin–biotin immunohistochemical technique was used forPCNA localization. Paraffin sections were collected on slides coatedwith 3-amino-propyltrieth-oxy-silane (Sigma Chemical Company, St.Louis, Missouri). Endogenous peroxidase activity was quenched with3% hydrogen peroxide in methanol for 20 min. Sections were immersedin antigen-retrieving solution (Antigen Unmasking Solution, VectorLaboratories, Burlingame, California) and treated twice at 700 W in amicrowave oven for 5 min, with a resting time of 1 min between in-cubations. Nonspecific binding was blocked with phosphate-bufferedsaline (PBS) containing 5% normal goat serum and 1.5% nonfat drymilk for 30 min. Sections were incubated overnight with biotinylatedmouse monoclonal antibody (mAb) anti-human PCNA, clone PC10(Pharmingen, San Diego, California), at 4 C in a humidity chamber,followed by avidin–peroxidase complex (Vectastain Elite ABC, VectorLaboratories). Color was developed with 3,39-diaminobenzidine tetra-hydrochloride and hydrogen peroxide chromogen substrate. PAS andHarris’ hematoxylin were used to counterstain the sections. Heart serialsections obtained from each animal incubated with mouse anti-rat IgGmAb or PBS were used as negative controls. Breast tumor sections wereincluded in each batch of stains as the positive control.

Quantification of PCNA-positive myocytes

The number of PCNA-labeled myocytes was evaluated in four mi-croscopic fields at the inflamed and spared RV, LV, and septal myocar-dium of infected and control rats, using the reticle described above.Intact myocytes were identified morphologically with PAS staining, tak-ing care not to include myocardial cells undergoing necrosis, fibroblasts,pericytes, mononuclear inflammatory cells, and capillary endothelialcells in the analysis.

Statistical analysis

Data were expressed as the mean calculated from the average mea-surement obtained from each rat and wall 6 SD. Statistical analysis wasperformed by analysis of variance and Bonferroni’s methods. Compar-ison of data from infected rats and control rats was performed using acompletely aleatorized design; the data of inflamed compared with non-inflamed myocardium from infected animals were analyzed using com-plete blocks. Values of P , 0.05 were considered significant.

RESULTS

Histopathological analysis of the heart of Wistar rats infectedwith the T. cruzi clone Sylvio-X10/4 showed a small numberof intracellular parasite nests, degenerating and necrotic myo-cardial cells, inflammatory cell infiltration, and fibrosis of var-iable intensity, as described in other animal models (Postan etal., 1987). Multifocal inflammatory infiltrates consisting mainlyof lymphocytes and macrophages, with fewer polymorphonu-clear and mast cells, were more severe during the acute stageof infection. Interstitial thickening and patches of fibrous tissuewere observed throughout the infection, being more extensiveand severe during the chronic stage.

The transversal diameter of myocytes at INI was significantlyhigher than that of myocytes in control hearts, with cardiomy-ocyte width increases ranging from 26 to 75% with respect tocontrols (Tables I, II). MTD values at the RV and septal II siteswere similar to control values, whereas they decreased 25% inthe LV at 375 days PI. The comparison between II and INImyocardial sites from infected rats showed significantly higher

ARNAIZ ET AL.—CARDIAC REMODELING IN T. CRUZI INFECTION 921

TABLE II. Percentage of change in histomorphometric measurements from Wistar rats infected with Trypanosoma cruzi with respect to controls.*

Days PI

INI

NTA MTD N(n)v

II

NTA MTD N(n)v

RV 30375515

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149.96154.37174.91

220.71225.82236.45

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239.26265.73258.02

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10.23131.9819.48

140.23126.10132.12

28.07238.67227.69

NSNSNS

NSNSNS

236.65254.74242.74

LV 30375515

19.92111.53136.22

140.61131.62148.00

NSNSNS

210.00221.87210.79

NS224.76

NS

236.05252.83254.62

* Abbreviations: RV, right ventricle; LV, left ventricle; PI, postinfection; INI, infected-noninflamed myocardium; II, infected-inflamed myocardium; NTA, nucleartransectional area; MTD, myocyte transversal diameter; N(n)v, number of nuclei per unit volume of myocardium; NS, non-significant.

FIGURE 1. Photomicrograph showing PCNA-labeled nuclei in mononucleated and binucleated myocytes (arrows) in inflammatory foci 30days PI with Trypanosoma cruzi. PCNA labeling is also present in cellular infiltrates and interstitial cells. Bar in the lower right cornerrepresents 100 mm.

MTD values in the latter at 30 and 375 days PI. In addition,age-related MTD increases occurred at the INI myocardium ofinfected rats and the controls.

The transectional area of the nucleus at INI sites of the freeLV wall and the septum was significantly higher than in thecontrols, regardless of time of infection (Tables I, II). In con-trast, NTA at II sites of the LV was significantly smaller thanin the respective controls. Significant NTA differences werealso observed between II and INI sites of infected rat hearts,depending on the wall of the heart and the time of infection.

A 36–66% decrease in N(n)v values was found at II sitesfrom T. cruzi–infected rats with reference to the controls, de-

pending on both the topographic location within the heart andthe time of infection. N(n)v values at the RV and septum INIsites from infected rats were also smaller (21–36% and 8–39%,respectively) than those in the controls (Tables I, II). The de-crease in N(n)v values at II sites was more pronounced than atINI sites, regardless of time of infection.

Labeling of PCNA protein was detected in mononucleatedand binucleated cardiac myocytes, interstitial cells, and inflam-matory mononuclear cells infiltrating the myocardium of T. cru-zi–infected rats (Fig. 1). A low level of PCNA nuclear stainingwas detected also in myocytes and interstitial cells from non-infected control rats, as reported by other authors (Marino et

922 THE JOURNAL OF PARASITOLOGY, VOL. 88, NO. 5, OCTOBER 2002

FIGURE 2. Photomicrograph showing a binucleated myocyte positively stained for PCNA. Note the presence of PCNA-positive interstitial andinflammatory cells. Bar in the lower right corner represents 30 mm.

al., 1996). The frequency of PCNA-positive myocytes at II sitesof infected animals was significantly higher than in the controls,regardless of the topographical location within the heart and thetime of infection (Fig. 2; Table III). Myocyte PCNA labelingwas more frequent also at INI sites of the LV wall and theseptum with respect to the controls, in all periods of the infec-tion analyzed (Figs. 3, 4). Comparison between PCNA labelingin II and INI sites of infected animals showed a higher fre-quency of PCNA-positive myocytes at II myocardium of theRV during the 3 periods of infection and of the LV wall andthe septum at 375 days PI.

DISCUSSION

The major finding of the present study is the distinctive adap-tive response of the Wistar rat ventricular myocardium to T.cruzi–induced injury, depending on the region of the heart, thetime of infection, and the presence of inflammatory cells. Inthis animal model, myofibers tend to retain their normal widthat inflammation sites but enlarge significantly in the sparedmyocardium. Nuclei followed inconsistent patterns in relationto the dimension of myocytes. For example, myocytes with ex-panded cytoplasm had enlarged nuclei at the LV- and septal-spared myocardium but not at the RV wall. Paradoxically, thesize of nuclei at the RV-spared myocardium remained withinthe same range as that of the normal controls, in spite of beingat the level at which the highest myocyte width increases wereregistered. The values of N(n)v decreased throughout the myo-cardium of T. cruzi–infected rats, a change indicative of myo-cyte hypertrophy. But the equation N(n)v was designed pri-

marily to detect myocyte enlargement in pure hypertrophicheart conditions. The presence of a strong inflammatory com-ponent may make the equation unsuitable for estimation of thecell volume per nucleus in the inflamed myocardium. The de-creased cell volume per nucleus found at the inflammation siteprobably results from the expansion of the interstitial space byedema, inflammatory cells, and fibrosis rather than from an in-crease in myocyte size (Baandrup and Olsen, 1981). The in-dependent analyses of the number of nuclear profiles per unitarea and the mean nuclear length indicate that the cell volumeper nucleus changes at these sites were related to modificationsin nuclear density, whereas the length of myocyte nuclei tendsto remain constant (data not shown). Changes in nuclear densitywere described also in hypertensive Wistar rats, although nu-clear polymorphism also occurred (Mandarim-de-Lacerda andPereira, 2001).

The pattern of myocyte growth exhibited at the level of non-inflamed myocardium from T. cruzi–infected rats is similar tothe hypertrophic response described in the spared myocardiumduring infarction (Anversa et al., 1985). Myocyte hypertrophyin the spared myocardium of T. cruzi–infected rats developsearly after infection and persists throughout the chronic stage.On the other hand, the cells remaining viable within inflam-matory and fibrotic foci appear to be unable to undergo hyper-trophy. The behavior of individual myocytes during T. cruziinfection might be related to the altered atrial natriuretic factorplasma levels described previously (Scaglione et al., 2001).

Aging and pathological hearts respond to the loss of myo-cardial cells with myocellular hypertrophy, DNA synthesis, and

ARNAIZ ET AL.—CARDIAC REMODELING IN T. CRUZI INFECTION 923

FIGURE 3. Photomicrograph showing a PCNA-labeled myocyte in the margin of a fibrotic scar 375 days PI. Bar in the lower right cornerrepresents 30 mm.

FIGURE 4. Myocyte PCNA labeling in an unaffected ventricular site from a rat 375 days PI with Trypanosoma cruzi. Bar in the lower rightcorner represents 30 mm.

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nuclear mitotic division (Olivetti et al., 1991; Quaini et al.,1994; Beltrami et al., 1997, 2001; Anversa and Kajstura, 1998;Kanoh et al., 1999; Petrovic et al., 2000). Necrosis in chagasicmyocarditis is characteristically associated with inflammationand fibrosis. The enhanced expression of PCNA protein foundat these sites probably reflects both DNA damage repair andreentry into the cell cycle (Shivji et al., 1992; Quaini et al.,1994; Soonpaa and Field, 1998). Although mitotic figures wererarely observed in the rats studied, the presence of binucleatedmyocytes positively labeled for PCNA in inflamed tissues sug-gests nuclear division. Mononucleated and binucleated myo-cytes expressing PCNA protein were observed previously inadult patients and also in animal models of other myocardialdiseases and after cardiac transplantation (Arbustini et al., 1993;Quaini et al., 1994; Liu et al., 1995; Beltrami et al., 1997).Whether the abnormal expression of PCNA protein representsa beneficial or detrimental effect in the development of chagasiccardiomyopathy remains to be determined.

There is increasing evidence that cytokines released locallyby both cardiac myocytes and myocardial inflammatory cellsplay an important role in the development of cardiovasculardisease (Matsumori, 1996; Meldrum, 1998). Tumor necrosisfactor alpha (TNF-a) and interleukin 1 beta (IL-1b) have a neg-ative effect on the contractility of cardiac myocytes throughinduction of nitric oxide (NO) synthase and NO release (Bal-ligand et al., 1993). NO is also a mediator for myocyte cellnecrosis (Pinsky et al., 1995; Meldrum 1998). Furthermore, itis known that IL-1b induces cardiac myocyte hypertrophy andreinitiates DNA synthesis (Palmer et al., 1995). TNF-a, IL-1b,IL-6, interferon-g, and transformation growth factor beta aresome of the cytokines known to be produced by inflammatorycells in the heart of chagasic patients and experimentally in-fected animals (Zhang and Tarleton, 1996; Reis et al., 1997;Huang et al., 1999). The enhanced expression of PCNA in theheart of T. cruzi–infected rats suggests that some of these cy-tokines may play an important role in cardiac remodeling dur-ing Chagas’ disease.

In summary, our study documents that ventricular myocytehypertrophy and hyperplasia participate in the cardiac remod-eling process during T. cruzi–induced myocarditis. Myocyte hy-pertrophy is the hallmark of the noninflamed myocardium. Onthe other hand, myocellular nuclear division and DNA repairprevail at inflammation sites. Both myocyte hypertrophy andhyperplasia can be considered as growth reserve mechanismsderived by the viable myocardial fraction to compensate for theloss of myocytes in the chagasic heart.

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———, AND J. KAJSTURA. 1998. Ventricular myocytes are not termi-nally differentiated in the adult mammalian heart. Circulation Re-search 83: 1–14.

ARBUSTINI, E., M. DIEGOLI, M. GRASSO, R. FASANI, A. D’ARMINI, L.MARTINELLI, C. GOGGI, C. CAMPANA, A. GAVAZZI, AND M. VIGANO.1993. Expression of proliferating cell markers in normal and dis-eased human hearts. American Journal of Cardiology 72: 608–614.

BAANDRUP, U., AND E. G. J. OLSEN. 1981. Critical analysis of endo-myocardial biopsies from patients suspected of having cardiomy-

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