Trypanosoma cruzi Infection Induces Myocardial Nitric Oxide Synthase

Trypanosoma cruzi Infection Induces Myocardial Nitric Oxide Synthase Huan Huang, MD,* John Chan, MD,† Murray Wittner, MD, PhD,* Louis M. Weiss, MD, MPH,* † Cyrus J. Bacchi, PhD,‡ Nigel Yarlett, PhD,‡ Martha Martinez, BA,‡ Stephen A. Morris, MD, PhD,* Vicki L. Braunstein, BS,* Stephen M. Factor, MD,* and Herbert B. Tanowitz, MD* † Departments of *Pathology and †Medicine, Albert Einstein College of Medicine, Bronx, New York; ‡Haskins Laboratory and the Biology Department, Pace University, New York, New York

11 Chagas’ disease caused by Trypanosoma cruzi, is an important cause of myocarditis and cardiomyopathy. Acute and chronic infection is associated with myocardial dysfunction, including dysrhythmias, conduction disturbances, and congestive heart failure. Nitric oxide (NO) has been implicated in the myocardial dysfunction associated with diseases of the myocardium. The inducible form of nitric oxide synthase (iNOS) mediates the synthesis of NO and L-citrulline from L-arginine. An abundance of iNOS mRNA by Northern blot and a marked expression of iNOS protein by Western blot was demonstrated in the myocardium of mice 30 days postinfection with the Brazil strain of T. cruzi. Immunocytochemical staining of the myocardial sections from infected mice also revealed the expression of iNOS. Consistent with these observations, the myocardial L-citrulline content was higher in infected mice, confirming NO expression in vivo. In addition, Northern blot analysis revealed that interleukin-1b (IL-1b) and tumor necrosis factor-a (TNF-a) mRNA were induced during infection. These data suggest that the myocardial cytokine–iNOS pathway may be an important factor in the pathogenesis of chagasic heart disease. In addition, this pathway may be a potential target of future pharmacologic intervention. Cardiovasc Pathol 1997;6:161–166 © 1997 by Elsevier Science Inc.

Chagas’ disease, caused by the hemoflagellate Trypanosoma cruzi, is an important cause of heart disease in endemic areas of Latin America where millions of people are at risk (1). Acute and chronic infection is associated with myocardial dysfunction, including dysrhythmias, conduction disturbances, and congestive heart failure. The mortality of Chagas’ disease often is related to the severity of the underlying heart disease, and the survival rate is dramatically reduced once myocardial dysfunction occurs (1). The etiology of the pathogenesis of chagasic heart disease is multifactorial. Among the important factors contributing to the pathogenesis of chagasic heart disease is the comproManuscript received July 24, 1996; accepted September 10, 1996. All decisions concerning review and acceptance of this manuscript were made by an Associate Editor of the Journal. Address for correspondence: Dr. Herbert B. Tanowitz, Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461; telephone: (718) 430-3342; fax: (718) 430-8543. Cardiovascular Pathology Vol. 6, No. 3, May/June 1997:161–166  1997 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

mised coronary microvasculature (2–4). It has been hypothesized that these alterations result in focal ischemia, inflammation, and biochemical changes leading to myocardial injury (2). Reports have demonstrated that T. cruzi infection is associated with increased expression of endothelin (5); cytokines from both the myocardium and the endothelium (6,7); and vascular adhesion molecules (8) (Tarleton, personal communication). The cytokines elevated during the course of T. cruzi infection (6,9,10), are reported to be a major stimulus for the induction of the inducible form of nitric oxide synthase (iNOS), the enzyme that mediates the formation of nitric oxide (NO). The reported negative inotropic effects of cytokines are believed to be mediated by NO because treatment with NOS inhibitors prevents cytokine-mediated decrease in contractility (11–13). iNOS activity also has been demonstrated in cardiac myocytes exposed to cytokines (14). Thus, the cardiac myocyte is both the target and a po-

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tential source of NO. Inflammation and myonecrosis are associated with the synthesis of proinflammatory cytokines and increased production of NO, which activates soluble guanylate cyclase, resulting in an increased synthesis of cGMP, thereby attenuating myocardial contractility (15– 17). This cytokine–NO pathway has been implicated in the pathogenesis of the myocardial dysfunction associated with myocarditis and cardiomyopathy (17). Consequently, we examined the induction of cytokines and iNOS in the myocardium of T. cruzi–infected mice.

Materials and Methods Infection of animals. The Brazil strain of T. cruzi was maintained in C3H mice (Jackson Laboratories, Bar Harbor, ME). Male CD-1 mice (Charles River, Wilmington, MA) were infected IP at approximately 9–11 weeks of age with 5 3 104 trypomastigotes. Preparation of heart tissue RNA. Mouse hearts were rapidly excised from mice anesthetized with pentobarbital and washed in ice-cold PBS over ice and immediately placed in ice-cold GIBCO BRL Trizol reagent. Following polytron homogenization (Brinkmann, Westbury, NY), total RNA was extracted according to the manufacturer’s protocol (GIBCO, Grand Island, NY). For the protection from RNAse activity, the final RNA pellets were solubilized in formazol (stabilized formamide; Molecular Research Center, Inc., Cincinnati, OH). Northern blot analysis. Aliquots of total RNA (15 mL) were incubated at 558C for 15 minutes in loading buffer (total volume 230 mL containing 81 mL formaldehyde; 0.25 mg/mL bromophenol blue; 38 mL glycerol; and 24 mL 10 3 MOS buffer; filled up to the total volume with H2O), and the RNA was then electrophoresed on 1% formaldehyde agarose gel and then transferred to nitrocellulose filters by capillarity. Filters were prehybridized at 428C for 6 to 12 hours in prehybridization solution (25 mM KPO4, pH 7.4, 5 3 SSC, 5 3 Denhardt’s solution, 50 mg/ml salmon sperm DNA, 50% formaldehyde) followed by a hybridization (prehybridization solution plus 10% dextran sulfate) with the appropriate Random-primed labeled denatured cDNA probes at 1–3 3 106 dpm/ml for 12 to 24 hours. The following cDNA probes were used: murine iNOS (18,19), IL-1b (gift of Dr. Joan Berman, Albert Einstein College of Medicine, Bronx, NY), and tumor necrosis factor-a (TNF-a) (gift of Dr. Andrew J. Hapel, Australian National University, John Curtin School of Medical Research, Canberra, Australia). Next, the filters were rinsed 15 minutes at room temperature twice in 1 3 SSC and 0.1% SDS and 15 minutes at room temperature twice in 0.5 3 SSC and 0.1% SDS. Autoradiography was performed with X-ray film and intensifying screen at 2708C. Western blot analysis. Specific antibody for iNOS was obtained from Transduction Laboratories (Lexington, KY). ECL Western blot kits were obtained from Amersham (Arling-

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ton Park, IL). For Western blot analysis, 150 mg of wholeheart homogenization protein was dissolved in sample buffer, boiled 2 minutes, and electrophoresed by 10% PAGE-SDS gel. The proteins were transferred to nitrocellulose gels, which were incubated with anti-iNOS antibody. Detection processing was done according to the Amersham ECL Western blot kit’s protocol. Quantification. Films were quantified by optical scanning expressed as optical units, as previously described (20). Immunocytochemistry. Immunocytochemistry on paraffin sections of infected and uninfected myocardium was performed by modifying methods previously described by Flynn et al. from this laboratory (21). The anti-iNOS antibody was used at a dilution of 1:100, and the secondary antibody (Peroxidase Vectastain ABC Kit, Vector Laboratories, Burlingame, CA) was used at a dilution of 1:100. Photomicrographs were taken by one observer without prior knowledge of the experimental groups. Cardiac citrulline analysis. Each heart was homogenized in 2 mL of buffer containing 20 mM Tris-HCL, 0.1 mM PMSF, 1 mM EDTA, 1 mM DTT, and 1 mL/mL aprotonin and leupeptin. Protein concentration was determined by BioRad assay. Cardiac citrulline was separated by reverse-phase high-performance liperiol chromatography (HPLC) analysis using a series LC 410 pump (Perkin-Elmer Corp., Norwalk, CT) coupled to a C-18 10-mm column (4.5 3 250 mm) at a flow rate of 1 mL min21. The method employed a 70-minute discontinuous gradient starting with 85% buffer A: 2.5 gL21 lithium citrate (pH 2.65) containing 0.22 gL21 octane sulfonic acid and ending with 85% of buffer B (acetonitrile). Standards and samples were precolumn derivatized with 0.8 gL21 o-ophthalaldehyde (2:1) dissolved in 3 mL methanol and added to 30.9 gL21 boric acid containing 24 gL21 KOH (pH 10.4) 1 1 ml 2-mercaptoethanol. The derivatized samples were quantitated with a fluorescence monitor using an excitation wavelength of 320 nm and absorption wavelength of 455 nm. Areas under the peaks were integrated using an LCI-100 data station (Perkin-Elmer Corp., Norwalk, CT) (22).

Results All studies were performed on myocardial tissue 30 days postinfection. Northern blot analysis revealed an induction of iNOS mRNA, but no expression was demonstrated in myocardial tissue obtained from uninfected mice (Figure 1). Northern blot analysis also revealed that TNF-a mRNA (Figure 2) and interleukin-1b (IL-1b) mRNA (Figure 3) were expressed in the hearts of infected mice. These cytokine mRNAs were not detectable in the hearts of uninfected mice. A murine rRNA cDNA probe was utilized to confirm the loading equivalency. Western blot analysis revealed a 130-kDa MW protein consistent with iNOS. This was not demonstrated in the hearts obtained from uninfected mice (Figure 4). Northern and Western blots were performed on at least three different samples.

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Figure 1. Representative Northern blot analysis demonstrating that iNOS mRNA was expressed in murine myocardial tissue obtained from T. cruzi– infected mice 30 days postinfection (I). iNOS mRNA was not expressed in myocardial tissue obtained from uninfected, control mice (C). In the lower panels a 18s rRNA cDNA probe was used to demonstrate the loading equivalency of each lane.

Consistent with the Northern and Western blot analyses HPLC analysis revealed that there was a significant increase in L-citrulline content in hearts obtained from infected mice compared to uninfected mice, indicating a high output of NO in the hearts of the infected mice (Figure 5). The levels are expressed as nmoles/100 mL of sample. Immunocytochemical localization revealed expression of iNOS protein in the myocardium of infected mice 30 days postinfection. Interstitial and cardiac myoctye cells were stained (Figure 6).

Discussion In the present study Northern and Western blot analyses demonstrated the induction of iNOS in the myocardium during the course of acute murine T. cruzi infection. This finding was confirmed by the HPLC analysis demonstrating a significant elevation of L-citrulline in the myocardium of infected mice. We are reporting on mice 30 days postinfection. At this time point we have shown that there is tissue parasitism and intense myonecrosis and inflammation (23). Many of the mice exhibited signs of congestive heart failure. Preliminary data demonstrated that iNOS mRNA is al-

Figure 2. Representative Northern blot analysis demonstrating that TNF-a mRNA was expressed in murine myocardial tissue obtained from T. cruzi–infected mice 30 days postinfection (I). TNF-a mRNA was not expressed in myocardial tissue obtained from uninfected, control mice (C). In the lower half of the top panel and in the lower panel a 18s rRNA cDNA probe was used to demonstrate the loading equivalency of each lane.

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Figure 3. Representative Northern blot analysis demonstrating that IL-1b mRNA was expressed in murine myocardial tissue obtained from T. cruzi– infected mice 30 days postinfection (I). IL-1b mRNA was not expressed in myocardial tissue obtained from uninfected, control mice (C). In the lower half of the top panel and in the lower panel a 18s rRNA cDNA probe was used to demonstrate the loading equivalency of each land.

ready induced 14 to 21 days postinfection. Although we only examined TNF-a and IL-1b, T. cruzi–associated increases in myocardial TNF-a, IL-12, IL-1b, and interferon-g (IFN-g) have also been reported (7). In addition, T. cruzi infection has been associated with an increase in circulatory and endothelial cell cytokines (6,9,10). Many of these cytokines are potent inducers of iNOS (12,14,17). An increase in myocardial iNOS and L-citrulline also was reported in mice infected with coxsackievirus B3 (24). The inducible form of iNOS mediates the conversion of L-arginine to L-citrulline and NO (15,17). iNOS gene expression induced by cytokines is accompanied by the generation of NO that exerts antiparasitic activity (25–27). In addition, NO plays an important role in modulating the contractile effects of b-adrenergic receptor stimulation. Upregulation of iNOS elevates cGMP, activates cGMP-dependent protein kinase, and inhibits the b-adrenergic receptor-mediated increase in Ca21, thus reducing myocardial contractility (28). T. cruzi infection may damage the myocardium in several ways. The reported increases in endothelin (5) and

Figure 4. Representative Western blot analysis demonstrating that iNOS antibody recognized a 130-kDa band in a homogenate from murine hearts obtained 30 days postinfection (I). There was no expression of iNOS protein in control uninfected hearts (C).

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Figure 5. HPLC analysis of myocardial L-citrulline (nmoles/ 100 ml). There was a significant increase in L-citrulline content in hearts obtained from infected mice (30 days postinfection, n 5 5). Bars represent standard error (p , .05). Abbreviations: C 5 control; I 5 infected mice.

thromboxane A2 (29) may be associated with microvascular vasospasm and reduction of blood flow to areas of the myocardium subserved by a particular portion of the circulation, resulting in focal ischemia. The parasite also may directly invade and damage myocardial cells. Both of these processes lead to the recruitment of inflammatory cells in this infection (3,8, and Tarleton, unpublished observations). The lesional and circulatory cytokines are capable of inducing

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iNOS expression in cardiac myocytes. The immunocytochemistry studies demonstrated an induction of iNOS expression in both inflammatory cells and cardiac myocytes during infection. Several pseudocysts also were positively stained. This most likely represents iNOS expression in infected cardiac myocytes, although it is also possible that this may represent an iNOS-like cross-reactivity to the parasite. Consistent with Northern blot and immunohistochemical analysis, the myocardial L-citrulline content was increased in infected mice confirming NO expression in vivo. Others also have shown that iNOS mRNA expression is increased in the spleens of infected mice (26,30) and that serum levels of NO are increased as early as 8 days after infection (26). NO has been implicated in the pathogenesis of myocardial dysfunction associated with a variety of cardiomyopathies and myocarditis. NO-associated changes in myocardial contractility may be important factors in a number of disease states, such as septic shock, rejection of heart transplants, and viral and antimyosin-induced myocarditis models in animals (31–33). One common feature shared by these disease states and models is the inflammation associated with the increased expression of myocardial iNOS. NO, implicated as an important factor that can modulate the signal transduction pathways involved in the control of myocardial contractlity, exerts a negative inotropic effect. De Belder et al. (32,33) demonstrated iNOS in endomyocardial biopsies from patients with heart failure. Circulating cytokines have been detected in patients with acute myocarditis (34). Notably, increased TNF-a has been reported in acute Chagas’ disease (9). Our in vitro studies

Figure 6. Immunocytochemical staining of representative myocardial sections obtained from uninfected (A) and infected (B) mice. There is a virtual absence of staining in the uninfected myocardium. The infected myocardium has foci that are heavily stained in the interstitium (thin arrows) and in infected cardiac myocytes (bold arrow). (A) 3150; (B) 3320. (See Materials and Methods.)

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(6) revealed that T. cruzi infection of endothelial cells resulted in increased expression of mRNA and protein for IL-1b, IL-6, and colony-stimulating factor-1 (CSF-1). Thus, local increases in inflammatory cytokines following early infection of the microvasculature and cardiac myocytes may result in iNOS induction and NO production. Infection of the cells of the microvasculature also may induce iNOS in cardiac myocytes even in the absence of parasitism of the cardiac myocytes. Cardiac myocytes express both constitutive (cNOS) and iNOS activities (14,17). Infection-associated inappropriate activation of cNOS/endothelial(e) NOS, or excessive or prolonged induction of iNOS in the myocardium may contribute to cardiac dysfunction (17). Cytokine-induced activation of the L-arginine/NO cascade in the myocardium is one of the important cellular signaling events associated with cardiac contractile failure. In this regard, Roberts et al. (12) have demonstrated that IL-1b, lipopolysaccharide (LPS), or the combination of IL-1b and IFN-g induced iNOS in cardiac myocytes with a reduction in contractility. TGF-b reduced the activity of iNOS, thereby suppressing the release of NO and reversing the action of IL-1b. Interestingly, in a coculture system composed of macrophages and cardiac monocytes (35), cytokine-induced increased expression of iNOS resulted in increased myocyte death that was blocked by an iNOS antagonist or TGF-b. This observation may explain cardiac myocyte death observed in acute and chronic myocarditis. In addition, TNF-a, IL-6 and IL-1b inhibited the contractility of a hamster papillary muscle preparation by generating NO and the addition of a NOS inhibitor blocked the cytokine-induced negative inotropic effect (36). Cardiac myocytes expressed iNOS in vitro after treatment with cytokines (IL-1b, IL-6, IFN-g, and TNF-a) (13,17). Therefore it seems likely that cytokines may be partly responsible for the increased expression of myocardial iNOS. In summary, this report documents for the first time the induction of iNOS in the myocardium during the acute phase of murine T. cruzi infection. We are currently investigating the expression of iNOS in the myocardium of mice with chronic cardiomyopathy and the kinetics of iNOS expression. This pathway may be a potential target of future pharmacologic intervention. Supported in part by NIH grants AI-12770, AI-29747, and AR-43018 and by a Grant-in-Aid from the American Heart Association.

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