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Electrocardiographic and Structural
Studies in Infected Mice with Two
Strains of Trypanosoma cruzi

Rivarola, Hector; Bustamante, J. Manuel;
Fernández A, Ruth; Enders, Julio;
Palma, José; Paglini-Oliva, Patricia

Cátedra de Física Biomédica, Facultad de Ciencias Médicas,
Universidad Nacional de Córdoba, Córdoba, Argentina

SUMMARY
The present work studied the electrocardiography and structural heart alterations provoked by two T. cruzi strains (Tulahuen and other which belongs to zimodema 12 (Z12), isolated from a chronic chagasic patient) in mice. Survival, parasitemia and cardiac beta receptors density and affinity were also studied. Mice were divided in two groups: one infected with 50 tripomastigotes, Tulahuen strain/mouse (GT, n=50) and the other infected with 50 tripomastigotes, Z 12 strain/mouse (GZ, n=50). Studies were performed in the acute, indeterminate and chronic phase of T. cruzi infection. GT presented higher parasitemias than GZ, p<0.01, for this survival was significantly higher in the last group. Electrocardiography abnormalities observed, specially auricle-ventricle and intraventricular were more frequent in GT than in GZ (35%, 50% and 90% and 14%, 40% and 40% respectively for each group and for acute, indeterminate and chronic phase, p<0.01 when analyzed by Chi square test for categorical variables).
Histopathologic studies showed higher structural damages in GT than in GZ (50 sections of each group and at least 30 areas from each section were studied and analyzed by chi square); higher functional compromise was observed in affinity and density of cardiac beta receptors of GT, measured using 3H dihidroalprenolol tritiado, than in GZ (analyzed by multiple comparison Tuckey test). Present work shows that T. cruzi strain defines the severity of cardiac compromise in the experimental Chagas´disease.

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   Chagas' disease, a parasitic infection caused by Trypanosoma cruzi, affects nearly 20 million people throughout South and Central America [1, 2].

   There are three stages in the human disease [3,4]: the acute phase, in which the infected patients suffer a short period with or without simptomatology. The intermediate phase generally symptomless, and the chronic phase in which 30 % of infected people develop cardiac symptoms of Chagas' disease [5-6]. Symptoms' diversity and severity go from a mild electrocardiography alteration to sudden death. This great variability and severity in cardiac Chagas' disease symptoms may be due to many factors, some of them could be: the parasite strains [7-9], the number of T. cruzi that infected the host [10], the reinfections, the host immunity [11-13], the nutritional aspects of host at the moment of infection or reinfection, and finally the action of some or all of them together.

   In the present work, we studied in a murine model, two different T. cruzi strains, in order to elucidate how relevant are different strains, both cardiotropic, in determining the variability of cardiac symptoms and the irreversible damage characteristic of Chagas' disease.

MATERIAL AND METHODS
   Albino Swiss male mice were challenged with 55 tripomastigote forms of T. cruzi, Tulahuen strain (n:50) (GT) and 500 tripomastigote forms of T. cruzi, which belong to zimodema 12 (Z12), isolated from a chronic chagasic patient, (n:50) (GZ) by an intraperitoneal injection. Parasitemias were determined in Neubawer hemocytometer from blood samples obtained from the tail 2 times a week.

   Studies were performed in the acute (35 days post infection (pi)), indeterminate (75 days pi) and chronic phase (135 days pi) of T. cruzi infection.

   Electrocardiograms were obtained with an electrocardiography apparatus (Fukuda), under Ketalar HCl (Parke Davis) anesthesia 10 mg/kg. The six standard leads(dipolar leads D1, D2, D3 and unipolar leads aVR, aVL, aVF) were studied recorded at 50 mm/s.

   b adrenergic receptors binding was performed in microsomal fraction of right and left ventricles from the groups under study. 3H/ dihydroalprenolol (NEN, USA) was used as radioligand and the experiments were carried out as previously (10). Dissociation constant (Kd) and maximum 3H/DHA binding (B max) was determined by a saturation curve and Scatchard analysis using GraFit (Erithacus Software Limited).

   Histopathologic studies were made 30, 75 and 135 days post-infection fixing the organs and staining them with hematoxylin-eosin technique. A total of 50 slices from each group were analyzed. At least 30 areas from each slice were examined with a 40 x objective.

Statistical analysis
   Data were compared by analysis of variance, Student's t test and multiple comparison by Tuckey Test and Chi square for categoric variables; significance level was set at 0.05.

RESULTS
   GT presented higher parasitemias than GZ, p<0.01, for this survival was significantly higher in the last group. Electrocardiography abnormalities observed, especially auricle-ventricle and intraventricular were more frequent in GT than in GZ (see table I).Histopathologic studies in the chronic phase showed higher structural damages with profuse fibrosis areas in hearts from GT than from GZ (see Figure 1, 2). Functional compromise of heart was studied through affinity (Kd) and density (Bmax) of cardiac beta receptors. GT showed the following results: Kd in nM: 5.63±0.26; 6.85±0.20; 11.21±0.25 and Bmax in fmol/mg.prot: 78.24; 77.28; 53.32. GZ showed the following results: Kd in nM:5.71±0.56; 6.28±0.39; 7.32±0.19, and Bmax in fmol/mg.prot: 207.63±8.15; 228.12±6.02; 184±2.10 respectively for acute, indeterminate and chronic phase. Non infected mice Kd: 3.61±0.05 nM and Bmax 71.96 fmol/mg prot.

Figure 1: Histological sections of hearts from T. cruzi infected mice in the chronic phase (135days pi). Tulahuen strain.


Figure 2: Histological sections of hearts from T. cruzi infected mice in the chronic phase (135days pi). Z12 strain, isolated from chronic chagasic patient.

DISCUSSION
   Only some more than 30% of all T. cruzi infected persons progress to manifest the clinical symptoms of Chagas'disease, and these present a great variability and severity. It has been described a correlation between the parasite strains (7-9) with the clinical forms of Chagas'disease. In the present we studied two strains described as cardiotrophic. Both provoked cardiac damages, but Get presented higher parasitemias, less survival, significantly higher electrocardiography abnormalities and beta receptors density lower than GZ and than normal (p<0.001). GZ presented lower beta receptors affinity than normal (p<0.001) and higher density than normal and GT (p<0.001) probably as a compensatory mechanism to protect cardiac function. Cardiac fibrosis areas were also fewer in GZ.

   Present work shows that T. cruzi strain defines the severity of cardiac compromise in the experimental Chagas´disease.

CONCLUSION
   The possible role of the exposure to different parasites strains, reinfections, malnutrition, vector species, individual resistance, etc in the progression of Chagas'disease morbility is still unclear. We believe that this work clearly showed that different cardiotropic parasites strains are important in determining the great variability and severity of symptoms.

REFERENCES

1. World Health Organization., Control of Chagas' Disease., Technical Report Series. 811 (1991).

2. Schofield C.J., Dias J.C., The Southern cone initiative against Chagas´disease, Adv Parasitol. 42 (1999) 1-27.

3. Tanowitz H.B., Kirchhoff L.V., Simon D., Morris S.A., Weiss L.M., Chagas' disease, Clin Microbiol Rev. 5 (1992) 400-419.

4. Madoery R., Madoery C., Cámera I., (Eds.), Actualizaciones en la Enfermedad de Chagas, Córdoba, Argentina, 1993.

5. Laguens R.P., Cabeza Meckert P., Vigliano C., Patogenia de la miocardiopatía chagásica crónica humana, Medicina (Bs Aires). 59 (1999) 63-68.

6. Andrade Z., Immunopathology of Chagas' disease, Mem Inst Oswaldo Cruz. 94 (1999) 71-80

7. Montamant E.E., D'oro G.M.D., Gallerano R.H., Sosa R., Blanco A., Characterization of Trypanosoma cruzi population by zymodemes: Correlation with clinical picture, Am J Trop Med Hyg. 55 (1996) 625-628.

8. Espinoza B., Vera-Cruz J.M., González H., Ortega E., Hernández R., Genotype and virulence correlation within Mexican stocks of Trypanosoma cruzi isolated from patients, Acta Tropica. 70 (1998) 63-72.

9. Vago A.R., Andrade L.O., Leite A.A., D'Avila Reis D., Macedo A.M., Adad S.J., Tostes S.Jr., Moreira C.V., Brasileiro G., Pena S.D.J., Genetic Characterisation of Trypanosoma cruzi directly from tissue of patients with chronic Chagas disease. Differential distribution of genetic types into diverse organs, Am J Pathol. 156 (2000) 1805-1809.

10. Fernández A.R., Enders J.M., Rivarola H.W., Paglini P.A., Palma J.A., Cardiac b receptors' density or affinity modified by different Trypanosoma cruzi amount, Acta Physiol Pharm et Therap Lat. 47 (1996) 137-143.

11. Koberle F., Chagas´disease and Chagas syndromes: The pathology of American Trypanosomiasis, Adv Parasitol. 6 (1968) 63-116.

12. Kalil J., Cunha-Neto E., Autoimmunity in Chagas´cardiomiopathy: fulfilling the criteria at last?, Parasitol Today. 12 (1996) 396-399.

13. Tomlinson S., Raper J., Natural immunity to Trypanosomes, Parasitol Today. 14 (1998) 354-359.

 

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2nd Virtual Congress of Cardiology

Dr. Florencio Garófalo
Steering Committee
President
Dr. Raúl Bretal
Scientific Committee
President
Dr. Armando Pacher
Technical Committee - CETIFAC
President
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