Insights from the use of erythropoietin in experimental Chagas disease

Abstract

In addition to the long-established role in erythropoiesis, erythropoietin (Epo) has protective functions in a variety of tissues, including the heart. This is the most affected organ in chronic Chagas disease, caused by the protozoan Trypanosoma cruzi. Despite seven million people being infected with T. cruzi worldwide, there is no effective treatment preventing the disease progression to the chronic phase when the pathological involvement of the heart is often observed. Chronic chagasic cardiomyopathy has a wide variety of manifestations, like left ventricular systolic dysfunction, dilated cardiomyopathy, and heart failure. Since Epo may help maintain cardiac function by reducing myocardial necrosis, inflammation, and fibrosis, this study aimed to evaluate whether the Epo has positive effects on experimental Chagas disease. For that, we assessed the earlier (acute phase) and also the later (chronic phase) use of Epo in infected C57BL/6 mice. Blood cell count, biochemical parameters, parasitic load, and echocardiography data were evaluated. In addition, histopathological analysis was carried out. Our data showed that Epo had no trypanocide effect nor did it modify the production of anti-T. cruzi antibodies. Epo-treated groups exhibited parasitic burden much lower in the heart compared to blood. No pattern of hematological changes was observed combining infection with treatment with Epo. Chronic Epo administration reduced CK-MB serum activity from d0 to d180, irrespectively of T. cruzi infection. Likewise, echocardiography and histological results indicate that Epo treatment is more effective in the chronic phase of experimental Chagas disease. Since treatment is one of the greatest challenges of Chagas disease, alternative therapies should be investigated, including Epo combined with benznidazole.

Keywords: Cardioprotection; Erythropoietin; Mice; Therapeutic drug; Trypanosoma cruzi.

Graphical abstract

Fig. 1

Experimental design. A. Distribution of experimental groups. B. Experiment timeline. Epo: erythropoietin. I_Epo_AP: infected with T. cruzi and treated with Epo in the acute phase (d1 to d30). I_S_AP: infected and treated with saline in the acute phase. NI_Epo_AP: uninfected and treated with Epo between d1 and d30. NI_S_AP: uninfected and treated with saline between d1 and d30. I_Epo_CP: infected with T. cruzi and treated with Epo in the chronic phase (d90 to d120). I_S_CP: infected and treated with saline in the chronic phase. NI_Epo_CP: uninfected and treated with Epo between d90 and d120. NI_S_CP: uninfected and treated with saline between d90 and d120. d represents the experimental day (0–180). The acute and chronic phases represent the stage of Chagas disease.

Fig. 2

Degree of cardiac involvement at the end of the experimental period (d180) using the left ventricular ejection fraction (EF) as a parameter. The left Y-axis indicates the EF. Each mouse is represented by a black circle. The right Y-axis represents the frequency of mice in each category of cardiac impairment. According to the American Society of Echocardiography's Guidelines EF ≥ 55% indicates absence of impairment, EF 45–54% is mildly abnormal, EF 30–44% is moderately abnormal, and EF <30% means a heart severely abnormal.

Fig. 3

Parasite burden of Trypanosoma cruzi infected groups. Blood (A), heart (B), large intestine (C), and spleen (D) samples were obtained after 180 days post-infection. DNAs were used as template in qPCR reactions using specific primers for T. cruzi nuclear DNA. Data points represent individual mice. Symbols indicate absolute levels and colored bars indicate mean and standard deviation. * Indicate that the parasite quantification is similar between groups under bars and significantly different from the other groups. p < 0.05. In B, the parasite quantification of groups under the dotted bars is not significantly different. No statistical difference was found in intestine (C).

Fig. 4

Histological sections of the cardiac tissue of mice not infected and infected with Trypanosoma cruzi treated or not with erythropoietin. The upper panel shows images of the heart without changes in uninfected groups: 4A: Mouse from the NI_S_AP group; 4B: Mouse from the NI_S_CP group; 4C: Mouse from the NI_Epo_AP group, and 4D: Mouse from the NI_Epo_CP group. In the lower panel, changes in the cardiac tissue are observed in infected groups: 4E: Mouse from the I_S_AP group showing multifocal inflammatory infiltrate in the interstitial region (black arrow); 4F: Mouse from the I_S_CP group showing multifocal inflammatory infiltrates in the perivascular (blue arrow) and interstitial regions (black arrow); 4G: Mouse from the I_Epo_AP group showing multifocal inflammatory infiltrate in the interstitial region, and 4H: Mouse from the I_Epo_CP group showing multifocal inflammatory infiltrate in the perivascular (blue arrow) and interstitial regions (black arrow). H&E. Bars: 100 μm in the upper panel and 50 μm in the bottom panel. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Heat map with the correlation analysis between the different variables evaluated. The heat map shows the correlation of production of anti-T. cruzi antibodies (IgG), hematocrit (Hct), Red blood cells (RBC), hemoglobin concentration (HGB), white blood cells (WBC), lymphocytes (LYN), monocytes (MON), segmented neutrophils (SEG), eosinophils (EOS), aspartate aminotransferase (AST), total creatine kinase (CK), creatine kinase myocardial band (CK-MB), ejection fraction (EF), fractional shortening (FS), left ventricule posterior wall thickness in diastole (LVPWD), parasitemia (qPCR Blood), parasitic load in the heart (qPCR Heart), parasitic load in the large intestine (qPCR Gut), parasitic load in spleen (qPCR Spleen), cardiac necrosis (Card_Necro), cardiac fibrosis (Card_Fib), inflammatory infiltrate in the perivascular region of the heart (Card_Inf_P), inflammatory infiltrate in the interstitial region of the heart (Card_Inf_I), follicular hyperplasia in the spleen (Spleen_Hyper), inflammatory infiltrate in intestine (Gut_Inf). *p < 0.05, **p < 0.01, ***p < 0.001, ns: non significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)