Genetic Ablation and Pharmacological Blockade of Bradykinin B1 Receptor Unveiled a Detrimental Role for the Kinin System in Chagas Disease Cardiomyopathy

Abstract

Chagas disease, the parasitic infection caused by Trypanosoma cruzi, afflicts about 6 million people in Latin America. Here, we investigated the hypothesis that T. cruzi may fuel heart parasitism by activating B1R, a G protein-coupled (brady) kinin receptor whose expression is upregulated in inflamed tissues. Studies in WT and B1R^-/- mice showed that T. cruzi DNA levels (15 days post infection-dpi) were sharply reduced in the transgenic heart. FACS analysis revealed that frequencies of proinflammatory neutrophils and monocytes were diminished in B1R^-/- hearts whereas CK-MB activity (60 dpi) was exclusively detected in B1R^+/+ sera. Since chronic myocarditis and heart fibrosis (90 dpi) were markedly attenuated in the transgenic mice, we sought to determine whether a pharmacological blockade of the des-Arg⁹-bradykinin (DABK)/B1R pathway might alleviate chagasic cardiomyopathy. Using C57BL/6 mice acutely infected by a myotropic T. cruzi strain (Colombian), we found that daily treatment (15-60 dpi) with R-954 (B1R antagonist) reduced heart parasitism and blunted cardiac injury. Extending R-954 treatment to the chronic phase (120-160 dpi), we verified that B1R targeting (i) decreased mortality indexes, (ii) mitigated chronic myocarditis, and (iii) ameliorated heart conduction disturbances. Collectively, our study suggests that a pharmacological blockade of the proinflammatory KKS/DABK/B1R pathway is cardioprotective in acute and chronic Chagas disease.

Keywords: Chagas disease; GPCRs; Trypanosoma cruzi; bradykinin; kallikrein.

Figure 1

Heart parasitism and severity of cardiac inflammation are attenuated in B1R-deficient mice acutely infected by Dm28c T. cruzi. C57BL/6 and B1R^−/− mice were infected systemically (i.p.) with Dm28c TCTs (10⁶). At 15 days pi., the intracardiac levels of B1R mRNA (A) and T. cruzi DNA (B) were analyzed by qPCR. Frequencies of CD11b⁺ total cells (C) and subsets of inflammatory monocytes (CD11b⁺Gr1^loLy6C^hiF4/80^lo) (E) and neutrophils (CD11b⁺Gr1^hiLy6C^int) (F) in cardiac tissue (15 dpi) by flow cytometry. (D) Dot plot representative of gate strategy in CD11b⁺ cells. (G,H) Extent of heart parasitism (qPCR) (G) and serum activity of CK-MB (H) measured at 60 dpi. The results are representative of at least three independent experiments; bar shows media ± SD and each symbol represents one mouse (n = 5 mice/group/experiment). In H, results represent the sum of 2 independent experiments. Statistical analysis was performed by t-test or one-way analysis of variance (ANOVA) test. p values < 0.05 were considered significant. NI, non-infected.

Figure 2

Chronic myocarditis and fibrosis are blunted in T. cruzi-infected B1R^−/− mice. C57BL/6 (WT) and B1R^−/− mice were infected systemically (i.p.) with a lower dose of Dm28c TCTs (1 × 10³ TCT). Hearts collected at 90 dpi were fixed in formalin (5%) and paraffin-embedded tissue. Hearts sections (4 μm) were stained by H&E to quantify inflammatory infiltrating cells (A) or to measure fibrotic areas (collagen fibers) by Picrosirius Red staining (B). Controls were hearts from non-infected mice (received PBS ip.; NI). The results are representative of three independent experiments (n = 7–10 mice/group/experiment). Statistical analysis was performed by one-way analysis of variance (ANOVA) test. p values < 0.05 were considered significant. nd, non-detected.

Figure 3

Beneficial effects of early B1R targeting in WT mice systemically infected by the myotropic Colombian T. cruzi strain. (A) Schematic figure of the infection model and treatment protocol. C57BL/6 (WT) mice were systemically infected (i.p.) with bloodstream Col TCTs (10² parasites). Daily B1R antagonist (R-954) treatment (1.6 mg/Kg; s.c.) started at 15 dpi and lasted up to 60 dpi., whereas the control mice received PBS. (B) Parasitemia was followed daily. After 60 dpi, heart tissues were fixed and sections (3 μm) were stained with T. cruzi-specific antibody for detection of amastigote nests. Results expressed in 100 fields (C). (D) Electrocardiogram records obtained at 60 dpi in diazepam sedated mice C57BL/6 mice: heart rate (bpm), PR interval, QRS, QTc, AVB2, and frequency of arrhythmias. (E) CK-MB activity was quantified in the serum and the results expressed by fold change. The results are representative of two independent experiments; bar shows media ± SD and each symbol represents one mouse (n = 8–10 mice/group/experiment). Statistical analyses were performed by one-way analysis of variance (ANOVA) test. p values < 0.05 were considered significant. nd, non-detected.

Figure 4

Therapeutic application of B1R antagonist reverses chronic chagasic cardiomyopathy (A) Schematic figure of the infection model and treatment protocol. C57BL/6 (WT) mice were systemically infected (i.p.) with Col TCTs (10² parasites). Daily R-954 treatment (1.6 mg/kg—s.c.) started at 120 days pi. and lasted up to 160 days pi., whereas the control mice received PBS. (B,D) Electrocardiogram registers were performed at 120 ((B); before treatment) and 160 dpi ((D); after treatment) in awake mice. The parameters were heart rate (bpm), PR interval, QRS, QTc, AVB2, and frequency of arrhythmias. (C) Survival rate was followed daily. (E–G) At 160 dpi, hearts sections were stained by H & E to evaluate the area of cardiac tissue presenting cellular nucleus. The yellow arrows indicated the presence of inflammatory infiltrate. (F) or were stained by Picrosirius Red to evaluate fibrosis (collagen area) (G). (H) CK-MB activity was quantified in the serum and the results expressed by fold change. The results are representative of two independent experiments; the bar shows media ± SD and each symbol represents one mouse (n = 8–10 mice/group/experiment). Statistical analysis was performed by one-way analysis of variance (ANOVA) test. p values < 0.05 were considered significant.

Figure 4

Therapeutic application of B1R antagonist reverses chronic chagasic cardiomyopathy (A) Schematic figure of the infection model and treatment protocol. C57BL/6 (WT) mice were systemically infected (i.p.) with Col TCTs (10² parasites). Daily R-954 treatment (1.6 mg/kg—s.c.) started at 120 days pi. and lasted up to 160 days pi., whereas the control mice received PBS. (B,D) Electrocardiogram registers were performed at 120 ((B); before treatment) and 160 dpi ((D); after treatment) in awake mice. The parameters were heart rate (bpm), PR interval, QRS, QTc, AVB2, and frequency of arrhythmias. (C) Survival rate was followed daily. (E–G) At 160 dpi, hearts sections were stained by H & E to evaluate the area of cardiac tissue presenting cellular nucleus. The yellow arrows indicated the presence of inflammatory infiltrate. (F) or were stained by Picrosirius Red to evaluate fibrosis (collagen area) (G). (H) CK-MB activity was quantified in the serum and the results expressed by fold change. The results are representative of two independent experiments; the bar shows media ± SD and each symbol represents one mouse (n = 8–10 mice/group/experiment). Statistical analysis was performed by one-way analysis of variance (ANOVA) test. p values < 0.05 were considered significant.