Functional antibodies against G-protein coupled receptors in Beagle dogs infected with two different strains of Trypanosoma cruzi

Abstract

The interaction of the anti-beta1-adrenergic receptor autoantibodies (β1ARAb) and the anti-muscarinic M2 receptor autoantibodies (M2RAb) with cardiac neurotransmitter receptors were identified in human chronic Chagas cardiomyopathy (CCC) related to the ECG and dysautonomia disturbances. Dogs are considered gold model to the study of Trypanosoma cruzi infection due the clinical similarities with CCC. This study aims to evaluate whether anti-β1ARAb, anti-β2ARAb, and anti-muscarinic M2RAb are generated in Beagle dogs infected by T. cruzi using Y and Berenice-78 strains of T. cruzi. Animals were infected with 4.0 x 10³ bloodstream trypomastigotes/kg of body weight and, after 25 months of infection, blood sample was collected, and serum stored at -80°C. Dog serum was treated by ammonium sulphate precipitation and the IgG antibodies isolated and added to the beating neonatal rats' cardiomyocytes. All T. cruzi-infected dogs developed agonistic β1ARAb, β2ARAb, and M2RAb. Animals infected by Berenice strain presented less β2ARAb and M2RAb activities than dogs infected by Y strain of the parasite. In cardiomyocytes culture, the antibodies recognized an epitope on the second extracellular loop of the receptors which were similar to findings in human Chagas disease. There was no detection of antibody against G protein-coupled receptor in serum from uninfected dogs. In conclusion, both Y and Berenice-78 strains of T. cruzi induced dog antibodies, whose targets located in the second extracellular loop of the adrenergic and muscarinic receptors were similar to those observed in individuals with CCC. Therefore, our findings highlight dogs as a promisor model to investigate pathogenic roles of functional Ab against G-protein coupled receptors.

Keywords: Cardiomyocytes; Trypanosoma cruzi; autoantibodies; chagas disease; dog model; g-protein coupled receptors.

Figure 1

(A) Positive chronotropic response of the cardiomyocytes after the addition of the immunoglobulin G (IgG) preparation, obtained from dogs infected with the Y-strain, in the presence of atropine. The cardiomyocytes show an accelerated beating frequency, and the effect of IgG was blocked by specific antagonists of the β2-adrenergic receptor (AR) (ICI118.551) and antagonist of the β1-AR (bisoprolol). The IgG of uninfected control animals exert no effect. (B) IgG preparation from dogs infected by Y-strain under blockade of the β1-AR and β2-AR by bisoprolol and ICI118.551, respectively. Under these conditions, the cardiomyocytes response with a negative chronotropic effect induced via the muscarinic M2 receptor and blocked by atropine.

Figure 2

Activity of the β1-adrenoceptor, β2-adrenoceptor and muscarinic M2 receptor antibodies (Abs) on serum obtained from animals infected by Y and Berenice-78 T. cruzi strains and on serum from uninfected dogs (controls). In the control samples, no functional Abs against G-protein coupled receptor were identified. (A) β1-adrenoceptor Abs activity; (B) β2-adrenoceptor Abs activity; and (C) the negative chronotropic activity of the muscarinic M2 receptor Abs. N.S., not significant (p>0.05).

Figure 3

Tagging of the binding epitopes of the antibodies (Abs) on the extracellular structures of the receptors. (A) The binding site of the β1-adrenoceptor Abs on the second extracellular loop of receptor represented by the peptide RAESDE. The β2-adrenoceptor Abs recognize the peptide sequence ATHQEAI on the second extracellular loop (B) and the muscarinic M2 receptor Abs recognize the sequence EDGECY localized on the second extracellular loop of the M2 receptor (C). The other short overlapping peptides of the three receptors did not develop any neutralizing activity. Cells were pre-treated with bisoprolol, a specific β1- receptor antagonist, ICI118551, a specific β2- receptor antagonist and atropine, a muscarinic M2 receptor antagonist.