Cardiac effects of long-term active immunization with the second extracellular loop of human β1- and/or β3-adrenoceptors in Lewis rats

Abstract

β1- and β3-adrenoceptor (AR) auto-antibodies were detected in patients with dilated cardiomyopathy. Many studies have shown that β1-AR auto-antibodies with partial agonist-like effect play an important role in the pathogenesis of this disease. Moreover, a recent study carried out in our laboratory has shown that β3-AR antibodies (β3-ABs), produced in rats, were able to reduce cardiomyocyte contractility via β3-AR activation. The aims of this study were (1) to investigate, in isolated cardiomyocytes from rabbit, the role of Gi proteins in the β3-ABs-induced cardiac negative inotropy, (2) to determine whether β3-ABs may exhibit β3-AR antagonistic property which is characteristic of partial agonists, and (3) to determine whether long-term active immunization producing both β1-ABs and/or β3-ABs leads to the development of cardiac dysfunction in Lewis rats. Lewis rats were immunized for 6 months with peptidic sequences corresponding to the second extracellular loop of human β3-AR and/or β1-AR. Agonistic effect of β3-ABs was evaluated on electrically field-stimulated isolated cardiomyocytes from adult rabbit by measuring the cell shortening. Echocardiography and ex vivo isolated perfused heart studies were conducted on immunized rats. Finally, β-AR expression was quantified by immunofluorescence and RT-qPCR. SR58611A (10 nM), a preferential β3-AR agonist, and purified β3-ABs (25 μg/ml) induced a decrease in cell shortening (-39.71±4.9% (n=10) and -17.06±3.9% (n=10) respectively). This effect was significantly inhibited when the cardiomyocytes were preincubated with pertussis toxin (0.3 μg/ml), a Gi protein inhibitor (p<0.05). In addition, SR58611A-mediated negative inotropic effect was decreased when cardiomyocytes were preincubated with β3-ABs (p<0.0001). Echocardiography revealed a decrease in the fractional shortening and ejection fraction in rats immunized against β1-AR and both β1- and β3-AR. However, the study on isolated heart showed a decrease of the isoproterenol-induced lusitropic and inotropic effects in the 3 groups of immunized rats. These systolic and diastolic dysfunctions are correlated with a decrease in the expression of β1-ARs and an increase of β3-ARs in rats immunized against the β1-AR and an increase of both β3-AR and β1-AR in rats immunized against the β3-AR. For the first time, these results showed that β3-ABs had a β3-AR partial agonist-like activity which might play a role in the pathogenesis of cardiac dysfunction.

Keywords: Auto-antibodies; Cardiac contractility; Dilated cardiomyopathy; Rats; β-Adrenoceptor.