Impact of human autoantibodies on β1-adrenergic receptor conformation, activity, and internalization

Abstract

AimsAutoantibodies against second extracellular loops of β1-adrenergic receptors frequent in dilated cardiomyopathy confer myocardial dysfunction presumably via cAMP stimulation. Here, we investigate the autoantibody impact on receptor conformation and function.Methods and resultsIgG was prepared from patients with dilated cardiomyopathy, matched healthy donors (10 each) or commercial IgG preparations (2). IgG binding to β1-adrenergic receptor peptides was detected in 5 of 10 patients and 2 of 10 controls. IgG colocalization with the native receptor was detected in 8 of 10 patients and 1 of 10 controls (10 of 10 patients and 7 of 10 controls at >30 mg IgG/L). All IgGs exhibiting receptor colocalization triggered changes in receptor conformation (determined with fluorescent sensors) not stringently correlated to cAMP stimulation, suggesting the induction of more or less active receptor conformations. Receptor-activating IgG was detected in 8 of 10 patients but only 1 of 10 controls. In addition, IgG from 8 of 10 patients and 3 of 10 controls attenuated receptor internalization (measured by total internal reflection fluorescence microscopy). IgG-inducing inactive receptor conformations had no effect on subsequent cAMP stimulation by isoproterenol. IgG-inducing active receptor conformations dampened or augmented subsequent cAMP stimulation by isoproterenol, depending on whether receptor internalization was attenuated or not. Corresponding IgG effects on the basal beating rate and chronotropic isoproterenol response of embryonic human cardiomyocytes were observed.Conclusions(i) Autoantibodies trigger conformation changes in the β1-adrenergic receptor molecule. (ii) Some also attenuate receptor internalization. (iii) Combinations thereof increase the basal beating rate of cardiomyocytes and optionally entail dampening of their chronotropic catecholamine responses. (iv) The latter effects seem specific for patient autoantibodies, which also have higher levels.

Angiotensin II type 1 receptor autoantibody as a novel regulator of aldosterone independent of preeclampsia

“…Thus, AT 1 -AA may regulate ALD through other mechanisms. It is known that some autoantibodies can regulate endocytosis of their receptors, resulting in increased and non-desensitizing receptor activation [29]. Earlier studies have shown that AT 1 -AA induces excessive Ca 2þ influx in the vascular smooth muscle cells [30].…”

A standardised FACS assay based on native, receptor transfected cells for the clinical diagnosis and monitoring of β1-adrenergic receptor autoantibodies in human heart disease

“…In humans, agonistic β 1 AR-autoantibodies are strongly associated with a sub-entity of dilated cardiomyopathy (DCM) [4][5][6], and their presence in DCM patients is a powerful predictor of cardiovascular morbidity and mortality [5,[7][8][9]. Agonistic β 1 AR-autoantibodies are also found in 10%-15% of patients with ischemic cardiomyopathy [10] and in a small number of healthy individuals [7,11,12]. However, the available studies on the occurrence of β 1 ARautoantibodies in patients with heart disease and healthy subjects report highly divergent results, which is blamed on the use of non-standardised detection procedures that differ vastly with respect to sensitivity and specificity [1,2,4,13].…”

“…Unfortunately, a diagnostic tool suitable for that purpose is currently not available because the receptor domain targeted by β 1 AR-autoantibodies is predicted to form an extra-cellular α-helix [21]. Cardionoxious β 1 AR-autoantibodies bind to distinct conformations of that structure and, thereby, induce or stabilize an active or activated conformation of the receptor molecule [12]. Due to targeting a labile and polymorphic conformational epitope, β 1 AR-autoantibodies cross-react poorly with linear peptide-mimics or denatured versions of the target domain [1,7,12].…”

Questionable Validity of Peptide-Based ELISA Strategies in the Diagnostics of Cardiopathogenic Autoantibodies That Activate G-Protein-Coupled Receptors

“…The receptor domain targeted by β 1 -AR aabs is predicted to form an extracellular α-helix [12,13]. β 1 -AR aabs bind to specific conformations of that structure and thereby induce or stabilise activation-associated conformational changes within the receptor molecule [14]. By targeting a metastable, polymorphic conformational epitope, β 1 -AR aabs only poorly cross-react with linear peptide mimics or denatured versions of that target domain.…”

“…By targeting a metastable, polymorphic conformational epitope, β 1 -AR aabs only poorly cross-react with linear peptide mimics or denatured versions of that target domain. Thus, measurements based on interactions with immobilised (linear) peptide homologues of the target domain are only poorly or not at all correlated with measurements based on interactions with native membrane-bound β 1 -AR [9,14,15,16]. Epidemiological studies using peptide-based immunoassays [17,18] differ significantly from studies employing native cell assays [15,19,20,21].…”