Unmasking features of the auto‐epitope essential for β₁‐adrenoceptor activation by autoantibodies in chronic heart failure

Abstract

Aims Chronic heart failure (CHF) can be caused by autoantibodies stimulating the heart via binding to first and/or second extracellular loops of cardiac β 1-adrenoceptors. Allosteric receptor activation depends on conformational features of the autoantibody binding site. Elucidating these features will pave the way for the development of specific diagnostics and therapeutics. Our aim was (i) to fine-map the conformational epitope within the second extracellular loop of the human β 1adrenoceptor (β 1 EC II) that is targeted by stimulating β 1-receptor (auto)antibodies and (ii) to generate competitive cyclopeptide inhibitors of allosteric receptor activation, which faithfully conserve the conformational auto-epitope. Methods and results Non-conserved amino acids within the β 1 EC II loop (compared with the amino acids constituting the EC II loop of the β 2-adrenoceptor) were one by one replaced with alanine; potential intra-loop disulfide bridges were probed by cysteine-serine exchanges. Effects on antibody binding and allosteric receptor activation were assessed (i) by (auto)antibody neutralization using cyclopeptides mimicking β 1 EC II ± the above replacements, and (ii) by (auto)antibody stimulation of human β 1-adrenoceptors bearing corresponding point mutations. With the use of stimulating β 1-receptor (auto)antibodies raised in mice, rats, or rabbits and isolated from exemplary dilated cardiomyopathy patients, our series of experiments unmasked two features of the β 1 EC II loop essential for (auto)antibody binding and allosteric receptor activation: (i) the NDPK 211-214 motif and (ii) the intra-loop disulfide bond C 209 ↔C 215. Of note, aberrant intra-loop disulfide bond C 209 ↔C 216 almost fully disrupted the functional auto-epitope in cyclopeptides. Conclusions The conformational auto-epitope targeted by cardio-pathogenic β 1-receptor autoantibodies is faithfully conserved in cyclopeptide homologues of the β 1 EC II loop bearing the NDPK 211-214 motif and the C 209 ↔C 215 bridge while lacking cysteine C 216. Such molecules provide promising tools for novel diagnostic and therapeutic approaches in β 1-autoantibodypositive CHF.

Association of Anti-β1 and Anti-M2 Antibodies with Autonomic Nervous System Modulation in Patients with Chronic Chagas Cardiomyopathy

“…These studies support the hypothesis that β1 adrenoceptor G protein-coupled autoantibodies are a decisive cofactor in chronic HF. 36,37 The present study showed that increased anti-β1 and anti-M2 receptor antibody titers seem to alter nighttime HRV (2:00 to 6:00 a.m.) and exert modulating effects on membrane receptors. A decrease in sympathetic activity was observed during this period in patients with high anti-β1 antibodies, paradoxically to their expected agonist effect.…”

Autoantibodies and Cardiomyopathy: Focus on Beta-1 Adrenergic Receptor Autoantibodies

“…141 However, there is increasing appreciation that β1AR autoantibodies may bind to the ECL2 of β1AR regulating receptor function through allosteric modulation without binding to the orthosteric agonist/antagonist site. 10,52,115,122,133 This would allow β1AR autoantibodies to noncanonically modulate agonist and/or antagonist signaling and subsequent cardiac outcomes. The differential signaling outcomes observed with the β1AR autoantibodies from various patients support this scientific premise.…”

“…115 These studies are seminal because they have identified the amino acid epitopes recognized by the autoantibody and leveraged this knowledge to provide insights on the potential use of cyclopeptides as therapeutic vehicles to mitigate deleterious effects of b1AR autoantibody. 115 The studies identified the binding sites through the use of monoclonal b1AR autoantibody, but however, patients could harbor a repertoire of autoantibodies recognizing all or a specific composition of these amino acids in the ECL2. In this context, binding of the autoantibody to different extracellular regions of the GPCR could determine differential downstream signaling events resulting in pathogenic versus beneficial outcomes.…”

“…133 It is important to note that the b-blockers bind close to or at the orthosteric agonist binding site, [137][138][139] while b1AR autoantibodies bind to the ECL2 of the b1AR. 52,115 Despite binding to the ECL, the b1AR autoantibodies are able to bias b1AR agonist/antagonist signaling through yet to be understood mechanisms. 10,52 Furthermore, pretreatment of cells with IgG3+ b1AR autoantibodies biases b1AR-blocker signaling toward G-proteins as measured by adenylyl cyclase activity that leads to generation of cAMP 133 (Fig.…”

“…Mutation of unique amino acids from positions 208–215 including the cysteines (209 & 215) present in the ECL2 of the β1AR could be the antigenic determinants because cyclopeptides harboring these amino acids were able to significantly alter cAMP responses mediated by β1AR autoantibody. 115 These studies are seminal because they have identified the amino acid epitopes recognized by the autoantibody and leveraged this knowledge to provide insights on the potential use of cyclopeptides as therapeutic vehicles to mitigate deleterious effects of β1AR autoantibody. 115 The studies identified the binding sites through the use of monoclonal β1AR autoantibody, but however, patients could harbor a repertoire of autoantibodies recognizing all or a specific composition of these amino acids in the ECL2.…”