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

Chronic heart failure (CHF) can be caused by autoantibodies stimulating the heart via binding to first and/or second extracellular loops of cardiac β₁ -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 β₁ -adrenoceptor (β₁ EC_II ) that is targeted by stimulating β₁ -receptor (auto)antibodies and (ii) to generate competitive cyclopeptide inhibitors of allosteric receptor activation, which faithfully conserve the conformational auto-epitope.

Non-conserved amino acids within the β₁ EC_II loop (compared with the amino acids constituting the EC_II loop of the β₂ -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 β₁ EC_II  ± the above replacements, and (ii) by (auto)antibody stimulation of human β₁ -adrenoceptors bearing corresponding point mutations. With the use of stimulating β₁ -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 β₁ 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 C²⁰⁹ ↔C²¹⁵ . Of note, aberrant intra-loop disulfide bond C²⁰⁹ ↔C²¹⁶ almost fully disrupted the functional auto-epitope in cyclopeptides.

The conformational auto-epitope targeted by cardio-pathogenic β₁ -receptor autoantibodies is faithfully conserved in cyclopeptide homologues of the β₁ EC_II loop bearing the NDPK^211-214 motif and the C²⁰⁹ ↔C²¹⁵ bridge while lacking cysteine C²¹⁶ . Such molecules provide promising tools for novel diagnostic and therapeutic approaches in β₁ -autoantibody-positive CHF.