ANTI-BETA(1)-ADRENOCEPTOR AUTOANTIBODIES WITH CHRONOTROPIC ACTIVITY FROM THE SERUM OF PATIENTS WITH DILATED CARDIOMYOPATHY - MAPPING OF EPITOPES IN THE FIRST AND 2ND EXTRACELLULAR LOOPS

In a preceding communication (Wallukat et al., 1992, Z Kardiol 81 [Sup pl. 4]: 79-83), it was reported that synthetic peptides, corresponding in amino acid sequence to either the first or the second extracellula r loop of the human beta(1)-adrenoceptor, selectively suppressed the m etoprolol- and bisoprolol-sensitive positive chronotropic action exert ed in cultures of beating neonatal rat cardiomyocytes by the serum imm unoglobulin fraction of patients with myocarditis and idiopathic dilat ed cardiomyopathy (DCM) and by affinity-purified autoantibodies from t hat fraction. These observations added to existing evidence that these antibodies were directed against. the beta(1)-adrenoceptor and might thus contribute to the harmful chronic cardiac adrenergic drive to whi ch patients with DCM are believed to be exposed. Specifically, they po inted to the putative first and second extracellular loops of this rec eptor (these loops are each identical in man and the rat) as the sites of epitopes recognized by the chronotropically active, beta(1)-agonis tic autoantibodies. Now we report on the mapping of these epitopes wit h the help of two series of short synthetic overlap peptides, one seri es forming part of the first and the other of the second extracellular loop of the beta(1)-adrenoceptor. Inhibition of the positive chronotr opic response of cultured rat cardiomyocytes to the anti-beta(1)-recep tor autoantibodies (EC(50) = 0.14 +/- 0.01 nM) from the serum immunogl obulin fraction of patients with DCM was taken as reflecting the neutr alization of these antibodies by a particular overlap peptide. In this way the sequences S-F-FC-E-L (residues 129-134) and A-R-R-C-Y-N-D (re sidues 206-212) emerged as the dominant epitopes in the first and seco nd extracellular loops, respectively, followed with respect to neutral izing ability by the first loop sequence E-Y-G-S-F-F (residues 126-131 ) and the second loop sequences H-W-W-R-A-E (residues 197-202) and P-K -C-C-D-F (residues 213-218). Synthetic peptides corresponding to the s equences of the third extracellular loop of the beta(1)-receptor (resi dues 346-356) and of the second extracellular loop of the human beta(2 )-receptor (residues 172-197) failed to neutralize the beta(1)-agonist ic autoantibodies. Using dithiothreitol as a reducing agent a disulfid e bridge between cysteine 132 in the first and cysteine 209 in the sec ond extracellular loop was considered to be essential for the chronotr opic action of these autoantibodies. The observed neutralizing power o f some of the short synthetic peptides employed in the present study a rgues for their introduction as potentially valuable agents into the i mmunotherapy of DCM.