Modulation of the beta-adrenergic response of cardiomyocytes by specific lipoxygenase products involves their incorporation into phosphatidylinositol and activation of protein kinase C

Arachidonic acid and its 15-lipoxygenase metabolite (15S)-hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid ((15S)-HETE) modulates the beta-adrenergic response of cultured rat neonatal cardiomyocytes as indicated by an increase in the beating rate following stimulation of the cells with suboptimal isoproterenol concentrations. The effect of 15-HETE was enantioselective for the S-isomer and could be detected at concentrations as low as 10(-12) M. (11S)-HETE and (5S,15S)-dihydroxy-(6E,8Z,11Z,13E)-eicosatetraen oic acid did also exhibit this effect, whereas other mono-, di-, and trihydroxyeicosanoids as well as the 15-lipoxygenase products of 11,14-eicosadienoic acid, of two eicosatrienoic acid isomers and of 5,8,11,14,17-eicosapentaenoic acid were ineffective. Immunohistochemical studies indicated the expression of a 15-lipoxygenase in cardiomyocytes and in resident heart mast cells. Induction of the beta-adrenergic supersensitivity is paralleled by a selective incorporation of (15S)-HETE into the cellular phosphatidylinositol pool. In contrast, (12S)-HETE, which did not induce beta-adrenergic supersensitivity, was incorporated preferentially into phosphatidylcholine and phosphatidylethanolamine. Calphostin C, an inhibitor of protein kinase C, blocked both the induction of supersensitivity by (15S)-HETE and its incorporation into phosphatidylinositol. These data suggest that in cardiomyocytes 15-lipoxygenase metabolites specifically induces a signal transduction cascade leading to a supersensitivity of the cells toward beta-adrenergic agonists, which involves the phosphatidylinositol cycle and a protein kinase C

Anti-beta1-adrenoceptor autoantibodies with chronotropic activity from the serum of patients with dilated cardiomyopathy: mapping ofepitopes in the first and second extracellular loops

In a preceding communication (Wallukat et al., 1992, Z Kardiol 81 [Suppl. 4]: 79-83), it was reported that synthetic peptides, corresponding in amino acid sequence to either the first or the second extracellular loop of the human beta 1-adrenoceptor, selectively suppressed the metoprolol- and bisoprolol-sensitive positive chronotropic action exerted in cultures of beating neonatal rat cardiomyocytes by the serum immunoglobulin fraction of patients with myocarditis and idiopathic dilated cardiomyopathy (DCM) and by affinity-purified autoantibodies from that 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 which patients with DCM are believed to be exposed. Specifically, they pointed to the putative first and second extracellular loops of this receptor (these loops are each identical in man and the rat) as the sites of epitopes recognized by the chronotropically active, beta 1-agonistic autoantibodies. Now we report on the mapping of these epitopes with the help of two series of short synthetic overlap peptides, one series forming part of the first and the other of the second extracellular loop of the beta 1-adrenoceptor. Inhibition of the positive chronotropic response of cultured rat cardiomyocytes to the anti-beta 1-receptor autoantibodies (EC50 = 0.14 +/- 0.01 nM) from the serum immunoglobulin fraction of patients with DCM was taken as reflecting the neutralization of these antibodies by a particular overlap peptide. In this way the sequences S-F-F-C-E-L (residues 129-134) and A-R-R-C-Y-N-D (residues 206-212) emerged as the dominant epitopes in the first and second extracellular loops, respectively, followed with respect to neutralizing 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 sequences of the third extracellular loop of the beta 1-receptor (residues 346-356) and of the second extracellular loop of the human beta 2-receptor (residues 172-197) failed to neutralize the beta 1-agonistic autoantibodies. Using dithiothreitol as a reducing agent a disulfide bridge between cysteine 132 in the first and cysteine 209 in the second extracellular loop was considered to be essential for the chronotropic action of these autoantibodies

Autoantibodies against the beta- and muscarinic receptors in cardiomyopathy

The sera of patients with idiopathic dilated cardiomyopathy and the Chagas' disease contain agonist-like autoantibodies directed against the {beta} 1-adrenoceptor and/or the muscarinic M2-receptor. The anti-{beta} 1- adrenoceptor antibodies could be directed against amino acid sequences of the first or second extracellular loop. In patients with dilated cardiomyopathy the first as well as the second extracellular loop was identified as an antibody epitope. In Chagas' disease the anti-{beta} 1-adrenoceptor antibody recognizes only 1 epitope on the second extracellular loop. The anti-{beta} 1- adrenoceptor antibodies acting like the {beta}-adrenergic agonist isoprenaline and exert a positive chronotropic effect in cultured rat cardiomyocytes. In contrast to isoprenaline the antibody caused no downregulation of the {beta}- adrenergic signal transduction cascade within 6 hours. The anti-M2-receptor antibodies recognize in both diseases an epitope on the second extracellular loop. The anti-M2-receptor antibody exert a negative chronotropic response in cultured cardiomyocytes. This antibody induced no downregulation of the muscarinic M2-receptor. The negative chronotropic effect was unabated for 6 hours. Based on these findings it is believed that the agonist-like autoantibodies that act against the {beta} 1-adrenoceptor and the muscarinic M2- receptor may play a role in the pathogenesis of dilated cardiomyopathy and Chagas' disease

[Autoantibodies against the beta 1-adrenergic receptor in myocarditis and dilated cardiomyopathy: localization of two epitopes] Autoantikörper gegen den beta 1-adrenergen Rezeptor bei Myokarditis und dilatativer Kardiomyopathie: Lokalisation von zwei Epitopen

Sera of patients with myocarditis and dilated cardiomyopathy contain stimulatory autoantibodies directed specifically against the beta 1-adrenergic receptor. The binding of the antibodies could be localized to either the first or the second extracellular loop of the beta 1-adrenoceptor. In 73% of the cases investigated the antibodies recognized the second extracellular loop. The agonistic effects of the antibodies were abolished by beta-adrenergic antagonists. Furthermore, the antagonists were able to remove the antibodies from their binding sites