Unmasking Features of the Auto-Epitope Essential for β1-Adrenoceptor Activation by Autoantibodies in Chronic Heart Failure

Figures

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  Figure 1. Neutralization of the stimulatory effects of rabbit anti‐β1ECII‐abs by pre‐incubation with cyclopeptides containing intra‐loop bonds C209↔C215 (18 C/C/S) or C209↔C216 (18 C/S/C): Stimulatory effects of polyclonal rabbit anti‐β1ECII‐abs on the human β1‐AR coupled to a CFP/YFP‐FRET sensor for intracellular cAMP, normalized to maximal stimulatory effects obtained by 1 μM (−)‐isoproterenol. Rabbit anti‐β1ECII‐abs were pre‐absorbed with the indicated cyclopeptides (40 mol CP/mol IgG). Unblocked: effect of stimulating rabbit anti‐β1ECII‐abs alone. Data are given as mean ± SEM (n ≥ 5 per experiment; differences between the conditions were analysed by one‐way ANOVA with subsequent Dunnett's post‐hoc test for multiple comparisons; **P < 0.01).

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  Figure 2. Schematic representation of the 22‐mer C/C/B‐β1 cyclopeptide mimicking the human β1ECII bearing the essential motif of the clinically relevant auto‐epitope: circles with capitals specify amino acids (single letter code). Numbers specify their position in the sequence of the human β1‐AR. ‘B' stands for the artificial amino acid α‐aminobutyrate substituting C216 in order to prevent a non‐physiological S‐S‐bond C209↔C216. Boxed letters N and C indicate the termini of the predicted β1ECII loop. The full line between C209 and C215 indicates the essential intra‐loop disulfide bridge; black circles depict amino acid residues essential for the neutralization of stimulating rodent anti‐β1ECII‐abs. Grey circles represent additional amino acid residues supposed to be relevant for the neutralization of stimulating human anti‐β1ECII‐aabs. Amino acid residues R207, E205, S203, and T220 were not found essential by directed mutational analysis.

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  Figure 3. Neutralization of the functional effects of polyclonal rat anti‐β1ECII‐IgG by 22‐mer cyclopeptides mimicking the human β1‐ECII with non‐conserved amino acids (compared with the amino acids constituting the ECII loop of the β2‐AR) sequentially replaced by alanine: sera of 20 cardiomyopathic Lewis rats (immunized with β1ECII/GST fusion proteins) were pre‐absorbed with the indicated cyclopeptide mutants (40 mol CP/mol IgG, 4°C, 16 h). Grey bars: IgG binding to the linear 25 AA (199–223) β1ECII peptide as determined by ELISA (triplicates). Black bars: β1‐AR‐mediated cAMP stimulation in HEK293 cells expressing native β1‐AR functionally coupled to a cAMP FRET sensor. Decreases in ELISA reactivity (grey bars) or receptor activation/cAMP stimulation (black bars) following pre‐absorption with the different cyclopeptide mutants are shown, normalized to the values obtained without blocking cyclopeptides. Columns represent mean values ± SEM of n = 20 rat sera. Differences between the non‐mutated 22 C/C/B‐β1 cyclopeptide and CP mutations were analysed by one‐way ANOVA with subsequent Dunnett's post‐hoc test for multiple comparisons; *P < 0.05; **P < 0.01; ***P < 0.001; †P < 0.0001. Internal…

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  Figure 4. Neutralization of human anti‐β1‐aabs from DCM patients by 22‐mer cyclopeptides mimicking β1ECII with non‐conserved amino acids (compared with the amino acids constituting the ECII loop of the β2‐AR) sequentially replaced by alanine: human anti‐β1‐aabs (IgG fractions) were pre‐absorbed with the indicated cyclopeptide mutants (40 mol/mol IgG, 4°C, 16 h). Anti‐β1‐aab‐induced cAMP production was measured in HEK293 cells expressing the native β1‐AR functionally coupled to a cAMP FRET sensor. (A) Representative recordings of FRET ratios obtained upon addition of anti‐β1‐aabs prepared from a male DCM patient followed by the maximal signal achieved with 1.0 μM of (−)‐isoproterenol (Iso). (B) Prevention of cAMP stimulation after pre‐absorption of patient anti‐β1‐aabs. Results are normalized to the values without pre‐absorption. Columns represent mean ± SEM from at least three to four independent experiments with IgG prepared from different exemplary DCM patients (two men and one woman). Differences between the non‐mutated 22 C/C/B‐β1 cyclopeptide and CP mutations were analysed by one‐way ANOVA with subsequent Dunnett's post‐hoc test for multiple comparisons; *P < 0.05; **P < 0.01; ***P <…

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  Figure 5. Stimulation of mutant β1‐ARs bearing point mutations within or flanking the presumed auto‐epitope of functional anti‐β1ECII antibodies: to ensure reproducibility of the test agent anti‐β1ECII antibody, HEK293 cells expressing human β1‐AR with the indicated point mutations were exposed to monoclonal anti‐β1ECII‐abs raised in mice (mouse Mab 23‐6‐7, 26 white columns) or rats (rat Mab 13F6, 26 grey columns). cAMP levels were determined in the cell lysates by radio‐immunoassay. For each experiment, increases in cAMP following antibody exposure were normalized to the maximal responses achieved with 1.0 μM of the full agonist (−)‐isoproterenol. Data are given as mean ± SEM of at least six independent experiments per column. Differences between the wild‐type β1‐AR and the indicated mutants were analysed by one‐way ANOVA with subsequent Dunnett's post‐hoc test for multiple comparisons; **P < 0.01; †P < 0.0001.