Myocardial Recovery in Patients With Systolic Heart Failure and Autoantibodies Against β 1 -Adrenergic Receptors

Abstract

BACKGROUND Among various cardiac autoantibodies (AAb), those recognizing the β1 adrenergic receptor (β1AR) demonstrate agonist-like effects and induce myocardial damage that can be reversed by β-blockers and immunoglobulin G3 (IgG3) immunoadsorption. OBJECTIVES We investigated the role of β1AR-AAbs belonging to the IgG3 subclass in patients with recent-onset cardiomyopathy. METHODS Peripheral blood was drawn at enrollment in subjects with recent-onset cardiomyopathy (left ventricular ejection fraction [LVEF] ≤0.40; <6 months). Presence of IgG and IgG3-β1AR-AAb was determined and echocardiograms assessed at baseline and 6 months. Subjects were followed for up to 4 years. RESULTS Among the 353 enrolled subjects, 62 (18%) were positive for IgG3-β1AR-AAb (IgG3), 58 (16%) were positive for IgG but not IgG3 (non-IgG3), and the remaining were negative. There were no significant differences in baseline systolic blood pressure, heart rate, or LVEF among the groups at baseline. LV end-diastolic and end-systolic (LVEDD and LVESD, respectively) diameters were significantly larger in the non-IgG3 group compared to the other groups (LVEDD: p < 0.01; LVESD: p = 0.03). At 6 months, LVEF was significantly higher in the IgG3 group (p = 0.007). Multiple regression analysis demonstrated IgG3-β1AR-AAb was an independent predictor of LVEF at 6 months and change in LVEF over 6 months, even after multivariable adjustment (LVEF at 6 months, β = 0.20, p = 0.01; change in LVEF, β = 0.20, p = 0.008). In the subjects with high New York Heart Association functional class (III or IV) at baseline, the IgG3 group had lower incidence of the composite endpoint of all-cause death, cardiac transplantation, and hospitalization due to heart failure, whereas the non-IgG3 group had the highest incidence of the composite endpoint. CONCLUSIONS IgG3-β1AR-AAb was associated with more favorable myocardial recovery in patients with recent-onset cardiomyopathy.

Antigen-free control wells in an ELISA set-up for the determination of autoantibodies against G protein-coupled receptors—a requisite for correct data evaluation

“…1Comparison of the binding of different IgG samples onto specific (target-peptide-antigen-coated) and non-specific (non-coated, coating buffer activated) 96-well ELISA plates. Specific wells (dark grey columns) were coated with 2.5 μg target-peptide/well (target-peptide corresponding to the second extracellular loop of the beta1-adrenoceptor, HWWRAESDEARRCYNDPKCCDFVTNR, for trapping autoantibodies directed at this second extracellular loop) freshly dissolved in 0.1 M carbonate coating buffer overnight at 4 °C according to Nagatomo et al [3]. Control wells (non-coated, activated wells) were incubated with the peptide-free coating buffer only (light grey columns).…”

“…According to the discussion forum about this topic in ResearchGate [1], different blocking/buffer conditions were tested. One, the conventional blocking buffer system consisting of 1% BSA/Tween 20 in PBS and the dilution buffer consisting of 20 mmol/L Tris-HCl, 150 mmol/L NaCl, 4 mmol/L KCl, and 0.05% Tween 20, pH 7.2 (TVP) ( a – c ) and a second, published by Nagatomo et al [3], consisting of PBS containing 3% skim milk, 0.1% Tween 20, and 0.01% merthiolate (PMT) ( d – f ). Washing conditions were comparable, at each washing step 3 × 200 μL/well washing buffer: 20 mmol/L Tris-HCl, 150 mmol/L NaCl, 4 mmol/L KCl, and 0.1% Tween 20, pH 7.2.…”

“…Specific wells were coated with 2.5 μg target-peptide/well (target-peptide corresponding to the second extracellular loop of the beta1-adrenoceptor, HWWRAESDEARRCYNDPKCCDFVTNR, for trapping autoantibodies directed at this second extracellular loop) freshly dissolved in 50 μL/well 0.1 M carbonate coating buffer overnight at 4 °C according to Nagatomo et al [3]. Control wells (non-coated) were incubated with the peptide-free coating buffer only.…”

“…After removing the coating buffers and washing (3 × 200 μL, washing buffer: 20 mmol/L Tris-HCl, 150 mmol/L NaCl, 4 mmol/L KCl, and 0.1% Tween 20, pH 7.2), the wells were blocked using ( a – c ) 1% bovine serum albumin (BSA) and 0.05% Tween 20 in phosphate buffered saline (PBS) or ( d – f ) PBS containing 3% skim milk, 0.1% Tween 20, and 0.01% merthiolate (PMT) according to Nagatomo et al [3]. The plates were incubated at 22 °C for 1 h, the blocking solutions were removed, the plates were washed again, and the samples were applied in ( a – c ) conventional dilution buffer consisting of 20 mmol/L Tris-HCl, 150 mmol/L NaCl, 4 mmol/L KCl, and 0.05% Tween 20, pH 7.2 (TVP) or ( d – f ) the PMT buffer again and were incubated at 22 °C for 2 h. After decanting the samples and washing, the wells were incubated with the secondary anti-human IgG (H+L)-POD antibody diluted 1:10,000 in 1% BSA/PBS ( a – c ) and PMT ( d – f ) and after washing the tetramethylbenzidine/H 2 O 2 detection at 450 nm (reference filter 620 nm) followed, using an Anthos HTII plate reader.…”

“…Here, we simulated such a constellation using a target-peptide ELISA developed and published by Nagatomo et al [3] for the detection of autoantibodies against the beta1-adrenoceptor.…”