Direct evidence for a beta 1-adrenergic receptor-directed autoimmune attack as a cause of idiopathic dilated cardiomyopathy - PubMed (original) (raw)

Direct evidence for a beta 1-adrenergic receptor-directed autoimmune attack as a cause of idiopathic dilated cardiomyopathy

Roland Jahns et al. J Clin Invest. 2004 May.

Abstract

Today, dilated cardiomyopathy (DCM) represents the main cause of severe heart failure and disability in younger adults and thus is a challenge for public health. About 30% of DCM cases are genetic in origin; however, the large majority of cases are sporadic, and a viral or immune pathogenesis is suspected. Following the established postulates for pathogenesis of autoimmune diseases, here we provide direct evidence that an autoimmune attack directed against the cardiac beta(1)-adrenergic receptor may play a causal role in DCM. First, we immunized inbred rats against the second extracellular beta(1)-receptor loop (beta(1)-EC(II); 100% sequence identity between human and rat) every month. All these rats developed first, receptor-stimulating anti-beta(1)-EC(II) Ab's and then, after 9 months, progressive severe left ventricular dilatation and dysfunction. Second, we transferred sera from anti-beta(1)-EC(II)-positive and Ab-negative animals every month to healthy rats of the same strain. Strikingly, all anti-beta(1)-EC(II)-transferred rats also developed a similar cardiomyopathic phenotype within a similar time frame, underlining the pathogenic potential of these receptor Ab's. As a consequence, beta(1)-adrenergic receptor-targeted autoimmune DCM should now be categorized with other known receptor Ab-mediated autoimmune diseases, such as Graves disease or myasthenia gravis. Although carried out in an experimental animal model, our findings should further encourage the development of therapeutic strategies that combat harmful anti-beta(1)-EC(II) in receptor Ab-positive DCM patients.

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Figures

Figure 1

Figure 1

Study protocol and anti–β1-AR Ab’s. (A) The plotted curve indicates mean titers plus or minus SEM (error bars) of specific rat anti–β1-ECII (IgG subclass) during the immunization experiment. Time points of subcutaneous immunizations are indicated by arrows. The inset depicts the immunoreactivities of IgG prepared from all 15 β1-ECII–injected rats 3 months after the first immunization determined by ELISA. (B) Scheme depicting concentrations of rat anti–β1-ECII (serum pool and transferred amounts) during the transfer experiment. Time points of intravenous serum transfer are indicated by arrows. The inset shows representative mean titers plus or minus SEM after transfer of 0.7 ∝g anti–β1-ECII/g BW over a 4-week period and the derived half-life for rat anti–β1-ECII.

Figure 2

Figure 2

Immunological and functional properties of rat anti–β1-ECII. (A) ELISA immunoreactivities of different rat and rabbit Ab’s (see key) with peptide antigens corresponding to selected domains of the β1- or β2-AR (N terminus (N); C terminus (C); and ECII-domain (ECII)) (14). (B) IFM with unfixed (IgG diluted 1:200) or (C) Western blots (IgG diluted 1:2,000) with lysates of Sf9 insect cells expressing recombinant human β1-AR, β2-AR, or the WT vector. (D) IFM colocalization experiments with HEK 293 cells transiently expressing β1-AR, β2-AR, or flag-tagged AT1a receptors (IgG diluted 1:200). Examples given are representative for IgG from β1-ECII–injected rats and a GST-injected rat (control); domain and β1-AR/β2-AR subtype-specific rabbit Ab’s (27) and a monoclonal mouse anti–AT1a flag Ab (28) served to immunostain the corresponding receptors (β1-AR, β2-AR, and AT1a receptor). (E) Increases in basal (–isoprenaline) or isoprenaline-stimulated (+10 ∝M/l isoprenaline) cAMP levels in Chinese hamster fibroblasts expressing human β1-AR (CHW-β1 cells) upon incubation with rat IgG (100 ∝g/ml). Columns represent amount of accumulated cAMP plus or minus SEM (error bars) obtained without (white, control) or in the presence of IgG from rats negative (light gray, GST/NaCl injected) or positive for functionally active anti–β1-ECII (black, β1-ECII injected). The stimulatory effects of anti–β1-ECII were blocked by 5 ∝M bisoprolol, a β1-selective receptor antagonist (dark gray). **P < 0.001 (ANOVA and Scheff– F post hoc test).

Figure 3

Figure 3

In vivo short-term effects of rat anti–β1-ECII. (A) Single representative tracing and (B) panels depicting the time course of systolic LV pressure, heart rate, and dp/_dt_max from naive Lewis/CrlBR rats after i.v. application of pooled sera from NaCl/GST-injected (control) or β1-ECII–injected rats (50 ∝l each). Arrows in A indicate time points of i.v. serum application. Error bars in B indicate mean plus or minus SEM of the indicated parameters normalized to the baseline (100%) at 0, 1, 2, 4, 6, and 8 minutes after serum application. *P < 0.05 (ANOVA and Bonferroni post hoc test). bpm; beats per minute.

Figure 4

Figure 4

Echocardiography of rat hearts. Digitally stored representative M-mode and Doppler images demonstrating (A) nonmarked and (B) online marked M-mode tracings from the same animal, off-line measured end-diastolic and end-systolic LV diameters (LVED/LVES) from (C) an anti–β1-ECII-positive and (D) a corresponding control animal (immunization experiment, month 15), and on-line determination of (E) LV outflow tract diameter (LVOT; M-mode tracings) and (F) aortic VTI in a same animal (Doppler tracings).

Figure 5

Figure 5

Echocardiographic follow-up. (A) Immunization and (B) transfer experiment. The panels depict the time course of selected echocardiographic parameters. Upper panels: LVED and end-systolic diameters (LVES). Middle panels: fractional shortening (%), derived from (LVED-LVES/LVED ∞ 100), and heart rate (bpm). Lower panels: Cardiac index corresponding to CO/BW. CO (milliliters per minute) was assessed by echocardiography (see Methods). Error bars indicate mean plus or minus SEM.*P < 0.05; **P < 0.01; ***P < 0.001 (ANOVA and Bonferroni post hoc test).

Figure 6

Figure 6

Anatomic measurements and histology of rat hearts. (A) The panel shows representative H&E-stained 1-∝m cross-sections of hearts from an anti–β1-ECII-positive rat and a control animal (left row, ∞10 magnification). The middle and right rows indicate schematically how LV diameter, wall thickness, and different cardiac areas were determined by computer-aided image analysis. IVS, interventricular septum; LVD, LV-diameter; PW, posterior wall; RV, right ventricular; RVCA, right ventricular cavity area. (B) Immunization and transfer experiment. Left panels: Columns represent heart wet weight (HW) and the relative heart weight (HW/BW). The insets show correlations between wet weight and echocardiographically predicted HW for all animals (Ab-positive and Ab-negative rats) in each experiment. Right panels: Columns represent LV diameter (LVD), LVCA, and LVWA. The insets show correlations between the anatomic and the echocardiographic LV diameter for all animals in each experiment. Error bars indicate mean values plus or minus SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student t test). (C) Immunization and transfer experiment. Left panels: H&E-stained 1-∝m sections of hearts from anti–β1-ECII-positive rats and corresponding control animals at ∞50 (upper rows) or ∞120 magnification (lower rows) with representative myocytes and corresponding nuclei. Right panels: Columns indicate the respective myocyte area (MA), nuclear area (NA), and the calculated nucleus-to-myocyte ratio (NA/MA). Error bars indicate mean values plus or minus SEM.

Figure 7

Figure 7

β1-AR/β2-AR subtypes and urine catecholamines. (A) Immunization and (B) transfer experiment. Columns represent total β-AR density and the amount of β1-AR and β2-AR subtypes (femtomoles per milligram of protein) in cardiac membranes of β1-ECII–injected (n = 15, aged 18 months) or anti–β1-ECII-transferred rats (n = 10, aged 15 months), and corresponding control animals at the end of each experiment (n = 20 or n = 10, respectively). Error bars indicate mean values plus or minus SEM. **P < 0.01; ***P < 0.001. The inset in A depicts a representative radioligand displacement curve using the β1-selective antagonist CGP 20712A (immunization experiment). (C) Immunization and (D) transfer experiment. Columns correspond to urine catecholamine concentrations determined at study end. Error bars indicate mean values plus or minus SEM.

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