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Cardiovascular Research 2007 76(1):51-60; doi:10.1016/j.cardiores.2007.05.022
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Copyright © 2007, European Society of Cardiology

Anti-β1-adrenergic receptor autoantibodies are potent stimulators of the ERK1/2 pathway in cardiac cells

Antonio S. Tutor1, Petronila Penela and Federico Mayor, Jr.*

Departamento de Biología Molecular and Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid, 28049 Madrid, Spain

*Corresponding address. Tel.: +34 91 497 4865; fax: +34 91 497 4799. fmayor{at}cbm.uam.es

Received 19 February 2007; revised 3 May 2007; accepted 21 May 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results and discussion
 Supplementary data
 References
 
Objective Antibodies specific for the β1-adrenergic receptor (β1AR) are highly prevalent in patients with idiopathic dilated cardiomyopathy (DCM) and known to contribute to the pathogenesis of heart failure, though the precise molecular mechanisms involved are largely unknown.

Methods We have explored the effects of β1AR autoantibodies obtained from DCM patients on extracellular signal-regulated kinase (ERK) activation in murine cardiomyocytes.

Results We find that human β1AR autoantibodies potently stimulate ERK1/2 in cardiac cells by using signalling pathways different from those triggered by the classic β-agonist isoproterenol, also leading to a different pattern of activated ERK subcellular localization. The extent of ERK stimulation by endogenous cardiac β1AR is markedly enhanced in the presence of both β1AR-autoantibodies and isoproterenol. Interestingly, β1AR-autoantibody-mediated ERK activation is not blocked by some βAR antagonists used in the treatment of heart failure.

Conclusions Our results suggest that these antibodies elicit a distinct β1AR active conformation that would lead to the engagement of signaling effectors different from those recruited by classic β-agonists, a finding that could lead to better understanding of DCM pathogenesis and aid in designing diagnostic and therapeutic strategies.

KEYWORDS Adrenergic receptors; Autoantibodies; MAP kinase; Cardiomyopathy; Signal transduction

This article is referred to in the Editorial by T.E. Hébert (pages 5–7) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results and discussion
 Supplementary data
 References
 
Dilated cardiomyopathy (DCM), a heart condition characterized by left ventricular dilatation and progressive loss of cardiac function, represents the main cause of severe heart failure in younger adults in Western countries (American Heart Association, 2005). Mutations in myocyte structural protein genes and cardiotoxic substances account for about one third of DCM cases, whereas the etiology of the remaining 70% is poorly understood [[1,2] and references therein]. Persistent viral infection or autoimmune damage to cardiomyocytes have been hypothesized to play a major role in the triggering or progression of idiopathic DCM [reviewed in [1–5]]. Interestingly, specific antibodies against the second extracellular loop of the β1-adrenergic receptor (anti-β1AR autoantibodies) are highly prevalent in DCM patients (30 to 90%, depending on clinical studies or screening method [6–8], and, to a lesser extent (10%) in patients with ischemic cardiomyopathy [9]. Antibodies cross-reacting with this β1AR region are also present in about 30% of patients with autoimmune Chagas disease [10], one of the most common causes of chronic heart failure. On the contrary, anti-β1AR autoantibodies (β1AR-Ab), show very low prevalence in healthy individuals, or in patients with heart failure due to valvular or hypertensive heart disease [2,3,11].

Overall, these data suggested that these antibodies may contribute to the pathogenesis of DCM and heart failure of certain etiologies. Consistent with this notion, the presence of anti β1AR-Ab is associated with a higher prevalence of ventricular arrhythmias [12], incidence of sudden cardiac death [13], increased mortality [14] and poor left ventricular function [9], whereas removal of anti-β1AR-Ab by immunoadsorption therapy improves hemodynamic parameters in DCM patients [15–17].

Recently, Jahns et al. have presented direct evidence that β1AR autoantibodies play a causal role in DCM and not merely correlate or are a consequence of myocardial inflammation and cardiac tissue injury [1]. Immunization of inbred rats against the second extracellular β1AR loop raised stimulating β1AR-Abs and promoted selective β1AR downregulation and progressive left ventricular dilatation and dysfunction, features characteristic of human DCM. Moreover, this phenotype was also reproduced in healthy isogenic rats by transferring sera from anti-β1AR-Abs-positive animals, thus fulfilling the criteria of an autoimmune disease [1,4]. β1AR-Abs have been shown to promote selective downregulation of cardiac β1AR and mild activation of the cAMP/PKA pathway in heterologous cell systems expressing β1AR [1,9,18]. However, very little is known about the functional effects of human β1AR-Abs on key signaling pathways triggered by endogenous βAR in cardiac cells, what is essential to better understand their pathological role.

Given the important role of the ERK1/2 kinase cascade in cardiac hypertrophy and heart failure [19–22], we have explored the effect of β1AR-Abs from DCM patients on ERK1/2 activation in cardiomyocytes. We report that anti-β1AR autoantibodies induce a potent MAPK stimulation by mechanisms different from that triggered by the β-agonist isoproterenol. Moreover, their effect is synergic with that of β-agonists and is differentially affected by certain βAR antagonists used in the treatment of heart failure.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results and discussion
 Supplementary data
 References
 
2.1 Cell culture
Primary cultures of mouse cardiomyocytes were prepared from 1-day old animals as reported [23].All animal experiments conformed with the guidelines of the "European Convention for the protection of vertebrate animals used for experimental and other scientific purposes" (Directive 86/609/EEC) and with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). The murine cardiomyocyte cell line HL-1, that displays phenotypic features typical of adult cardiomyocytes [24–26], were obtained from Dr. W.C. Claycomb, Louisiana State University Medical Center, New Orleans, LA. It should be noted that there is 100% sequence identity in the second extracellular loop of mouse and human β1ARs. Cells were grown as monolayers in Claycomb MediaTM (JRH Biosciences, Lenexa, KS) and supplemented with 10% fetal bovine serum, 0.1 mM norepinephrine, 2 mM L-glutamine, and 1x antibiotic/antimycotic solution. All culture dishes and flasks were pre-coated with 0.00125% fibronectin (Sigma) in 0.02% gelatin (BD Biosciences). HEK293 cells purchased from the American Type Culture Collection (Manassas, VA) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum. Cells were maintained at 37 °C in a humidified atmosphere of 95% air plus 5% CO2.

2.2 Transfections
HEK293 cells were transfected with a cDNA coding for human β1-adrenergic receptor in the pcDNA3 expression vector with LipofectAMINE (Life Technologies). Cells were selected with 200 µg/ml hygromycin B, and resistant cells were screened for receptor expression by radioligand binding, immunofluorescence and flow cytometry. After subcloning by serial dilution, subclones with high receptor expression (800–1300 fmol/mg of total protein) were propagated.

2.3 Detection of autoantibodies against beta 1 adrenergic receptor in idiophatic dilated cardiomyopathy patient's sera
Sera obtained from 20 healthy donors and twenty patients diagnosed with idiopathic dilated cardiomyopathy admitted at the COMET trial were generously provided by Dr. I. Terol (Hospital Carlos III, Madrid). The investigation conforms with the principles outlined in the Declaration of Helsinki. Patients had a mean ejection fraction of 0.26, New York Heart Association chronic heart failure class II to IV, and ≥ cardiovascular admission within the last 2 years. Blood was collected and fractioned, and the serum was stored at –70°C until needed. HEK-293 cells stably transfected or not with human β1AR were coated on 96 well microtiter plates. After blocking with PBS plus 3% BSA, human serum (different dilutions in PBS/1%BSA/0.05% Tween 20) was added (1 hr at 37°C) and antigen–antibody complexes were detected using peroxidase-conjugated goat anti-human IgG as secondary antibody OPD was used as the chromogen. Optical density was measured on a BioRad spectrophotometer at 405 nm. Sera were considered positive to β1AR antibodies when the signal obtained with β1AR-expressing cells was at least two-fold that obtained with wild-type cells. When needed, purified serum IgG was prepared by caprylic acid precipitation as reported [1] and its purity analyzed by SDS-PAGE and Coomassie staining.

2.4 Cell treatment and analysis of ERK activation
The media of cells grown at 70–80% confluence was replaced with serum-and norepinephrine-free medium. After overnight culture, cells were treated with isoproterenol and/or DCM or control IgGs for the indicated periods of time and at the desired concentrations. In order to dissect the signal transduction pathways involved in ERK activation, HL-1 cells were pretreated with H-89 (a PKA inhibitor, 10 µM for 1 h), pertussis toxin (100 ng/ml for 16 h), PP2 (a Src tyrosine kinase inhibitor, 10 µM for 15 min) or monodansylcadaverine (a receptor internalization inhibitor, 300 µM for 1 h). Cells were re-suspended in 0.4 ml of RIPA buffer (0.5% deoxycholate, 50 mM Tris–HCl, pH 7.4, 200 mM NaCl, 1% Triton x100, 0.1% SDS, 2 mM Na3VO4, 10 mM NaF and protease inhibitors). After incubation for 2 h at 4°C, the lysates were clarified by centrifugation at 14,000 xg for 15 min. Aliquots corresponding to 25 µg of protein were mixed with SDS loading buffer, and resolved using 12% SDS-Polyacrylamide gel electrophoresis. MAPK activation was detected by Western blot analysis using an anti-phospho-p42/p44 MAP kinase (Thr 202/Tyr 204) polyclonal antibody (Cell Signaling). Results were normalized for total ERK by re-probing the same blots with anti-ERK polyclonal antibodies (Santa Cruz Biotechnology). In some experiments, cells were preincubated for 3 min with or without different β-blockers: Atenolol (10–5 M), Bisoprolol (10–5 M), Propanolol (10–5 M), Carvedilol (10–5 M), Metoprolol (10–5 M) or Labetolol (10–4 M), kindly provided by Dr. M. Bouvier (University of Montreal, Canada), or with the specific β1AR antagonist CGP20712A (10 µM) or the β2AR antagonist ICI118551 (1 µM),before challenge with isoproterenol or DCM IgGs and analysis of ERK activation as above. Concentrations of beta-blockers known to completely inhibit the β1AR-stimulated adenylyl cyclase pathway were used.

For the analysis of nuclear and extranuclear pools of endogenous ERK1/2, HL-1 cells we stimulated with Isoproterenol (10 µM) or DCM IgGs (50 µg/ml) for 10 min. Monolayers were washed twice with ice-cold phosphate-buffered saline and collected in 2 ml of hypotonic lysis buffer (10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.3% (v/v) Nonidet P-40, 2 mM Na3V04, 10 mM NaF and protease inhibitors). Cells were incubated on ice for 10 min to allow lysis, and were then centrifuged at 500 xg for 5 min to obtain the nuclear fraction. The supernatant (containing a mixture of plasma membrane, microsomal vesicles, cytoskeleton, and cytosol) represented the extranuclear fraction. Pellets containing cell nuclei were washed in lysis buffer without Nonidet P-40 and again pelleted at 500 xg. Both the extranuclear and nuclear fractions were solubilized in 2x Laemmli sample buffer and phospho-ERK1/2 was determined by protein immunoblotting as above. Correct cell fractionation was verified by immunoblot analysis with antibodies to the cytoplasmic marker tubulin and the nuclear marker PSF (protein-associated splicing factor). As an alternative approach to investigate the subcellular localization of activated ERK, HL-1 cells were seeded onto fibronectin and gelatin-coated 35-mm glass bottom dishes and serum-starved overnight. Cells were challenged with agonists as above. Immunofluorescent staining of endogenous activated ERK1/2 was assessed by incubation with a rabbit polyclonal anti-phospho-ERK1/2 antibody in PBS containing 1% BSA (1:500 dilution, 1 h at room temperature), followed by a Fluorescein-conjugated polyclonal anti-rabbit IgG antibody (1:500) in 1% BSA, 1% FBS for 1 h at room temperature. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Labelling was then analyzed using a confocal microscope.


   3. Results and discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results and discussion
Supplementary data
 References
 
In order to analyze the effects of human β1AR-Abs on MAPK signaling pathways in cardiac cells, we first screened the sera of 20 DCM patients and 20 healthy donors for the presence of such antibodies, by using a method based on the recognition of native human β1AR expressed in intact HEK-293 cells (see Methods). In line with the prevalence reported by others using similar screening procedures [[3] and references therein], β1AR-Ab were found to be present in 6 out of 20 DCM patients, and absent in all healthy individuals. Purified IgG fractions from healthy controls, β1AR-Ab-positive and β1AR-Ab-negative DCM patients were then tested for their ability to stimulate the ERK1/2 pathway using the HL-1 mouse cardiomyocyte cell line, which express endogenous βAR, predominantly of the β1AR subtype [24,25]. Interestingly, the presence of IgG from most (5 out of 6) β1AR-Ab-positive DCM patients (DCM+) promoted a marked (3 to 5-fold) increase in endogenous ERK1/2 activation, that was not observed with similar samples from healthy controls or DCM patients negative for β1AR-Ab (DCM-) (Fig. 1A and B). Such stimulatory effect was strongly inhibited in the presence of CGP20712A, a specific β1AR antagonist, thus indicating that it was mediated by endogenous β1AR receptors (Fig. 1C). β1AR antibodies also promoted a strong activation of the ERK pathway in HEK 293 cells transfected with the human β1AR, again completely blocked in the presence of CGP20712A (Suppl. Fig. 1A), suggesting that β1AR from different species respond similarly to β1AR antibodies to trigger the ERK cascade. Activation of ERK by purified DCM+ IgGs was dose-dependent (Suppl. Fig. 1B), and readily observable in the range of 5 to 50 µg/ml, concentrations similar or lower than those previously used to test their effects in vitro or attained in vivo [1]. Fold-stimulation of ERK by such concentrations of DCM+ IgGs in HL-1 cells was similar or higher to that triggered by the presence of 1–10 µM isoproterenol, a potent beta-adrenergic agonist (Suppl. Fig. 1C), thus stressing their potential physiological relevance. Similar results were obtained when using a primary culture of mouse cardiomyocytes instead of HL-1 cells (Fig. 1D), including the inhibition of IgG-mediated ERK stimulation by a β1AR antagonist. Overall, these data strongly suggest that β1AR autoantibodies present in DCM patients are able to potently trigger the ERK cascade by activating endogenous β1ARs in cardiac cells.


Figure 1
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  		Fig. 1 β1AR autoantibodies are potent stimulators of the ERK 1/2 pathway in cardiac cells. A and B) HL-1 cells were incubated for 5 min with IgGs (50 µg/ml) obtained from different DCM patients either positive (β1AR-Ab-positive), or negative (β1AR-Ab-negative) for β1AR autoantibodies, or from healthy controls, and ERK 1/2 activation assessed as detailed in Methods using specific anti-phospho ERK antibodies (P-ERK). Blots were subsequently reprobed for total cellular ERK 1/2 levels to normalize the results. Data are represented as fold-stimulation of ERK activity over basal conditions (no IgG added), and are mean (±SE) of 4–5 independent experiments. ERK stimulation by IgGs from β1AR-Ab-positive patients was significantly different (p<0.01) when compared with the two other groups of samples (ANOVA followed by Fisher's or Scheffé's test). In panel B, ERK activation by IgGs from control (C1, C2) or DCM patient positive (P8, P12, P18) or negative (P3, P4) for β1AR-antibodies was compared in the same gel. C and D). The stimulatory effect of β1AR-autoantibodies is mediated by endogenous β1AR. HL-1 cells (C) or mouse cardiomyocytes (D) were challenged for 5 min with the indicated concentration of IgGs obtained from a β1AR-Ab-positive DCM patient (number P18) in the absence or presence of the β1AR antagonist CGP20712A (10 µM), and ERK activation analysed as in panel A. Data are expressed as percentage of ERK activation in the absence of antagonist (C) or as fold-stimulation over basal in the different experimental conditions (D), and are mean (±SE) of 3 independent experiments. *p<0.05 when compared to DCM-stimulated cells in panels C and D (paired t-test, two-tailed). Representative blots are shown for all the panels.

 
In order to characterize this new effect, we performed a detailed comparative analysis of ERK stimulation by IgGs from DCM+ patients or the β-agonist isoproterenol (Iso) in HL-1 cells. Consistent with the predominant expression of β1AR vs β2AR reported in these cells[24,25], Iso-mediated ERK stimulation was markedly inhibited in the presence of the specific β1AR antagonist CGP20712A (Suppl. Fig. 2A) and not affected by co-incubation with the β2AR antagonist ICI118551, that also did not alter DCM IgGs-mediated ERK activation (Suppl. Fig. 2B). Overall, these data clearly established that both Iso and β1AR-Abs stimulate the ERK pathway in HL-1 cells by triggering β1ARs. As shown in Figs. 2A and B, both agents promoted a similar time-dependent and transient ERK activation pattern. More interestingly, activation of the ERK pathway by Iso or DCM IgGs was differentially affected by the presence of pharmacological inhibitors of several signaling pathways (Fig. 2C). In HL-1 cells ERK activation by DCM IgGs was partially inhibited in the presence of the PKA inhibitor H-89, unaffected by incubation with pertussis toxin or when blocking receptor endocytosis with monodansylcadaverine (MDC), and markedly reduced in the presence of the Src tyrosine kinase inhibitor PP2. Similar results were obtained when using IgGs from other β1AR-antibodies-positive DCM patients (Suppl. Fig. 3). In contrast, ERK stimulation by the classic agonist Iso was markedly inhibited by H-89, unaffected by pertussis toxin or PP2 treatment and partially inhibited in the presence of MDC.


Figure 2
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  		Fig. 2 Comparative analysis of ERK stimulation by β1AR autoantibodies or isoproterenol in cardiac cells. A and B) Time-course of ERK 1/2 activation. HL-1 cells were incubated for different times with the indicated concentrations of IgGs obtained from a β1AR-Ab-positive DCM patient (P18) (panel A) or the β-agonist isoproterenol (panel B), and ERK 1/2 stimulation assessed as detailed in Methods and Fig. 1A. Results are expressed as fold-activation over basal conditions and are mean (±SE) of 4 independent experiments. *p<0.05 versus basal conditions (paired t-test, two-tailed). Representative blots are shown. C) β1AR autoantibodies and isoproterenol promote ERK 1/2 activation by different intracellular signaling pathways. HL-1 cells were incubated for 5 min with the desired agonist as in panel A (control conditions) or after treatment with the PKA inhibitor H-89, the tyrosine kinase inhibitor PP2, the receptor internalization inhibitor monodansylcadaverine (MDC) or pertussis toxin (PTX), as detailed in Methods. ERK 1/2 stimulation was assessed as in Fig. 1A. Data are expressed as percentage of ERK activation in control conditions and are mean (±SE) of 3 independent experiments. *p<0.05, **p<0.01 when compared to stimulation by DCM IgGs in the absence of inhibitors; #p<0.05, ##p<0.01 when compared to stimulation by Iso in the absence of inhibitors (paired t-test, two-tailed).

 
These results indicate that activation of β1AR by Iso or autoantibodies trigger different signal transduction pathways in order to stimulate ERK1/2. Both pathways involve PKA stimulation, that apparently is not related to the PKA-phosphorylation mediated coupling switch from Gs to Gi previously reported for the β2AR [27] or for β1AR expressed in heterologous systems [28,29], since pertussis toxin does not affect this process in either case. However, the signaling route triggered by the two stimuli clearly differs in their requirement for Src-like tyrosine kinase activity (essential for autoantibodies, not required for Iso) and in the effect of receptor internalization inhibitors (that only reduces Iso-mediated ERK stimulation).

Overall, these data suggested that binding of autoantibodies to β1AR would elicit or stabilize an active receptor conformation different from that promoted by classic agonists such as isoproterenol, what would result in changes in the composition and/or stability of β1AR-associated signaling complexes.

Since activation of the ERK pathway by different receptor-stimulated mechanisms has also been reported to lead to preferential stimulation of subcellular ERK pools [30], we analysed this parameter by both subcellular fractionation and confocal microscopy approaches. Consistent with the involvement of distinct signaling pathways in Iso or β1ARs-Abs-mediated ERK stimulation, we find that such β1AR agonists also promote a different pattern of activated ERK subcellular localization. In cultured cardiac HL-1 cells, β1AR-Abs trigger a larger ERK1/2 stimulation in nucleus vs cytoplasm, whereas the opposite is true for Iso (Fig. 3A and B). Both nuclear and cytoplasmic staining was noted by confocal microscopy upon dual stimulation. Thus, it is tempting to suggest that ERK activation by receptor autoantibodies would lead to regulation of a different set of ERK targets, compared to physiological activation of β1AR by catecholamines, thus contributing to cardiac failure.


Figure 3
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  		Fig. 3 β1AR autoantibodies and isoproterenol promote a different pattern of activated ERK subcellular localization in cardiac cells. HL-1 cells were challenged with DCM IgGs or isoproterenol as in Fig. 2C, and the presence of activated ERK was assessed in nuclear and cytoplasmic fractions as detailed in Methods and in previous figures (panel A) or by immunofluorescence labelling and confocal microscopy analysis (panel B). Correct cell fractionation was verified by immunoblot analysis with antibodies for tubulin and PSF markers (see Methods). In panel B, cell nuclei were stained with DAPI. Data in A are mean (±SE) of three independent experiments. Representative blots or immmunofluorescence fields are shown.

 
The participation of β-arrestin as a MAPK module scaffold in response to stimulation of different GPCR has been suggested to result in more efficient activation of a cytoplasmic ERK pools [[31,32] but see also [33]]. In addition, differential coupling of receptor variants to downstream effectors (as reported for Angiotensin receptor mutants) also leads to change in the proportion of nuclear versus cytoplasmic ERK activation [34]. The detailed mechanisms by which β1AR-Abs and Iso promote a different subcellular pattern of ERK activation remain to be investigated.

Heart failure is characterized by chronic stimulation of heart β1AR as a consequence of increased sympathetic activity and elevated catecholamine levels [35]. Many in vitro and in vivo studies indicate that β1AR hyperstimulation, in addition to promoting a variety of changes in the βAR signaling and regulatory system in cardiomyocytes, is critical in promoting the cardiomyopathic phenotype [[4], and references therein]. In fact, a variety of β-AR antagonists are currently used for the treatment of heart failure patients [36–38]. Although anti-β1AR-Abs-induced experimental immune cardiomyopathy has been suggested to mimic low-dose isoproterenol-induced heart failure models [1, 39], the potential functional interplay between the chronic catecholamine override and the circulating β1AR-Abs present in many patients had not been addressed, nor the effects of β-blockers on β1AR-Abs-mediated signalling.

In this context, we tested the effects of the simultaneous presence of low doses of both β1AR-autoantibodies and isoproterenol on ERK activation in cardiac cells. Fig. 4 indicates that Iso and two different DCM IgGs display a marked "sensitising" effect on ERK activation in both HL-1 cells and cultured mouse cardiomyocytes. A strong ({approx}3-fold over basal values) ERK stimulation is observed when both types of agonists are present, whereas such activation is barely detectable when only one of them is added to the cell media at these doses. The time-course of ERK activation upon dual stimulation (Suppl. Fig. 4) is transient, with the peak activation attained at 5–10 min, consistent with a situation integrating signals from β-agonists (peak at 5 min, Fig. 2B) and β1AR-Abs (peak at 10 min, Fig. 2A). The increased extent of ERK signaling could be a consequence of the simultaneous triggering of the different receptor downstream pathways used by Iso and β1AR-Abs to stimulate ERK. Alternatively, or in addition, conformational changes induced in endogenous β1AR by either of the agonists may favour receptor activation by the other stimuli. From the physiological point of view, these data suggest that the concurrence of elevated catecholamines and β1AR-autoantibodies that is likely to occur in DCM patients during the progression of the disease would reinforce each other and result in enhanced β1AR signaling and heart damage. How these pathways operate during the progression to heart failure when β1AR are downregulated and other components and regulators of the β1AR transduction system are altered [4] remains to be investigated.


Figure 4
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  		Fig. 4 Effect of the simultaneous challenge of cardiac cells with β1AR autoantibodies and isoproterenol on ERK activation. HL-1 cells (panel A) were incubated for 5 min with low doses of isoproterenol (0.1 µM) or IgGs (2.5 µg/ml) obtained from two different β1AR-Ab-positive DCM patients (P12 and P18), or with both stimuli simultaneously as indicated. ERK stimulation was assessed as in previous figures. Similar experiments were performed in cultured mouse cardiomyocytes (panel B), using IgGs from patient P18. In both panels results are expressed as fold-stimulation over basal conditions (no agonist added), and are mean (±SE) of 4–5 independent experiments. *, #p<0.05 when compared to cells stimulated with Iso or DCM IgG's alone (paired t-test, two-tailed). Representative blots are shown.

 
Given the important clinical implications of β-blockers in the treatment of cardiovascular diseases, we also addressed whether different drugs would similarly modulate isoproterenol or β1AR-Abs-induced ERK activation in cardiac cells. Interestingly, whereas some β-blockers (such as labetolol, metoprolol or propanolol) inhibited Iso or antibody-mediated ERK1/2 stimulation of endogenous β1AR in HL-1 cells to a similar extent, similar high doses of bisoprolol (which by itself increased ERK activation) or atenolol did not attenuate signaling to ERK by β1AR-autoantibodies obtained from two different patients (Fig. 5A and Suppl. Fig. 5). Similar results were obtained when performing these experiments in cultured cardiomyocytes (Fig. 5B). Iso-mediated ERK activation was potently inhibited in the presence or atenolol, carvedilol or metoprolol, whereas atenolol had no effect on ERK stimulation by β1AR-Abs from three different DCM patients. Therefore, it appears that distinct active conformations promoted by isoproterenol or autoantibodies in the β1AR are not equally modulated by some β-blockers.


Figure 5
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  		Fig. 5 Effect of β-adrenergic receptor blockers on β1AR-autoantibodies or isoproterenol-mediated ERK activation in cardiac cells. HL-1 cells (panel A) or cultured mouse cardiomyocytes (panel B) were incubated for 5 min in the absence (none) or presence of the indicated β-blockers as detailed in Methods. ERK stimulation was assessed and quantified as in previous figures in the presence of the β-agonist isoproterenol or IgGs obtained from different β1AR-Ab-positive DCM patients (P18, P12 and P8). Representative blots are shown. Data from Iso and patient P18 are also represented as mean (±SE) of 3 independent experiments.

 
In sum, although the detailed mechanisms involved remain to be established and the notion that autoantibodies and classic β1AR agonists promote distinct receptors conformations need to be confirmed by other experimental approaches, our results indicate that β1AR-autoantibodies stimulate the ERK1/2 pathway through endogenous β1AR in cardiac cells by triggering different signaling effectors than classic β1AR agonists. In this regard, emerging evidence suggest that binding of different GPCR ligands can promote distinct conformational changes leading to specific signaling outcomes [40]. The order of potency/efficacy of compounds acting through a given receptor can vary depending of the effector system analyzed [41–44]. Different active conformations may favour activation of one effector pathway over another [45] or promote different patterns of GRK phosphorylation and β-arrestin recruitment, leading to altered receptor signaling and internalisation [46–49]. Very recently, Galandrin and Bouvier, have shown distinct signaling profiles of β1AR and β2AR ligands towards adenylyl cyclase and MAPK [40]. Overall, these data back a multistate model of receptor activation in which the stability and composition of the receptor-associated signaling complexes would vary depending on the specific conformations stabilized by the ligand [43].

The β1AR-autoantibodies directed against the second extracellular loop of the β1AR are known to be very sensitive to the conformational state of the receptor, since they preferentially recognize native β1AR in different immunological assays [[2,3] and references therein]. The second extracellular loop of the β1AR is predicted to form a β-hairpin which dips partly into the ligand binding site, and its correct folding is essential for the formation of the ligand binding pocket [2,3]. This may explain why autoantibodies directed against this loop can alter receptor conformation and/or interfere with ligand binding. Alternatively, β1AR dimerization may also underlie the agonist properties of the autoantibodies [4,50]. Future experiments should be directed to explore those possibilities.

The notion that β1AR-autoantibodies promote a different "signaling state" of β1AR is consistent with previous observations showing their modest efficacy compared to classic β-agonists in promoting adenylyl cyclase activation [1,9,18], as well as differences in agonist-induced receptor desensitisation and internalisation [6]. The fact that β1AR-Abs are distinct and potent stimulators of the ERK1/2 pathway may play a role in the transition from cardiac hypertrophy to heart failure, although its clinical significance should be further established. Activation of the MEK/ERK1/2 pathway is involved in the development of cardiac hypertrophy [19], and elevated ERK activation has been reported in failing human hearts with dilated cardiomyopathy [22]. In this regard, it would be of interest to investigate whether such β1AR-Abs-triggered β1AR conformation affects other signaling pathways relevant to DCM pathophysiology.

Finally, the presence of β1AR-autoantibodies could also prove relevant for therapeutic strategies in heart failure patients. The variable clinical outcomes observed with βAR antagonists [51] could also be related, among other reasons, to their different ability to modulate β1AR and β2AR effector pathways [40], and to inhibit classic β-agonist or β1AR-autoantibodies-triggered ERK1/2 activation, as we now report. Thus, in patients positive for β1AR-autoantibodies, the use of beta-blockers able to attenuate or prevent the distinct antibody-induced activation of β1AR, in combination with new therapeutic approaches based on immunoadsorption or reduction of circulating antibodies [reviewed in [2,3]] will be indicated.


   Supplementary data
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results and discussion
 Supplementary data
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2007.05.022.

Time for primary review 22 days


   Acknowledgements
 
We thank Dr. I. Terol, J. Seguí and M. Bouvier for experimental tools, and S. Rojo and L. Horrillo for expert technical and secretarial assistance, respectively. This work was funded by grants from Ministerio de Educación y Ciencia (SAF2005-03053), Fundación Ramón Areces, The Cardiovascular Network (RECAVA) of Ministerio Sanidad y Consumo-Instituto Carlos III (RD06-0014/0037), Comunidad de Madrid (S-SAL-0159-2006), and the MAIN European Network (LSHG-CT-2003-502935). P.P. is a recipient of a "Ramón y Cajal" contract.


   Notes
 
[1] Current address. Centro Nacional de Investigaciones Cardiovasculares, 28034, Madrid, Spain. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results and discussion
 Supplementary data
 References
 

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E. H. Medei, J. H.M. Nascimento, R. C. Pedrosa, L. Barcellos, M. O. Masuda, S. Sicouri, M. V. Elizari, and A. C. Campos de Carvalho
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