Novel Receptor-Derived Cyclopeptides to Treat Heart Failure Caused by Anti-β1-Adrenoceptor Antibodies in a Human-Analogous Rat Model

Despite recent therapeutic advances the prognosis of heart failure remains poor. Recent research suggests that heart failure is a heterogeneous syndrome and that many patients have stimulating auto-antibodies directed against the second extracellular loop of the β1 adrenergic receptor (β1EC2). In a human-analogous rat model such antibodies cause myocyte damage and heart failure. Here we used this model to test a novel antibody-directed strategy aiming to prevent and/or treat antibody-induced cardiomyopathy. To generate heart failure, we immunised n = 76/114 rats with a fusion protein containing the human β1EC2 (amino-acids 195–225) every 4 weeks; n = 38/114 rats were control-injected with 0.9% NaCl. Intravenous application of a novel cyclic peptide mimicking β1EC2 (β1EC2-CP, 1.0 mg/kg every 4 weeks) or administration of the β1-blocker bisoprolol (15 mg/kg/day orally) was initiated either 6 weeks (cardiac function still normal, prevention-study, n = 24 (16 treated vs. 8 untreated)) or 8.5 months after the 1st immunisation (onset of cardiomyopathy, therapy-study, n = 52 (40 treated vs. 12 untreated)); n = 8/52 rats from the therapy-study received β1EC2-CP/bisoprolol co-treatment. We found that β1EC2-CP prevented and (alone or as add-on drug) treated antibody-induced cardiac damage in the rat, and that its efficacy was superior to mono-treatment with bisoprolol, a standard drug in heart failure. While bisoprolol mono-therapy was able to stop disease-progression, β1EC2-CP mono-therapy -or as an add-on to bisoprolol- almost fully reversed antibody-induced cardiac damage. The cyclo¬peptide acted both by scavenging free anti-β1EC2-antibodies and by targeting β1EC2-specific memory B-cells involved in antibody-production. Our model provides the basis for the clinical translation of a novel double-acting therapeutic strategy that scavenges harmful anti-β1EC2-antibodies and also selectively depletes memory B-cells involved in the production of such antibodies. Treatment with immuno-modulating cyclopeptides alone or as an add-on to β1-blockade represents a promising new therapeutic option in immune-mediated heart failure.


Introduction
Heart failure (HF) is a major cause of hospitalization and death; overall 50% of the patients die within four years of diagnosis [1]. HF may result from various causes and pathologies and is therefore considered a heterogeneous syndrome rather than a single disease entity. The presence of auto-antibodies directed against the β 1 -adrenergic receptor (β 1 -AR) apparently identifies a subgroup of patients at risk [2]. The β 1 -AR mediates most of the cardiac effects of the catecholamines adrenaline and noradrenaline, which are often highly elevated and predict unfavorable prognosis in HF [3,4]. Whereas short-term β 1 -AR stimulation improves cardiac performance, its chronic activation leads to progressive deterioration of cardiac structure and function [5].
During the past decade evidence has accumulated that many HF patients have functionally active autoantibodies directed against and stimulating the cardiac β 1 -AR (anti-β 1 -abs) [6,7,8]. Such anti-β 1 -abs are found particularly in patients with idiopathic dilated cardiomyopathy (DCM), which is characterised by dilatation and impaired contraction of the left or both ventricles [9]. The presence of stimulating anti-β 1 -abs is associated with reduced cardiac function [10], ventricular arrhythmias [2], sudden cardiac death [2,11], and increased cardiovascular mortality [2]. This suggests a potential for strategies to counteract such harmful receptorantibodies.
Stimulating anti-β 1 -abs almost exclusively target the second extracellular loop of the β 1 -AR (β 1 EC2), which is the largest and most structured of the three extracellular receptor loops and, thus, may represent a readily accessible target on the cell surface [12,13]. Moreover, β 1 EC2 contains T-and B-cell epitopes [14,15]. Recent data derived from the receptor's crystal structure underscore that β 1 EC2 is essential for the stabilisation and locking of the receptor's agonist binding pocket [13,16]. Thus, it seems conceivable that anti-β 1 EC2 may allosterically induce an active state of the β 1 -AR [12,17]. Immunisation of Lewis rats against the β 1 EC2 gives rise to stimulating anti-β 1 EC2, and within 8 months antibody-positive rats develop progressive cardiac dilatation, wall-thinning, and loss of contractile function typical for DCM [18]. Isogenic transfer of anti-β 1 EC2 to naїve Lewis rats likewise induced HF in recipients [6,18].
To target such harmful antibodies, we conceived a novel peptide-based strategy aiming to specifically neutralise disease-inducing autoantibodies, in particular anti-β 1 EC2. In this aim we generated peptide-homologs of β 1 EC2 and cyclised them to increase their stability in vivo [19] and to better mimic the epitope-structure, and then investigated whether they might prevent or have a therapeutic effect (alone or -to better mimic the clinical situation-as add-on to β 1blocker therapy) in our rat model of anti-β 1 EC2-induced HF.

Materials and Methods
Generation and characterisation of β 1 EC2-cyclopeptides Linear peptides comprising 24 amino-acids of the human β 1 EC2-sequence (AA199 to 222; ARAESDEARRCYNDPKCCDFVTNRG) [20] were synthesised commercially on a Multiple Peptide Synthesizer (SYROII, MultiSynTech GmbH, Witten, Germany) using the solid phase Fmoc protocol with side chain protected Fmoc amino-acid derivatives on Rink Amide MBHA resins (Novabiochem-Merck Biosciences GmbH, Bad Soden, Germany). For cyclisation of the peptide on the solid phase, an additional Fmoc-Glu-ODmab was incorporated at the C-terminal end of the linear peptide; after selective removal of the Dmab side chain, the resin-bound linear peptide was treated with diisopropyl-carbodiimide and N-hydroxy-9-azabenzotriazole in N,N'-dimethyl-formamide for several hours. The cyclisation process was monitored by repeated Kaiser'-tests [59]. Cleavage from the synthesis resin generated a peptide amide; the protective groups of the cyclopeptide were removed by treating the resin with trifluoro-acetic acid/ triisopropylsilane/ ethandithiole/water for 2 hours. The generated cyclopeptide β 1 EC2-CP was analysed by high pressure liquid chromatography (HPLC), and by mass spectrometry (MALDI-MS).
A cyclic peptide corresponding to the β 2 EC2-sequence (comprising AA182 to 204; RATH-QEAINCYANETCCDFFTNQG) [16] was synthesized and purified along the same lines and served as a control for specificity.
The effects of anti-β 1 EC2 on β 1 AR-mediated intracellular cAMP-production were assessed by measuring fluorescence resonance energy transfer (FRET) in HEKβ 1 -cells transiently transfected with a FRET-sensor for cAMP, Epac1-camps [8]. The sensor consists of the cAMPbinding protein Epac1 flanked by enhanced cyan or yellow fluorescent protein. 48 h after transfection with Epac1-camps, cAMP measurements were performed microscopically as described [8]. The cells were maintained in FRET-buffer supplemented with 50 nM ICI 118551 (Sigma, Deisenhofen, Germany) to block the small level of endogenous β 2 -AR (*0.1 pmol/mg membrane protein). IgG-preparations were added to the cells at 0.13 μg/μl protein concentration; 2μM (-)isoproterenol (Sigma) was used as a reference to determine the maximal cAMP-response. To test the blocking-efficacy of β 1 EC2-CP on anti-β 1 EC2-induced adrenergic signaling, the different IgG-preparations were pre-incubated with β 1 EC2-CP (20 μg/μl) for 6 h at 4°C; for pharmacological blockade of anti-β 1 EC2-induced signals we utilised 5.0 μM bisoprolol.

Echocardiography and haemodynamic measurements
Echocardiograms were obtained from lightly anaestetised rats (30 mg/kg ketamine-HCl and 5 mg/kg xylazine i.p.) with a Vevo770 system (Visual Sonics Inc., Toronto, Canada) equipped with a 17.5 MHz transducer as previously described [18], always by the same experienced echocardiographer, who was blinded to the treatment groups. In brief, the rats were lightly anaestetised (30 mg/kg ketamine-HCl and 5 mg/kg xylazine i.p.), shaved (chest), and placed supine on a special table. M-mode tracings were recorded at baseline (before immunisation), and subsequently every four months in the parasternal long and short axis views according to the the guidelines of the American Society for Echocardiography [22]. Pulsed-wave Doppler spectra were recorded from the apical five-chamber view and the velocity-time integral (VTI) of the transaortic flow served to calculate cardiac output (CO [ml/min] = Aortic VTI x (π [LVoutflow tract diameter/2] 2 ) x bpm). Reproducibility of the echocardiographic measurements was assessed as previously described [18]; intra-and interobserver variabilities were <2% or <5%.
Fourty-eight to 72 h after the final echo-Doppler examinations the rats underwent left heart catheterisation using a 3.5 F high-fidelity catheter (Millar Instruments, Houston, Texas) as described in [18]. LV-pressure tracings were recorded digitally over 15 min and analysed off-line (PowerLab, A.D. Instruments, Castle Hill, Australia) [18].

Macroanatomy and histology of rat tissues
After further deep anesthesia (70 mg/kg sodium pentobarbital i.p.), the hearts were quickly removed, rinsed with ice-cold relaxing-buffer (5% dextrose, 25 mM KCl in PBS), and weighed. The apex was cut, frozen in isopentane (-56°C), and stored at -80°C for further analysis (binding and gene expression studies); the remainig tissue was fixed in 10% PBS-buffered formalin (24-28 h). After the hearts all other relevant inner organs (e.g., lung, liver, spleen, kidneys, brain, and eyes) were removed, rinsed with ice-cold PBS, weighed, sectioned, and fixed for further histologic analysis.
Heart morphometry, histology, and TUNEL assay From paraffin-embedded heart preparations cavity-and wall-dimensions were determined by computer-aided analysis of H&E-stained mid-ventricular 2μm-sections as previously described [18]. H&E-stained paraffin-sections also served to quantify damaged and fibrotic cardiac areas (scars). For detection of mast cells, deparaffinised cardiac sections were stained with acidified (pH<2.5) toluidine blue 0.1% (Sigma). The number of toluidine-positive cells was normalized to square millimeter of cardiac tissue. TUNEL-positive cells were quantified in 2μm midventricular paraffin-sections using a TMR Red in Situ Death Detection Kit (Roche, Basel, Switzerland). Only the total number of TUNEL-postive cells/section was determined without further differentiation of the specific apoptotic cell type (e.g., fibroblasts, endothelial cells, cardiomyocytes).

Membrane preparation and radioligand binding studies
Membranes from apical cardiac tissues from rats of each study-group were prepared as previously described [18]. To determine total β-AR density (B max ), 35μg membrane protein were incubated (1.5 h, 25°C) in binding buffer with 200pM of the non-selective β-AR antagonist 125 I-cyanopindolol ( 125 I-CYP; Perkin Elmer Life and Analytical Sciences, Billerica, MA). Nonspecific binding was determined with 5μM unlabeled L-propranolol. The proportion of β 1 -and β 2 -AR subtypes was estimated from biphasic competition-curves with 10 -10 to 10 -2 M of the unlabeled β 1 -selective antagonist CGP20712A (Sigma). The reaction was stopped by rapid filtration (Whatman GF/C filters) and washing with ice-cold buffer. Filter-bound radioactivity was measured by γ-counting. Estimates of maximal binding (B max ) and the proportion of β 1and β 2 -AR-subtypes were calculated with GraphPad Prism 5.00 (San Diego, CA).

Cardiac gene expression
Total RNA was isolated from frozen myocardium by RNeasy mini Kit (Qiagen, Hilden, Germany). Reverse transcription of total RNA isolated from frozen myocardium was performed in 96-well plates utilising a high capacity RNA-to-cDNA master mix (Applied Biosystems, Foster City, CA). PCR reactions were conducted in the presence of the fluorescent dye Sybr-Green (Cambrex BioScience, East Rutherford, NY) and the reference-dye 6-carboxy-X-rhodamine (ROX) using an ABI PRISM sequence detection system 7700 (Applied Biosystems). All amplification products were controlled for specificity by running a melting curve analysis; results were calculated using the 2 -ΔΔCT method. The relative expression levels were derived from a standard curve and normalised to GAPDH as an endogenous control. Quantitative real-time PCR (qRT-PCR) analyses are presented as fold change compared to untreated (0.9% NaClinjected) control hearts.
Detection of antigen-specific CD4 + T-cells and memory B-cells as well as antibody-secreting plasma cells, and plasmablasts (a) Recall-assays with T-cells from spleens of immunised untreated vs. β 1 EC2-CP-treated animals were conducted as described in [60]. In brief, to purify CD4 + T-cells from the splenic cell preparations, B-cells and CD8 + T-cells were depleted by commercially available magnetic beads (MACS, Miltenyi Biotec, Bergisch-Gladbach, Germany) yielding a purity >85%. 1x10 5 of the purified CD4 + T-cells were then co-incubated in 96-well plates with 1x10 6 irradiated thymic antigen presenting cells (prepared from a younger rat). Reagents added in the different assays were 1.0 μg/ml β 1 EC2-CP, 1.0 μg/ml tuberculin purified protein derivative (PPD, internal control), 1.0 μg/ml glutathion-S-transferase (GST), as well as 1.0 and 0.1 μg/ml GST/β 1 EC2-fusion proteins (FP), respectively. Measured T-cell reactivities were normalised to medium. After 48 h of incubation the cells were pulsed with 1.25 μCi/well [ 3 H]-thymidine and further incubated for 16 h before the cells were harvested; the DNA-incorporated radioactivity was measured using a beta-plate counter.
(d) To detect functional β 1 EC2-specific memory B-cells in vivo, B-cells were first purified from total splenocytes by negative depletion using mAb V65, R73, 10/78 and WT.5 plus magnetic beads (MACS, Miltenyi Biotec, Bergisch-Gladbach, Germany) yielding average purities of 87%. 2.6x10 7 purified B cells were injected in 250 μl PBS i.v. into antigen-naïve syngeneic male Lewis rats. To selectively trigger the transferred memory B-cells, recipient rats were immunised after five days with only 12.5 μg GST/β 1 EC2-FP in adjuvans per animal. Serum was collected for antibody detection just before as well as three and seven days after immunisation. Rats without (memory) B-cell transfer did not produce anti-β 1 EC2 IgG within the seven-day observation period (data not shown).

Statistical analyses
Data are given as mean ±SEM of at least four animals per treatment-group if not otherwise stated. Statistical analyses were performed using GraphPad Prism 5.00 (San Diego, CA). Functional assays (FRET), molecular parameters (qRT-PCR), as well as haemodynamic and morphometric parameters of antibody-positive vs. corresponding control rats or untreated vs. treated animals, respectively, were analysed by Student's t-test (where applicable) or repeated measurements one-way ANOVA; significance between the different groups was analysed by Dunnett's post-test.
Kinetics of anti-β 1 EC2-antibodies and/or the different peptides (where applicable) and differences between functional and echocardiographic parameters (long-term follow-up) were analysed by two-way ANOVA followed by Bonferroni post-hoc testing. Values of p<0.05 were considered statistically significant.
Specificity and conformational character of the resultant anti-β 1 EC2 towards β 1 -ARs was ascertained along several lines: by larger ELISA signals with cyclic vs. linear β 1 EC2-peptides ( Fig. 1A), by a better recognition of cyclic vs. linear β 1 EC2-peptides in blocking assays (together with a clear preference for β 1 -over β 2 -EC2 sequence (Fig. 1B)), and by a better concentrationdependent neutralization in vitro achieved with cyclic vs. linear β 1 EC2-peptides (Fig. 1C). In addition, immunofluorescence-studies confirmed that all rat anti-β 1 EC2 stained native human β 1 -AR in the membrane of stably transfected human embryonic kidney cells (HEKβ 1 -cells), and co-localised with purified β 1 -specific amino-terminal rabbit antibodies [21] (Fig. 1D). Finally, the anti-β 1 EC2 stimulated β 1 -AR-mediated signaling in HEKβ 1 -cells, as evidenced by an increase in cAMP monitored with a co-transfected sensor that shows a decrease in fluorescence resonance energy transfer (FRET) upon binding of cAMP [8]; these signals varied in amplitude and in some cases almost reached the effects induced by the β-AR agonist isoproterenol (Fig. 1E, right panel). No such cAMP-signals were detected with IgG prepared from 0.9% NaCl-injected control rats (Fig. 1E, left panel). Also, control IgG reacted neither with β 1 EC2peptides in ELISA or competition assays (not shown), nor with β 1 -AR expressed in HEK-cells (Fig. 1D). Stimulation of β 1 -AR/ cAMP signaling by anti-β 1 EC2 was inhibited by pre-incubation with β 1 EC2-peptides, again better by cyclic than by linear peptides (Fig. 1F, top panels); interestingly, this inhibition was more efficient than that achieved by the specific β 1 -AR antagonist bisoprolol (Fig. 1F, bottom right). As internal controls, we also generated cyclic EC2-peptides of the β 2 -AR (β 2 EC2-CP). In ELISA, competition-and FRET-assays, anti-β 1 EC2 was not blocked at all by these β 2 -AR-derived peptides, documenting the β 1 -AR-specificity of the generated antibodies ( Fig. 1B and Fig. 1F, bottom left).
In both studies cardiac function was followed every 4 months by echocardiography, and was assessed by left ventricular (LV) catheterisation at month 16 (prevention-study) or month 20 (therapy-study) as previously described. [18] After 8 months anti-β 1 EC2-positive untreated rats developed LV-dilatation and -dysfunction that progressed continuously in the course of both studies. In the prevention-study, echocardiography and cardiac catheterisation ( Fig. 2C and D, and S2 Fig.: A, C, E and G) as well as histomorphology of the hearts (Table 1) of untreated vs. treated animals revealed that both bisoprolol and β 1 EC2-CP were able to prevent development of cardiomyopathy and heart failure.
In the therapy-study, β 1 EC2-CP (injected either alone or as add-on to bisoprolol-treatment) almost fully reversed the cardiomyopathic phenotype that had developed before the initiation of therapy, whereas mono-therapy with bisoprolol only stopped further disease-progression. With β 1 EC2-CP (alone or as add-on), echocardiographic LV-dimensions, LV-ejection fraction and cardiac index (Fig. 2G), LV end-diastolic pressure and systolic contraction (Fig. 2H) as well as the heart weight of cardiomyopathic rats returned to control values ( Table 2). In contrast, β 1 EC1-Lin or β 1 EC2-Lin failed to elicit any cardiopotective effects (S2 Fig.: B, D, and F). Unlike bisoprolol (alone or as add-on), neither linear peptides nor β 1 EC2-CP decreased heart rate or blood pressure of treated animals (S2 Fig.: G and H).
Morphometry and immunohistology of midventricular 2μm-sections of the hearts analysed at the end of the therapy-study underscored the beneficial effects of β 1 EC2-CP (alone or as add-on). The number of myocardial fibrotic scars ( Fig. 3A and B) and TUNEL-positive apopotic cells (Fig. 3C) returned to normal levels in β 1 EC2-CP-treated rats. Such a reversal was not seen with bisoprolol mono-therapy ( Fig. 3A-C). By contrast, the increases in cardiac transcripts of distinct profibrotic markers (IL1-β, TGF-β1) observed in immunisation-rats as negative controls. Time-course of anti-β1EC2-titers in the (B) prevention-and (F) therapy-study. Titers were measured before and 24h after CPinjection. Values in (B) correspond to the anti-β1EC2-titers in mg/ml over 16 study-months (black circles, untreated; diamonds, β1EC2-CP (1.0 mg/kg/month i.v.); triangles, bisoprolol (15 mg/kg/day orally)). Values in (F) correspond to the anti-β1EC2-titers in mg/ml over 20 study-months (black circles, untreated; green diamonds, β1EC2-CP mono-treated; blue triangles, bisoprolol mono-treated; purple diamonds, β1EC2-CP/bisoprolol co-treated). For better readability error-bars are not shown in the graph. Echocardiography in the (C) prevention-and (G) therapy-study. Graphs show the time-course of the LV end-diastolic diameter (LVED). Lower panels: Time-course of the cardiac index derived from cardiac output/ body weight. Error bars indicate mean ±SEM in the indicated groups; *p<0.01; **p<0.001 (two way ANOVA and Bonferroni post-hoc test). Invasively obtained parameters in the (D) prevention-and (H) therapy-study. Upper panels show LV end-diastolic pressures (mmHg), lower panels contractilility (+dP/dt, mmHg/s). Error bars indicate mean ±SEM of the indicated groups; ns, not significant; *p<0.01; **p<0.001 (one way ANOVA and Dunnett`s post-hoc test). doi:10.1371/journal.pone.0117589.g002 Cyclopeptide-Therapy in Immune Heart Failure induced HF were reduced by *50% with either β 1 EC2-CP or bisoprolol mono-treatment, and even by >70% in the co-treatment group (indicating an synergistic anti-inflammatory effect, Fig. 3D). Also the documented increase in cardiac mast cells in immunized rats was reversed to control levels by both substances (either as mono-or as co-treatment, Fig. 3E). Further morphometric analysis of the hearts revealed enlarged LV-cavities and wall thinning in untreated cardiomyopathic rats. All these features, including the heart weight, were returned to normal in β 1 EC2-CP-treated animals ( Table 2 and Table 3). Moreover, the relative wet weight (Table 2), histology (S3 Fig.), and selected laboratory parameters (S4 Fig.) of other organs than the heart revealed an increase in lung and liver weight in untreated anti-β 1 EC2positive rats (accompanied by a significant increase in GLDH). These signs of congestion were almost reverted in β 1 EC2-CP-treated, but not in bisoprolol mono-treated animals. Importantly, no β 1 EC2-CP-related pathologies were noted in treated vs. control animals. In particular, neither the kidneys nor other inner organs, nor the eyes of β 1 EC2-CP-treated rats had any signs of damage or organ-toxicity attributable to an accumulation or deposition of anti-β 1 EC2/β 1 EC2-CP-complexes (S3 Fig.).
Cardiac failure is usually accompanied by downregulation of cardiac β 1 -but not β 2 -ARs, and by upregulation of cardiac GRKs [4,23,24]. Radioligand-binding studies with 125 I-cyanopindolol and selective antagonists showed that β 1 -specific downregulation of β-ARs also occurred in our immunisation-induced HF-model [18], and that this downregulation was largely prevented by (mono-)application of β 1 EC2-CP alone ( Fig. 4A and B). As typical for a β-AR antagonist, bisoprolol (mono-)treatment even induced a small increase in cardiac β 1 -ARs, an effect that was fully preserved with β 1 EC2-CP/bisoprolol co-treatment (Fig. 4B). No changes were seen for β 2 -AR under any of the study conditions. Corresponding to the downregulation of cardiac β 1 -AR protein, β 1 -AR mRNA-levels were also significantly reduced in untreated cardiomyopathic rats and returned to normal levels with either β 1 EC2-CP or bisoprolol treatment alone or with β 1 EC2-CP/bisoprolol combination-therapy (Fig. 4C). Moreover, qPCR-analysis of the expression of G protein-coupled receptor kinases (GRKs) involved in counter-balancing sympathetic activity [4,24,25] revealed an upregulation of GRK2 and GRK5 in immunisationinduced HF, which was reverted by bothβ 1 EC2-CP and (to a somewhat lesser extent) by bisoprolol mono-therapy (Fig. 4C), whereas co-treatment with both substances had a clear synergistic effect, resulting even in a slight (non-significant) down-regulation of both GRK's ( Fig. 4C). The upper part of the table shows the relative (wet) weights in g/kg body weight of selected organs in the groups of the therapy-study. Non-immunised 0.9% NaCl-injected rats had no intervention (Control) or received treatment with either bisoprolol (Cont. Immunomodulating effects of β 1 -EC2-cyclopeptides To assess whether preventive and/or therapeutic treatment of anti-β 1 EC2-positive rats with the different peptides interfered with the immunopathology of antibody-induced HF, we determined the kinetics of anti-β 1 EC2-abs before and 24 h after peptide-application by ELISA (measuring only free anti-β 1 EC2-abs). In immunised untreated animals, anti-β 1 EC2-titers continuously increased after each antigen-boost ( Fig. 2B and F). In line with an antibody-scavenging effect, treatment with β 1 EC2-CP already after two injections significantly decreased, and after 5 injections almost fully suppressed a rise in free anti-β 1 EC2, while bisoprolol treatment did not affect the amount of free anti-β 1 EC2-abs. With preventive and also with (repeated) therapeutic application of β 1 EC2-CP, anti-β 1 EC2-levels remained low despite continued boosting with β 1 EC2/GST-FPs ( Fig. 2B and F). This unexpected titer-course, monitored more closely in the first 6 months of the prevention-study (Fig. 5A), suggested that beside its action as a simple scavenger, β 1 EC2-CP also negatively impacted on B-and/or T-cells as the lymphocyte populations responsible for anti-β 1 EC2-production. Moreover, as even preventive injection was initiated after the priming-immunisation and production of class-switched antiβ 1 EC2 of the IgG isotype, β 1 EC2-CP-treatment putatively interfered with the function not of naïve, but of already differentiated memory B-cells, plasma cells (PC), and/or T-cells.
For T-cells, treatment with antigenic peptides has been shown to either induce dominant unresponsiveness of the CD4 + T cell-compartment via induction of CD4 + CD25 + regulatory T-cells (T reg cells) [26] or to functionally impair non-T reg CD4 + T helper cells [27]. We, therefore, tested the in vitro recall responses of purified CD4 + T-cells isolated from the spleens of preventively ( Fig. 5B and C) or therapeutically (Fig. 5D) treated and untreated rats to the β 1 EC2/GST-FP and its components. CD4 + T-cells from both groups clearly responded to the FP but not to β 1 EC2-CP, indicating that the relevant CD4 + T-cell epitopes were contained within GST (Fig. 5B-D). Further in vitro testing did also not provide any evidence for an induction of β 1 EC2-specific T reg cells by β 1 EC2-CP in vivo (not shown).
As the T cell-compartment appeared not to be targeted by β 1 EC2-CP we further analysed the cells directly involved in antibody-production, i.e. B-cells and PC. ELISPOT assays using splenocytes or bone marrow cells freshly isolated from rats two weeks after the last antigenboost allowed us to determine the frequencies of (mostly) long-lived PC in these organs; however, these experiments revealed that β 1 EC2-CP-treatment did not reduce the frequencies of anti-β 1 EC2-producing PC (Fig. 5E and F).
While long-lived PC express very little or no immunoglobulins on the cell surface [28], Bcells do and could thus serve as direct targets of β 1 EC2-CPs. To detect the few antigen- Morphometric data of rat hearts harvested at the end of the therapy-study were obtained by computer-aided analysis of H&E-stained mid-ventricular 2 μm cross-sections as previously described in detail [18]. specific memory B-cells within splenocytes of treated vs. untreated rats we differentiated memory B-cells into short-lived plasmablasts by boosting the rats with β 1 EC2/GST-FPs three days prior to the analysis [29]. This allowed us to detect them together with long-lived PC by ELISPOT. Preventive ( Fig. 5G and H) as well as therapeutic ( Fig. 5I and J) application of β 1 EC2-CPs resulted in a *80% reduction in the frequencies of splenocytes secreting anti-β 1 EC2-abs, which was not achieved with β 1 EC2-Lin (S5A Fig.). As long-lived PC were not targeted by β 1 EC2-CPs (Fig. 5E), this means that more than 80% of the β 1 EC2-specific memory B-cells were affected by this kind of treatment impairing B-cell receptor (BCR)-mediated β 1 EC2specific memory B-cell expansion and differentiation into anti-β 1 EC2-producing PC. Direct detection of β 1 EC2-specific memory B-cells by flow cytometry (Fig. 5K and L) further revealed complete depletion of this cell population by β 1 EC2-CP treatment. Accordingly, transfer of purified B-cells from immunised β 1 EC2-treated rats into antigen-naïve recipients followed by suboptimal immunisation with β 1 EC2/GST-FP (12.5μg instead of 50μg in Cardiac adrenergic system and molecular markers. Columns represent the relative amount of β 1 /β 2 -AR subtypes (fmol/mg protein) in cardiac membranes of anti-β 1 EC2-positive immunised rats (A) preventively or (B) therapeutically treated with either bisoprolol or β 1 EC2-CP alone, or with a combination of both, and corresponding control animals (prevention-arm, month 16; therapy-arm, month 20). Error bars indicate mean values ±SEM of the indicated treatment groups; *p<0.05. (C) Molecular analysis of hearts (apical segments) from treated anti-β 1 EC2-positive rats and corresponding control animals in the therapy-study. Columns ± error bars represent the level of expression of the indicated markers ±SEM in cardiac tissues from the indicated treatment-groups compared to values from 0.9% NaCl-injected rats (controls, set at 100%), as determined by qRT-PCR normalised to GAPDH (one way ANOVA and Dunnett`s post-hoc test; *p<0.05; **p<0.001).
doi:10.1371/journal.pone.0117589.g004 adjuvans) did not induce detectable amounts of anti-β 1 EC2-abs in the serum of the recipient rats while B-cells from immunised untreated rats did (Fig. 5M). In summary, our results suggest that β 1 EC2-CP protects rats from immunisation-induced HF, both, by specifically scavenging free anti-β 1 EC2 and by depleting β 1 EC2-specific memory B-cells. It should be noted, however, that the overall numbers of IgG-producing cells were not at all affected by β 1 EC2-CP-treatment (Fig. 5F, H, and J), precluding a general immuno-suppressant effect of the cyclic peptide.

Autoantibody-induced diseases and current treatment approaches
Autoantibodies directed against self-antigens are the hallmark of many autoimmune diseases, and some of them may even directly cause the disease [30,31]. In Graves'disease [32], myasthenia gravis [33], and a sub-entity of insulin-resistant diabetes [30] functional autoantibodies (abs) directed against membrane receptors have been recognised as main pathogenetic factors. This illustrates that receptors, proteins that are generally considered as not very immunogenic, can serve as targets for disease-causing abs. This appears to be the case also in a significant number of HF patients, where anti-β 1 AR-abs are suspected to contribute to the development of DCM [2,6,7,8,10,11,18,34,35,36].
Current treatment approaches in autoantibody-induced diseases comprise either targeting the abs themselves, as is the case for immunoadsorption [36], or the cells producing the abs, i.e. short-lived plasmablasts and/or long-lived plasma cells. While plasmablasts and their precursors, i.e. memory B-cells, may be deleted by cytostatic drugs like cyclophosphamide [37] or anti-CD20 + -abs [38], ablation of long-lived plasma cells requires the use of proteasome-inhibitors [39]. All these therapies present specific problems in terms of unwanted effects and outcome; in particular, proteasome-inhibitors are quite cardiotoxic, rendering them not suitable for the treatment of anti-β 1 AR-mediated HF [40].
Here we present a novel approach to address such disease-causing abs based on cyclic peptides mimicking the relevant target-epitope. We and others have previously shown that immunisation against the 2 nd extracellular loop of the β 1 -AR gives rise to receptor-stimulating anti-β 1 EC2 in various animal models [12,14,18,35,41]. Such antibodies appear to allosterically activate the receptors and their signaling cascade, and this activation occurs both in the absence and in the presence of catecholamines [6,12,18]. In the long run, such anti-β 1 EC2 cause myocardial tissue injury, characterised by myocyte apoptosis and fibrosis, myocyte dysfunction, cardiac dilatation, and in the end congestive HF [5,6]. The most likely explanation is that stimulating anti-β 1 EC2 abs cause partial, but chronic activation of cardiomyocyte β 1 -ARs, and thus potentiate the vicious circle of sympathetic activation and HF-progression [3,4,6].
Mode of action of β 1 -receptor mimicking cyclic peptides In our study, monthly injections of β 1 EC2-CP either prevented (preventive application) or even reversed (therapeutic application) the detrimental consequences of stimulating anti-β 1 EC2. These injections were tolerated well by both immunised anti-β 1 EC2-positive rats and antibody-naïve control animals, and over one year of treatment did not elicit any apparent β 1 EC2-CP-related adverse effects, neither in routine blood laboratory tests nor in a series of organs (S3 and S4 Figs.). In addition, the protection achieved with monthly β 1 EC2-CP-injections was clearly superior to daily applications of 15 mg/kg bisoprolol alone, although mono-treatment with bisoprolol was able to stop the progression of immunisation-induced heart failure. Unlike bisoprolol, β 1 EC2-CP mono-treatment affected neither heart rate nor blood pressure. The beneficial effects of β 1 EC2-CP must at least in part be due to scavenging of anti-β 1 EC2, as is evident from the specific and high-affinity recognition of these antibodies which reflects both the primary sequence of the epitope (by comparison with the β 1 EC1-or β 2 EC2-peptides) as well as the structure (by comparison with the linear β 1 EC2-peptide). In addition, β 1 EC2-CP had a long half-life in blood, which contributes to its scavenging-efficacy.
In addition to its anti-β 1 EC2-neutralizing action, the most intriguing effect of β 1 EC2-CP was the ability to essentially block further anti-β 1 EC2 antibody-production despite continuous antigen-boosts. As detailed in the results-section this failure to respond to the antigen boosts is due to depletion or functional inactivation of β 1 EC2-specific memory B-cells. On a molecular level this depletion is explained by monomeric, e.g. non-productive, stimulation of the BCR [42] precluding an expansion of anti-β 1 EC2-expressing memory B-cells in β 1 EC2-CP -treated animals.
As in the case of direct scavenging, the effect on anti-β 1 EC2-expressing memory B-cells was sequence-and epitope-specific, and was, in particular, not elicited by the corresponding linear peptide (S2 and S5 Figs.).
While direct targeting of PC in anti-β 1 EC2-mediated HF remains a challenge, the cyclopeptide-approach presented here neutralises the products of short-lived plasmablasts as well as long-lived PC (i.e. existing anti-β 1 EC2-antibodies) and hits β 1 EC2-specific memory B-cells. Although antibody-neutralisation appears sufficient to mediate protection in this model, we hypothesise that depletion of the β 1 EC2-specific memory B-cells may be important for maintaining low antibody-titers and, therefore, enhances the long-term therapeutic efficacy of our approach. By this double action β 1 EC2-CPs not only prevent antibody-induced β 1 -AR activation, but also address the site of antibody-generation, and thereby prevent or revert antiβ 1 EC2-induced cardiac damage. These positive effects were seen in terms of cardiac morphology and function as well as different microscopic, laboratory, and molecular parameters. Moreover, in β 1 EC2-CP-treated animals, together with the restauration of cardiac membrane β 1 -AR, also the increases in GRK2-and GRK5-expression were almost fully reverted. These effects were even more pronounced, when injecting β 1 EC2-CP (as an add-on) to bisoprolol-treatment, resulting in a slight (non-significant) downregulation of both GRK's. This is of particular interest, as increases in cardiac GRK-transcripts are thought to represent an early adaptation to adrenergic stress preceding β 1 -AR desensitisation [4,43,44]. In addition, increases in cardiac GRK-transcripts have been shown to correlate well with disease severity in HF patients [44], suggesting that β 1 EC2-CP treatment alone or-on a molecular level even more efficient-combined with a β 1 -receptor blocking agent (which corresponds better to the current clinical requirements and treatment guidelines) might also act beneficially in anti-β 1 EC2-mediated human heart failure.
Chronic heart failure, relevance of the immune system, and new therapeutic strategies Various factors may contribute to non-ischaemic DCM, including persistent viral infection [45] and autoimmune-mediated damage to myocytes [46,47,48]. These factors may coincide, because autoimmune-reactions to myocardial proteins may be virus-triggered [31,47]. In the last decade a growing number of heart-directed abs and alterations of the immune system have been described in heterogeneous subsets of patients with DCM [34,46,49], indicating that multiple mechanisms may play a role in the pathogenesis of autoimmune-mediated heart failure (HF). Amongst others, abnormalities have been found in cytokines [46], T lymphocyte subsets, and in cells mediating myocardial inflammation [50]. Moreover, in >30% of DCM patients abnormal immune-reactions against distinct cardiac self-antigens have been described, including autoantibodies against myocyte contractile proteins [47,51], mitochondrial proteins [52], and membrane receptors [10,53,54]. However, only a few of them have been shown to cause, in fact, myocardial tissue injury and congestive HF by themselves [18,35,51]. In humans the individual genetic predisposition also significantly influences the susceptibility to self-directed immune-reactions [34,49,55], but the so far available clinical data underscore the pathophysiological and clinical importance of stimulating anti-β 1 AR-abs in HF and the need for novel antibody-directed therapeutic strategies [6,36,56]. We demonstrate here that anti-β 1 EC2-mediated cardiostimulatory effects in vivo cannot be efficiently neutralised with β 1 -receptor blockers alone. This finding fits perfectly with in vitro data using anti-β 1 EC2 isolated from DCM-patients [8]. Other experimental antibody-directed strategies consist in their removal from the blood by immunoadsorption [36,57,58] or, more recently, in the development of anti-β 1 ARneutralising small oligonucleotides currently assayed in vitro [56].
As a simple alternative, we present here a novel specific and very efficient strategy to treat autoimmune-mediated HF in vivo with cyclopeptides mimicking the target-domain of stimulating anti-β 1 EC2-antibodies (administered alone or as an add-on to β 1 -receptor blocker treatment). β 1 EC2-CPs acted as antibody-scavengers in the circulation, but also depleted or functionally inhibited anti-β 1 EC2-expressing memory B-cells involved in antibody-production. Our data suggest that β 1 EC2-CPs (alone or combined with a β 1 -receptor blocking drug) might evolve into a novel save and efficient strategy to neutralise stimulating anti-β 1 EC2 also in human HF. Furthermore, it will be interesting to investigate whether such cyclic peptides might prove to be beneficial also in other autoantibody-mediated diseases.

Conclusions
By taking advantage of a human-analogous rat model the here presented in vivo-experiments provide the basis for the clinical translation of a novel double-acting therapeutic strategy for immune-mediated heart failure. Cyclic peptides mimicking the target epitope of functionally active antibodies stimulating the cardiac β 1 -adrenergic receptor on the one hand act as direct antibody-scavengers in the circulation thereby precluding antibody-induced harm from the heart; in addition, the cyclic peptides were found to have a long-term effect by selectively depleting memory B-cells involved in the production of cardio-noxious receptor antibodies. Besides the prevention and/or treatment of immune heart failure (either as a mono-substance or combined with a β 1 -receptor blocking agent) the here presented approach might be helpful also in other autoantibody-mediated diseases.