Autoantibodies against the β3-Adrenoceptor Protect from Cardiac Dysfunction in a Rat Model of Pressure Overload

Jin Wang; Meixia Li; Xiurui Ma; Kehua Bai; Li Wang; Zi Yan; Tingting Lv; Zhiqing Zhao; Rongrui Zhao; Huirong Liu

doi:10.1371/journal.pone.0078207

Abstract

β₃-adrenoceptors (β₃-ARs) mediate a negative inotropic effect in human ventricular cardiomyocytes, which is opposite to that of β₁- and β₂-ARs. It has been previously demonstrated that autoantibodies against the β_1/β₂-AR exist in the sera of some patients with heart failure (HF) and these autoantibodies display agonist-like effects. Our aim in this study was to observe whether autoantibodies against the β₃-AR (β₃-AR Abs) exist in the sera of patients with HF and to assess the effects of β₃-AR Abs on rat model of pressure overload cardiomyopthy. In the present study, the level of β₃-AR Abs in the sera of HF patients was screened by ELISA. β₃-AR Abs from HF patients were administrated to male adult rats with abdominal aortic banding (AAB), and the cardiac function was measured by echocardiographic examination and hemodynamic studies. The biological effects of this autoantibody on cardiomyocytes were evaluated using a motion-edge detection system, intracellular calcium transient assay, and patch clamp techniques. Compared to healthy subjects, the frequency of occurrence and titer of β₃-AR Abs in the sera of HF patients were greatly increased, and β₃-AR Abs could prevent LV dilation and improve the cardiac function of rats with AAB. β₃-AR Abs exhibited negative chronotropic and inotropic effects and were accompanied by a decreased intracellular Ca²⁺ transient and membrane L-type Ca²⁺ current in cardiomyocytes. Our results demonstrated the existence of β₃-AR Abs in the sera of patients with HF and found that this autoantibody could alleviate the cardiac dysfunction induced by pressure-overload in AAB rats.

Citation: Wang J, Li M, Ma X, Bai K, Wang L, Yan Z, et al. (2013) Autoantibodies against the β₃-Adrenoceptor Protect from Cardiac Dysfunction in a Rat Model of Pressure Overload. PLoS ONE 8(10): e78207. https://doi.org/10.1371/journal.pone.0078207

Editor: Wolfgang Rudolf Bauer, University Hospital of Würzburg, Germany

Received: October 29, 2012; Accepted: September 17, 2013; Published: October 11, 2013

Copyright: © 2013 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This research is supported in part by grants from Key Laboratory of Cellular Physiology Director Fund of Shanxi Medical University (2010-06) to Jin Wang, Youth Fund of Shanxi Medical University to Jin Wang, and NSFC (National Nature Science Foundation of People’s Republic of China, 30973163) to Huirong Liu. And the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Heart failure (HF) is a life-threatening clinical condition in which the heart cannot pump enough blood to the rest of the body. The clinical symptoms of HF include water-sodium retention, decreased perfusion of peripheral tissues and organs, which are the common final phase of many cardiovascular diseases [1]. Despite the improvement of medical therapy, the clinical outcome is extremely poor [2]. The main problem is its heterogeneous in etiology and pathogenesis. Among them, dysregulation of the β-adrenergic system has been considered to play a critical role in the development of cardiac dysfunction associated with HF [3-5].

In recent years, autoantibodies against the β₁- and β₂- adrenoceptor (AR) have been detected in the sera of patients with chronic HF [6-9]. These autoantibodies are specifically directed against the second extracellular loop of human β₁- and β₂-ARs and display agonist-like activities [6,9-13]. Furthermore, immunization by peptides corresponding to the target sequences of the anti-receptor autoantibodies induced morphological and functional changes in the rat or rabbit heart similar to those observed in patients with HF [14-18]. These studies suggest that autoanbibodies against the G-protein-coupled receptors have important pathophysiologic role in the occurrence and development process of HF [19].

β₃-AR is a newly-identified cardiac adrenoceptor that belongs to the superfamily of G protein-coupled-receptors [20]. However, β₃-AR differs from classical β₁- and β₂-AR by its opposite roles in the regulation of cardiac functions. The β₃-AR has been found in the human ventricular myocardium where they produce a negative inotropic effect that was mediated through Gi proteins [21]. Moreover, in contrast to the downregulation of β₁- and β₂-AR during the development of HF [22], β₃-AR proteins were markedly increased (2- to 3- fold) in failing compared with non-failing hearts, and a similar increase was also observed for Gi proteins that coupled β₃-AR to their negative inotropic effect [23-26]. Furthermore, Rasmussen [27] and Niu [28] et al. have demonstrated that β₃-AR agonists could improve cardiac function of HF patients and a lack of β₃-AR could exacerbate LV dilation and dysfunction [29]. These results suggest that activation of β₃-AR may also play an important role in the modulation of cardiac function in HF. As β₃-AR also belongs to the G protein-coupled receptor family [20], we speculated that it may have similar immunological characteristics with β₁-AR/β₂-AR, and that autoantibodies against the β₃-AR (β₃-AR Abs) may also exist in the sera of HF patients. If this assumption is valid, what is the effect of β₃-AR Abs on cardiac function?

Therefore, the purposes of the present study were 1) to determine whether HF patients could produce β₃-AR Abs, 2) to investigate whether β₃-AR Abs could affect the cardiac function in rats with abdominal aortic banding, and 3) to study the biological activities of these autoantibodies on cardiomyocytes in an attempt to explore its possible mechanisms.

Methods

The research protocol was approved by the Institutional Committee for the Protection of Human Subjects of Shanxi Medical University Hospital. All patients were informed about the purpose and protocol of the study, and written consent was obtained. The study adheres to the principles of the Declaration of Helsinki and Title 45, US Code of Federal Regulations, Part 46, Protection of Human Subjects, revised 13 November 2001, effective from 13 December 2001. All experimental procedures and protocols were approved by the Ethics Committee and Animal Welfare Committee of Shanxi Medical University.

Patient Characteristics

Sera from 76 patients with HF were collected from the First and Second Hospitals of Shanxi Medical University, Taiyuan, China. The diagnosis of HF was based on the patients’ clinical history, physical examination, echocardiography, left ventriculography, electrocardiogram, chest radiography, and coronary angiography according to WHO/ISFC [30]. For purposes of comparison, sera from 100 healthy subjects were obtained from normal healthy volunteers at the First Hospital of Shanxi Medical University. The protocol was approved by the Research Committee of Shanxi Medical University. The sera were collected and stored at −20°C for subsequent detection of β₃-AR Abs. The characteristics of patients with HF and healthy subjects are summarized in Table 1.

Characteristics	HF(n=76)	Control(n=100)
Age(y)	58.8±8.6	52.8±10.6
Gender(male/female)	48/28	66/34
Heart rate (beats/min)	76±11	72±13
SBP(mmHg)	117±18	119±15
DBP(mmHg)	73±13	75±13
Functional class (NYHA)*
I	9	100
II	32	0
III	21	0
IV	14	0
Ejection fraction (%)	37.2±6.7^*	70.5±8.6
Plasma BNP (pg/ml)	328±45^*	32.4±16.7
Medications
ACE inhibitors / ARBs	63	0
β-Blockers	58	0
Diuretics	32	0

Table 1. Characteristics of patients with HF and healthy subjects.

HF: Heart Failure; NYHA: New York Heart Association. ^*P < 0.05 vs. healthy subjects.

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Peptides

A peptide corresponding to the sequence (residues 176-202) of the second extracellular loop of the human β₃-AR [31] with a cysteine as carboxy terminus (QWWRVGADAEAQRCHSNPRCCAFASNMC) was synthesized by Meilian Bioengineering Company, Xian, China.

ELISA

50 μl of peptide (5 μg/ml) in a 0.1 mol/L Na₂CO₃ solution (pH 11.0) were coated on a 96-well microplate overnight at 4°C. The wells were then saturated with PBS supplemented with 5% bovine serum and 0.1% Tween 20 (PMT). 50 μl of sera dilutions from 1:10 to 1:160 in PMT were allowed to react with the peptide for 1 hour at 37°C. After washing three times with PBS, 0.05 ml of biotinylated rabbit anti-human IgG antibody (1:1000 dilution in PMT) was allowed to react for 1 hour at 37°C. After three washings, the bound biotinylated antibody was detected by incubation of the plates for 1 hour with streptavidin-peroxidase (1 μg/ml) solution in PMT. This was followed by three washings in PBS and addition of substrate (2.5 mmol/L H₂O₂, 2 mmol/L ABTS, Sigma Immunochemicals). Optical densities (O.D.) were read after 30 min at 405 nm in a microplate reader. The positivity of the sera to the peptide was defined as P/N≥2.1 (P/N=specimen O.D.- blank O.D./negative control O.D.-blank control O.D.). The antibody titer was determined by the continuous double dilution of the samples from 1:10 and expressed as the maximum dilution when P/N≥2.1 [32].

Purification of IgG

Based on a seropositive response in enzyme immunoassay to peptide 176-202 of the β₃-AR, immunoglobulin fractions (IgGs) from these positive sera were prepared using a MabTrap Kit (Amersham) by following the manufacturer’s instructions. The concentration of purified IgGs was determined by using a Coomassie blue detection kit (Jiancheng Bioengineering Company, Nanjing, China). The specificity of the purified IgGs was determined by ELISA.

Immunofluorescence Staining

Cultured H9c2 cells were washed with PBS (pH 7.4) and fixed with 4% paraformaldehyde for 20min at 37°C. Cells were blocked with 5% bovine serum albumin (BSA) in PBS (w/v) for 1 hour at 37°C. Then, the cells were incubated overnight at 4°C with β₃-AR antibody (Abcam, UK) and the IgG fractions (25 μg/ml) from β₃-AR Abs positive HF patients, respectively. Following three PBS washes, cells were incubated with FITC-labeled secondary antibodies for 1 hour in the dark at 37°C. After being rinsed with PBS, cover slips with mounting medium containing DAPI stain nuclei were coated. Negative controls were performed by omitting primary antibodies. Fluorescence images were acquired and analyzed using an Olympus FV 1000 Confocal microscope.

Abdominal aortic banding surgery

Abdominal aortic banding (AAB) was induced by standard methods [33]. Briefly, Wistar rats (10 weeks old, weighing 200-220g) were chosen and anaesthetized with 10% chloral hydrate solution (30 mg/kg i.p.) and with aseptic surgical procedures. For the banding model, we opened the abdomen and separated the abdominal aorta, placed a 0.7 mm needle adjacent to the isolated aortic segment, tightly banded the aorta with an adjacent needle, and then drew out the needle. The sham control group underwent the same procedures without constriction of the aorta.

Group-I: control, sham-operated; Group-II: Untreated abdominal aortic banding (AAB) rats; Group-III: AAB rats treated with β₃-AR Abs via tail vein injection, 2 μg/g; Group-IV: AAB rats treated with negative IgGs via tail vein injection, 2 μg/g. IgGs were administered once every 10 days and the total period was 8 weeks.

Echocardiographic examination

In vivo cardiac function and geometry were assessed by transthoracic echocardiography (VIVID 7 dimension system, General Electric-Vingmed Ultrasound). The rats were anesthetized with methoxyflurane by inhalation. Left ventricular (LV) end-systolic and end-diastolic cross-sectional diameter (LVESD, LVEDD), and the mean of septal and posterior wall thicknesses were recorded from M-mode images. LV fractional shortening (FS) and LV ejection fraction (EF) were determined as previously described [34] .

Hemodynamic Studies

Hemodynamic parameters were measured by cardiac catheterization [35]. Left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), and maximal rate of rise and decline of ventricular pressure (±dp/dt_max) were recorded and analyzed.

Culture of neonatal beating cardiomyocytes

The hearts were removed aseptically from 1- to 2-day-old Wistar rats and the isolated cardiomyocytes were cultured as previously described [36]. Briefly, single cells were dissociated from the minced ventricles with a 0.25% solution of trypsin and were cultured at 37°C for 4 days as monolayers. On the day of the experiment, the medium was replaced and the cells were incubated at 37°C for 2 hours. Thereafter, the beating frequency of the spontaneously beating cardiomyocytes was measured on a heated stage of an inverted microscope at 37°C. The number of beats of a selected isolated myocardial cell or a cluster of synchronously contracting cells in each of 10 fields was counted for 15 sec each time. The changes of beating frequency were measured 5 min after the addition of the tested agents. The basal beating rate was 92.67±10.86 beats per minute. The data represented observations on 10 to 30 cells or cell clusters of synchronously beating cardiomyocytes in three different cultures.

Cell isolation procedure

Single ventricular cardiomyocytes were enzymatically isolated from the rat hearts as described previously [37]. Briefly, the rats were decapitated and the hearts were rapidly excised and mounted onto a Langendorff perfusion apparatus and were immediately perfused with Ca²⁺-free Tyrode solution (in mmol/L: 143 NaCl, 5.4 KCl, 0.5 MgCl₂, 0.3 NaH₂PO₄, 5.0 HEPES, 5.0 glucose, pH 7.4) equilibrated with O₂ until spontaneous contractions ceased. Subsequently the heart was perfused with Ca²⁺-free Tyrode solution containing 0.4 g/L collagenase II (270U/mL) and 0.7 g/L bovine serum albumin (BSA) for about 20 min until it became soft and then followed by 5 min perfusion with Ca²⁺-free Tyrode solution to remove the enzyme. Ventricles were separated and minced in Krebs solution (in mmol/L: 70 L-glutamic acid, 25 KCl, 20 Taurine, 10 KH₂PO₄, 3.0MgCl₂, 0.5 EGTA, 10 HEPES, 10 glucose, pH 7.4) supplemented with 2% BSA before being filtered through a nylon mesh (200 mesh). The viable cells were subsequently separated by sedimentation for 10 min, twice. The ventricular cardiomyocytes were then re-suspended in the Krebs solution supplemented with 2% BSA, and Ca²⁺ was slowly added to the cell suspension until it reached a final concentration of 1.8 mmol/L. Typically, about 70-80% rod-shaped cardiomyocytes were obtained.

Cell shortening/re-lengthening assay

The contraction and intracellular Ca²⁺ transient of ventricular cardiomyocytes were assessed by a video-based motion edge detection system (IonOptix, USA) [38]. Cells were placed in a chamber mounted on the stage of an inverted microscope (Olympus) and superfused (1 ml/min, 25°C) with Tyrode solution. The cells were field-stimulated at a frequency of 0.5 Hz at a 5 ms duration using a pair of platinum electrodes placed on the opposite sides of the chamber. The cardiomyocyte being studied was displayed on the computer monitor imaged through a 40× objective using an IonOptix Myocam camera. Criteria for choosing cardiomyocytes for the experiment include: i) a rod shape, ii) clearly defined sarcomeric striations, iii) steadily contracted in response to electrical stimulation and without spontaneous contractions, and iv) a stable steady-state contraction amplitude for at least 5 min before drug administration. Cell shortening and re-lengthening were assessed by the following indices: peak twitch amplitude (PTA, % cell length), time to 90% peak shortening (TPS), time to 90% re-lengthening (TR₉₀), and velocities of shortening (-dL/dt) and re-lengthening (+dL/dt).

Intracellular fluorescence measurement

Cardiomyocytes were loaded with fura-2/AM (0.5 µmol/L) for 30 min in the dark at room temperature. The fluorescence measurement was then recorded by a dual-excitation fluorescence photomultiplier system (IonOptix). Cells were exposed to light emitted by a 75 W lamp and passed through either a 360- or a 380-nm filter, while being stimulated to contract at 0.5 Hz. Fluorescence emission was detected at 510 nm by a photomultiplier tube after first illuminating the cells at 360 nm then at 380 nm for the duration of the recording protocol. The 360 nm excitation scan was repeated at the end of the protocol and qualitative changes in intracellular Ca²⁺ concentration were inferred from the ratio of the fluorescence intensity at the two wavelengths [39].

Electrophysiological measurements

Single ventricular cardiomyocytes were obtained using an enzymatic dissociation procedure similar to that described previously [40]. The whole-cell clamp technique was used for recording the membrane currents. An aliquot of the cell suspension was placed in a recording chamber on the stage of an inverted microscope (XDP-1, Shanghai) and perfused constantly at a rate of 1-2 ml/min with Tyrode’s solution. The electrodes were pulled in two stages by the two-step progress patch pipette puller (Narishige, Japan). For the measurement of I_Ca-L, the pipette solution (in mmol/L) contained egtazic acid (EGTA) 10.0, KCl 140.0, Na₂-ATP 2.0, HEPES 5.0, 4-aminopyridine (4-AP, Sigma) 5.0, MgCl₂ 1.0 (PH 7.3 adjusted with KOH). The glass pipette has a resistance of 2-4 megaohms after filling with the pipette solution. A whole-cell ‘giga ohm seal’ recording was used as described by Hamill et al. [41]. The pipette was connected to a patch-clamp amplifier (Axopatch-200A, Axon Instrument, USA). Computer program pClamp 5.51 (Axon Instrument, USA) was used to produce voltage clamping signals. Analysis was carried out by program pClampfit 8.0.

Reagents

BRL37344 (a selective β₃-AR agonist), Bupranolol, a nonselective β-AR antagonist, Nadolol (a β₁- and β₂- AR antagonist) was provided by Dr. Zhi-Qing Zhao (Mercer University School of Medicine, USA) and Dr. Che-Ping Cheng (Bowman Gray Medical College, USA).

Statistical Analysis

All data are expressed as means ± SD. Results were analyzed by Independent Samples t Test, chi square test or one-way ANOVA where appropriate using SPSS 11.5 statistics software. Probabilities of 0.05 or less were considered statistically significant.

Results

Prevalence and β₃-AR Abs titers in sera from HF and healthy subjects

A scatter diagram of optical density (OD) values was displayed in Figure 1. In the sera of 100 healthy subjects, 11 (11%) were positive for β₃-AR Abs. However, 31 (40.8%) of 76 HF patient sera was positive for β₃-AR Abs, a significantly greater prevalence than in healthy subjects (P < 0.001). The geometric mean of positive β₃-AR Abs titers in HF patients was also significantly greater than that of healthy subjects (1:75.9 ± 1.89 in HF patients vs. 1:13.8 ± 1.72 in control group, P < 0.001).

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Figure 1. Detection of β₃-AR Abs in HF patients and healthy subjects.

Scatter diagram demonstrating optical density values (dilution of 1:10) for detecting β₃-AR Abs in each serum sample from 76 HF patients and 100 healthy subjects.

https://doi.org/10.1371/journal.pone.0078207.g001

β₃-AR Abs could bind to β₃-ARs on the surface of H9c2 cells

Immunofluorescence staining was used to determine whether the IgG fraction isolated from the β₃-AR Abs positive sera of HF patients could bind to β₃-ARs of rats. The result showed that β₃-AR Abs from HF patients was mainly bound to the cell membrane, and the binding pattern of β₃-AR Abs with β₃-AR was virtually identical to commercial β₃-AR specific antibodies (Figure 2A, B, C).

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Figure 2. β₃-AR Abs from HF patients bound to β₃-AR on the surface of H9c2 cells.

A. The negative control. Nuclei were labeled with DAPI (blue). B. The positive control. Nuclei (blue), β₃-AR was identified with anti-β₃-AR antibody (Abcam, UK) (green). C. The experiment group. Nuclei (blue), β₃-AR was identified with β₃-AR Abs from HF patients (green).

https://doi.org/10.1371/journal.pone.0078207.g002

β₃-AR Abs could improve cardiac function in rats undergoing abdominal aortic banding (AAB) surgery

Echocardiography data showed that LV end-diastolic dimensions (LVEDD) and end-systolic dimensions (LVESD) progressively increased in AAB rats at 4 weeks (LVEDD 5.32±0.27 mm vs. 4.28±0.19 mm, LVESD 3.62±0.23 mm vs. 2.66±0.21 mm, P<0.05 vs. sham-operated group) and increased further at 8 weeks (Figure 3A, 3B and 3C). The fractional shortening (FS%) and ejection fraction (EF%) significantly decreased at 4 weeks and decreased further at 8 weeks (Figure 3D, 3E) compared with the sham-operated rats. In contrast, AAB rats treated with β₃-AR Abs prevented LV dilation (P<0.05 for both LVEDD and LVESD), and ameliorated the decrease in FS% and EF% compared with the AAB group at 4 and 8 weeks. However, the AAB rats treated with negative IgGs did not show significant effect on cardiac function. (Figure 3B, 3C, 3D, 3E)

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Figure 3. Effects of β₃-AR Abs on LV dilation and systolic function in AAB rats by echocardiography.

A. M-mode echocardiography images from Sham-operated (Sham), 8wk post-AAB, and 8wk post-AAB treated with β₃-AR Abs. B. C. β₃-AR Abs prevented LV chamber dilation induced by AAB. D. E β₃-AR Abs ameliorated LV systolic dysfunction induced by AAB.

^*P<0.05 vs. Sham-operated group, ^#P<0.05 vs. AAB group. Sham n=5, AAB n=5, AAB+β₃-AR Abs n=8, AAB+Negative IgGs n=8.

FS: fractional shortening, EF: ejection fraction.

https://doi.org/10.1371/journal.pone.0078207.g003

In addition, cardiac function was evaluated by LV hemodynamic analysis at 8 weeks after AAB. The AAB rats showed a lower left ventricular systolic pressure (LVSP), and a higher left ventricular end diastolic pressure (LVEDP) compared to the sham-operated rats, and the AAB rats also showed a depressed left ventricular ±dp/dt_max. Conversely, the AAB rats treated with β₃-AR Abs showed an increased LVSP and a reduced LVEDP accompanied with a higher ±dp/dt_max compared with AAB rats, and AAB rats treated with negative IgGs showed no obvious changes. (Figure 4A, 4B, 4C, 4D)

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Figure 4. Effects of β₃-AR Abs on cardiac function in AAB rats by LV hemodynamic analysis.

The change of LVSP (A), +dp/dt_max (B), LVEDP (C), and -dp/dt_max (D) in AAB rats treated with β₃-AR Abs.

^*P<0.05, ^**P<0.01 vs. Sham-operated group, ^#P<0.05, ^##P<0.01 vs. AAB group. Sham n=5, AAB n=5, AAB+β₃-AR Abs n=8, AAB+Negative IgGs n=8.

https://doi.org/10.1371/journal.pone.0078207.g004

Taken together, these data indicate that β₃-AR Abs could improve cardiac function in rats with abdominal aortic banding.

β₃-AR Abs had a negative chronotropic and negative inotropic effects on rat cardiomyocytes

To evaluate the potential role played by β₃-AR Abs in HF pathogenesis and associated cardiac dysfunction, we examined the chronotropic effects of β₃-AR Abs on the spontaneous beating frequency of neonatal rat cardiomyocytes. As shown in Figure 5A, purified IgGs (0.1 µmol/L) from HF patient sera positive for β₃-AR Abs markedly decreased cardiomyocyte beating frequency from 93.56±5.47/min to 64.32±8.13/min after 1 hour of exposure, suggesting a negative chronotropic effect of β₃-AR Abs. The negative chronotropic effects of β₃-AR Abs reached statistical significance within 1 hour and remained unabated during measurements for up to 6 hours, suggesting a cellular lack of desensitization to β₃-AR Abs’ chronotropic effects. As summarized in Figure 5B, seronegative IgGs had no effect upon cardiomyocyte beating frequency. However, β₃-AR Abs exerted a negative chronotropic effect comparable to that of β₃-AR agonist BRL37344 (0.1 µmol/L). Moreover, the negative chronotropic effect of β₃-AR Abs was abolished by bupranolol, a nonselective β-AR antagonist, but not by nadolol, a selective β₁- and β₂-AR antagonist. Neither nadolol nor bupranolol had any effects upon cardiomyocyte beating frequency. These results provided direct evidence that the negative chronotropic effect was mediated by β₃-AR, and not β₁- or β₂-AR. The specificity of β₃-AR Abs’ chronotropic effects was also verified by pre-incubation with its corresponding antigen peptide; the negative chronotropic effects of β₃-AR Abs were completely eliminated by the β₃-AR antigen peptide.

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Figure 5. Negative chronotropic effects of β₃-AR Abs in cultured neonatal rat cardiomyocytes.

A. Negative chronotropic effects of β₃-AR Abs (0.1 µmol/L) upon beating frequency of neonatal rat cardiomyocytes at different time points. ^*P < 0.05, ^**P < 0.01. B. Negative chronotropic actions of β₃-AR Abs (0.1 µmol/L) on beating frequency of neonatal rat cardiomyocytes after one hour exposure. The effects of nadolol, bupranolol, and β₃-AR antigen peptide on the actions of β₃-AR Abs are shown. ^**P < 0.01 vs. control. (n=10).

https://doi.org/10.1371/journal.pone.0078207.g005

We concomitantly investigated the inotropic effects of β₃-AR Abs on isolated adult rat cardiomyocytes. Representative contraction profiles were shown in Figures 6A and 6B, demonstrating the effects of β₃-AR Abs upon amplitude and velocity of shortening/re-lengthening in isolated rat cardiomyocytes. As summarized in Figure 6C, purified IgGs (0.1 µmol/L) from β₃-AR Abs positive HF sera manifested agonist-like effects upon cardiomyocyte contraction similar to β₃-AR agonist BRL37344 (0.1 μmol/L), as evidenced by a decrease in PTA (peak twitch amplitude, % cell length). Again, the nonselective β₃-AR antagonist bupranalol successfully blocked the inotropic effect of purified IgGs from β₃-AR Abs positive HF sera, but nadolol, a selective antagonist of β₁- and β₂-AR, did not, suggesting the mediation of observed IgG inotropic effects through β₃-ARs rather than β₁- or β₂-ARs. Neither nadolol nor bupranolol had any effects upon cell length. Furthermore, similar to the chronotropic effects experiment, the inotropic effects of purified IgGs from β₃-AR Abs positive HF patients were completely abolished after pre-incubation with its corresponding β₃-AR antigen peptide. This result strongly suggests that the negative inotropic effects were induced by the purified IgGs from β₃-AR Abs positive HF patients and were mediated by β₃-AR Abs rather than other IgGs present in the patient sera.

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Figure 6. Negative inotropic effect of β₃-AR Abs on isolated adult rat cardiomyocytes.

A and B. Representative profiles of cell length and dL/dt. C. Negative inotropic agonist-like activities of β₃-AR Abs (0.1 µmol/L, n=10) and the effects of nadolol, bupranolol, and β₃-AR antigen upon β₃-AR Abs-induced activities in isolated adult rat cardiomyocytes. (^**P < 0.01 vs. control, n=10).

https://doi.org/10.1371/journal.pone.0078207.g006

To clarify the mechanisms underlying the negative inotropic effects of β₃-AR Abs, we investigated changes in intracellular calcium transient ([Ca²⁺]_iT) and L-type Ca²⁺ current (I_Ca-L) of cardiomyocytes induced by β₃-AR Abs. A representative [Ca²⁺]_iT profile was shown in Figure 7A, demonstrating the effects of β₃-AR Abs upon cardiomyocytes [Ca²⁺]_iT. Figure 7B showed decreasing effects of β₃-AR Abs on [Ca²⁺]_iT in isolated rat cardiomyocytes. Purified IgGs (0.1 μmol/L) from β₃-AR Abs positive HF sera decreased the peak systolic [Ca²⁺]_iT from control value of 48.54 ± 12.41% to 30.26 ± 3.34% (P < 0.01, n=10) in isolated rat cardiomyocytes. This effect was comparable with that induced by β₃-AR agonist BRL37344 (0.1 μmol/L, which decreased peak systolic [Ca²⁺]_iT from control value of 48.54 ± 12.41% to 32.97 ± 5.81%, P < 0.01, n=10). Figure 7C showed an inhibitory effect of β₃-AR Abs (0.1 µmol/L) on ventricular membrane L-type Ca²⁺ current (I_Ca-L). As shown in Figure 7D, it was evident that β₃-AR Abs markedly decreased the peak inflow I_Ca-L from control value of 1467 ± 223 to 626 ± 138 pA (P < 0.01, n=5). This effect was comparable to that observed with β₃-AR agonist BRL37344 (0.1 µmol/L), which decreased peak inward I_Ca-L from control value of 1467 ± 223 to 574 ± 129 (P < 0.01, n=5).

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Figure 7. Effects of β₃-AR Abs on intracellular calcium transient ([Ca²⁺]_iT) and I_Ca-L in isolated adult rat cardiomyocytes.

A. Representative [Ca²⁺]_iT profile in isolated rat cardiomyocytes demonstrating decreased [Ca²⁺]_iT induced by β₃-AR Abs (0.1 μmol/L). B. Summary data (n=10, ^**P < 0.01 vs. control.) C. Representative I_Ca-L profile in isolated rat ventricular cardiomyocytes demonstrating inhibitory effects of β₃-AR Abs (0.1 µmol/L) on I_Ca-L. a. control; b. β₃-AR Abs; c. after β₃-AR Abs wash out. D. Group mean data demonstrating effects of β₃-AR Abs (0.1 µmol/L) and BRL37344 (0.1 µmol/L) on I_Ca-L in isolated rat ventricular cardiomyocytes. ^**P < 0.01 vs. control. n=5.

https://doi.org/10.1371/journal.pone.0078207.g007

Discussion

The novel findings in this study were that 1) the occurrence frequency and the OD value of β₃-AR Abs in patients with HF were much higher than that of healthy subjects, and 2) β₃-AR Abs could ameliorate cardiac dysfunction in AAB rats, and its negative inotropic and chronotropic effects and inhibition of L-type calcium channels may be responsible for the protective effects.

In this study, the peptide with a sequence corresponding to the second extracellular loop of human β₃-AR was used as the antigen to screen for β₃-AR Abs in the sera from patients with HF and healthy control subjects. The reason for choosing the second extracellular loop of the receptor is that this loop has been shown to be highly antigenic, immunogenic and important for receptor function in many G-protein-coupled receptors [6,9,42]. Furthermore, this loop contains the T and B cell epitopes necessary for induction of an immune response [10,43].

Our results showed that 40.8% of patients with HF were β₃-AR Abs positive. Moreover, the occurrence frequency and the OD value of β₃-AR Abs in patients with HF were much higher than that of healthy subjects, which were similar to those for anti-β₁- and β₂-adrenoceptors or muscarinic M₂-receptor autoantibodies reported previously by Chiale [9], Liu [42] and Fu [44]. Although autoantibody-producing B-cell clones exist in healthy organisms, they are generally suppressed or activated to a limited extent in normal conditions and are thus insufficient to cause damage or a disease state. Therefore, the presence of autoantibodies at a lower titer does not necessarily reflect a pathological state [42,45]. The higher frequency of occurrence of β₃-AR Abs in HF patients suggested that this novel autoantibody may play a significant role in the pathogenesis of HF.

In most cases, HF is accompanied by cardiac hypertrophy which is an appropriate adaptive response to maintain adequate function in the presence of chronic pathological stress [46,47]. Although this myocardial enlargement is initially beneficial [48], prolonged hypertrophy may accelerate cardiac dysfunction and HF [49,50]. Consequently, it is necessary to determine whether β₃-AR Abs could mitigate or accelerate the transition from compensatory cardiac hypertrophy to HF. And a common cause of cardiac hypertrophy is chronic pressure overload due to hypertension or aortic stenosis [51].

Therefore, in the present study, the pressure overload rat model was set up through abdominal aortic banding (AAB), and β₃-AR Abs purified from HF patients were passively administered to AAB rats to observe whether these autoantibodies could affect the cardiac function. In fact, numerous studies [32,52,53] adopted human-derived antibodies in animal models to examine the biological characteristics of these antibodies. Furthermore, the immunofluorescence technique was used to confirm that β₃-AR Abs purified from HF patients could recognize β₃-ARs expressed on rat cardiomyocytes.

In the present study, we observed that administering the β₃-AR Abs to AAB rats for 4 and 8 weeks could prevent LV chamber dilation and ameliorate cardiac dysfunction, showing the cardioprotective role of β₃-AR Abs in AAB rats. In order to explore the underlying mechanisms responsible for its cardioprotective effects, we investigated the biological activities of β₃-AR Abs on cardiomyocyte. Compared with the complex factors affecting the cardiac functions in vivo, the use of the isolated cells was easily manipulated and suitable for the study of direct functional effects of autoantibodies on cardiomyocytes. Our results confirmed the agonist-like activities of these β₃-AR Abs as evidenced by their negative chronotropic effects in cultured cardiomyocytes without desensitization and negative inotropic effects with decreasing [Ca²⁺]_iT and I_Ca-L in isolated cardiomyocytes. And the inhibitory effects of β₃-AR Abs on I_Ca-L and [Ca²⁺]_iT may be the molecular mechanisms that are responsible for its negative inotropic effects. In addition, our current experimental results demonstrated that the agonist-like effects of β₃-AR Abs were mediated by β₃-AR because the chronotropic and inotropic effects of β₃-AR Abs were completely blocked by bupranolol, a nonselective β-AR antagonist and by β₃-AR antigen peptide, but not by nadolol, a selective β₁- and β₂-AR antagonist. These results suggested that the cardioprotective role of β₃-AR Abs in pressure-overload hypertrophy may be attributable to its negative chronotropic and negative and inhibition of L-type calcium channels which could reduce myocardial oxygen consumption and intracellular calcium overlod, hence may ameliorate the pressure overload-induced pathological remodeling and cardiac dysfunction.

Previous reports and the present study show that both autoantibodies against the β₁-AR (β₁-AR Abs) and β₃-AR (β₃-AR Abs) exist in the sera of patients with HF and it is of significance to compare the role of β₁-AR Abs and β₃-AR Abs in the development of HF. In recent years, a large number of investigations have demonstrated the involvement of β₁-AR Abs in the pathogenesis of HF [6-8]. These autoantibodies display a stimulatory agonist-like activity on the target receptors without desensitization [11,12]. Hence, β₁-AR Abs would permanently overstimulate the β₁-ARs which result in a large amount of energy consumption and finally lead to HF. In contrast to β₁-AR, stimulation of β₃-AR could induce a negative inotropic effect [21]. Moreover, the β₃-AR is markedly up-regulated in failing heart [23-26] which may reflect a compensatory mechanism to protect the heart from overstimulation of β₁-AR. Thus the β₃-AR Abs may serve as a “brake” to protect the heart from catecholamine overstimulation by means of its stimulatory agonist-like activities on β₃-ARs. In the current study, we confirmed that β₃-AR Abs from patients with HF exerted a cardioprotective role in the rat failing heart induced by pressure overload. We observed that administering the β₃-AR Abs to rats with AAB could prevent LV dilation and improve the cardiac function of rats. Our findings are partly supported by several recent reports that β₃-AR agonists had cardioprotective effects in pressure overload hypertrophy and HF [27,28] and lacking of β₃-AR showed exacerbated pathological remodeling and impaired cardiac function [29].

In conclusion, we have shown that β₃-AR Abs had substantial cardioprotective effects in pressure overload hypertrophy, which may alleviate the development of HF. These findings provide new insights concerning the significance of β₃-AR Abs in the pathogenesis of HF.

Limitation

Our work leaves some unanswered questions for future study. Although we observed that β₃-AR Abs could improve heart function, the mechanisms require further research.

Acknowledgments

We are indebted to Lindsey Devillier for his assistance with English-language editing.

Author Contributions

Conceived and designed the experiments: HL. Performed the experiments: JW ML XM KB LW. Analyzed the data: ZY TL ZZ RZ. Contributed reagents/materials/analysis tools: ZZ. Wrote the manuscript: JW.

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Figures

Abstract

Introduction

Methods

Patient Characteristics

Peptides

ELISA

Purification of IgG

Immunofluorescence Staining

Abdominal aortic banding surgery

Echocardiographic examination

Hemodynamic Studies

Culture of neonatal beating cardiomyocytes

Cell isolation procedure

Cell shortening/re-lengthening assay

Intracellular fluorescence measurement

Electrophysiological measurements

Reagents

Statistical Analysis

Results

Prevalence and β3-AR Abs titers in sera from HF and healthy subjects

β3-AR Abs could bind to β3-ARs on the surface of H9c2 cells

β3-AR Abs could improve cardiac function in rats undergoing abdominal aortic banding (AAB) surgery

β3-AR Abs had a negative chronotropic and negative inotropic effects on rat cardiomyocytes

Discussion

Limitation

Acknowledgments

Author Contributions

References

Prevalence and β₃-AR Abs titers in sera from HF and healthy subjects

β₃-AR Abs could bind to β₃-ARs on the surface of H9c2 cells

β₃-AR Abs could improve cardiac function in rats undergoing abdominal aortic banding (AAB) surgery

β₃-AR Abs had a negative chronotropic and negative inotropic effects on rat cardiomyocytes