Pone.0064711 1.15

Increased Beta2-Adrenoceptors in Doxorubicin-InducedCardiomyopathy in Rat Nolwenn Merlet1,2, Nicolas Piriou1,3, Bertrand Rozec1,4, Amandine Grabherr1, Benjamin Lauzier1,2, Jean- Noe¨l Trochu1,2,3, Chantal Gauthier1,2* 1 l'institut du thorax, Unite´ Inserm UMR 1087/CNRS UMR 6291, Nantes, France, 2 Universite´ de Nantes, Nantes, France, 3 CHU Nantes, l'institut du thorax, Nantes, France, 4 CHU Nantes, Department of Anaesthesiology, Nantes, France Background: The toxicity of doxorubicin, leading to an irreversible heart failure, limits its use as chemotherapeutic agent.
The beneficial effects of early administration of b-blocker were reported in patients with heart failure due to doxorubicin,suggesting an important role of b-adrenoceptors (b-ARs). This study aimed to identify a putative target (b-AR and/or itseffectors) at the early phase of a chronic doxorubicin-induced cardiomyopathy (Dox-CM) in a rat model.
Methodology: Dox-CM was induced by six doxorubicin injections (cumulative dose: 15 mg.kg21) and validated byechocardiography and left ventricle (LV) catheterization. The b-AR protein expressions in LV were evaluated by western-blotat days 35 (d35) and 70 (d70) after the first doxorubicin injection. Ex vivo cardiac contractility (dP/dtmax, dP/dtmin) wasevaluated on isolated heart in response to specific b-AR stimulations at d35.
Results: At d35, Dox-CM hearts were characterized by mild LV systolic and diastolic dysfunctions, which were exacerbated atd70. In Dox-CM hearts, b3-AR expression was only decreased at d70 (-3768%). At d35, b1-AR expression was decreased by6866%, but ex vivo b1-AR function was preserved due to, at least in part, an increased adenylyl cyclase response assessed byforskolin. b2-AR expression was increased both at d35 (+58622%) and d70 (+174635%), with an increase of ex vivo b2-ARresponse at d35. Inhibition of Gi protein with pertussis toxin did not affect b2-AR response in Dox-CM hearts, suggesting adecoupling of b2-AR to Gi protein.
Conclusion: This study highlights the b1/b2-AR imbalance in early Dox-CM and reveals the important role that b2-AR/Gicoupling could play in this pathology. Our results suggest that b2-AR could be an interesting target at early stage of Dox-CM.
Citation: Merlet N, Piriou N, Rozec B, Grabherr A, Lauzier B, et al. (2013) Increased Beta2-Adrenoceptors in Doxorubicin-Induced Cardiomyopathy in Rat. PLoSONE 8(5): e64711. doi:10.1371/journal.pone.0064711 Editor: Philippe Rouet, I2MC INSERM UMR U1048, France Received January 29, 2013; Accepted April 17, 2013; Published May 31, 2013 Copyright: ß 2013 Merlet et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the ‘‘Association Franc¸aise contre les Myopathies'', the ‘‘Fe´de´ration Franc¸aise de Cardiologie'', the ‘‘Fondation de France''and the ‘‘Fondation Genavie''. 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.
* E-mail: [email protected] As in different HF etiologies, Dox-CM is characterized by an alteration of adrenergic system [8]. However, at the present time, Anthracyclines, like doxorubicin (Dox), epirubicin and dauno- only few studies have examined the role of cardiac b1- and b2- rubicin, are among the most effective drugs used in chemotherapy adrenoceptor (b-AR) subtypes in the pathogenesis of Dox- for cancer patients. Since the late 60s, Dox is frequently used cardiotoxicity [9,10] and only one study, at late-onset Dox-CM, against a variety of cancers including Hodgkin's lymphoma [1], assessed b3-AR subtype [11], which is recently described as a new soft-tissue sarcomas [2], leukemia and solid tumors. However, Dox target for some b-blockers such as nebivolol [12,13]. Despite this administration is limited due to severe cardiotoxic effects leading lack in experimental data, some clinical studies investigated b- to dilated cardiomyopathy [3]. Prognosis of heart failure (HF) due blocker therapies in Dox-CM. Kalay et al., demonstrated that left to Dox-cardiotoxicity is poor and even worse than ischemic or ventricular (LV) diameters remained constant and diastolic idiopathic dilated cardiomyopathy. Although several mechanisms function was better preserved after Dox-treatment in patients have been proposed to describe the mechanisms by which Dox receiving carvedilol, compared to placebo [14]. However, induces cardiotoxicity (generation of free radicals, mitochondrial Georgakopoulos et al., demonstrated that metoprolol, a b-blocker disruption, alteration of cellular energetic, and initiation of without antioxidative properties, failed to give cardioprotection in apoptotic cascades), these mechanisms are still not fully under- lymphoma-treated doxorubicin patients [15]. It was reported that stood [4–6] and there is no specific treatment for Dox-induced an early start of treatment with angiotensin-converting enzyme cardiomyopathy (Dox-CM) [6,7]; treatments classically used for inhibitors (ACEIs), in association or not with b-blockers, both other HFs with systolic dysfunction induce only limited beneficial improves myocardial contractility [16] and patients' prognosis effects in Dox-CM.
PLOS ONE www.plosone.org May 2013 Volume 8 Issue 5 e64711 Beta-Adrenoceptors in Doxorubicin Cardiomyopathy Table 1. Antibodies used for western-blot.
Secondary antibody Rabbit polyclonal antibody, Sigma-Aldrich Goat anti-rabbit immunoglobulin G, Santa-Cruz Biotechnology (sc-2054) Rabbit polyclonal antibody, AbCam Ltd Goat anti-rabbit immunoglobulin G, Santa-Cruz Biotechnology (sc-2054) Rabbit polyclonal antibody, Santa Cruz Goat anti-rabbit immunoglobulin G, Santa-Cruz Biotechnology (sc-50436) Biotechnology (sc-2054) Rabbit polyclonal antibody, Merk Chemicals Ltd 1/1,000 Goat anti-rabbit immunoglobulin G, Santa-Cruz Biotechnology (sc-2054) Rabbit polyclonal antibody, Merk Chemicals Ltd 1/5,000 Goat anti-rabbit immunoglobulin G, Santa-Cruz Biotechnology (sc-2054) Mouse monoclonal antibody, Santa-Cruz Goat anti-mouse immunoglobulin G, Santa-Cruz Biotechnology (sc-32233) Biotechnology (sc-2055) Primary antibodies were diluted in TBS-T, excepted for Gia2 protein detection which was diluted in 5% non-fat dry milk in TBS-T. All secondary antibodies were diluted in1% non-fat dry milk in TBS-T.
doi:10.1371/journal.pone.0064711.t001 [17]. Although the exact mechanism is still poorly understood, theauthors highlighted the importance of an early diagnosis, becausea delayed treatment (.6 months after the end of chemotherapy) isinefficient [17].
The aim of the present study was to identify a putative target (b- AR and/or its effectors) involved at the early phase of a chronicDox-CM in a rat model. This study demonstrated for the first timethat b2-AR expression was increased from 35 days after the firstDox-injection, this effect was maintained until 70 days after thefirst Dox-injection, resulting in an increase of b2-AR-inducedcontractility. In addition, b1-AR function was preserved, in spite ofdecreased b1-AR protein expression. This discrepancy could beexplained by an increase of adenylyl cyclase (AC) expression and/or activity as illustrated by an increased forskolin-induced responsein Dox-CM rats.
All experiments were performed in accordance with the 1996 Guide for the Care and Use of Laboratory Animals published bythe U.S. National Institute of Health. The protocol was approvedby the Direction De´partementale de la Protection des Populations(agreement number C-44 015) and all efforts were made tominimize suffering.
One hundred and fifty eight male Sprague-Dawley rats (225– 250 g) were purchased from Janvier (Le Genest St Isle, France)and were housed under standard conditions of room temperature,humidity (40–60%) and 12 h light/dark cycle. Food and waterwere available ad libitum.
Doxorubicin (AdriblastineH 50 mg/25 mL, Pfizer, France) was administered intraperitoneally in six equal injections (eachcontaining 2.5 mg.kg21) over a period of two weeks, with a totalcumulative dose of 15 mg.kg21 body weight (Dox-CM: n = 100).
Age-matched rats injected with saline were used as controls (Ctrl:n = 58). Rats were then bred in animal housing until three weeksafter the last injection (day 35 (d35); Ctrl: n = 46, Dox-CM: n = 76) Figure 1. Body weight (A) and survival curves (B) of Ctrl and or until eight weeks after the last injection (day 70 (d70); Ctrl: Dox-CM rats. Values are means 6 sem **: P,0.001 vs Ctrl. Ctrl: n = 12, Dox-CM: n = 24). At d35, twenty Dox-CM rats and twenty control, Dox-CM: Doxorubicin-induced cardiomyopathy.
Ctrl rats were randomly selected to be hemodynamically explored PLOS ONE www.plosone.org May 2013 Volume 8 Issue 5 e64711 Beta-Adrenoceptors in Doxorubicin Cardiomyopathy Table 2. Cardiac morphological parameters in Ctrl and Dox-CM rats, at d35 and d70.
Heart wt/Body wt (mg.g21) LV wt/Body wt (mg.g21) Heart wt/Tibia length (mg.cm21) LV wt/Tibia length (mg.cm21) Ctrl: control; Dox-CM: Doxorubicin-induced cardiomyopathy; LV: left ventricle; wt: weight. Values are means 6 sem. *: P,0.05 vs respective Ctrl. **: P,0.001 vsrespective Ctrl.
doi:10.1371/journal.pone.0064711.t002 by echocardiography-Doppler. Then either rats were hemody- 2D speckle-tracking method on every medial myocardial segment namically assessed by LV catheterization, or rat hearts were removed either to test ex vivo cardiac contractile function or toperform biochemical studies. At d70, rats were used to perform Left Ventricle Catheterization echocardiography-Doppler and biochemical studies.
LV catheterization was performed, at d35, by a 2F microtip pressure catheter (SPR 838, Millar instruments Inc, Houston, Texas). Anaesthesia maintenance on spontaneously breathing rats Transthoracic echocardiography was performed using a com- was performed with an inhalational anaesthesia system for small mercially available ultrasound system (VIVID7, GE Healthcare, animal (TEM anaesthesia, Lormont, France). Isoflurane was Horton, Norway) equipped with a 10 MHz sectorial probe. Rats delivered through a nose mask at a concentration of 2% volume were anaesthetized with a gas-mixture of 1% isoflurane (ForeneH, and 1 L.min21 O2 flow to limit hemodynamic repercussion. Body Abbott France, Rungis, France) in O2. The chest was shaved and temperature was monitored by rectal probe and maintained the animal was positioned on a heating pad in a supine position.
constant (37uC) by warming-blanket. The right carotid artery was All recordings were monitored under a continuous single-channel isolated, ligated at the proximal part and the pressure catheter was electrocardiogram obtained on the imaging system by fixing the inserted in. Signals were recorded using an Analogic/Digital electrodes to the limbs. Using two-dimensional imaging, a short converter (EMKA Technologies, Paris, France), stored and axis view of the LV at the level of the papillary muscles was displayed on a computer by the IOX1.5.7 Software System obtained and the two-dimensionally guided M-mode recording (EMKA Technologies). Data were analysed using Datanalyst through the anterior and posterior walls of the LV was taken as software (EMKA Technologies). The following parameters were recommended by the American Society of Echocardiography [18].
obtained: LV end diastolic pressure (LVEDP), LV contraction and Then, trans-mitral inflow in pulsed-wave Doppler from apical four relaxation velocities (dP/dtmax and dP/dtmin, respectively), the chamber view and tissue Doppler imaging (TDI) on basal index of LV relaxation constant (Tau).
segments of septal and lateral walls in apical four chamber view The animals were thereafter sacrificed by injection of a were taken as previously described [19]. A cine-loop of LV parasternal short axis view with high frame rate was obtained. Allacquisitions were performed by the same operator.
All images were digitally stored on hard disks for off-line analysis The expressions of b1-AR, b2-AR, b3-AR, Gsa, Gia2 and (EchoPac Q-analysis software, GE Healthcare). Measurements GAPDH were examined by western-blot, at d35 and d70. Hearts were made on five cardiac cycles and averaged for each data were rapidly isolated and placed in a cold Tyrode solution value. The following parameters were determined as recom- composed as followed (in mM): NaCl, 137; KCl, 5.4; MgCl2, 1.2; mended by the American Society of Echocardiography [18]: LV Na2HPO4, 1.2; Hepes, 20; CaCl2, 1.0; pH 7.4 (Sigma-Aldrich, St end diastolic and systolic diameters (LVEDD and LVESD), Quentin Fallavier, France). LV and septum free walls were diastolic posterior wall thicknesses (dPWth). LV end diastolic and carefully separated and freezed in liquid nitrogen and stored at systolic volumes (LVEDV and LVESV) were calculated from the 280uC until used. LV and septum free walls samples were Teichholz method in order to assess LV ejection fraction (LVEF), homogenized in 3 mL of Ripa buffer for 1 g of tissue plus 1X whereas LV shortening fraction (LVSF) was calculated from PMSF (Interchim, Montluc¸on, France) and 2X protease inhibitor LVEDD and LVESD previously measured. LV diastolic function cocktail (Roche, Mannheim, Germany). Protein samples (25 mg) parameters were derived from pulsed-wave trans-mitral inflow were submitted to electrophoresis on a 10% polyacrylamide/ pattern and TDI off-line analyses as previously described [20]: the sodium dodecyl sulfate gel and then were run 150 min at 20 mA peak of E wave velocities, the isovolumic relaxation time (IVRT), per membrane in TG-SDS buffer (Interchim). Gels and nitrocel- the mean of peak velocities of basal septal and lateral walls (pulsed lulose membranes (Hybond C super membrane, Amersham, Saclay, wave TDI) during systole (Sa) and in early diastole (Ea) to calculate France) were equilibrated in TG-SDS buffer with 20% ethanol, E/Ea ratio. Radial 2D strain analyses were performed using the and protein fractions were transferred using an electroblottingapparatus (Bio-Rad, Marnes La Coquette, France). Nonspecific PLOS ONE www.plosone.org May 2013 Volume 8 Issue 5 e64711

Beta-Adrenoceptors in Doxorubicin Cardiomyopathy Figure 2. Representative images obtained by echocardiography at day 35 in Ctrl (A) and Dox-CM (B) rats. Images were obtained with ashort axis view of a two-dimensionally directed M-mode. Ctrl: control, dAWth: diastolic anterior wall thickness, Dox-CM: Doxorubicin-inducedcardiomyopathy, dPWth: diastolic posterior wall thickness, LVEDD: left ventricular end diastolic diameter, LVESD: left ventricular end systolic diameter,sAWth: systolic anterior wall thickness, sPWth: systolic posterior wall thickness.
doi:10.1371/journal.pone.0064711.g002 PLOS ONE www.plosone.org May 2013 Volume 8 Issue 5 e64711 Beta-Adrenoceptors in Doxorubicin Cardiomyopathy PLOS ONE www.plosone.org May 2013 Volume 8 Issue 5 e64711 Beta-Adrenoceptors in Doxorubicin Cardiomyopathy Figure 3. In vivo cardiac parameters obtained by echocardiography-Doppler in Ctrl and Dox-CM rats. Ctrl are showed in empty bar andDox-CM in full bar. Values are means 6 sem *: P,0.05 vs respective Ctrl **: P,0.001 vs respective Ctrl. d35: day 35, d70: day 70, Dox-CM: Doxorubicin-induced cardiomyopathy, dPWth: diastolic posterior wall thickness; E: E wave velocity; Ea: peak velocity of basal and lateral walls in early diastole;IVRT: isovolumic relaxation time; LV: left ventricle; LVEF: left ventricular ejection fraction; LVEV: left ventricular end volume; LVSF: left ventricularshortening fraction; Sa: peak velocity of basal and lateral walls in systole.
doi:10.1371/journal.pone.0064711.g003 binding was blocked by incubating membranes in 5% non-fat dry period, specific b1-, b2- or b3-AR functions and AC stimulation milk in Tris-buffered saline (TBS) (200 mM Trizma base, 1.4 M were assessed by constructing concentration-response curves to NaCl, pH = 7.5) with 0.1% Tween 20 added (TBS-T) and then different pharmacological agents. Thus, b1- and b2-AR functions membranes were incubated with the primary antibody. Mem- were assessed by perfusing isoproterenol (a non selective b-AR branes were washed in 5% non-fat dry milk in TBS-T and agonist, 1029 to 1025 M) in the presence of 1026 M L-748,337 (a hybridized with the secondary antibody in 1% non-fat dry milk in selective b3-AR antagonist) associated to 1026 M ICI-118,551 (a TBS-T. Finally, membranes were washed with 5% non-fat dry selective b2-AR antagonist) or to 1026 M CGP-20712A (a milk in TBS-T then TBS, and antibody complexes were revealed selective b1-AR antagonist), respectively. b3-AR function was by the enhanced chemiluminescence detection process (Bio-Rad).
assessed by perfusing growing concentrations (1029 to 1025 M) of Chemiluminescence was visualized using an Amersham Image- SR 58611A (a b3-AR agonist) and AC response was assessed by Quant RT-ECL camera (GE Healthcare) and band signals were perfusing growing concentrations (3.1028 to 1025 M) of forskolin assessed by densitometry with ImageQuant TL software (GE (an AC activator). In order to assess the role of Gi in the b2-AR Healthcare). For each lane, a ratio to the corresponding GAPDH pathway, we used pertussis toxin (PTX) (a selective Gi inhibitor).
band intensity was calculated; the use of GAPDH as reference was Classically, PTX is administered to rats two or three days before validated by checking the abundance stability of GAPDH protein experiments [22]. However, due to high mortality in Dox-CM rats between Ctrl and Dox-CM groups.
pre-treated with PTX, we were not able to apply this protocol and Antibody references and conditions used in this study are used another one consisting in pre-treating heart with PTX summarized in Table 1.
(4 mg.L21) in a closed system during 30 min, as described by othergroups [23]. Data were analysed using Datanalyst software Whole Heart Contractility (EMKA Technologies). After Langendorff experiments, hearts At d35, after removal, hearts were quickly flushed into a cold were weighed, as well as LV free walls which were carefully Tyrode buffer, and then were mounted on a cannula via the aorta separated from the heart.
on a Langendorff apparatus (EMKA Technologies) and perfusedwith a Krebs-modified solution (in mM: NaCl, 116; KCl, 5; Data Analysis and Statistics Data are presented as means 6 standard error to the mean 1.1; NaH2PO4, 0.35; NaHCO3, 27; glucose, 10; mannitol, 16; Na-Pyruvate, 2; CaCl (sem) of n experiments obtained from n different animals. In vivo 2, 1.8) continuously oxygen- ated with a 95% O and ex vivo cardiac parameters in baseline as well as protein 2, 5% CO2 gas mixture to maintain a pH 7.4.
Hearts were perfused at a constant flow-rate of 14 mL.min21 at expressions were compared using Student's t test for unpaired 37.060.4uC. After heart beatings became regular, left auricle was data. Concentration-response curves were compared with a two- gently cut off to insert a small latex balloon, connected to a way ANOVA (concentration, treatments) for repeated measures pressure transducer to record LV pressure, into the LV via mitral followed, when appropriated, by a Bonferroni's multiple compar- valves. Before starting record, the minimum pressure into the isons test. Differences were considered significant if P,0.05.
balloon was adjusted to 8–15 mmHg. Cardiac parameters wererecorded using IOX1.5.7 software (EMKA Technologies): inotro- Drugs and Chemicals pism, lusitropism and chronotropism were respectively evaluated by measuring the contraction velocity (dP/dt max), the relaxation min) and the heart rate (HR). After the stabilization dro chloride, L-748,337, N-[[3-[(2S)-2-Hydroxy-3-[[2-[4-[(phenylsulfo- Table 3. In vivo cardiac parameters obtained by LV catheterization in Ctrl and Dox-CM rats, at d35.
forskolin and PTX were obtained from Tocris Bioscience (Bristol,UK), isoproterenol was obtained from Sigma-Aldrich (St QuentinFallavier, France) and SR 58611A, [(RS)-N-[(25)-7-ethxycarbonyl- ethanamide hydrochloride] was a generous gift from Sanofi- Synthe´labo (Montpellier, France). All drugs were dissolved in distilled water, excepted isoproterenol which was solubilised in 1% acid ascorbic and L-748,337 and forskolin which were solubilised in LV diastolic function dimethylsulfoxide (Sigma-Aldrich). The final concentration of the solvent in the organ bath was less than 0.1% v.v21 and was used as controls for the effect of the active drug.
LV loading conditions LVEDP (mmHg) Ctrl: control; Dox-CM: Doxorubicin-induced cardiomyopathy; dP/dtmax: leftventricular contraction velocity; dP/dtmin: left ventricular relaxation velocity; LV: Mortality and General Characteristics of Animals left ventricle; LVEDP: left ventricular end diastolic pressure; Tau: index of leftventricular relaxation constant. Values are means 6 sem. *: P,0.05 vs Ctrl.
Dox-CM rats did not gain weight during the Dox-treatment, whereas the body weight of Ctrl rats increased regularly. After the PLOS ONE www.plosone.org May 2013 Volume 8 Issue 5 e64711 Beta-Adrenoceptors in Doxorubicin Cardiomyopathy Figure 4. b3-AR expression and function in Dox-CM hearts. A. Representative b3-AR immunoblotting at days 35 (d35) and 70 (d70). B. b3-ARprotein quantification at day 35. C. b3-AR protein quantification at day 70. Protein levels were quantified using Amersham ImageQuant RT-ECLcamera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TL software (GE Healthcare) and a ratio to thecorresponding GAPDH band intensity was calculated. D. Cardiac inotropic (dP/dtmax), E. lusitropic (dP/dtmin) and F. chronotropic (HR) effects ofincreasing concentrations of SR 58611A (1029 to 1025 M) were evaluated on isolated perfused hearts. **: P,0.001 vs Ctrl. Ctrl: control, Dox-CM:Doxorubicin-induced cardiomyopathy.
doi:10.1371/journal.pone.0064711.g004 PLOS ONE www.plosone.org May 2013 Volume 8 Issue 5 e64711 Beta-Adrenoceptors in Doxorubicin Cardiomyopathy end of Dox-treatment, the rat body weight increased again, but intensity in Dox-CM and Ctrl (P = 0.192), whereas, at d70, b3-ARs remained significantly lower than those of Ctrl rats, and reached a were down-expressed in Dox-CM hearts compared to Ctrl hearts plateau from 5 weeks after the end of Dox-treatment (day 49; (237.267.9%; P,0.001) (Figures 4A–C). In Ctrl isolated heart, at Figure 1A). In addition, Dox-CM rats became lethargic compared d35, b3-AR stimulation performed by cumulative increasing to Ctrl one.
concentrations of SR 58611A produced no significant effect on The mortality rate was 25.0% at d35 and 58.3% at d70 in Dox- heart rate and dP/dtmax at any concentration (P = 0.239 and CM when no mortality was observed in Ctrl (Figure 1B). At d35, P = 0.385, respectively) (Figures 4D, F) and decreased dP/dtmin all surviving Dox-CM rats presented a hepatomegaly and 79.4% only at the higher concentration of SR 58611A (1025M) of them presented ascites (30.865.8 mL), whereas at d70 no (P = 0.004) (Figure 4E). Compared to Ctrl hearts, b3-AR hepatomegaly was observed and ascites was present in 38.9% of stimulation in Dox-CM hearts produced a significant decrease in Dox-CM rats (35.764.9 mL). Whole heart and LV weights from dP/dtmax only at the higher concentration of SR 58611A (1025M) Dox-CM rats were significantly smaller than those of Ctrl animals (P,0.001) (Figure 4D) and had no effect on dP/dtmin nor heart both at d35 and d70, and they were still smaller after rate (P = 0.406 and P = 0.107, respectively) (Figures 4E, F).
normalization to the tibia length. However, heart/body weightratios and LV/body weight ratios were similar between Ctrl and b1-adrenoceptor Expression and Function Dox-CM rats at d35 when it was increased in Dox-CM group By western-blot, the antibody directed against b1-AR detected a compared to Ctrl at d70 (Table 2).
band at 72 kDa (Figure 5A) whose intensity was significantlydecreased in Dox-CM LV at d35 by 268.266.1% (P = 0.002 vs Cardiac Function Obtained by Echocardiography- Ctrl) (Figure 5B) and at d70 by 275.3614.0% (P,0.001 vs Ctrl) (Figure 5C). In Ctrl heart, at d35, b1-AR stimulation induced a Echocardiography-Doppler analyses were performed at d35 concentration-dependant increase in dP/dtmax, dP/dtmin and and d70. Heart rate, measured before all echocardiographic heart rate with a maximal effect observed at a concentration of acquisitions, was monitored in order to be similar between both 1025 M (P,0.001, each) (Figures 5D–F). The heart rate and the groups (d35: Ctrl: 36466 bpm, Dox-CM: 37166 bpm, P = 0.427; positive inotropic and lusitropic effects were unchanged in Dox- d70: Ctrl: 36866 bpm, Dox-CM: 337617 bpm, P = 0.079).
CM isolated hearts compared to Ctrl hearts (P = 0.544, P = 0.772 Representative images obtained by echocardiography in short and P = 0.667, respectively) (Figures 5D–F).
axis view of a two-dimensionally directed M-mode at d35 areshown in Figure 2.
b2-adrenoceptor Expression and Function The dPWth decreased in Dox-CM compared to Ctrl hearts at By western-blot, the antibody directed against b2-AR detected a d35 (29.563.3%, P = 0.055) and was significantly thinner at d70 band at 55 kDa (Figure 6A) whose intensity was significantly (220.564.4%, P,0.001) (Figure 3A). LVESV was similar in both increased in Dox-CM LV at d35 by +57.9621.6% (P = 0.039 vs groups at d35 as at d70 (P = 0.199 and P = 0.116, respectively). On Ctrl) (Figure 6B) and at d70 by +173.7635.1% (P,0.001 vs Ctrl) the contrary, LVEDV was reduced in Dox-CM compared to Ctrl (Figure 6C). At d35, b2-AR stimulation induced a similar at d35 (212.661.9%, P,0.001) as at d70 (221.768.7%, concentration-dependant increase of heart rate between Ctrl and P = 0.050) (Figure 3B).
Dox-CM hearts (P = 0.695) (Figure 6F). In Ctrl isolated hearts, b2- Compared to Ctrl, Dox-CM LVs were characterized at d35 by AR stimulation induced a concentration-dependant increase of mild but significant decreases in LVEF (-14.262.0%; P,0.001) dP/dtmax and dP/dtmin (P,0.001 and P = 0.005, respectively) and LVSF (223.462.7%; P,0.001), that were more pronounced (Figures 6D, E) but at a lesser extent than b1-AR one. In Dox-CM at d70 (225.266.2%, P,0.001 and 234.967.4%, P,0.001, hearts, the maximal effects induced by b2-AR stimulation on dP/ respectively) (Figures 3C, D). Moreover, Dox-CM hearts presented dtmax and dP/dtmin, compared to Ctrl, were both increased by an alteration in longitudinal deformation, since Sa was decreased +108.0618.2% (P,0.001) and +155.3626.8% (P,0.001), respec- at d35 (231.266.5%; P = 0.001) as at d70 (248.6610.3%, tively (Figures 6D, E), and reached a similar level than this P,0.001) (Figure 3E) whereas radial deformation was only produced by b1-AR stimulation.
observed at d70 (238.764.9%, P = 0.031) (Figure 3F). In Dox-CM rats, IVRT value was significantly increased at d35 Gs Protein Expression and Adenylyl Cyclase Stimulation (+25.763.4%, P,0.001) as at d70 (+31.1610.6%, P = 0.003) By western-blot, we detected two bands for Gsa protein (Figure 3G) albeit the LV filling pressures, evaluated by E/Ea expression: a short one at 45 kDa and a long one at 52 kDa ratio, were similar in Ctrl and Dox-CM rats at both d35 and d70 (Figure 7A). The expression of both forms was unchanged in Dox- (P = 0.776 and P = 0.071, respectively) (Figure 3H).
CM hearts at both d35 and d70 (Figures 7B, C). In Ctrl and Dox-CM isolated hearts, at d35, forskolin induced a similar concen- Cardiac Function Obtained by Left Ventricle tration-dependant increase of heart rate between Ctrl and Dox- CM hearts (P = 0.941) (Figure 7F). In Ctrl group, forskolin induced As our study consists to identify molecular target at early stage a concentration-dependant increase of dP/dtmax (P,0.001) and of Dox-CM, we only performed LV catheterization at d35. Basal dP/dtmin (P = 0.010) (Figures 7D, E). In Dox-CM isolated hearts heart rate, measured before all pressure acquisitions, was increased compared to Ctrls, the forskolin effects on dP/dtmax and dP/dtmin in Dox-CM rats compared to Ctrl (P = 0.007) (Table 3). Dox-CM were increased by +43.2610.3% (P = 0.004) and +61.1610.7% rats presented a decreased Tau (210.062.2%; P = 0.009) albeit (P = 0.002), respectively (Figures 7D, E).
dP/dtmax and dP/dtmin were unchanged (Table 3). LVEDP wasunchanged between both groups (P = 0.216).
Gi Protein Expression and its Involvement in b2-ARCardiac Contractility b3-adrenoceptor Expression and Function Gia2 protein expression was detected by one band of 35 kDa Using western-blot, we detected b3-AR protein at a band of (Figure 8A) whose intensity was similar both in Ctrl and Dox-CM 68 kDa (Figure 4A). At d35, the detected band presented a similar hearts at d35 (P = 0.184 vs Ctrl) (Figure 8B), but was increased in PLOS ONE www.plosone.org May 2013 Volume 8 Issue 5 e64711 Beta-Adrenoceptors in Doxorubicin Cardiomyopathy Figure 5. Impaired b1-AR expression in Dox-CM hearts was not associated to an impaired cardiac b1-AR function. A. Representative b1-AR immunoblotting at days 35 (d35) and 70 (d70). B. b1-AR protein quantification at day 35. C. b1-AR protein quantification at day 70. Protein levelswere quantified using Amersham ImageQuant RT-ECL camera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TLsoftware (GE Healthcare) and a ratio to the corresponding GAPDH band intensity was calculated. D. Cardiac inotropic (dP/dtmax), E. lusitropic (dP/dtmin) and F. chronotropic (HR) effects of increasing concentrations of isoproterenol (1029 to 1025 M) in the presence of 1026 M ICI-118,551 a b2-ARantagonist and 1026 M of L-748,337 a b3-AR antagonist were evaluated on isolated perfused hearts. B: Baseline; B+A; Baseline in presence ofantagonists. **: P,0.001 vs Ctrl. Ctrl: control, Dox-CM: Doxorubicin-induced cardiomyopathy.
doi:10.1371/journal.pone.0064711.g005 PLOS ONE www.plosone.org May 2013 Volume 8 Issue 5 e64711

Beta-Adrenoceptors in Doxorubicin Cardiomyopathy PLOS ONE www.plosone.org May 2013 Volume 8 Issue 5 e64711 Beta-Adrenoceptors in Doxorubicin Cardiomyopathy Figure 6. b2-AR expression and function are increased in Dox-CM hearts. A. Representative b2-AR immunoblotting at days 35 (d35) and 70(d70). B. b2-AR protein quantification at day 35. C. b2-AR protein quantification at day 70. Protein levels were quantified using Amersham ImageQuantRT-ECL camera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TL software (GE Healthcare) and a ratio to thecorresponding GAPDH band intensity was calculated. D. Cardiac inotropic (dP/dtmax), E. lusitropic (dP/dtmin) and F. chronotropic (HR) effects ofincreasing concentrations of isoproterenol (1029 to 1024 M) in presence of 1026 M of CGP-20712A a b1-AR antagonist and 1026 M of L-748,337 a b3-AR antagonist were evaluated on isolated perfused hearts. B: Baseline; B+A; Baseline the in presence of antagonists. **: P,0.001 vs Ctrl; Ctrl: control,Dox-CM: Doxorubicin-induced cardiomyopathy.
doi:10.1371/journal.pone.0064711.g006 Dox-CM hearts at d70 by +118.1637.6% (P = 0.009 vs Ctrl) compared to controls but a significantly reduced longitudinal (Figure 8C). Ex vivo, at d35, Gi protein inhibition by PTX pre- strain with preserved radial contractility [34].
treatment produced no effect in heart rate in both groups In human, Dox administered to adults (for breast cancer, for (P = 0.346) (Figure 8F). PTX-pretreatment induced a significant example) is well known to induce chronic cardiotoxicity where increase of b2-AR stimulation in Ctrl isolated hearts (dP/dtmax: cardiac morphologic and functional alterations are close to those +64.469.8%; P,0.001; dP/dtmin: +128.0620.8%; P,0.001) of dilated cardiomyopathy [3]. However, when administered in (Figures 8D, E) but induced no change of these two parameters children, Dox can induce restrictive cardiomyopathy with normal in Dox-CM hearts (P = 0.173 and P = 0.451, respectively) LV dimensions [35]. Those findings, are consistent with those of (Figures 8D, E).
other long-term follow-up studies conducted in other groups ofanthracycline-treated childhood cancer survivors [36]. In our study, Dox was administered to young rats (7 week-old). At d35,we observed diastolic dysfunction with normal systolic function This study demonstrated for the first time in Dox-cardiotoxicity, that could support the hypothesis of a restrictive-cardiomyopathy.
b2-AR expression was increased at the early stage of the pathology, At d70, the development of systolic dysfunction could suggest with an increase of b2-AR-induced contractility. Furthermore, b1- restrictive cardiomyopathy worsening with increase LV dimen- AR function was preserved in spite of decreased b1-AR protein sions. Indeed, although LVEDDs were not statistically different expression and could be explained by an increase of AC between Ctrl and Dox-CM rats at d70, it is important to note that expression and/or activity as illustrated by an increased for- LV dPWth was significantly decreased in Dox-CM compared to skolin-induced contractility in isolated hearts from Dox-CM rats.
Ctrl rats, strengthening the notion of LV morphologic changes in In our study, the body weight of Dox-CM rats was smaller than those rats. We could hypothesize that a period of 56 days post- in Ctrl one as previously described by other studies [8,24]. This treatment was a too short duration of the disease evolution to has been related to a decrease in food consumption [25,26]. In our observe a significant LV dilation in Dox-CM rats. Indeed, in experimental conditions, we have, 35 days after the beginning of human HF, the dilation is considered as the last stage of ischemic Dox-treatment, 25% of mortality and rats presented a large or non ischemic cardiomyopathy evolution as in Dox-CM. In this amount of ascites. Those observations were in agreement with latter disease, it is well known that cardiomyopathy and thus LV other studies that reported a mortality rate fluctuating between dilation occur up to decades after exposure [37].
19% and 45% [27,28] and a volume of ascites generally varying In our Dox-CM model, we reported a remodeling in b-AR from 30 mL to 140 mL [24,28–30]. However, as Dox induced a expression and function. Three weeks after last Dox-injection multi-organ toxicity, it could be not excluded that mortality and (d35), b3-AR protein expression was unchanged and a slight ascites observed in rats could be due, at least in part, to liver and negative inotropic effect was obtained in Dox-CM hearts at the kidney damages. Seventy days after the beginning of Dox- higher concentration of SR 58611A (1025 M). Our data suggest treatment, mortality rate increased to 58% and ascites was still that in early chronic Dox-cardiotoxicity, b3-AR could not play a observed. Very few studies reported long term effects of Dox- major role in the cardiac alterations. At a later stage (d70), we treatment and data on mortality and ascites are very heteroge- observed a decreased b3-AR protein expression in Dox-CM hearts neous. Whereas a study observed 50% mortality rate 6 weeks after albeit another team reported an overexpression of b3-AR protein the last Dox-injection, associated with a very large amount of at the same stage [11]. Several hypotheses could explain those ascites [25] others did not observed mortality eight weeks after the discrepancies. First, the rat strain used in the study is different last Dox-injection [11]. Also, others did not reported mortality and (Wistar vs Sprague-Dawley) and several studies reported that observed a decrease in ascites amount between days 40 and 70 protein expression could be different between Sprague-Dawley and Wistar rats [38,39] albeit no study compared b-AR subtypes During echocardiographic acquisitions, heart rate was moni- expression between these two rat strains. Second, in the Sun's tored in order to have similar heart rate in both groups, allowing a study, cardiac b-ARs expressions were assessed in Ctrl rats comparison of echocardiographic parameters. At d35, Dox-CM compared to Dox-treated rats which were also Sham-castrated rats presented a slight alteration of systolic function as illustrated (scrotum incision); no surgery being performed in Ctrl rats.
by a mild decrease in LVEF and LVSF and in LV longitudinal Therefore, b-AR protein expressions were assessed in a Dox-CM deformation. Dox-CM rats presented also a mild diastolic model different from our one used in our study.
dysfunction as suggested by the increased IVRT and Tau values.
In our Dox-CM model, b1-AR protein expression was At d70, decreases in LVEF, LVSF and LV longitudinal decreased at both stages. This result is in agreement with those deformation were exacerbated whereas diastolic dysfunction was reported for b1-AR protein expression both in rabbits [40] and in unchanged. As diastolic dysfunction frequently occurs before rats [8,11]. Surprisingly, albeit a decreased b1-AR expression at systolic dysfunction [31–33], we could suggest that in our animal d35, the cardiac b1-AR responses were preserved. Several model, at d35, systolic function just began to be altered, meaning hypotheses could explain this feature. Firstly, a b1-AR reserve that this model corresponds to an early chronic Dox-cardiotox- could be present and recruited in such conditions and, secondly, icity. It is important to note that our results are very close to that an increase in b1-AR signaling pathway can be evoked. We observed in women who received anthracycline-treatment for showed that Dox-treatment did not change Gsa protein expres- breast cancer. Indeed, these patients had preserved LVEF sion, as it was reported in other study [8,11], but increased PLOS ONE www.plosone.org May 2013 Volume 8 Issue 5 e64711 Beta-Adrenoceptors in Doxorubicin Cardiomyopathy Figure 7. Gsa protein expression and forskolin response. A. Representative Gsa immunoblotting at days 35 (d35) and 70 (d70). B. Gsa proteinquantification at day 35. C. Gsa protein quantification at day 70. Protein levels were quantified using Amersham ImageQuant RT-ECL camera (GE Healthcare).
The band signals were assessed by densitometry with ImageQuant TL software (GE Healthcare) and a ratio to the corresponding GAPDH band intensity wascalculated. D. Cardiac inotropic (dP/dtmax), E. lusitropic (dP/dtmin) and F. chronotropic (HR) effects of increasing concentrations of the adenylyl cyclase activatorforskolin (3.1028 to 1025 M) were evaluated on isolated perfused hearts. *: P,0.05 vs Ctrl. Ctrl: control, Dox-CM: Doxorubicin-induced cardiomyopathy.
doi:10.1371/journal.pone.0064711.g007 PLOS ONE www.plosone.org May 2013 Volume 8 Issue 5 e64711 Beta-Adrenoceptors in Doxorubicin Cardiomyopathy PLOS ONE www.plosone.org May 2013 Volume 8 Issue 5 e64711 Beta-Adrenoceptors in Doxorubicin Cardiomyopathy Figure 8. Gi protein expression and involvement in b2-AR cardiac contractility. A. Representative Gia2 immunoblotting at days 35 (d35)and 70 (d70). B. Gia2 protein quantification at day 35. C. Gia2 protein quantification at day 70. Protein levels were quantified using AmershamImageQuant RT-ECL camera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TL software (GE Healthcare) and aratio to the corresponding GAPDH band intensity was calculated. D. Cardiac inotropic (dP/dtmax), E. lusitropic (dP/dtmin) and F. chronotropic (HR)effects of increasing concentrations of isoproterenol (1029 to 1024 M) in the presence of 1026 M of CGP-20712A and 1026 M of L-748,337 wereevaluated on isolated perfused hearts pre-treated or not with 4 mg.L21 of pertussis toxin (PTX) a Gi protein inhibitor. B: Baseline; B+A; Baseline in thepresence of antagonists. *: P,0.05 vs Ctrl; **: P,0.001 vs Ctrl. Ctrl: control, Dox-CM: Doxorubicin-induced cardiomyopathy.
doi:10.1371/journal.pone.0064711.g008 forskolin response, suggesting an increase in AC expression and/or HF [49–54]. Concerning a putative detrimental effect of b2-AR, it activity. This surprising data contrasts with other studies which could be proposed to use b2-AR blocker. However, it is important reported a decreased AC activity [41,42] or no change [43].
to note that b2-AR antagonists by blocking other physiological However, those differences seem to be due to the cardiotoxicity functions regulated by b2-AR, could also have detrimental effects: level since the Dox cumulative doses used were 24, 24.75 and (i) a vasoconstriction because b2-AR is one of the most important 6 mg.kg21, respectively. Thus, the increased AC response to vascular b-AR subtype whose activation induces a vasodilation in forskolin observed in our study could be a compensatory several vascular beds (including coronary vessels), (ii) a broncho- mechanism at early stage of Dox-cardiotoxicity to maintain constriction due to the reduction of bronchodilation induced by cardiac contractility and could explain, at least in part, the pulmonary b2-AR,… preserved b1-AR response. However, as b1-ARs are involved in Thus, we rather suggest that further experiments must be pro-apoptotic effects, b1-AR could be also responsible for some performed to identify the most suitable effector of the cardiac b2- Dox cardiotoxic effects as previously suggested [9,10].
AR pathway. Then, according to the results of this next important Finally, we reported, for the first time, in chronic Dox- study, we hope that we could propose a new therapeutic target for cardiotoxicity, a b2-AR protein overexpression. At d35, in Ctrl isolated heart, specific b2-AR stimulation induced positive In conclusion, we have shown for the first time in rat Dox-CM, inotropic and lusitropic responses but to a lesser extent than b1- an increase of b2-AR protein expression at early stage of the AR one. This lower response could be explained by a more pathology, associated to an increased contractility. Furthermore, compartmentalized cAMP signaling of b2-AR [44]. Surprisingly, we observed a preserved b1-AR function albeit b1-AR was in Dox-CM hearts, b2-AR inotropic effect was increased and reduced. Clinical studies have suggested no beneficial effect of reached a similar level than b1-AR stimulation. This increase could be due to several mechanisms. Firstly, we show that b 1-AR blocker, in Dox-treated lymphoma patients [15] but a beneficial effect of nebivolol, a b protein expression was increased in Dox-CM LV and could lead to 1-AR blocker associated 3-AR agonistic properties against anthracycline-induced 2-AR response. Secondly, cardiac b2-AR could be cardiomyopathy in patients with breast cancer [55]. However, the linked both to Gs and Gi proteins [45,46]. In Dox-CM LV, Gia2 clinical roles of b protein expression was not modified at d35 but it was overex- 2-AR remain to elucidate in order to determine pressed at d70 of Dox-CM, as reported previously at this latter 2-AR activation or blockade could prevent cardiac alterations due to anthracycline.
stage by Sun et al. [11]. Surprisingly, at d35, in Ctrl hearts, the Giinhibition by PTX allowed to increase b2-AR response in order toreach the same level that obtained in Dox-CM in the absence of PTX treatment, whereas Gi inhibition in Dox-CM hearts slightly We thank Chrystelle Bailly, Ste´phanie Lemarchand-Minde´, Amandine increased b2-AR response. These data suggest that, in Dox-CM, Lefebvre and Patricia Charpentier for animal care, and Morte´za Erfanian b2-AR was mainly linked to Gs.
for its technical assistance.
To know whether enhanced b2-AR/Gs signaling observed in our study is a beneficial compensatory mechanism or a Author Contributions detrimental effect is still to be determined. To support the Conceived and designed the experiments: NM BR CG. Performed the hypothesis of a beneficial effect of b2-AR agonist, it has been experiments: NM NP AG BL. Analyzed the data: NM NP AG BL BR CG.
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