Available online at www.sciencedirect.com

Journal of Molecular and Cellular Cardiology 45 (2008) 240–249www.elsevier.com/locate/yjmcc

Original article

Intracellular mechanisms of specific β-adrenoceptor antagonists involved inimproved cardiac function and survival in a genetic model of heart failure

Jan B. Bartholomeu a, Andréa S. Vanzelli a, Natale P.L. Rolim a, Julio C.B. Ferreira a,Luiz R.G. Bechara a, Leonardo Y. Tanaka a, Kaleizu T. Rosa b, Márcia M. Alves c,Alessandra Medeiros a, Katt C. Mattos a, Marcele A. Coelho a, Maria C. Irigoyen b,Eduardo M. Krieger b, Jose E. Krieger b, Carlos E. Negrão a,b, Paulo R. Ramires a,

Silvia Guatimosim c, Patricia C. Brum a,⁎

a School of Physical Education and Sport, University of São Paulo, São Paulo, Brazilb Heart Institute (InCor), Medical School, University of São Paulo, Brazil

c Department of Physiology and Biophysics, Federal University of Minas Gerais, Belo Horizonte, Brazil

Received 5 December 2007; received in revised form 16 May 2008; accepted 16 May 2008Available online 27 May 2008

Abstract

β-blockers, as class, improve cardiac function and survival in heart failure (HF). However, the molecular mechanisms underlying thesebeneficial effects remain elusive. In the present study, metoprolol and carvedilol were used in doses that display comparable heart rate reduction toassess their beneficial effects in a genetic model of sympathetic hyperactivity-induced HF (α2A/α2C-ARKO mice). Five month-old HF mice wererandomly assigned to receive either saline, metoprolol or carvedilol for 8 weeks and age-matched wild-type mice (WT) were used as controls. HFmice displayed baseline tachycardia, systolic dysfunction evaluated by echocardiography, 50% mortality rate, increased cardiac myocyte width(50%) and ventricular fibrosis (3-fold) compared with WT. All these responses were significantly improved by both treatments. Cardiomyocytesfrom HF mice showed reduced peak [Ca2+]i transient (13%) using confocal microscopy imaging. Interestingly, while metoprolol improved [Ca2+]itransient, carvedilol had no effect on peak [Ca2+]i transient but also increased [Ca2+] transient decay dynamics. We then examined the influence ofcarvedilol in cardiac oxidative stress as an alternative target to explain its beneficial effects. Indeed, HF mice showed 10-fold decrease in cardiacreduced/oxidized glutathione ratio compared with WT, which was significantly improved only by carvedilol treatment. Taken together, we providedirect evidence that the beneficial effects of metoprolol were mainly associated with improved cardiac Ca2+ transients and the net balance ofcardiac Ca2+ handling proteins while carvedilol preferentially improved cardiac redox state.© 2008 Elsevier Inc. All rights reserved.

Keywords: Heart failure; Sympathetic nervous system; β-adrenergic receptor antagonists; Calcium transients; Oxidative stress

1. Introduction

β-adrenoceptor (AR) antagonist therapy substantiallyreduces mortality and sudden death, and improves symptomsin patients with heart failure (HF) [1,2]. The rationale for the useof β-AR antagonists in HF is based on the observation that

⁎ Corresponding author. Escola de Educação Física e Esporte da Universidadede São Paulo, Departamento de Biodinâmica do Movimento do Corpo Humano,Av. Professor Mello Moraes, 65 - Butantã — São Paulo — SP, 05508-900 —Brazil. Tel.: +5511 3091 3136; fax: +5511 3813 5921.

E-mail address: pcbrum@usp.br (P.C. Brum).

0022-2828/$ - see front matter © 2008 Elsevier Inc. All rights reserved.doi:10.1016/j.yjmcc.2008.05.011

sympathetic efferent neuronal activity is increased in HF and thesympatho-excitation has independent prognostic value in HFpatients. In fact, large-scale trials have demonstrated that β-ARantagonists bisoprolol, metoprolol, and carvedilol improveclinical outcome in HF patients compared with placebo [1,3,4].In this regard, carvedilol has been shown to improve cardiacfunction and survival to a greater extent than metoprolol [5], butthese findings have not been consistent [6].

Differences in several pharmacological properties (e.g. β1/β2-selectivity, intrinsic sympathomimetic activity, antioxidant effectsand additional vasodilating properties) are expected to producedifferent outcome in the clinical use of these β-AR antagonists in

241J.B. Bartholomeu et al. / Journal of Molecular and Cellular Cardiology 45 (2008) 240–249

HF. Within the β-AR antagonists available for clinical use [7,8],metoprolol (a second generationβ-AR antagonist) is selective forβ1-AR preserving β2-AR cardiac signaling pathways. β2-ARshave anti-apoptotic effects potentially favorable in HF [9]. Inaddition, recent studies have demonstrated that supplementationwith β2-AR agonist enhances and extends therapeutic effects ofβ1-AR antagonist on cardiac remodeling [10]. Carvedilol, a thirdgeneration non-selective β-AR antagonist with anti-α1-ARvasodilatory effect, has potent antioxidant activity associatedwith anti-cardiac remodeling and improved cardiac function [11].

The underlying mechanisms responsible for the specificeffects of most used β-AR antagonists are not completely under-stood. In HF, an abnormal Ca2+ homeostasis appears to beprimarily responsible for depression of myocyte contractility.Reduced peak systolic Ca2+ and slower rate of decay of the Ca2+

transient together with elevated diastolic Ca2+ at faster heart ratesare common changes observed in cardiomyocytes from differentmodels of heart failure [12,13]. Even though chronic HF treat-ment withmetoprolol or carvedilol is associatedwith an increasedexpression profile of Ca2+ handling proteins as sarcoplasmicreticulum Ca2+ ATPase (SERCA2) [14,15], little is known abouttheir respective effects on cardiac Ca2+ transients.

We have previously reported that mice lacking both α2A- andα2C-adrenoceptors (α2A/α2C-ARKO) develop sympathetichyperactivity-induced HF [16], which is associated withabnormalities in Ca2+ handling proteins [17,18]. Therefore,these mice provide a unique model for better understanding ofthe mechanisms underlying the cardiac deleterious effect ofsympathetic hyperactivity, as well as, for testing different β-ARantagonist therapy in HF.

The present study was undertaken to test whether metoprololand carvedilol would have similar impact on cardiac contrac-tility and survival of α2A/α2C-ARKO mice. In addition, westudied specific effects of metoprolol and/or carvedilol oncardiac Ca2+ transient and redox balance. Here, we report thatmetoprolol and carvedilol were equally efficient in improvingα2A/α2C-ARKO survival and cardiac contractility, while re-ducing cardiac remodeling. Of interest, beneficial effects ofmetoprolol were mainly associated with improved cardiac Ca2+

transients and the net balance of cardiac Ca2+ handling proteinswhile carvedilol preferentially improved cardiac redox state.

2. Materials and methods

2.1. Sampling

2.1.1. Animals and drug treatmentsA cohort of male wild-type (control WT group) and congenic

α2A/α2C-ARKO (HF group) mice in a C57Bl6/J geneticbackground from Medical School — University of São Paulowere studied from 5 to 7 months of age. At this age, these micedisplay advanced stage cardiomyopathy as previously described[16]. Genotypes were determined by polymerase chain reactionon genomic DNA obtained from tail biopsies using primers todetect the intact and disrupted genes.

Mice were maintained in a light- (12-h light cycle) and tem-perature- (22 °C) controlled environment and were fed a pellet

rodent diet (Nuvital Nutrientes S/A, Curitiba, PR, Brazil) adlibitum and had free access to water. HF mice were randomlyassigned to daily receive by gavage either placebo (saline),metoprolol (165 mg/kg, Sigma Aldrich Corporation, SaintLouis, MO, USA) or carvedilol (38 mg/kg, Baldacci S.A., SP,Brazil). The doses of metoprolol and carvedilol were optimizedto achieve comparable heart rate levels observed in control age-matched WT group. The drug regimens did not change cardiacfunction, structure, Ca2+ or cardiac oxidative index in controlmice. Therefore, we used only one control group for furthercomparisons with HF groups. Finally, mice were assigned into 4groups: control, HF placebo, HF metoprolol-treated, and HFcarvedilol-treated. This study was accorded to Ethical Principlesin animal research adopted by Brazilian College of AnimalExperimentation (www.cobea.org.br). In addition this study wasapproved by the University of São Paulo Ethical Committee(CEP#004).

2.2. Measurements and procedures

2.2.1. Cardiovascular measurementsHeart rate (HR) and blood pressure were determined non-

invasively using a computerized tail-cuff system (BP-2000,Visitech System, Apex, NC, USA) described elsewhere [19].Mice were acclimatized to the apparatus during daily sessionsover 6 days, one week before starting the experimental period.HR measurements were obtained serially, 24 h after β-ARantagonists or saline administration, in control and HF miceonce a week throughout the 8 weeks of experiment.

Non-invasive cardiac function was assessed by two-dimen-sional guided M-mode echocardiography, in halothane-anesthe-tized control and HF mice, before and after 8 weeks of saline orβ-blocker therapy. Briefly, mice were positioned in the supineposition with front paws wide open, and an ultrasoundtransmission gel was applied to the precordium. Transthoracicechocardiography was performed using an Acuson Sequoiamodel 512 echocardiographer (Acuson Corporation, MountainView, CA, USA) equipped with a 14 MHz linear transducer.Left ventricle systolic function was estimated by fractionalshortening as follows:

Fractional Shortening kð Þ ¼ LVEDD� LVESDð Þ=LVEDD½ �� 100

where, LVEDD means left ventricular end-diastolic dimension,and LVESD means left ventricular end-systolic dimension.

2.2.2. Structural analysisTwenty-four hours after the last drug administration, all mice

were killed and their tissues were harvested. The heart wasstopped at diastole (KCl, 14 mM) and dissected to obtain the leftventricle, which corresponds to the remaining organ uponremoval of both atria and free wall of the right ventricle. Formorphometric analysis, left ventricle samples obtained from thefree wall, at the level of papillary muscle, were fixed in 4%buffered formalin and embedded in paraffin, cut in 4 μmsections and subsequently stained with hematoxylin and eosin.

Fig. 1. Heart rate (HR, A), survival (B) and fractional shortening (FS, C) incontrol wild-type (WT) and, heart failure (HF) placebo, metoprolol andcarvedilol groups. FS was evaluated before (■) and after (□) 8 weeks of salineorβ-AR antagonist therapy. Note that bothβ-AR antagonists reducedmortalityrate and improved FS in HF mice. Of interest, both β-AR antagonists decreasedto the same extent the baseline HR of HF treated mice, which became similar toHR of control group. Data are presented as mean±SE. ⁎ Pb0.05 vs controlWT, metoprolol and carvedilol groups, † Pb0.05 vs. control WT group,‡ Pb0.05 vs before treatment period. The number of animals studied is shownbetween parenthesis (Panels A and B) or indicated by numerals on the abscissa(Panel C).

242 J.B. Bartholomeu et al. / Journal of Molecular and Cellular Cardiology 45 (2008) 240–249

Two randomly selected sections from each animal were vi-sualized by light microscopy using an objective with a cali-brated magnification (400×). Myocytes with visible nucleus andintact cellular membranes were chosen for diameter determina-tion. The width of individually isolated cardiomyocyte dis-played on a viewing screen was manually traced, across themiddle of the nuclei, with a digitizing pad and determined by acomputer assisted image analysis system (Quantimet 520;Cambridge Instruments, UK). For each animal approximately15 visual fields were analyzed.

Quantification of left ventricular fibrosis was achieved bySirius red staining. Two randomly selected sections from eachanimal were visualized by light microscopy using an objectivewith a calibrated magnification of 200×. Interstitial collagenarea was quantified by a computer assisted image analysissystem (Quantimet 520; Cambridge Instruments, UK). For eachanimal approximately 5 visual fields were analyzed.

2.2.3. RT-PCRIn order to verifywhetherα2A/α2C-ARKOmicewould present

reactivation of fetal genes involved in cardiac remodeling andfailure, as well as the effect of metoprolol and carvedilol, weevaluated atrial natriuretic peptide (ANP) and skeletal α-actingene expression. Therefore, RNAwas isolated from left ventricletissue with Trizol reagent (GIBCO-BRL, Invitrogen Corp.,Carlsbad, CA, USA). cDNA was synthesized using SuperscriptIII Reverse Transcriptase (200 U/ml, Invitrogen) at 42 °C for50 min. Taq DNA Polymerase (Fermentas, Burlington, ON, Ca-nada) was used for DNA amplification in the presence of specificprimers for cardiac isoforms of skeletal α-actin and ANP. Thespecific primer sequences were: α-actin sense, 5′GCTCTTC-CAGCCTTCCTTT; α-actin antisense, 5′ACGTTGTTGGCG-TACAGGT; ANP sense, 5′CTTCGGGGGTAGGATTGAC; andANP antisense, 5′CTTGGGATCTTTTGCGATCT. For eachcDNA species, the number of amplification cycles was thatnecessary for 50% saturation, as determined in preliminaryassays. mRNA levels were normalized to that of β-actin mRNA(which is not changed in this model of HF) in the same assay.

2.2.4. Cardiomyocyte isolation and Ca2+ recordingTo verify the cardiomyocyte Ca2+ transients, animals re-

ceived an intraperitoneal injection of pentobarbital sodium(100 mg/kg), and after full anesthesia the heart was rapidlyremoved. Cardiac ventricular myocytes were isolated, andimaged for [Ca2+]i as previously described [20]. The Ca2+ levelmeasured with confocal microscopy was reported as F/F0,where F0 is the resting Ca

2+ fluorescence. Images were obtainedusing the ZEISS Meta confocal microscope from CEMEL(Biological Sciences Institute, UFMG, Brazil).

2.2.5. AntibodiesMouse monoclonal antibodies to SERCA2 (1:2500), Phos-

pholamban (PLN, 1:500) and Na+–Ca2+ exchanger (1:2000)were obtained from Affinity BioReagents (Golden, CO, USA);rabbit polyclonal antibody to protein phosphatase type 1 (PP1,1:1000) were obtained from Upstate (Lake Placid, NY, USA);phospho-Ser16-PLN (1:5000) and phospho-Thr17-PLN (1:5000)

were obtained from Badrilla (Leeds, UK). Troponin I (1:500)was obtained from Santa Cruz Biotechnology (Santa Cruz, CA,USA). Phospho-Ser23/24-troponin I (1:1000), calmodulin kinaseII (CaMKII, 1:750), phospho-Thr286-CaMKII (1:1000) wereobtained from Cell Signaling Technology (Danvers, MA, USA).Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:2000)was obtained from Advanced Immunochemical (Long Beach,CA, USA). Targeted bands were normalized to cardiac GAPDH

Fig. 2. Cardiac myocyte cross-sectional diameter (A) and cardiac collagenvolume fraction (B) in control wild-type (WT) and, heart failure (HF) placebo,metoprolol and carvedilol groups. Data are presented as mean±SE. ⁎ Pb0.05 vscontrol WT group, † Pb0.05 vs. placebo HF group; ‡ Pb0.05 vs metoprololgroup. The number of animals studied is indicated by numerals on the abscissa.

Fig. 3. Intracellular Ca2+ transient in isolated ventricular myocytes. A: (Top)Representative line-scan images showing electrically stimulated-Ca2+ transientfrom cardiomyocytes isolated from control wild-type (WT), heart failure (HF)placebo, metoprolol and carvedilol groups. B: Averaged data showing peak ofCa2+ transient. C: Bar graph shows a comparison of Ca2+ transient kinetics (timefrom peak to 90% decay) between the different groups of cells. Cardiomyocytesfrom HF placebo group cardiomyocytes develop significantly smaller (by 13%)[Ca2+]i transients than control cells. Note that only HF metoprolol group showedsignificantly increased peak Ca2+ transient. Ca2+ transient kinetics of decay wasalso faster in cardiomyocytes from HF metoprolol group. Data are presented asmean±SD. ⁎ Pb0.05 vs control WT group, † Pb0.05 vs. placebo group;‡ Pb0.05 vs metoprolol group. The number of cardiomyocytes evaluated isindicated by numerals on the abscissa.

243J.B. Bartholomeu et al. / Journal of Molecular and Cellular Cardiology 45 (2008) 240–249

obtained in the same blot after stripping and reprobing withGAPDH antibody.

2.2.6. Western blot analysisSince it has been demonstrated that β-AR antagonists

improve cardiac function in human HF that is paralleled bychanges in expression of Ca2+ handling proteins [21,22], weinvestigated whether metoprolol or carvedilol would differen-tially change Ca2+ handling protein expression profile in our HFmodel. Therefore, left ventricular homogenates were analyzedbyWestern blotting to compare SERCA2, PLN, phospho-Ser16-PLN, phospho-Thr17-PLN, PP1, and Na+–Ca2+ exchanger.Briefly, liquid nitrogen frozen ventricles isolated from controland HF mice were homogenized in a buffer containing 50 mMpotassium phosphate buffer (pH 7.0), 0.3 M sucrose, 0.5 mMDTT, 1 mM EDTA (pH 8.0), 0.3 mM PMSF, 10 mM NaF, andphosphatase inhibitor cocktail (1:100, Sigma Aldrich Corpora-tion). Samples were subjected to SDS-PAGE in polyacrylamidegels (6% or 10% depending on protein molecular weight). Afterelectrophoresis, proteins were electro-transferred to nitrocellu-lose membrane (Amersham Biosciences; Piscataway, NJ,USA). Equal loading of samples (50 μg) and even transferefficiency were monitored with the use of 0.5% Ponceau Sstaining of the blot membrane. The blotted membrane was thenblocked (5% nonfat dry milk, 10 mM Tris–HCl, pH 7.6,

150 mMNaCl, and 0.1% Tween 20) for 2 h at room temperatureand incubated with specific antibodies overnight at 4 °C.Binding of the primary antibody was detected with the use ofperoxidase-conjugated secondary antibodies (rabbit or mouse

244 J.B. Bartholomeu et al. / Journal of Molecular and Cellular Cardiology 45 (2008) 240–249

depending on the protein, 1:10,000, for 1:30 h at room tem-perature) and developed using enhanced chemiluminescence(Amersham Biosciences) detected by autoradiography. Quan-tification analysis of blots was performed with the use ofScion Image software (Scion Corporation based on NIHimage).

2.2.7. Myocardial redox statusAs reactive oxygen species are produced in failing myo-

cardium [23,24] and related to injury in cardiac myocytes[25,26], we measured myocardial tissue reduced glutathione(GSH): oxidized glutathione (GSSG) ratio in all mice studied.Heart samples were homogenized in cold buffer (1:20 w/v)containing 0.32 M sucrose, 10 mMHEPES, and 1 mM EDTA atpH 7.4, and immediately centrifuged at 12,000 g for 20 min at4 °C. Proteins were precipitated with trichloroacetic acid (10%w/v) and supernatant was injected into a high performanceliquid chromatography (HPLC) system with electrochemicaldetection [27] for GSH measurement, using a standard curve ofpurified GSH. Total GSH was measured after GSSG wasregenerate to GSH by NADP and glutathione reductase at pH7.5. The concentration of GSSG was estimated by subtractingthe measured GSH from the measured total GSH. Redox statuswas described as calculated GSH:GSSG ratio.

2.2.8. Myocardial lipid hydroperoxide measurementAs an index of cardiac oxidative injury, lipid hydroperoxides

were evaluated by the ferrous oxidation-xylenol orangetechnique (FOX2) [28]. Heart samples were homogenized(1:20 w/v) in cold phosphate-buffered saline (100 mM, pH 7.4)and immediately centrifuged at 12,000 g for 20 min at 4 °C.Proteins were precipitated with trichloroacetic acid (10% w/v)and supernatant was mixed with FOX reagent and incubated for30 min. The absorbance of the sample was read at 560 nm.

2.2.9. Statistical analysisData are presented as mean±SE. Kaplan–Meier method was

used for constructing survival curves and Cox proportionalhazards model for estimating risk factors in survival analysis.One-way ANOVA for repeated measurements with post-hoctesting by Tukey (Statistica software, StatSoft, Inc.,Tulsa, OK,USA) was used to compare the effect of treatment (placebo,metoprolol and carvedilol) on HR and fractional shorteningmeasurements along the experiment period. One-way ANOVAwith post-hoc testing by Tukey (Statistica software) was used tocompare the effect of treatment (placebo, metoprolol andcarvedilol) on cardiac myocyte width, ventricular fibrosis,mRNA and protein expression levels, Ca2+ transients, and oxi-dative stress. Statistical significance was considered achievedwhen the value of P was b0.05.

Fig. 4. SERCA2, Na+–Ca2+ exchanger (NCX), phospho-Ser16-PLN, phospho-Thr17-Pcontrol wild-type (WT) and, heart failure (HF) placebo, metoprolol and carvedilol grophospho-Ser16-PLN, phospho-Thr17-PLN, PP1, phospho-Ser23/24-troponin, phosphmetoprolol and carvedilol groups. B, C, D, E, F, G and H: SERCA2, NCX, phosphoThr286-CaMKII expression, respectively. Targeted bands were normalized to cardiacnumber of animals studied is indicated by numerals on the abscissa.

3. Results

3.1. Effects of β-AR antagonists on survival, hemodynamics,and cardiac contractility

Placebo HF mice displayed baseline tachycardia whencompared with age-matched control mice (Fig. 1A) even thoughresting blood pressure was similar among all groups studied(Supplementary data). Upon chronic metoprolol and carvediloltreatments, blood pressure remained unchanged while baselineHR was restored in HF mice to control mice levels from the 4thweek to the 8th week of treatment (Fig. 1A). While HF micepresented 50% mortality rate at the 8th week of study (Fig. 1B),metoprolol or carvedilol treatments significantly reduced HFmice mortality to (31%) (Fig. 1B). Similarly both treatmentssignificantly increased fractional shortening of HF mice tosimilar WT mice levels (Fig. 1C).

3.2. Myocardial structure

Consistent with the detrimental cardiac function observed inthe HF mice, the quantitative morphometrical analysis showedthat cardiac myocyte cross-sectional diameter was significantlygreater in these animals compared with WT mice (Fig. 2A).Cardiac myocyte cross-sectional diameter was significantlysmaller in metoprolol- or carvedilol-treated HF mice than inHF mice, but only metoprolol reduced it to WT mice levels(Fig. 2A). Increased cardiac myocyte cross-sectional diameterin HF mice was associated with an increase in ventricularfibrosis, as indicated by a 3-fold increase in cardiac collagenvolume fraction compared with WT mice (Fig. 2B). Metopro-lol-treated HF mice displayed reduced ventricular fibrosiscompared with HF mice, but they were significantly greaterthan WT mice (Fig. 2B). Carvedilol treatment tended to reduceleft ventricular fibrosis in HF mice but this result did not reachsignificant values (Fig. 2B).

The cardiac ultrastructural changes observed in HFmice wereassociated with increased ANP (18±1.9%, P=0.0002) andskeletal α-actin (23±4.0%, P=0.03) mRNA levels comparedwithWTmice. Therefore, both treatments were equally efficientin reducing ANP (85.3±2 and 80.7±2.2% for metoprolol andcarvedilol, respectively) and skeletal α-actin (73±3.4% and69.1±5.9% for metoprolol and carvedilol, respectively) geneexpression in HF mice to similar WT mice levels.

3.3. Myocyte Ca2+ transients and expression of cardiac Ca2+

handling proteins

Since Ca2+ handling is an essential regulator of cardiac con-tractile function, we first examined the Ca2+ transients in isolated

LN, PP1, phospho-Ser23/24-troponin, and phospho-Thr286-CaMKII expression inups. Data are presented as mean±SE. A: Representative blots of SERCA2, NCX,o-Thr286-CaMKII and GAPDH from control WT, heart failure (HF) placebo,-Ser16-PLN, phospho-Thr17-PLN, PP1, phospho-Ser23/24-troponin, and phospho-GAPDH. ⁎ Pb0.05 vs control WT group, † Pb0.05 vs. placebo HF group. The

245J.B. Bartholomeu et al. / Journal of Molecular and Cellular Cardiology 45 (2008) 240–249

Fig. 5. Effect of β-AR blocker therapies on cardiac GSH:GSSG ratio (A), and onlipid hydroperoxidation (B) in control wild-type (WT), heart failure (HF)placebo, metoprolol and carvedilol groups. Note that carvedilol increased GSH:GSSG ratio in HF group to a greater extent than metoprolol. Both drugssimilarly decreased myocardial lipid hydroperoxides. Data are presented asmean±SE. ⁎ Pb0.05 vs control WT group. The number of animals studied isindicated by numerals on the abscissa.

246 J.B. Bartholomeu et al. / Journal of Molecular and Cellular Cardiology 45 (2008) 240–249

cardiomyocytes from HF mice. Cardiomyocytes from HF miceshowed reduced peak [Ca2+]i transient (Figs. 3A and B).Metoprolol treatment significantly increased peak [Ca2+]i andreduced [Ca2+]i transient decay. Surprisingly, carvedilol did notimprove peak [Ca2+]i transient but also increased [Ca

2+] transientdecay dynamics (Fig. 3).

To gain further insight into this response, we investigated theexpression of key cardiac Ca2+ handling proteins. First, weobserved that SERCA2 expression levels were significantlyreduced in HF mice compared with WT mice and both treat-ments significantly increased SERCA2 expression to the levelsobserved in WT mice (Figs. 4A and B). As expected, Na+–Ca2+

exchanger expression levels were significantly increased in HFmice and both treatments also significantly reduced Na+–Ca2+

exchanger expression to the same levels observed in WT mice(Fig. 4A and C).

Cardiac PLN (data not shown) and phospho-Ser16-PLNexpressions were similar between HF and WT mice. Upon car-vedilol treatment, the phospho-Ser16-PLN expression decreasedsignificantly in HF mice (Figs. 4A and D). Phospho-Thr17-PLNexpression was significantly increased in HFmice compared withWT mice and both treatments significantly reduced phospho-Thr17-PLN levels in HF mice to levels similar to WT mice(Figs. 4A and E).

The PP1 expression, which is involved mainly in depho-sphorylation of PLN at Ser16 residue, was significantly higherin HF mice than WT mice and both β-blockers signifi-cantly reduced PP1 expression to levels similar to WT mice(Figs. 4A and F).

Troponin I and CaMKII expression levels were similar amongall four groups studied (data not shown). Phospho-Ser23/24-troponin I expression was increased in HF mice versus WT miceand upon treatment only carvedilol significantly reducedphospho-Ser23/24-PLN levels in HF mice to levels similar toWT mice (Figs. 4A and G). Phospho-Thr286-CaMKII expressionwas increased in HF mice and carvedilol treatment led to asignificant reduction phosphorylation levels, whereas metoprololled to smaller response, although not significant (P=0.08)(Figs. 4A and H).

GAPDH protein levels remained unchanged in all blotsanalyzed and among the four groups studied (Fig. 4A) and wereused to normalize the cardiac Ca2+ handling protein levels.

3.4. Effects of β-AR antagonists on markers of cardiacoxidative stress

The metoprolol-induced changes in Ca2+ handling proteinsand transients are consistent with the beneficial effects of thedrug on cardiac function in this genetic model of HF, whereas thesame changes cannot fully explain the similar improvementsobserved with carvedilol treatment, specially considering thatthis drug did not improve Ca2+ transients. Thus, as an alternativetarget, we investigated the influence of carvedilol in cardiacoxidative stress in HF mice. Interestingly, HF mice showedsignificant decrease in cardiac GSH:GSSG ratio, indicatingreduced redox balance, and increase in cardiac lipid hydroper-oxides (Fig. 5) compared withWT.While metoprolol-treated HF

mice did not change cardiac GSH:GSSG ratio, carvedilolincreased cardiac GSH:GSSG ratio to levels similar to WTmice (Fig. 5A). In contrast, when lipid hydroperoxides wereexamined, both treatments displayed comparable reduction incardiac lipid hydroperoxides (Fig. 5B).

4. Discussion

We provide evidence for differences in the underlying intra-cellular cardiac mechanisms when different β-AR antagonistswere used to treat heart failure in this geneticmodel. It is importantto emphasize that this differences were observed even though thetwo agents led to comparable cardiac remodeling, improvement ofcardiac function and survival in a genetic model characterized bysympathetic hyperactivity-induced HF. Metoprolol preferentiallyimproved cardiomyocyte Ca2+ transient while carvedilol had abeneficial effect on global myocardial redox balance.

Sympathetic hyperactivity plays a prominent role in the pa-thogenesis of HF [29,30]. Sustained sympathetic nervous systemstimulation leads to a downregulation ofβ1-ARs and uncouplingof β2-ARs, which are paralleled by a desensitization of proteinkinase A (PKA) pathway and increased CaMKII signalingpathways [31–33]. Moreover, shift from PKA to CAMKII path-way is associated with cardiac dysfunction and remodeling

247J.B. Bartholomeu et al. / Journal of Molecular and Cellular Cardiology 45 (2008) 240–249

[34,35]. The increased phospho-Thr286-CaMKII expressionparalleled by increased expression of phospho-Thr17-PLN inthe untreated α2A/α2C-ARKO mice (HF group) is consistentwith activated CaMKII pathway. Bothmetoprolol and carvedilolreestablished phospho-Thr17-PLN expression to WT levels.Indeed, metoprolol and carvedilol phospho-Thr286-CaMKIIexpression was similar to WT mice.

PP1 expression levels were significantly higher in HF groupand both metoprolol and carvedilol decreased PP1 expression toWT mice levels. As increased PP1 expression in heart failurehas been linked to depressed sarcoplasmic Ca2+ pump activity[36], the decreased PP1 expression by metoprolol and car-vedilol might contribute to increased cardiac function in treatedHF mice.

Even though both metoprolol and carvedilol seem to atte-nuate PP1 expression and CaMKII signaling in HF mice, onlymetoprolol improved cardiac Ca2+ transients. In fact, onlymetoprolol is known to prevent downregulation of β-ARs in-duced by β-agonists [37] that would favor the signaling switchtowards a predominant PKA pathway.

Carvedilol treatment appeared not to improve [Ca2+]i tran-sient dynamics, since it failed to increase peak and prolongedthe decay of [Ca2+]i transients. However, carvedilol signifi-cantly attenuated phospho-Ser16-PLN and phospho-Ser23/24-troponin I expression levels. These responses might be asso-ciated with the prolonged decay of [Ca2+]i transients, but it isimportant to highlight that decreasing the phosphorylation oftroponin I in heart failure might also have positive impact oncardiac function since troponin I phosphorylation by PKA isdecreased while it is increased by PKC [38]. Increased troponinphosphorylation desensitizes the myofilament to Ca2+, butkinase-specific phosphorylations appear to have different andopposing effects on kinetic properties of the cross bridge cycle[39]. While PKA phosphorylation of troponin I on Ser 23/24

residues is associated with improved cardiac function [40],elevated troponin phosphorylation by PKCβII decreases Ca2+

responsiveness and cardiac contractility [41].The mechanisms underlying the effect of carvedilol on re-

ducing phospho-Ser23/24-troponin I expression were not ad-dressed in the present study, but they seem to be related to theantioxidant properties of carvedilol [42]. In fact, carvedilol has awell recognized antioxidant activity, which led us to investigatewhether it would improve myocardial redox balance in HFgroups. Indeed, only carvedilol improved redox balance that ledto greater global antioxidant effects (Fig. 5). The increased GSH:GSSG ratio in carvedilol-treated HF mice might have directeffects on cardiac contractility, since increased reactive oxygenspecies upregulate kinases involved in the phosphorylation oftroponin T and I that culminates in reduced actin–myosininteraction [43]. Indeed, carvedilol, but not metoprolol, inhibitsCa2+ overload-induced augmentation of mitochondrial oxygenconsumption [44], which may affect cardiac contractility.

In the present study the improved survival and cardiaccontractility after metoprolol or carvedilol treatment in HF micewere paralleled by an anti-remodeling effect. Both treatmentsreduced ANP and skeletal α-actin gene expression and myocytewidth in HF mice. However, metoprolol had greater impact on

reducing ventricular collagen. The mechanisms involved in anti-remodeling effects of metoprolol and carvedilol may be relatedto their pharmacological properties. While the greater antiox-idant effect of carvedilol might be involved in their reverseremodeling effect, the selective blockade of β1-AR bymetoprolol is of particular interest. Cardiac β2-AR preventscatecholamine-, hypoxia- and reactive oxygen species-inducedapoptotic death[45–48]. Indeed, the beneficial effects of pre-serving β2-AR signaling pathways in HF have been demon-strated by an improved cardiac function in moderateoverexpression of β2-AR transgenic mouse hearts [49], and inβ2-AR agonist treatment of ischemia-induced HF rats [10].

The evidence for differences in efficacy of carvedilol versusmetoprolol in improving cardiac function and survival is ham-pered by known limitations in clinical studies including patientnumber, cardiomyopathy etiology, dosage of β-AR antagonists,co-morbidities, and genetic makeup of the studied population toname a few. In the present study, we took advantage of a geneticmodel of sympathetic hyperactivity-induced HF to assess po-tential differences in cardiac structure and function when theanimals were treated with doses of carvedilol and metoprololoptimized to achieve comparable HR exhibited by the WTgroup for two months. We cannot exclude that some of thesedifferences may be influenced by dose-specific effects for eachof the drugs, but within this time window, both treatments wereequally effective in improving cardiac function and survival.Under these conditions, we provide compelling evidence thatthe underlying mechanisms involved with these responses maydiffer. It will be important, however, to further explore whetherlife long-treatment of these animals leads to different efficacy inreducing mortality and if so, what is the potential role of thepreferential Ca2+ transient improvement associated to metopro-lol versus the greater antioxidant effects mediated by carvedilol.

5. Conclusion

Taken together, we provided evidence that doses of meto-prolol and carvedilol treatments that are equally effective inimproving survival and cardiac contractility and reducing leftventricle remodeling in a genetic model of HF may involvedifferent underlying molecular mechanisms. Thus, differencesin life long clinical use of these agents may have to beinvestigated in light of preferential intracellular targets includ-ing cardiomyocyte calcium handling or global myocardialredox balance.

Acknowledgments

The authors want to thank the Fundação de Amparo àPesquisa do Estado de São Paulo, São Paulo — SP (FAPESP# 2002/04588-8), FAPEMIG and Instituto Milênio (S.G),CNPq (S.G. and M.M. A) for funding the present investiga-tion, and Laboratórios Baldacci S.A. for providing carvedilol.We also want to express our gratitude to Fundação Zerbini,and Vascular Biology Laboratory, Heart Institute, School ofMedicine, University of São Paulo — SP, for the support inthis study.

248 J.B. Bartholomeu et al. / Journal of Molecular and Cellular Cardiology 45 (2008) 240–249

J.B.B. held a master scholarship from FAPESP (2003/10297-9). P.C.B, S.G, and C.E.N hold scholarship from ConselhoNacional de Pesquisa e Desenvolvimento (CNPq-BPQ).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.yjmcc.2008.05.011.

References

[1] Domanski MJ, Krause-Steinrauf H, Massie BM, Deedwania P, FollmannD, Kovar D, et al. A comparative analysis of the results from 4 trials ofbeta-blocker therapy for heart failure: BEST, CIBIS-II, MERIT-HF, andCOPERNICUS. J Card Fail 2003 Oct;9(5):354–63.

[2] Hjalmarson A. Prevention of sudden cardiac death with beta blockers. ClinCardiol 1999 Oct;22(Suppl 5):V11–5.

[3] Effect of metoprolol CR/XL in chronic heart failure: Metoprolol CR/XLRandomised Intervention Trial in Congestive Heart Failure (MERIT-HF).Lancet 1999 Jun 12;353(9169):2001–7.

[4] Eichhorn EJ, Bristow MR. The Carvedilol Prospective RandomizedCumulative Survival (COPERNICUS) trial. Curr Control Trials Cardio-vasc Med 2001;2(1):20–3.

[5] Poole-Wilson PA, Swedberg K, Cleland JG, Di Lenarda A, Hanrath P,Komajda M, et al. Comparison of carvedilol and metoprolol on clinicaloutcomes in patients with chronic heart failure in the Carvedilol orMetoprolol European Trial (COMET): randomised controlled trial. Lancet2003 Jul 5;362(9377):7–13.

[6] Sanderson JE, Chan SK, Yip G, Yeung LY, Chan KW, Raymond K, et al.Beta-blockade in heart failure: a comparison of carvedilol with metoprolol.J Am Coll Cardiol 1999 Nov 1;34(5):1522–8.

[7] Hunt SA. ACC/AHA 2005 guideline update for the diagnosis andmanagement of chronic heart failure in the adult: a report of the AmericanCollege of Cardiology/American Heart Association Task Force on PracticeGuidelines (Writing Committee to Update the 2001 Guidelines for theEvaluation and Management of Heart Failure). J Am Coll Cardiol 2005Sep 20;46(6):e1–e82.

[8] Swedberg K, Cleland J, Dargie H, Drexler H, Follath F, Komajda M, et al.Guidelines for the diagnosis and treatment of chronic heart failure:executive summary (update 2005): The Task Force for the Diagnosis andTreatment of Chronic Heart Failure of the European Society of Cardiology.Eur Heart J 2005 Jun;26(11):1115–40.

[9] Patterson AJ, Zhu W, Chow A, Agrawal R, Kosek J, Xiao RP, et al.Protecting the myocardium: a role for the beta2 adrenergic receptor in theheart. Crit Care Med 2004 Apr;32(4):1041–8.

[10] Ahmet I, Krawczyk M, Heller P, Moon C, Lakatta EG, Talan MI.Beneficial effects of chronic pharmacological manipulation of beta-adrenoreceptor subtype signaling in rodent dilated ischemic cardiomyo-pathy. Circulation 2004 Aug 31;110(9):1083–90.

[11] Feuerstein GZ, Ruffolo Jr RR. Carvedilol, a novel vasodilating beta-blocker with the potential for cardiovascular organ protection. Eur Heart J1996 Apr;17(Suppl B):24–9.

[12] Piacentino III V, Weber CR, Chen X, Weisser-Thomas J, Margulies KB,Bers DM, et al. Cellular basis of abnormal calcium transients of failinghuman ventricular myocytes. Circ Res 2003 Apr 4;92(6):651–8.

[13] Janse MJ. Electrophysiological changes in heart failure and theirrelationship to arrhythmogenesis. Cardiovasc Res 2004 Feb 1;61(2):208–217.

[14] Sun YL, Hu SJ, Wang LH, Hu Y, Zhou JY. Effect of beta-blockers oncardiac function and calcium handling protein in postinfarction heartfailure rats. Chest 2005 Sep;128(3):1812–21.

[15] Sun YL, Hu SJ, Wang LH, Hu Y, Zhou JY. Comparison of low and highdoses of carvedilol on restoration of cardiac function and calcium-handlingproteins in rat failing heart. Clin Exp Pharmacol Physiol 2005 Jul;32(7):553–60.

[16] Brum PC, Kosek J, Patterson A, Bernstein D, Kobilka B. Abnormalcardiac function associated with sympathetic nervous system hyperactivityin mice. Am J Physiol Heart Circ Physiol 2002 Nov;283(5):H1838–45.

[17] Rolim NP, Medeiros A, Rosa KT, Mattos KC, Irigoyen MC, Krieger EM,et al. Exercise training improves the net balance of cardiac Ca2+ handlingprotein expression in heart failure. Physiol Genomics 2007 May 11;29(3):246–52.

[18] Medeiros A, Rolim NP, Oliveira RS, Rosa KT, Mattos KC, Casarini DE,et al. Exercise training delays cardiac dysfunction and prevents calciumhandling abnormalities in sympathetic hyperactivity-induced heart failuremice. J Appl Physiol 2007 Nov 1.

[19] Johns C, Gavras I, Handy DE, Salomao A, Gavras H. Models ofexperimental hypertension in mice. Hypertension 1996;28(6):1064–9.

[20] Guatimosim S, Sobie EA, dos Santos Cruz J, Martin LA, Lederer WJ.Molecular identification of a TTX-sensitive Ca(2+) current. Am J PhysiolCell Physiol 2001 May;280(5):C1327–39.

[21] Kubalova Z, Terentyev D, Viatchenko-Karpinski S, Nishijima Y, Gyorke I,Terentyeva R, et al. Abnormal intrastore calcium signaling in chronic heartfailure. Proc Natl Acad Sci U S A 2005 Sep 27;102(39):14104–9.

[22] Shannon TR, Pogwizd SM, Bers DM. Elevated sarcoplasmic reticulumCa2+ leak in intact ventricular myocytes from rabbits in heart failure. CircRes 2003 Oct 3;93(7):592–4.

[23] Ide T, Tsutsui H, Kinugawa S, Suematsu N, Hayashidani S, IchikawaK, et al.Direct evidence for increased hydroxyl radicals originating from superoxidein the failing myocardium. Circ Res 2000 Feb 4;86(2):152–7.

[24] HeymesC,Bendall JK, Ratajczak P, CaveAC, Samuel JL,Hasenfuss G, et al.Increased myocardial NADPH oxidase activity in human heart failure. J AmColl Cardiol 2003 Jun 18;41(12):2164–71.

[25] von Harsdorf R, Li PF, Dietz R. Signaling pathways in reactive oxygenspecies-induced cardiomyocyte apoptosis. Circulation 1999 Jun 8;99(22):2934–41.

[26] Cesselli D, Jakoniuk I, Barlucchi L, Beltrami AP, Hintze TH, Nadal-Ginard B, et al. Oxidative stress-mediated cardiac cell death is a majordeterminant of ventricular dysfunction and failure in dog dilated car-diomyopathy. Circ Res 2001 Aug 3;89(3):279–86.

[27] Hiraku Y, Murata M, Kawanishi S. Determination of intracellularglutathione and thiols by high performance liquid chromatography witha gold electrode at the femtomole level: comparison with a spectroscopicassay. Biochim Biophys Acta 2002 Feb 15;1570(1):47–52.

[28] Nourooz-Zadeh J, Tajaddini-Sarmadi J, Wolff SP. Measurement ofplasma hydroperoxide concentrations by the ferrous oxidation-xylenolorange assay in conjunction with triphenylphosphine. Anal Biochem1994 Aug 1;220(2):403–9.

[29] Curtis BM, O'Keefe Jr JH. Autonomic tone as a cardiovascular risk factor:the dangers of chronic fight or flight. Mayo Clin Proc 2002 Jan;77(1):45–54.

[30] Francis GS, Cohn JN, Johnson G, Rector TS, Goldman S, Simon A. Plasmanorepinephrine, plasma renin activity, and congestive heart failure. Relationsto survival and the effects of therapy in V-HeFT II. The V-HeFT VA Co-operative Studies Group. Circulation 1993 Jun;87(6 Suppl):VI40–8.

[31] Tilley DG, Rockman HA. Role of beta-adrenergic receptor signaling anddesensitization in heart failure: new concepts and prospects for treatment.Expert Rev Cardiovasc Ther 2006 May;4(3):417–32.

[32] Zhang R, Khoo MS, Wu Y, Yang Y, Grueter CE, Ni G, et al. Calmodulinkinase II inhibition protects against structural heart disease. Nat Med 2005Apr;11(4):409–17.

[33] WangW,ZhuW,WangS,YangD, CrowMT,XiaoRP, et al. Sustained beta1-adrenergic stimulation modulates cardiac contractility by Ca2+/calmodulinkinase signaling pathway. Circ Res 2004 Oct 15;95(8):798–806.

[34] Ponicke K, Heinroth-Hoffmann I, Brodde OE. Role of beta 1- and beta 2-adrenoceptors in hypertrophic and apoptotic effects of noradrenaline andadrenaline in adult rat ventricular cardiomyocytes. Naunyn SchmiedebergsArch Pharmacol 2003 Jun;367(6):592–9.

[35] Zhu WZ, Wang SQ, Chakir K, Yang D, Zhang T, Brown JH, et al. Linkageof beta1-adrenergic stimulation to apoptotic heart cell death throughprotein kinase A-independent activation of Ca2+/calmodulin kinase II.J Clin Invest 2003 Mar;111(5):617–25.

[36] Gupta RC, Mishra S, Rastogi S, Imai M, Habib O, Sabbah HN. CardiacSR-coupled PP1 activity and expression are increased and inhibitor 1

249J.B. Bartholomeu et al. / Journal of Molecular and Cellular Cardiology 45 (2008) 240–249

protein expression is decreased in failing hearts. Am J Physiol Heart CircPhysiol 2003 Dec;285(6):H2373–81.

[37] Flesch M, Ettelbruck S, Rosenkranz S, Maack C, Cremers B, Schluter KD,et al. Differential effects of carvedilol and metoprolol on isoprenaline-induced changes in beta-adrenoceptor density and systolic function in ratcardiac myocytes. Cardiovasc Res 2001 Feb 1;49(2):371–80.

[38] Bilchick KC, Duncan JG, Ravi R, Takimoto E, Champion HC, Gao WD,et al. Heart failure-associated alterations in troponin I phosphorylation impairventricular relaxation-afterload and force-frequency responses and systolicfunction. Am J Physiol Heart Circ Physiol 2007 Jan;292(1):H318–325.

[39] Biesiadecki BJ, Kobayashi T, Walker JS, John Solaro R, de Tombe PP. Thetroponin C G159D mutation blunts myofilament desensitization induced bytroponin I Ser23/24 phosphorylation. CircRes 2007May25;100(10):1486–93.

[40] Pena JR, Wolska BM. Troponin I phosphorylation plays an important rolein the relaxant effect of beta-adrenergic stimulation in mouse hearts.Cardiovasc Res 2004 Mar 1;61(4):756–63.

[41] Takeishi Y, Chu G, Kirkpatrick DM, Li Z, Wakasaki H, Kranias EG, et al.In vivo phosphorylation of cardiac troponin I by protein kinase Cbeta2decreases cardiomyocyte calcium responsiveness and contractility intransgenic mouse hearts. J Clin Invest 1998 Jul 1;102(1):72–8.

[42] Itoh S, Ding B, Bains CP, Wang N, Takeishi Y, Jalili T, et al. Role of p90ribosomal S6 kinase (p90RSK) in reactive oxygen species and proteinkinase C beta (PKC-beta)-mediated cardiac troponin I phosphorylation.J Biol Chem 2005 Jun 24;280(25):24135–42.

[43] He X, Liu Y, Sharma V, Dirksen RT, Waugh R, Sheu SS, et al.

ASK1 associates with troponin T and induces troponin T phosphor-ylation and contractile dysfunction in cardiomyocytes. Am J Pathol2003 Jul;163(1):243–51.

[44] Kametani R, Miura T, Harada N, Shibuya M, Wang R, Tan H, et al.Carvedilol inhibits mitochondrial oxygen consumption and superoxideproduction during calcium overload in isolated heart mitochondria. Circ J2006 Mar;70(3):321–6.

[45] Chesley A, Lundberg MS, Asai T, Xiao RP, Ohtani S, Lakatta EG, et al. Thebeta(2)-adrenergic receptor delivers an antiapoptotic signal to cardiacmyocytes throughG(i)-dependent coupling to phosphatidylinositol 3′-kinase.Circ Res 2000 Dec 8;87(12):1172–9.

[46] Communal C, Singh K, Sawyer DB, Colucci WS. Opposing effects ofbeta(1)- and beta(2)-adrenergic receptors on cardiac myocyte apoptosis:role of a pertussis toxin-sensitiveGprotein. Circulation 1999Nov 30;100(22):2210–2.

[47] ZauggM, XuW, Lucchinetti E, Shafiq SA, Jamali NZ, Siddiqui MA. Beta-adrenergic receptor subtypes differentially affect apoptosis in adult ratventricular myocytes. Circulation 2000 Jul 18;102(3):344–50.

[48] Bernstein D, Fajardo G, Zhao M, Urashima T, Powers J, Berry G, et al.Differential cardioprotective/cardiotoxic effects mediated by beta-adrener-gic receptor subtypes. Am J Physiol Heart Circ Physiol 2005 Dec;289(6):H2441–2449.

[49] Tevaearai HT, Koch WJ. Molecular restoration of beta-adrenergic receptorsignaling improves contractile function of failing hearts. Trends Cardio-vasc Med 2004 Aug;14(6):252–6.