Nandrolone decanoate impairs exercise-induced ...

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Marcus F. Oliveirab,∗,1, José Hamilton Matheus Nascimentoa,1 a Instituto de ..... 207–212. [24] B. Trifunovic, G.R. Norton, M.J. Duffield, P. Avraam, A.J. Woodi-.
Journal of Steroid Biochemistry & Molecular Biology 99 (2006) 223–230

Nandrolone decanoate impairs exercise-induced cardioprotection: Role of antioxidant enzymes Elen Aguiar Chaves a,b,∗ , Pedro Paulo Pereira-Junior a , Rodrigo Soares Fortunato a , Masako Oya Masuda a , Antˆonio Carlos Campos de Carvalho a , Denise Pires de Carvalho a , Marcus F. Oliveira b,∗,1 , Jos´e Hamilton Matheus Nascimento a,1 a

Instituto de Biof´ısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Cidade Universit´aria, Rio de Janeiro, Brazil b Instituto de Bioqu´ımica M´ edica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro, Cidade Universit´aria, Rio de Janeiro, Brazil Received 19 December 2005; accepted 27 January 2006

Abstract The beneficial effects of exercise in reducing the incidence of cardiovascular diseases are well known and the abuse of anabolic androgenic steroids (AAS) has been associated to cardiovascular disorders. Previous studies showed that heart protection to ischemic events would be mediated by increasing the antioxidant enzyme activities. Here, we investigated the impact of exercise and high doses of the AAS nandrolone decanoate (DECA), 10 mg kg−1 body weight during 8 weeks, in cardiac tolerance to ischemic events as well as on the activity of antioxidant enzymes in rats. After a global ischemic event, hearts of control trained (CT) group recovered about 70% of left ventricular developed pressure, whereas DECA trained (DT), control sedentary (CS) and DECA sedentary (DS) animals recovered only about 20%. Similarly, heart infarct size was significantly lower in the CT group compared to animals of the three other groups. The activities of the antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) were significantly higher in CT animals than in the other three groups, whereas catalase activity was not affected in any group. Together, these results indicate that chronic treatment with DECA cause an impairment of exercise induction of antioxidant enzyme activities, leading to a reduced cardioprotection upon ischemic events. © 2006 Elsevier Ltd. All rights reserved. Keywords: Exercise; Anabolic steroid; Heart; Ischemia–reperfusion; Cardioprotection; Antioxidant enzymes

1. Introduction Regular physical exercise has been associated with reduced incidence of heart diseases by decreasing plasma cholesterol, high blood pressure, obesity and glucose intolerance [1]. Among the adaptations related to acute and chronic exercise, the improvement of heart tolerance to ischemic events represents one of the most important and clinically relevant features [2–9]. Exercise adaptation has ∗ Corresponding authors at: bloco D, sub-solo sala 005, Instituto de Bioqu´ımica M´edica, Universidade Federal do Rio de Janeiro, Cidade Universit´aria, Rio de Janeiro, RJ 21941-590, Brazil. Tel.: +55 21 2562 6755; fax: +55 21 22708647. E-mail addresses: [email protected] (E.A. Chaves), [email protected] (M.F. Oliveira). 1 These authors have equally contributed to this work.

0960-0760/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2006.01.004

been related with enhanced cardiac contractile function and reduced infarct size post-ischemia, which depends on the exercise regularity and intensity [2,4,5,10]. The molecular mechanisms involved in these adaptive responses include increased expression of heat shock proteins [4,6], induction of nitric oxide synthase [11], protein kinase C activation [9] as well as increased antioxidant enzyme activities [3,5,7,8,12]. Reactive oxygen species (ROS) are by-products of aerobic cellular metabolism and antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, play a crucial role in order to circumvent their deleterious effects. The imbalance between ROS generation and the intracellular levels of antioxidant defenses leads to oxidative stress, a condition that has been associated with apoptosis, neurodegenerative diseases and ischemia–reperfusion (I/R) injury [13,14]. Several lines of evidence indicate a massive production of ROS upon

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I/R events in different tissues such as brain, vascular endothelial cells and heart [8,15]. Among the mechanisms involved in the exercise cardioprotection, myocardial enzymatic antioxidants are particularly important since they inhibit or delay the oxidative damage to several biomolecules [16]. In fact, it has been widely accepted that increased oxidation of low-density lipoprotein (LDL) would be a crucial event in the progression of atherosclerosis [17]. However, despite the numerous reports supporting the beneficial effects of antioxidants preventing the LDL oxidation, large-scale clinical studies have shown inconsistent results about the efficacy of antioxidants against atherosclerosis [18,19]. Anabolic androgenic steroids (AAS) are derivatives of testosterone, which were synthesized aiming the improvement of a myriad of human pathologies, including those associated with catabolic states, osteoporosis, starvation, burns, and others [20]. The first case of AAS abuse was described in 1950, in weight lifters and other “strength” athletes, and later in non-athletes aiming to increment in physical performance and muscular mass gain [20,21]. Despite the positive effects of AAS in improving physical performance, many studies demonstrated that high doses of AAS raises the risk of cardiovascular diseases, e.g. increased levels total cholesterol and low density lipoprotein, decreased levels of high density lipoprotein, increased blood pressure, thrombosis, myocardial infarct and heart failure [20,21]. Interestingly, Du Toit et al. have recently shown that swimming rats treated with AAS lost the improvement of cardiac tolerance to ischemic events induced by exercise [22]. However, no molecular mechanism was proposed as determinants of the impairment of cardioprotection in AAS exercised animals. Thus, the scarcity of information in this issue led us to investigate the effects of nandrolone decanoate (DECA) treatment on heart tolerance to ischemic events as well as on the activity of antioxidant enzymes in sedentary and in treadmill exercised rats.

35 m min−1 on the 8th week. Active recovery time was progressively decreased to reach 1 min at 10 m min−1 at the beginning of the 8th week. In the 7th week 5% grade was adopted, and increased to 10% on the 8th week. The two last weeks of training were characterized by maintenance of load (10 min of warm-up at 10 m min−1 , six bouts at 35 m min−1 with an active recovery of 1 min at 10 m min−1 and 10% grade). From the 3rd to 10th week of the experimental protocol, DS and DT rats received a weekly intramuscular injection of 10 mg kg−1 of DECA (Deca durabolin, Organon do Brasil, Brazil). Control animals (CS and CT) were injected with the same volume of vehicle, composed of peanut oil with 10% (v/v) benzyl alcohol, as previously described [24]. 2.2. In vitro I/R experiments

2. Materials and methods

Rats were anaesthetized with ethyl ether and sacrificed by cervical dislocation. Hearts were rapidly excised, and placed in Krebs Henseleit modified buffer (118 mM NaCl, 4.7 mM KCl, 25 mM NaHCO3 , 1.2 mM MgSO4 , 1.2 mM KH2 PO4 , 11 mM glucose, and 1.25 mM CaCl2 , aerated with 95% O2 and 5% CO2 ). Heart aortas were hung on the cannula of a modified Langendorff apparatus at a constant flow of 10 ml min−1 at 37 ± 0.5 ◦ C. A latex balloon was placed into the left ventricle chamber through the mitral valve, and developed pressure waveforms were digitally acquired (MLT0380 BP transducer—ADInstruments; A/D Interface PowerLab/400—ADInstruments; ML110/D Bridge Amplifier—ADInstruments). Hearts were kept immersed in perfusion solution and end diastolic pressure was set in 10 mmHg, as previously described [9]. After approximately 30 min of control period, where heart rate, diastolic and systolic pressures were stable, peristaltic pump was stopped and hearts were submitted to 30 min of global ischemia and subsequent 60 min of reperfusion. Pressure acquisition and analysis were performed in PowerLab Chart 4 for Windows (ADInstruments), and values of developed pressure were plotted as % of control.

2.1. Exercise training program and steroid treatment

2.3. Measurement of myocardial infarct size

Adult male Wistar rats (weighing 220 ± 20 g) were kept at 25 ± 2 ◦ C with 12:12 h dark/light cycles with free access to rat chow and water ad lib following the institutional animal care and use committee. Animals were randomly allocated into four experimental groups: control sedentary (CS), DECA sedentary (DS), control trained (CT) and DECA trained (DT). A program of exercise training was adapted from previously described methods [23]. Rats were submitted to exercise 5 days week−1 for 10 weeks. In the 1st week, rats were familiarized to motor-driven treadmill (3 days: 30 min at 10 m min−1 ) followed by sudden speed increments (2 days: three bouts of 30 s at 15–20 m min−1 with 2.5 min of active recovery at 10 m min−1 ). The number of bouts was increased to reach 6 on the 3rd week, and the same to the speed, reaching

After the I/R procedure, hearts were weighted and atria were removed. Infarct size determination was performed as previously described with some modifications [9]: ventricles were sliced into approximately 1.5 mm cross-sections from apex to base and incubated in 1% (w/v) triphenyl tetrazolium chloride (TTC) in phosphate buffer (pH 7.4) for 4 min at 37 ◦ C. Then, the slices were placed in a 10% (v/v) formaldehyde solution for 24 h to improve contrast between stained (viable) and unstained (necrotic) tissues. Ventricle slices were placed between two glass slides and their images were digitally acquired in a scanner. Infarct size was determined by planimetry, using ImageJ Program (NIH ImageJ: National Institute of Health, USA, version 1.22). Values were expressed in % of total area.

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2.4. Preparation of heart homogenates

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priated, Tukey’s post hoc test was applied. Significance was established at P < 0.05.

Hearts were quickly removed and washed in an ice-cold saline. Ventricles were quickly dissected, frozen in N2 , and stored at −80 ◦ C until analysis. Frozen cardiac tissues were cut into small pieces, minced in N2 and homogenized with a Potter-Elvehjem in nine volumes of 10 mM Tris–HCl pH 7.4 containing 0.9% NaCl, 1 mM phenylmethylsulphonyl fluoride (PMSF) and 0.5 ␮g ml−1 aprotinine. Homogenate was centrifuged at 720 × g at 4 ◦ C for 10 min and the supernatant was used for enzymatic measurements. All enzymatic assays were performed in a UV–vis spectrophotometer (GBC 920—GBC Scientific Equipment Pty Ltd.). Protein content was determined by method of Bradford [25], using bovine serum albumin as standard.

3. Results Based on the abuse of AAS in humans and the lack of information regarding the effects of high doses of AAS on exercise cardioprotection, the aim of this study was to determine some of the physiological and biochemical changes promoted by DECA treatment in hearts of sedentary and exercised rats. Table 1 summarizes the effects of chronic exercise training and DECA administration on body and heart weights. No significant differences were observed in body or absolute heart weights among all groups. Noteworthy, when heart weights were normalized by body weights, a significant increase of heart weights was observed in DS and DT rats when compared to CS and CT, respectively. In agreement with previous reports, DECA treatment also caused a significant reduction in testis weights in sedentary and trained animals, suggesting

2.5. Enzyme activities All enzyme activity assays were performed at 37 ◦ C in duplicate following the methods described previously in the literature. Total superoxide dismutase (SOD, EC 1.15.1.1) activity was determined by reduction of cytochrome c at 550 nm as described by Crapo et al. [26] and expressed as units per milligram of protein. Glutathione peroxidase (GPx, EC 1.11.1.9) activity was assayed following NADPH oxidation at 340 nm in the presence of an excess of glutathione reductase, reduced glutathione and tert-butyl hydroperoxide as substrate [27] and expressed as nmol of oxidized NADPH per milligram of protein. Glutathione reductase (GR, EC 1.6.4.2) activity was performed following NADPH oxidation at 340 nm as described by Calberg and Mannervik [28] and expressed as nmol of oxidized NADPH per milligram of protein. Catalase (EC 1.11.1.6) activity was assayed following the method of Aebi [29] and activity was expressed as units per milligram of protein. 2.6. Statistical analysis

Fig. 1. Effects of exercise and DECA treatment on left ventricular contractile function during I/R protocol. C, control period. Time course of changes are expressed as percentage of initial (pre-ischemia) values of left ventricular developed pressure (% LVDP) during the 30 min global ischemia and subsequent 60 min reperfusion. % LVDP (means ± S.E.) at 60 min reperfusion: CS, 21.4 ± 5% (n = 7); DS, 26.1 ± 7% (n = 7); CT, 77.9 ± 13% (n = 9); DT, 19 ± 3% (n = 8). #P < 0.01 at 40 min vs. CS, DS and DT. *P < 0.001 from 45 to 90 min vs. CS, DS and DT.

Myocardial contractility (expressed as a percentage of baseline values of left ventricular developed pressure) was analyzed by two-way analysis of variance. Infarct size and enzyme activities measurements were analyzed by one-way analysis of variance, using Graphpad Prism, 3.02 version (Graphpad Software Inc., San Diego, USA). When appro-

Table 1 Rat body weights measured at the beginning (week 1) and at the end (week 10) of the experimental period, whereas heart weights and heart-to-body weight ratio were obtained at week 10 (the n column indicates the number of animals from each group utilized in this experiment. Values are expressed as mean ± S.E.M.) Groups

Body weight (g) Week 1

CS DS CT DT *

227 221.8 218.8 222.1 P < 0.05 vs. CS and CT.

± ± ± ±

Heart weight (g)

Heart weight/body weight (g/kg)

n

Week 10 4.9 4.1 2.5 5.6

340 315.2 323 312.6

± ± ± ±

10 7.2 6.9 11.6

1.31 1.35 1.22 1.31

± ± ± ±

0.06 0.03 0.03 0.05

3.8 4.3 3.7 4.2

± ± ± ±

0.11 0.08* 0.10 0.19*

12 19 10 11

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a testicular atrophy associated to high doses of AAS (data not shown). Fig. 1 shows the time course of left ventricular developed pressure (LVDP), expressed as a percentage of baseline values, during the I/R protocol. During global ischemia % of LVDP declined to zero in the first 5 min in all groups. In agreement with other studies [4,5], exercised rats showed increased % LVDP at the end of reperfusion compared to sedentary animals. A partial recovery (20–25%) of contrac-

tile function was achieved in SC, SD and DT rats at the end of the reperfusion period, but only CT rats showed a significant improvement (about 70%) of contractile function, indicating that DECA treatment abrogated the exercise-induced cardioprotection upon I/R. Our next step was to determine the infarct size area, which was calculated as percentage of the risk area after 30 min of global ischemia and 60 min of reperfusion (Fig. 2). CT rats exhibited a drastic reduction of infarct size when compared

Fig. 2. Effect of exercise and DECA treatment on myocardial infarct size of isolated hearts after I/R protocol. (A) CS; (B) DS; (C) CT; (D) DT; (E) quantification of infarct size areas. Dark gray areas represent the preserved heart tissue whereas the light gray ones the infarcted damaged tissue. Infarct size was expressed as percentage of risk area and values are means ± S.E. CS, 46.3 ± 4% (n = 10); DS, 53.5 ± 3.2% (n = 12); CT, 15 ± 2.4% (n = 12); DT, 35.1 ± 4.6% (n = 11).*P < 0.001 vs. CS, DS and DT. **P < 0.01 vs. DS.

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Fig. 3. Effect of DECA treatment on the antioxidant enzymes activities. (A) Superoxide dismutase (SOD) activities (U/mg protein) are expressed as means ± S.E. Control sedentary (CS), 49.4 ± 5.9 (n = 10); DECA sedentary (DS), 51.2 ± 4.3 (n = 14); control trained (CT), 84 ± 6.3 (n = 7); DECA trained (DT), 56.7 ± 9.4 (n = 8). *P < 0.05 vs. CS, DS and DT. (B) Glutathione peroxidase (GPx) activities (nmol NADPH oxidized/mg protein/min) are expressed as means ± S.E. CS, 100.9 ± 6 (n = 10); DS, 83.9 ± 5.1 (n = 14); CT, 132.3 ± 9 (n = 7); DT, 92.8 ± 5.6 (n = 8). *P < 0.05 vs. CS, DS and DT. (C) Glutathione reductase (GR) activities (nmol NADPH oxidized/mg protein/min) are expressed as means ± S.E. CS, 34.3 ± 1.6 (n = 10); DS, 31.7 ± 1.5 (n = 14); CT, 36.7 ± 1.2 (n = 7); DT, 28.8 ± 2.3 (n = 8). **P < 0.05 vs. CT. (D) Catalase activity (U/mg protein) are expressed as means ± S.E. CS, 6.6 ± 0.6 (n = 10); DS, 5.4 ± 0.4 (n = 14); CT, 7.5 ± .4 (n = 7); DT, 5.9 ± 0.6 (n = 8).

with CS group, which was reversed in DT rats indicating that exercise-induced tolerance to I/R, observed in CT animals, was somehow impaired by DECA treatment in this group. Despite insignificant differences were observed between CS (45% of infarct size) versus DT (35% of infarct size) and CS versus DS (55% of infarct size) groups, infarct size of DS animals was significantly higher than in DT group, suggesting that training provided a small protection against infarction in face of the steroid treatment compared to non-exposed group. In order to unravel one of the possible molecular mechanisms involved in the impairment of cardioprotection induced by DECA treatment, the activities of four antioxidant enzymes (SOD, GPx, GR and catalase) were evaluated (Fig. 3). As previously described, exercise induced a significant increase of SOD and GPx activities in CT animals [3,5,8,12]. Interestingly, DECA treatment abolished the exercise-induced increase of SOD (Fig. 3A), and GPx (Fig. 3B) activities, showing levels very close to the sedentary groups. Differently from SOD and GPx, exercise itself did not induce a significant increase on GR activity, but DECA treatment was able to significantly inhibit it in DT group (Fig. 3C). Moreover, we also observed that in sedentary rats, DECA promoted a slight, but not significant, decrease in both GPx and

GR activities. No statistic differences were observed in catalase activity among all experimental groups, but again DECA caused a small, but non-significant, decrease in DS and DT rats (Fig. 3D). The overall data indicate that exercise-induced improvement of antioxidant enzymes activities was impaired by DECA treatment and this would be implicated as one of the causes related to the impairment of cardioprotection in exercised rats treated with DECA.

4. Discussion The data presented here show for the first time that exercise-induced cardioprotection is impaired by supraphysiological doses of DECA in treadmill-exercised rats. The major finding of this work is that the increased antioxidant enzyme levels promoted by exercise is impaired by DECA treatment, which could be associated with the cardiac deleterious effects of this drug. The hearts of DT animals exhibited lower SOD, GPx and GR activities when compared with CT group indicating that in DT animals, DECA could be acting through the blockage or down regulation of the mechanism(s) involved in the improved antioxidant defenses, which would

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explain the reduced % LVDP (Fig. 1) and increased infarct size (Fig. 2) in this group. It is very well established that there is a relationship between exercise training and oxidative stress [30,31]. Several lines of evidence suggest that exercise induces adaptations in many tissues against an oxidative insult, due mainly to a mild oxidative stress, which would upregulate antioxidant enzymes gene expression through redox-regulated transcription factors [32–34]. In this way, Hollander et al. demonstrated that an acute bout of exercise increased binding of the transcription factors NF-␬B and AP-1 in skeletal muscle. These factors stimulated Mn–SOD mRNA transcription and increased CuZn–SOD protein levels upon exercise [35]. Moreover, Ji et al. showed recently that exercise induces the activation of NF-␬B signalling cascade in a redox-sensitive manner during muscular contraction and this would be associated with increased production of free radicals [36]. In fact, this sounds quite plausible based on previous reports showing a significant increase of ROS levels in muscle of exercised rats compared to resting animals [37]. Additionally, rats depleted of reduced glutathione (GSH) showed an expressive reduction of endurance performance, suggesting a central role of antioxidant defenses not only in the adaptative response against exercise-induced oxidative stress but also as regulators of exercise performance [34]. The overall physiological significance of the antioxidant adaptive response triggered by exercise is more evident in the I/R tissue damage context [3,5,7,12]. Myocardial ischemia occurs when the requirement for oxygen exceeds the supply and may arise in a diversity of conditions such as physical exertion, coronary artery disease, reduction in blood pressure and coronary bypass surgery [38,39]. Depending on the intensity and duration of myocardial ischemia, ventricular contractile dysfunction may result [2,4,5,8–10]. Several studies have proposed the essential role of ROS in the pathogenesis of myocardial I/R injury. In this regard, it has been shown that superoxide, hydrogen peroxide and hydroxyl radicals are formed upon reperfusion in several tissues and they seem to be involved in the subsequent injuries [40–42]. This is supported by experiments using ROS generating systems, which produced patterns of alterations in cardiac function very similar to hearts subjected to I/R [15]. Once produced, the excessive levels of ROS would mediate a myriad of tissue molecular changes such as lipid peroxidation, enzyme inhibition and DNA strand breaks. The targets of ROS generated

upon I/R in heart would include the calcium handling proteins, such as the calcium-activated protease calpain, which has been shown to play a deleterious role during I/R since it degrades the sarco/endoplasmic reticulum calcium ATPase (SERCA) [43]. Thus, antioxidant mechanisms involved in both prevention and suppression of ROS would attenuate their deleterious effects upon ischemia. In line with these observations, many reports have shown the protective role of antioxidants in I/R injury as these compounds prevent the changes in myocardial function and calcium handling in isolated hearts subjected to global I/R [43]. Despite the intensive research efforts, the molecular mechanisms associated to exercise-induced cardioprotection are still controversial [2–12]. The divergences found in the literature may be explained by the variables such as the rat strain used and the exercise type, which would determine the level and the pathways involved in these adaptive responses [2–12]. Although the mechanisms by which DECA promote the deleterious effects in rat heart are not known, this drug might be acting in one of the events involved in the improved cardiac protection against I/R injury triggered by exercise. The main outcome would be that DECA treated animals do not show the adaptive response of the exercise-induced increase of antioxidant enzymes activities establishing a chronic oxidative stress condition, which would explain the cardiac injuries frequently found in AAS users [20,21]. Our data are in agreement with a recent study showing that in sedentary and swim exercised rats, nandrolone laurate treatment increased the susceptibility of hearts to I/R injuries [22]. In our study sedentary rats showed no statistical differences in all the functional and biochemical parameters analyzed (Figs. 1–3) whereas in that work sedentary treated animals showed a significant reduction in the rate pressure product after reperfusion compared to non-treated sedentary animals. This could be explained by differences in the AAS used (laurate instead of decanoate) or in the rat strain (Long-Evans instead of Wistar). Mechanistically, however, the Du Toit et al.’s work explored only effects of nandrolone laurate on the sedentary rats demonstrating the increased basal levels of myocardial cAMP, and TNF-␣ concentrations in basal and postischemia periods [22]. To date, the present report is the first to show a mechanistic explanation for the observed impairment of exercise-induced cardioprotection promoted by an AAS. The informations presented here suggest that the cascade of events that lead to cardioprotection is impaired by DECA

Fig. 4. Schematic diagram of the possible effects of DECA in the exercise-induced cardioprotection in rat heart. Gray shaded box indicates the point affected by DECA in the cascade of events, which lead to cardioprotection suggested in this work.

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at least the one involved in cellular ability to detoxify ROS (Fig. 4). In conclusion, the present study evaluated the influence of high doses of AAS in treadmill exercised rat heart tolerance to I/R injuries and the involvement of antioxidant enzymes in the lack of cardioprotection observed in exercised AAS treated rats. Further research is necessary to determine whether other biochemical pathways would be affected by AAS in heart tolerance to I/R events.

Acknowledgements We would like to thank Mrs. Daisy Avanzi for the excellent technical assistance. To Luciane Barcellos, Escola de Educac¸a˜ o F´ısica e Desportos (UFRJ), to Joyce Carvalho, to Prof. Antonio Galina and to Prof. Leopoldo De Meis at the Instituto de Bioqu´ımica M´edica (UFRJ) for the treadmill use. To Fabr´ıcio Passos, Fredson Serejo and Ana Carolina Carvalho, Instituto de Biof´ısica Carlos Chagas Filho (UFRJ) for the valuable help with Langendorff apparatus. This study was supported by Coordenac¸a˜ o de Aperfeic¸oamento de Pessoal de N´ıvel Superior (CAPES), Conselho Nacional de Desenvolvimento Cient´ıfico e Tecnol´ogico (CNPq), and Fundac¸a˜ o de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ). MFO, ACCC and DPC are research scholars of CNPq.

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