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Journal of Steroid Biochemistry & Molecular Biology 171 (2017) 34–42

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Administration of anabolic steroid during adolescence induces long-term cardiac hypertrophy and increases susceptibility to ischemia/reperfusion injury in adult Wistar rats Fernando de Azevedo Cruz Searaa,b , Raiana Andrade Quintanilha Barbosac, Dahienne Ferreira de Oliveirab , Diorney Luiz Souza Gran da Silvad, Adriana Bastos Carvalhoc , Andrea Claudia Freitas Ferreirad,e, José Hamilton Matheus Nascimentob , Emerson Lopes Olivaresa,* a Laboratory of Cardiovascular Physiology and Pharmacology, Department of Physiological Sciences, Institute of Biology, Federal Rural University of Rio de Janeiro, 23890-000 Seropedica, RJ, Brazil b Laboratory of Cardiac Electrophysiology, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, 21941-902 RJ, Brazil c Laboratory of Cellular and Molecular Cardiology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, 21941-902 RJ, Brazil d Laboratory of Endocrine Physiology, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, 21941-902 RJ, Brazil e NUMPEX-Bio, Pólo de Xerém, Federal University of Rio de Janeiro, Rio de Janeiro, 21941-902 RJ, Brazil

A R T I C L E I N F O

Article history: Received 9 November 2016 Received in revised form 9 January 2017 Accepted 19 January 2017 Available online 6 February 2017 Keywords: Anabolic steroids Myocardial infarction IR injury Catalase

A B S T R A C T

Chronic administration of anabolic androgenic steroids (AAS) in adult rats results in cardiac hypertrophy and increased susceptibility to myocardial ischemia/reperfusion (IR) injury. Molecular analyses demonstrated that hyperactivation of type 1 angiotensin II (AT1) receptor mediates cardiac hypertrophy induced by AAS and also induces down-regulation of myocardial ATP-sensitive potassium channel (KATP), resulting in loss of exercise-induced cardioprotection. Exposure to AAS during adolescence promoted long-term cardiovascular dysfunctions, such as dysautonomia. We tested the hypothesis that chronic AAS exposure in the pre/pubertal phase increases the susceptibility to myocardial ischemia/reperfusion (IR) injury in adult rats. Male Wistar rats (26 day old) were treated with vehicle (Control, n = 12) or testosterone propionate (TP) (AAS, 5 mg kg 1 n = 12) 5 times/week during 5 weeks. At the end of AAS exposure, rats underwent 23 days of washout period and were submitted to euthanasia. Langendorffperfused hearts were submitted to IR injury and evaluated for mechanical dysfunctions and infarct size. Molecular analysis was performed by mRNA levels of a-myosin heavy chain (MHC), bMHC and brainderived natriuretic peptide (BNP), ryanodine receptor (RyR2) and sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) by quantitative RT-PCR (qRT-PCR). The expression of AT1 receptor and KATP channel subunits (Kir6.1 and SURa) was analyzed by qRT-PCR and Western Blot. NADPH oxidase (Nox)-related reactive oxygen species generation was assessed by spectrofluorimetry. The expression of antioxidant enzymes was measured by qRT-PCR in order to address a potential role of redox unbalance. AAS exposure promoted long-term cardiac hypertrophy characterized by increased expression of bMHC and bMHC/ aMHC ratio. Baseline derivative of pressure (dP/dt) was impaired by AAS exposure. Postischemic recovery of mechanical properties was impaired (decreased left ventricle [LV] developed pressure and maximal dP/dt; increased LV end-diastolic pressure and minimal dP/dt) and infarct size was larger in the

Abbreviations: AAS, anabolic androgenic steroids; TP, testosterone propionate; LV, left ventricle; LVDP, LV developed pressure; LVSP, LV systolic pressure; LVEDP, LV enddiastolic pressure; min. dP/dt, minimal derivative of pressure; max. dP/dt, maximal derivative of pressure; NADPH, Nicotinamide dinucleotide phosphate oxidase; Nox, NADPH oxidase; qRT-PCR, quantitative real time RT-PCR; AT1R, angiotensin II type 1 receptor; KATP, ATP-sensitive potassium channel; Gpx, glutathione peroxidase; CAT, catalase; SOD, superoxide dismutase; GR, glutathione reductase; RAS, renin-angiotensin system; LDL, low-derived lipoprotein; IR, ischemia/reperfusion; TTC, triphenyltetrazolium chloride (TTC); BNP, brain-derived natriuretic peptide; MHC, myosin heavy chain; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MPTP, mitochondrial permeability transition pore. * Corresponding author. E-mail addresses: [email protected], [email protected] (E.L. Olivares). http://dx.doi.org/10.1016/j.jsbmb.2017.01.012 0960-0760/© 2017 Published by Elsevier Ltd.

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AAS group. Catalase mRNA expression was significantly decreased in the AAS group. In conclusion, chronic administration of AAS during adolescence promoted long-term pathological cardiac hypertrophy and persistent increase in the susceptibility to myocardial IR injury possible due to disturbances on catalase expression. © 2017 Published by Elsevier Ltd.

1. Introduction Growing misuse of AAS among individuals aiming an improvement at physical appearance and athletic performance has been extensively reported along the last decades [1,2]. Recent data estimates that approximately 2.9–4.0 million Americans have used AAS, a significant rise compared with data reported in the beginning of the 90 s [3,4]. In this context, a vast number of studies have demonstrated an alarming rate of AAS abuse among adolescents in occidental nations [2,3,5–9]. Approximately 22% of AAS users started to use steroids before age 20 [10]. The prevalence of AAS consumption among adolescents from American states ranged from 2.1% to 7.2%, according to the 2009 Youth Risk Behavior Surveillance System [11]. These epidemiological data are especially important given that adolescents are more susceptible to the effects of AAS abuse [12]. Hypogonadism is the most frequent adverse effect reported during AAS abuse [13,14]. In addition, clinical and experimental reports have shown a high mortality associated with cardiovascular dysfunctions in AAS users [15–17]. Administration of high doses of AAS promotes a broad spectrum of abnormalities that culminate on enhanced susceptibility to myocardial ischemia, such as hypercoagulability state, hyperlipidemia, rise on serum LDL, vasospasm, interstitial fibrosis, cardiac hypertrophy and increased thrombogenesis, resulting in coronary artery disease and myocardial infarction, as evidenced in animal studies, case reports and post-mortem studies [18–27]. In such a condition, reperfusion consists in the most effective therapeutic technique. However, reperfusion per se elicits several downstream pathways that culminate on cell death, a condition known as IR injury. Experimental findings revealed that chronic administration of AAS is sufficient to increase the susceptibility to myocardial IR injury, resulting in impaired postischemic recovery of left ventricular mechanical properties and increased infarct size in isolated hearts of adult rats [26]. Pharmacological and molecular evidences strongly suggest that overactivation of cardiac reninangiotensin system (RAS), redox unbalance and down-regulation of KATP channel are largely correlated with the loss of exerciseinduced cardioprotection and increased susceptibility to IR injury promoted by AAS in adult rats [27–29]. Recently, our group demonstrated that chronic administration of testosterone propionate (TP) strictly in the prepubertal/pubertal phase of Wistar rats elicited long-term behavioral, electrolytic and cardiovascular dysfunctions, such as dysautonomia, during adulthood [30]. However, the molecular mechanisms underlying these alterations were largely unknown so far. Herein, we tested the hypothesis that chronic administration of AAS during adolescence of Wistar rats induces long-term increase in the susceptibility to myocardial IR injury in adulthood. The possible role of antioxidant enzymes, myocardial AT1 receptor and KATP channel imbalance were also tested.

23, revised in 1996), and the experimental protocols were approved by the Ethics Committee on Animal Use of the Federal Rural University of Rio de Janeiro in accordance with Brazilian law (Law No. 11.794 of October 8, 2008). All animals were housed in cages (four animals/cage) under controlled temperature (21  2  C), with daily exposure to a 12-h light–dark cycle (lights off at 7:00 pm) and free access to water and standard chow. Experimental design is illustrated in Fig. 1. Twenty-four male Wistar rats, with bodyweights between 110 and 130 g, were used in the present study. Prepubertal (postnatal day 26, P26) rats were randomly distributed into two groups: Anabolic androgenic steroid group (AAS, n = 12) received testosterone propionate (TP; Hertape Carlier1, Hertape Carlier Animal Health Laboratory, 5 mg kg 1 bodyweight), and control group (Control, n = 12) received a proportional volume of vehicle (peanut oil with benzyl alcohol, 90:10, v/v). Five intramuscular injections were given per week (always between 15:00 and 17:00) during 5 weeks as previously described [31]. The choice to use TP and the dose regimen were based on previous reports [2,32,33]. Both treatments were discontinued after 5 weeks of AAS or vehicle injections, totalizing 25 injections per animal. Rats were euthanized 23 days after the last day of treatment in both groups (P82) to assess the long-term effects of AAS administration during in the adulthood. The susceptibility to IR injury was evaluated in isolated hearts using a Langedorff apparatus. Biological samples of heart, lungs, liver and testis were collected to perform molecular and pathological analysis. 2.2. Ex vivo IR experiments Methods for isolated rat heart experiments were similar to those previously described [27,29]. Excised hearts were weighted and placed in modified Krebs-Henseleit solution (KHS) (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 10 mM glucose, 1.8 mM CaCl2, aerated with 95% O2 and 5% CO2). Aortas were hung on the cannula of a modified Langendorff apparatus and hearts were artificially perfused with modified KHS adjusted for pH 7.4 and 37  C at a constant flow of 10 mL/min. A latex balloon was placed into the left ventricle (LV) chamber through the mitral valve. Hearts were kept immersed in perfusion solution and baseline LV end-diastolic pressure (LVEDP) was set in 10 mmHg. Left ventricular developed pressure (LVDP), left ventricular systolic pressure (LVSP), left ventricular end-diastolic

2. Material and methods 2.1. Experimental protocol The present study was conducted according to the “Guide for the Care and Use of Laboratory Animals” (NIH Publication No. 85–

Fig. 1. Schematic diagram of the experimental protocol. Treatments with testosterone propionate or vehicle were started in postnatal day 26 (P26). At the end of treatments, both experimental groups underwent a washout period of 3 weeks until the postnatal day 82 (P82). At the P82, rats were submitted to euthanasia and the isolated heart experiments were performed.

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pressure, left ventricular maximal derivative of pressure (max. dP/ dt) and left ventricular minimal derivative of pressure (min. dP/dt) waveforms were recorded. After 20 min of baseline period, wherein heart rate, diastolic and systolic pressures were stable, the peristaltic pump was stopped and hearts were submitted to 30 min of global ischemia and subsequent 60 min of reperfusion. 2.3. Measurement of infarct size Infarct size was determined as previously described with modifications [34]: LV 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 5 min at 37  C. 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 using ImageJ software (NIH ImageJ: National Institute of Health, USA, version 1.22). Values were expressed in % of total area. 2.4. H2O2 generation Frozen LV tissues were homogenized in a buffer (50 mM sodium phosphate, pH 7.2, 0.25 M sucrose, 0.5 mM dithiothreitol, 1 mM EGTA, and protease inhibitors: 5 mg/mL aprotinin, and 34.8 mg/mL phenylmethylsulphonyl fluoride), centrifuged at 100 000  g, 35 min, 4  C, and suspended in a 50 mM sodium phosphate buffer, pH 7.2, containing 0.25 M sucrose, 2 mM MgCl2 and protease inhibitors. Protein content was determined by Bradford method [35]. Samples were incubated in 150 mM sodium phosphate buffer, pH 7.4, containing 100 U/mL superoxide dismutase (Sigma), 0.5 U/ mL horseradish peroxidase (Roche), 50 mM Amplex red (Molecular Probes) and 1 mM EGTA, with or without 1.5 mM CaCl2. Then 0.1 mg/mL NADPH was added and the fluorescence was immediately measured in a microplate reader (Victor X4; PerkinElmer) at 30  C (excitation: 530 nm; emission: 595 nm). H2O2 production was determined using standard calibration curves. Calciumdependent H2O2 generation was obtained by subtracting H2O2 generation in the presence of CaCl2 from that obtained in the absence of CaCl2. Specific enzymatic activity was expressed as nmols H2O2 h 1 mg 1 protein. 2.5. Quantitative RT-PCR Total RNA was extracted from LV tissue samples using RNeasy1 Fibrous Tissue Mini Kit (QIAGEN) and cDNA was prepared from 1 mg of total RNA using High-Capacity Reverse Transcription kit (ThermoScientific) according to the manufacturer’s instructions. mRNA levels of target genes (Supplementary Table S1 in the online version at DOI: http://dx.doi.org/10.1016/j.jsbmb.2017.01.012) were evaluated by qRT-PCR. The amplification reactions containing 1 ng of cDNA were performed at 60  C during the anneling and extension cycles. The expression of the chosen genes was normalized to GAPDH as an internal control. Primer sequences were described in Supplementary Table S1 in the online version at DOI: http://dx.doi.org/10.1016/j.jsbmb.2017.01.012. The relative quantities of mRNA were determined by 2 (DDCT)method and expressed as fold change of AAS group compared to the control group where CT is the “threshold cycle” determined for each plate by the 7500 real-time PCR system sequence detection software (ThermoScientific) [36]. Relative mRNA level of AAS group was expressed as the fold change compared to the control group.

2.6. Western blot Frozen LV samples were homogenized using TissueRupter (QIAGEN) and total protein was extracted using RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1% Triton X-100, 5 mmol L 1 EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM NaF) containing protease inhibitors (1 x complete protease inhibitors cocktail [Roche]). Protein concentrations of the lysates were determined using the BCA method. Proteins were separated on SDS-PAGE and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% non-fat dry milk and incubated with primary antibodies against AT1R (1:1000, sc-1173, Santa Cruz Biotechnology, Inc.), SUR2a (1:500, sc-32461, Santa Cruz Biotechnology, Inc.); Kir6.1 (1:200, sc-11228, Santa Cruz Biotechnology, Inc.) followed by incubation with fluorescent anti-goat secondary antibody (1:15000). Fluorescence was measured with Odissey Fc Imaging System (Licor). Densitometry was normalized using GAPDH as a loading control. 2.7. Statistical analysis Data are presented as mean  standard error of mean (S.E.M.). Normal statistical distribution of all data was determined Q-Q Plot test (Statext v2.7, http://www.statext.com/index.php). Repeatedmeasures two-way ANOVA followed by Bonferroni post-hoc test were used to analyze changes in LVDP, LVSP, LVEDP, maximal and minimal dP/dt over time (Prism 7.02, GraphPad Software Inc.). Infarct size, gene and protein expressions were analyzed using Student’s T Test. Statistical differences were considered significant when P < 0.05. 3. Results 3.1. Pathology Table 1 shows pathology results at the end of the washout period. Mean body weight was not statistically different between the experimental groups. AAS group showed a significant decrease in both absolute (P < 0.001) and relative (P < 0.001) testicular weight in comparison to the Control group, suggesting that AAS exposure resulted in persistent testicular atrophy. Absolute heart weight was significantly increased by AAS when compared to the vehicle treatment (P < 0.001). Given that the mean body weight did not differ between the experimental groups, the relative heart weight was significantly increased by AAS administration in comparison to the Control group (P < 0.001). Neither relative nor absolute weights of lung and liver were significantly different between the AAS and Control groups. Table 1 AAS exposure during adolescence promoted long-term cardiac hypertrophy and testicular atrophy in adult rats. Parameters

Initial body weight (g) Final body weight (g) Heart weight (mg) Relative heart weight (mg/g) Testicle weight (mg) Relative testicle weight (mg/g) Lung weight (mg) Relative lung weight (mg/g) Liver weight (mg) Relative liver weight (mg/g) Data are mean  SEM. N = 10–12/group. *** P < 0,001 vs. control.

Groups Control

AAS

102.9  0.9857 269.8  7.679 1.245  0.03879 4.733  0.1802 2.393  0.07700 8.892  0.2281 1.967  0.1283 7.274  0.3846 8.400  0.3891 31.33  1.552

102.6  1.009 265.0  8.966 1.658  0.08511*** 6.528  0.3550*** 1.727  0.03911*** 6.558  0.3369*** 2.058  0.1381 7.901  0.6309 8.758  0.5310 32.96  1.465

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3.2. Molecular characterization of cardiac hypertrophy To address the potential phenotype of the cardiac hypertrophy observed in the Section 3.1, we assessed the mRNA expression profile of genes that are commonly modified in the process of hypertrophy. Fig. 2 demonstrates the normalized mRNA expression levels of bMHC, aMHC, BNP, SERCA2a and RyR2, where the data related of AAS group were expressed as fold change of Control group. AAS exposure along the prepubertal/pubertal phase significantly increased the mRNA expression level of bMHC in approximately 2.25 fold compared to the values exhibited by the Control group (Fig. 2B, P < 0.01), whereas mRNA level of aMHC was not different between the experimental groups (Fig. 2A). As a consequence, the ratio of bMHC/aMHC was significantly increased in the AAS group (Fig. 2C, P < 0.001). The mRNA expression levels of BNP, RyR2 and SERCA-2a were not different between the AAS and Control groups (Fig. 2D–F).

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Table 2 Chronic exposure to AAS during adolescence promoted long-term down-regulation of myocardial catalase expression. Targets

Fold change

Catalase SOD1 SOD2 SOD3 Gpx1 Gpx3 AT1R Kir6.1 SURa Bcl2 Bax Bad

0,731071*** 0,769044 0,928141 1,121992 0,883311 0,681862 1.10 1.26 1.16 0.95 1.01 1.17

N = 5/group. *** P < 0,001 AAS vs. control.

3.4. Evaluation of myocardial susceptibility to IR injury 3.3. Expression of AT1 receptor and nox activity We assessed the expression of myocardial AT1 receptor and activity of Nox in order to investigate potential mechanisms associated with long-term cardiac hypertrophy exhibited by adult Wistar rats treated with AAS along prepubertal/pubertal phase. Quantitative RT-PCR analyses revealed that the mRNA expression level of AT1 receptor was not statistically different between the experimental groups (Table 2). Furthermore, AT1 receptor protein bioavailability also did not differ significantly, as evidenced by the Western blot analyses (Supplementary Fig. S1A and B in the online version at DOI: http://dx.doi.org/10.1016/j.jsbmb.2017.01.012). Nox activity was not statistically different between the experimental groups (Supplementary Table S2 in the online version at DOI: http://dx.doi.org/10.1016/j.jsbmb.2017.01.012), suggesting that this pathway is not correlated to persistent cardiac hypertrophy at the end of washout period.

Left ventricular mechanical performance is shown in Fig. 3, where parameters related to LV pressure were expressed in the baseline, ischemic and postischemic periods. Baseline recordings revealed that chronic exposure to AAS in the adolescence induced long-term impairment in the min. dP/dt when compared to Control group (Fig. 3E). Baseline LVDP (Fig. 3A), LVSP (Fig. 3B) and max. dP/dt (Fig. 3D) were not significantly different between the experimental groups. After the onset of global ischemia, LVEDP (Fig. 3C) gradually increased, indicating that ischemic hearts were developing a contracture state, although the amplitude of ischemic contracture was not different between the experimental groups (Fig. 3F). All parameters showed a notable improvement along postischemic period in relation to the ischemic period. However, AAS group showed significantly increased levels of LVEDP in comparison with the Control group, whereas LVSP was not significantly different. Consequently, the levels of LVDP were significantly

Fig. 2. AAS exposure during adolescence promoted long-term shift in the expression of MHC isoforms in adult rats. a-MHC (A), b-MHC (B), a-MHC/b-MHC (C), BNP (D), RyR2 (E) and SERCA2a (F) mRNA levels in Control group (circles, n = 5) and AAS group (squares, n = 5). MHC, myosin heavy chain; BNP, brain-derived natriuretic peptide; RyR2, ryanodine receptor; SERCA2a, sarcoplasmic reticulum calcium ATPase type 2a. Data are expressed as Mean  SEM. **P < 0.01 and ***P < 0.001.

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Fig. 3. Baseline and postischemic myocardial mechanical performance of adult rat hearts were impaired by AAS exposure during adolescence. Baseline, ischemic and postischemic left ventricle developed pressure (A, LVDP), left ventricle systolic pressure (B, LVSP), left ventricle end-diastolic pressure (C, LVEDP), maximal derivative of pressure (D, max. dP/dt), minimal dP/dt (E, min. dP/dt) and the amplitude of ischemic contracture (F) were measured in isolated rat hearts. Data are expressed as Mean  SEM. *P < 0.05, **P < 0.01 and ***P < 0.001.

decreased in the AAS group. Furthermore, AAS group also showed significantly decreased levels of max. dP/dt and min. dP/dt in comparison with Control group. Induction of IR resulted in the formation of infarcted tissue in both groups, as demonstrated in Fig. 4B. However, AAS group exhibited a significant increase in the percentage of infarct area related to total ventricular area compared to the Control group (Fig. 4A, P < 0.05). 3.5. Expression of proapoptotic and antiapoptotic molecules, antioxidant enzymes and KATP channel To address the potential mechanism involved in the increased susceptibility to myocardial IR injury showed by AAS group, we

assessed the expression of Bcl-2, Bax, BAD, antioxidant enzymes and KATP channel. As demonstrated in Table 2, relative mRNA expression levels of Bcl-2, Bax and Bad were not different between the experimental groups. Regarding antioxidant enzymes, AAS group exhibited a significant down-regulation in the expression of Catalase when compared to the Control group (0.7310 fold, P < 0.001). Conversely, the mRNA expression levels of Gpx1, Gpx3, SOD1, SOD2 and SOD3 were not statistically different between the experimental groups. Similarly, mRNA expression levels of KATP channel subunits, Kir6.1 and SURa were not statistically different between the experimental groups. Although SURa protein bioavailability was strongly down-regulated in the AAS group (Supplementary Fig. S1E-F in the online version at DOI: http://dx.doi.org/10.1016/j.jsbmb.2017.01.012, P = 0.0692), Western blot analyses revealed that Kir6.1 (Supplementary Fig. S1C-D in the online version at DOI: http://dx.doi.org/10.1016/j. jsbmb.2017.01.012) and SURa expressions were not significantly different between the experimental groups. 4. Discussion

Fig. 4. AAS administration during adolescence increased the infarct size in adult rat hearts after IR protocol. (A) Percentage of infarcted area in relation to total LV area of Control group (white boxes, n = 6) and AAS group (black boxes, n = 6). (B) Representative images of Control group (right) and AAS group (left). Data are expressed as Mean  SEM. *P < 0.05.

The current study was designed to analyze the long-term effects of chronic administration of AAS in the prepubertal/pubertal phases in the susceptibility to IR injury in adult rats, as well as to provide potential molecular evidence to support the observed cardiac effects. The major findings of this study was the existence of persistent cardiac abnormalities elicited by chronic administration of TP in the adolescence, resulting in impaired postischemic mechanical dysfunction and increased infarct size in adult life, even when the AAS administration were interrupted for 23 days. Long-term detrimental cardiovascular effects of AAS overdose were associated, at least in part, to the downregulation of catalase expression. As far as we know, this is the first report showing adult consequences of juvenile AAS administration in rats in the context of myocardial IR injury. In order to determine if the protocol used in the present study was efficient in establishing a condition of AAS overdose, testicular weight was analyzed at the end of the experimental protocol. As demonstrated in Table 1, testicular weight of the AAS group was

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significantly reduced in comparison to Control group, supporting our model of AAS overdose. Given that our protocol was effective to elicit AAS overdose, the next step was to evaluate whether chronic administration of TP promoted long-term cardiovascular abnormalities. Pathological analyses revealed that absolute and relative heart weights of the AAS group were significantly higher than the values from Control group. Interestingly, these findings suggest that AAS overdose promoted long-term cardiac hypertrophy, despite the interruption of AAS administration. Indeed, cardiac hypertrophy in conditions of AAS abuse has been frequently reported by clinical and post-mortem data and is characterized by thickening of LV posterior wall and interventricular septum, as well as diffuse or focal fibrosis [16,37–39]. Previous studies in rats reported increased heart weight, collagen deposition, cardiomyocyte hypertrophy, mitochondrial and sarcomeric injury, and microvascular rarefaction after chronic administration of AAS in adult phase [29,40–43]. Furthermore, Vasilaki et al. observed that rabbits chronically treated with high doses of stanozolol presented myocardial fibrosis after 4 months of wash-out period although no change in cardiac weight was found [44]. In conjunction, these findings support previous studies in which AAS abuse in humans were related to irreversible cardiac hypertrophy assessed by pathological and imaging methods [39,45,46], and add new insights into AAS consequences in adult rats treated during adolescence. Corroborating the pathological findings, AAS-induced hypertrophy was marked by a robust up-regulation in the levels of b-MHC mRNA when compared to the Control group. Since mRNA expression of aMHC was not altered, TP-treated group showed a significant increase in the bMHC/aMHC ratio compared to the Control group. As evidenced elsewhere, cardiac hypertrophy is characterized by changes in the pattern of MHC expression [47]. Specifically, pathological hypertrophy is marked by up-regulation of bMHC and bMHC/aMHC ratio in rat hearts, suggesting that TP administration induced long-term pathological hypertrophy, even though the levels of BNP, RyR2 and SERCA-2a did not change between groups [48]. Recently, the study of Pirompol and Teekabut demonstrated that hearts chronically exposed to AAS first developed cardiac hypertrophy characterized by increased aMHC expression and lack of fibrosis, consistent with a phenotype of physiological hypertrophy [49]. Overtime, cardiac hypertrophy turned into the phenotype of pathological hypertrophy. These evidences strongly suggest that AAS-induced cardiac hypertrophy in adolescent phase is marked by a shift in the hypertrophy phenotype and, as demonstrated by the present study, is persistent even after interruption of exposure in adult phase [44]. The underlying mechanisms correlating the administration of AAS to the development of cardiac hypertrophy are not clear. Previous studies suggested a prominent role of cardiac RAS in the development of detrimental effects induced by AAS. The upregulation of AT1 is a consequence of direct action of AAS in cardiomyocytes and not secondary to other systemic dysfunctions [50]. In addition, activity of cardiac angiotensin-converting enzyme is significantly increased after chronic administration of nandrolone [42]. Indeed, angiotensin II is thought to promote cardiac remodeling characterized by cardiomyocyte hypertrophy and increased collagen deposition independently of mechanical load. Blockade of AT1 receptor prevented the development of cardiac hypertrophy induced by chronic administration of AAS [27]. Nox-induced oxidative stress has been suggested to mediate the detrimental effects of AT1 receptor in the development of cardiac hypertrophy [51–53]. Furthermore, recent studies reported that rats chronically exposed to high doses of AAS exhibited increased expression of cardiac Nox and Nox activity [28,29]. Based on these evidences, we hypothesized that cardiac RAS was

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involved in the pathological cardiac hypertrophy induced by chronic TP exposure. Surprisingly, cardiac expression of AT1 receptor did not change between the experimental groups, as evidenced by mRNA and protein levels. Moreover, the levels of Nox activity were equivalent between the experimental groups. It is out of question that RAS exerts a pivotal role in the development of cardiac hypertrophy along the period of AAS administration. However, the current findings suggest that AT1 receptor was not up-regulated, at least after the interruption of AAS exposure, resulting in unaltered of Nox activity. Thus, other mechanism might be related to the progression of cardiac hypertrophy after interruption of AAS exposure, such as inflammatory cytokines and sympathetic activity [30,50]. From the clinical point of view, these pathophysiological singularities are especially important in the attempt to reverse the long-term cardiac hypertrophy in former AAS users, which might be resistant to AT1 receptor blockers therapy. In addition to myocardial remodeling, rats exposed to AAS developed long-term cardiac dysfunction. TP-treated rats exhibited impaired myocardial relaxation, as evidenced by the reduction in minimal dP/dt in the baseline period. Consistent with this finding, isolated papillary muscle of adult Wistar rats chronically exposed to AAS exhibited decreased minimal derivative of tension [49,54]. Conceptually, delayed myocardial relaxation is a primary cause of diastolic dysfunctions [55]. As a consequence, myocardial stiffness is significantly increased, resulting in inability of LV to accept an adequate volume of blood along the diastolic period. Together, these findings support the notion that chronic use of AAS is sufficient to promote long-term myocardial mechanical dysfunctions as evidenced in bodybuilders examined several years after the last AAS exposure [56]. During the ischemic period, both experimental groups exhibited a robust impairment of mechanical properties, such as downregulation of LVSP and progressive increase of LVEDP. After the onset of ischemia, the metabolic changes imposed to the heart induce a condition known as myocardial stunning, characterized by a significant impairment of mechanical performance, especially in the inotropic properties. This aspect is mainly explained by a drop in the ATP bioavailability, pH acidification and calcium overload [57–59]. In such a condition, reestablishment of blood flow is the most efficient therapeutic approach, as it restores the availability of metabolic fuels and the production of ATP, and also washes out the metabolic byproducts produced in the ischemic period [60,61]. However, reperfusion per se recruits several pathophysiological pathways that culminate in cell death and impede the complete recovery of mechanical performance [62]. The abrupt washout of the extracellular H+ promotes a robust increase in the extrusion of H+ ions by the Na+/H+ exchanger in order to reestablish the ion gradient, resulting in increased subsarcolemmal levels of sodium. The extrusion of Na+ ions by the Na+/Ca+2 exchanger increases and results in calcium overload and an intense myocardial contracture [63,64]. In this context, AAS significantly impaired the recovery of LVEDP and LVDP along the 60 min of reperfusion. At the end of reperfusion, LVEDP in the AAS group was approximately 26.68% higher than the control values, resulting in a decrease of 31.66% in LVDP versus the Control group. In addition, the kinetics of the cardiac contraction-relaxation cycle in the post-ischemic period was also impaired by AAS. At the end of reperfusion, the values of max. dP/dt and min. dP/dt of the AAS group were approximately 35.93% and 23.91% lower when compared to the Control group. In addition to mechanical impairment, hearts of AAS group exhibited an infarct size approximately 46% higher than the Control group. These evidences support previous findings that chronic administration of AAS significantly impairs the post-ischemic recovery of

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myocardial mechanical properties and the cell survival [26]. Furthermore, to the best of our knowledge, this is the first evidence that AAS promote long-term increase in the susceptibility to myocardial IR injury. The molecular mechanisms associated with the increased susceptibility to myocardial IR injury elicited by AAS are complex, especially the long-term pathways. In this condition, calcium overload activates several downstream pathways involving the down-regulation of Bcl-2 and up-regulation of Bax and Bad, resulting in opening of the mitochondrial permeability transition pore (MPTP), cytochrome c release and apoptosis [65,66]. Molecular analyses revealed that both endogenous and exogenous testosterone elevated the levels of proapoptotic protein BAX in vivo and in vitro, whereas Bcl-2 was down-regulated [67,68]. In contrast, the current findings demonstrate the chronic administration of AAS did not change the level of Bcl-2, BAX and BAD mRNA expression in comparison with the Control group. However, further analyses are needed in order to evaluate the level of protein expression and phosphorylation of these molecules. Recently, Marques-Neto et al. reported that chronic AAS exposure significantly decreased the expression of KATP channel subunits, resulting in blunted cardioprotection elicited by chronic treadmill exercise and increased susceptibility to myocardial IR injury in rats [27]. Sarcolemmal KATP channel exerts a pivotal role in the cardiac resistance to IR injury by reduction of calcium overload, whereas mitochondrial KATP channel activation results in delayed opening of MPTP during ischemia and reperfusion [69– 72]. Although the evidences aforementioned highlight that the down-regulation of myocardial KATP supports a pivotal role in the loss of exercise-induced cardioprotection during AAS administration, it does not seem to be the case in the context of long-term effects of AAS. Consistent with this hypothesis, Marques-Neto et al. demonstrated that blockade of AT1R prevented the downregulation of KATP channel, suggesting that AAS-induced RAS hyperactivity negatively modulates the KATP channel [27]. Given that AT1R expression was not altered in the present study, these findings suggest that the RAS—KATP pathway progressively returns to the physiological level and might not be involved with the longterm effects of AAS in the pathophysiological progression of myocardial IR injury. The involvement of ROS in the pathophysiology of IR injury has been extensively studied. At physiological levels, ROS exert essential roles in the maintenance of cell homeostasis by several signaling pathways [73,74]. In fact, moderately and punctual bursts of ROS exert beneficial effects in cardioprotection against IR injury [75]. However, high bioavailability of ROS provokes damage in several cell structures, such as mitochondria, plasma membrane and DNA [76]. Furthermore, ROS increase the likelihood of MPTP opening and release of proapoptotic molecules [77]. Endogenous testosterone per se reduces mRNA expression of SOD2 (or Mn-SOD), an enzyme that catalyzes the conversion of superoxide anion to the less reactive H2O2 [68]. Consonant with this aspect, administration of stanozolol promoted long-term increase in the level of myocardial malodyaldehyde, a lipid peroxidation marker [78]. Furthermore, Chaves et al. previously demonstrated that the increased susceptibility to myocardial IR injury in AAS-treated rats was correlated with down-regulation of glutathione reductase, a critical component of the antioxidant machinery that degrades H2O2 [29]. In the current study, chronic administration of AAS promoted long-term down-regulation in the gene expression of catalase, compared to the control group, although SOD1-3 and Gpx1-3 did not differ. Catalase exerts a pivotal role in the control of ROS bioavailability by catalyzing the conversion of H2O2 to H2O and O2. Isolated hearts of mice overexpressing catalase exhibit a reduction of approximately 40% in the infarct size after IR, whereas mechanical performance is

improved 300%, compared to wild-type [79]. Taken together, these findings suggest that AAS overdose impairs the expression of antioxidant machinery even after the interruption of drug administration, resulting in long-term increase in the susceptibility to redox unbalance and myocardial IR injury. The present findings demonstrated that chronic administration of TP promoted long-term pathological hypertrophy and impaired baseline diastolic properties in isolated hearts. The susceptibility to myocardial IR injury was significantly increased by exposure to TP, resulting in increased infarct size and impaired post-ischemic mechanical performance. Molecular evidences support a potential role of redox unbalance, since the expression of catalase mRNA was decreased in the AAS group. In conjunction, these findings suggest that chronic administration of AAS might worsen myocardial infarction prognosis and reduces the efficacy of therapeutic maneuvers. Further analyses are necessary in order to demonstrate if the impaired therapeutic efficacy of cardioprotective maneuvers remains after the interruption of AAS exposure. 4.1. Limitations Although case reports and post-mortem studies have reported AMI in AS abusers and the National Institute of Drug Abuse recognized AMI as a severe consequence of this condition, there are no epidemiological surveys investigating the prevalence of AMI and coronary dysfunction among AS abusers. In addition, we studied the pathophysiological features of myocardial IR injury in an ex vivo model in order to investigate the intrinsic cardiac abnormalities. However, myocardial IR injury can be affected by several systemic inputs that were not considered in the present study, such as autonomic nervous system activity. In the present study, expressions of antioxidant enzymes, BNP, RyR, MHC isoforms, Bax, Bcl-2 and Bad were analyzed by quantitative RT-PCR, which inform just the level of mRNA expression, whereas protein bioavailability was not evaluated. Furthermore, although we observed that catalase expression was down-regulated in AAS group, the functional significance of this abnormality was not investigated. Conflict of interest None declared. Source of funding The present work was supported by grants from the National Research Council (CNPq—Brazil) and the Rio de Janeiro State Research Agency (FAPERJ). References [1] D. Sagoe, C. Schou Andreassen, S. Pallesen, The aetiology and trajectory of anabolic- androgenic steroid use initiation: a systematic review and synthesis of qualitative research, Substance (2014) 1–14, doi:http://dx.doi.org/10.1186/ 1747-597X-9-27. [2] J. Cohen, R. Collins, J. Darkes, D. Gwartney, A league of their own: Demographics, motivations and patterns of use of 1,955 male adult nonmedical anabolic steroid users in the United States, J. Int. Soc. Sports Nutr. 4 (2007) 1–14, doi:http://dx.doi.org/10.1186/1550-2783-4. [3] W.E. Buckley, C.E. Yesalis, K.E. Friedl, W.A. Anderson, A.L. Streit, J.E. Wright, Estimated prevalence of anabolic steroid use among male high school seniors, JAMA 260 (1988) 3441–3445. (Accessed 16 August 2016). http://jama. jamanetwork.com/article.aspx?articleid=375628. [4] C.E. Yesalis, N.J. Kennedy, A.N. Kopstein, M.S. Bahrke, Anabolic-androgenic steroid use in the United States, JAMA 270 (1993) 1217–1221. (Accessed 16 August 2016) http://jama.jamanetwork.com/article.aspx?articleid=408303. [5] M.S. Bahrke, C.E. Yesalis, K.J. Brower, J. Kirk, Anabolic-androgenic steroid abuse and performance-enhancing drugs among adolescents, Child Adolesc. Psychiatr. Clin. N. Am. 7 (1998) 821–838. (Accessed 16 August 2016) http:// psycnet.apa.org/psycinfo/1998-12461-008.

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