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reperfusion injury in aged rats
Journal of Steroid Biochemistry and Molecular Biology 191 (2019) 105335
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Paradoxical effect of testosterone supplementation therapy on cardiac ischemia/reperfusion injury in aged rats
T
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Fernando A.C. Searaa,c, ,1, Raiana A.Q. Barbosaa,1, Marcus V.N. Santosa, Ainá E. Domingosa, Gustavo Monnerata, Adriana B. Carvalhoa, Emerson L. Olivaresc, José G. Millb, Jose H.M. Nascimentoa, Antonio C. Campos de Carvalhoa,d a
Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES, Brazil c Department of Physiological Sciences, Federal Rural University of Rio de Janeiro, Seropedica, RJ, Brazil d National Center for Structural Biology and Bioimaging, Federal University of Rio de Janeiro, RJ, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Testosterone Aging Myocardial infarction Ischemia Reperfusion
Aging is followed by numerous physiological limitations that reduce health span, particularly cardiovascular and metabolic disorders. Testosterone supplementation therapy (TST) has been widely used in the treatment of aging dysfunctions in either adult or aged patients, although recent evidence have suggested that the incidence of myocardial infarction might be increased in elderly patients. So far, though, the effects of TST in the progression of cardiac ischemia/reperfusion (IR) injury in aged hearts remain unclear. Male aged (23–24 months old) and adult (6 months old) Wistar rats were treated with placebo (Old + Placebo n = 5 / Adult + Placebo n = 5) or TST (Old + TST n = 7 / Adult + TST n = 5) for 30 days. After euthanasia, artificially-perfused isolated rat hearts were submitted to IR. Cardiac expression levels of genes encoding α and β myosin heavy chain (MHC), ryanodine receptor (RyR), brain-natriuretic peptide (BNP), sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), glucose-regulated protein 78 kDa (GRP78), eukaryotic initiation factor 2α (eIF2α), C/EBP-homologous protein (CHOP), caspase 3 and B cell lymphoma 2 (Bcl-2) were accessed by qRT-PCR. Protein levels of CHOP, p-Akt, and p-glycogen synthase kinase 3β (p-GSK-3β) were measured by Western Blot. Compared to placebo-treated aged rats, Old + TST group exhibited increased heart weight and up-regulation of αMHC mRNA expression levels, whereas βMHC mRNA expression (p < 0.05). During reperfusion, left ventricular developed pressure, dP/dt+, dP/dt-, and cardiac contractile function index were increased in Old + TST rat hearts (p < 0.05), whereas infarct size was increased (p < 0.05) in comparison with Old + Placebo group. p-Akt levels of Old + TST rat hearts were decreased when compared to Old + Placebo group. Conversely, TST did not promote significant effects in adult rat hearts. Taken together, these findings suggest that myocardial stunning and infarct size of aged hearts were distinctly affected by TST.
1. Introduction Life expectancy has rapidly increased worldwide. Recently, the World Health Organization estimated 617 million people aged 65 and older, which accounts for 8.5% of the global population [1]. Moreover, it is estimated that 20% of the global population will be 65 and over by 2030 [2]. However, the increase in life expectancy has not been followed by a proportional increase in healthspan [2]. Biological senescence is characterized by time-related deterioration of several physiologic mechanisms necessary for homeostatic maintenance, resulting in
allostatic overload and development of a disease. Most aging-associated physiological limitations include muscle and bone waste, cognitive decline, sexual dysfunction and cardiovascular and metabolic disorders [3]. In the search for new methods to improve healthspan during aging, the physiologist Charles Édouard Brown-Séquard reported an improvement in his physical and cognitive performances after injections of testicular extracts, which he named the elixir of life [4]. Although most of these findings have later been proven to be placebo effects, testosterone, the main androgen hormone produced by male gonads,
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Corresponding author. E-mail address: [email protected] (F.A.C. Seara). 1 These authors had an equivalent contribution. https://doi.org/10.1016/j.jsbmb.2019.03.012 Received 24 January 2019; Received in revised form 18 March 2019; Accepted 19 March 2019 Available online 28 March 2019 0960-0760/ © 2019 Elsevier Ltd. All rights reserved.
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testosterone propionate (TP, Hertape Carlier® Hertape Carlier Animal Health Laboratory) at 1 mg.kg-1 daily for 30 days, or an equivalent volume of vehicle (peanut oil and benzyl alcohol, 90:10, v/v). The animals were divided into two experimental groups randomly treated with vehicle (Old + Placebo, N = 5) or TST (Old + TST, N = 7). The susceptibility to cardiac IR injury was assessed in isolated hearts using a Langendorf apparatus after the 30 days. Hearts were weighted to estimate pathological abnormalities and heart samples were stored at -80 °C. In a separate cohort, adult rats (6 months old) were submitted to placebo (Adult + Placebo, n = 5) or TST (Adult + TST, n = 5) treatments and to the same experimental protocols of aged rats. All data of adult rats are shown in the supplemental material.
has arisen as a potential therapeutic intervention to treat health disabilities in male elderly patients, either as replacement or supplementation therapies. Testosterone supplementation therapy (TST) has been used in the treatment of obesity, cachexia, lack of libido and infertility, and to improve erectile dysfunction, mood and cognitive disorders, and overall well-being in patients without hypogonadism [5–10]. Since testosterone promotes numerous effects on the heart, cardiovascular outcomes have been under intense investigation. It has been demonstrated that eugonadal elderly patients with chronic heart failure have experienced improvements in peak oxygen consumption, muscle strength, insulin sensitivity, and baroreflex control of the heart when treated with TST [11–15]. Noteworthy, heart failure symptoms can be reduced by at least one functional class in patients under testosterone supplementation [16]. On the other hand, the use of TST has been challenged by clinical evidence associating this therapeutic intervention with increased cardiovascular risk, including cerebrovascular and ischemic heart diseases [17,18]. It has been reported that administration of testosterone and other anabolic steroids can increase the susceptibility to cardiac ischemia/reperfusion (IR) injury, either in eugonadal or hypogonadal rats, while the efficacy of cardioprotective maneuvers is blunted [19–23]. These evidences have suggested that not only the risk for myocardial ischemia is increased, but post-ischemic recovery of cardiac function might be impaired by TST. This is particularly important in the case of elderly patients since aging per se is a dominant risk factor for cardiovascular dysfunction. Indeed, the prevalence of cardiovascular diseases among elderlies can be significantly enhanced after chronic testosterone supplementation [24]. However, the effects of TST in the progression of IR injury in aged hearts remain largely unknown. During prolonged periods of IR, cardiomyocyte survival depends mostly upon the balance between pro-apoptotic and anti-apoptotic downstream signaling. Decreased Akt phosphorylation and activation has been proposed as a key pathophysiological mechanism whereby testosterone impairs cardiomyocyte survival in adult hearts [25]. In addition, recent studies have demonstrated that testosterone regulates cell survival by recruitment of the endoplasmic reticulum (ER) stressactivated unfolded protein response (UPR) [26–28]. Chronic UPR activation promotes cardiac hypertrophy, cardiomyocyte apoptosis, increased susceptibility to cardiac IR injury, and heart failure [29–32]. So far, though, there is no evidence of whether ER stress and UPR can be modulated by TST, either in adult or aged hearts. Despite the growing use of TST, particularly by elderlies, there are few studies about the effects of TST on cardiovascular homeostasis in more advanced ages, and most reports have focused on anabolic steroid overdose or testosterone replacement in adult rats. In the present study, we investigated the effects of TST in the progression of cardiac IR injury in aged rat hearts.
2.3. Ex vivo experiments All methods for isolated heart experiments were based on previous studies from our laboratory [19]. Isolated hearts were connected to a Langendorff apparatus and retrogradely perfused with modified KrebsHenseleit solution (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, saturated with 95% O2 and 5% CO2) adjusted for pH 7.4 and 37 °C at constant flow (10 ml/min). A latex balloon was placed inside the left ventricle cavity through an incision in the left atrial appendage. Baseline left ventricular (LV) end-diastolic pressure (LVEDP) was set at 10 mmHg. After stabilization of heart rate and systolic and diastolic pressures, perfusion was interrupted, and hearts were submitted to 30 min of global ischemia, and subsequent 60 min of reperfusion. LVEDP, LV systolic pressure (LVSP), LV developed pressure (LVDP), maximal (dP/ dt+) and minimal (dP/dt-) pressure derivatives, and contractile function index (CI) waveforms were recorded with a pressure transducer (PT 300, Grass Technologies). The transducer was connected to an amplifier (ML 110 ADInstruments), and all signals were converted to digital data by an analogic/digital converter (PowerLab 400, ADInstruments). All recordings were digitized and stored on a computer for posterior analysis (LabChart 7.0, ADInstruments). 2.4. Measurement of infarct size
2. Material and methods
Atria were discarded and ventricles were sliced into approximately 1.5 mm thick section from apex to base. A small piece of the ventricle apex was stored for future biochemical and molecular analyses. All other slices were incubated in 1% triphenyltetrazolium chloride in phosphate buffer (pH 7.4) for 5 min at 37 °C. All ventricular slices were incubated in 10% (v/v) formaldehyde solution for 24 h to improve the 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 as a percentage of total ventricular area, as previously described [19].
2.1. Animals
2.5. Quantitative polymerase-chain reaction (qRT-PCR)
This study followed the standards and ethical guidelines of the Ethics Committee for Research of the Federal University of Rio de Janeiro, and it was approved by the commission under the number 023/14. All standards proposed by the Guide for the Care and Use of Laboratory Animals (U.S. National Institute of Health (NIH) Publication No. 85-23, revised 1996) were observed.
Total RNA was extracted from LV apex tissue using RNeasy® Fibrous Tissue Mini Kit (QIAGEN), and cDNA was prepared from 1 μg of total RNA using High-Capacity Reverse Transcription Kit (Thermo Fisher Scientific), following manufacturer’s instructions. Messenger RNA (mRNA) levels of sarcoplasmic reticulum calcium ATPase (SERCA2a), ryanodine receptor (RyR), myosin heavy chain isoforms α (αMHC) and β (βMHC), brain natriuretic peptide (BNP), glucose-regulated protein 78 (GRP78), eukaryotic translation initiation factor 2A (eIF2A), CCAAT-enhancer-binding protein homologous protein (CHOP), Bcl-2 and caspase 3 were measured by qRT-PCR. Amplification reactions containing 5 ng of cDNA were performed at 60 °C during annealing and extension cycles. The expression was normalized to glyceraldehyde-3phosphate dehydrogenase (GAPDH) as an internal control. The
2.2. Experimental protocol Male aged (23–24 months-old) Wistar rats were housed in cages under controlled temperature (21 ± 2 °C), daily exposed to 12-hour light-dark cycle (lights off at 7:00 pm), and water and standard chow ad libitum. TST administration consisted of subcutaneous injections of 2
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increased heart weight relative to tibia length (Fig. 1A, HW/TBL, p < 0.05), consistent with cardiac hypertrophy. Decreased mRNA expression of αMHC (Fig. 1B, p < 0.05), increased βMHC mRNA expression levels (Fig. 1C, p < 0.05), and decreased SERCA-2a mRNA expression levels (Fig. 1F, p < 0.05) were also observed. Neither BNP (Fig. 1D), nor RyR (Fig. 1E) mRNA expression levels were significantly affected TST (p > 0.05).
quantification of selected mRNA was determined by 2-(ΔΔCt) method in a Viia7 Software v1.2.4 (Thermo Fisher Scientific) and expressed as fold change compared to Old + Placebo group. 2.6. Western blot 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 EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM NaF) supplemented with complete protease inhibitor cocktail (Roche), and complete phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich). Protein concentration was determined by BCA method (Bio-Rad Protein Assay, Bio-Rad), according to manufacturer’s instruction. Proteins were separated on SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes with a wet transfer system (BioRad). PVDF membranes were incubated in 5% non-fat dry milk diluted in Tris-buffered saline with 0.1% Tween® 20 (TBST) for 2 h at room temperature. After blocking, all PVDF membranes were incubated with primary antibodies against p-Akt (1:1000, Cell Signalling, Inc.), C/EBP homologous protein (CHOP, 1:500 Santa Cruz, Inc), phosphorylated glycogen synthase kinase 3 beta (p-GSK3β, 1:500, Cell Signalling, Inc.) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:2000, Santa Cruz, Inc), followed by incubation with anti-rabbit secondary antibody (1:5000, Cell Signalling). The signals were detected using an enhanced chemiluminescence kit (ECL Prime, GE Healthcare), with Odissey Fc Imaging System (Licor), and were analyzed using ImageJ. Densitometry was normalized using GAPDH as a loading control.
3.2. TST did not affect baseline LV performance nor ischemic contracture Pre-ischemic LV function is demonstrated in Fig. 2. LVSP (Fig. 2A), LVDP (Fig. 2B), dP/dt+ (Fig. 2C), dP/dt- (Fig. 2D), and CI (Figure E) levels were equivalent between Old + Placebo and Old + TST groups (p > 0.05). The amplitude of ischemic contracture was equivalent between Old + Placebo and Old + TST groups (p > 0.05).
3.3. Post-ischemic recovery of LV hemodynamic properties of aged rat hearts was ameliorated by TST Post-ischemic LVSP (Fig. 3A) and LVEDP (Fig. 3B) levels were equivalent (p > 0.05), whereas LVDP (Fig. 3C), dP/dt+ (Fig. 3D), dP/ dt- (Fig. 3E), and CI (Fig. 3F) of Old + TST group were increased when compared to Old + Placebo group (p < 0.05). Infarct size (Fig. 4) was significantly increased in Old + TST rat hearts in comparison with Old + Placebo hearts (p < 0.05).
3.4. p-Akt, but not p-GSK3β or ER stress levels, was attenuated by TST in aged hearts
2.7. Statistical analysis Data are presented as mean ± standard error of the mean (S.E.M.). Student’s t-test was used to compare the means of Placebo versus TST groups. Two-way ANOVA followed by Sidak post-hoc test (Prism 6.00, GraphPad Software) were used for temporal analyses of ex vivo data. Statistical differences were considered significant when P < 0.05.
Cardiac levels of p-Akt were significantly decreased in Old + TST versus Old + Placebo group (Fig. 5A, p < 0.05), whereas p-GSK3β levels were not significantly different (Fig. 5B, p > 0.05). Furthermore, caspase 3 mRNA levels tended to increase in Old + TST versus Old + Placebo group but did not reach statistical significance (Fig. 5C, p = 0.1). Bcl-2 mRNA levels were similar in both groups (Fig. 5D, p > 0.05). Compared to Old + Placebo group, Old + TST hearts exhibited equivalent mRNA levels of GRP78 (Fig. 6A), CHOP (Fig. 6B), and eIF2α (Fig. 6C), and equivalent protein levels of CHOP (Fig. 6D) (p > 0.05).
3. Results 3.1. TST promoted cardiac hypertrophy in aged rats Compared to Old + Placebo group, Old + TST rats exhibited
Fig. 1. TST promoted cardiac hypertrophy in aged rats. (A) Heart weight (HW) relative to tibia length (TBL), and mRNA expression levels of (B) αMHC, (C) βMHC, (D) BNP, (E) RyR, and (F) SERCA-2a were compared between Old + Placebo (White box, n = 5) and Old + TST (Black box, n = 7) groups. Data are expressed as mean ± S.E.M. *p < 0.05 versus Old + Placebo group. 3
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Fig. 2. Baseline LV function and amplitude of ischemic contracture were not affected by TST. LVSP (A), LVDP (B), dP/dt+ (C), dP/dt- (D), CI(E), and the amplitude of ischemic contracture (F) were compared between Old + Placebo (White box, n = 5) and Old + TST (Black box, n = 7) groups. Data are expressed as mean ± S.E.M.
Fig. 3. Post-ischemic recovery of LV function was improved by TST. LVSP (A), LVEDP (B), LVDP (C), dP/dt+ (D), dP/dt- (E), and CI (F) were compared between Old + Placebo (n = 5) and Old + TST (n = 7) groups during cardiac ischemia and reperfusion. Data are expressed as mean ± S.E.M. *p < 0.05 and **p < 0.01 versus Old + Placebo group.
CHOP (Figure S6C), nor protein levels of CHOP (Figure S6D) of adult hearts were affected by TST.
3.5. Effects of TST in in adult rats In adult hearts, HW/TBL (Figure S1A) and mRNA expression levels of αMHC, βMHC, RyR, BNP, and SERCA-2A (Figure S1B-S1 F) were equivalent between Adult + Placebo and Adult + TST groups (p > 0.05). Baseline LV function (Figure S2A-S2E) and the amplitude of ischemic contracture (Figure S2F) were equivalent between Adult + Placebo and Adult + TST (p > 0.05) as well. There was no significant difference in cardiac function (Figure S3A – Figure S3 F) during reperfusion period or in infarct size (Figure S4) between Adult + Placebo and Adult + TST groups (p > 0.05). Levels of p-Akt (Figure S5A) and p-GSK3β (Figure S5B) of adult hearts were not affected by TST in comparison with placebo (p > 0.05). Caspase 3 and Bcl-2 mRNA levels were equivalent between Adult + Placebo and Adult + TST groups (Figure S5C and S5D, p > 0.05). In addition, neither mRNA levels of GRP78 (Figure S6A), eIF2α (Figure S6B), and
4. Discussion This report provides experimental evidence that TST distinctly affected the susceptibility to cardiac IR injury in adults and aged rats. As far as we know, this is the first report on the effects of TST in the susceptibility of aged hearts to cardiac IR injury. Despite its therapeutic benefits, anabolic steroids (AS) can promote adverse cardiac remodeling depending on the dose regimen and AS compound. At supraphysiological doses, AS can promote cardiomyocyte death and fibrosis [33]. Moreover, previous reports have demonstrated that chronic testosterone propionate overdose can promote cardiac hypertrophy early after exposure, that can persist even after AS discontinuation [19,34]. Using supplementation protocols, however, 4
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cypionate [36]. This is particularly important for elderly patients, who exhibit a higher prevalence of risk factors for cardiac hypertrophy when compared to adults. Adult rats treated with testosterone supplementation, on the other hand, did not exhibit signs of cardiac hypertrophy, suggesting that aged hearts are more susceptible to hypertrophic stimuli elicited by testosterone [34]. When analyzed for baseline LV hemodynamic properties, Old + TST group performed equally to Old + Placebo group. These findings imply that LV function of Old + TST group was preserved despite cardiac hypertrophy, at least under ex vivo assessment. Longer treatment periods than used in the present study can result in deterioration of LV function, even with testosterone replacement therapeutic protocols. Similarly, baseline LV hemodynamic performance of adult rat hearts was not significantly affected by TST [37]. In adult rats, longer supplementation with TP has been reported to increase overall LV contractile function [38]. However, it must be noted that testosterone can impair LV function by extrinsic mechanisms, such as autonomic imbalance. Sympathetic hyperactivity and down-regulation of parasympathetic activity have been largely reported during chronic administration of anabolic steroids [39]. These abnormalities have long been associated with increased susceptibility to LV electrophysiological and mechanical dysfunctions in the progression of cardiovascular diseases, particularly in advanced ages [40]. As expected, LV contractile function ceased after the onset of global ischemia and amplitude of ischemic contracture progressively increased in all groups, a condition termed stone heart [41]. Indeed, contractile properties can be impaired as early as 10 s after the onset of ischemia, according to the landmark study by Katz and Tada [42]. Ischemic contracture depends mostly upon ATP levels and the shift towards glycolytic metabolism [43]. The resultant cytosolic acidification is counter-balanced by Na+/H + exchanger, whereby H + is transported to the extracellular milieu in exchange for Na+. This, in turn, stimulates Na + transport to the extracellular milieu in exchange for Ca+2 by the Na+/Ca+2 exchanger, resulting in up-regulation of cytosolic
Fig. 4. Infarct size was increased by TST. (A) Percentage infarct size relative to ventricular area was determined after global ischemia/reperfusion in Old + Placebo (White box, n = 5) and Old + TST (Black box, n = 7) groups. (B) Representative slices of infarcted hearts. Data are expressed as mean ± S.E.M. *p < 0.05 versus Old + Placebo group.
there are few pieces of evidence on the repercussion of testosterone treatment, particularly in aged hearts. We found that TST promoted hypertrophy in aged rats. Furthermore, TST also decreased αMHC mRNA levels, while βMHC was up-regulated in the aged animals. In rodents, increased HW and βMHC/αMHC ratio are consistent with pathologic cardiac hypertrophy [35]. In agreement with these findings, previous reports have suggested that aged rabbits can also exhibit cardiac hypertrophy after chronic supplementation with testosterone
Fig. 5. p-Akt but not p-GSK3β levels of aged hearts were decreased by TST. Levels of pAkt (A, n = 4/group) and p-GSK3β (B, n = 3/ group) were compared between Old + Placebo (White box) and Old + TST (Black box) groups. Representative bands of p-Akt and p-GSK3β are demonstrated above each respective graph. Caspase 3 (C) and Bcl-2 (D) mRNA expression levels were compared between Old + Placebo (n = 5) and Old + TST groups (n = 7). Data are expressed as mean ± S.E.M. *p < 0.05 versus Old + Placebo group.
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Fig. 6. TST did not promote ER stress in aged hearts. mRNA expression levels of GRP78 (A), CHOP (B), and eIF2α were compared between Old + Placebo (White box, n = 5), and Old + TST (Black box, n = 7) groups. Protein levels of CHOP (D) were measured by Western Blot (n = 4/group), and representative bands are exhibited in E. Data are expressed as mean ± S.E.M.
androgen receptor antagonists or testosterone deficiency might prevent LV dysfunction during IR in adults [25], our findings suggest that TST does not affect post-ischemic LV hemodynamic properties or cardiomyocyte integrity of adult hearts. In contrast, previous reports have demonstrated that testosterone replacement can provide cardioprotection in adult rat hearts [52–55]. Taken together, these findings suggest that testosterone supplementation and replacement induce distinct effects in the progression of cardiac IR injury. Additionally, previous evidence has suggested that testosterone can impair cardiomyocyte survival during IR by decreasing the activation of anti-apoptotic signaling, at least in adult rat hearts. Accordingly, either castration or administration of androgen receptor blockers enhances Akt phosphorylation, and thereby activation of anti-apoptotic signals during cardiac reperfusion [25]. Furthermore, down-regulation of pAkt levels during cardiac reperfusion was reported after chronic overdose with nandrolone decanoate, a class II anabolic steroid [20]. Downregulation of p-Akt levels has likewise been correlated with a worsened renal ischemic injury in testosterone-treated mice [56]. We found that p-Akt levels of aged hearts were down-regulated in Old + TST rat hearts in comparison to Old + Placebo, whereas Adult + TST levels were not affected when compared to Adult + Placebo. Decreased Akt activation has long been correlated with impaired cardiomyocyte survival during reperfusion [57]. Numerous evidences have demonstrated that Akt-induced cardioprotection involves phosphorylation, and thereby inactivation of GSK3β. However, p-GSK3β levels were equivalent among all experimental groups, suggesting that down-regulation of p-Akt might have affected cardiomyocyte survival by other signaling pathways [25]. Caspase 3 mRNA levels exhibited a trend toward up-regulation in Old + TST group when compared to Old + Placebo group, suggesting that caspase 3 might be involved in the decreased cell viability after reperfusion in Old + TST group. Penna et al. reported similar findings during the administration of nandrolone decanoate to adult rats, i.e., decreased levels of cardiac p-Akt and unchanged p-GSK3β levels after IR, followed by increased infarct size [20]. Recently, it has been reported that cardiomyocyte survival during IR can be modulated by ER homeostasis [29,32]. UPR activation can affect cardiomyocyte apoptosis by several mechanisms that concatenate into cell survival or death. GRP78 is a chaperone protein located within ER lumen that binds to newly correctly synthesized proteins during protein folding, as well as to abnormal proteins targeted for degradation [58].
calcium concentration and contracture [44]. This is further aggravated by down-regulation of Ca+2 transport into SR by SERCA-2a, and decreased actomyosin turnover following ATP depletion. We found that the amplitude of ischemic contracture was equivalent between Placebo versus TST groups. However, Callies et al. demonstrated that cytosolic calcium concentration at the end of myocardial ischemia was significantly decreased after just one injection of testosterone undecanoate in eugonadal rats, which was administered two weeks before the induction of IR [45]. It remains elusive, however, whether down-regulation of cytosolic calcium by testosterone in this period would be enough to decrease the amplitude of cardiac ischemic contracture. LV function was just partially restored after the onset of reperfusion, in keeping with the reports of Braunwald and Kloner [46]. Although naturally reversible, myocardial stunning can promote hemodynamic instability and might contribute to the development of pulmonary edema and cardiogenic shock after IR. In severe cases, myocardial stunning can be treated with inotropic agents. Whereas most reports have demonstrated the effects in adult hearts, the repercussion in aged hearts was unclear so far. Old + TST rat hearts exhibited increased LVDP, CI, dP/dt+, and dP/dt- during reperfusion when compared to Old + Placebo group, clearly indicating that myocardial stunning was ameliorated in some degree by TST. Somewhat surprisingly, infarct size was significantly increased in Old + TST rat hearts when compared to Old + Placebo group. Interestingly, TST was shown to promote ambivalent cardiovascular and metabolic effects in adult and aged rats during the progression of systemic hypertension [47]. However paradoxical as it might seem, clinical and experimental reports have demonstrated similar findings in conditions of increased workload and energy demand, resulting in infarct expansion, such as after administration of inotropic drugs [48,49]. Since aged hearts have impaired adaptation capacity to conditions of energetic imbalance, such as those imposed by IR [50,51], this condition might be further aggravated by the increased hemodynamic stress and energetic demand promoted by TST, since contractile properties in TST aged hearts were increased during ex vivo reperfusion. These distinctions may explain in part why Old + TST exhibited increased infarct size when compared to Old + Placebo. This finding also raises the question of whether the down-regulation of cardiac contractile function during reperfusion would be an allostatic mechanism to preserve cardiomyocyte integrity in aged hearts. On the other hand, infarct size was equivalent between Adult + Placebo and Adult + TST. Thus, although 6
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Since GRP78 is a master regulator of ER stress and its levels are remarkably increased under conditions characterized by the accumulation of unfolded proteins within ER lumen, GRP78 has been used as a biomolecular marker of ER stress [58]. Surprisingly, no evidence on whether testosterone can modulate cardiac ER stress and UPR in cardiomyocytes has been hitherto reported. Herein, we found that expression levels of genes encoding GRP78, eIF2α, and CHOP were not affected by TST, neither in adult nor in aged hearts. Additionally, CHOP protein levels, the main pro-apoptotic signal evoked during UPR, were not affected by TST as well, when compared with placebo. These findings imply that most of the effects of TST in the progression of cardiac IR injury do not involve significant changes in the ER stress levels or UPR. Taken together, the present findings demonstrated that TST promoted ambivalent effects during IR in aged rat hearts, while adult hearts were not significantly affected. TST promoted cardiac hypertrophy without significant LV hemodynamic repercussions in aged hearts. Ex-vivo post-ischemic recovery of LV contractile function of aged rat hearts was enhanced by TST, whereas infarct size was increased. Aged hearts exposed to TST exhibited decreased p-Akt levels, though p-GSK3β levels were not affected. Additional studies will be necessary to investigate why aged hearts were more susceptible to TST effects.
[10]
[11]
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Funding
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This study was supported by the Department of Science and Technology - Brazilian Ministry of Health (DECIT/SCTIE/MS), the Brazilian Council for Scientific and Technological Development (CNPq), and the Rio de Janeiro State Research Foundation (FAPERJ).
[18]
[19]
Disclosure summary The authors have nothing to disclose. Appendix A. Supplementary data
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Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jsbmb.2019.03.012. [21]
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Paradoxical effect of testosterone supplementation therapy on cardiac ischemia/reperfusion injury in aged rats
T
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Fernando A.C. Searaa,c, ,1, Raiana A.Q. Barbosaa,1, Marcus V.N. Santosa, Ainá E. Domingosa, Gustavo Monnerata, Adriana B. Carvalhoa, Emerson L. Olivaresc, José G. Millb, Jose H.M. Nascimentoa, Antonio C. Campos de Carvalhoa,d a
Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES, Brazil c Department of Physiological Sciences, Federal Rural University of Rio de Janeiro, Seropedica, RJ, Brazil d National Center for Structural Biology and Bioimaging, Federal University of Rio de Janeiro, RJ, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Testosterone Aging Myocardial infarction Ischemia Reperfusion
Aging is followed by numerous physiological limitations that reduce health span, particularly cardiovascular and metabolic disorders. Testosterone supplementation therapy (TST) has been widely used in the treatment of aging dysfunctions in either adult or aged patients, although recent evidence have suggested that the incidence of myocardial infarction might be increased in elderly patients. So far, though, the effects of TST in the progression of cardiac ischemia/reperfusion (IR) injury in aged hearts remain unclear. Male aged (23–24 months old) and adult (6 months old) Wistar rats were treated with placebo (Old + Placebo n = 5 / Adult + Placebo n = 5) or TST (Old + TST n = 7 / Adult + TST n = 5) for 30 days. After euthanasia, artificially-perfused isolated rat hearts were submitted to IR. Cardiac expression levels of genes encoding α and β myosin heavy chain (MHC), ryanodine receptor (RyR), brain-natriuretic peptide (BNP), sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a), glucose-regulated protein 78 kDa (GRP78), eukaryotic initiation factor 2α (eIF2α), C/EBP-homologous protein (CHOP), caspase 3 and B cell lymphoma 2 (Bcl-2) were accessed by qRT-PCR. Protein levels of CHOP, p-Akt, and p-glycogen synthase kinase 3β (p-GSK-3β) were measured by Western Blot. Compared to placebo-treated aged rats, Old + TST group exhibited increased heart weight and up-regulation of αMHC mRNA expression levels, whereas βMHC mRNA expression (p < 0.05). During reperfusion, left ventricular developed pressure, dP/dt+, dP/dt-, and cardiac contractile function index were increased in Old + TST rat hearts (p < 0.05), whereas infarct size was increased (p < 0.05) in comparison with Old + Placebo group. p-Akt levels of Old + TST rat hearts were decreased when compared to Old + Placebo group. Conversely, TST did not promote significant effects in adult rat hearts. Taken together, these findings suggest that myocardial stunning and infarct size of aged hearts were distinctly affected by TST.
1. Introduction Life expectancy has rapidly increased worldwide. Recently, the World Health Organization estimated 617 million people aged 65 and older, which accounts for 8.5% of the global population [1]. Moreover, it is estimated that 20% of the global population will be 65 and over by 2030 [2]. However, the increase in life expectancy has not been followed by a proportional increase in healthspan [2]. Biological senescence is characterized by time-related deterioration of several physiologic mechanisms necessary for homeostatic maintenance, resulting in
allostatic overload and development of a disease. Most aging-associated physiological limitations include muscle and bone waste, cognitive decline, sexual dysfunction and cardiovascular and metabolic disorders [3]. In the search for new methods to improve healthspan during aging, the physiologist Charles Édouard Brown-Séquard reported an improvement in his physical and cognitive performances after injections of testicular extracts, which he named the elixir of life [4]. Although most of these findings have later been proven to be placebo effects, testosterone, the main androgen hormone produced by male gonads,
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Corresponding author. E-mail address: [email protected] (F.A.C. Seara). 1 These authors had an equivalent contribution. https://doi.org/10.1016/j.jsbmb.2019.03.012 Received 24 January 2019; Received in revised form 18 March 2019; Accepted 19 March 2019 Available online 28 March 2019 0960-0760/ © 2019 Elsevier Ltd. All rights reserved.
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testosterone propionate (TP, Hertape Carlier® Hertape Carlier Animal Health Laboratory) at 1 mg.kg-1 daily for 30 days, or an equivalent volume of vehicle (peanut oil and benzyl alcohol, 90:10, v/v). The animals were divided into two experimental groups randomly treated with vehicle (Old + Placebo, N = 5) or TST (Old + TST, N = 7). The susceptibility to cardiac IR injury was assessed in isolated hearts using a Langendorf apparatus after the 30 days. Hearts were weighted to estimate pathological abnormalities and heart samples were stored at -80 °C. In a separate cohort, adult rats (6 months old) were submitted to placebo (Adult + Placebo, n = 5) or TST (Adult + TST, n = 5) treatments and to the same experimental protocols of aged rats. All data of adult rats are shown in the supplemental material.
has arisen as a potential therapeutic intervention to treat health disabilities in male elderly patients, either as replacement or supplementation therapies. Testosterone supplementation therapy (TST) has been used in the treatment of obesity, cachexia, lack of libido and infertility, and to improve erectile dysfunction, mood and cognitive disorders, and overall well-being in patients without hypogonadism [5–10]. Since testosterone promotes numerous effects on the heart, cardiovascular outcomes have been under intense investigation. It has been demonstrated that eugonadal elderly patients with chronic heart failure have experienced improvements in peak oxygen consumption, muscle strength, insulin sensitivity, and baroreflex control of the heart when treated with TST [11–15]. Noteworthy, heart failure symptoms can be reduced by at least one functional class in patients under testosterone supplementation [16]. On the other hand, the use of TST has been challenged by clinical evidence associating this therapeutic intervention with increased cardiovascular risk, including cerebrovascular and ischemic heart diseases [17,18]. It has been reported that administration of testosterone and other anabolic steroids can increase the susceptibility to cardiac ischemia/reperfusion (IR) injury, either in eugonadal or hypogonadal rats, while the efficacy of cardioprotective maneuvers is blunted [19–23]. These evidences have suggested that not only the risk for myocardial ischemia is increased, but post-ischemic recovery of cardiac function might be impaired by TST. This is particularly important in the case of elderly patients since aging per se is a dominant risk factor for cardiovascular dysfunction. Indeed, the prevalence of cardiovascular diseases among elderlies can be significantly enhanced after chronic testosterone supplementation [24]. However, the effects of TST in the progression of IR injury in aged hearts remain largely unknown. During prolonged periods of IR, cardiomyocyte survival depends mostly upon the balance between pro-apoptotic and anti-apoptotic downstream signaling. Decreased Akt phosphorylation and activation has been proposed as a key pathophysiological mechanism whereby testosterone impairs cardiomyocyte survival in adult hearts [25]. In addition, recent studies have demonstrated that testosterone regulates cell survival by recruitment of the endoplasmic reticulum (ER) stressactivated unfolded protein response (UPR) [26–28]. Chronic UPR activation promotes cardiac hypertrophy, cardiomyocyte apoptosis, increased susceptibility to cardiac IR injury, and heart failure [29–32]. So far, though, there is no evidence of whether ER stress and UPR can be modulated by TST, either in adult or aged hearts. Despite the growing use of TST, particularly by elderlies, there are few studies about the effects of TST on cardiovascular homeostasis in more advanced ages, and most reports have focused on anabolic steroid overdose or testosterone replacement in adult rats. In the present study, we investigated the effects of TST in the progression of cardiac IR injury in aged rat hearts.
2.3. Ex vivo experiments All methods for isolated heart experiments were based on previous studies from our laboratory [19]. Isolated hearts were connected to a Langendorff apparatus and retrogradely perfused with modified KrebsHenseleit solution (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, saturated with 95% O2 and 5% CO2) adjusted for pH 7.4 and 37 °C at constant flow (10 ml/min). A latex balloon was placed inside the left ventricle cavity through an incision in the left atrial appendage. Baseline left ventricular (LV) end-diastolic pressure (LVEDP) was set at 10 mmHg. After stabilization of heart rate and systolic and diastolic pressures, perfusion was interrupted, and hearts were submitted to 30 min of global ischemia, and subsequent 60 min of reperfusion. LVEDP, LV systolic pressure (LVSP), LV developed pressure (LVDP), maximal (dP/ dt+) and minimal (dP/dt-) pressure derivatives, and contractile function index (CI) waveforms were recorded with a pressure transducer (PT 300, Grass Technologies). The transducer was connected to an amplifier (ML 110 ADInstruments), and all signals were converted to digital data by an analogic/digital converter (PowerLab 400, ADInstruments). All recordings were digitized and stored on a computer for posterior analysis (LabChart 7.0, ADInstruments). 2.4. Measurement of infarct size
2. Material and methods
Atria were discarded and ventricles were sliced into approximately 1.5 mm thick section from apex to base. A small piece of the ventricle apex was stored for future biochemical and molecular analyses. All other slices were incubated in 1% triphenyltetrazolium chloride in phosphate buffer (pH 7.4) for 5 min at 37 °C. All ventricular slices were incubated in 10% (v/v) formaldehyde solution for 24 h to improve the 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 as a percentage of total ventricular area, as previously described [19].
2.1. Animals
2.5. Quantitative polymerase-chain reaction (qRT-PCR)
This study followed the standards and ethical guidelines of the Ethics Committee for Research of the Federal University of Rio de Janeiro, and it was approved by the commission under the number 023/14. All standards proposed by the Guide for the Care and Use of Laboratory Animals (U.S. National Institute of Health (NIH) Publication No. 85-23, revised 1996) were observed.
Total RNA was extracted from LV apex tissue using RNeasy® Fibrous Tissue Mini Kit (QIAGEN), and cDNA was prepared from 1 μg of total RNA using High-Capacity Reverse Transcription Kit (Thermo Fisher Scientific), following manufacturer’s instructions. Messenger RNA (mRNA) levels of sarcoplasmic reticulum calcium ATPase (SERCA2a), ryanodine receptor (RyR), myosin heavy chain isoforms α (αMHC) and β (βMHC), brain natriuretic peptide (BNP), glucose-regulated protein 78 (GRP78), eukaryotic translation initiation factor 2A (eIF2A), CCAAT-enhancer-binding protein homologous protein (CHOP), Bcl-2 and caspase 3 were measured by qRT-PCR. Amplification reactions containing 5 ng of cDNA were performed at 60 °C during annealing and extension cycles. The expression was normalized to glyceraldehyde-3phosphate dehydrogenase (GAPDH) as an internal control. The
2.2. Experimental protocol Male aged (23–24 months-old) Wistar rats were housed in cages under controlled temperature (21 ± 2 °C), daily exposed to 12-hour light-dark cycle (lights off at 7:00 pm), and water and standard chow ad libitum. TST administration consisted of subcutaneous injections of 2
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increased heart weight relative to tibia length (Fig. 1A, HW/TBL, p < 0.05), consistent with cardiac hypertrophy. Decreased mRNA expression of αMHC (Fig. 1B, p < 0.05), increased βMHC mRNA expression levels (Fig. 1C, p < 0.05), and decreased SERCA-2a mRNA expression levels (Fig. 1F, p < 0.05) were also observed. Neither BNP (Fig. 1D), nor RyR (Fig. 1E) mRNA expression levels were significantly affected TST (p > 0.05).
quantification of selected mRNA was determined by 2-(ΔΔCt) method in a Viia7 Software v1.2.4 (Thermo Fisher Scientific) and expressed as fold change compared to Old + Placebo group. 2.6. Western blot 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 EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM NaF) supplemented with complete protease inhibitor cocktail (Roche), and complete phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich). Protein concentration was determined by BCA method (Bio-Rad Protein Assay, Bio-Rad), according to manufacturer’s instruction. Proteins were separated on SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes with a wet transfer system (BioRad). PVDF membranes were incubated in 5% non-fat dry milk diluted in Tris-buffered saline with 0.1% Tween® 20 (TBST) for 2 h at room temperature. After blocking, all PVDF membranes were incubated with primary antibodies against p-Akt (1:1000, Cell Signalling, Inc.), C/EBP homologous protein (CHOP, 1:500 Santa Cruz, Inc), phosphorylated glycogen synthase kinase 3 beta (p-GSK3β, 1:500, Cell Signalling, Inc.) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:2000, Santa Cruz, Inc), followed by incubation with anti-rabbit secondary antibody (1:5000, Cell Signalling). The signals were detected using an enhanced chemiluminescence kit (ECL Prime, GE Healthcare), with Odissey Fc Imaging System (Licor), and were analyzed using ImageJ. Densitometry was normalized using GAPDH as a loading control.
3.2. TST did not affect baseline LV performance nor ischemic contracture Pre-ischemic LV function is demonstrated in Fig. 2. LVSP (Fig. 2A), LVDP (Fig. 2B), dP/dt+ (Fig. 2C), dP/dt- (Fig. 2D), and CI (Figure E) levels were equivalent between Old + Placebo and Old + TST groups (p > 0.05). The amplitude of ischemic contracture was equivalent between Old + Placebo and Old + TST groups (p > 0.05).
3.3. Post-ischemic recovery of LV hemodynamic properties of aged rat hearts was ameliorated by TST Post-ischemic LVSP (Fig. 3A) and LVEDP (Fig. 3B) levels were equivalent (p > 0.05), whereas LVDP (Fig. 3C), dP/dt+ (Fig. 3D), dP/ dt- (Fig. 3E), and CI (Fig. 3F) of Old + TST group were increased when compared to Old + Placebo group (p < 0.05). Infarct size (Fig. 4) was significantly increased in Old + TST rat hearts in comparison with Old + Placebo hearts (p < 0.05).
3.4. p-Akt, but not p-GSK3β or ER stress levels, was attenuated by TST in aged hearts
2.7. Statistical analysis Data are presented as mean ± standard error of the mean (S.E.M.). Student’s t-test was used to compare the means of Placebo versus TST groups. Two-way ANOVA followed by Sidak post-hoc test (Prism 6.00, GraphPad Software) were used for temporal analyses of ex vivo data. Statistical differences were considered significant when P < 0.05.
Cardiac levels of p-Akt were significantly decreased in Old + TST versus Old + Placebo group (Fig. 5A, p < 0.05), whereas p-GSK3β levels were not significantly different (Fig. 5B, p > 0.05). Furthermore, caspase 3 mRNA levels tended to increase in Old + TST versus Old + Placebo group but did not reach statistical significance (Fig. 5C, p = 0.1). Bcl-2 mRNA levels were similar in both groups (Fig. 5D, p > 0.05). Compared to Old + Placebo group, Old + TST hearts exhibited equivalent mRNA levels of GRP78 (Fig. 6A), CHOP (Fig. 6B), and eIF2α (Fig. 6C), and equivalent protein levels of CHOP (Fig. 6D) (p > 0.05).
3. Results 3.1. TST promoted cardiac hypertrophy in aged rats Compared to Old + Placebo group, Old + TST rats exhibited
Fig. 1. TST promoted cardiac hypertrophy in aged rats. (A) Heart weight (HW) relative to tibia length (TBL), and mRNA expression levels of (B) αMHC, (C) βMHC, (D) BNP, (E) RyR, and (F) SERCA-2a were compared between Old + Placebo (White box, n = 5) and Old + TST (Black box, n = 7) groups. Data are expressed as mean ± S.E.M. *p < 0.05 versus Old + Placebo group. 3
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Fig. 2. Baseline LV function and amplitude of ischemic contracture were not affected by TST. LVSP (A), LVDP (B), dP/dt+ (C), dP/dt- (D), CI(E), and the amplitude of ischemic contracture (F) were compared between Old + Placebo (White box, n = 5) and Old + TST (Black box, n = 7) groups. Data are expressed as mean ± S.E.M.
Fig. 3. Post-ischemic recovery of LV function was improved by TST. LVSP (A), LVEDP (B), LVDP (C), dP/dt+ (D), dP/dt- (E), and CI (F) were compared between Old + Placebo (n = 5) and Old + TST (n = 7) groups during cardiac ischemia and reperfusion. Data are expressed as mean ± S.E.M. *p < 0.05 and **p < 0.01 versus Old + Placebo group.
CHOP (Figure S6C), nor protein levels of CHOP (Figure S6D) of adult hearts were affected by TST.
3.5. Effects of TST in in adult rats In adult hearts, HW/TBL (Figure S1A) and mRNA expression levels of αMHC, βMHC, RyR, BNP, and SERCA-2A (Figure S1B-S1 F) were equivalent between Adult + Placebo and Adult + TST groups (p > 0.05). Baseline LV function (Figure S2A-S2E) and the amplitude of ischemic contracture (Figure S2F) were equivalent between Adult + Placebo and Adult + TST (p > 0.05) as well. There was no significant difference in cardiac function (Figure S3A – Figure S3 F) during reperfusion period or in infarct size (Figure S4) between Adult + Placebo and Adult + TST groups (p > 0.05). Levels of p-Akt (Figure S5A) and p-GSK3β (Figure S5B) of adult hearts were not affected by TST in comparison with placebo (p > 0.05). Caspase 3 and Bcl-2 mRNA levels were equivalent between Adult + Placebo and Adult + TST groups (Figure S5C and S5D, p > 0.05). In addition, neither mRNA levels of GRP78 (Figure S6A), eIF2α (Figure S6B), and
4. Discussion This report provides experimental evidence that TST distinctly affected the susceptibility to cardiac IR injury in adults and aged rats. As far as we know, this is the first report on the effects of TST in the susceptibility of aged hearts to cardiac IR injury. Despite its therapeutic benefits, anabolic steroids (AS) can promote adverse cardiac remodeling depending on the dose regimen and AS compound. At supraphysiological doses, AS can promote cardiomyocyte death and fibrosis [33]. Moreover, previous reports have demonstrated that chronic testosterone propionate overdose can promote cardiac hypertrophy early after exposure, that can persist even after AS discontinuation [19,34]. Using supplementation protocols, however, 4
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cypionate [36]. This is particularly important for elderly patients, who exhibit a higher prevalence of risk factors for cardiac hypertrophy when compared to adults. Adult rats treated with testosterone supplementation, on the other hand, did not exhibit signs of cardiac hypertrophy, suggesting that aged hearts are more susceptible to hypertrophic stimuli elicited by testosterone [34]. When analyzed for baseline LV hemodynamic properties, Old + TST group performed equally to Old + Placebo group. These findings imply that LV function of Old + TST group was preserved despite cardiac hypertrophy, at least under ex vivo assessment. Longer treatment periods than used in the present study can result in deterioration of LV function, even with testosterone replacement therapeutic protocols. Similarly, baseline LV hemodynamic performance of adult rat hearts was not significantly affected by TST [37]. In adult rats, longer supplementation with TP has been reported to increase overall LV contractile function [38]. However, it must be noted that testosterone can impair LV function by extrinsic mechanisms, such as autonomic imbalance. Sympathetic hyperactivity and down-regulation of parasympathetic activity have been largely reported during chronic administration of anabolic steroids [39]. These abnormalities have long been associated with increased susceptibility to LV electrophysiological and mechanical dysfunctions in the progression of cardiovascular diseases, particularly in advanced ages [40]. As expected, LV contractile function ceased after the onset of global ischemia and amplitude of ischemic contracture progressively increased in all groups, a condition termed stone heart [41]. Indeed, contractile properties can be impaired as early as 10 s after the onset of ischemia, according to the landmark study by Katz and Tada [42]. Ischemic contracture depends mostly upon ATP levels and the shift towards glycolytic metabolism [43]. The resultant cytosolic acidification is counter-balanced by Na+/H + exchanger, whereby H + is transported to the extracellular milieu in exchange for Na+. This, in turn, stimulates Na + transport to the extracellular milieu in exchange for Ca+2 by the Na+/Ca+2 exchanger, resulting in up-regulation of cytosolic
Fig. 4. Infarct size was increased by TST. (A) Percentage infarct size relative to ventricular area was determined after global ischemia/reperfusion in Old + Placebo (White box, n = 5) and Old + TST (Black box, n = 7) groups. (B) Representative slices of infarcted hearts. Data are expressed as mean ± S.E.M. *p < 0.05 versus Old + Placebo group.
there are few pieces of evidence on the repercussion of testosterone treatment, particularly in aged hearts. We found that TST promoted hypertrophy in aged rats. Furthermore, TST also decreased αMHC mRNA levels, while βMHC was up-regulated in the aged animals. In rodents, increased HW and βMHC/αMHC ratio are consistent with pathologic cardiac hypertrophy [35]. In agreement with these findings, previous reports have suggested that aged rabbits can also exhibit cardiac hypertrophy after chronic supplementation with testosterone
Fig. 5. p-Akt but not p-GSK3β levels of aged hearts were decreased by TST. Levels of pAkt (A, n = 4/group) and p-GSK3β (B, n = 3/ group) were compared between Old + Placebo (White box) and Old + TST (Black box) groups. Representative bands of p-Akt and p-GSK3β are demonstrated above each respective graph. Caspase 3 (C) and Bcl-2 (D) mRNA expression levels were compared between Old + Placebo (n = 5) and Old + TST groups (n = 7). Data are expressed as mean ± S.E.M. *p < 0.05 versus Old + Placebo group.
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Fig. 6. TST did not promote ER stress in aged hearts. mRNA expression levels of GRP78 (A), CHOP (B), and eIF2α were compared between Old + Placebo (White box, n = 5), and Old + TST (Black box, n = 7) groups. Protein levels of CHOP (D) were measured by Western Blot (n = 4/group), and representative bands are exhibited in E. Data are expressed as mean ± S.E.M.
androgen receptor antagonists or testosterone deficiency might prevent LV dysfunction during IR in adults [25], our findings suggest that TST does not affect post-ischemic LV hemodynamic properties or cardiomyocyte integrity of adult hearts. In contrast, previous reports have demonstrated that testosterone replacement can provide cardioprotection in adult rat hearts [52–55]. Taken together, these findings suggest that testosterone supplementation and replacement induce distinct effects in the progression of cardiac IR injury. Additionally, previous evidence has suggested that testosterone can impair cardiomyocyte survival during IR by decreasing the activation of anti-apoptotic signaling, at least in adult rat hearts. Accordingly, either castration or administration of androgen receptor blockers enhances Akt phosphorylation, and thereby activation of anti-apoptotic signals during cardiac reperfusion [25]. Furthermore, down-regulation of pAkt levels during cardiac reperfusion was reported after chronic overdose with nandrolone decanoate, a class II anabolic steroid [20]. Downregulation of p-Akt levels has likewise been correlated with a worsened renal ischemic injury in testosterone-treated mice [56]. We found that p-Akt levels of aged hearts were down-regulated in Old + TST rat hearts in comparison to Old + Placebo, whereas Adult + TST levels were not affected when compared to Adult + Placebo. Decreased Akt activation has long been correlated with impaired cardiomyocyte survival during reperfusion [57]. Numerous evidences have demonstrated that Akt-induced cardioprotection involves phosphorylation, and thereby inactivation of GSK3β. However, p-GSK3β levels were equivalent among all experimental groups, suggesting that down-regulation of p-Akt might have affected cardiomyocyte survival by other signaling pathways [25]. Caspase 3 mRNA levels exhibited a trend toward up-regulation in Old + TST group when compared to Old + Placebo group, suggesting that caspase 3 might be involved in the decreased cell viability after reperfusion in Old + TST group. Penna et al. reported similar findings during the administration of nandrolone decanoate to adult rats, i.e., decreased levels of cardiac p-Akt and unchanged p-GSK3β levels after IR, followed by increased infarct size [20]. Recently, it has been reported that cardiomyocyte survival during IR can be modulated by ER homeostasis [29,32]. UPR activation can affect cardiomyocyte apoptosis by several mechanisms that concatenate into cell survival or death. GRP78 is a chaperone protein located within ER lumen that binds to newly correctly synthesized proteins during protein folding, as well as to abnormal proteins targeted for degradation [58].
calcium concentration and contracture [44]. This is further aggravated by down-regulation of Ca+2 transport into SR by SERCA-2a, and decreased actomyosin turnover following ATP depletion. We found that the amplitude of ischemic contracture was equivalent between Placebo versus TST groups. However, Callies et al. demonstrated that cytosolic calcium concentration at the end of myocardial ischemia was significantly decreased after just one injection of testosterone undecanoate in eugonadal rats, which was administered two weeks before the induction of IR [45]. It remains elusive, however, whether down-regulation of cytosolic calcium by testosterone in this period would be enough to decrease the amplitude of cardiac ischemic contracture. LV function was just partially restored after the onset of reperfusion, in keeping with the reports of Braunwald and Kloner [46]. Although naturally reversible, myocardial stunning can promote hemodynamic instability and might contribute to the development of pulmonary edema and cardiogenic shock after IR. In severe cases, myocardial stunning can be treated with inotropic agents. Whereas most reports have demonstrated the effects in adult hearts, the repercussion in aged hearts was unclear so far. Old + TST rat hearts exhibited increased LVDP, CI, dP/dt+, and dP/dt- during reperfusion when compared to Old + Placebo group, clearly indicating that myocardial stunning was ameliorated in some degree by TST. Somewhat surprisingly, infarct size was significantly increased in Old + TST rat hearts when compared to Old + Placebo group. Interestingly, TST was shown to promote ambivalent cardiovascular and metabolic effects in adult and aged rats during the progression of systemic hypertension [47]. However paradoxical as it might seem, clinical and experimental reports have demonstrated similar findings in conditions of increased workload and energy demand, resulting in infarct expansion, such as after administration of inotropic drugs [48,49]. Since aged hearts have impaired adaptation capacity to conditions of energetic imbalance, such as those imposed by IR [50,51], this condition might be further aggravated by the increased hemodynamic stress and energetic demand promoted by TST, since contractile properties in TST aged hearts were increased during ex vivo reperfusion. These distinctions may explain in part why Old + TST exhibited increased infarct size when compared to Old + Placebo. This finding also raises the question of whether the down-regulation of cardiac contractile function during reperfusion would be an allostatic mechanism to preserve cardiomyocyte integrity in aged hearts. On the other hand, infarct size was equivalent between Adult + Placebo and Adult + TST. Thus, although 6
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Since GRP78 is a master regulator of ER stress and its levels are remarkably increased under conditions characterized by the accumulation of unfolded proteins within ER lumen, GRP78 has been used as a biomolecular marker of ER stress [58]. Surprisingly, no evidence on whether testosterone can modulate cardiac ER stress and UPR in cardiomyocytes has been hitherto reported. Herein, we found that expression levels of genes encoding GRP78, eIF2α, and CHOP were not affected by TST, neither in adult nor in aged hearts. Additionally, CHOP protein levels, the main pro-apoptotic signal evoked during UPR, were not affected by TST as well, when compared with placebo. These findings imply that most of the effects of TST in the progression of cardiac IR injury do not involve significant changes in the ER stress levels or UPR. Taken together, the present findings demonstrated that TST promoted ambivalent effects during IR in aged rat hearts, while adult hearts were not significantly affected. TST promoted cardiac hypertrophy without significant LV hemodynamic repercussions in aged hearts. Ex-vivo post-ischemic recovery of LV contractile function of aged rat hearts was enhanced by TST, whereas infarct size was increased. Aged hearts exposed to TST exhibited decreased p-Akt levels, though p-GSK3β levels were not affected. Additional studies will be necessary to investigate why aged hearts were more susceptible to TST effects.
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Funding
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This study was supported by the Department of Science and Technology - Brazilian Ministry of Health (DECIT/SCTIE/MS), the Brazilian Council for Scientific and Technological Development (CNPq), and the Rio de Janeiro State Research Foundation (FAPERJ).
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Disclosure summary The authors have nothing to disclose. Appendix A. Supplementary data
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Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jsbmb.2019.03.012. [21]
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