Mol Cell Biochem DOI 10.1007/s11010-015-2326-1
Aerobic exercise training improves oxidative stress and ubiquitin proteasome system activity in heart of spontaneously hypertensive rats Luiz Henrique Soares de Andrade • Wilson Max Almeida Monteiro de Moraes Eduardo Hiroshi Matsuo Junior • Elizabeth de Orleans Carvalho de Moura • Hanna Karen Moreira Antunes • Jairo Montemor • Ednei Luiz Antonio • Danilo Sales Bocalini • Andrey Jorge Serra • Paulo Jose´ Ferreira Tucci • Patricia Chakur Brum • Alessandra Medeiros
•
Received: 14 August 2014 / Accepted: 16 January 2015 Ó Springer Science+Business Media New York 2015
Abstract The activity of the ubiquitin proteasome system (UPS) and the level of oxidative stress contribute to the transition from compensated cardiac hypertrophy to heart failure in hypertension. Moreover, aerobic exercise training (AET) is an important therapy for the treatment of hypertension, but its effects on the UPS are not completely known. The aim of this study was to evaluate the effect of AET on UPS’s activity and oxidative stress level in heart of spontaneously hypertensive rats (SHR). A total of 53 Wistar and SHR rats were randomly divided into sedentary and trained groups. The AET protocol was 59/week in
Luiz Henrique Soares de Andrade and Wilson Max Almeida Monteiro de Moraes have contributed equally to this study. L. H. S. de Andrade W. M. A. M. de Moraes E. H. Matsuo Junior E. de Orleans Carvalho de Moura H. K. M. Antunes A. Medeiros (&) Universidade Federal de Sa˜o Paulo- Departamento de Biocieˆncias, Silva Jardim, 136-Vl. Mathias, Santos, SP 11015-020, Brazil e-mail: [email protected] J. Montemor E. L. Antonio D. S. Bocalini A. J. Serra P. J. F. Tucci Cardio-Physiology and Pathophysiology Laboratory, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil D. S. Bocalini Department of Post-Graduation in Physical Education, Sa˜o Judas Tadeu University, Sa˜o Paulo, Brazil
treadmill for 13 weeks. Exercise tolerance test, non-invasive blood pressure measurement, echocardiographic analyses, and left ventricle hemodynamics were performed during experimental period. The expression of ubiquitinated proteins, 4-hydroxynonenal (4-HNE), Akt, phosphoAktser473, GSK3b, and phospho-GSK3bser9 were analyzed by western blotting. The evaluation of lipid hydroperoxide concentration was performed using the xylenol orange method, and the proteasomal chymotrypsin-like activity was measured by fluorimetric assay. Sedentary hypertensive group presented cardiac hypertrophy, unaltered expression of total Akt, phospho-Akt, total GSK3b and phospho-GSK3b, UPS hyperactivity, increased lipid hydroperoxidation as well as elevated expression of 4-HNE but normal cardiac function. In contrast, AET significantly increased exercise tolerance, decreased resting systolic blood pressure and heart rate in hypertensive animals. In addition, the AET increased phospho-Akt expression, decreased phospho-GSK3b, and did not alter the expression of total Akt, total GSK3b, and ubiquitinated proteins, however, significantly attenuated 4-HNE levels, lipid hydroperoxidation, and UPS’s activity toward normotensive group levels. Our results provide evidence for the main effect of AET on attenuating cardiac ubiquitin proteasome hyperactivity and oxidative stress in SHR rats. Keywords Hypertension Aerobic exercise training Cardiac remodeling Ubiquitin proteasome system Oxidative stress
A. J. Serra Postgraduate Program in Biophotonics Applied to Health Sciences, Universidade Nove de Julho, Sa˜o Paulo, Brazil
Introduction
P. C. Brum School of Physical Education and Sport, University of Sa˜o Paulo, Sa˜o Paulo, Brazil
Arterial hypertension (AH) is an independent risk factor and one of the most relevant risk factors for cardiovascular
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disease [1]. Its high prevalence associated with low control rates is reflected in international statistics becoming a serious public health problem [2]. In AH, the sustained elevation of pressure levels may result in left ventricular hypertrophy (LVH), followed by an excessive collagen accumulation, which increases cardiac stiffness. This shift from stable LVH to decompensated state may increase the odds for cardiac complications as arrhythmias, myocardial infarction, and heart failure [3], besides being considered a predictor of all cardiac deaths in hypertensive adults [4]. Cardiac hypertrophy in response to pressure overload is one of the main morbidities in AH [5]. One of the mechanisms that might be involved in AH associated cardiac remodeling is an impairment in the ubiquitin proteasome system (UPS). UPS is a system, which the main function is to maintain the protein quality control. Additionally, UPS is considerate a major proteolytic system responsible for removing oxidative stress-induced damage of proteins in mammalian cells [6]. In this regard, when the heart is overloaded with oxidative stress-induced misfolded and dysfunctional proteins, an increased UPS activity is observed to remove these damaged proteins. This is observed in compensated cardiac hypertrophy [7, 8]. However, a failure in UPS removal of damaged proteins is observed in severe cardiac dysfunction, since reactive oxygen species can directly affect UPS, decreasing its activity. This will result in misfolded proteins aggregation forming aggresomes that are not degraded by UPS [7, 9, 10]. Although several studies have demonstrated alterations on the cardiac UPS activity in different models of pressure overload [7, 8, 11–13], to our knowledge, only one study evaluated the activity of the UPS in cardiac tissue in SHR, but this study did not use normotensive control animals [14]. Therefore, there is a limited knowledge about the UPS activity in hearts of SHR, which exhibits a progression from stable LVH with normal cardiac function to heart failure similar to those observed in hypertensive patients [15]. Another mechanism involved with cardiac injury in heart disease progression is the oxidative stress, an unbalance between pro-oxidants and anti-oxidants in favor of oxidants, which may aggravate cardiac remodeling and hypertension [16]. Furthermore, redox imbalance may negatively influence the activity of the UPS, since it can directly modulate UPS activity or it can change protein structure affecting its function. These responses will preclude UPS from degrading these dysfunctional proteins [10, 17]. Campos et al. recently demonstrated that the accumulation of 4-hydroxynonenal (4-HNE), an aldehyde accumulated from lipid peroxidation, inhibits the proteasome peptidase activity worsening cardiac remodeling in rats with heart failure [10].
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In contrast, aerobic exercise training (AET) is a wellestablished non-pharmacological approach that can, among others, lower blood pressure [18, 19], promote physiological cardiac hypertrophy [20], improve autonomic control of circulation [21, 22], and reduce oxidative stress [16, 23]. We have previously demonstrated that AET is able to positively modulate the UPS with improved cardiac remodeling in a model of HF [10], but it is still unknown whether AET would improve cardiac UPS in SHR. Thus, the present study was undertaken to determine whether AET 1) would delay progression of hypertension attenuating cardiac hypertrophy in SHR and 2) would affect the relationship between the activation of UPS and oxidative stress in hearts of SHR.
Methods Animals’ care A cohort of male SHR and Wistar rats (WR) was studied from 8 to 21 weeks of age. Adult male rats were housed under controlled environmental conditions (temperature, 22 °C; 12-h dark period starting at 08:00 h) and had free access to standard laboratory chow (Nuvital Nutrients, Brazil) and water. The animals were randomly assigned into four experimental groups: sedentary WR (WR, n = 14), exercise training WR (WR ? EX, n = 10), sedentary SHR (SHR, n = 15), and exercise training SHR (SHR ? EX, n = 14). This study was carried out in accordance with National Research Council’s Guidelines for the Care and Use of Laboratory Animals [24] and was approved by the Ethics and Research Committee (CEP) of the UNIFESP (CEP #1576/11). Aerobic exercise training Moderate-intensity AET was performed on a motor treadmill over 13 weeks, 5 days/week. The running speed and duration of exercise were progressively increased to elicit 55 % of maximal speed, achieved during a graded treadmill exercise protocol, for 60 min from the 5th week. Exercise capacity, estimated by total distance run, was evaluated with a graded treadmill exercise protocol for rats. Briefly, after being adapted to treadmill exercises over a week (10 min of exercise session), rats were placed in the treadmill streak and allowed to acclimatize for at least 30 min. Intensity of exercise was increased by 5 m/min (5–50 m/min) every 3 min at 0 % grade until exhaustion, when rats were no longer able to run. A single observer, blinded to rat’s identity, carried out the progressive exercise testing in the following stages of
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the experimental period: at the initial (1st week), during (between 6 and 7 weeks) and at the final (13th week). Blood pressure measurements Blood pressure and heart rate were performed by tail plethysmography, using a specific system for rats (Visitech Systems: BP-2000—Series II—Blood Pressure Analysis System). Rats were acclimatized to the apparatus during daily sessions over 4 days, 1 week before starting the experimental period. The measurement was performed once a week throughout the experimental period; on days that trained groups were not subjected to AET. The average values for systolic blood pressure were subsequently obtained from ten sequential cuff inflation–deflation cycles. Echocardiography Analyses of echocardiography were performed in two moments of the experimental protocol, the initial (week 1) and final (week 13). After ketamine–xylazine anesthesia (i.p.), transthoracic echocardiography was performed by an observer blinded to the animal’s group, as previously described [25], using an HP Sonos-5500 echocardiograph (Hewlett Packard, Andover, MA, USA) with a 12-MHz linear transducer. The rats were imaged in the left lateral decubitus position with three electrodes placed on their paws for the electrocardiogram. Two-dimensional parasternal long- and short-axis views and 2D-targeted M-mode tracings throughout the anterior and posterior left ventricular (LV) walls were recorded. Fractional shortening (FS) and E/A relationships were obtained. Left ventricle hemodynamics Immediately after echocardiography at the final of the experimental period, the rats were intubated, ventilated (Rodent Ventilator, Harvard Apparatus Mod 683; Holliston, MA, USA), and a 2-F Millar catheter-tip micromanometer was inserted through the right carotid artery into the LV cavity. Measurements of LV parameters, including LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), and maxima positive (?dP/dt) and negative (-dP/dt) time derivatives of the developed pressure, were studied using AcqKnowledge 3.5.7 software (Biopac Systems Inc., Santa Barbara, CA, USA) [26]. Cardiac structural analysis Forty-eight hours after the last bout of AET, the rats were sacrificed by decapitation and their tissues were harvested. Cardiac chambers were dissected and the left ventricle then fixed by immersion in 4 % buffered formalin and
embedded in paraffin for routine histological processing. Sections (4 lm) were stained with hematoxylin-eosin for the quantification of the cardiomyocyte diameter. The image was magnified 4009, and myocytes with visible nuclei and intact cell membrane were chosen for analysis. These measurements were analyzed with a computerassisted morphometric system (Leica Quantimet 500, Cambridge, UK, England), as described previously [27]. Lipid hydroperoxidation Lipid hydroperoxides were evaluated using the ferrous oxidation–xylenol (FOX) orange technique [28]. Left ventricles samples were homogenized (1:20 wt/vol) in phosphate buffered saline (PBS; 100 mM, pH 7.4) and centrifuged at 12,000 g for 20 min at 4 °C. Pellet was discarded and supernatant was precipitated with trichloroacetic acid (10 wt%/vol) and centrifuged at 12,000 g for 20 min at 4 °C. Supernatant was mixed with FOX reagent containing 250 mM ammonium ferrous sulfate, 100 mM xylenol orange, 25 mM H2SO4, and 4 mM butylated hydroxytoluene in 90 % methanol and incubated at room temperature for 30 min. Absorbance of samples was read at 560 nm. Assay of 26S proteasome activity Proteasomal chymotrypsin-like activity was assayed in the total lysate from heart using the fluorogenic peptide SucLeu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Biomol International, USA). Peptidase activities were measured in the absence and presence (20 lM) of the proteasome-specific inhibitor epoxomicin, and the difference between the two rates was attributed to the proteasome. Details from this method have been described before [5]. Western blot Polyubiquitinated proteins, 4-HNE, total Akt, phosphoAktser473, total GSK3 b, phospho-GSK3bser9, and GAPDH expression levels were evaluated by western blotting in total extracts from the ventricle. Briefly, samples were subjected to SDS-PAGE in polyacrylamide gels (10 %) depending upon protein molecular weight. After electrophoresis, proteins were electrotransferred to nitrocellulose membranes (BioRad Biosciences; Piscataway, NJ, USA). Equal gel loading and transfer efficiency were monitored using 0.5 % Ponceau S staining of blot membrane. Blotted membrane was then blocked (5 % nonfat dry milk, 10 mM Tris–HCl pH 7.6, 150 mM NaCl, and 0.1 % Tween 20) for 2 h at room temperature and then incubated overnight at 4 °C with specific antibodies against polyubiquitinated proteins (Biomol Int., PA, USA), 4-HNE (Calbiochem, HE,
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Germany), total Akt, phospho-Aktser473 phosphoGSK3bser9 (Cell Signaling Technology, MA, USA), and total GSK3b and GAPDH (Thermo Fisher Scientific Inc., MA, USA). Binding of the primary antibody was detected with the use of peroxidase-conjugated secondary antibodies (rabbit or mouse, depending on the protein, for 2 h at room temperature) and developed using enhanced chemiluminescence (Amersham Biosciences, NJ, USA) detected by autoradiography. Quantification analysis of blots was performed with the use of Scion Image software (Scion based on NIH image). Statistical analysis The data are expressed as mean ± standard error of the mean. A two-way ANOVA was used to determine the differences among the groups followed by Newman–Keuls’s post hoc test. Blood pressure and heart rate data were analyzed by repeated measures ANOVA with post hoc Newman–Keuls’s tests. Statistical analyses were performed using Graphic Pad Prism software (version 5.0, San Diego, CA, USA). Values of p \ 0.05 were considered statistically significant.
Results Aerobic exercise training increases exercise tolerance, reduces blood pressure, and promotes resting bradycardia in SHR As expected hypertensive groups (SHR and SHR ? EX) displayed significant higher systolic blood pressure, heart rate, and exercise tolerance than normotensive groups (WR and WR ? EX) at the beginning of the protocol (Fig. 1a– c). AET decreased systolic blood pressure and heart rate in SHR rats from 9 and 10 weeks of aerobic exercise protocol, respectively, (Fig. 1b–c). In addition, AET further increased the exercise tolerance (Fig. 1a). Aerobic exercise training does not alter cardiac mass and cardiomyocyte diameter but activates Akt/GSK3b pathway in SHR In order to evaluate cardiac hypertrophy, the weight of the heart chambers and the cardiomyocyte cross-sectional diameter were evaluated at the end of the protocol. The heart chamber weight was normalized by the tibial length. SHR displayed increased cardiac hypertrophy, as assessed by the ratio of left ventricle mass/tibia length (Fig. 2a), and cardiomyocyte croos sectional diameter (Fig. 2b). In order to evaluate the activation of the prosurvival Akt/GSK3b
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Fig. 1 The effects of aerobic exercise training on exercise tolerance, systolic blood pressure and heart rate. a Exercise tolerance (time run), b systolic blood pressure, c heart rate in sedentary WR (WR), exercise training WR (WR ? EX), sedentary SHR (SHR) and exercise training SHR (SHR ? EX) during 13 weeks of either sedentary or exercise training protocol. Ampersand symbol indicates p \ 0.05 within-group differences; omega symbol indicates p \ 0.05 versus WR and WR ? EX at same moment; number sign symbol indicates p \ 0.05 versus SHR at same moment; asterisk symbol indicates p \ 0.05 versus WR
pathway, the expression of phospho-Akt and phosphoGSK3b were evaluated. SHR showed unaltered total Akt, phospho-Akt, total GSK3b, and phospho-GSK3b expression. AET had no effect on ratio of left ventricle mass/tibia length (Fig. 2a), cardiomyocyte diameter (Fig. 2b), total Akt (Fig. 3a), and total GSK3b expression (Fig. 3a) but increased phospho-Aktser473 expression (Fig. 3a–b) and decreased phospho-GSK3bser9 expression (Fig. 3a–c).
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Aerobic exercise training does not alter cardiac function in SHR In order to evaluate cardiac function, echocardiographic analyses were performed before and after experimental period, and left ventricle hemodynamics was performed after experimental period. SHR presented no alteration in echocardiographic parameters when compared with WR and AET had no effect on any of these parameters (Table 1). Aerobic exercise training decreases cardiac oxidative stress and re-establishes cardiac ubiquitin–proteasome system activity in SHR
Fig. 2 The influence of aerobic exercise training on cardiac hypertrophy: a left ventricle mass/tibia length ratio, b cardiomyocyte diameter in sedentary WR (WR), exercise training WR (WR ? EX), sedentary SHR (SHR) and exercise training SHR (SHR ? EX) before and after 13 weeks of either sedentary or exercise training protocol. omega symbol indicates p \ 0.05 versus WR and WR ? EX; asterisk symbol indicates p \ 0.05 versus WR
SHR showed increases in cardiac oxidative stress as assessed by lipid hydroperoxidation and 4-HNE expression (Fig. 4a–c). AET significantly reduced the cardiac lipid hydroperoxidation and the levels of 4-HNE in SHR toward WR group levels (Fig. 4a–c). SHR presented proteasomal chymotrypsin-like overactivity but normal levels of ubiquitinated proteins in the heart (Fig. 4d–f). AET significantly reduced the cardiac proteasomal chymotrypsin-like activity in the SHR toward WR group levels (Fig. 4d).
Fig. 3 Effect of aerobic exercise training on Akt/GSK3b pathway. a Representative blots of total Akt, phospho-Aktser473, total GSK3b, phospho-GSK3bser9, and GAPDH expression in total extracts from sedentary WR (WR), exercise training WR (WR ? EX), sedentary SHR
(SHR) and exercise training SHR (SHR ? EX), b phospho-Aktser473, c phospho-GSK3bser9 expression levels in total extracts from WR, WR ? EX, SHR and SHR ? EX. Ampersand symbol indicates p \ 0.05 versus WR ? EX; number sign symbol indicates p \ 0.05 versus SHR
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Mol Cell Biochem Table 1 Cardiac function in sedentary WR (WR), exercise training WR (WR ? EX), sedentary SHR (SHR) and exercise training SHR (SHR ? EX) before and after 13 weeks of either sedentary or Variables
exercise training assessed by echocardiography analyses or just after 13 weeks of either sedentary or exercise training protocol assessed by left ventricle hemodynamics
Experimental groups WR
WR ? EX
SHR
SHR ? EX
Echocardiography FS (%) Initial
45 ± 4.77
50 ± 7.26
47 ± 4.09
46 ± 4.63
Final
43 ± 4.99
45 ± 4.57
51 ± 6.46
48 ± 5.58
Initial
0.79 ± 0.22
0.59 ± 0.12
0.73 ± 0.27
0.62 ± 0.20
Final
0.76 ± 0.13
0.82 ± 0.06
0.66 ± 0.20
0.68 ± 0.08
116.97 ± 7.92
138.62 ± 8.39
127.14 ± 3.80
1.24 ± 0.79
4.12 ± 1.62
4.28 ± 0.85
E/A
Basal hemodynamics LVSP (mmHg) LVEDP (mmHg)
121.25 ± 15.52 3.70 ± 0.76
?dP/dt (mmHg/sec)
10,297 ± 3,127
7,612 ± 647
12,189 ± 891
8,406 ± 807
-dP/dt (mmHg/sec)
-6,483 ± 1,366
-6,018 ± 509
-5,680 ± 467
-6,471 ± 631
Results are expressed as the mean ± SD FS fractional shortening; E/A speed ratio of the wave E/A; LVSP left ventricle systolic pressure; LVEDP left ventricle end-diastolic pressure; ?dP/dt maximum positive time derivative of developed pressure; -dP/dt maximum negative time derivative of developed pressure
Discussion The present study shows that in SHR, moderate-intensity AET exerts several cardiovascular benefits since it reduced blood pressure levels, increased exercise tolerance, activated physiological cardiac hypertrophy pathway, and prevented some of the cardiac alterations associated with hypertension, such as tachycardia. In parallel, we observed that AET prevented cardiac oxidative stress and proteasomal chymotrypsin-like overactivity, which highlights AET as an important therapeutic strategy to hypertension. AET promoted hemodynamic adaptations in SHR as resting bradycardia and reduced systolic blood pressure similar to that observed in humans [19, 29, 30] and other animal studies using SHR as a model of AH [16, 23, 31]. This resting bradycardia observed in trained animals is probably due to the decreased cardiac sympathetic overactivity [32, 33] and/or increased vagal control of the heart rate [34, 35]. Considering that the increased sympathetic activity is a hallmark for AH, the reduction in sympathetic activity post-exercise training program besides improvement of vagal control of heart can beneficially affect not only the heart rate but also leads to improvement in autonomic balance [31, 36]. Hypotension post-AET in hypertension has also been another consistent finding in the literature [23, 31, 32, 34, 37]. It is important to note that small reductions in systolic blood pressure can reduce the risk of stroke by 6 %, chronic heart disease by 4 %, and overall mortality by 3 % [38].
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The SHR is an established model of human hypertension and cardiac hypertrophy, which progresses to heart failure only about the last 6 months of their lifespan [39]. In vivo studies have shown that, in the early stages of hypertension, SHRs have a normal cardiac function [40]. In fact, the present study showed that SHR presented cardiac hypertrophy but normal cardiac function at 21st weeks of age, and AET was not able to attenuate the cardiac hypertrophy, since AET did not reduce the ratio of left ventricle mass/ tibia length and cardiomyocyte cross-sectional diameter. Nevertheless, AET increased phospho-Aktser473 expression and decreased GSK3bser9 expression, which indicates an activation of physiological cardiac hypertrophy pathway, since phosphatidylinositol-3 kinase (PI3K)/Akt/GSK3b pathway has been reported to mediate physiological hypertrophy associated with exercise training [41]. In fact, Garciarena et al. demonstrated the effectiveness of swimming training to convert pathological into physiological hypertrophy in SHR [20]. They showed that swimming training increased myocardial hypertrophy assessed by left ventricular weight/tibial length and myocyte cross-sectional area, and decreased collagen volume fraction and the mRNA abundance of atrial natriuretic factor and myosin light chain 2, which are markers of fetal reprogramming program and pathological cardiac hypertrophy [20]. Another recently stud by Jia et al. have recently demonstrated that 16 weeks of moderate AET decreased the expression of atrial natriuretic peptide [36]. They found reduction on the ratio of heart mass/body mass, but the
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Fig. 4 The impact of aerobic exercise training on oxidative stress and ubiquitin–proteasome system function in rat. a Lipid hydroperoxidation expressed, b 4-hydroxynonenal expression, c 4-hydroxynonenal blot image, d proteasomal chymotrypsin-like activity, e ubiquitinated proteins expression, f ubiquitinated proteins blot image in sedentary
WR (WR), exercise training WR (WR ? EX), sedentary SHR (SHR) and exercise training SHR (SHR ? EX) before and after 13 weeks of either sedentary or exercise training protocol. omega symbol indicates p \ 0.05 versus WR and WR ? EX; number sign symbol indicates p \ 0.05 versus SHR
protocol of AET used by these researchers was 3 weeks longer, which can account for this contrasting results. Therefore, we cannot exclude that other factors that were not evaluated in the present study may have been improved by AET (i.e., collagen volume fraction and expression of markers of pathological cardiac hypertrophy). Besides cardiac hypertrophy, SHR presented increased cardiac oxidative stress, more precisely, in markers of lipid peroxidation as lipid hydroperoxides and increased protein expression of adducts modified by 4-HNE. Several reports indicated that oxidative stress is increased in hypertensive patients and SHR, and that oxidative stress defense system,
which includes vitamin E, glutathione peroxidase, and superoxide dismutase, is reduced [16, 42, 43]. The formation and accumulation of aldehydes resulting from oxidative stress are toxic and contribute to the onset and/or aggravation of cardiovascular diseases [10, 16]. Among the various aldehydes accumulated in cardiac tissue, the 4-HNE, originated from the oxidation of unsaturated lipids present in the membranes, has serious cardiac deleterious power. This electrophilic aldehyde is able to attack nucleophilic amino acids and form adducts with proteins, resulting in inactivation of target proteins [44]. Grune et al. (1994) reported that in the isolated hearts of
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the SHR, the 4-HNE degradation rate is reduced and suggested that the low degradation of the cytotoxic lipid peroxidation products in hypertrophic hearts may contribute to reduce antioxidant defense in those hearts [42]. Exercise-induced-higher antioxidant defense is a great interest since elevated levels of reactive oxygen species can alter the UPS functioning, leading to an ‘‘overload’’ [45], especially in advanced stages of cardiac dysfunction, culminating in a significant reduction of UPS activity [10]. Considering that UPS function is to prevent accumulation of damaged, misfolded and mutant proteins by proteolysis, dysfunctional UPS can induce additional cardiac stress. In fact, impaired UPS activity may be insufficient for degrading accumulated misfolded proteins, which will induce cardiac proteotoxicity. Furthermore, dysfunctional UPS leads to the activation of signaling pathways, such as calcineurin-NFAT [46], and mitogen-activated protein kinase (MAPK) [47] that will further contribute to the hypertrophic growth. At our knowledge, the current study shows, for the first time, that SHR presents compensated cardiac hypertrophy associated with increased oxidative stress and UPS activity and that AET prevented oxidative stress and UPS overactivity in cardiac tissue. AET prevented increased cardiac 4-HNE protein expression induced by hypertension, probably related to its ability of upregulating aldehyde dehydrogenase 2 [48], one the major mitochondrial matrix enzymes responsible for the elimination of 4-HNE. In this regard, the expression of several metabolic enzymes, such as glutathione peroxidase and superoxide dismutase from LV, was shown to be altered after AET protocol in SHR animals [16]. Thus, maintaining reduced levels of oxidative stress by increasing endogenous antioxidant defense systems in response to AET not only protects cardiac tissue from the attack of reactive oxygen species but also contributes to maintain UPS function preventing the accumulation of ubiquitinated and damaged proteins. These effects of AET are important to slow the progression of hypertension to heart failure since Meiners et al. have shown that inhibition of the ubiquitin–proteasome system suppresses expression of matrix metalloproteinases and collagens in rat cardiac fibroblasts and effectively prevents myocardial remodeling in spontaneously hypertensive rats [14]. Therefore, the effects of AET observed in the present study in SHR rats are extremely relevant and can, at least in part, contribute to the prevention of the hemodynamic changes observed in hypertension. However, to confirm this preventive effect, more studies are acknowledged. Acknowledgments This work was supported by Conselho Nacional de Pesquisa e Desenvolvimento—CNPq (#474085/2011-2). L. H. S. de Andrade and W. M. A. M. de Moraes had a master degree and PhD
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scholarship from CAPES, respectively. CNPq had no role in the design, analysis or writing of this article. Conflict of interest
The authors declare no conflict of interests.
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Aerobic exercise training improves oxidative stress and ubiquitin proteasome system activity in heart of spontaneously hypertensive rats Luiz Henrique Soares de Andrade • Wilson Max Almeida Monteiro de Moraes Eduardo Hiroshi Matsuo Junior • Elizabeth de Orleans Carvalho de Moura • Hanna Karen Moreira Antunes • Jairo Montemor • Ednei Luiz Antonio • Danilo Sales Bocalini • Andrey Jorge Serra • Paulo Jose´ Ferreira Tucci • Patricia Chakur Brum • Alessandra Medeiros
•
Received: 14 August 2014 / Accepted: 16 January 2015 Ó Springer Science+Business Media New York 2015
Abstract The activity of the ubiquitin proteasome system (UPS) and the level of oxidative stress contribute to the transition from compensated cardiac hypertrophy to heart failure in hypertension. Moreover, aerobic exercise training (AET) is an important therapy for the treatment of hypertension, but its effects on the UPS are not completely known. The aim of this study was to evaluate the effect of AET on UPS’s activity and oxidative stress level in heart of spontaneously hypertensive rats (SHR). A total of 53 Wistar and SHR rats were randomly divided into sedentary and trained groups. The AET protocol was 59/week in
Luiz Henrique Soares de Andrade and Wilson Max Almeida Monteiro de Moraes have contributed equally to this study. L. H. S. de Andrade W. M. A. M. de Moraes E. H. Matsuo Junior E. de Orleans Carvalho de Moura H. K. M. Antunes A. Medeiros (&) Universidade Federal de Sa˜o Paulo- Departamento de Biocieˆncias, Silva Jardim, 136-Vl. Mathias, Santos, SP 11015-020, Brazil e-mail: [email protected] J. Montemor E. L. Antonio D. S. Bocalini A. J. Serra P. J. F. Tucci Cardio-Physiology and Pathophysiology Laboratory, Federal University of Sa˜o Paulo, Sa˜o Paulo, Brazil D. S. Bocalini Department of Post-Graduation in Physical Education, Sa˜o Judas Tadeu University, Sa˜o Paulo, Brazil
treadmill for 13 weeks. Exercise tolerance test, non-invasive blood pressure measurement, echocardiographic analyses, and left ventricle hemodynamics were performed during experimental period. The expression of ubiquitinated proteins, 4-hydroxynonenal (4-HNE), Akt, phosphoAktser473, GSK3b, and phospho-GSK3bser9 were analyzed by western blotting. The evaluation of lipid hydroperoxide concentration was performed using the xylenol orange method, and the proteasomal chymotrypsin-like activity was measured by fluorimetric assay. Sedentary hypertensive group presented cardiac hypertrophy, unaltered expression of total Akt, phospho-Akt, total GSK3b and phospho-GSK3b, UPS hyperactivity, increased lipid hydroperoxidation as well as elevated expression of 4-HNE but normal cardiac function. In contrast, AET significantly increased exercise tolerance, decreased resting systolic blood pressure and heart rate in hypertensive animals. In addition, the AET increased phospho-Akt expression, decreased phospho-GSK3b, and did not alter the expression of total Akt, total GSK3b, and ubiquitinated proteins, however, significantly attenuated 4-HNE levels, lipid hydroperoxidation, and UPS’s activity toward normotensive group levels. Our results provide evidence for the main effect of AET on attenuating cardiac ubiquitin proteasome hyperactivity and oxidative stress in SHR rats. Keywords Hypertension Aerobic exercise training Cardiac remodeling Ubiquitin proteasome system Oxidative stress
A. J. Serra Postgraduate Program in Biophotonics Applied to Health Sciences, Universidade Nove de Julho, Sa˜o Paulo, Brazil
Introduction
P. C. Brum School of Physical Education and Sport, University of Sa˜o Paulo, Sa˜o Paulo, Brazil
Arterial hypertension (AH) is an independent risk factor and one of the most relevant risk factors for cardiovascular
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disease [1]. Its high prevalence associated with low control rates is reflected in international statistics becoming a serious public health problem [2]. In AH, the sustained elevation of pressure levels may result in left ventricular hypertrophy (LVH), followed by an excessive collagen accumulation, which increases cardiac stiffness. This shift from stable LVH to decompensated state may increase the odds for cardiac complications as arrhythmias, myocardial infarction, and heart failure [3], besides being considered a predictor of all cardiac deaths in hypertensive adults [4]. Cardiac hypertrophy in response to pressure overload is one of the main morbidities in AH [5]. One of the mechanisms that might be involved in AH associated cardiac remodeling is an impairment in the ubiquitin proteasome system (UPS). UPS is a system, which the main function is to maintain the protein quality control. Additionally, UPS is considerate a major proteolytic system responsible for removing oxidative stress-induced damage of proteins in mammalian cells [6]. In this regard, when the heart is overloaded with oxidative stress-induced misfolded and dysfunctional proteins, an increased UPS activity is observed to remove these damaged proteins. This is observed in compensated cardiac hypertrophy [7, 8]. However, a failure in UPS removal of damaged proteins is observed in severe cardiac dysfunction, since reactive oxygen species can directly affect UPS, decreasing its activity. This will result in misfolded proteins aggregation forming aggresomes that are not degraded by UPS [7, 9, 10]. Although several studies have demonstrated alterations on the cardiac UPS activity in different models of pressure overload [7, 8, 11–13], to our knowledge, only one study evaluated the activity of the UPS in cardiac tissue in SHR, but this study did not use normotensive control animals [14]. Therefore, there is a limited knowledge about the UPS activity in hearts of SHR, which exhibits a progression from stable LVH with normal cardiac function to heart failure similar to those observed in hypertensive patients [15]. Another mechanism involved with cardiac injury in heart disease progression is the oxidative stress, an unbalance between pro-oxidants and anti-oxidants in favor of oxidants, which may aggravate cardiac remodeling and hypertension [16]. Furthermore, redox imbalance may negatively influence the activity of the UPS, since it can directly modulate UPS activity or it can change protein structure affecting its function. These responses will preclude UPS from degrading these dysfunctional proteins [10, 17]. Campos et al. recently demonstrated that the accumulation of 4-hydroxynonenal (4-HNE), an aldehyde accumulated from lipid peroxidation, inhibits the proteasome peptidase activity worsening cardiac remodeling in rats with heart failure [10].
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In contrast, aerobic exercise training (AET) is a wellestablished non-pharmacological approach that can, among others, lower blood pressure [18, 19], promote physiological cardiac hypertrophy [20], improve autonomic control of circulation [21, 22], and reduce oxidative stress [16, 23]. We have previously demonstrated that AET is able to positively modulate the UPS with improved cardiac remodeling in a model of HF [10], but it is still unknown whether AET would improve cardiac UPS in SHR. Thus, the present study was undertaken to determine whether AET 1) would delay progression of hypertension attenuating cardiac hypertrophy in SHR and 2) would affect the relationship between the activation of UPS and oxidative stress in hearts of SHR.
Methods Animals’ care A cohort of male SHR and Wistar rats (WR) was studied from 8 to 21 weeks of age. Adult male rats were housed under controlled environmental conditions (temperature, 22 °C; 12-h dark period starting at 08:00 h) and had free access to standard laboratory chow (Nuvital Nutrients, Brazil) and water. The animals were randomly assigned into four experimental groups: sedentary WR (WR, n = 14), exercise training WR (WR ? EX, n = 10), sedentary SHR (SHR, n = 15), and exercise training SHR (SHR ? EX, n = 14). This study was carried out in accordance with National Research Council’s Guidelines for the Care and Use of Laboratory Animals [24] and was approved by the Ethics and Research Committee (CEP) of the UNIFESP (CEP #1576/11). Aerobic exercise training Moderate-intensity AET was performed on a motor treadmill over 13 weeks, 5 days/week. The running speed and duration of exercise were progressively increased to elicit 55 % of maximal speed, achieved during a graded treadmill exercise protocol, for 60 min from the 5th week. Exercise capacity, estimated by total distance run, was evaluated with a graded treadmill exercise protocol for rats. Briefly, after being adapted to treadmill exercises over a week (10 min of exercise session), rats were placed in the treadmill streak and allowed to acclimatize for at least 30 min. Intensity of exercise was increased by 5 m/min (5–50 m/min) every 3 min at 0 % grade until exhaustion, when rats were no longer able to run. A single observer, blinded to rat’s identity, carried out the progressive exercise testing in the following stages of
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the experimental period: at the initial (1st week), during (between 6 and 7 weeks) and at the final (13th week). Blood pressure measurements Blood pressure and heart rate were performed by tail plethysmography, using a specific system for rats (Visitech Systems: BP-2000—Series II—Blood Pressure Analysis System). Rats were acclimatized to the apparatus during daily sessions over 4 days, 1 week before starting the experimental period. The measurement was performed once a week throughout the experimental period; on days that trained groups were not subjected to AET. The average values for systolic blood pressure were subsequently obtained from ten sequential cuff inflation–deflation cycles. Echocardiography Analyses of echocardiography were performed in two moments of the experimental protocol, the initial (week 1) and final (week 13). After ketamine–xylazine anesthesia (i.p.), transthoracic echocardiography was performed by an observer blinded to the animal’s group, as previously described [25], using an HP Sonos-5500 echocardiograph (Hewlett Packard, Andover, MA, USA) with a 12-MHz linear transducer. The rats were imaged in the left lateral decubitus position with three electrodes placed on their paws for the electrocardiogram. Two-dimensional parasternal long- and short-axis views and 2D-targeted M-mode tracings throughout the anterior and posterior left ventricular (LV) walls were recorded. Fractional shortening (FS) and E/A relationships were obtained. Left ventricle hemodynamics Immediately after echocardiography at the final of the experimental period, the rats were intubated, ventilated (Rodent Ventilator, Harvard Apparatus Mod 683; Holliston, MA, USA), and a 2-F Millar catheter-tip micromanometer was inserted through the right carotid artery into the LV cavity. Measurements of LV parameters, including LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), and maxima positive (?dP/dt) and negative (-dP/dt) time derivatives of the developed pressure, were studied using AcqKnowledge 3.5.7 software (Biopac Systems Inc., Santa Barbara, CA, USA) [26]. Cardiac structural analysis Forty-eight hours after the last bout of AET, the rats were sacrificed by decapitation and their tissues were harvested. Cardiac chambers were dissected and the left ventricle then fixed by immersion in 4 % buffered formalin and
embedded in paraffin for routine histological processing. Sections (4 lm) were stained with hematoxylin-eosin for the quantification of the cardiomyocyte diameter. The image was magnified 4009, and myocytes with visible nuclei and intact cell membrane were chosen for analysis. These measurements were analyzed with a computerassisted morphometric system (Leica Quantimet 500, Cambridge, UK, England), as described previously [27]. Lipid hydroperoxidation Lipid hydroperoxides were evaluated using the ferrous oxidation–xylenol (FOX) orange technique [28]. Left ventricles samples were homogenized (1:20 wt/vol) in phosphate buffered saline (PBS; 100 mM, pH 7.4) and centrifuged at 12,000 g for 20 min at 4 °C. Pellet was discarded and supernatant was precipitated with trichloroacetic acid (10 wt%/vol) and centrifuged at 12,000 g for 20 min at 4 °C. Supernatant was mixed with FOX reagent containing 250 mM ammonium ferrous sulfate, 100 mM xylenol orange, 25 mM H2SO4, and 4 mM butylated hydroxytoluene in 90 % methanol and incubated at room temperature for 30 min. Absorbance of samples was read at 560 nm. Assay of 26S proteasome activity Proteasomal chymotrypsin-like activity was assayed in the total lysate from heart using the fluorogenic peptide SucLeu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Biomol International, USA). Peptidase activities were measured in the absence and presence (20 lM) of the proteasome-specific inhibitor epoxomicin, and the difference between the two rates was attributed to the proteasome. Details from this method have been described before [5]. Western blot Polyubiquitinated proteins, 4-HNE, total Akt, phosphoAktser473, total GSK3 b, phospho-GSK3bser9, and GAPDH expression levels were evaluated by western blotting in total extracts from the ventricle. Briefly, samples were subjected to SDS-PAGE in polyacrylamide gels (10 %) depending upon protein molecular weight. After electrophoresis, proteins were electrotransferred to nitrocellulose membranes (BioRad Biosciences; Piscataway, NJ, USA). Equal gel loading and transfer efficiency were monitored using 0.5 % Ponceau S staining of blot membrane. Blotted membrane was then blocked (5 % nonfat dry milk, 10 mM Tris–HCl pH 7.6, 150 mM NaCl, and 0.1 % Tween 20) for 2 h at room temperature and then incubated overnight at 4 °C with specific antibodies against polyubiquitinated proteins (Biomol Int., PA, USA), 4-HNE (Calbiochem, HE,
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Germany), total Akt, phospho-Aktser473 phosphoGSK3bser9 (Cell Signaling Technology, MA, USA), and total GSK3b and GAPDH (Thermo Fisher Scientific Inc., MA, USA). Binding of the primary antibody was detected with the use of peroxidase-conjugated secondary antibodies (rabbit or mouse, depending on the protein, for 2 h at room temperature) and developed using enhanced chemiluminescence (Amersham Biosciences, NJ, USA) detected by autoradiography. Quantification analysis of blots was performed with the use of Scion Image software (Scion based on NIH image). Statistical analysis The data are expressed as mean ± standard error of the mean. A two-way ANOVA was used to determine the differences among the groups followed by Newman–Keuls’s post hoc test. Blood pressure and heart rate data were analyzed by repeated measures ANOVA with post hoc Newman–Keuls’s tests. Statistical analyses were performed using Graphic Pad Prism software (version 5.0, San Diego, CA, USA). Values of p \ 0.05 were considered statistically significant.
Results Aerobic exercise training increases exercise tolerance, reduces blood pressure, and promotes resting bradycardia in SHR As expected hypertensive groups (SHR and SHR ? EX) displayed significant higher systolic blood pressure, heart rate, and exercise tolerance than normotensive groups (WR and WR ? EX) at the beginning of the protocol (Fig. 1a– c). AET decreased systolic blood pressure and heart rate in SHR rats from 9 and 10 weeks of aerobic exercise protocol, respectively, (Fig. 1b–c). In addition, AET further increased the exercise tolerance (Fig. 1a). Aerobic exercise training does not alter cardiac mass and cardiomyocyte diameter but activates Akt/GSK3b pathway in SHR In order to evaluate cardiac hypertrophy, the weight of the heart chambers and the cardiomyocyte cross-sectional diameter were evaluated at the end of the protocol. The heart chamber weight was normalized by the tibial length. SHR displayed increased cardiac hypertrophy, as assessed by the ratio of left ventricle mass/tibia length (Fig. 2a), and cardiomyocyte croos sectional diameter (Fig. 2b). In order to evaluate the activation of the prosurvival Akt/GSK3b
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Fig. 1 The effects of aerobic exercise training on exercise tolerance, systolic blood pressure and heart rate. a Exercise tolerance (time run), b systolic blood pressure, c heart rate in sedentary WR (WR), exercise training WR (WR ? EX), sedentary SHR (SHR) and exercise training SHR (SHR ? EX) during 13 weeks of either sedentary or exercise training protocol. Ampersand symbol indicates p \ 0.05 within-group differences; omega symbol indicates p \ 0.05 versus WR and WR ? EX at same moment; number sign symbol indicates p \ 0.05 versus SHR at same moment; asterisk symbol indicates p \ 0.05 versus WR
pathway, the expression of phospho-Akt and phosphoGSK3b were evaluated. SHR showed unaltered total Akt, phospho-Akt, total GSK3b, and phospho-GSK3b expression. AET had no effect on ratio of left ventricle mass/tibia length (Fig. 2a), cardiomyocyte diameter (Fig. 2b), total Akt (Fig. 3a), and total GSK3b expression (Fig. 3a) but increased phospho-Aktser473 expression (Fig. 3a–b) and decreased phospho-GSK3bser9 expression (Fig. 3a–c).
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Aerobic exercise training does not alter cardiac function in SHR In order to evaluate cardiac function, echocardiographic analyses were performed before and after experimental period, and left ventricle hemodynamics was performed after experimental period. SHR presented no alteration in echocardiographic parameters when compared with WR and AET had no effect on any of these parameters (Table 1). Aerobic exercise training decreases cardiac oxidative stress and re-establishes cardiac ubiquitin–proteasome system activity in SHR
Fig. 2 The influence of aerobic exercise training on cardiac hypertrophy: a left ventricle mass/tibia length ratio, b cardiomyocyte diameter in sedentary WR (WR), exercise training WR (WR ? EX), sedentary SHR (SHR) and exercise training SHR (SHR ? EX) before and after 13 weeks of either sedentary or exercise training protocol. omega symbol indicates p \ 0.05 versus WR and WR ? EX; asterisk symbol indicates p \ 0.05 versus WR
SHR showed increases in cardiac oxidative stress as assessed by lipid hydroperoxidation and 4-HNE expression (Fig. 4a–c). AET significantly reduced the cardiac lipid hydroperoxidation and the levels of 4-HNE in SHR toward WR group levels (Fig. 4a–c). SHR presented proteasomal chymotrypsin-like overactivity but normal levels of ubiquitinated proteins in the heart (Fig. 4d–f). AET significantly reduced the cardiac proteasomal chymotrypsin-like activity in the SHR toward WR group levels (Fig. 4d).
Fig. 3 Effect of aerobic exercise training on Akt/GSK3b pathway. a Representative blots of total Akt, phospho-Aktser473, total GSK3b, phospho-GSK3bser9, and GAPDH expression in total extracts from sedentary WR (WR), exercise training WR (WR ? EX), sedentary SHR
(SHR) and exercise training SHR (SHR ? EX), b phospho-Aktser473, c phospho-GSK3bser9 expression levels in total extracts from WR, WR ? EX, SHR and SHR ? EX. Ampersand symbol indicates p \ 0.05 versus WR ? EX; number sign symbol indicates p \ 0.05 versus SHR
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Mol Cell Biochem Table 1 Cardiac function in sedentary WR (WR), exercise training WR (WR ? EX), sedentary SHR (SHR) and exercise training SHR (SHR ? EX) before and after 13 weeks of either sedentary or Variables
exercise training assessed by echocardiography analyses or just after 13 weeks of either sedentary or exercise training protocol assessed by left ventricle hemodynamics
Experimental groups WR
WR ? EX
SHR
SHR ? EX
Echocardiography FS (%) Initial
45 ± 4.77
50 ± 7.26
47 ± 4.09
46 ± 4.63
Final
43 ± 4.99
45 ± 4.57
51 ± 6.46
48 ± 5.58
Initial
0.79 ± 0.22
0.59 ± 0.12
0.73 ± 0.27
0.62 ± 0.20
Final
0.76 ± 0.13
0.82 ± 0.06
0.66 ± 0.20
0.68 ± 0.08
116.97 ± 7.92
138.62 ± 8.39
127.14 ± 3.80
1.24 ± 0.79
4.12 ± 1.62
4.28 ± 0.85
E/A
Basal hemodynamics LVSP (mmHg) LVEDP (mmHg)
121.25 ± 15.52 3.70 ± 0.76
?dP/dt (mmHg/sec)
10,297 ± 3,127
7,612 ± 647
12,189 ± 891
8,406 ± 807
-dP/dt (mmHg/sec)
-6,483 ± 1,366
-6,018 ± 509
-5,680 ± 467
-6,471 ± 631
Results are expressed as the mean ± SD FS fractional shortening; E/A speed ratio of the wave E/A; LVSP left ventricle systolic pressure; LVEDP left ventricle end-diastolic pressure; ?dP/dt maximum positive time derivative of developed pressure; -dP/dt maximum negative time derivative of developed pressure
Discussion The present study shows that in SHR, moderate-intensity AET exerts several cardiovascular benefits since it reduced blood pressure levels, increased exercise tolerance, activated physiological cardiac hypertrophy pathway, and prevented some of the cardiac alterations associated with hypertension, such as tachycardia. In parallel, we observed that AET prevented cardiac oxidative stress and proteasomal chymotrypsin-like overactivity, which highlights AET as an important therapeutic strategy to hypertension. AET promoted hemodynamic adaptations in SHR as resting bradycardia and reduced systolic blood pressure similar to that observed in humans [19, 29, 30] and other animal studies using SHR as a model of AH [16, 23, 31]. This resting bradycardia observed in trained animals is probably due to the decreased cardiac sympathetic overactivity [32, 33] and/or increased vagal control of the heart rate [34, 35]. Considering that the increased sympathetic activity is a hallmark for AH, the reduction in sympathetic activity post-exercise training program besides improvement of vagal control of heart can beneficially affect not only the heart rate but also leads to improvement in autonomic balance [31, 36]. Hypotension post-AET in hypertension has also been another consistent finding in the literature [23, 31, 32, 34, 37]. It is important to note that small reductions in systolic blood pressure can reduce the risk of stroke by 6 %, chronic heart disease by 4 %, and overall mortality by 3 % [38].
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The SHR is an established model of human hypertension and cardiac hypertrophy, which progresses to heart failure only about the last 6 months of their lifespan [39]. In vivo studies have shown that, in the early stages of hypertension, SHRs have a normal cardiac function [40]. In fact, the present study showed that SHR presented cardiac hypertrophy but normal cardiac function at 21st weeks of age, and AET was not able to attenuate the cardiac hypertrophy, since AET did not reduce the ratio of left ventricle mass/ tibia length and cardiomyocyte cross-sectional diameter. Nevertheless, AET increased phospho-Aktser473 expression and decreased GSK3bser9 expression, which indicates an activation of physiological cardiac hypertrophy pathway, since phosphatidylinositol-3 kinase (PI3K)/Akt/GSK3b pathway has been reported to mediate physiological hypertrophy associated with exercise training [41]. In fact, Garciarena et al. demonstrated the effectiveness of swimming training to convert pathological into physiological hypertrophy in SHR [20]. They showed that swimming training increased myocardial hypertrophy assessed by left ventricular weight/tibial length and myocyte cross-sectional area, and decreased collagen volume fraction and the mRNA abundance of atrial natriuretic factor and myosin light chain 2, which are markers of fetal reprogramming program and pathological cardiac hypertrophy [20]. Another recently stud by Jia et al. have recently demonstrated that 16 weeks of moderate AET decreased the expression of atrial natriuretic peptide [36]. They found reduction on the ratio of heart mass/body mass, but the
Mol Cell Biochem
Fig. 4 The impact of aerobic exercise training on oxidative stress and ubiquitin–proteasome system function in rat. a Lipid hydroperoxidation expressed, b 4-hydroxynonenal expression, c 4-hydroxynonenal blot image, d proteasomal chymotrypsin-like activity, e ubiquitinated proteins expression, f ubiquitinated proteins blot image in sedentary
WR (WR), exercise training WR (WR ? EX), sedentary SHR (SHR) and exercise training SHR (SHR ? EX) before and after 13 weeks of either sedentary or exercise training protocol. omega symbol indicates p \ 0.05 versus WR and WR ? EX; number sign symbol indicates p \ 0.05 versus SHR
protocol of AET used by these researchers was 3 weeks longer, which can account for this contrasting results. Therefore, we cannot exclude that other factors that were not evaluated in the present study may have been improved by AET (i.e., collagen volume fraction and expression of markers of pathological cardiac hypertrophy). Besides cardiac hypertrophy, SHR presented increased cardiac oxidative stress, more precisely, in markers of lipid peroxidation as lipid hydroperoxides and increased protein expression of adducts modified by 4-HNE. Several reports indicated that oxidative stress is increased in hypertensive patients and SHR, and that oxidative stress defense system,
which includes vitamin E, glutathione peroxidase, and superoxide dismutase, is reduced [16, 42, 43]. The formation and accumulation of aldehydes resulting from oxidative stress are toxic and contribute to the onset and/or aggravation of cardiovascular diseases [10, 16]. Among the various aldehydes accumulated in cardiac tissue, the 4-HNE, originated from the oxidation of unsaturated lipids present in the membranes, has serious cardiac deleterious power. This electrophilic aldehyde is able to attack nucleophilic amino acids and form adducts with proteins, resulting in inactivation of target proteins [44]. Grune et al. (1994) reported that in the isolated hearts of
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Mol Cell Biochem
the SHR, the 4-HNE degradation rate is reduced and suggested that the low degradation of the cytotoxic lipid peroxidation products in hypertrophic hearts may contribute to reduce antioxidant defense in those hearts [42]. Exercise-induced-higher antioxidant defense is a great interest since elevated levels of reactive oxygen species can alter the UPS functioning, leading to an ‘‘overload’’ [45], especially in advanced stages of cardiac dysfunction, culminating in a significant reduction of UPS activity [10]. Considering that UPS function is to prevent accumulation of damaged, misfolded and mutant proteins by proteolysis, dysfunctional UPS can induce additional cardiac stress. In fact, impaired UPS activity may be insufficient for degrading accumulated misfolded proteins, which will induce cardiac proteotoxicity. Furthermore, dysfunctional UPS leads to the activation of signaling pathways, such as calcineurin-NFAT [46], and mitogen-activated protein kinase (MAPK) [47] that will further contribute to the hypertrophic growth. At our knowledge, the current study shows, for the first time, that SHR presents compensated cardiac hypertrophy associated with increased oxidative stress and UPS activity and that AET prevented oxidative stress and UPS overactivity in cardiac tissue. AET prevented increased cardiac 4-HNE protein expression induced by hypertension, probably related to its ability of upregulating aldehyde dehydrogenase 2 [48], one the major mitochondrial matrix enzymes responsible for the elimination of 4-HNE. In this regard, the expression of several metabolic enzymes, such as glutathione peroxidase and superoxide dismutase from LV, was shown to be altered after AET protocol in SHR animals [16]. Thus, maintaining reduced levels of oxidative stress by increasing endogenous antioxidant defense systems in response to AET not only protects cardiac tissue from the attack of reactive oxygen species but also contributes to maintain UPS function preventing the accumulation of ubiquitinated and damaged proteins. These effects of AET are important to slow the progression of hypertension to heart failure since Meiners et al. have shown that inhibition of the ubiquitin–proteasome system suppresses expression of matrix metalloproteinases and collagens in rat cardiac fibroblasts and effectively prevents myocardial remodeling in spontaneously hypertensive rats [14]. Therefore, the effects of AET observed in the present study in SHR rats are extremely relevant and can, at least in part, contribute to the prevention of the hemodynamic changes observed in hypertension. However, to confirm this preventive effect, more studies are acknowledged. Acknowledgments This work was supported by Conselho Nacional de Pesquisa e Desenvolvimento—CNPq (#474085/2011-2). L. H. S. de Andrade and W. M. A. M. de Moraes had a master degree and PhD
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scholarship from CAPES, respectively. CNPq had no role in the design, analysis or writing of this article. Conflict of interest
The authors declare no conflict of interests.
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