Aerobic exercise training improves oxidative stress and ubiquitinproteasome system activity in heart of spontaneously hypertensiverats

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 / Published online: 28 January 2015

� 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 hyper-

tension, 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 Wi-

star and SHR rats were randomly divided into sedentary

and trained groups. The AET protocol was 59/week in

treadmill for 13 weeks. Exercise tolerance test, non-inva-

sive blood pressure measurement, echocardiographic

analyses, and left ventricle hemodynamics were performed

during experimental period. The expression of ubiquiti-

nated proteins, 4-hydroxynonenal (4-HNE), Akt, phospho-

Aktser473, 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 hyperten-

sive group presented cardiac hypertrophy, unaltered

expression of total Akt, phospho-Akt, total GSK3b and

phospho-GSK3b, UPS hyperactivity, increased lipid hy-

droperoxidation 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 expres-

sion of total Akt, total GSK3b, and ubiquitinated proteins,

however, significantly attenuated 4-HNE levels, lipid hy-

droperoxidation, 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

Introduction

Arterial hypertension (AH) is an independent risk factor

and one of the most relevant risk factors for cardiovascular

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 Sao Paulo- Departamento de

Biociencias, Silva Jardim, 136-Vl. Mathias, Santos,

SP 11015-020, Brazil

e-mail: a.medeiros@unifesp.br

J. Montemor � E. L. Antonio � D. S. Bocalini �A. J. Serra � P. J. F. Tucci

Cardio-Physiology and Pathophysiology Laboratory, Federal

University of Sao Paulo, Sao Paulo, Brazil

D. S. Bocalini

Department of Post-Graduation in Physical Education,

Sao Judas Tadeu University, Sao Paulo, Brazil

A. J. Serra

Postgraduate Program in Biophotonics Applied to Health

Sciences, Universidade Nove de Julho, Sao Paulo, Brazil

P. C. Brum

School of Physical Education and Sport, University

of Sao Paulo, Sao Paulo, Brazil

123

Mol Cell Biochem (2015) 402:193–202

DOI 10.1007/s11010-015-2326-1

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 car-

diac stiffness. This shift from stable LVH to decompen-

sated 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 mecha-

nisms 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 over-

loaded with oxidative stress-induced misfolded and dys-

functional proteins, an increased UPS activity is observed to

remove these damaged proteins. This is observed in com-

pensated 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 unbal-

ance 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 pre-

clude 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 protea-

some peptidase activity worsening cardiac remodeling in

rats with heart failure [10].

In contrast, aerobic exercise training (AET) is a well-

established non-pharmacological approach that can, among

others, lower blood pressure [18, 19], promote physiolog-

ical 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), sed-

entary 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 tread-

mill 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 tread-

mill 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

194 Mol Cell Biochem (2015) 402:193–202

123

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 para-

sternal long- and short-axis views and 2D-targeted M-mode

tracings throughout the anterior and posterior left ventric-

ular (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 sys-

tolic 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 computer-

assisted 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 trichloro-

acetic 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 Suc-

Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Biomol

International, USA). Peptidase activities were measured in

the absence and presence (20 lM) of the proteasome-spe-

cific 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, phospho-

Aktser473, 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 electro-

phoresis, 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,

Mol Cell Biochem (2015) 402:193–202 195

123

Germany), total Akt, phospho-Aktser473 phospho-

GSK3bser9 (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 chemilumi-

nescence (Amersham Biosciences, NJ, USA) detected by

autoradiography. Quantification analysis of blots was per-

formed 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–Ke-

uls’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 statisti-

cally 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 proto-

col, 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/GSK3bpathway 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

pathway, the expression of phospho-Akt and phospho-

GSK3b were evaluated. SHR showed unaltered total Akt,

phospho-Akt, total GSK3b, and phospho-GSK3b expres-

sion. 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).

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

196 Mol Cell Biochem (2015) 402:193–202

123

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

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 overac-

tivity 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. 2 The influence of aerobic exercise training on cardiac hypertrophy:

a leftventriclemass/tibia length ratio,b cardiomyocytediameter 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

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

Mol Cell Biochem (2015) 402:193–202 197

123

Discussion

The present study shows that in SHR, moderate-intensity

AET exerts several cardiovascular benefits since it reduced

blood pressure levels, increased exercise tolerance, acti-

vated 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 proteaso-

mal 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 over-

activity [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 improve-

ment of vagal control of heart can beneficially affect not

only the heart rate but also leads to improvement in auto-

nomic 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].

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 hyperten-

sion, SHRs have a normal cardiac function [40]. In fact, the

present study showed that SHR presented cardiac hyper-

trophy 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/GSK3bpathway has been reported to mediate physiological

hypertrophy associated with exercise training [41]. In fact,

Garciarena et al. demonstrated the effectiveness of swim-

ming 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-sec-

tional 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 demon-

strated 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

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

exercise training assessed by echocardiography analyses or just after

13 weeks of either sedentary or exercise training protocol assessed by

left ventricle hemodynamics

Variables 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

E/A

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

Basal hemodynamics

LVSP (mmHg) 121.25 ± 15.52 116.97 ± 7.92 138.62 ± 8.39 127.14 ± 3.80

LVEDP (mmHg) 3.70 ± 0.76 1.24 ± 0.79 4.12 ± 1.62 4.28 ± 0.85

?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

198 Mol Cell Biochem (2015) 402:193–202

123

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 tis-

sue, the 4-HNE, originated from the oxidation of unsatu-

rated 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

Fig. 4 The impact of aerobic exercise training on oxidative stress and

ubiquitin–proteasome system function in rat. a Lipid hydroperoxida-

tion 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

Mol Cell Biochem (2015) 402:193–202 199

123

the SHR, the 4-HNE degradation rate is reduced and sug-

gested that the low degradation of the cytotoxic lipid per-

oxidation 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, cul-

minating 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 overac-

tivity in cardiac tissue. AET prevented increased cardiac

4-HNE protein expression induced by hypertension, prob-

ably related to its ability of upregulating aldehyde dehy-

drogenase 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

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.

References

1. Sliwa K, Stewart S, Gersh BJ (2011) Hypertension: a global

perspective. Circulation 123(24):2892–2896. doi:10.1161/CIR

CULATIONAHA.110.992362

2. Mendis S, Lindholm LH, Anderson SG, Alwan A, Koju R, On-

wubere BJ, Kayani AM, Abeysinghe N, Duneas A, Tabagari S,

Fan W, Sarraf-Zadegan N, Nordet P, Whitworth J, Heagerty A

(2011) Total cardiovascular risk approach to improve efficiency

of cardiovascular prevention in resource constrain settings. J Clin

Epidemiol 64(12):1451–1462. doi:10.1016/j.jclinepi.2011.02.001

3. Lorell BH, Carabello BA (2000) Left ventricular hypertrophy:

pathogenesis, detection, and prognosis. Circulation 102(4):470–479

4. Bombelli M, Facchetti R, Carugo S, Madotto F, Arenare F,

Quarti-Trevano F, Capra A, Giannattasio C, Dell’Oro R, Grassi

G, Sega R, Mancia G (2009) Left ventricular hypertrophy

increases cardiovascular risk independently of in-office and out-

of-office blood pressure values. J Hypertens 27(12):2458–2464.

doi:10.1097/HJH.0b013e328330b845

5. Petriz BA, Franco OL (2014) Effects of hypertension and exer-

cise on cardiac proteome remodelling. BioMed Res Int

2014:634132. doi:10.1155/2014/634132

6. Depre C, Powell SR, Wang X (2010) The role of the ubiquitin-

proteasome pathway in cardiovascular disease. Cardiovasc Res

85(2):251–252. doi:10.1093/cvr/cvp362

7. Tsukamoto O, Minamino T, Okada K, Shintani Y, Takashima S,

Kato H, Liao Y, Okazaki H, Asai M, Hirata A, Fujita M, Asano

Y, Yamazaki S, Asanuma H, Hori M, Kitakaze M (2006)

Depression of proteasome activities during the progression of

cardiac dysfunction in pressure-overloaded heart of mice. Bio-

chem Biophys Res Commun 340(4):1125–1133. doi:10.1016/j.

bbrc.2005.12.120

8. Depre C, Wang Q, Yan L, Hedhli N, Peter P, Chen L, Hong C,

Hittinger L, Ghaleh B, Sadoshima J, Vatner DE, Vatner SF, Madura K

(2006) Activation of the cardiac proteasome during pressure overload

promotes ventricular hypertrophy. Circulation 114(17):1821–1828.

doi:10.1161/CIRCULATIONAHA.106.637827

9. Predmore JM, Wang P, Davis F, Bartolone S, Westfall MV, Dyke

DB, Pagani F, Powell SR, Day SM (2010) Ubiquitin proteasome

dysfunction in human hypertrophic and dilated cardiomyopathies.

Circulation 121(8):997–1004. doi:10.1161/CIRCULATIONAHA.

109.904557

10. Campos JC, Queliconi BB, Dourado PM, Cunha TF, Zambelli

VO, Bechara LR, Kowaltowski AJ, Brum PC, Mochly-Rosen D,

Ferreira JC (2012) Exercise training restores cardiac protein

quality control in heart failure. PLoS One 7(12):e52764. doi:10.

1371/journal.pone.0052764

11. Takaoka M, Ohkita M, Itoh M, Kobayashi Y, Okamoto H,

Matsumura Y (2001) A proteasome inhibitor prevents vascular

hypertrophy in deoxycorticosterone acetate-salt hypertensive rats.

Clin Exp Pharmacol Physiol 28(5–6):466–468

12. Demasi M, Laurindo FR (2012) Physiological and pathological

role of the ubiquitin-proteasome system in the vascular smooth

muscle cell. Cardiovasc Res 95(2):183–193. doi:10.1093/cvr/

cvs128

13. Ludwig A, Fechner M, Wilck N, Meiners S, Grimbo N, Baumann

G, Stangl V, Stangl K (2009) Potent anti-inflammatory effects of

low-dose proteasome inhibition in the vascular system. J Mol

Med (Berl) 87(8):793–802. doi:10.1007/s00109-009-0469-9

200 Mol Cell Biochem (2015) 402:193–202

123

14. Meiners S, Hocher B, Weller A, Laule M, Stangl V, Guenther C,

Godes M, Mrozikiewicz A, Baumann G, Stangl K (2004)

Downregulation of matrix metalloproteinases and collagens and

suppression of cardiac fibrosis by inhibition of the proteasome.

Hypertension 44(4):471–477. doi:10.1161/01.HYP.0000142772.

71367.65

15. Trippodo NC, Frohlich ED (1981) Similarities of genetic (spon-

taneous) hypertension. Man and rat. Circ Res 48(3):309–319

16. Bertagnolli M, Schenkel PC, Campos C, Mostarda CT, Casarini

DE, Bello-Klein A, Irigoyen MC, Rigatto K (2008) Exercise

training reduces sympathetic modulation on cardiovascular sys-

tem and cardiac oxidative stress in spontaneously hypertensive

rats. Am J Hypertens 21(11):1188–1193. doi:10.1038/ajh.2008.

270

17. Shang F, Taylor A (2011) Ubiquitin-proteasome pathway and

cellular responses to oxidative stress. Free Radic Biol Med

51(1):5–16. doi:10.1016/j.freeradbiomed.2011.03.031

18. Cornelissen VA, Buys R, Smart NA (2013) Endurance exercise

beneficially affects ambulatory blood pressure: a systematic

review and meta-analysis. J Hypertens 31(4):639–648. doi:10.

1097/HJH.0b013e32835ca964

19. Brook RD, Appel LJ, Rubenfire M, Ogedegbe G, Bisognano JD,

Elliott WJ, Fuchs FD, Hughes JW, Lackland DT, Staffileno BA,

Townsend RR, Rajagopalan S (2013) Beyond medications and

diet: alternative approaches to lowering blood pressure: a scien-

tific statement from the american heart association. Hypertension

61(6):1360–1383. doi:10.1161/HYP.0b013e318293645f

20. Garciarena CD, Pinilla OA, Nolly MB, Laguens RP, Escudero

EM, Cingolani HE, Ennis IL (2009) Endurance training in the

spontaneously hypertensive rat: conversion of pathological into

physiological cardiac hypertrophy. Hypertension 53(4):708–714.

doi:10.1161/HYPERTENSIONAHA.108.126805

21. Neto OB, Abate DT, Marocolo M Jr, Mota GR, Orsatti FL, Silva

RC, Reis MA, da Rossi e Silva VJ (2013) Exercise training

improves cardiovascular autonomic activity and attenuates renal

damage in spontaneously hypertensive rats. Journal of sports

science medicine 12(1):52–59

22. Edwards KM, Wilson KL, Sadja J, Ziegler MG, Mills PJ (2011)

Effects on blood pressure and autonomic nervous system function

of a 12-week exercise or exercise plus DASH-diet intervention in

individuals with elevated blood pressure. Acta Physiol (Oxf)

203(3):343–350. doi:10.1111/j.1748-1716.2011.02329.x

23. Roque FR, Briones AM, Garcia-Redondo AB, Galan M, Marti-

nez-Revelles S, Avendano MS, Cachofeiro V, Fernandes T,

Vassallo DV, Oliveira EM, Salaices M (2013) Aerobic exercise

reduces oxidative stress and improves vascular changes of small

mesenteric and coronary arteries in hypertension. Br J Pharmacol

168(3):686–703. doi:10.1111/j.1476-5381.2012.02224.x

24. Council NR (2011) Guide for the care and use of laboratory

animals. The National Academies Press, Washington DC,

pp 1–209

25. Couser WG, Remuzzi G, Mendis S, Tonelli M (2011) The con-

tribution of chronic kidney disease to the global burden of major

noncommunicable diseases. Kidney Int 80(12):1258–1270.

doi:10.1038/ki.2011.368

26. Dos Santos L, Antonio EL, Souza AF, Tucci PJ (2010) Use of

afterload hemodynamic stress as a practical method for assessing

cardiac performance in rats with heart failure. Can J Physiol

Pharmacol 88(7):724–732

27. Ferreira JC, Bacurau AV, Evangelista FS, Coelho MA, Oliveira

EM, Casarini DE, Krieger JE, Brum PC (2008) The role of local

and systemic renin angiotensin system activation in a genetic

model of sympathetic hyperactivity-induced heart failure in mice.

Am J Physiol Regul Integr Comp Physiol 294(1):R26–R32.

doi:10.1152/ajpregu.00424.2007

28. Petriz BA, Franco OL (2014) Application of cutting-edge pro-

teomics technologies for elucidating host-bacteria interactions.

Adv Protein Chem Struct Biol 95:1–24. doi:10.1016/B978-0-12-

800453-1.00001-4

29. Cornelissen VA, Smart NA (2013) Exercise training for blood

pressure: a systematic review and meta-analysis. J Am Heart

Assoc 2(1):e004473. doi:10.1161/JAHA.112.004473

30. Huang G, Shi X, Gibson CA, Huang SC, Coudret NA, Ehlman

MC (2013) Controlled aerobic exercise training reduces resting

blood pressure in sedentary older adults. Blood Press

22(6):386–394. doi:10.3109/08037051.2013.778003

31. Moraes-Silva IC, De La Fuente RN, Mostarda C, Rosa K, Flues

K, Damaceno-Rodrigues NR, Caldini EG, De Angelis K, Krieger

EM, Irigoyen MC (2010) Baroreflex deficit blunts exercise

training-induced cardiovascular and autonomic adaptations in

hypertensive rats. Clin Exp Pharmacol Physiol 37(3):e114–e120.

doi:10.1111/j.1440-1681.2009.05333.x

32. Gava NS, Veras-Silva AS, Negrao CE, Krieger EM (1995) Low-

intensity exercise training attenuates cardiac beta-adrenergic tone

during exercise in spontaneously hypertensive rats. Hypertension

26(6.2):1129–1133

33. Medeiros A, Rolim NP, Oliveira RS, Rosa KT, Mattos KC, Ca-

sarini DE, Irigoyen MC, Krieger EM, Krieger JE, Negrao CE,

Brum PC (2008) Exercise training delays cardiac dysfunction and

prevents calcium handling abnormalities in sympathetic hyper-

activity-induced heart failure mice. J Appl Physiol

104(1):103–109. doi:10.1152/japplphysiol.00493.2007

34. Masson GS, Costa TS, Yshii L, Fernandes DC, Soares PP,

Laurindo FR, Scavone C, Michelini LC (2014) Time-dependent

effects of training on cardiovascular control in spontaneously

hypertensive rats: role for brain oxidative stress and inflammation

and baroreflex sensitivity. PLoS One 9(5):e94927. doi:10.1371/

journal.pone.0094927

35. Medeiros A, Oliveira EM, Gianolla R, Casarini DE, Negrao CE,

Brum PC (2004) Swimming training increases cardiac vagal

activity and induces cardiac hypertrophy in rats. Braz J Med Biol

37(12):1909–1917

36. Jia LL, Kang YM, Wang FX, Li HB, Zhang Y, Yu XJ, Qi J, Suo

YP, Tian ZJ, Zhu Z, Zhu GQ, Qin DN (2014) Exercise training

attenuates hypertension and cardiac hypertrophy by modulating

neurotransmitters and cytokines in hypothalamic paraventricular

nucleus. PLoS One 9(1):e85481. doi:10.1371/journal.pone.

0085481

37. Veras-Silva AS, Mattos KC, Gava NS, Brum PC, Negrao CE,

Krieger EM (1997) Low-intensity exercise training decreases

cardiac output and hypertension in spontaneously hypertensive

rats. Am J Physiol 273(6.2):H2627–H2631

38. Whelton PK, He J, Appel LJ, Cutler JA, Havas S, Kotchen TA,

Roccella EJ, Stout R, Vallbona C, Winston MC, Karimbakas J

(2002) Primary prevention of hypertension: clinical and public

health advisory from the National High Blood Pressure Education

Program. JAMA 288(15):1882–1888

39. Doggrell SA, Brown L (1998) Rat models of hypertension, car-

diac hypertrophy and failure. Cardiovasc Res 39(1):89–105

40. Osterholt M, Nguyen TD, Schwarzer M, Doenst T (2013)

Alterations in mitochondrial function in cardiac hypertrophy and

heart failure. Heart Fail Rev 18(5):645–656. doi:10.1007/s10741-

012-9346-7

41. Kemi OJ, Ceci M, Wisloff U, Grimaldi S, Gallo P, Smith GL,

Condorelli G, Ellingsen O (2008) Activation or inactivation of

cardiac Akt/mTOR signaling diverges physiological from path-

ological hypertrophy. J Cell Physiol 214(2):316–321. doi:10.

1002/jcp.21197

42. Grune T, Schonheit K, Blasig I, Siems W (1994) Reduced

4-hydroxynonenal degradation in hearts of spontaneously

Mol Cell Biochem (2015) 402:193–202 201

123

hypertensive rats during normoxia and postischemic reperfusion.

Cell Biochem Funct 12(2):143–147. doi:10.1002/cbf.290120210

43. Crowley SD (2014) The cooperative roles of inflammation and

oxidative stress in the pathogenesis of hypertension. Antioxid

Redox Signal 20(1):102–120. doi:10.1089/ars.2013.5258

44. Isom AL, Barnes S, Wilson L, Kirk M, Coward L, Darley-Usmar

V (2004) Modification of cytochrome c by 4-hydroxy-2-nonenal:

evidence for histidine, lysine, and arginine-aldehyde adducts.

J Am Soc Mass Spectrom 15(8):1136–1147. doi:10.1016/j.jasms.

2004.03.013

45. Cunha TF, Bacurau AV, Moreira JB, Paixao NA, Campos JC,

Ferreira JC, Leal ML, Negrao CE, Moriscot AS, Wisloff U, Brum

PC (2012) Exercise training prevents oxidative stress and ubiq-

uitin-proteasome system overactivity and reverse skeletal muscle

atrophy in heart failure. PLoS One 7(8):e41701. doi:10.1371/

journal.pone.0041701

46. Tang M, Li J, Huang W, Su H, Liang Q, Tian Z, Horak KM,

Molkentin JD, Wang X (2010) Proteasome functional insuffi-

ciency activates the calcineurin-NFAT pathway in cardiomyo-

cytes and promotes maladaptive remodelling of stressed mouse

hearts. Cardiovasc Res 88(3):424–433. doi:10.1093/cvr/cvq217

47. Chen B, Ma Y, Meng R, Xiong Z, Zhang C, Chen G, Zhang A,

Dong Y (2010) MG132, a proteasome inhibitor, attenuates

pressure-overload-induced cardiac hypertrophy in rats by modu-

lation of mitogen-activated protein kinase signals. Acta Biochim

Biophys Sin 42(4):253–258

48. Gu Q, Wang B, Zhang XF, Ma YP, Liu JD, Wang XZ (2014)

Chronic aerobic exercise training attenuates aortic stiffening and

endothelial dysfunction through preserving aortic mitochondrial

function in aged rats. Exp Gerontol 56:37–44. doi:10.1016/j.

exger.2014.02.014

202 Mol Cell Biochem (2015) 402:193–202

123