J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 3 5 1 36 5

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NanoUPLC/MS E proteomic analysis reveals

modulation on left ventricle proteome from
hypertensive rats after exercise training

Bernardo A. Petriza,b , Jeeser A. Almeidaa,b,d , Clarissa P.C. Gomesa , Rinaldo W. Pereiraa, f ,

Andr M. Muradc , Octavio L. Francoa,e, f,
a
Centro de Anlises Protemicas e Bioqumicas, Programa de Ps-Graduao em Cincias Genmicas e Biotecnologia, Universidade Catlica de Braslia,
Braslia-DF, Brazil
b
UDF Centro Universitrio, Braslia, DF, Brazil
c
Embrapa Recursos Genticos e Biotecnologia Laboratrio de Biologia Sinttica, Braslia-DF, Brazil
d
Programa de Ps Graduao em Cincias e Tecnologias em Sade, Universidade de Braslia UnB, Ceilndia-DF, Brazil
e
S-Inova, Ps-graduao em Biotecnologia, Universidade Catlica Dom Bosco, Campo Grande MS, Brazil
f
Programa de Ps-Graduao em Educao Fsica, Universidade Catlica de Braslia, Braslia-DF, Brazil

AR TIC LE I N FO ABS TR ACT

Article history: NanoUPLC/MSE was used to verify the effects of 8 weeks of low (SHR-LIT = 4) and high
Received 20 August 2014 (SHR-HIT = 4) intensity training over the left ventricle proteome of hypertensive rats (SHR-C = 4).
Accepted 17 October 2014 Training enhanced the aerobic capacity and reduced the systolic blood pressure in all exercised
rats. NanoUPLC/MSE identified 250 proteins, with 233 in common to all groups and 16 exclusive to
SHR-C, 2 to SHR-LIT, and 2 to the SHR-HIT. Cardiac hypertrophy related proteins appeared only in
Keywords: SHR-C. The SHR-LIT enhanced the abundance of 30 proteins and diminished 6, while SHR-HIT
Hypertension enhanced the abundance of 39 proteins and reduced other 7. The levels of metabolic ( and
Heart proteome -enolase, adenine phosphoribosultransferase, and cytochrome b-c1), myofibril (myosin light
Left ventricle chain 4, tropomyosin and -chain), and transporter proteins (hemoglobin, serum albumin,
NanoUPLC-MSE and hemopexin) were increased by both intensities. Transcription regulator and histone variants
Exercise were enhanced by SHR-LIT and SHR-HIT respectively. SHR-LIT reduced the concentration of

Abbreviations: Alb, serum albumin; AMP, adenosine monophosphate; ANT1, ADPATP translocase 1; APRT, adeninse phos-
phoribosyltransferase; BP, blood pressure; CAP1, protein DJ 1; CEP52, ubiquitin 60S ribosomal protein L40; Chtf8, chromosome
transmission fidelity protein 8 homolog isoform 2; CTE1, acyl coenzyme A thioesterase 1; CyC2, cytochrome c testis specific; DBP, diastolic
blood pressure; DC147, basigin; Des, desmin; DLDH, dihydrolipoyl dehydrogenase mitochondrial; FHL-2, four and a half LIM domains
protein 2; FUMH, fumarate hydratase mitochondrial; GO, gene ontology; GPx-1, glutathione peroxidase 1; H2A.Z, histone H2Az; H2A1,
histone H2A type 1; H2A1C, histone H2A type 1C; H2A1E, histone H2A type 1 E; H2A1F, histone H2A type 1 F; H2A2A, histone H2A type 2AE;
H2A3, histone H2A type 3; H2A4, histone H2A type 4; H2AJ, histone H2AJ; Hbb1, hemoglobin subunit beta 1; Hbb2, hemoglobin subunit
beta 2; Hpx, hemopexin; HSP70-1/HSP70-2, heat shock protein 70 1A1B; IT, maximal velocity incremental text; LV, left ventricle; LVH, left
ventricle hypertrophy; MLSS, maximum lactate steady state; MSE, -enolase; MtCk, creatine kinase U mitochondrial type; MyBP-C,
myosin binding protein C cardiac type; MYL4, myosin light chain 4; NSE, -enolase; PGK1, phosphoglycerate kinase 1; PKLR, pyruvate
kinase isozymes RL; Prdx6, peroxiredoxin 6; PRG-2, prostaglandin-2; QCR6, cytochrome b c1 complex subunit 6 mitochondrial; RS27A,
ubiquitin 40S ribosomal protein S27a;SBP, Systolic blood pressure;SHR-C, spontaneously hypertensive rat - control group; SHR-HIT, high
intensity training; SHR-LIT, low intensity training; TAGL2, transgelin-2; TIM, pyruvate kinase isozymes RL; TnTc, troponin T cardiac;
TPM1, 3 and 4, tropomyosin alpha 1, 3 and 4 chain; Tpm2, tropomyosin beta chain; UBB, polyubiquitin B; UBC, polyubiquitin C; VDAC3,
voltage dependent anion selective channel protein 3; VIM, vimentin
Corresponding author at: Centro de Anlises Protemicas e Bioqumicas, Programa de Ps-Graduao em Cincias Genmicas e Biotecnologia, Universidade
Catlica de Braslia SGAN, Quadra 916, Mdulo B, Av. W5 Norte, CEP 70.790-160 Braslia-DF, Brazil. Tel.: +55 61 3448 7220; fax: +55 61 3347 4797.
E-mail addresses: ocfranco@pos.ucb.br, ocfranco@gmail.com (O.L. Franco).

http://dx.doi.org/10.1016/j.jprot.2014.10.010
1874-3919/ 2014 Elsevier B.V. All rights reserved.
352 J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 3 5 1 36 5

myosin binding protein C, while desmin and membrane voltage dependent anion selective
channel protein-3 were reduced only by SHR-HIT. In addition, polyubiquitin B and C, and
transcription regulators decreased in both intensities. Exercise also increased the concentration
of anti-oxidant proteins, peroxiredozin-6 and glutathione peroxidase-1.

Biological significance
Pathologic left ventricle hypertrophy if one of the major outcomes of hypertension being a
strong predictor of heart failure. Among the various risk factors for cardiovascular disorders,
arterial hypertension is responsible for the highest rates of mortality worldwide. In this way,
this present study contribute to the understanding of the molecular mechanisms involved in
the attenuation of hypertension and the regression of pathological cardiac hypertrophy
induced by exercise training.

2014 Elsevier B.V. All rights reserved.

1. Introduction By this means, it is well established that exercise training

Among the various risk factors for cardiovascular disorders
(CVD), arterial hypertension is responsible for the highest rates
of mortality worldwide, being considered a global epidemic by the
World Health Organization [1]. With 95% of its cases attributed to
unknown factors, untreated hypertension leads to pathologic
cardiac remodelling, which chronically compromises cardiovas-
cular function [2]. With a direct response to enhanced peripheral
vascular resistance and hemodynamic overload, pathologic
heart hypertrophy occurs with greater maladaptive remodelling
in the left ventricle myocardium [2]. Therefore, maladaptive left
ventricle hypertrophy (LVH) resulting from hypertension is a
strong marker for cardiac events and strongly associated
with abnormal electrical conduction (e.g. arrhythmia) and
development of congestive heart failure [3].
Considering heart plasticity, several aspects have been
linked specifically to pathologic hypertrophy in contrast to
physiologic hypertrophy (e.g., pregnancy and exercise), where
cardiac structure and function are maintained normally
without progression to heart failure [4]. Hypertension has
been associated with a concentric hypertrophy pattern linked
to structural dysfunction within sarcomere organization and
abnormalities in the regulation of contractile proteins,
especially seen in the shift of myosin heavy chain (MyHC)
protein species, from -MyHC to -MyHC [5]. In addition,
alteration of calcium handling [6] and impaired regulation of
ion transporter proteins [7], collagen infiltration, and fibrosis
within myocytes are also strong contributors to a diminished
contractive performance of concentric hypertrophied heart.
Furthermore, a substantive alteration of heart metabolism with
reduced free fatty acid metabolism and increased glycolytic
metabolism are also peculiar aspects [8,9]. In animal models,
enhanced apoptosis signalling can play a central role in
pathologic hypertrophy and heart failure progression [10,11].
Despite that, maladaptive modulations are chronically
progressive with sustained high blood pressure. The litera-
ture describes myocardium hypertrophy as a rapid response
to hemodynamic stress due to heart plasticity. The heart
proteome is also acutely regulated in a few hours followed by
afterload insult, leading to rapid heart phenotype remodel-
ling [12], as well as to other stimuli such as exercise [13],
which can restore maladaptive remodelling and delay heart
failure [14,15].
maintains and improves physical capacity and improves health
status. It is also considered a non-pharmacologic agent with
strong evidence supporting its recommendation as one of the
main clinical interventions for preventing and attenuating
several pathologic conditions such as cardiovascular disease
and correlated metabolic disorders [16]. The benefits of exercise
for the cardiovascular system are not limited to the myocardi-
um, but extend intensively to the peripheral vasculature as well
as to systemic effects on the metabolism [17], immune system
[18], cognition, and others [19]. Among the cardiovascular
diseases, hypertension has received attention due to a rapid
and sustained response to exercise training, leading exercise to
be seen as an important co-therapy agent in its treatment
[20,21]. Therefore, exercise is successfully used in cardiac
rehabilitation programs and is also associated with diminished
mortality rates [22,23].
Several studies are being conducted in order to demonstrate
how exercise training may convert pathological to physiological
hypertrophy by re-establishing the several dysfunctional param-
eters which are directly resultant from hypertension-derivate
cardiac remodelling [14,24,25]. Much of this research has
been performed on spontaneously hypertensive rats (SHR),
since this animal model closely mimics the effects of
essential hypertension with massive data demonstrating its
left ventricle hypertrophy (LVH) [20,24]. Thus, exercise is
shown to reduce apoptosis profile [14], improve systolic and
diastolic function [20], restore metabolic profile [26], calcium
handling [27], and contractile function [24]. Furthermore, it is
important to consider that many of these researches have
focused only on a few targets and there is a wide range of
animal ages and types of exercise stimulus. Thus, consider-
ing heart plasticity, proteomic investigations might reveal a
collection of significant data.
Despite the large body of heart proteome research in
response to a number of diseases, little research has been
conducted in response to exercise stimulus on the non-
pathologic heart [2832] and even less on pathologic conditions
such as ischemic insult [33], infarction [34], and obesity [13].
Although the majority of these data are gathered by gel-based
techniques, LC-MS methods are the most up-to-date, being
used as qualitative and quantitative analytical approaches to
complement gel-based methods and considerably enhance
proteomic data [3537].
 J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 3 5 1 36 5
The restorative effect of exercise on cardiac hypertrophy is
still not fully understood, and proteomic analysis plays a
central role in the investigation of the mechanisms related to
the transition of pathologic to physiologic cardiac hypertro-
phy. In this matter, the present study aimed to analyze the
effect of exercise training on the LV proteome of spontane-
ously hypertensive rats by nanoUPLC-MSE. Moreover, two
different intensities of endurance exercise were used to verify
the effect of exercise dose-response on hypertensive hemo-
dynamic and cardiac proteome. In addition, to date this has
been the first study to contrast different exercise intensities
with proteomic data from a hypertensive rat model.
2. Material and methods
2.1. Animals
All the procedures in this work were conducted according
to international ethical standards [38], and were approved
by the local committee on animal care and use. The animals
were kept in collective cages in controlled temperature
(23 2 C) with 12:12 h dark-light cycle with water and food
provided ad libitum. Fifteen male hypertensive rats (SHR)
were obtained from the Federal University of So Paulo with
21 weeks of age.
2.2. Exercise adaptation and training
Prior to exercise training, animals were familiarized with the
treadmill device (Li 870, Letica Scientific Instruments, Barce-
lona, Spain) for 5 days per week for 2 weeks. During this
period, speed and duration were increased progressively, in
which the velocities and duration of exercise were 10 m.min1
for 10 min, 15 m.min1 for 12 min and 20 m.min 1 for 15 min
[39]. The adaptation protocol aims to minimize stress and
physiological changes related to physical exercise. After the
adaptation period, 15 animals were divided in 3 groups: SHR
control group (SHR-C; n = 5), SHR low intensity exercise group
(SHR-LIT; n = 5) and SHR high intensity exercise group
(SHR-HIT; n = 5). After the adaptation period, all animals
from the exercise group underwent 30 min of continuous
running exercise, 5 days per week for 8 weeks. Exercise
intensities were based on the maximal lactate steady state
(MLSS) previously identified at 20 m.min1 in this animal
model by our group [39]. The SHR-LIT group trained at 20%
under MLSS (16 m.min1), corresponding to a low intensity
exercise and the SHR-HIT group trained at 15% above MLSS
(24 m.min1), considered a high exercise intensity. A maximal
velocity incremental test (IT) was performed pre-exercise
training and at the end of the 4th and the 8th week of exercise
training. The IT was performed with increments of 3 m.min1
every 3 min, starting at 8 m.min 1 until animal exhaustion as
described previously [39]. The IT at the 4th week of exercise
was used to adjust the exercise intensities for both animal
groups, and at the end of the 8 weeks of exercise, to verify
cardiovascular adaptation to both exercise-training intensi-
ties. During the experiment, 2 rats died (from SHR-C and
SHR-LIT respectively). Exercise experimental design is presented
in Fig. 1.
353
2.3. Arterial systolic and diastolic blood measures
Determination of systolic blood pressure (SBP) was performed
by the tail cuff method [40] (Panlab NIPB system, Barcelona
Spain) with an mild sedation with 2% xylazine (10 mg.kg1) and
10% ketamine (10 mg.kg1). Eight consecutive measurements
were performed, where the first three were not considered.
Blood pressure (BP) was measured before exercise training
period (animals with 22 weeks of age) and at the end of the eight
weeks of exercise training (30 weeks of age).
2.4. Tissue extraction
Twenty-four hours after the last session of exercise, all animals
were sedated with 2% xylazine (50 mg.kg1) and 10% ketamine
(80 mg.kg1) and euthanized. Hearts were excised, immedi-
ately rinsed with chilled PBS solution and weighed. The left
ventricle free wall was dissected and washed again in chilled
PBS solution to remove blood and external contaminants.
Subsequently, the LV fragments were all immediately frozen in
liquid nitrogen and stored at 80 C until protein extraction and
proteomic analysis.
2.5. Total soluble protein extraction from left ventricle
Left ventricle soluble proteins were extracted according to
Comunian et al. [36] with adaptations. LV tissue from SHR-C
(n = 4; 0.14 0.02 g), SHR-LIT (n = 4; 0.13 0.02 g) and SHR-HIT
(n = 4; 0,12 0.01 g) were individually homogenized in ice-cold
lysate buffer (300 mM Sucrose, 10 mM Tris, 2 mM EDTA, 10 mM
DTT, 1 mM PMSF and 0.1% SDS) at the proportion of 0.150 g per
0.5 ml (w/v). After homogenization, sample was centrifuged at
1000 g for 10 min, supernatant was collected and concentrated
by speedvac (Eppendorf) to adjust the volume to RapiGEST
surfactant concentration. Sample was then resuspended in
1000 mM ammonium bicarbonate and 0.5% RapiGEST
(Waters, Milford, MA, USA) and heated at 95 C for 5 min to
extract membrane proteins. Protein concentration from each
extraction was assayed by Qubit, then protein concentration
was adjusted to 50 l for trypsin digestion.
2.6. Sample preparation for nanoUPLC-MSE acquisition
An aliquot of 50 L at 1 g.L1 of total soluble protein from each
sample was added to 10 L of 50 mM ammonium bicarbonate in
a microcentrifuge tube. Next, 25 l of RapiGEST (0.2% v/v) was
added, and samples were vortexed and incubated in a dry bath
at 80 C for 15 min. The sample was briefly centrifuged, and
2.5 L of 100 mM DTT was added. The sample was vortexed
gently and incubated at 60 C for 30 min, followed by centrifu-
gation. Iodoacetamide (2.5 L of a 300 mM solution) was added,
and the sample was vortexed slightly and incubated in the dark
at room temperature for 30 min. Next, 10 l of trypsin (with
400 L of 100 mM ammonium bicarbonate added per 20 g vial
of trypsin) was added, and the sample was homogenized
slightly. The sample was digested at 37 C in a dry bath
overnight. To precipitate the RapiGEST, 10 L of a 5% TFA
solution was added, and samples was homogenized, incubated
for 90 min at 37 C in a dry bath, and centrifuged at 10,000 g at
6 C for 30 min. The supernatant was transferred to a Waters
354 J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 3 5 1 36 5

Total Recovery vial (Waters, USA), freeze dried, and resus-
pended in 100 L of 200 mM of ammonium formate solution
containing 50 fmol.L 1 of Rabbit Phosphorilase B (PhosB)
digested standard (Waters, USA). The final concentration of
the protein was 0.5 g.L 1, and the final concentration of
PhosB was 50 fmol.L 1.
2.7. NanoUPLC-MSE acquisition
Nanoscale LC separation of tryptic peptides from TSP was
performed using a nanoACQUITY system (Waters Corp., USA)
with 2D dilution technology, where each individual sample was
treated separately and two injections of each individual was
carried out. The first dimension (FD) was performed using an
XBridge 300 mm x 50 mm nanoEase BEH130 C18, 5 m
particle. The second dimension (SD) was performed with a
Symmetry C18 5 m, 5 mm x 300 m pre-column and a HSST3
C18, 1.8 m particle, 75 m x 150 mm analytical reversed phase
column (Waters, Milford, USA). In the FD, the mobile phase A was
20 mM ammonium formate, and mobile phase B was acetoni-
trile. In the SD mobile phase A was 0.1% formic acid in water, and
mobile phase B was 0.1% formic acid in acetonitrile. Two runs
were performed. The first one was a 1D simulation lasting 70 min
to check digestion and quantification of sample. The second was
a full 2D run using 5 fractions taking 5 h to complete. For the first
run, 2 ul of the samples were initially transferred to the FD
column in 0.5 min at 2 L.min1 and 0.1% B. Peptides were eluted
from FD at 2 L.min1 and 65% of mobile phase B for 4 min and
diluted to the pre-column on SD using an aqueous 0.1% formic
acid solution with a flow rate of 20 L.min1 for 20.5 min. The
peptides were separated using a gradient of 740% mobile
phase B for 54 min with a flow rate of 500 L.min 1 followed

Fig. 1 Experimental design. Schematic illustration of the methodological steps in the following study.
 J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 3 5 1 36 5
by a 10 min rinse with 85% of mobile phase B. The column
was re-equilibrated to the initial conditions for 10 min. The
column temperature was maintained at 35 C. The lock mass
Glu-fibrino peptide (GFP) was delivered from the fluidics system
of SynaptG2 using a constant flow rate of 500 L.min1 at a
concentration of 320 fmol of GFP to the reference sprayer of the
NanoLockSpray source of the mass spectrometer. After acqui-
sition, samples were quantified using the protein identification
method described below, and the second run using 5 fractions
was performed by adjusting the injected volume to a concen-
tration of 500 g. The samples were initially transferred to the
FD column in 0.5 min at 2 L.min 1 and 0.1% B. Peptides for
the first fraction were eluted from FD at 2 L.min 1 and 10.8%
of mobile phase B for 2 min and diluted to the pre-column on
SD using an aqueous 0.1% formic acid solution with a flow
rate of 10 L.min 1 for 8.5 min. The peptides were separated
using a gradient of 735% mobile phase B for 37 min with a
flow rate of 500 L.min 1 followed by a 5 min rinse with 85%
of mobile phase B. The column was re-equilibrated to the
initial conditions for 5 min. The column temperature was
maintained at 35 C. Peptides for the second, third, fourth
and fifth fractions were eluted using 14, 16.7, 20.4 and 50% of
mobile phase B respectively. The conditions for dilution, flow
rate and SD analysis were the same as for the first fraction.
Lock mass was delivered in all fractions in the same conditions
described above. All samples were analyzed in duplicate. The
tryptic peptides were analyzed using a Synapt G2 HDMS
mass spectrometer (Waters, Manchester, UK) with a hybrid
quadrupole/ion mobility/orthogonal acceleration time-of-flight
(oa-TOF) geometry. For all measurements, the mass spec-
trometer was operated in the resolution mode of analysis
with a typical resolving power of at least 20,000 full-width
half-maximum (FWHM). All analyses were performed using a
positive nanoelectrospray ion mode (nanoESI +). The time-
of-flight analyzer of the mass spectrometer was externally
calibrated with GFP b + and y + ions from m/z 50 to 1990 with
the data post acquisition lock mass corrected using the GFP
double charged precursor ion [M + 2H]2 + = 785.8426. The
reference sprayer was sampled at a frequency of 30 s. Exact
mass retention time (EMRT) nanoUPLC-MSE data were col-
lected in an alternating low energy and elevated energy mode
of acquisition [41]. The continuum spectra acquisition time,
in each mode, was 1.5 s with a 0.1 s interscan delay. In the
low energy MS mode, data were collected at constant
collision energy of 3 eV. In the elevated energy MS mode,
the collision energy was ramped from 12 to 45 eV during each
1.5 s spectrum. The radio frequency applied to the quadru-
pole mass analyzer was adjusted so that the ions from m/z
200 to 2000 were efficiently transmitted, which ensured that
any ions less than m/z 200 that were observed in the LC-MS
data only arose from dissociations in the TRAP T-wave
collision cell.
2.8. Data processing and protein identification
The MS data that were obtained from the nanoUPLC-MSE were
processed and searched using the ProteinLynx Global Server
(PLGS) version 2.5.2 (Waters, Manchester, UK). Proteinlynx soft-
ware has dedicated algorithms to validate and curates the
data to deliver confident data. Proteins were identified using
355
the softwares embedded ion accounting algorithm and a
search of the Uniprot Rattus novergicus database with MassPREP
digestion standards (MPDS) UniProtKB/Swiss-Prot sequences
(Phosphorylase P00489 PHS2_RABIT, Bovine Hemoglobin
P02070 HBB_BOVIN, ADH P00330 ADH1_YEAST, BSA
P02769 ALBU_BOVIN) appended to the database. Identifications
and quantitative data packaging were generated using dedicated
algorithms [4144] and a search against a species-specific Uniprot
database. The ion detection, clustering, and log-scale parametric
normalizations were performed in PLGS with an ExpressionE
license installed. The intensity measurements were typically
adjusted on these components, i.e., deisotoped and charge state-
reduced EMRTs that were replicated throughout the
complete experiment for the analysis at the EMRT cluster
level (Supplementary data 1). The fixed modification of
carbamidomethyl-C was specified, and the variable modifica-
tions included were amidation C-terminus, deamidation N,
deamidation Q, methyl N-teminus, methyl C-terminus, glycation
N-terminus, O-GlcNac ST and oxidation M. Components were
typically clustered with a 10 ppm mass precision and a 0.25 min
time tolerance against the database-generated theoretical pep-
tide ion masses with a minimum of one matched peptide
(Supplementary data 2). The alignment of elevated energy ions
with low energy precursor peptide ions was conducted with
an approximate precision of 0.05 min. One missed cleavage
site was allowed. The precursor and fragment ion tolerances
were determined automatically. The protein identification
criteria also included the detection of at least three fragment
ions per peptide, six fragments per protein, the determination
of at least one peptide per protein, and the identification of
the protein was allowed with a maximum 4% false positive
discovery rate in at least three technical repeatability injections.
Using protein identification replication as a filter, the false
positive rate was minimized because false positive protein
identifications, i.e., chemical noise, have a random nature and
do not tend to replicate across injections. For the analysis of
protein identification and quantification level, the observed
intensity measurements were normalized to the intensity
measurement of the identified peptides of the digested internal
standard. The total protein identification was considered
after filtering results to the minimum of two replicates,
where proteins were only considered if its identification was
observed across both injections for each individual. More-
over, only proteins with up regulation of Student t test P 0.95
and down regulation of P 0.05 were considered, with a
delta () of at least () 0.5-fold change in Log(e) ratio between
the treatments.
2.9. Statistical analysis
Normality analysis was verified through Shapiro-Wilk test
(P > 0.05) and data are presented as mean and standard
deviation. For comparison of variables (eg. Vmax and SBP)
between groups an ANOVA one-way with post hoc test by
Bonferroni was used. For the inter-group comparative analysis
of protein abundance levels, only proteins with up regulation of
Student t test P 0.95 and down regulation of P 0.05 were
considered, with a delta () of at least () 0.5-fold change in
Log(e) ratio between the treatments. Among these proteins,
P-values less than 0.05 were considered significant [45].
356 J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 3 5 1 36 5

3.2. Functional proteome annotations

3. Results
NanoUPLC-MSE analysis of left ventricle proteome resulted in
3.1. The effect of exercise on hypertensive phenotype the identification of 250 distinct proteins (Supplementary data
3). Despite being part of this list, the proteins used as
In the present study, a systematic analysis of the effects of two quantitation standards (Phosphorylase P00489 PHS2_RABIT,
distinct exercise-training intensities on the LV myocardium Bovine Hemoglobin P02070 HBB_BOVIN, ADH P00330
proteome of SHR rats was performed. Initially, in order to ADH1_YEAST, BSA P02769 ALBU_BOVIN) were not consid-
characterize the hypertension degree of 22-week-old SHR rats, ered for comparative analysis. Moreover, the identified proteins
the SBP of all animals was measured prior to the experiment were classified by their cellular localization, molecular function
beginning. A mean value of SBP (169.8 13.8 mmHg-1) indicated and biologic process according to Gene Ontology (GO) annota-
the hypertensive phenotype of all animals before exercise tions. Supplementary data 2 shows the identified proteins
training (Table 1). Furthermore, all rats were randomly according to their cellular localization (A), biologic process
allocated to one of the three experimental groups; untrained (B) and metabolic function (C). In this study, the majority of
control (SHR-C), low intensity training (SHR-LIT) and high the identified proteins were derived from mitochondrion (38%)
intensity training (SHR-HIT) as shown in the experimental followed by cytoplasm (23%), cytoskeleton/myofibril (14%) and
design in Fig. 1. nucleus (12%) (Supplementary data 4A). Hence, some proteins
The positive effects of eight weeks of exercise training in may be allocated in a distinct cellular locus, which also reveals
high BP was confirmed by the reduction of the SBP compared the nature of their biologic and molecular function. Following
to pre-exercise period in the SHR-LIT group (168.5 9.2 vs. the enhanced number of mitochondrial proteins, the biological
156.1 5.6 mmHg 1) and in the SHR-HIT group (168.5 9.2 vs process annotation also indicated that the majority of the
139.4 10.5 mmHg1). The untrained rats (SHR-C) presented a identified proteins were related to metabolic processes (38%),
continuous elevation in the SBP levels (from 168.5 9.2 to followed by transport activity (11%), muscle contraction (8%),
182 8.9 mmHg1) (Table 1). cell organization (8%) and oxidation-reduction-process (7%)
The incremental test (IT) of maximal velocity demonstrated (Supplementary data 4B). Lastly, proteins were classified accord-
that 4 and 8 weeks of exercise were sufficient to improve ing to their molecular function, where proteins with enzymatic
the aerobic capacity of both exercised groups; SHR-LIT activity represented almost 50% of all molecular functions (45%),
(pre-exercise; 27.2 2.5 m.min1 vs. 8 weeks; 32.8 4.5 m.min1, followed by binding (20%), structural (16%), transport (8%), and
P < 0.05), SHR-HIT (pre-exercise; 26 4.2 m.min1 vs. 4 weeks; chaperone activity (5%) (Supplementary data 4C).
33.3 1.7 m.min1 vs. 8 weeks; 39.8 3.4 m.min1 P < 0.05*) A Venn diagram was used to show the distribution of the
(Fig. 2). It was observed that 4 weeks of SHR-HIT presented an identified proteins into the three experimental groups (Fig. 3).
enhanced velocity in the IT compared to the SHR-C group From the 250 identified proteins, 223 were common to all
(pre-exercise, 4th and 8th weeks) (P < 0.05 ), while 8 weeks of three groups, with 16 proteins being excluFsive to the non-
SHR-HIT enhanced the velocity in the IT compared to SHR-C exercised control group (SHR-C), 2 proteins exclusive to the
(pre-exercise, 4th and 8th weeks) and SHR-LIT group by the 4th LIT (chromosome transmission fidelity protein 8 homolog
and 8th weeks of training (P < 0.05 ). Considering morpholog- isoform 2 and basigin) and 2 proteins exclusive to the HIT
ical parameters, no significant difference between groups was group (prostaglandin reductase 2 and transgelin 2) (Fig. 3). The
observed in the heart-to-body weight (HW/BW) ratio, usually identified proteins exclusive to the SHR-C group were mainly
used as a physiologic marker of cardiac hypertrophy (Table 1). related to metabolic processes (38%) followed by nucleosome
Sample characterization is presented in Table 1. assembly proteins (25%). The proteins exclusive to LIT are
related with apoptosis (prostaglandin reductase 2) and muscle
Table 1 Experimental rat group characterization. development (transgelin 2), while in the HIT group one protein
is associated with cell communication (basigin), and the
SHR-C SHR-LIT SHR-HIT
biologic process of chromosome transmission for protein 8 is
N 4 4 5 not predicted by Gene Ontology annotations (Fig. 3). Moreover,
Age pre-training 22 22 22 the majority of the 223 proteins shared between all groups are
Age euthanasia 33 33 33
responsible for diverse metabolic processes (Supplementary
BW pre-training (g) 306.3 16.6 315.3 5.8 299 10.9
BW post-training (g) 317.5 22.7* 337 18.8 305 27
data 4).
Heart weight (g) 1.4 0.1 1.4 0.1 1.3 0.2
HW/BW (mg/g) 4.5 0.2 4.2 0.2 4.3 0.2 3.3. Evaluation of different exercise intensities over ventricular
SBP pre-training (mmHg1) 168.5 9.2 168.5 9.2 168.5 9.2 proteome
SBP post-training (mmHg1) 182 8.9a 156.1 5.6b 139.4 10.5c Hence, the ratio of SHR-LIT:SHR-C and the ratio of SHR-HIT:
SBP pre/post training 13.5 12.4 29.1
SHR-C were compared. This analysis aimed to assess the
Footnote: SPB (systolic blood pressure); BW (body weight), HW/BW effect of high and low intensity exercise training over non-
(heart weight to body weight ratio), SBP (systolic blood pressure), exercised LV proteome (SHR-C). In addition, both exercised
SBP (delta of blood pressure). Symbols indicate significant difference groups were also compared between them (SHR-LIT:SHR-HIT).
where; * different from BW pre-training (0.01); different from From all identified proteins, the abundance of 36 proteins
SHR-LIT and SHR-HIT (P = 0.0001), a different from SBP pre-training
was shown to be followed by low intensity training in the
(P = 0.04), kdifferent from SBP pre-training (P = 0.02) and c different
from SBP pre-training (P = 0.003).
SHR-LIT:SHR-C analysis (Supplemental data 5) compared
to 44 altered proteins after SHR-HIT in the SHR-HIT:SHR-C
 J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 3 5 1 36 5 357

Fig. 2 Incremental text (IT) of maximal velocity. Comparison between the TI velocities (m.min1) applied to all three animal
groups (SHR-C, SHR-L and SHR-H) before the training program (pre-training 0 white bars), after four weeks (weeks 4 grey
bars) and at the end of eight weeks of exercise training (week 8 black bars). Symbols are related to: * P 0.05 from week 0 in
each respective group; # P 0.05 from week 4; 0.05 vs. SHR-H in week 4; P 0.05 vs. SHR-H in week 8.

Fig. 3 Venn analysis. Venn analysis of all identified proteins interplayed in SHR-C (gray), SHR-L (blue) and SHR-H (red) groups.
Exclusive proteins are listed and assigned their biologic process according to Gen Ontologies (GO) annotations.
358 J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 3 5 1 36 5
 J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 3 5 1 36 5 359

analysis (Supplemental data 6). The effects of SHR-LIT and
SHR-HIT on LV proteins were grouped according to their
biologic processes and are presented all together in Fig. 4.
A list of all identified proteins in each group is presented
in Supplemental Data 7 (SHC-C), 8 (SHR-L) and 9 (SHR-H)
in addition to peptide sequences of all identified proteins
(Supplementary data 10).
From the 36 proteins altered after SHR-LIT, the concentration
of 30 of them were enhanced and 6 were shown to be reduced.
In this analysis (SHR-LIT:SHR-C), proteins were mainly related
to metabolic processes (12 enhanced and 2 reduced), muscle
contraction and cell component organization (7 enhanced and 1
reduced), transport (5 enhanced) and translation, and transcrip-
tion regulation (1 enhanced and 3 reduced). Five remaining
proteins were all enhanced, related to nucleosome assembly,
proteolysis, biologic regulation, oxidation-reduction process, and
stress response (Supplementary data 5 and Fig. 4).
From the 44 proteins altered after SHR-HIT, 37 proteins
were enhanced (P 0.95) while another 7 were reduced (P 0.05).
Different to SHR-LIT, proteins altered by SHR-HIT were
mainly related to Contraction and cell organization process
(12 increased and 1 reduced), metabolic process (8 increased
and 2 reduced), nucleosome assembly (8 increased), transport
(4 increased and 1 reduced), transcription and translation
regulation (3 reduced). The remaining 5 proteins were all
enhanced, being related to biologic regulation, oxidative-
reduction process and immune response (Supplementary
data 6 and Fig. 4).
In addition, when the SHR-LIT group was compared to
the SHR-HIT group, the ration of protein abundance (SHR-LIT:
SHR-HIT Log(e) ratio) revealed that hemoglobin subunit alpha 12
was increased by low exercise intensity towards high intensity
exercise (P 0.95) and adenine phosphoribosyltransferase was
reduced (P 0.05).
4. Discussion
Systemic hypertension is one of the most prevalent diseases
worldwide [46,47] with LVH being a major outcome and a
significant predictor of heart failure [47]. Hence, LVH is
shown to be attenuated by blood pressure control [4851],
contributing to myocardium normalization, and decreasing
cardiovascular morbidity. Exercise training is shown to im-
prove systolic dysfunction [20] and regress pathologic LVH
induced by pressure-overload [24,48,49]. Moreover, anti-
apoptotic effects, improvement in microcirculatory profile
and endothelial vasodilation contribute to peripheral vascular
resistance reduction [49,50,5254]. However, the molecular
mechanisms underlying these events in LVH under hyperten-
sion are still poorly understood.
Here SHR was used, since this is the most common
experimental model of essential hypertension shown to
develop elevated levels of BP in early stages of life and
consistently developing LVH [51,55]. Eight weeks of aerobic
training performed at low and high intensity enhanced
aerobic capacity in all animals, where, as expected, SHR-HIT
presented a higher cardiovascular improvement (Fig. 2). After
the training period the SBP was significantly reduced in
both groups compared to pre-training levels with a greater
reduction observed in SHR-HIT group (SHR-L; 12.4 and
SHR-HIT; 29.1 mmHg 1). Moreover, the SBP of untrained
rats presented a significant elevation (SHR-C; 13.5 mmHg 1)
at the end of the experimental period (Table 1). This response
is in accordance with several studies, where different exercise
regimens and intensities are shown to reduce SBP and improve
cardiovascular fitness [48,49,5658], also contributing to
interrupt pathological hypertrophy in SHR rats [59]. Besides
LVH, other pathologic parameters such as collagen and wall-
to-lumen ratio were also shown to be reduced in hyperten-
sive rats after treadmill training [60]. Moreover, low intensity
training is also reported to significantly reduce SBP in
hypertensive phenotype [6163].
The proposed exercise protocol displayed no alteration
in heart weight and HW/BW ratio, indicating that by these
parameters cardiac morphology was not significantly altered
by training (Table 1). However, alteration in HW/BW ratio
has been evidenced between non-pathologic and SHR rats
submitted to swimming training [64,65]. In an opposite way,
heart weight was decreased with no alteration in the HW/BW
ratio in SHR after treadmill training at moderate-to-high
intensity [14]. Although no cardiac mass alteration was
observed (Table 1), our training regimen led to significant
up-regulation of structural and contractile proteins classi-
cally associated with LVH [66]. Moreover, our proteomic
analysis revealed that 36 proteins were altered by SHR-LIT,
while SHR-HIT altered 44. From these proteins, 24 were
commonly modulated by intensities, the majority being
enhanced rather than reduced (Fig. 4).
Alteration in the pattern of structural and contractile
proteins is one of the main outcomes in pathologic LVH,
with a significant impact on cardiomyocyte contractility [8].
Myocyte fibrosis and impairment contractility affects the
Ca2+-dependent cross-bridge interaction with actin, influenc-
ing the progression of hypertensive LVH to HF. Differentially,
hypertrophy induced by exercise improves force and contrac-
tion regulators, leading to positive cardiac adaptations
[13,28,30]. In contrast to the down-regulation of myosin light

Fig. 4 Effects of exercise training on LV proteome. Histogram of protein abundance levels from intergroup analysis
considering only proteins with up-regulation (P 0.95) and down-regulation (P 0.05), with a delta () of at least () 0.5-fold
change. The X axis represents the Log(e) ratio between the treatments (SHR-L:SHR-C ratio and SHR-H:SHR-C). Enhanced protein
concentrations are represented in blue, where the solid blue represents the effect of HIT on untrained rats (SHR-H:SHR-C) and
striped blue represents the effect of LIT on untrained rats (SHR-L:SHR-C). Reduction in protein concentration are represented in
red, where solid red represents the effect of HIT on untrained rats (SHR-H:SHR-C) and striped red represents the effect of LIT on
untrained rats (SHR-L:SHR-C). All altered proteins are grouped according to their biologic process as noted in Gen Ontology
(GO). Muscle contraction and cell organization process (A), Metabolic process and respiratory electron transport (B), nucleosome
assembly and transcription regulation (C), transport (D) and miscellaneous (E).
360 J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 3 5 1 36 5
chain 1 observed in LV in SHR [67], the abundance of proteins
related to force regulation (myosin light chain 4) and thin
filament structure (tropomyosin beta and alpha chains 1,2, 3,
and troponin T) was enhanced by both training intensities
(Fig. 4A). These alterations indicated positive exercise aspects
over the intrinsic mechanisms of force and contraction reg-
ulation in hypertension. However, troponin T, an indicator
of myocardial cell damage, reported to be released after
prolonged bouts of exercise [68], was observed to be increased
in SHR-HIT animals.
In addition, only myosin binding C cardiac (MyBP-C) (SHR-LIT)
and desmin (SHR-HIT) concentrations were reduced (Fig. 4A). The
MyBP-C protein contributes to hold thick filaments, also being a
substrate of cAMP-dependent protein kinase [69], where MyBP-C
phosphorylation is shown to reduce its interaction with myosin,
evidencing its role in contraction regulation [69]. Therefore,
SHR-LIT might have displayed a negative effect on contraction
regulation. However, the role of MyBP-C phosphorylation induced
by exercise has not yet been investigated, are the exact functional
consequences of this change are still unknown. In contrast,
desmin down-regulation is seen as an effect of exercise on
LVH, since its expression is enhanced in several studies with
pathologic myocyte hypertrophy [67,7072].
Under cardiac hypertrophy, glucose oxidation is enhanced
towards FAO [67,71,7375]. The impairment in mitochondrial
oxidative capacity is shown to play a fundamental role in
early LVH, even before the onset of hypertension [76]. In
contrast, alterations in mitochondrial enzymes induced by
endurance exercise are associated with improvement in
cardiovascular capacity [17,32,66,77,78] and are generally
opposite to proteomic changes seen in heart failure induced
by hypertension [7981]. With the exception of polyubiquitin
B and C, related to ubiquitin-proteosome-mediated muscle
protein degradation [82], all metabolic proteins related with
PCr, nucleotide, FAO, and glucose oxidation were enhanced
(Fig. 4B). Reduced abundance of ubiquitin-proteasome pro-
teins might indicate a negative effect of exercise on cardiac
protein targeting and degradation, since these proteins
are associated with muscle atrophy [83,84]. Curiously, SHR-
LIT altered more metabolic proteins (n = 14) than SHR-HIT
(n = 12), such as acyl coenzyme A thioesterase 1 (FAO),
creatine kinase B type (PCr metabolism) and fumarate
hydratase mitochondrial (Krebs cycle enzyme), including
glycolytic enzymes (Fig. 4B). On the other hand, the exclusive
up-regulation of nucleotide metabolism enzyme (adenine
phosphoribosyltransferase) and bicarbonate formation enzyme
(carbonic anhydrase 2) was only observed after SHR-HIT. These
responses reveal that the alteration in LV metabolic proteome
may be exercise-intensity dependent.
From all metabolic proteins, -enolase, adenine phos-
phoribosyltransferase and cytochrome b c1 were significant-
ly more increased, indicating their sensitive response to
exercise. The glycolytic enzyme -enolase is shown to be
increased by different stimuli of cardiac hypertrophy such as
exercise [66] and hypertension [71,75], indicating the main
role of glucose oxidation in LV remodelling. In addition, the
phosphorylation of -enolase is considered to play a key role
in the development of LVH in SHR rats [67]. However, this fact
has not been investigated in exercise yet. Heart tissue of rat is
shown to contain both protein species and an intermediate
form of -enolase. It is thought that reduction of -enolase
toward -enolase might be associated with beneficial energy
changes in cardiac hypertrophy [85]. However, contrary results
have also been found in right ventricle of rat submitted to
right ventricle pressure overload [79]. In addition, high levels
of -enolase were observed after endurance exercise, being
considered a serum marker of exercise-induced muscle dam-
age [86]. The up-regulation of enzymes involved in nucleotide
metabolism, especially adenine phosphoribosyltransferase
(Fig. 4B), may be associated with the increase of purine
metabolism after exercise [87]. Since this enzyme partici-
pates in the formation of nucleotides [88], this synthesis
might be a response to elevated energy demands in cardio-
myocyte submitted to SHR-HIT. Lastly, cytochrome b c1
complex subunit 6 mitochondrial was also highly enhanced
at both intensities. Elevated concentrations of this protein
class are found in skeletal muscle after training [89], despite
its pro-apoptotic role observed in LV myocyte of SHR. The
up-regulation of cytochrome b c1 seems to be consistent with
ATP synthesis through the generation of proton gradient in
response to enhanced energy demanded during exercise.
Furthermore, exercise also enhances ADPATP transporter 1,
which promotes the transport of ADP and ATP through the
mitochondrial membrane (ATP to cytoplasm and ADP to
mitochondrial matrix) [90]. Thus, the up-regulation of this
protein (SHR-LIT, Fig. 4D) seems to be a cardiomyocyte
response to enhanced energy demands, and its response is
dependent on exercise intensity.
Incorporation of histone variants, histone modification
and DNA methylation are interconnected epigenetic factors
acting on eukaryotic chromatin assembly, making it more or
less prone to transcription [9193]. Pathologic hypertrophy
stimuli are shown to affect nucleosome assembly, inducing
chromatin remodelling influencing transcription regulation
[94,95]. Moreover, the replacement of core histones with
specialized variants is involved in transcriptional regulation,
where the up-regulation of histone 2A.Z was observed during
cardiac hypertrophy, and its knock-down was followed by
hypertrophy reduction [96]. Here, H2A.Z was identified
exclusively in the SHR-C group, suggesting the role of this
protein in LVH in SHR rats (Fig. 3). This protein controls
chromatin remodelling, playing an important role in DNA
repair [97]. The relationship between H2A.Z and untrained
SHR rats might be linked to a physiological status that
produces more oxidative components, increasing DNA lesion
and demanding more DNA repair. Moreover, we found several
nucleosome assembly components from the histone H2A
family enhanced by SHR-HIT. Only H2A type 3 was altered by
both intensities (Fig. 4C). The epigenetic role played by histone
variants showed to be exercise-dose dependent, as most of
the H2A variants were increased in SHR-HIT. The chromatin
remodelling by the increased abundance of H2A variants and
their relationship with gene expression and the improvement
in LVH from pathological to a more physiological phenotype
should be more deeply investigated. In contrast to these
nucleosome assembly proteins, both exercise intensities
played a negative regulatory effect in some transcription and
translation regulation proteins (four and a half LIM domains
protein 2, ubiquitin 40S ribosomal protein S27a and ubiquitin
60S ribosomal protein L40) except for the protein DJ-1, which
 J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 3 5 1 36 5 361

was enhanced only by SHR-LET (Fig. 4C). This protein is
highly expressed in heart and other tissues, playing a
cytoprotective role against oxidative damage and inflammation
process [98,99].
Enhanced apoptotic signalling is characterized in LVH
induced by hypertension [100]. Here, we observed a down-
regulation of voltage-dependent anion selective channel
protein 3 (VDAC3) after SHR-HIT (Fig. 4D), where its protein
specie was also shown to be reduced in myocardial infarcted
rats submitted to exercise training [34]. This protein is
increased in LV in aged rats [101], with a given role in
apoptosis [102], where there is a correlation between VACD2
down-regulation and improved cardiac function. Our data
indicate that VDAC3 and VACD2 respond similarly to exercise
stimuli, which indicates their regulatory effect in pathologic
affected cardiomyocytes.
Furthermore, disturbed ATP synthesis is associated with
mitochondrion Ca2 + overload and enhancement of reactive
oxygen species (ROS) contributing to the pathogenesis
of hypertension [100]. Exercise training was effective in
up-regulating anti-oxidant proteins such as peroxiredoxin 6,
glutathione peroxisade 1 and dihydrolipoyl dehydrogenase
mitochondrial 1 (Fig. 4E). Peroxiredoxin 6 is considered the
main enzyme to exert hydrogen peroxide reductase activity,
where its plasma levels are shown to be induced by
exercise [103]. Moreover, mice lacking peroxiredoxin 6 are
more susceptible to oxidative stress [104]. Regarding gluta-
thione peroxidase-1, a similar proteomic profile was ob-
served in exercised rats with infarcted myocardium [34].
Together, these findings link the redox-based mechanisms
with cardioprotection induced by exercise.
Finally, we have identified some well-known proteins altered
in cardiac hypertrophy (SR-Ca2+-ATPase, phospholamban and
GAPDH) in all three experimental groups (Supplemental data 3, 7,
8 and 9). Nevertheless, no significant alterations were observed
following exercise (Fig. 3). Analysing the proteins that appeared
exclusively in each group, 16 were shown only in untrained rats,
among them cytochrome c testis, vimentin and Histone 2HA.z
have all been associated respectively with apoptosis induction,
fibrosis and hypertrophy induction in cardiomyocytes [105,106].
Furthermore, 2 proteins were found exclusively in each exercised
group (Fig. 3). While the biologic role of chromosome transmis-
sion fidelity proteins is unknown, basigin (SHR-LIT), participates
in several cellular communication processes, including sper-
matocyte differentiation, inflammation and chaperone activi-
ty [107], also being induced by diverse stimuli of metabolic

Fig. 5 Modulation in cardiomyocyte proteome. The main results found in this study are presented in this cardiomyocyte
representation. *: proteins found exclusively to an experimental group, (): increased concentration, (): reduced concentration.
Proteins without any experimental group designation (SHR-LIT or SHR-HIT) were altered by both exercise intensives. Gray arrows
indicate proteins expressed in different subcellular localizations. Protein nomenclature: Alb (serum albumin), AMP; (adenosine
monophosphate), ANT1 (ADPATP translocase 1), APRT (adeninse phosphoribosyltransferase), CAP1 (protein DJ 1), CEP52 (ubiquitin
60S ribosomal protein L40), Chtf8 (chromosome transmission fidelity protein 8 homolog isoform 2), CTE1 (acyl coenzyme A
thioesterase 1), CyC2 (cytochrome c testis specific), DC147 (basigin), Des (desmin), DLDH (dihydrolipoyl dehydrogenase
mitochondrial), FHL-2 (four and a half LIM domains protein 2), FUMH (fumarate hydratase mitochondrial), GPx-1 (glutathione
peroxidase 1), H2A.Z (histone H2Az), H2A1 (histone H2A type 1), H2A1C (histone H2A type 1C), H2A1E (histone H2A type 1 E), H2A1F
(histone H2A type 1 F), H2A2A (histone H2A type 2AE), H2A3 (histone H2A type 3), H2A4 (histone H2A type 4), H2AJ (histone H2AJ),
Hbb1 (hemoglobin subunit beta 1), Hbb2 (hemoglobin subunit beta 2), Hpx (hemopexin), HSP70-1/HSP70-2 (heat shock protein 70
1A1B), MSE (-enolase), MtCk (creatine kinase U type mitochondrial), MyBP-C (myosin binding protein C cardiac type), MYL4
(myosin light chain 4), NSE (-enolase), PGK1 (phosphoglycerate kinase 1), PKLR (pyruvate kinase isozymes RL), Prdx6
(peroxiredoxin 6), PRG-2 (prostaglandin-2), QCR6 (cytochrome b c1 complex subunit 6 mitochondrial), RS27A (ubiquitin 40S
ribosomal protein S27a), TAGL2 (transgelin-2), TIM (pyruvate kinase isozymes RL), TnTc (troponin T cardiac), TPM1, 3 and 4
(tropomyosin alpha 1, 3 and 4 chain), Tpm2 (tropomyosin beta chain), UBB (polyubiquitin B), UBC (polyubiquitin C), VDAC3 (voltage
dependent anion selective channel protein 3), VIM (vimentin).
362 J O U RN A L OF P ROT EO M IC S 1 1 3 ( 2 01 5 ) 3 5 1 36 5
activation [108], which may suggest its involvement in the
metabolic alteration promoted by exercise. On the other
hand, prostaglandin reductase 2 and transgelin 2 were found
only in SHR-HIT. The first protein is related to prostaglandin
metabolism, and its up-regulation is involved in the transcrip-
tion regulation of peroxisome proliferator-activated receptor
gamma activation (PPARgama), also inhibiting adipocyte differ-
entiation [109]. However, the physiologic significance of this
relation in the cardiomyocyte is unclear. Differently, transgelin
2 protein specie (transgelin) was shown in a recent proteomic
study to be reduced after exercise training in infarcted
myocardial rat model [34] suggesting a suppressing effect of
exercise on this protein. Moreover, transgelin 2 belongs to the
calponin family, where these proteins are shown to inhibit the
ATPase activity of myosin in smooth muscle, affecting myosin-
actin binding [110]. Thus, to date, this was the first study to
verify the effect of exercise on transgelin-2 in hypertensive LV.
Furthermore, a cardiomyocyte representation was used to
better visualise and understand the role of the main proteins
identified and discussed by their response to exercise as well as
showing them to be exclusive to any one of the experimental
groups. These proteins are all displayed in Fig. 5.
5. Conclusions
Here, low and high intensity training improved aerobic capacity
and was shown to significantly diminish BP levels in a
hypertensive rat model. These responses indicated that low
and high intensity training based upon MLSS can be potential
therapeutic strategies in the treatment of essential hyperten-
sion. Following these positive hemodynamic changes, the LV
proteome was significantly modulated, where both intensities
altered several proteins related to muscle contraction, meta-
bolic process and transport in a similar way, while SHR-HIT
displayed a more significant alteration in nucleosome proteins.
The specific down-regulation of desmin, poliubiquitin B and C,
and voltage-dependent anion selective channel protin-3 and
the up-regulation of protein DJ-1 and anti-oxidant proteins
represent important cardioprotective effects of exercise in LVH.
Moreover, the sensitive response of histone 2HA subtype family
to SHR-HIT also contributes to the view of exercise as a potent
chromatin modulator with great potential to influence tran-
scription regulation process. As expected, exercise modulated
several cytosolic and mitochondrial metabolic enzymes. How-
ever, protein -enolase and adenine phosphoribosyltransferase
were significantly more altered, indicating a sensitive role
of these glucose oxidation and nucleoside synthesis enzymes
to exercise. In addition, exercise displayed a positive effect
on myofibril proteins, despite a negative regulation in the
contraction regulator proteins; myosin binding protein C
cardiac. Thus, these data represent some more insights into
how the hypertensive LV proteome responds to different
controlled exercise intensities. However, several molecular
mechanisms underlying the attenuation of hypertension
and LVH through exercise are still poorly understood, and
therefore we suggest more studies especially regarding different
exercise regimens.
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.jprot.2014.10.010.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The authors acknowledge grant support from CNPq, CAPES
and Catholic University of Brasilia Brazil.
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