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Aerobic Exercise Promotes a Decrease in Right Ventricle

Apoptotic Proteins in Experimental Cor Pulmonale
Rafael Colombo, MSc, Rafaela Siqueira, MSc, Adriana Conzatti, BSc, Tânia Regina Gattelli Fernandes,
Angela Maria Vicente Tavares, PhD, Alex Sander da Rosa Araújo, PhD, and
Adriane Belló-Klein, PhD

RV to increased afterload.2 An epidemiological study showed

Abstract: Pulmonary arterial hypertension is characterized by that 20–50 persons per million suffer from PAH, and the
progressive increases in resistance and pressure in the pulmonary survival rate in those individuals is 3.8 and 5.6 years in fe-
artery and Cor pulmonale. The effect of exercise on hydrogen per- males and males, respectively.3 Untreated patients with PAH
oxide–dependent signaling in the right ventricle (RV) of Cor pulmo- have 1-, 3-, and 5-year survival rates of 68%, 48%, and 34%,
nale rats was analyzed. Rats were divided into sedentary control respectively.4
(SC), sedentary monocrotaline (SM), trained control (TC), and Oxidative stress is closely related to the functional
trained monocrotaline (TM) groups. Rats underwent exercise train- decline and adverse remodeling of the heart observed in Cor
ing (60% of VO2 max) for 5 weeks, with 3 weeks after monocrota- pulmonale induced by monocrotaline (MCT).5 Increasing evi-
line injection (60 mg/kg intraperitoneally). Pulmonary resistance was dence suggests that reactive oxygen species (ROS) also serve
enhanced in SM (2.0-fold) compared with SC. Pulmonary artery as second messengers, mediating both physiological and path-
pressure was increased in SM (2.7-fold) and TM (2.6-fold) compared ological responses.6 It is known that a very large increase in
with their respective controls (SC and TC). RV hypertrophy indexes the levels of ROS can deregulate basic cellular functions and
increased in SM compared with SC. Hydrogen peroxide was higher cause adverse cardiac remodeling, influenced by proapoptotic
in SM (1.7-fold) than SC and was reduced by 47% in TM compared signaling. Mitochondria can also participate in redox signal-
with SM. p-Akt was increased in TM (2.98-fold) compared with SM. ing, being the main cellular organelles for H2O2 formation.7
The Bax/Bcl-2 ratio and caspase 3 were also increased (2.9-fold and In situations of oxidative stress, thiol oxidation can become
3.9-fold, respectively) in SM compared with SC. Caspase 3 was an irreversible event, modifying protein structure and apopto-
decreased in TM compared with SM (P , 0.05). Therefore, exercise tic signaling in cardiomyocytes. The classical signal transduc-
training promoted a beneficial response by decreasing hydrogen per- tion of stimulatory G protein (caused by endothelin 1,
oxide concentrations, and consequently, apoptotic signaling in RV. mechanical overload, angiotensin 2, and adrenergic stimula-
Key Words: monocrotaline, hydrogen peroxide, endurance training, tion) leads to cardiomyocyte apoptosis, facilitating the open-
pulmonary arterial hypertension, cardiac hypertrophy ing of membrane mitochondrial transition pores by Bax
(Bcl-2–associated X protein) activation and the development
(J Cardiovasc Pharmacol  2015;66:246–253) of a pathological hypertrophic phenotype.8 Moreover, cellular
stress induced by ROS can initiate the activation of mitochon-
drial proteins that will interact with domains of the Bax and
INTRODUCTION Bcl-2 proteins. This interaction may open transition pores in
Pulmonary arterial hypertension (PAH) is a severe the outer mitochondrial membrane, facilitating the relocation
disease characterized by progressive increases in resistance of cytochrome c that is released by mitochondria. The release
and pressure in the pulmonary artery because of pulmonary of cytochrome c is crucial to the formation of apoptosomes
arteriole remodeling.1 Pulmonary vascular obstruction usually and activation of pro-caspase-3, a protein directly involved in
progresses and imposes an overload on the right ventricle cellular apoptosis.9 In a study published in 2010 using umbil-
(RV) and may lead to a worsening of right ventricular func- ical vein endothelial cells, it was noticed that expression of
tion known as Cor pulmonale. The disease outcome is pre- the antiapoptotic protein Bcl-2 was decreased by hydrogen
dominantly determined by the hypertrophic response of the peroxide, whereas the expression of Bax (proapoptotic) was
increased.10 Likewise, the use of an antioxidant was able to
Received for publication November 18, 2014; accepted April 3, 2015. activate a protein involved in cell survival and physiologic
From the Laboratory of Cardiovascular Physiology and Reactive Oxygen Spe- hypertrophy induced by exercise (protein kinase B–Akt) and
cies, Physiology Department, Federal University of Rio Grande do Sul,
Porto Alegre, Brazil. inactivate signaling for Bax-dependent apoptosis.10
Supported by CNPq, CAPES, and FAPERGS, Brazilian Research Agencies. However, it is known that at low concentrations, the
The authors report no conflicts of interest. formation of ROS is important for an adaptive response that
Reprints: Adriane Belló-Klein, PhD, Laboratory of Cardiovascular Physiol- can improve cytoprotection, a phenomenon termed horme-
ogy and Reactive Oxygen Species, Federal University of Rio Grande do
Sul, Sarmento Leite Street, 500, 90.050-170 Porto Alegre, Brazil (e-mail:
sis.11 Exercise is a good example of a hormetic effect induced by ROS. It is believed that this response is related to an
Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved. increase in antioxidant protein expression induced by exercise

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J Cardiovasc Pharmacol   Volume 66, Number 3, September 2015 Exercise and Apoptotic Signaling in Cor Pulmonale

training.12 Furthermore, a small and transient increase in ROS 0.6 km/h and finished with a session of 60 minutes at 0.9
caused by exercise can trigger a beneficial adaptive response, km/h.20 At the end of the second week of training, all animals
which is characterized by a positive effect on cardiac remod- were anesthetized with ketamine and xylazine (90 mg/kg
eling and function, and activate cAMP-dependent protein intraperitoneally/10 mg/kg intraperitoneally, respectively)
kinase and Akt.13 According to the literature, there are 2 and assessed by basal echocardiography. After echocardio-
independent pathways for exercise activation of Akt. The first graphic analysis, the MCT groups (SM and TM) received
is the classical pathway dependent on insulin-like growth a single injection of MCT (60 mg/kg intraperitoneally) or
factor (IGF-1/PI3K/Akt). The other path is by increasing the same volume of saline (SC and TC groups).5 On the
the activity of cAMP-dependent protein kinase, also called day after injection, the animals underwent the maximum
protein kinase A (PKA).14–16 Thus, different levels of protein speed test, established according to Rodrigues et al,21 to read-
oxidation induced by ROS may mediate adaptive or malad- just the workload imposed (60% of maximal oxygen con-
aptive cardiac remodeling and could affect the progression of sumption). Twenty-four hours after the test, the animals
Cor pulmonale.17,18 started 3 weeks of exercise training. At the end of the second
There is a paucity of information on the possible effects week of training, the maximum speed test was repeated, and
induced by exercise training on redox-sensitive signaling and at the end of the third week, the animals were anesthetized
its relation to remodeling of RV in Cor pulmonale. Because with ketamine (90 mg/kg) and xylazine (10 mg/kg); final
of the major controversy in the scientific literature about the echocardiography was performed, and the rats were killed
effects of physical exercise on the treatment of Cor pulmo- by cervical dislocation even under the effects of anesthesia.
nale, the search for information that may answer these ques-
tions has proven to be crucial. Thus, this work has as its main Echocardiographic Assessment
objectives to evaluate the role of aerobic exercise on pulmo- For echocardiographic analysis, the EnVisor Philips
nary vascular resistance (PVR) and on the expression of pro- (Andover, MA) system with a 12- to 13-MHz transducer was
teins involved in the balance of cell survival and apoptosis used, for 3 cm deep, fundamental, and harmonic images. PVR
influenced by ROS. was measured by the acceleration time/ejection time (AT/ET)
ratio of pulmonary artery flow.22

METHODS Hemodynamic Measurements

For the hemodynamic recordings, the animals were
Ethical Approval anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/
All procedures used in this study were in accordance kg) administered intraperitoneally. Each animal’s jugular vein
with the Ethical Principles for Animal Experimentation was exposed, and a polyethylene catheter (PE-50) filled with
formulated by the Brazilian College of Animal Experimen- saline was introduced into the right atrium and ventricle. The
tation and in agreement with the Ethics in Research right ventricular systolic pressure (RVSP) was monitored
Committee of the Federal University of Rio Grande do Sul using a pressure transducer (Strain-Gauge, Narco Biosystem
(approval number 21398). Miniature Pulse Transducer PR-155, Houston, TX) connected
to a signal amplifier (HP 8805C Pressure Amplifier).20 The
Experimental Design analog pressure data were digitized (Windaq; Data Acquisi-
Male Wistar rats weighing 146 6 16 g were used. Ani- tion System, PC, Akron, OH) over a frequency range of
mals from the Reproduction Center and Laboratory Animal 1000 Hz and expressed in mm Hg. To estimate the pulmonary
Experimentation of Federal University of Rio Grande do Sul artery pressure (PAP), the following formula was used: mean
(CREAL) received water and food ad libitum and were housed PAP (in mm Hg) = 0.61 · systolic PAP + 2. The RVSP was
in a temperature- (20–258C) and humidity- (70%) controlled used to estimate systolic PAP. It is well known that RVSP is
environment with a regulated light/dark cycle of 12 hours. The similar to the systolic PAP, justifying their use in the formula
total number of rats used was 30. The sample size for each described above.23
experiment (n) is cited in the description of each outcome.
Animals were divided into 4 groups: (1) sedentary control Morphometric Analysis
(SC), animals that did not receive MCT and were not involved After final echocardiography, the animals, still anes-
in the aerobic exercise protocol; (2) trained control (TC), ani- thetized, were killed. The heart was quickly removed, and RV
mals that did not receive MCT but participated in the aerobic hypertrophy was assessed by the relationship between RV
exercise protocol; (3) sedentary monocrotaline (SM), animals mass/body mass (in milligrams per gram), RV mass/tibia
that received MCT and were not involved in the aerobic exer- length (milligrams per millimeter), or right ventricular mass/
cise protocol; and (4) trained monocrotaline (TM), animals that left ventricular mass (milligrams per milligram). Hearts were
received MCT and participated in the aerobic exercise immediately frozen in liquid nitrogen and homogenized to
protocol. perform molecular and biochemical evaluations.
Animals in the TC and TM groups were subjected to an
aerobic exercise protocol that consisted of 5 weekly sessions Cardiac Concentration of Hydrogen Peroxide
held for 5 weeks.19 Two weeks of training were established The assay was based on horseradish peroxidase–mediated
on a treadmill (Ibramed TK-01, Porto Alegre, Brazil) adapted oxidation of phenol red by hydrogen peroxide. The right
for rodents. This period was started with 15 minutes at ventricular samples were homogenized in potassium chloride

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Colombo et al J Cardiovasc Pharmacol   Volume 66, Number 3, September 2015

(1.15% wt/vol) and phenyl methyl sulphonyl fluoride (PMSF, temperature for 25 minutes. To each sample, 1 mol/L of
20 mmol/L) and centrifuged (1000·g for 20 minutes at 0 to NaOH was then added, a reading was taken at 610 nm, and
48C). The supernatants were used for further analysis. A stan- tissue hydrogen peroxide concentration was expressed as
dard curve of hydrogen peroxide concentrations of 10, 20, picomole per milligram protein.24
and 30 mmol/L was also constructed. After adding hydrogen
peroxide to the standard curve and the supernatants of the Western Blot Analysis
homogenates to microplate wells, the experiment was contin- The RV was homogenized in a homogenizer (Ultra-
ued with phenol red dextrose buffer and incubation with Turrax; Bosch, Atibaia, Brazil) with 10· cell lysis buffer
horseradish peroxidase buffer (phenol red solution). After (Cell Signaling Technology, Beverly, MA) diluted in Milli-
adding the PRS buffer, the microplate was shaken at room Q water, and PMSF was added. The suspension was

FIGURE 1. AT/ET ratio. Values are

expressed as mean 6 SD (n = 4–5).
(A) AT/ET ratio. (B) Representative
image of pulmonary artery outflow
by pulsed wave Doppler. *P , 0.05
compared with SC.

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J Cardiovasc Pharmacol   Volume 66, Number 3, September 2015 Exercise and Apoptotic Signaling in Cor Pulmonale

Statistical Analysis
All statistical tests were performed using the Statistical
Package for the Social Sciences 17.0 software (SPSS 17.0).
The sample size (n) was calculated a priori from data obtained
from the literature, considering a = 0.05 and a statistical
power of 80% (a = 0.8). The value of n obtained for each
analysis is indicated in individual figures and tables. Results
are presented as mean 6 SD. Comparisons among parametric
data were performed by 2-way analysis of variance, with
complementary post hoc Bonferroni’s test. Differences were
considered statistically significant when the alpha error prob-
ability was less than 0.05 (P , 0.05).

FIGURE 2. PAP in mm Hg. Values are expressed as mean 6 SD RESULTS

(n = 6). *P , 0.05 compared with SC; +P , 0.05 compared
with TC. Echocardiographic Parameters
Acceleration Time/Ejection Time
centrifuged at 8000·g for 15 minutes at 48C to remove the A significant decrease (50%) was noted in the AT/ET
nuclei and cell debris. The supernatants were then used for ratio in animals receiving MCT (SM) as compared with
Western blot analysis. Forty-eight micrograms of protein control animals (SC) (0.26 6 0.06 and 0.13 6 0.02, respec-
were subjected to 1-dimensional sodium dodecyl sulfate- tively). The decrease in this ratio shows that there was a sig-
polyacrylamide gel electrophoresis (SDS-PAGE) in a discon- nificant increase in PVR in SM animals. In contrast, the
tinuous system using different concentrations of separating animals that received MCT and participated in the aerobic
gel and stacking gel.25,26 The separated proteins were trans- exercise protocol (TM) did not show statistically significant
ferred electrophoretically to polyvinylidene difluoride changes in AT/ET ratio, which implies that the exercise
(PVDF) membranes using a running buffer (pH 8.2, Tris 25 may minimize the insults induced by increase in PVR in
mmol/L, glycine 192 mmol/L, and SDS 0.1%) in a cooled the TM group (Fig. 1A).
Bio-Rad Trans-Blot Unit (Bio-Rad, São Paulo, Brazil). Non-
specific protein binding was blocked by a 1 hour incubation Pulmonary Artery Pressure
with nonfat milk in Tris buffer. Membranes were processed The PAP was elevated in MCT animals. A significant
for immunodetection using the following primary antibodies: increase (2.7-fold) in SM compared with SC was noted.
anti-PI3K, anti-Akt 1/2/3, anti-p-Akt 1/2/3, anti-Bax, anti- Comparing TM animals with TC, a rise (2.6-fold) in this
Bcl-2, and anti-caspase-3, all obtained from Santa Cruz outcome was apparent. The PAP was not affected by aerobic
Biotechnology (Santa Cruz, CA). Binding of the primary anti- training (Fig. 2).
bodies was detected with secondary antibodies conjugated to
horseradish peroxidase, and membranes were developed
Morphometric Parameters
using chemiluminescence detection reagents. The autoradio-
graphs generated were analyzed quantitatively with an ImageJ Hypertrophy Indexes
densitometer (Wayne Rasband, Research Services Branch, In SM animals, a significant increase in RV hypertro-
National Institute of Mental Health, Bethesda, MD). The phy indexes was identified when compared with the SC
molecular weights of the bands were determined by reference group, a predominant characteristic of this drug-induced Cor
to a standard series of molecular weight markers (RPN 800 pulmonale. In contrast, in TM animals, no increase in hyper-
rainbow full range; Bio-Rad, Hercules, CA). The optical den- trophy ratios was noted when compared with the TC group. In
sity (OD) results from each sample were normalized accord- SM animals, this increase was 63% relative to their respective
ing to their immunocontent of b-tubulin. Protein content was control (SC), whereas for TM, the increase was 29% com-
determined by the method of Lowry et al,27 using bovine pared with its control (TC). No changes between the SM and
serum albumin as a standard. TM groups were noted (Table 1).

TABLE 1. Morphometric Parameters of the Different Experimental Groups Measured at the End of the Experimental Protocol
Morphometric Parameters SC SM TC TM
RV/body mass (mg/g) 0.54 6 0.04 0.98 6 0.18* 0.65 6 0.22 0.92 6 0.14
RV/tibia length (mg/mm) 4.42 6 0.54 7.23 6 1.44* 5.06 6 1.55 6.57 6 0.99
RV/LV (mg/mg) 0.28 6 0.03 0.48 6 0.10* 0.32 6 0.10 0.47 6 0.07
Values are expressed as mean 6 SD (n = 7–9) in each group.
*P , 0.05 compared with SC.

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Colombo et al J Cardiovasc Pharmacol   Volume 66, Number 3, September 2015

Proteins Involved in Apoptotic Signaling

The susceptibility of RV to apoptosis was verified by
analyzing proteins involved in this response. One of the
markers evaluated was the Bax/Bcl-2 ratio, which was found
to be significantly increased (2.9-fold) in the SM group as
compared with its respective control group (SC) (0.72 6 0.18
OD and 0.25 6 0.01 OD, respectively). Nevertheless, when
analyzing this ratio in the TM group, despite detecting
a minimization of this outcome caused by exercise training,
significant changes were not visualized when compared with
SM (Fig. 5). Caspase-3, which plays a central role in the
execution phase of apoptosis, had its immunocontent signif-
icantly increased (3.4-fold) in animals of the SM group as
compared with the SC group (3.79 6 0.73 OD and 0.95 6
0.25 OD, respectively). Analyzing the effect of training in the
FIGURE 3. RV hydrogen peroxide levels. Values are expressed
as mean 6 SD (n = 6–8). *P , 0.05 compared with SC; #P ,
MCT groups (SM and TM), there was a significant attenua-
0.05 compared with SM. tion (48%) in the signal for this protein after exercise training
(3.79 6 0.79 and 1.97 6 0.58, respectively). Participation by
animals receiving MCT in the exercise program was able to
Cardiac Concentration of Hydrogen Peroxide promote a decrease in the immunocontent of caspase-3,
Because of its stability and free flow through mem- equating this measurement to baseline (Table 2).
branes, hydrogen peroxide is the main ROS involved in
intracellular signaling. In SM animals, a significant increase
(71%) in hydrogen peroxide concentration was noted com- DISCUSSION
pared with SC (0.41 6 0.10 and 0.24 6 0.04, respectively). The main finding of our study was that aerobic exercise
Taking the effect of aerobic exercise on Cor pulmonale into can decrease intracellular signaling for apoptosis in a severe
account, a significant decrease (47%) in this outcome was experimental model of Cor pulmonale and that the decrease
visualized in TM animals compared with SM (0.22 6 0.05 in this signal is associated with the antiapoptotic role of Akt.
and 0.41 6 0.10, respectively) (Fig. 3).
Effects of Exercise on RV Remodeling
Immunocontent of Proteins Involved in In a previous study from our group, it was observed that
Physiologic and Pathologic Hypertrophy aerobic exercise was not able to minimize cardiac hypertro-
To visualize the phenotypic characteristics of hypertro- phy induced by MCT. However, a reduction in the extracel-
phy in each of the experimental groups, we sought to analyze lular matrix and an increase in intramyocardial vessel volume
intracellular proteins involved in adaptive and maladaptive were noted.19 Handoko et al28 demonstrated that exercise was
remodeling in the RV. able to increase cardiac capillarization by 25%, which may be
associated with an improvement in tissue perfusion and res-
Proteins Involved in Exercise-induced Hypertrophy toration of cardiac function after pressure overload. In this
The upstream Akt protein phosphoinositide 3-kinase study, pathologic hypertrophy observed in SM rats was gen-
(PI3K) did not show any significant changes at the end of the erated because of the increased afterload imposed by PAP
experimental protocol (Table 2). The Akt signaling pathway elevation, a common outcome of Cor pulmonale. In fact,
is involved in adaptive signaling of the heart against extrinsic our results showed that MCT decreased the AT/ET ratio,
or intrinsic stimuli. The p-Akt immunocontent had an suggesting an increased PVR. This result is reinforced by
increase (3.4-fold) in TM animals compared with SM the data on mean pulmonary pressure observed in this study.
(1.88 6 0.56 OD and 0.63 6 0.14 OD, respectively) However, in animals receiving MCT and undergoing physical
(Fig. 4). However, total Akt immunocontent did not change exercise, PVR was not significantly different from its control.
among the groups (Table 2). The percentage change in RV hypertrophy in TM animals

TABLE 2. Proteins involved in exercise-induced hypertrophy and apoptotic signaling

Evaluated Proteins (OD
Normalized by b-tubulin) SC SM TC TM
PI3K 0.53 6 0.42 0.10 6 0.06 0.19 6 0.09 0.31 6 0.12
Akt 2.37 6 1.13 1.11 6 0.59 1.72 6 0.09 2.34 6 1.19
Caspase-3 0.95 6 0.25 3.79 6 0.72* 2.18 6 0.49 1.98 6 0.58†
Values are expressed as mean 6 SD (n = 3–4) in each group.
*P , 0.05 between SM and SC.
†P , 0.05 between TM and SM.

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J Cardiovasc Pharmacol   Volume 66, Number 3, September 2015 Exercise and Apoptotic Signaling in Cor Pulmonale

FIGURE 4. p-Akt immunocontent. Values

are expressed as mean 6 SD (n = 3–4) ani-
mals. A representative gel from Western blot
experiments showing 3 and 4 bands for
each experimental group. #P , 0.05 com-
pared with SM.

compared with TC was increased (29%), which was not sta- cytochrome c. Cytochrome c release acts to activate
tistically significant, whereas it was significantly greater caspase-dependent apoptosis, a phenomenon that is directly
(63%) in SM animals when compared with SC. The AT/ET linked to a decrease in cardiac contractility.30 In our experi-
ratio showed the same pattern of response. Thus, it can be mental model, it was noted that Bax and caspase-3–dependent
suggested that exercise minimizes PVR and consequently RV signaling were increased in animals receiving MCT compared
hypertrophy generated by the increase in afterload imposed with control animals. This suggests that our model of right-
by MCT in this severe model of Cor pulmonale. sided heart failure follows the same molecular profile for
apoptosis as other pathological models of cardiovascular
Apoptotic Protein Immunocontent on RV diseases.29,30
It is well known that apoptosis is an event associated Besides Bax-dependent apoptotic signaling, other pro-
with various features of cardiac pathology. Apoptosis is teins are also involved in antiapoptotic events such as the
observed in pressure overload or ischemia and is linked to Bcl-2 protein, responsible for suppressing the opening of
progression to heart failure.29 In cardiac hypertrophy and mitochondrial membrane pores.31 This suppression is due to
heart failure, an increase in Bax expression is noted, which the antagonistic action of Bcl-2 over Bax.9 In our study, it was
promotes the formation of mitochondrial pores, releasing noted that animals receiving MCT that developed Cor

FIGURE 5. Bax/Bcl-2 immunocontent. Val-

ues are expressed as mean 6 SD (n = 3–4). A
representative gel from Western blot ex-
periments showing 3 and 4 bands for each
experimental group. *P , 0.05 compared
with SC.

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Colombo et al J Cardiovasc Pharmacol   Volume 66, Number 3, September 2015

FIGURE 6. Signaling proteins involved in

the influence of exercise on Cor pulmonale
resulting form pulmonary hypertension
induced by MCT.

pulmonale showed increases in their Bax/Bcl-2 ratio at the in the expression of antioxidant enzymes and antiapoptotic
end of the experimental protocol. This suggests that the apo- proteins in cardiac tissue, providing an improvement in car-
ptotic signal is strengthened in this disease. diac resistance to apoptosis in an ischemia/reperfusion situa-
There is clear evidence that redox status is involved in tion.34 One of the key proteins involved in this adaptive
the modulation of apoptosis. This event may be mediated by remodeling is Akt. Besides being involved in cell survival
hydrogen peroxide, which because of its high stability and signaling and improving cardiac metabolism, Akt is directly
free movement across cell membranes may act on proteins linked to inhibition of caspase-dependent apoptotic signal-
involved in apoptotic events. It is known that hydrogen ing.9,35,36 Moreover, it is already clear that exercise reduces
peroxide can stimulate the release of mitochondrial cyto- the formation of hydrogen peroxide, possibly by acting pos-
chrome c. Furthermore, treatment of endothelial cells with an itively on mitochondrial complex 1.37 It is suggested that the
antioxidant known as catalpol was able to reduce hydrogen decrease in ROS production promoted by exercise can influ-
peroxide concentrations and, at the same time, reduce ence a reduction in caspase-3 expression and, therefore, medi-
apoptosis and caspase-3 activity.10 Catalpol is also known ate a decrease in mitochondrial permeability and reduce
to stimulate the expression of antiapoptotic proteins such as apoptotic signaling mediated by Akt in TM rats.
Bcl-2.32 Thus, it was suggested that the increase in RV hydro- It is clear from the literature that activation of Akt on
gen peroxide concentrations of SM animals when compared physiologic hypertrophy induced by exercise training is
with the SC group is associated with the increase in the apo- dependent on the IGF1-PI3K pathway.36 This signaling is asso-
ptotic signal noted. Despite knowing that there is a relation- ciated with a series of intracellular responses; among them,
ship between intracellular hydrogen peroxide concentrations stimulation of protein synthesis, changes in gene expression,
and strengthening of the apoptotic signal, the mechanisms and improvements in tissue perfusion and energy metabolism
involved in this relationship are still not well elucidated. can be noted. Following this logic, PI3K is activated on IGF-1
binding to its tyrosine kinase receptor, causing an increase
Influence of Exercise on RV Signaling in the formation of phosphatidylinositol-3,4,5-trisphosphate
Our data suggest that the expression of proteins (PtdIns (3,4,5) P3). The formation of PtdIns (3,4,5) P3 is the
involved in apoptosis and the concentration of hydrogen main mechanism involved in Akt activation. However, PDK1
peroxide in the RV of Cor pulmonale rats are influenced by (phosphoinositide-dependent kinase) can also act together with
aerobic exercise. In our experimental model, 5 weeks of aer- the PtdIns (3,4,5) P3 by stimulating Akt-dependent signaling.38
obic exercise on a treadmill was able to decrease caspase-3 In addition, phosphatase and tensin homolog (PTEN) acts to
expression and reduce hydrogen peroxide concentrations in dephosphorylate PtdIns (3,4,5) P3, inhibiting Akt activation
TM animals when compared with SM. These data suggest and physiologic hypertrophy signaling.38,39 Although statisti-
that exercise plays an antiapoptotic role in this model of cally significant changes in the immunocontent of PI3K were
severe Cor pulmonale, which seems to be related to the not found and we did not explore all the pertinent factors, the
decrease in ROS concentrations. Besides exercise training current experimental findings essentially imply that this classi-
promoting an improvement in aerobic capacity, it is directly cal pathway may be involved in the increased Akt-dependent
involved in cellular adaptations, including mitochondrial bio- phosphorylation observed in TM animals.
genesis, an improvement in the efficiency of heart mitochon-
drial oxidative phosphorylation, a reduction in the transition
pores permeability of mitochondria, and an increase in enzy- CONCLUSIONS
matic antioxidant activity.33 One possible mechanism by In summary, our study suggests that exercise exerts
which aerobic exercise exerts cardioprotection may be its role a positive influence on Cor pulmonale progression after the

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J Cardiovasc Pharmacol   Volume 66, Number 3, September 2015 Exercise and Apoptotic Signaling in Cor Pulmonale

MCT-induced RV hypertrophy in rats. In addition to promot- 19. Colombo R, Siqueira R, Becker CU, et al. Effects of exercise on
ing a structural improvement, as described above, it is monocrotaline-induced changes in right heart function and pulmonary
artery remodeling in rats. Can J Physiol Pharmacol. 2013;91:38–44.
believed that this beneficial response is influenced by 20. Souza-Rabbo MP, Silva LF, Auzani JA, et al. Effects of a chronic exer-
a decrease in the concentration of hydrogen peroxide, conse- cise training protocol on oxidative stress and right ventricular hypertro-
quently promoting a decrease in apoptotic signaling in RV phy in monocrotaline-treated rats. Clin Exp Pharmacol Physiol. 2008;35:
(Fig. 6). Just by demonstrating that aerobic exercise promotes 944–948.
an improvement in the outcomes analyzed, the clinical signif- 21. Rodrigues B, Figueroa DM, Mostarda CT, et al. Maximal exercise test
is a useful method for physical capacity and oxygen consumption
icance of this adjuvant in the treatment of severe Cor pulmo- determination in streptozotocin-diabetic rats. Cardiovasc Diabetol.
nale should be investigated. 2007;6:38.
22. Jones JE, Mendes L, Rudd MA, et al. Serial noninvasive assessment of
REFERENCES progressive pulmonary hypertension in a rat model. Am J Physiol Heart
1. Malenfant S, Neyron AS, Paulin R, et al. Signal transduction in the Circ Physiol. 2002;283:H364–H371.
development of pulmonary arterial hypertension. Pulm Circ. 2013;3: 23. Koskenvuo JW, Mirsky R, Zhang Y, et al. A comparison of echocardi-
278–293. ography to invasive measurement in the evaluation of pulmonary arterial
2. Bogaard HJ, Abe K, Vonk Noordegraaf A, Voelkel NF. The right ven- hypertension in a rat model. Int J Cardiovasc Imaging. 2010;26:
tricle under pressure: cellular and molecular mechanisms of right-heart 509–518.
failure in pulmonary hypertension. Chest. 2009;135:794–804. 24. Pick E, Keisari Y. A simple colorimetric method for the measurement of
3. Peacock AJ, Murphy NF, McMurray JJ, et al. An epidemiological study hydrogen peroxide produced by cells in culture. J Immunol Methods.
of pulmonary arterial hypertension. Eur Respir J. 2007;30:104–109. 1980;38:161–170.
4. Waxman AB, Zamanian RT. Pulmonary arterial hypertension: new in- 25. Laemmli UK. Cleavage of structural proteins during the assembly of the
sights into the optimal role of current and emerging prostacyclin thera- head of bacteriophage T4. Nature. 1970;15:680–685.
pies. Am J Cardiol. 2013;111:1A–16A. 26. Araujo AS, Ribeiro MF, Enzveiler A, et al. Myocardial antioxidant
5. Farahmand F, Hill MF, Singal PK. Antioxidant and oxidative stress changes activities and concentration and glutathione metabolism in experimental
in experimental cor pulmonale. Mol Cell Biochem. 2004;260:21–29. hyperthyroidism. Mol Cell Endocrinol. 2006;249:133–139.
6. Rhee SG. Redox signaling: hydrogen peroxide as intracellular messen- 27. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement
ger. Exp Mol Med. 1999;31:53–59. with the Folin phenol reagent. J Biol Chem. 1951;193:265–275.
7. Murphy MP. Modulating mitochondrial intracellular location as a redox 28. Handoko ML, de Man FS, Happe CM, et al. Opposite effects of training
signal. Sci Signal. 2012;5:pe39. in rats with stable and progressive pulmonary hypertension. Circulation.
8. Bernardo BC, Weeks KL, Pretorius L, McMullen JR. Molecular distinc- 2009;120:42–49.
tion between physiological and pathological cardiac hypertrophy: exper- 29. Whelan RS, Kaplinskiy V, Kitsis RN. Cell death in the pathogenesis of
imental findings and therapeutic strategies. Pharmacol Ther. 2010;128: heart disease: mechanisms and significance. Annu Rev Physiol. 2010;72:
191–227. 19–44.
9. Dorn GW II. Molecular mechanisms that differentiate apoptosis from 30. Dorn GW II. Mechanisms of non-apoptotic programmed cell death in
programmed necrosis. Toxicol Pathol. 2013;41:227–234. diabetes and heart failure. Cell Cycle. 2010;9:3442–3448.
10. Hu L, Sun Y, Hu J. Catalpol inhibits apoptosis in hydrogen peroxide-induced 31. Sinha K, Das J, Pal PB, Sil PC. Oxidative stress: the mitochondria-
endothelium by activating the PI3K/Akt signaling pathway and modulating dependent and mitochondria-independent pathways of apoptosis. Arch
expression of Bcl-2 and Bax. Eur J Pharmacol. 2010;628:155–163. Toxicol. 2013;87:1157–1180.
11. Samjoo IA, Safdar A, Hamadeh MJ, et al. The effect of endurance 32. Li DQ, Bao YM, Li Y, et al. Catalpol modulates the expressions of Bcl-2
exercise on both skeletal muscle and systemic oxidative stress in pre- and Bax and attenuates apoptosis in gerbils after ischemic injury. Brain
viously sedentary obese men. Nutr Diabetes. 2013;3:1–10. Res. 2006;1115:179–185.
12. Gounder SS, Kannan S, Devadoss D, et al. Impaired transcriptional 33. Campos JC, Gomes KM, Ferreira JC. Impact of exercise training on
activity of Nrf2 in age-related myocardial oxidative stress is reversible redox signaling in cardiovascular diseases. Food Chem Toxicol. 2013;
by moderate exercise training. PLoS One. 2012;7:e45697. 62:107–119.
13. Frasier CR, Moore RL, Brown DA. Exercise-induced cardiac precondi- 34. Kavazis AN. Exercise preconditioning of the myocardium. Sports Med.
tioning: how exercise protects your achy-breaky heart. J Appl Physiol 2009;39:923–935.
(1985). 2011;111:905–915. 35. Chen YL, Loh SH, Chen JJ, Tsai CS. Urotensin II prevents cardiomyo-
14. Filippa N, Sable CL, Filloux C, et al. Mechanism of protein kinase B cyte apoptosis induced by doxorubicin via Akt and ERK. Eur J Phar-
activation by cyclic AMP-dependent protein kinase. Mol Cell Biol. 1999; macol. 2012;680:88–94.
19:4989–5000. 36. McMullen JR, Jennings GL. Differences between pathological and phys-
15. Yang J, Cron P, Thompson V, et al. Molecular mechanism for the reg- iological cardiac hypertrophy: novel therapeutic strategies to treat heart
ulation of protein kinase B/Akt by hydrophobic motif phosphorylation. failure. Clin Exp Pharmacol Physiol. 2007;34:255–262.
Mol Cell. 2002;9:1227–1240. 37. Starnes JW, Barnes BD, Olsen ME. Exercise training decreases rat heart
16. Song G, Ouyang G, Bao S. The activation of Akt/PKB signaling pathway mitochondria free radical generation but does not prevent Ca2+-induced
and cell survival. J Cell Mol Med. 2005;9:59–71. dysfunction. J Appl Physiol (1985). 2007;102:1793–1798.
17. Burgoyne JR, Oka S, Ale-Agha N, Eaton P. Hydrogen peroxide sensing 38. Maillet M, van Berlo JH, Molkentin JD. Molecular basis of physiological
and signaling by protein kinases in the cardiovascular system. Antioxid heart growth: fundamental concepts and new players. Nat Rev Mol Cell
Redox Signal. 2013;18:1042–1052. Biol. 2013;14:38–48.
18. Shao D, Oka S, Brady CD, et al. Redox modification of cell signaling in 39. Aoyagi T, Matsui T. Phosphoinositide-3 kinase signaling in cardiac
the cardiovascular system. J Mol Cell Cardiol. 2012;52:550–558. hypertrophy and heart failure. Curr Pharm Des. 2011;17:1818–1824.

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