Sport Sciences for Health (2021) 17:173–179

https://doi.org/10.1007/s11332-020-00670-2

ORIGINAL ARTICLE

Effects of high intensity interval training and aging on cardiac muscle

apoptosis markers in C57BL/6 Mice
Rahman Soori1 · Amine Ghram1,2 · Maryam Zare Shahneh1 · Siroos Choobineh1 · Pablo B. Costa3 ·
Fabrício A. Voltarelli4

Received: 28 February 2020 / Accepted: 26 June 2020 / Published online: 21 September 2020
© Springer-Verlag Italia S.r.l., part of Springer Nature 2020

Abstract
Background Apoptosis is a specific form of programmed cell death that plays an important role in tissue homeostasis and
also occurs during aging. In addition, exercise training may alter apoptosis-related signaling in muscles, including the cardiac
muscle. However, the effects of high-intensity interval training (HIIT) on apoptosis markers in the heart have not yet been
studied. Thus, the aim of the present study was to investigate the effects of HIIT on Bcl-2 and Bax expression protein levels
in young and old cardiac muscles.
Methods Four and 24-month old C57BL/6J mice were used as our young adult and old groups, respectively. Animals were
randomly assigned to four groups: young control (YC), young trained (YT), old control (OC), and old trained (OT). The
trained groups performed HIIT 5 sessions per week for 4 weeks. RNA extraction and synthesis of cDNA was performed,
and Bax and Bcl2 heart gene levels were determined by RT-PCR analysis.
Results Training induced significant increases in Bcl-2 gene expression in YT group comparing to the YC group (p = 0.010)
and in OT group comparing to OC group (p = 0.002). Training induced non-significant decrease (p > 0.05) in Bax gene
expression and in Bax/Bcl-2 ratio in YT group comparing to the YC group and in OT group comparing to OC group. Aging
showed no significant effect in Bcl-2 gene expression, Bax gene expression, and Bax/Bcl-2 ratio in CY group comparing to
the OC group and in YT group comparing to OT group (p > 0.05).
Conclusion In conclusion, 4 weeks of HIIT increased Bcl-2 level for young and old heart muscles, and no effect of aging
was revealed, indicating that HIIT ameliorated anti-apoptotic signaling in the young and old heart of mice.

Keywords Exercise · Age · Apoptosis · High-intensity interval training · Rat myocardium

Rahman Soori and Amine Ghram have contributed equally to this

work.

1
* Rahman Soori Department of Exercise Physiology, Faculty of Physical
soori@ut.ac.ir Education and Sport Sciences, University of Tehran, Tehran,
Iran
* Amine Ghram
2
amineghram.110@ut.ac.ir; ghram.amine@hotmail.fr Department of Cardiac Rehabilitation, Tehran Heart Center,
Tehran University of Medical Sciences, Tehran, Iran
Maryam Zare Shahneh
3
maryamza88@gmail.com Department of Kinesiology, California State University,
Fullerton, Fullerton, CA, USA
Siroos Choobineh
4
choobineh@ut.ac.ir Graduation Program in Health Sciences, Faculty
of Medicine, Federal University of Mato Grosso, Cuiabá,
Pablo B. Costa
Brazil
pcosta@fullerton.edu
Fabrício A. Voltarelli
voltarellifa@gmail.com

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Introduction
Aging is the functional deterioration of physiological
integrity which leads to pathophysiological alterations
in the cardiovascular system [1], increases vulnerability
to death [2], as well as reduces normal organism physi-
ological function [3]. Further, aging can increase apoptosis
in skeletal muscle [4] and elicit diverse diseases such as
myocardial infarction.
Apoptosis is a specific form of programmed cell death
which plays an important role in tissue homeostasis and
development, as well as tissue dysfunction, and is con-
trolled by two types of genes interacting with each other:
pro- and anti-apoptotic. The B-cell lymphoma-2 (Bcl-2)
family group consists of anti-apoptotic proteins such as
Bcl-2, and Bcl-xL regulating mitochondrial dysfunction,
and the pro-apoptotic proteins (e.g., Bax, Bim, Bad. Bax,
and Bcl-2) interact with each other to regulate apoptosis
occurrence and progression [5], and control the balance
between cell life and cell death [6]. The activation of Bax
expression will inhibit that of Bcl-2[7]. The ratio of pro-
to anti-apoptotic proteins (e.g., Bax/Bcl-2) can affect the
increase in the Bax/Bcl-2 ratio, which is considered to be
a reliable indicator of cells to undergo apoptosis [7], can
regulate the myonuclei and cell survival by controlling
mitochondrial membrane stability [8], and can be changed
during the aging process [9].
Previous study showed an increase in both the Bcl-2 and
Bax protein levels in aging rat cardiac muscle [10]. Other
studies also reported an increase in Bax and Bax/Bcl-2
ratio protein levels in aging rat cardiac muscle [11] and in
pro-apoptotic Bax and cleaved caspase-3 levels and DNA
fragmentation in aging rat skeletal and cardiac muscles
[12, 13]. However, there was no significant difference in
the Bax and Bcl-2 protein expression levels during aging,
even with increased cardiomyocyte apoptosis [14]. There
are some limitations concerning mechanisms responsible
for apoptotic signaling in the aged heart.
Exercise training has the potential to decrease apoptosis
through up-regulation of protective stress-sensitive pro-
teins [15, 16]. However, the mechanisms by which exer-
cise training improves heart function and cardiovascular
disease risk profile have not been fully elucidated. Regular
exercise is beneficial for cardiovascular health, thereby
also reducing cardiovascular mortality and developing car-
diovascular disease. Bcl‐2 mRNA levels increased in both
ventricle and soleus muscles of trained animals, and Bax
mRNA levels decreased in the soleus of trained animals
when compared with controls animals [17]. They suggest
that exercise training attenuates the extent of apoptosis in
cardiac and skeletal muscles [17]. In addition, the com-
bined effects of aging with exercise training on apoptosis
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Sport Sciences for Health (2021) 17:173–179
signaling have not been clearly explored thus far. Hence,
in order to understand the function of apoptosis sign-
aling and its relationship with aging, it is important to
assess the impact of exercise training on apoptosis cardiac
biomarkers.
Only a few studies have attempted to investigate the inci-
dence of apoptosis in young and old cardiac muscles [13,
17, 18]. Although aging increased Bax and Bax/Bcl-2 ratio
in skeletal muscle, exercise training resulted in a marked
reduction of pro-apoptotic Bax and elevated Bcl-2 protein
expression in skeletal muscle of old rats [13]. The appropri-
ate strategy and lifestyle to protect the heart against apopto-
sis-mediated damage and the improvement of the status of
muscle tissue in aging is substantial.
Recently introduced as an exercise model, high-intensity
interval training (HIIT) has been shown to be safe and effec-
tive for improving cardiovascular health in clinical popula-
tions [19]. Few studies have revealed contradictory results
regarding the effects of HIIT on the apoptosis processes [20,
21]. Markers of apoptosis (PI3K, AKT, AMPK) decreased
after HIIT and aerobic training in female rats [21]. Aero-
bic interval training increased caspase-9 gene expression
and reduced the cardiomyocytes apoptosis after myocardial
infarction [20]. To our knowledge, studies on the combined
effects of HIIT and aging on the apoptosis markers are very
limited and more studies are needed. Therefore, due to the
importance of selecting the beneficial and effective train-
ing program to improve cardioprotection, the purpose of the
present study was to investigate the effects of HIIT on Bcl-2
and Bax gene expression in young and old cardiac muscles
of mice.
Methods
Animals
4- and 24-month old C57BL/6J mice were used as younger
adult and older adult groups, respectively, and were pur-
chased from the Razi Institute (Tehran, Iran) and maintained
in the Central of Animal House, University of Tehran-Iran
[22]. The mice were maintained at 20–24 °C with 40–60%
humidity under a 12/12 h light/dark cycle, receiving stand-
ard chow and water ad libitum. Studies commenced follow-
ing one week of acclimatization. The animal body mass
and food intake were determined weekly during all of the
experimental period. After 2 weeks, weight-matched rats
were randomly divided into four groups: young trained (YT),
young control (YC), old trained (OT), and old control (OC)
groups. Each group included 5 C57BL/6J mice. The pro-
tocols used conformed to guidelines of animal studies and
were approved by the committee on the ethics of animal
Sport Sciences for Health (2021) 17:173–179 175

experiments in Faculty of Physical Education and Sport Sci-
ence, University of Tehran.
Maximal running speed test
After familiarization with the treadmill, C57BL/6J mice
were given a graded intensity treadmill test to determine
maximal running speed. After a warm-up of 6 min running
at 6 m/min, the treadmill speed was increased by 2 m/min
every 120 s until exhaustion was reached [23]. To encour-
age the mice to run, an electric shock grid at the base of
the treadmill was activated to deliver a 0.2-mA pulse [23].
Exhaustion (endpoint for treadmill cessation) was defined
as the point at which mice maintained continuous contact
with the shock grid for 5 s [24]. At baseline, the maximum
running speed average (meter/minute) was 33.66 ± 2.33 and
29.33 ± 3.26 for young and old mice, respectively.
Training intervention
chloroform (Germany, Qiagen) in accordance to manufac-
turers’ instructions. Approximately 50 mg of muscle tissue
was homogenized separately in 1–10 portions in ­QlAzol®
Lysis Reagent for total RNA extraction and for removing
protein components. The final product was centrifuged at
12,000×g for 10 min at 4 °C, and mixed with chloroform
in 1–5 portions and shaken severely for 15 s. Then the
supernatant was at 12,000×g for 10 min at 4 °C and its
mineral part and its water removed. Finally, its RNA con-
tained portion removed and mixed with isopropanol in 1–5
portions. It was left for 10 min in room temperature and
then centrifuged in 4 °C for 10 min with 12,000g revolu-
tion. RNA contained pellet was washed and resolved in
20 μL RNsa-free water. RNA concentration was measured
by UV spectrophotometry method (Eppendorf, Germany),
and 260–280 portions in 1.8–2 were determined as the
desired purification. cDNA synthesis was done by using
QuantiTect Reverse Transcription Kit (Qiagen, Germany)
in accordance to manufacturer’s manual.

As shown in Table 1, training consisted of 6–12 2-min inter-

vals (interspersed with 1 min of rest) performed 5 times/
week for 4 weeks. The intensity of the intervals was initially
set at 80% of the peak speed reached for each mouse during
the incremental test and was increased by 5% each week.
Tissue preparation
Mice were anesthetized with sodium pentobarbital (50 mg/
kg i.p.) following the exercise training period for all groups.
Animals in the exercise training groups were anesthetized
48 h following the last bout of exercise training. Sedentary
animals were euthanized at the same time as their trained
partner animals. The cardiac muscle tissue was removed and
frozen immediately in liquid nitrogen and stored at − 80 °C
until further analysis.
RNA extraction and cDNA synthesis
Total RNA was extracted from the heart’s myocardium
with ­Q lAzol ® Lysis Reagent (Germany, Qiagen) and
RT‑PCR analysis
Real-Time PCR quantity method was used by Premix
SYBR Green II (Qiagen, Germany) for measuring Kif5B
mRNA expression levels (Applied Biosystems Step One,
America). Reaction mixture was performed in the final
volume in 20 μL (includes 1 μL of cDNA, 1 μL of forward
primer, 1 μL of reverse primer, 7 μL of DEPC water, and
10 μL of Syber Green), and each reaction in a duplicate
form. Designing primers was performed according Bcl-2,
Bax and β-actin genes in a gene bank of NCBI. Total RNA
was extracted using RNeasy fibrous tissue mini kit (Qia-
gen, Germany) following the manufacturer’s instructions.
Primers for the specific genes are listed in Table 2, while
β-actin was used as control gene. Thermal program used
in Real Time-PCR included: 95 °C for 10 min, 95 °C for
15 s, 60 °C for 1 min (40cyclerepetitions). Reactions were
performed in triplicate and expression levels calculated
using the CT comparative method ( 2−ΔΔCT).

Table 1  Summary of the training performed by both old and young Table 2  Gene sequencing primer design used
mice during the experimental period
Genes Primer sequence Gene Bank
Weeks Number of Mean Mean running distance/
intervals/training speed/week week, m/min Bcl-2 F:TGA​GTA​CCT​GAA​CCG​GCA​TCT​ Ncbi
session (%) R:GCA​TCC​CAG​CCT​CCG​TTA​T
Old mice Young mice
Bax F:AAG​CTG​AGC​GAG​TGT​CTC​CGGCG​ Ncbi
1 6 80 24.9 28.6 R:GCC​ACA​AAG​ATG​GTC​ACT​GTC​TGC​
2 8 85 26.4 30.3 C
3 10 90 28.2 32.3 β-Actin F:GCC​CTG​AGG​CTC​TTT​TCC​AG NM_007393.4
4 10 95 28.1 32.4 R:TGC​CAC​AGG​ATT​CCA​TAC​CC

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176 Sport Sciences for Health (2021) 17:173–179

Statistics analysis (a) 0.7

*
**
Data are reported were as mean ± SEM (standard error 0.6

of the mean). The Shapiro–Wilk test was used to verify 0.5

normal statistic distributions. Statistical analysis was per- YC
0.4
formed by One-way analysis of variances (ANOVA) and

Bcl-2
YT
followed by the LSD post-hoc test comparison when was 0.3
OC
appropriated (YT vs. YC vs. OT vs. OC). Cohen’s d effect 0.2 OT
sizes, reported for all observations with ≤0.20 represent-
0.1
ing a small effect, 0.50 representing a medium effect, and
≥ 0.80 representing a large effect [25], were estimated to 0
compare the magnitude of training response. The level YC YT OC OT

of significance was set at p < 0.05.All data were analyzed (b)

0.3
using the SPSS 21.0 software package (USA).
0.25

0.2 YC
Results YT

Bax
0.15
OC
One way ANOVA showed a significant group effect 0.1 OT
(F = 8.1, p = 0.002) in Bcl-2 gene expression. Train-
ing induced significant increases in Bcl-2 gene expres- 0.05

sion in YT group comparing to the YC group (p = 0.010, 0

ES = − 1.37, 95% CI − 2.59 to 0.12) and in OT group com- YC YT OC OT
paring to OC group (p = 0.002, ES = − 5.18, 95% CI − 7.07 (c)
to − 2.28) (Fig. 1). However, aging showed no significant 8
effect in Bcl-2 gene expression in CY group comparing 7
to the OC group (p = 0.149, ES = 0.8, 95% CI − 0.55 to 6 YC
2.01) and in YT group comparing to OT group (p = 0.454,
5
Bax/Bcl-2

ES = 0.63, 95% CI − 0.69 to 1.84) (Fig. 1). YT

One-way ANOVA showed non-significant group effect
(F = 1.459, p = 0.263) in Bax gene expression. Training
induced non-significant decrease in Bax gene expres-
sion in YT group comparing to the YC group (p = 0.093,
ES = 2.57, 95% CI 0.72–3.92) and in OT group compar-
ing to OC group (p = 0.302, ES = − 0.5, 95% CI − 0.8 to
1.71) (Fig. 1). Aging showed no significant effect in Bax
gene expression in YC group comparing to the OC group
(p = 0.623, ES = 0.23, 95% CI − 1.03 to 1.45) and in YT
group comparing to OT group (p = 0.828, ES = − 0.38,
95% CI − 1.59 to 1.71) (Fig. 1).
One way ANOVA showed non-significant group effect
(F = 2.080, p = 0.143) in Bax/Bcl-2 ratio. Training induced
non-significant decrease in Bax/Bcl-2 ratio in YT group
comparing to the YC group (p = 0.066, ES = 1.17, 95% CI
− 0.27 to 2.3) and in OT group comparing to OC group
(p = 0.152, ES = − 0.05, 95% CI − 1.29 to 1.19) (Fig. 1).
Aging showed no significant effect in Bax/Bcl-2 ratio in
control young group comparing to the control old group
(p = 0.670, ES = 0.9, 95% CI − 0.48 to 2.1) and in YT
group comparing to OT group (p = 0.973, ES = − 0.83,
95% CI − 2.03 to 0.53) (Fig. 1).
4
OC
3
OT
2
1
0
YC YT OC OT
Fig. 1  Effect of age and HIIT on Bcl-2 (a) and Bax (b) gene
expression and Bax/Bcl-2 ratio (c) in the heart myocardium. Data
(mean ± Standard error) are presented for young control (YC), young
Trained (YT), old control (OC) and old training (OT). *, **Signifi-
cant exercise effect p < 0.05, p < 0.01; respectively
Discussion
The influence of aging and exercise training on apoptosis
has not been fully defined. To our knowledge, this is the
first study to investigate the effect of regular HIIT on apop-
tosis in young and old cardiac muscle tissue. We demon-
strate that HIIT increased Bcl-2 gene expression in young
and old rats and showed no effects on Bax gene expression
and Bcl-2/Bax ratio. Together, the data indicate that regu-
lar exercise training is associated with augmented levels of

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Sport Sciences for Health (2021) 17:173–179
anti-apoptotic genes in cardiac muscle and aging had no
effect on apoptotic genes.
Similarly to our results, Bcl-2 gene expression in the
ventricular muscles of trained animals was not significantly
higher in compared with control animals (11%) [26]. Bcl-2
gene expression transcript content was 48% and 35% higher
in soleus and ventricle samples, respectively, of trained ani-
mals than in muscles of control animals, which indicated
that running 5 days weekly for 8 weeks attenuates the extent
of apoptosis in cardiac and skeletal muscles [17]. Running
5 days weekly for 12 weeks resulted in a down-regulation
of caspase-9 (Bcl-2 family) levels in left ventricle samples
from both the young and old age groups [27]. Bcl‐2 protein
levels decreased after spontaneous exercise in muscle of mdx
mice [28]. Based on these discrepant results, it is possible to
affirm that Blc-2 expression levels does not always show the
same pattern of response to physical exercise.
Contrary to our results, long-term swimming exercise
did not change protein expression of Bcl-2 [29].On the
other hand, other studies demonstrated that Bcl-2 protein
expression in the retinas of STZ-induced diabetic rats
was increased compared with the control [30]. Based on
these discrepant results, it is possible to affirm that Blc-2
expression levels does not always show the same pattern
of response to physical exercise. Previous study showed an
increase in both the Bcl-2 and Bax protein levels in aging rat
cardiac muscle [10]. Other studies also reported an increase
in Bax and Bax/Bcl-2 ratio protein levelsin aging rat cardiac
muscle [11] and in pro-apoptotic Bax and cleaved caspase-3
levels and DNA fragmentation in aging rat skeletal and car-
diac muscles [12, 13]. However, there was no significant
difference in the Bax and Bcl-2 protein expression levels
during aging, even with increased cardiomyocyte apoptosis
[14]. There are some limitations concerning mechanisms
responsible for apoptotic signaling in the aged heart.
The non-significant decrease in pro-apoptotic Bax gene
expression after training in young and old cardiac muscles
is not in line with previous studies. The transcript content
of Bax in soleus muscles of trained animals decreased sig-
nificantly by 35% when compared to control animals [17].
Running 5 days weekly for 12 weeks resulted in a signifi-
cant decrease in Bax protein expression of the left ventricle
in the old trained group when compared with old controls
[27]. Transcript contents of Bax in ventricular muscles of
the trained group were significantly lower than the control
group (80%) [26]. Nuclear Bax levels were dramatically
reduced to levels of normotensive animals following 6 weeks
of treadmill training [31]. Treadmill training up-regulated
anti-apoptotic markers in the cardiac tissue of rats after
ischemia–reperfusion injury [32].
We did not observe significant effect of training on Bax/
Bcl-2 ratio in young and old rats. Contrarily to our results,
177
myocardial Bax/Bcl-2 ratio was significantly higher in the
control group compared to the trained group [26]. Bax/
Bcl-2 ratio increased by 98% in the white gastrocnemius
with age but decreased dramatically by 96.8% with exer-
cise training in old rats [13]. Bcl‐2/Bax ratio decreased in
skeletal muscles after a 16 h spontaneous wheel running in
normal healthy animals [33, 34]The present study showed
no effect of aging on apoptotic markers. Contradictory to
our result, protein expression of proapoptotic Bax in the
left ventricle was much higher in the old sedentary group
than in the young sedentary group [27].
These findings indicated that physical training sup-
ports the heart against apoptosis [35] and could be a useful
strategy to prevent cardiac problems in the elderly [18].
Physical training prevents apoptosis through mitochondrial
pathway. Physical activity could be effective in prevent-
ing cardiac apoptosis by lowering ROS to some extent
and preventing release of mitochondrial cytochrome C
[36]. Although HIIT has been used in the field of medical
rehabilitation, the appropriate exercise protocol to prevent
from cardiac events has remained under debate. The diver-
sity of strategies applied in previous studies also make it
difficult to perform crosswise comparative analysis among
them [21]. Therefore, future experiments will be con-
ducted to indicate the most effective exercise strategies.
Strength of the current study was that this was the first
study to test the effect of HIIT and aging on apoptosis
markers. The study has limitations. The relatively low
number of animals. In addition, we did not compare with
type of exercise. A control group with another type of
exercise training was lacking. In addition, other apopto-
sis markers and proteins expressions were not assessed.
Finally, we did not use other biological techniques such as
western blot analysis, immunohistochemistry. Thus, fur-
ther investigations should examine the HIIT on different
cardiac muscle markers and compare between HIIT and
other exercise regimens.
Conclusion
The present study demonstrated that 4-weeks of HIIT
increased Bcl-2 gene expression in young and old cardiac
muscles and no effects of aging were revealed. These novel
findings indicated that HIIT ameliorated anti-apoptotic
signaling in the young and old cardiac muscles. Future
studies should explore the complex role of exercise regard-
ing cell protection in aged heart.
Acknowledgements The study was supported by Department of Exer-
cise Physiology, Faculty of Physical Education and Sport Sciences,
University of Tehran, Tehran, Iran.
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178
Compliance with ethical standards
Conflict of interest None of the authors have financial or other conflict
of interest in regard to this research.
Statement of human and animal rights All applicable international,
national, and/or institutional guidelines for the care and use of animals
were followed. The study protocol was approved by the Institutional
Animal Ethics Committee of the University.
Informed consent For this type of study, formal consent was not
required.
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