Introduction
The heart consumes the most energy in the cardiovascular system, allowing it to
provide the necessary oxygen for various tissues and organs to maintain the normal
metabolic and functional activities of the human body [1]. The benefits of physical exercise to the cardiovascular system have
been reported [2]
[3].
However, unrestricted increase in the time and intensity of exercise might not
always bring about more benefits to the cardiovascular system [4]. The heart is also one of the most sensitive organs
to overtraining [1]. Many studies have confirmed that
exhaustive exercise (EE) has adverse effects on the heart [5]
[6]
[7].
Exhaustive exercise not only causes a reduction in cardiac function and electro
cardio-electric changes, but also leads to destruction of the myocardial
ultrastructure and abnormal energy metabolism [1].
Exercise-induced myocardial injury has become a current focus of sports
medicine.
Reactive oxygen species (ROS) and overproduction of free radicals are considered to
be the most important causes of multiple tissue injury during EE [1]
[8]. Mitochondria, as
the key cell organelles responsible for energy production and the control of many
processes from signaling to cell death, are also important sites of ROS and free
radical production [9]. Mitochondria are also
important target organelles of oxidative stress. The heart has some of the highest
mitochondrial densities of tissues found within the body [10]. As such, the higher oxidative capacity of the heart suggests it has
higher potential to oxidative stress. Oxidative stress and mitochondrial dysfunction
have been extensively studied and are considered targets of various
pathophysiological processes [11]
[12]. One of the explanations for ROS production by
mitochondria is an excessive increase in energy demand. The mitochondrial
respiratory chain constitutes the main intracellular source of ROS in most tissues
[13]. It has been reported that complex I and
complex III of the respiratory chain are responsible for ROS production, and also
a
number of other mitochondrial oxidoreductases producing hydrogen peroxide
and/or superoxide radical [14]. Thus, studying
the relationship between mitochondrial function and oxidative stress during EE is
beneficial to deepen our understanding of the mechanism of myocardial injury induced
by EE.
S100A1, also known as S100 calcium-binding protein A1, is highly expressed in cardiac
and skeletal muscle, and localizes to Z-discs and the sarcoplasmic reticulum. Early
studies showed that S100A1 overexpression enhances cardiac contractile performance
and established the concept of S100A1 as a regulator of myocardial contractility
[15]. S100A1 released from ischemic cardiomyocytes
can signal myocardial damage via Toll-like receptor 4 [16]. S100A1 has been used as a target for gene therapy in the rodent
model following acute myocardial infarction [17].
These studies suggested that S100A1 plays protective role when cardiomyocytes are
damaged. However, studies on EE, myocardial injury, S100A1 and oxidative stress are
limited.
Therefore, the aims of the present study were to explore: (1) the effect of EE on
myocardial injury, S100A1, and oxidative stress; (2) the role of the S100A1 in
oxidative stress-induced cardiomyocyte injury; and (3) the mechanism by which S100A1
regulates mitochondrial function and oxidative stress. Our results will increase our
understanding of the precise role of S100A1 in cardiomyocyte injury induced by EE
and will promote the potential clinical applications of this protein as a diagnostic
or prognostic biomarker.
Materials and Methods
Animals and treatments
This study was conducted with the approval of the Animal Care and Use Committee
of the Tianjin University of Sport, China. Animal care was performed in
accordance with the China Laboratory Animal Management Regulations, the Guide
for the Care and Use of Laboratory Animals (Institute for Laboratory Animal
Research, Washington, DC, USA), and the Ethical Standards in Sport and Exercise
Science Research: 2020 Update [18]. The protocol
was approved by the Committee on the Ethics of Animal Experiments of Tianjin
University of Sport. All surgery was performed under sodium pentobarbital
(1%, 1 ml/100 g weight, i.p.) anesthesia, and
all efforts were made to minimize animal suffering.
Male Wistar rats (56 days old, about 220 g) were used for the experiment,
and twelve rats were randomly divided into two groups (6 rats/group):
control and exhaustive exercise (EE). Rats were acclimatized to their
surroundings for 1 week before the start of the experiment. The animals were
maintained on a 12 h light/dark cycle under a controlled
temperature of 25±2°C. Food and water were available for the
duration of the experiments unless otherwise noted. All animal handling
procedures were performed in strict accordance with the guide for the use and
care of laboratory animals. Exhaustive exercise was performed as described in
previous publications [19]
[20]
[21]. Briefly, two groups followed
by 10 days of adaptive exercise training on the small animal platform. And on
the 11th day, rats in EE group were trained for exhaustion exercise. First, rats
were trained with a slope of 0° and a speed of 9 m/min
for 15 minutes. Then, the rats were trained with a slope of 5°
and a speed of 15 m/min for 15 minutes. Finally, the
slope of the rat training was adjusted to 10° and the speed was adjusted
to 20 m/min, until they were exhausted. When the rat could not
run further under the condition of electrical stimulation, it was determined to
be exhausted. After exhausted exercise, all rats were humanely euthanized with
sodium pentobarbital [22]. The plasma samples and
myocardial tissues were collected for western blotting, immunohistochemistry,
and enzyme linked immunosorbent assay (ELISA).
Cell culture and treatments
H9c2 cells (rat embryonic cardiomyoblast-derived H9c2 cardiomyocytes) were
maintained at 37°C in a 5% CO2 incubator with
Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich
Corporation, St. Louis, MO, USA; D6429) supplemented with 10% fetal
bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA, USA; S12450), 2 mmol
L-glutamine (GIBCO, Grand Island, NY, USA; 25030–081), and 1%
penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA;
15140163).
Oxidative stress is one of the most important ways of cardiomyocytes injury
induced by exhaustive exercise [23]. The oxidative
stress model induced by hydrogen peroxide (H2O2) is often
used to study the myocardial injury induced by exhaustive exercise [24]. For oxidative stress, H9c2 cells were exposure
to H2O2 (1 mM) for 2, 4, 6, 12, 24 hours
at approximately 80%–90% confluency [25]
[26].
Constructs and reagents
H9c2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences
(Shanghai, China). The S100A1 overexpression plasmid was purchased from Open
Biosystems, Inc. (Lafayette, CO, USA). Lipofectamine 2000 was purchased from
Life Technologies (Grand Island, NY, USA). The TRIzol reagent (15596026) and
First-Strand Synthesis system (18080051) was bought from Invitrogen Corporation
(Waltham, MA, USA). Sequences of the S100a1 siRNA oligonucleotides were
5′-CUU CUG UCA AGA ACC UGC UTT-3′ and 5′-AGC AGG UUC UUG
ACA GAA GTT-3′. The antibodies specific against S100A1 (5066 s)
were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibodies
specific against ANT1 (ab102032), PGC-1α (ab54481), and Tfam (ab131607)
were obtained from Abcam (Cambridge, MA, USA).
Quantitative real-time PCR (qPCR)
Total RNA was extracted from cells using the TRIzol reagent. Total cDNAs were
synthesized using the RT-PCR system (Invitrogen Corporation; 11146–057).
Real-time PCR was conducted following the protocol for the Fast SYBR Green
Master Mix kit (Applied Biosystems, Foster City, CA, USA; 4385614) in a 7900HT
Fast Real-Time PCR System (Applied Biosystems). The primers for Pgc1a
(encoding PPARG coactivator 1 alpha) were 5′-TGG AGT GAC ATA GAG TGT
GCTG-3′ and 5′-TAT GTT CGC GGG CTC ATT GT-3′; for
Tfam (encoding transcription factor A, mitochondrial) were
5′-TCA TGA CGA GTT CTG CCG TT-3′ and 5′-CTT CAC AAA CCC
GCA CGA AA-3′; for S100a1 were 5′-AAA GAC CTG CTA CAA ACT
GA-3′ and 5′-CAC CAG CAC AAC AAA CTC C-3′; for
Ant (encoding adenine nucleotide translocase) were 5′-CGC TAC
TTC GCT GGT AAC CT-3′ and 5′-ATG ATG CCC TGC ACA GAG
AC-3′; and for Gapdh (encoding glyceraldehyde-3-phosphate
dehydrogenase) were 5′-CCC CCA ATG AAT CCG TTG TG-3′ and
5′-TAG CCC AGG ATG CCC TTT AGT-3′. Quantitative analysis was
conducted as previously reported [27].
Fluorescence imaging of ROS generation
Thirty minutes before imaging, cells were fed with phenol red free DMEM, loaded
with Dichloro-dihydro-fluorescein diacetate (DCFH-DA) (10 μM:
Sigma Aldrich) in the dark, and kept in a CO2 incubator at
37°C. Cells were then washed with DMEM and examined under a fluorescence
microscope (Thermo Fisher Technology Co. LTD, Waltham, MA, USA) [28]
[29]
[30].
MDA, SOD, GSH-PX, and CK content analysis
The contents of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione
peroxidase (GSH-PX), and creatine kinase (CK) in the cell cultures were measured
using enzyme-linked immunosorbent assay (ELISA) kits (A003-1, A001-2, A005 and
A032, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). ELISA was
performed according to the manufacturer’s instructions: The cell culture
was added into a 96-well plate (100 μL/well), which was
then sealed with parafilm and incubated at 37°C for 90 min. The
antibody (100 μL/well) was added and incubated for
60 min. The enzyme-binding solution
(100 μL/well) was added and incubated for a further
30 min. The plate was washed four times and the optical absorption was
estimated at 450 nm using a microplate reader (Bio-Rad, Hercules, CA,
USA).
Hematoxylin-eosin staining (HE) and immunohistochemistry (IHC)
The left ventricle was used to hematoxylin-eosin staining as previously described
[31]. Antibody specific against S100A1 (Cell
Signaling, Cat NO: 5066 s) was used for IHC, and the protein expression
levels of the left ventricle were analyzed as previously described [32].
Electron microscopy analysis
Electron microscopy analysis was conducted as previously described [33]. Briefly, the myocardial tissue less than 1
cubic millimeter was fixed in 2.5% glutaraldehyde phosphate buffer for
2 hours. Then, wash with 0.1 M phosphoric acid rinse solution
three times (15 minutes / time), fix with 1% osmic acid
fixed solution for 2–3 hours, and wash with 0.1 M
phosphoric acid rinse solution (15 minutes / time). Next,
ethanol is dehydrated, acetone is embedded and dried. The slices were cut into
70 nm thick sections and stained with 3% uranyl acetate and lead
citrate. Finally, the images were observed and photographed by transmission
electron microscope JEOL JEM-1230 (80 kV).
Seahorse methods
Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were
measured as previously described [34] using a
Seahorse XFe24 (Agilent Technologies). Before testing, DMEM medium without
glucose, pyruvate, and glutamax was used to as cell culture medium, and the
cells was incubated at 37°C for 1 h without CO2 in a
dry incubator. For OCR and ECAR measurements, oligomycin is used to block ATP
synthesis and measure proton leakage, FCCP is used to uncouple respiration, and
antimycin A is used to suppress electron transfer. First, three basal
measurements of OCR and ECAR were recorded, then 1 mM oligocmycin,
1 mM FCCP, and 1 mM antimycin A were injected successively.
During this period, proton leak, maximal respiration, and non-mitochondrial
respiration were recorded in turn. Reserve capacity is the difference between
maximal respiration and basal respiration, while ATP-linked OCR is the
difference between basal and proton leak.
Western blotting
Antibodies specific for S100A1, ANT1, PGC-1α, and Tfam were obtained from
Abcam (Cambridge, MA, USA). Anti-tubulin and Anti-GAPDH antibodies were
purchased from Bioworld Technology, Inc. (Bloomington, MN, USA). Western
blotting was performed as described in a previous publication [35]. Specifically, tissues or cells were lysed in
RIPA buffer (Solaibao Biotechnology Co., Ltd., Beijing, China) supplemented with
phenylmethylsulphonylfluoride (PMSF, Solaibao Biotechnology Co., Ltd., Beijing,
China) to obtain total protein. Total protein concentrations were determined by
using BCA Assay Kit (Solaibao Biotechnology Co., Ltd., Beijing, China). Samples
in an equal volume of 5X sample loading buffer were boiled (100°C,
10 min) in loading buffer. Samples were loaded on the polyacrylamide gel
(15%) along with the standard marker proteins and the electrophoresis
was run with supply of 250 mA current (100 min), followed by
transfer to a PVDF membrane (Millipore, USA). After being blocked with
5% nonfat dry milk for 2 h at room temperature, the membranes
were incubated with 1:1000-diluted primary antibody overnight at 4°C.
After washed 10 min in triplicate with the TBS-T buffer, the membranes
were incubated with goat anti-rabbit secondary antibody at room temperature for
1 h. Membranes were washed three times for 10 min and treated
with enhanced chemiluminescence (ECL) reagent. Autoexposure settings were used
to get protein bands. Meanwhile, the optical intensity of the bands was analyzed
by the ImageJ software. GAPDH or Tublin was used to normalize the western spot
values.
Statistical analysis
All statistical analyses were performed using SPSS 17.0 software (IBM Corp.,
Armonk, NY, USA). Wilcoxon rank sum test was used to determine the significance
of the differences between two groups. For the three or more groups of data,
statistical analysis was performed using Kruskal-Wallis test. A two-sided value
of P<0.05 was considered statistically significant.
Results
Exhaustive exercise leads to decrease in mitochondrial antioxidants, increase
ROS production, and myocardial injury in rats
Cardiac cells require a continuous supply of energy for their function and thus
contain a higher number of mitochondria to achieve their energy requirements
[36]. Under conditions of EE, a highly
oxidative metabolic environment predisposes the cardiac cells to free radical
damage. Therefore, an EE model of rats was constructed to evaluate myocardial
injury and oxidative stress under EE. The results showed that the level of ROS
in the plasma of the EE group was significantly higher than that of the control
group ([Fig. 1a]) (P<0.05). We
also found that the level of SOD ([Fig. 1b]) and
GSH-PX ([Fig. 1c]) in the EE group was
significantly lower than that in the control group (P<0.05).
Considering the correlation between oxidative stress and myocardial injury [37], we then examined the extent of the injury to
cardiomyocytes. Exhaustive exercise led to a significant increase in plasma CK
([Fig. 1d]), cTnT ([Fig. 1f]), cTnI ([Fig. 1g]) and CK-MB
([Fig. 1h]) levels (P<0.05).
Hematoxylin and eosin staining showed that EE resulted in myocardial damage,
including disordered distribution of cardiomyocytes, blurred cell boundaries,
myocardial fibers fracture, reticulated cytoplasm, and vacuolation ([Fig. 1e]).
Fig. 1 Exhaustive exercise leads to oxidative stress and
myocardial injury in rats. (a) ROS levels were detected in the
plasma of rats in the control and EE groups using fluorescence.
(b-c) The plasma of rats in the control and EE groups
was subjected to ELISA to determine the SOD and GSH-PX concentrations.
(d) The CK concentration was detected in the plasma of rats
in control and EE groups using ELISA. (e) The myocardial tissues
in rats in the control and EE groups were detected by HE staining.
(f) Concentration of cTnT in rats’ serum; (g)
Concentration of cTnI in rat’s serum; (h) Concentration
of CK-MB in rats’ serum. Wilcoxon rank sum test was used to
determine the significance of differences between two groups
(n=5). An asterisk (*) indicates a significant change
compared with that in the control group (P<0.05). Each
experiment was repeated six times.
Exhaustive exercise promotes myocardial mitochondria injury and
downregulation of S100A1 in rats
Considering that mitochondria are one of the important sites for ROS production
[38], we detected the effect of exhausting
exercise on the structure of mitochondria in vivo. As shown in [Fig. 2a], EE induced disordered and sparse
myocardial fibers and swelling of mitochondria. S100A1 is a regulator of
myocardial contractility. However, the relationship between S100A1 and
mitochondrial function or oxidative stress is largely unknown. In the present
study, S100a1 expression was downregulated after EE in rats ([Fig. 2b], [2c]
& [d]). These results indicate that
S100A1 might be related to mitochondrial dysfunction and oxidative stress after
EE.
Fig. 2 Exhaustive exercise promotes myocardial mitochondria injury
and downregulation of S100A1 in rats. (a) The myocardial tissues
of rats in the control and EE groups were examined using electron
microscopy. (b) The myocardial tissue extracts were subjected to
western blotting to determine S100A1 levels. GAPDH was used as a protein
loading control. (c) Quantitative analysis of the S100A1 level in
(B) by Image J. An asterisk (*) indicates a significant change
compared with that in the control group (P<0.05, n=3).
(d) S100A1 levels detected using immunohistochemistry.
(e) Quantitative analysis of the S100A1 level in (D) by Image
J (https://imagej.en.softonic.com/). Calculate the integral optical
density of the brown area in the picture and the area of the target
distribution area to get the average optical density value, which is
used to represent the S100A1 level of the sample.
S100A1 protects H9c2 cells from oxidative stress in vitro
To further explore the relationship between the decrease in S100A1 expression and
oxidative stress induced by EE, we used an S100a1 overexpression vector
to increase the level of S100A1 in cells under oxidative stress. The results
showed that oxidative stress induced by H2O2 could
decrease the cell survival rate in a dose-dependent ([Fig. 3a]) and time-dependent ([Fig.
3b]) manner. The mRNA level of S100A1 was significantly increased in
H9c2 (S100A1) cells compared with H9c2 (Vector) cells under oxidative stress
([Fig. 3c]). S100a1 overexpression
significantly reduced the death rate ([Fig. 3d])
of H9c2 cells induced by oxidative stress. Intracellular ROS levels of H9c2
(S100A1) also decreased compared with those in H9c2 (Vector) cells under
oxidative stress ([Fig. 3e], [f]). In terms of intracellular antioxidant enzyme
levels, S100a1 overexpression led to a significant increase in SOD and
GSH-PX level in oxidatively stressed H9c2 cells ([Fig.
3g] & [h]). The results
illustrated that S100A1 could inhibit oxidative stress in H9c2 cells induced by
H2O2, and reduce the injury and mortality of oxidative
stress in H9c2 cells. S100a1 overexpression significantly reduced ROS
levels ([Fig. 3i]) and inhibited injury ([Fig. 3j]) in H9c2 cells treated by
H2O2.
Fig. 3 S100A1 protects H9c2 cells from oxidative stress in
vitro. (a) H9c2 cells (2 × 105) were
seeded into each well of 6-well plates and cultured until the cell
density reached 80–90%. The cells were then exposed to
0, 0.2, 0.5, 1.0, or 2.0 mM H2O2 for
24 h. The cells were subjected to a luminescent assay to
determine the cell viability. (b) The H9c2 cells were exposed to
1 mM H2O2 for 0 h, 2, 4, 6, 12, or
24 h. The cells were subjected to a luminescent assay to
determine the cell viability. An asterisk (*) indicates a
significant decrease compared with that in the control group
(P<0.05, n=3). (c) S100a1 overexpression
was identified in H9c2 cells transfected with the S100A1 vector in
comparison with H9c2 cells transfected with the control vector under
1 mM H2O2 treatment. (d)
Representative images of H9c2 (S100A1) and H9c2 (Vector) cells, with or
without oxidative stress, were captured under a microscope. (e)
Intracellular ROS levels in H9c2 (S100A1) and H9c2 (Vector) cells, with
or without oxidative stress, were detected using a DCFH-DA fluorescent
probe. The fluorescence intensity was scored and presented as relative
units (f). (g-h) The SOD and GSH-PX concentrations
of cell extracts were detected using ELISA. (i) ROS levels in
cell extracts of H9c2 (S100A1) and H9c2 (Vector) cells, with or without
oxidative stress, were detected using fluorescence. (j) CK
concentrations were detected using ELISA. An asterisk (*)
indicates a significant change (P<0.05, n=3)
S100A1 increases maximal respiration by Seahorse
The function of the mitochondrial respiration is coupled with the production of
ROS in the form of superoxide anions or hydrogen peroxide [9]. Therefore, we tested the effect of S100A1 on
mitochondrial respiratory function in oxidatively stressed cardiomyocytes. As
shown in [Fig. 4a], H2O2
induced a significant decrease in mitochondrial respiratory function including,
basal, ATP-linked, proton-leak, maximal respiration, and non-mitochondrial
respiration. Upregulation of S100a1 showed a protective effect on
mitochondrial respiration (maximal respiration and non-mitochondrial
respiration) of H9c2 cells under oxidative stress ([Fig. 4a] & b). Based off our previous data that EE and
H2O2 could lead to a decrease in S100a1
expression and the oxidative stress response in cardiomyocytes, we decided to
knock-down S100A1 in cardiomyocytes. Thus, we introduced si-S100A1 into H9c2
cells to explore the effect of S100A1 on respiratory function. The results
indicated that knockdown of S100a1 led to a significant decrease in
mitochondrial respiratory function, including basal, proton-leak, maximal
respiration, and non-mitochondrial respiration ([Fig.
4c] & [d]). These results
suggested that S100A1 is an important molecule in the redox balance role of the
mitochondria, which therefore allows for normal respiration to occur.
Fig. 4 S100A1 increases maximal respiration, as assessed using the
Seahorse system. (a) Oxygen consumption rate (OCR) of H9c2
(S100A1) and H9c2 (Vector) cells, with or without oxidative stress, was
detected using the Seahorse system. (b) OCR was analyzed in
several stages of respiration in H9c2 (S100A1) and H9c2 (Vector) cells
with oxidative stress, including basal, ATP-linked, proton-leak, Maximal
respiration, and non-mitochondrial respiration. (c) OCR of H9c2
(si-S100A1) and H9c2 (Nonsense) cells was detected using the Seahorse
system. (d) OCR was analyzed in several stages of respiration in
H9c2 (si-S100A1) and H9c2 (Nonsense) cells, including basal, ATP-linked,
proton-leak, Maximal respiration, and non-mitochondrial respiration. An
asterisk (*) indicates a significant change (P<0.05,
n=3)
S100A1 promotes the expression of ANT1, PGC-1α, and Tfam in H9c2
cells
For cells, the respiratory capacity of mitochondria is related to their quantity
and function. Therefore, we detected the effect of S100A1 on expression of
Ant1, Pgc1a, and Tfam, which are related to
mitochondrial oxidative phosphorylation, transcription of energy metabolism
genes, and mitochondrial genome replication, respectively. As shown in [Fig. 5], H2O2 led to a
significant reduction in Ant1, PGC-1α, and Tfam expression at the
mRNA ([Fig. 5a]) and protein ([Fig. 5b]) levels. Upregulation of S100a1 in
H9c2 cells (treated with H2O2) promoted the expression of
Ant1, Pgc1a, and Tfam ([Fig. 5b]–[f]. Furthermore, the expression patterns of ANT1,
PGC-1α, and Tfam in H9c2 cells (si-S100A1) were similar to those in H9c2
cells (treated with H2O2). Specifically, downregulation of
S100a1 in H9c2 cells inhibited the mRNA expression ([Fig. 6a]) and protein content ([Fig. 6b]–[f]) of Ant1, Pgc1a, and Tfam. These results indicated that
S100A1 is a key protein of mitochondrial oxidative phosphorylation, energy
metabolism gene transcription, and mitochondrial genome replication via its
regulation of ANT1, PGC-1α, and Tfam.
Fig. 5 Upregulation of S100A1 promotes the expression of
Ant, Pgc1a, and Tfam in H9c2 cells. (a)
Real-time PCR was carried out to determine the mRNA expression of
Ant, Pgc1a, and Tfam using cDNA samples
collected from H9c2 cells (1 mM H2O2 for
24 h), H9c2 cells (S100A1, 1 mM
H2O2 for 24 h), or H9c2 cells
(non-treated control). Values in the graphs represent the
mean±standard deviation; an asterisk (*) indicates a
significant change (P<0.05, n=3). (b) Extracts of
H9c2 cells (1 mM H2O2 for 24 h),
H9c2 cells (S100A1, 1 mM H2O2 for
24 h), or H9c2 cells (non-treated control) were analyzed using
western blotting. Tubulin was used as a protein loading control.
(c–f) Quantitative analysis of ANT,
PGC-1α, S100A1, and Tfam levels in (B) using Image J. Values in
the graphs represent the mean±standard deviation; an asterisk
(*) indicates a significant change (P<0.05,
n=3).
Fig. 6 Knockdown of S100a1 inhibits the expression of
Ant, Pgc1a, and Tfam in H9c2 cells. (a)
Real-time PCR was carried out to determine the mRNA expression of
Ant, Pgc1a, S100a1, and Tfam using cDNA
samples collected from H9c2 cells (1 mM
H2O2 for 24 h), H9c2 cells
(si-S100A1), H9c2 cells (Nonsense siRNA) or H9c2 cells (non-treated
control). Values in the graphs represent the mean±standard
deviation; an asterisk (*) indicates a significant change
(P<0.05, n=3). (b) Extracts of H9c2 cells
(1 mM H2O2 for 24 h), H9c2 cells
(si-S100A1), H9c2 cells (Nonsense siRNA), or H9c2 cells (non-treated
control) were analyzed using western blotting. Tubulin was used as a
protein loading control. (c-f) Quantitative analysis of
ANT, S100A1, PGC-1α and Tfam levels in (B) using Image J. Values
in the graphs represent the mean±standard deviation; an asterisk
(*) indicates a significant change (P<0.05,
n=3).
Discussion
Although free radicals were discovered in 1954, it was not until the 1970s that
oxidative stress caused by muscle exercise was linked to body damage [39]. In recent years, death caused by EE and excessive
fatigue has been gradually recognized, and the injury effects caused by EE have
become a research focus. ROS produced in mitochondria participate in a variety of
signaling and damaging pathways, regulating a variety of physiological and disease
processes [40]. In the present study, our results
showed that significantly increased ROS levels in the serum of exhausted rats were
accompanied by myocardial tissue damage. Further testing found that plasma CK also
increased significantly, while SOD and GSH-PX levels in plasma decreased
significantly ([Fig. 1]). These results suggest that
increased ROS after EE might be involved in the process of myocardial injury.
However, the mechanism of myocardial injury during EE remains unclear.
Mitochondria are not only the source of energy supply during EE, but also are one
of
the important sites of ROS production. In addition, mitochondria are an important
target of ROS. Ultrastructural observation of the mitochondria in the myocardium of
EE rats was performed using electron microscope. As shown in [Fig. 2a], EE induced disordered and sparse myocardial
fibers and swelling of mitochondria. In vitro, we also confirmed that
oxidative stress caused significant impairment of mitochondrial respiratory function
([Fig. 4a]). In view of the important role of
S100A1 in the protection of myocardial cell injury, and the relationship between
S100A1 and exercise, we detected the expression of S100A1 in the myocardium of EE
rats. The results showed that EE led to a significant decrease in S100A1 levels as
shown by immunohistochemistry ([Fig. 2d]) and western
blotting ([Fig. 2b]). Those results indicated that
S100A1 downregulation in EE rats seemed to be involved in mitochondria and
cardiomyocytes injury.
There are reports that S100A1 can regulate the inflammatory response and oxidative
stress in H9C2 cells via the TLR4/ROS/NF-κB pathway [41]. However, the regulatory effect of oxidative stress
on S100A1 and the feedback regulatory effect of S100A1 on ROS have not been
explained clearly. To further explore the relationship between ROS and S100A1
expression, H9c2 cells were employed to model oxidative stress in vitro. We
found that oxidative stress resulted in a significant decrease in cell survival
([Fig. 3a] & [b]) and S100a1 expression ([Fig.
3c] & [Fig. 5b]), and that
overexpression of S100a1 reduced the level of oxidative stress ([Fig. 3e] & [f])
and increased the cell survival rate ([Fig. 3d]) in
H9c2 cells. These results suggested mutual regulation between ROS and S100A1.
Mitochondrial function and ROS are also mutually regulated [42]
[43]. Thus, we hypothesized that the
relationship between ROS and S100A1 is related to mitochondrial function. The
results of Seahorse system analysis showed that oxidative stress led to
mitochondrial respiratory dysfunction including, basal, ATP-linked, proton-leak,
maximal respiration, and non-mitochondrial respiration, and S100a1
overexpression inhibited the effect of oxidative stress in H9c2 cells ([Fig. 4a] & [b]). Inhibition of S100a1 led to mitochondrial respiratory dysfunction
in H9c2 cells ([Fig. 4c] & [d]). These results suggested that S100A1 is partly
responsible for mitochondrial respiration dysfunction under oxidative stress in H9c2
cells. Other mechanisms that can lead to myocardial injury during exhausting
exercise are inhibiting autophagy, reducing mitochondrial function and increasing
the level of oxidative stress [44]
[45]. There are limitations in the conclusion drawn only
from oxidative stress cell model.
S100A1 is a regulator of Ca2+ in cardiomyocytes [46]
[47]
[48]. Specifically, enhancement of L-type calcium
channel trans-sarcolemmal calcium influx by S100A via protein kinase A has been
reported [49]. Considering the relationship between
calcium regulation and mitochondrial function in cells [50]
[51], it is not surprising that
Ca2+ is one of the pathways by which S100A1 regulates
mitochondrial function. To explore the non-Ca2+ mitochondrial
regulatory pathway of S100A1, we detected the expression of ANT1, PGC-1α,
and Tfam, which are related to mitochondrial oxidative phosphorylation [52], the transcription of energy metabolism genes [53], and mitochondrial genome replication [54] in oxidatively stressed H9c2 cells. The results
showed that the expression levels of ANT1, PGC-1α and Tfam were
significantly decreased in H9c2 (H2O2) cells compared with
those in H9c2 cells. Overexpression of S100a1 reversed the decrease in ANT1,
PGC-1α and Tfam expression induced by H2O2 ([Fig. 5]). Inhibition of S100a1 expression in
H9c2 cells achieved similar effects to those of oxidative stress ([Fig. 6]). These results demonstrate the regulatory
effect of S100A1 on ANT1, PGC1, and Tfam on the transcriptomic level.
