Exosomes derived from cardiac progenitor cells attenuate CVB3-induced apoptosis via abrogating the proliferation of CVB3 and modulating the mTOR signaling pathways
Li et al. Cell Death and Disease
Exosomes derived from cardiac progenitor cells attenuate CVB3-induced apoptosis via abrogating the proliferation of CVB3 and modulating the mTOR signaling pathways
Xin Li 0
Zuocheng Yang 0
Jie Jiang 0 1 2
Shentang Li 0
Zhuoying Li 0
Lang Tian 0
Xing Ma 3
0 Department of Pediatrics, the Third Xiangya Hospital, Central South University , Changsha , China
1 Department of Urology , Chinese People's Liberation Army , 89th Hospital , Weifang, Shandong , China
2 Department of Urology , Chinese People's Liberation Army , 89th Hospital , Weifang, Shandong , China
3 Sate Key Laboratory of Advanced Welding and Joining, Harbin Institute of Technology (Shenzhen) , Shenzhen , China
Viral myocarditis is potentially fatal and lacking a specific treatment. Exosomes secreted by cardiac progenitor cells (CPCs) have emerged as a promising tool for cardioprotection and repair. In this study, we investigated whether CPCsderived exosomes (CPCs-Ex) could utilize the mTOR signal pathway to reduce the apoptosis in viral myocarditis. In vitro, exosomes were, respectively, added to H9C2 cells after CVB3 infection to detect the anti-apoptosis effect of CPCs-Ex. Compared with the controls, the apoptosis rate was reduced, accompanied with the depressed expression of viral capsid protein 1 (VP1) and pro-apoptosis factors of Bim/caspase families. Meanwhile, the phosphorylation of Akt, mTOR, and p70S6K were promoted, but that of 4EBP1 was suppressed. In vivo, the results of apoptosis, expression of CVB3 and pro-apoptosis factors, and phosphorylation of Akt/mTOR factors of CVB3-infected cardiomyocytes were consistent with that of vitro. Following that, we use Rapamycin and MK-2206 to inhibit the Akt/mTOR signaling pathway, meanwhile, Rattus 4EBP1, p70S6K, Akt1 and Akt2 were transfected to H9C2 cells to establish the stably transfected cell lines. In the group with Rapamycin or MK-2206 pretreatment, CPCs-Ex also could decrease the apoptosis of H9C2 cells and expression of CVB3 mRNA, followed by decreased expression of apoptosis factors. In Akt2, p70S6K and 4EBP1 overexpression groups, CPCs-Ex promoted CVB3-induced apoptosis, VP1 expression and cleavage of caspase-3. Our results therefore identify CPCs-Ex exerts an anti-apoptosis effect in CVB3-infected cells by abrogating the proliferation of CVB3 and modulating the mTOR signaling pathways as well as the expression of Bcl-2 and caspase families. Viral myocarditis, mainly caused by CVB3 infection, is lacking a specific treatment. Our study identified an antiapoptosis role of CPCs-Ex in CVB3-infected cells and rats, which shown that CPCs-Ex may be an effective tool to treat viral myocarditis. We believe that with more in-depth research on the functionality of CPCs-Ex, there will be a breakthrough in the treatment of viral myocarditis.
Viral myocarditis (VMC) is a common cause of dilated
cardiomyopathy and sudden cardiac death1. Therefore,
finding effective therapies for VMC is still a big challenge,
and recent researches on cardiac progenitor cells (CPCs)
may provide a turning point for the treatment of VMC2.
CPCs are a group of heterogeneous cells distributed
throughout the heart and able to differentiate into several
cell types, such as cardiomyocytes (CMs), vascular smooth
muscle cells and endothelial cells (ECs), holding great
promise for cardiac regeneration and functional
reconstruction. After years of investigation, stem-cell therapy
has yielded encouraging results in heart repair3–5.
Moreover, CPCs from adult heart have emerged as better
candidates for cardiac cell therapy compared with stem
cells from bone marrow or adipose tissue6. Nevertheless,
direct transdifferentiating into cardiac tissues is
considered unlikely. The mechanism of adult stem-cell
therapy has been tested to be mediated through paracrine
release of extracellular vesicles containing growth factors
and cytokines to exert anti-apoptosis, suppress immunity,
and promote angiogenesis6,7. And exosomes may be the
major active components of extracellular vesicles derived
Exosomes are nano-sized (30–100 nm) particles secreted
via exocytosis from dendritic cells (DCs), macrophages,
T cells and cells of other tissue origins under physiological
and pathological conditions, carrying proteins, lipids, and
regulatory nucleic acids such as small and large non-coding
RNAs into recipient cells9. Several pre-clinnical studies
have suggested a better therapeutic role of CPCs-derived
exosomes (CPCs-Ex) for cardiac repair compared with cell
transplantation10. Indeed, CPCs-Ex containing specific
agents have been effectively used to treat cardio-vascular
disease. Chen et al.11 reported that Sca1 + CPCs-Ex can
reduce cell apoptosis via inhibiting caspase-3/7 activation
in a mouse model of acute myocardial
ischemia/reperfusion. CPCs-Ex enriched with miR-21 were reported to
prevent cardiomyocytes apoptosis by targeting PDCD412.
Recently, a deep proteomic analysis of neonatal CPCs-Ex
conducted by Sudhish Sharma et al.13 identified a high level
of insulin-like growth factor-1 (known to promote
cardioprotection, inhibit apoptosis), suggesting an anti-apoptosis
role of CPCs-Ex.
Coxsackievirus B (CVB), a member of the
Picornaviridae family, is a common enterovirus that can cause
various human systemic inflammatory disease such as
myocarditis, meningitis, and pancreatitis and the six CVB
serotypes are each responsible for different diseases and
syptomes14, of which the infants and children are more
VMC still lacks effective treatments in this aria. Several
preclinical stem-cell therapies have made some progress
in reducing inflammation and improving myocardial
function, but they are still not satisfactory4,18,19. As a
cellfree therapeutic method, exosomes could avoid many of
the limits of cell therapy. Barile et al.20 has demonstrated
that CPCs-Ex could prevent staurosporine-induced
cardiomyocyte apoptosis and they were more
cardioprotective than MSCs-secreted exosomes. But the role of
CPCsEx in VMC was still unexplored. Here we investigated the
cardioprotective effect of the CPCs-Ex for myocardial
cells in CVB3-induced myocarditis model, which is
mainly through abrogating the CVB3 proliferation as well
as regulating the expressions of mTOR signaling pathway
and Bcl-2, caspase families. The fruitful work offers
a possible cell therapy approach for viral myocarditis
Materials and methods
Cell isolation and culture
Cardiac progenitor cells (CPCs) were generated from the
hearts of 2-month-old male Sprague-Dawley (SD) rats
following these steps. Briefly, the first, the rat heart tissue
was aseptically isolated and chopped with scissors and
scalpel (finer as possible), and the tissue debris were loaded
into 15 ml tube. Second, 5 ml type IV collagenase digestion
(1 mg/mL, containing Dnase I) was added, digested 5 mins
in 37 °C, three times in total. After that, discarding the
supernatant by standing or briefly centrifuged. Then, after
cleaned with PBS, the tissue block was re-suspended with
CEM medium and noculated in 20 µg/mL FN coated Petri
dish. After 14 days, the dishes were gently washed with PBS
and then digested for 1–2 min with a 0.05% trypsin
(preheated at 37 °C). The collected cells were cultured and
maintained in complete media containing M199 (Corning,
Corning, NY, USA), EGM-2 (Lonza, Walkersville, MD,
USA), 10% exosomes-depleted FBS, 10 nM b-FGF, 1%
MEM nonessential amino acids (Gibco, USA), and
penicillin–streptomycin (Gibco, USA).
Exosome isolation and purification
The CPCs-Ex isolation and purification were followed
by the procedure of ExoQuick-TCTM Exosome Isolation
Reagent (System Biosciences, USA). When exosomes
were prepared from media, the media was first
concentrated from 50 mL to 130 µL with Amicon Ultra filter
(Millipore, Billerica, MA) with a 100000-molecular weight
cutoff before ExoQuick treatment21,22.
Transmission electron microscopy
As Hinescu et al.23 described before, exosome pellet was
re-suspended and fixed with 2.5% glutaraldehyde,
postfixed in buffered 1% OsO4 with 1.5% K4Fe(CN)6,
embedded in 1% agar, and processed according to the
standard Epon812 embedding procedure. The presence
and the size of exosomes were determined using
transmission electron microscopy (TEM, FEI Company,
Netherlands) at 80 kV. Micrographs were used to measure
the diameter of exosomes.
Exosome labeling with DioC18(3) and uptake study
To assess in vitro uptake of CPCs-Ex by H9C2 cells, the
purified CPCs-Ex were labeled with DioC18(
perchlorate, Dio) green fluorescent labeling kit (Yeasen
Company, China) according to the procedure. The Dio
concentration is 0.5 µM per microliter exosomes from 1 ×
104 cells. The labeled exosomes were stained with Dio dye
in 100 µL DMSO diluted by DMEM for 20 min at 37 °C,
and another equal volume of serum without exosomes was
added to stop the labeling. Then, the labeled CPCs-Ex
were incubated with H9C2 cells for 12 h at 37 °C and
determined with the fluorescence microscope.
All rats (male SD rats, 0–3 days after birth) used in our
study were obtain form the department of Laboratory
Animals, Central South University, China. And all
experimental protocols were approved by the Institutional
Review Board (IRB) of Third Xiangya Hospital, Central
South University, China. All animal procedures
conformed to the guidelines from Directive 2010/63/EU of
the European Parliament on the protection of animals
used for scientific purposes. In CVB3 group (n = 12), rats
were injected CVB3 with 104 TCID50 enterocoelialy to
establish the viral myocarditis modal. In addition, the rats
infected with CVB3 were injected with CPCs-Ex
intravenously at 24 h p.i. in CPCs-Ex group (n = 12). Afterward,
all rats were sacrificed by cervical dislocation at 7 days p.i.
and acquired the heart tissue. Then, the tissue structural
modification was observed using hematoxylin-eosin (HE)
staining and TEM. The apoptosis of myocardial cells was
detected by TdT-mediated dUTP nick-end labeling
(TUNEL) as well as the immunofluorescence (IF).
Realtime fluorescence quantitative PCR and western blot
analysis were used to examine the expression of mTOR
signaling factors and apoptosis proteins.
Plasmid construction and cells stable transfection
The empty vector (pcDNA3.1-myc-HisA(-)) were the
generous gifts from Dr. Qiaojia Huang (Molecular
Medicine Research Center of Fuzhou General Hospital,
Nanjing Military Region) preserved in our library. The
sequences of Rattus 4EBP1, p70S6K, Akt1, and Akt2 were
acquired from NCBI and amplified from cDNA of H9C2
cell line. The pcDNA3.1-myc-HisA(-)-4EBP1,
pcDNA3.1myc-HisA(-)-p70S6K, pcDNA3.1-myc-HisA(-)-Akt1, and
pcDNA3.1-myc-HisA(-)-Akt2 constructs expressing
Rattus 4EBP1, p70S6K, Akt1, and Akt2, respectively, were
expressed and extracted from Escherichia coli. After that,
H9C2 cells were obtained from the Institute of Oncology,
Central South University and grown in Dulbecco’s
modified Eagle’s medium (DMEM, Gibco, Life Technologies,
Inc.) containing 10% heat-inactivated fetal bovine serum
(FBS, Gibco, Life Technologies, Inc.) which was extracted
exosome by ultracentrifuging at 37 °C in a humidified
incubator with 5% CO2. H9C2 cells at 60% confluence
were transfected with pcDNA3.1-myc-HisA(-)-4EBP1/
p70S6K/Akt1/Akt2 or an empty vector, respectively. For
stable transfection of H9C2 cells, 4 μg of expression
plasmid was introduced by using Lipofectamine 2000
reagent (Invitrogen, Life Technologies) according to the
manufacturer’s instructions. At 5–6 h post transfection,
the cells were refreshed by the DMEM containing 10%
exosome-free FBS. And then, after 12–24 h, Geneticin
(G418) (Gibco, Life Technologies, Inc.) was added as a
selective marker at the final concentration of 800 μg mL−1
for selecting the transfected clones and at the final
concentration of 400 μg/mL for the maintenance of
transfection during the course of experiments24.
Coxsackievirus B3 (CVB3) Nancy strain was obtained
from Shanghai Jiao Tong University School of Medicine,
propagated in H9C2 cells and stored at −80 °C in our
laboratory. The titer of virus was examined prior to each
experiment. Cells in CVB3 groups were infected at 100
TCID50 with CVB3 or with DMEM containing 2%
exosome-free FBS for Sham. After 1 h infection, cells were
washed with phosphate-buffered saline (PBS) and
replenished with fresh DMEM containing 2% exosome-free FBS,
kept growing in a humidified incubator with 5% CO2.
Cell apoptosis analysis
Apoptosis in different groups was determined by
fluorescence-activated cell sorting (FACS) analysis of cells
stained with Annexin-V FITC and propidium iodide (PI,
Promega) by flow cytometer (FCM). At 12 and 24 h p.i.,
cells were harvested using 0.25% EDTA-Trypsin (Gibco,
Life Technologies, Inc.). After centrifugation, cell pellets
were washed twice with cold PBS, and then the cells
pellets were incubated with Annexin-V FITC and
propidium iodide to achieved double staining, according to the
manufacturer’s instructions. The mixture was incubated
in the dark for 15 min at room temperature. Afterward,
400 μL of 1 × binding buffer was added to each tube and
cells were immediately analyzed by FACS Calibur flow
cytometry (Becton Dickinson, USA)24.
Real-time fluorescence quantitative PCR
H9C2 cells in different groups were harvested at 12 and
24 h p.i. First, mRNA was extracted following the method
of RNA Extracted Kit (Omega, USA). Next, mRNA was
reverse transcribed into cDNA following the
RevertAidTM First Strand cDNA Synthesis Kit (Thermo, USA).
At last, real-time fluorescence quantitative PCR (RT-PCR)
amplification was done following the SYBGREEN PCR
Master Mix Kit (ABI, USA). The primer sequences are on
the table below (Table 1). The amplification profile was
10 min at 95 °C, 15 s at 95 °C, 30 s at 60 °C for 40 cycles.
Signal of a gene was normalized with β-actin using the
formula ΔCT = CT target − CT reference. And ΔΔCT =
mean value of ΔCT control − ΔCT sample. At last,
2-ΔΔCt method was used to calculate the differences of
mRNA transcript level21.
Western blot analysis
CPCs-Ex, rat lymphocytes and H9C2 cells in different
groups were washed twice with ice-cold PBS and then
kept on ice for 10 min. In short, 80 μL lysis buffer
(Beyotime Institute of Biotechnology) containing 0.1%
phenylmethylsulfony (PMSF, Cwbio, China) was added in
each well. Afterward, cell lysates were collected, and the
precipitate was discarded after centrifugation. Then 30 μg
extracted protein were fractionated on sodium dodecyl
sulfate—10 to 12% polyacrylamide gels,
electrophoretically transferred to 0.45 μm PVDF membranes
(Millipore Corporation), and blocked with PBS containing
0.5‰ Triton-100 (Cwbio, China) and 5% nonfat dry milk
for 1 h. Afterward, the membrane was incubated with the
primary antibody (monoclonal anti-CD63 (Abcam, USA)),
monoclonal anti-CD-81 (Santa Cruz Biotechnology, Inc.,
USA), monoclonal anti-4EBP1 antibody (Cell Signaling
Technology), monoclonal anti-p70S6K antibody (Cell
Signaling Technology), monoclonal anti-enterovirus
antibody (Dako Co.), monoclonal anti-bim antibody
(Cell Signaling Technology), polyclonal anti-bax
antibodies (Cell Signaling Technology), monoclonal
anti-caspase-9 antibody (Cell Signaling Technology),
polyclonal anti-caspase-3 antibodies (Cell Signaling
Technology), ponoclonal anti-Akt antibody (Cell
Signaling Technology, USA), monoclonal anti-pAkt antibody
(Cell Signaling Technology, USA), monoclonal
antimTOR antibody (Cell Signaling Technology, USA),
monoclonal anti-pmTOR antibody (Cell Signaling
Technology, USA), monoclonal anti-BNP antibody (ab19645),
monoclonal anti-c-Kit antibody (Biotin, ab25022),
ponoclonal anti-CK-MB antibody (Proteintech: 15137-1-AP),
ponoclonal anti-cTnI antibody (Proteintech: 21652-1-AP)
and monoclonal anti-β-actin antibody (Proteintech
Group, Inc., USA) at 4 °C overnight, followed by
incubation with horseradish peroxidase-conjugated secondary
antibodies (Proteintech Group, Inc., USA). At last, protein
expression was detected by enhanced chemiluminescence
(Pierce Professional Resources, USA).
Heart tissue was washed thrice with cold PBS at each
time point, fixed in cold paraformldehyde for 30 min, and
then blocked with PBS containing 5% Tween (Cwbio,
China) and 3% bovine serum albumin (BSA) for 2 h at
4 °C, then incubated with monoclonal anti-enterovirus
antibody (Dako Co.) for 4 h at room temperature.
Followed by fluorophore-labeled donkey anti-mouse IgG
(H + L) antibody (Invitrogen, Life Technologies), DAPI
(Roche Group, Switzerland) was incubated for 3 min at
room temperature to dye the nucleus at last and observed
under Olympus microscope (Olympus Corporation,
Japan) equipped with a Metamorph image acquisition
system (DP2-BSW software).
Two-way analysis of variance with multiple
comparisons and paired Student’s t-tests were performed. Data
were presented as the mean ± standard error (S.E.). The
P value of <0.05 was considered significant.
CPCs-Ex identification, labeling, and uptake in H9C2 cells
To extract the exosomes, the CPCs were isolated and
cultured from the SD rats heart tissue firstly (Fig. 1a), then
identified by c-kit makers (Fig. 1b). The isolated CPCs-Ex
were generated as described above and were visualized
and detected using TEM and FCM. As we shown,
exosomes were 30–100 nm in diameter on average (Fig. 1c).
Compared with the lymphocyte lysates, samples have
been demonstrated to contain a large number of
exosomes, revealed that the tetraspanin molecule CD63 and
CD81 were abundant in CPCs-Ex (Fig. 1d). And FCM also
showed that CPCs-Ex containe a lot of CD63 (Fig. 1e).
Furthermore, we labeled exosomes with DioC18(
fluorescent cell linker compound that is incorporated into
the cell membrane via selective partitioning. After
incubating the labeled CPCs-Ex with H9C2 cells, we observed
a green fluorescence in the cytoplasm in almost every
H9C2 (Fig. 1f).
CPCs-Ex reduce the CVB3-induced H9C2 cell apoptosis via
depressing the expression of VP1, blocking CVB3-induced
Bim and Bax activation and cleavage of caspase-9 and caspase-3 and promoting the expression of BcL-2 in vitro
To illustrate the relation between CPCs-Ex and the
CVB3-induced apoptosis, we established the CVB3
infection cell modal, moreover, CPCs-Ex was added with
200 ng/mL at 1 h later after the CVB3 infection. After 24
and 48 h p.i., the expression of CVB3, Bim, Bcl-2, caspase-3,
caspase-9 using real-time fluorescence quantitative PCR
(RT-PCR) and western blot (WB) analysis and apoptosis
rate was detected by flow cytometry (FCM). We found that
compared with the control, CVB3-induced apoptosis was
inhibited by the CPCs-Ex at 12, 24, and 48 h p.i. (Fig. 2a &
supplementary file 1, P < 0.05), following the abrogated
VP1 expression (Fig. 2b, c, P < 0.05). As the anti-apoptosis
factor, the activation of Bcl-2 induced by CVB3 infection
was further activated, while the expressions of the
proapoptosis factors Bim and Bax were inhibited. It was also
noteworthy that like the effect of caspase-9 and caspase-3,
the provoked cleavage of them was prevented during the
infection course (Fig. 2d, P < 0.05).
CPCs-Ex stimulate the Akt/mTOR signaling pathway and its phosphorylation suppressed by CVB3 infection
Our previous studies have shown that the Akt/mTOR
signaling pathway plays an important role in
antiapoptosis and regulation of cellular transcription by
activating p70S6K and 4EBP1. To further elucidate
whether the mTOR signaling pathway is involved in the
antiapoptotic effect of CPCs-Ex, we measured the expression
levels of related proteins and their phosphorylation levels.
After adding CPCs-Ex to the H9C2 cells, protein
expression of Akt, mTOR, p70S6K, and 4EBP1 was
increased at 12 and 24 h, but at 48 h, protein expression of
Akt and p70S6K was decreased, 4EBP1 was increased, and
mTOR had no significant changes (Fig. 3a, P < 0.05). In
the process of infection, the phosphorylation levels of
mTOR and p70S6K were suppressed by CVB3 infection
and repromoted by CPCs-Ex, but that change of 4EBP1
was on the contrary (Fig. 3b, P < 0.05).
Injection of CPCs-Ex in CVB3-induced myocarditis rats can attenuate cardiomyocyte apoptosis, repair the cardiomyocyte function and reduce CVB3 replication by regulating the Akt/mTOR pathway
To further explore the anti-apoptotic effect of
CPCsEx in vivo, we established a rat model of CVB3-induced
myocarditis. The rats were injected with CPCs-Ex
through the tail vein and imaged under IVIS Lumina
III In Vivo Imaging System (Fig. 4a). Forty-eight hours
later, the rats were sacrificed and dissected for
heart tissues, then analized by HE staining and TEM
(Fig. 4b, c). The apoptosis rate of cardiomyocytes and
the expression of VP1, apoptosis-related proteins and
Akt/mTOR pathway-related proteins of each group
were measured. After injection of CPCS-EX, the
apoptotic rate of rat cardiomyocytes was significantly lower
than that of the control group (Fig. 4d, P < 0.05). The
expression of VP1 in cardiomyocytes of EXO group was
significantly lower than that of the control group by
western blot and immunofluorescence (Fig. 4e, f). In the
meantime, through detecting the myocardial enzyme
CK-MB and cTnI, we found that CPCs-Ex drop
the elevated enzyme CK-MB and cTnI, improve the
myocardial function (supplementary file 2, P < 0.05).
Furthermore, the injection of CPCs-Ex promoted the
expression of BcL-2, depressed that of Bim, and
also inhibited the cleavage of caspase-3 and caspase-9
(supplementary file 3, P < 0.05). Different from the
results of H9C2 cell line, the protein expression levels of
Akt, mTOR, p70S6K, and 4EBP1 in the rats had not
changed much after the injection of CPCs-Ex. The
results of phosphorylation were consistent with the
cell experiments, which is the phosphorylation of
Akt, mTOR, and p70S6K was enhanced and that of
4EBP1 was inhibited compared with the control groups
(supplementary file 4, P < 0.05).
CPCs-Ex could decrease CVB3-induced apoptosis and suppress CVB3 expression with MK-2206 or rapamycin pretreatment by decreasing Bim/Bax expression and cleavage of caspase-3 in H9C2 cells
In this study, we used 10 nmol/L Rapamycin (Rap) and
2.5 µmol/L MK-2206 to pretreat H9C2 cells, respectively,
for 30 min, then cells were infected with CVB3 for 1 h,
and finally 200 ng/mL of CPCs-Ex were added to cells.
After cultivating for 48 h, cells were measured for
apoptosis rate, CVB3 expression and protein levels of
proapoptosis factors. We found that CPCs-Ex could
significantly decrease cell apoptosis with Rap or MK-2206
pretreatment compared with control (Fig. 5a, P < 0.05).
The results of RT-PCR showed that expression of CVB3
mRNA decreased after CPCs-Ex treatment in Rap or
MK-2206 groups (Fig. 5b, P < 0.05). Compared with
control, the VP1 expressions were suppressed by
CPCsEX in MK-2206 groups, but promoted by CPC-EX in Rap
groups at 12 and 24 h p.i. (Fig. 5c, P < 0.05). In addition,
CPCs-Ex decreased Bim, BcL-2, and Bax expression and
cleavage of caspase-3 but increased cleavage of caspase-9
in Rap or MK-2206 groups compared with control
(supplementary file 5, P < 0.05).
CPCs-Ex suppressed the phosphorylation of 4EBP1 and p70S6K with MK-2206 pretreatment but strengthened that with the pretreatment of Rapamycin
To further explore the mechanism under the change of
CVB3 expression with MK or Rap pretreatment, we then
measured the phosphorylation of Akt, mTOR, 4EBP1, and
p70S6K in H9C2 cells. After the treatment of CPCs-Ex,
the phosphorylation of Akt, 4EBP1, and p70S6K
was strengthened in Rap groups compared with control
(P < 0.05). And the phosphorylation levels of mTOR,
4EBP1, and p70S6K were decreased at 48 h p.i. by
CPCsEx in MK-2206 groups compared with controls
(supplementary file 6, P < 0.05).
CPCs-Ex promoted CVB3-induced apoptosis in Akt1 Akt2
4EBP1 and p70S6K overexpression groups by promoting
VP1 expression and cleavage of caspase-3 at 48 h p.i.
As we described above, CPCs-Ex seems utilized the
mTOR signaling pathway to mediate the CVB3-induced
apoptosis. To further investigate the relationship between
mTOR signaling pathway and CPCs-Ex during the
process of apoptosis, we established the eukaryotic expression
plasmids overexpressed the Ratus Akt1, Akt2, 4EBP1, or
p70S6K, which were stable transfected to H9C2 cells. The
expressions of Akt1, Akt2, 4EBP, and p70S6K were
determined by western blot analysis (Fig. 6a). After
infected with CVB3 for 1 h, CPCs-Ex were added with
200 ng/mL. Unexpectedly, CPCs-Ex decreased apoptotic
rate at 12 h but increase that at 48 h p.i. in the four
overexpression groups compared with controls (Fig. 6b,
P < 0.05). RT-PCR shown that the expression of CVB3
mRNA decreased in the overexpression groups with
CPCs-Ex treatment compared with controls p.i. (Fig. 7c,
P < 0.05). And western blot assays showed VP1 expression
were decreased at 12 and 24 h p.i. but increased
significantly at 48 h p.i. in Akt2, 4EBP1, and p70S6K
overexpression groups after CPCs-Ex treatment (Fig. 6d, P <
0.05). Besides, Bax and Bim expression was both
decreased in the four overexpression groups by CPCs-Ex
at 24 and 48 h p.i. But at the late stage of infection, the
cleavage of caspase-3 was promoted and the cleavage of
caspase-9 was repressed in Akt2, 4EBP1, and p70S6K
overexpression groups (supplementary file 7, P < 0.05).
CPCs-Ex promoted phosphorylation of Akt mTOR and
P70S6K at early stage p.i. and stimulated phosphorylation of 4EBP1 at late stage of infection in overexpression groups
We then measured the phosphorylation levels of Akt,
mTOR, 4EBP1, and P70S5K in the overexpression groups.
After the treatment of CPCs-Ex, the phosphorylation
levels of Akt, mTOR, and p70S6K were increased at 12
and 24 h in almost all the four overexpression groups.
However, the phosphorylation of 4EBP1 was suppressed
at early stage but stimulated at 48 h p.i. in Akt1, Akt2, and
P70S6K groups (supplementary file 8, P < 0.05).
As a major cause of sudden cardiac death in youth,
VMC still lack specific and effective treatment25. Years of
research have confirmed that the role of CPCs in the
repair of the heart is mainly through the paracrine, rather
than directly differentiate into cardiomyocytes6,26. As an
important component of paracrine secretion, exosomes
participate in processes such as immune response, antigen
presentation, cell migration, cell differentiation and tumor
invasion9,27. Cardiac-, plasma- and stem-cell-derived
exosomes were reported to be cardioprotective by
numerous studies, among which CPCs-Ex was the most
promising one28,29. A recent research has showed that the
efficacy of rat-derived CPCs-Ex stimulates after hypoxia30.
Just as our results shown, the CVB3-induced increase of
CK-MB and cTnI in rats were repaired after the injection
of CPCs-Ex, which directly demonstrated the
cardioprotection of CPCs-Ex. Besides, the miRNA content in the
exosomes also changed with hypoxia, implying that there
are some possible compensatory pathways exist in the
CPCs to alter exosome secretion on the basis of
microenvironmental clues31. Arslan et al.32 have reported that
exosomes derived from stem cells improve myocardial
viability after injury and alleviate adverse remodeling of
the damaged heart due to the activation of AKT pathway
and the reduction in oxidative stress in a myocardial
infarction (MI) model in vivo. It is precisely because this
lack of mechanism and effective treatment in the VMC,
based on our previous data, our present study devoted to
illuminate the relationship between CPCs-Ex and the Akt/
mTOR in CVB3-induced apoptosis, proving that CPCs-Ex
could suppress the CVB3-induced apoptosis and
replication in H9C2 cells and myocarditis rat model, which
indicating that CPCs-Ex could be a novel and significant
therapeutic strategy for VMC.
The dysregulation of the apoptotic pathway is an
important pathological process of CVB3-induced VMC33,
during which BcL-2 family and the caspase family play
important regulatory roles in the transduction pathway,
especially the mitochondrial pathway34. In this study, we
measured the expression of BcL-2 and caspase families, to
reflect the change of apoptosis pathways under CVB3
infection and CPCs-Ex treatment. In present research, the
data implied that CPCs-Ex could alleviate the apoptosis
induced by CVB3 both in vivo and in vitro. Further, we
found that the expressions of Bim and Bax in CPCs-Ex
groups were lower than those in CVB3 groups. But just the
opposite, as an anti-apoptosis protein, the expression of
Bcl-2 in CPCs-Ex was increased. In the meantime,
compared with the CVB3 group, the cleaved caspase-9 and
caspase-3 were inhibited in the CPCs-Ex group. Bim and
Bax are well known to be the pro-apoptosis, which can
promote the major core of intrinsic apoptosis signal
pathway. Caspase family is another common mediator of
apoptosis, of which caspase-9 contributes to the apoptosis
origination while caspase-3 participates in the execution. It
is the common concept that the activated Bax contributes
to form channels on the mitochondrial membrane leading
to cytochrome C release35,36, which can lead to caspase-9
self-cleave, then further processes other caspase members,
such as initiating a caspase cascade including caspase-3.
Those two families are interacted. Accordingly, our data
indicate that CPCs-Ex could reduce the CVB3-induced
apoptosis via inhibiting the virus replication, activating
Bcl2 expression and suppressing the activation of Bim, Bax
and the self-cleavage of caspase-9 and caspase-3 induced by
CVB3 infection simultaneously.
Several studies have shown that stem-cell-derived
exosomes activate the Akt/mTOR pathway to counteract
apoptosis or promote cell growth during the repair of cell
damage37–39. And Akt/mTOR pathway were also reported
to play a pivotal role in CBV3 replication and
CVB3induced apoptosis by our precious work24,40. Therefore,
we further detected the expression of the Akt/mTOR
pathway factors in the present study. We found that
protein expression and phosphorylation of Akt, mTOR,
and p70S6K were increased after addition of CPCs-Ex to
H9C2 cells, suggesting that CPCs-Ex may activate Akt/
mTOR pathway during CVB3-induced apoptosis. It is
commonly held the concepts that Akt/mTOR pathway is
an anti-apoptosis factor, which is also an important
mediator in the angiogenesis, protein synthesis and other
cell growth process. Interestingly, when CPCs-Ex were
added both in cells and rats after CVB3 infection, there
was an increase in the expression of 4EBP1 and decrease
of its phosphorylation, in consort with the reduced VP1
expression. It had been proved that CVB3 replication can
be suppressed by non-phosphorylated 4EBP1, which can
lead to suppression of translation initiation of 5′TOP
mRNA41. In an early study, mTOR/4EBP1 signaling
pathway has been demonstrated to be hosted and utilized
by CVB3 to promote its own replication during the
pathogenesis of VMC42. Therefore, CPCs-Ex could inhibit
CVB3 replication therefore reduce the CVB3-induced
apoptosis by inhibiting the phosphorylation of 4EBP1,
which might be a novel mechanism utilized by CPCs-Ex.
To further explore the relationship of Akt/mTOR and
CPCs-Ex in CVB3-induced apoptosis, we used Akt/
mTOR inhibitors MK-2206 and Rap, along with the
construction of stable cell lines using Akt1, Akt2, 4EBP1,
and p70S6K. Compared with the control, apoptosis rate in
CPCs-Ex group pretreated with Rap or MK was obviously
reduced followed by the suppression of VP1 expression.
While in the overexpression groups, CPCs-Ex reduced the
CVB3-induced apoptosis and VP1 expression at 24 h p.i.,
whereas, both of that were increased at 48 h p.i. In the
meantime, we found that CPCs-Ex could suppressed the
phosphorylation of mTOR, 4EBP1, and p70S6K after with
the pretreatment of MK, but strengthened that with the
pretreatment of Rap which resulting in the decreased VP1
expression in MK groups and increased VP1 expression in
Rap groups. This result may also suggest that the
antiCVB3 replication effect of CPCs-Ex may be mainly
mediated by Akt/4EBP1 pathway. It is well known
that mTOR has two functionally distinct complexes:
mTOR complex 1 (mTORC1) and mTOR complex 2
(mTORC2)43. When mTORC1 is activated, it then
phosphorylates the 4EBP1 and p70S6K. The
phosphorylation of 4EBP1 allows cap-dependent translation to
proceed. Meanwhile, the activation of p70S6K enhances
the translation of mRNAs, promoting the cell growth
consequently44. When mTORC1 was inhibited by the
Rap, CPCs-Ex could increase the phosphorylation of
p70S6K via the surviving mTORC2 pathway. Yet, the
phosphorylation of 4EBP1 was strengthened by CPCs-Ex,
which resulting in the increase replication of CVB3. An
early study has demonstrated that gastric cancer exosome
could supress Jurkat T cells apoptosis by stimulating
downstream Akt activity45. Like that, our data shown
CPCs-Ex enhanced Akt and its downstream
phosphorylation, which decreased the viral replication and reduce
CVB3-induced apoptosis consequently. In our present
study, apoptosis rate and VP1 expression in CPC-Ex
group were both reduced at 12 h p.i. in the groups of
overexpression. Whereas, apoptosis was exacerbated by
CPCs-Ex at 48 h p.i. followed by the rebound of viral
replication. By adding CPCs-Ex to Akt1, Akt2, 4EBP1, and
p70S6K overexpressed cells, we found that CPCs-Ex could
enhance the anti-apoptosis effect of Akt1, Akt2, and
p70S6K overexpression by stimulate phosphorylation of
Akt, mTOR, and p70S6K. Meanwhile Akt2 and p70S6K
overexpression also stimulated the phosphorylation of
4EBP1, which might contribute to CVB3 replication. It is
commonly held the concepts that Akt is an anti-apoptosis
factor. Besides, a recent study has reported that Akt1
activation may prevent apoptosis through upregulating of
the survivin46. Akt2 is implicated in diverse process of
cardiomyocyte signaling including survival and
metabolism, whose deficiency may cause retardation of
cardiomyocyte development47, implying a pivotal role in the
cardiomyocyte survival. At early stage after viral infection,
CPCs-Ex reduced the viral replication and apoptosis via
decreasing the phosphorylation of 4EBP1 and p70S6K.
Along with the infected time, the increased effect of
CPCs-Ex on p70S6K was translated to decrease, yet the
phosphorylation of 4EBP1 was stimulated, which may be
utilized by CVB3 to promote the replication and then
reinforce the apoptosis process.
On the other hand, we detected the Bcl-2 and caspase
family members when Akt/mTOR signaling pathway was
inhibited or overexpressed, respectively. We found that
along with the infected time, CPCs-Ex could suppress the
activated cleaved caspase-3, while keeping the cleavage of
caspase-9 with the Rap or MK pretreatment. In the
overexpressed groups, however, the expression of cleaved
caspase-3 was further increased and the cleavage of
caspase-9 was repressed. As previously mentioned, during
the apoptosis process, caspase-9 contributes to the
apoptosis origination and caspase-3 participates in the
execution. Zhang et al.48 have observed that the
mitochondrial membrane potential collapses and the ratio of
Bax/Bcl-2 in the cytoplasm increases, inducing
cytochrome c release from the mitochondria to the cytoplasm,
activates caspase-9/-3 and finally induces apoptosis,
implying caspase-9 and caspase-3 are responsible for the
activation of apoptosis. Another study has reported that
bone mesenchymal stem cells (BMSCs) derived exosome
could suppress the apoptosis via reducing the cleavage of
caspase-3, caspase-8, and caspase-9 directly in colitis rats.
Therefore, we speculate that when Akt/mTOR signaling
pathway is inhibited, CPC-Ex can synergistically mitigate
CVB3-induced apoptosis via a caspase-3 dependent
pathway. Exosome, theoretically treated to be an inhibitor
of apoptosis and accelerator of proliferation, have been
demonstrated to induce the apoptosis in Jurkat T cells via
inhibiting the PI3K/Akt pathway and mediating the
caspase family45. Thus, when more substrates of Akt/mTOR
pathway were provided, CPCs-Ex was turned into the
accomplice of CVB3, promoting cell apoptosis via
promoting the activation of cleaved caspase-3 which
induced by CVB3 infected.
In conclusion, based on our data, we suggest that
CPCsEx could mitigate the CVB3-induced apoptosis and block
CVB3 replication by inhibiting the phosphorylation of
4EBP1 and suppressing pro-apoptosis factors (Fig. 7).
Moreover, the synergetic anti-apoptosis effect of CPCs-Ex
rely on the Akt/4EBP1 and caspase-3 dependent
pathways. Our work may provide new insights into the role of
exosomes in the pathogenic mechanism and treatment on
VMC, yet still requires our more in-depth research.
We appreciate that Dr. Qiaojia Huang (Molecular Medicine Research Center of
Fuzhou General Hospital, Nanjing Military Region) provided us the necessary
empty vector, and the Center Laboratory at the Third Xiangya Hospital of the
Central South University provided us with the experimental equipment and
technical guidance necessary to complete our work. This work was supported
by the National Natural Science Foundation (No. 81500225).
Dr. Li Xin designed and completed most of the research. Professor Yang
Zuocheng guided and solved some of the problems encountered in the
research. Dr. Nie Wenyuan completed the data collation and analysis. Dr. Li
Shentang, Li Zhuoying, and Jiang Jie completed some experiments. Professor
Ma Xing guided the writing of the article.
Conflict of interest
The authors declare that they have no conflict of interest.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Supplementary Information accompanies this paper at (https://doi.org/
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