ELA-11 Protects The Heart Against Oxidative Stress

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TYPE Original Research

PUBLISHED 08 September 2022


DOI 10.3389/fphar.2022.873614

ELA-11 protects the heart against


OPEN ACCESS oxidative stress injury induced
EDITED BY
Zhi-Ren Zhang,
Harbin Medical University, China
apoptosis through ERK/MAPK
REVIEWED BY
Yong Fen Qi,
and PI3K/AKT signaling pathways
Peking University, China
Tamer M. A. Mohamed,
University of Louisville, United States
Xuejun Wang 1,2†, Li Zhang 1†, Mengwen Feng 2, Zhongqing Xu 1,
Firdos Ahmad, Zijie Cheng 1,2* and Lingmei Qian 1,2*
University of Sharjah, United Arab
Emirates 1
Hongqiao International Institute of Medicine, Tongren Hospital, Shanghai Jiao Tong University School
of Medicine, Shanghai, China, 2Department of Cardiology, The First Affiliated Hospital of Nanjing
*CORRESPONDENCE
Medical University, Nanjing, China
Zijie Cheng,
[email protected]
Lingmei Qian,
[email protected]

These authors have contributed equally Increasing evidence revealed that apoptosis and oxidative stress injury were
to this work
associated with the pathophysiology of doxorubicin (DOX)-induced myocardial
SPECIALTY SECTION injury. ELABELA (ELA) is a newly identified peptide with 32 amino acids, can
This article was submitted to
Cardiovascular and Smooth Muscle reduce hypertension with exogenous infusion. However, the effect of 11-
Pharmacology, residue furn-cleaved fragment (ELA-11) is still unclear. We first administrated
a section of the journal
ELA-11 in DOX-injured mice and measured the cardiac function and
Frontiers in Pharmacology
investigated the effect of ELA-11 in vivo. We found that ELA-11 alleviated
RECEIVED 11 February 2022
ACCEPTED 29 July 2022
heart injury induced by DOX and inhibited cardiac tissues from apoptosis. In
PUBLISHED 08 September 2022 vitro, ELA-11 regulated the sensitivity towards apoptosis induced by oxidative
CITATION stress with DOX treatment through PI3K/AKT and ERK/MAPK signaling pathway.
Wang X, Zhang L, Feng M, Xu Z, Cheng Z Similarly, ELA-11 inhibited oxidative stress-induced apoptosis in cobalt chloride
and Qian L (2022), ELA-11 protects the
heart against oxidative stress injury
(CoCl2)-injured cardiomyocytes. Moreover, ELA-11 protected cardiomyocyte
induced apoptosis through ERK/MAPK by interacting with Apelin receptor (APJ) by using 4-oxo-6-((pyrimidin-2-ylthio)
and PI3K/AKT signaling pathways. methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221). Hence, our results indicated
Front. Pharmacol. 13:873614.
doi: 10.3389/fphar.2022.873614 a protective role of ELA-11 in oxidative stress-induced apoptosis in DOX-
induced myocardial injury.
COPYRIGHT
© 2022 Wang, Zhang, Feng, Xu, Cheng
and Qian. This is an open-access article KEYWORDS
distributed under the terms of the
Creative Commons Attribution License ELA-11, doxorubicin, heart failure, apoptosis, oxidative stress
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms.
Abbreviations: LDH, lactate dehydrogenase; ROS, relative oxygen species; TUNEL assay, terminal-
deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling assay; ELISA, enzyme-
linked immunosorbent assay; DOX, doxorubicin; CoCl2, cobalt chloride; MDA, malondialdehyde; SOD,
superoxide dismutase; ERK, extracellular signal-related kinase; JNK, Jun amino-terminal kinase; PI3K,
phosphatidylinositol 3 kinase; AKT, protein kinase B; JC-1, tetrechloro-tetraethylbenzimidazol
carbocyanine iodide; ML221, 4-oxo-6-((pyrimidin-2-ylthio) methyl)-4H-pyran-3-yl 4-
nitrobenzoate; Scr, scramble; AA, amino acid; EF, ejection fraction; FS, fractional shortening;
LVEDs, left ventricular end-systolic dimension; APJ, Apelin receptor (APJ).

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Wang et al. 10.3389/fphar.2022.873614

Introduction pressure in heart and kidney (Johnson et al., 2017). Furthermore,


natriuretic peptide family members can be used to diagnose heart
Myocardial injury caused by chemotherapy drugs is a major failure (Brady et al., 2019). In conclusion, peptides play
reason that affecting the prognosis of tumor patients (Zhang et al., indispensable roles in regulating physiological function and
2019). Statistically, 70% of patients in worldwide have different pathophysiological process.
degrees of cardio-toxic reactions during chemotherapy, and these It has been reported that a conserved gene, AK092578, is
symptoms will accompany them for life (Leemasawat et al., 2020). In predicted to encode a 54aa hormone with a signal peptide (Yang
China, nearly 30% of cancer patients die from cardiovascular et al., 2017). This hormone is highly conserved among multiple
diseases. The most common cardio-toxicity caused by species which is named ELABELA (ELA) or toddler (Read et al.,
anthracycline is heart failure, with an incidence of 48% (Wu 2019). As an early endogenous ligand of the Apelin receptor
et al., 2017). It is known that the mechanism of doxorubicin (APJ), ELA is essential for heart development. In recent studies, it
(DOX)-induced cardiotoxicity is very complex, which involving has been suggested that a 32-amino-acid mature peptide (ELA-
pathological processes such as cell apoptosis, oxygen free radical 32) can be decomposed to produce endogenous fragments
damage, iron ion metabolism disorder, calcium overload and including ELA-21 and ELA-11 (Chen et al., 2017). Studies
metabolic disorder (Kostrzewa-Nowak et al., 2005; Govender have proved that ELA-32 and ELA-11 could inhibit renal
et al., 2014; Eid et al., 2021). Oxygen free radical injury is one of ischemia-reperfusion (I/R) injury, and ELA-21 could
the representative theories of cardiotoxic injury caused by DOX (Ma significantly increase angiogenesis, promoted cardiomyocyte
et al., 2020). Cytochrome P450 reductase and a variety of reductases proliferation and reduced apoptosis and heart fibrosis near the
can produce superoxide free radicals (O2-) and reduce infarct area (Xu, 2021). Recent study has clarified that Elabela
anthraquinone of DOX to form quinone-semiquinone cycle, (19–32) could ameliorate doxorubicin (DOX)-induced
leading to lipid peroxidation of mitochondria and microsomes, cardiotoxicity by promoting autophagic flux through TFEB
then damages myocardial cells (Yuan et al., 2020). Due to the pathway (Chen et al., 2022). However, the effect and
decreased content of antioxidant enzymes in cardiomyocytes, a large mechanism of ELA-11 in DOX-induced cardiac injury is unclear.
number of reactive oxygen species and free radicals are generated to The PI3K-AKT signaling pathway is a classical signaling
induce the oxidative stress response of cardiomyocytes and pathway that regulates apoptosis (Brady et al., 2019). PI (3, 4, 5)
aggravate the damage of cardiomyocytes (Carrasco et al., 2021). P3 is an intracellular second messenger in the cell that is required
Free radicals generated by DOX activated NAD(P)H oxidases for the transfer of protein kinase B (AKT) to the membrane for
[NAD(P)H oxidases, NOXs] can also activate the apoptosis activation (Lin et al., 2019). Phosphorylation of AKT mediates
pathway of cardiac myocytes and cause cell death (Wang et al., insulin and various growth factors to induce cell growth and
2018). Furthermore, Pharmacological strategies that inhibit promotes cell survival through numerous channels (Song et al.,
apoptosis and oxidative stress injury can protect patients from 2018). ELA-11 induced ERK/MAPK is a classical anti-apoptotic
chemotherapeutic drug-induced myocardial damage. signaling pathway. When the downstream phosphorylation of
Oxidative stress is an accompaniment of apoptosis through ERK is activated, it inhibits the process of apoptosis.
activating mitochondrial dysfunction, the death receptor In present study, we found that ELA-11 could attenuate
pathway or endoplasmic reticulum stress (Dai et al., 2014). DOX-induced free radical production, which protected
When cells are stimulated by oxidative stress, the cardiomyocytes against oxidative stress-induced apoptosis.
accumulation of oxidizing substances such as free radicals can Mechanistically, ELA-11 inhibited oxidative stress-induced
damage organelles and activate cell death program (Lüscher, apoptosis by suppressing mitochondrial membrane potential
2015). Recent studies have demonstrated that the increase of mediated by ERK/MAPK and PI3K/AKT signaling pathways.
intracellular ROS levels could put cells in a state of constitutive Moreover, ELA-11 acted as a protective role in DOX-induced
oxidative stress, leading to cell apoptosis (Lee et al., 2019). cardiac injury by targeting APJ.
Targeting cell death pathways before oxidative stress
manifestation can alleviate oxidative impairment by prevented
free radicals which could potentially pave the way for new Materials and methods
therapeutics through against oxidative stress induced
apoptosis (Münzel et al., 2010; Malla et al., 2020). Cell culture
It is well known that peptides produced by proteasomes
degradation can play a protective role in vivo and act as Rat primary cardiomyocytes were extracted from rats 24 h
endogenous ligands or receptors to mediate a variety of after birth. The blood, fat, connective tissues and heart tissue
signaling pathways (Ye et al., 2019). For example, endorphins sections were separated and digested with trypsin at 37°C at
as an endogenous peptide which can ease pain, like morphine 60 rpm for 15 min. The solution was removed, and the digestion
and analgesic (Paes et al., 2019). Angiotensin II binds to was repeated three times. The cell-containing digested fluids were
angiotensin receptors to regulate water-salt balance and blood placed in a centrifuge tube and centrifuged together after passing

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Wang et al. 10.3389/fphar.2022.873614

through a 180-mesh filters. The cells were resuspended in 10 ml Lactate dehydrogenase level detection
DMEM containing 10% horse serum (HS, Gibco, United States)
and incubated in a 10 cm2 dish for 1.5 h. The cell suspension was Levels of lactate dehydrogenase (LDH) released were
removed, and the cells (5–6 × 105 cells per dish) were inoculated detected in serum using an LDH release assay kit according to
into a new dish as previously described. When cell adhered to the manufacturer’s protocol.
about 80%, 1uM ELA-11 was added to the culture media. After
12 h of co-incubation with DOX, experimental verification was
carried out. Terminal-deoxynucleotidyl transferase-
mediated dUTP-biotin nick end-labeling
staining assay
Animals
The rate of apoptosis can be was detected by a TUNEL
Male C57BL/6J mice (6–8 weeks of age, 20–22 g) were obtained staining kit. The cells were seeded (1 × 105 cells per well) in 6-well
from the Model Animal Research Center of Nanjing University dishes. After the treatments described above were performed, the
(Nanjing, Jiangsu, China), and all procedures used were approved by cells were washed once with phosphate-buffered saline (PBS) and
the ethical committee of Nanjing Medical University. All animals fixed with 4% paraformaldehyde. Apoptotic cells were visualized
were housed at 20–25°C and 50%–70% relative humidity. The with TUNEL staining according to the manufacturer’s protocol.
experimental mice were randomly divided into four groups. TUNEL fluorescence intensity/DAPI fluorescence density was
Before injected with 5 mg/kg DOX by intraperitoneal injection used to calculate the percentage of positive cells, and the density
for five consecutive weeks, 10 mg/kg ELA-11 was injected to was evaluated using ImageJ software 1.26 (Wayne Rasband,
mice through veil tail for 7 days, after which electrocardiograms National Institutes of Health, Bethesda, MD, United States).
were obtained and the mice were sacrificed. The mice were treated
according to the experimental requirements. All animal experiments
complied with the Guide for the Care and Use of Laboratory Tetrechloro-tetraethylbenzimidazol
Animals published by the National Institutes of Health (NIH carbocyanine iodide assay
Publications No. 85-23, revised 1996).
The mitochondrial membrane potential was measured by a
mitochondrial membrane potential assay kit with JC-1 according
Cell-in-cell experiment to the manufacturer’s instructions. The cells were cultured in
serum-free DMEM containing (×1) JC-1 staining working fluid
We chemically synthesized FITC-labeled, incubated cells at 37°C for 20 min. Then, the cells were washed twice with JC-1
with ELA-11 for 1 h at 37°C and 5% CO2 atmosphere. The buffer, after which 2 ml DMEM was added, and then the cells
localization of ELA-11 was observed by fluorescence microscopy. were photographed with a fluorescence microscope (BX61;
Olympus Corporation, Tokyo, Japan). The JC-1 density was
assessed by ImageJ software and calculated upon
Cell counting kit-8 assay normalization to the control.

Thousand cells were inoculated into each well of the 96-well


plate, ELA-11, DOX or CoCl2 was added into the plates Reactive oxygen species measurement
respectively after cells were adhered. Measured the absorbance
at 450OD. Cell viability (%) = [OD (experimental group)–OD The levels of intracellular ROS were determined using a
(blank group)]/[OD (control group)–OD (blank group)] * 100%. relative oxygen species assay kit following the instructions.
Cells were incubated in serum-free DMEM containing 0.1%
DCFH-DA at 37°C for 20 min, washed with serum-free
Cell death rates DMEM three times and photographed with a fluorescence
microscope.
Trypan blue staining was used to calculate the mortality of
the primary cells. The cells were collected at different times (0, 6,
12, 18, 24 or 36 h) and stained with the dye from a trypan blue Western blot
staining cell viability assay kit to determine the cell death rate. At
different concentrations of DOX (0.1, 0.5, 1, 2, and 5 µM) and Proteins were isolated from cells using lysis buffer
CoCl2 (200, 400, 600, 800 and 1,000 µM), measurements were (containing RIPA and 1% PMSF). Protein quantification was
taken according to the manufacturer’s instructions. performed using a BCA protein detection kit (23229; Thermo

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Wang et al. 10.3389/fphar.2022.873614

FIGURE 1
Function of ELA-11 in doxorubicin-injured model. (A) The biological characteristics of ELA. (B) Schematic map for DOX-induced mice cardiac
injury model. (C) The body weight of mice in four groups during the experiment. (D) The representative echocardiograms of DOX-induced cardiac
injured group. (E) EF value for echocardiography. (F) FS value for echocardiography. (G) LVEDs value for echocardiography. ***p < 0.001 and ### p <
0.001, one-way ANOVA, N = 5 mice per group.

Fisher Scientific). Protein samples of the same mass were standard substance was prepared and diluted according to the
separated on 10% SDS-PAGE gels and transferred to instructions, the concentration of each well was 600, 400, 200,
nitrocellulose membranes (Millipore, Billerica, MA, 100, and 50 U/L, respectively. The tested samples were added to
United States), which were blocked with 5% skim milk and the plate and mixed, then incubated at 37°C for 30 min. Wash
then incubated with specific primary antibodies. Caspase-3 plates, add enzyme and chromogenic reaction were proceeded
(14220T, 1:1000), PARP (9532T, 1:1000), β-actin (3700S, 1: according to the instructions. After incubating at 37°C for
1000), GAPDH (5174T, 1:1000), AKT (4685S, 1:1000), p-AKT 15 min, the absorbance of each well was measured at 450 nm
(4060T, 1:2000), PI3K (4249T, 1:1000), ERK (4695T, 1:1000) and wavelength.
p-ERK (4370T, 1:1000) were purchased from Cell signaling
Technology. The anti-rabbit (SA00001-2, 1:10000), PI3K
(20584-1-AP, 1:1000) and anti-mouse (SA00001-1, 1:10000) Echocardiography
secondary antibody were purchase from Proteintech.
FluorChem M system was used to quantify the positive bands Post DOX and ELA-11 administration, all the mice were
representing proteins involved in the orchestrated immune subjected to M-mode echocardiography to assess heart
responses (ProteinSimple, San Jose, CA, United States). function. All animal experiments complied with the Guide
for the Care and Use of Laboratory Animals published by the
National Institutes of Health (NIH Publications No. 85-23,
Malondialdehyde, superoxide dismutase, revised 1996).
and glutathione peroxidase assay

Cellular oxidative stress was determined by detecting the Enzyme-linked immunosorbent assay
state of intracellular oxidation and reduction. Lipid peroxidation
Malondialdehyde (MDA) assay (Beyotime, Nanjing, China) and Serum Creatine kinase isoenzyme (CKMB) and B-type
total superoxide dismutase assay (Beyotime, Nanjing, China) natriuretic peptide (BNP) level in mice heart tissue was
were measured by following the manufacturer’s instructions. determined by a commercially available Enzyme-linked
glutathione peroxidase (GSH-Px) assay kit was purchased immunosorbent assay (ELISA) kit (Mlbio, Shanghai, China)
from Elabscience. The cell supernatant was collected, and the according to the manufacturer’s instructions.

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Wang et al. 10.3389/fphar.2022.873614

FIGURE 2
ELA-11 protects doxorubicin-injured heart in vivo. (A) Serum CKMB release in DOX-injured model. (B) Serum BNP release in DOX-injured
model. (C) Representative photographs of HE staining. (D) Representative photographs of Masson staining. (E,F) Representative photographs of WGA
staining and its quantitative data. (G,H) Representative photographs of TUNEL staining and its quantitative data. ***p < 0.001 and ### p < 0.001, one-
way ANOVA, N = 5 mice per group.

Hematoxylin & Eosin (H&E) Staining and ponceau acid fuchsin solution was used for 5–10 min after
washing. Soaked with 2% glacial acetic acid solution for 30 s, and
Obtained the cardiac tissues and fixed them in 4% differentiate with 1% phosphomolybdate solution for 3–5 min.
paraformaldehyde for 7 days after the blood in the heart Then, the slices were dyed with aniline blue for 5 min, and
cavity was pumped out. The tissue was transparent with bathed again with 0.2% glacial acetic acid solution for 30 s.
ethanol and xylene. Soak the transparent tissue into melted Finally, the slices were sealed with 95% alcohol, anhydrous
paraffin for embedding. After cooling and solidification, they alcohol, xylene transparent and neutral gum.
were cut into five micron slices and placed in hot water to flatten
and then pasted onto slides and dried in a 45°C incubator. Before
staining, paraffin was removed again, and Hematoxylin and eosin Wheat germ agglutinin staining
dyes were respectively used for staining, which made the nucleus
and intracellular ribosomes stained blue and purple by Obtained and sectioned tissue according to the above
Hematoxylin (H). The cytoplasm is stained red or reddish method. Placed the section in the antigen repair buffer of
with Eosin (E). pH 8.0, then the section was decolorized with PBS after
cooled naturally. A histochemical pen was used to draw
circles around the tissues and Wheat germ agglutinin (WGA)
Masson staining staining buffer was added into the circles, and the cells were
incubated for 30 min at 37°C. The nucleus was stained with
Obtained and sectioned tissue according to the above method. DAPI. Sealed the section, then collected the images under
Weigert sapwood semen was used for staining nuclear for 5–10 min, fluorescence microscope.

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FIGURE 3
ELA-11 attenuates doxorubicin-induced injury in rat primary cardiomyocytes. (A) The location of ELA-11 in rat primary cardiomyocytes. (B)
Trypan blue assay for cell death with different concentration of DOX with different duration (N = 9 per group). (C) The cell viability of 1 uM DOX in
different duration (N = 9 per group). (D) Cell viability of ELA-11 with different concentration for 12 h (N = 9 per group). (E) Cell viability in DOX-induced
cardiomyocyte model (N = 9 per group). (F) LDH release in DOX-induced cardiomyocyte model (N = 9 per group). ***p < 0.001 and ### p <
10.001, one-way ANOVA.

Statistical analysis echocardiographic results suggested that DOX reduced


ejection fraction (EF) and fractional shortening (FS), but
All results are expressed as the mean ± SD. Comparisons increased left ventricular end-systolic dimension (LVED)
between multiple groups were performed by one-way analysis of significantly, however, ELA-11 increased EF and FS, and
variance (ANOVA), and p < 0.05 (*), p < 0.01 (**) and p < 0.001 decreased left ventricular end-systolic dimension (LVED)
(***) were considered significant. All experiments were repeated significantly compared with DOX + Scr group (Figures
at least three times unless otherwise specified. All data was 1D–G). These results demonstrated that ELA-11 could inhibit
analyzed by GraphPad Prism software. DOX-induced injury in vivo.

Results ELA-11 protects doxorubicin-injured heart


in vivo
Function of ELA-11 in doxorubicin-injured
model Then, our results demonstrated that the release of CKMB and
BNP in blood serum increased significantly in DOX + Scr treated
To understand the shape of ELA-11 in molecular chain, we group compared with Scr group, but ELA-11 decreased CKMB
predicted its structure by I-TASSER server (https://zhanggroup. and BNP release significantly (Figures 2A,B). To further identify
org/I-TASSER/) and analyzed the biological characteristics the effect of ELA-11 in vivo, we collected organs to perform
through Expasy (https://www.expasy.org/) (Figure 1A). We pathological examination. HE staining results indicated that
found that ELA-11 possessed the advantages of light myocardial fibers in DOX-treated group were in disordered
molecular weight and high lipid solubility. Hence, we arrangement and cardiomyocytes were changed into
investigated the effect of ELA-11 in DOX-induced cardiac vacuolated compared with Scr group, but there was less
injury in vivo (Figure 1B). We weighed the mice every disarrayed myocardial fibers and vacuolated cells in DOX +
2 weeks for 10 weeks, the result demonstrated that the weight ELA-11 treated group (Figure 2C). The evidence from Masson
of DOX-induced group began to decrease after 6 weeks, scramble staining suggested that fibrosis was significantly increased by
(scr) peptide could not affect cell death, but DOX increased cell DOX but reduced by ELA-11 (Figures 2D,E). To figure out the
death since week 5. Compared with DOX, ELA-11 could increase cardiomyocyte areas of the four groups, WGA staining results
weight index significantly (Figure 1C). Next, the demonstrated that the cardiomyocyte area decreased

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FIGURE 4
ELA-11 inhibits DOX-induced cardiac injury through oxidative stress -induced apoptosis. (A) Representative photographs of TUNEL staining in
DOX- induced cardiomyocyte injury (N = 9 per group). (B) Representative photographs of JC-1 in DOX-induced cardiomyocyte injury. (C) The
expression of PARP and caspase-3 in DOX-induced cardio-myocyte injury by western blot. (D) Representative photographs of ROS in DOX-induced
cardio-myocyte injury. (E) Detection of MDA production, SOD activity and GSH-PX release in DOX-induced cardiomyocyte injury (N = 9 per
group). ***p < 0.001 and ### p < 0.001, one-way ANOVA.

significantly in DOX-treated group compared with the Scr group, was no statistic difference between the two groups (Figure 3C).
but ELA-11 significantly rescued cardiomyocyte area To figure out whether ELA-11 could affect cell survival, we
(Figures 2F,G). TUNEL staining showed a significant increase cultured ELA-11 with different duration times (0, 6, 12, and
in the apoptosis rate in the myocardium in the DOX + Scr group, 24 h) and concentrations (0.1, 0.5, 1, 2, and 5 uM). Then trypan
and it generally decreased to the level in DOX mice administered blue dyeing results demonstrated that 0.5 uM ELA-11 decreased
with ELA-11 (Figures 2H,I). These results demonstrated that cell death at 24 h, 1 uM, and 5 uM ELA-11 both could decrease
ELA-11 might have a protective role in DOX-induced cardiac cell death at 12 h. But there was no significant difference among
injury. the groups (Figure 3D). Next, we cultured 1 uM ELA-11 before
DOX treatment for 12 h and found ELA-11 could significantly
increase cell viability compared with DOX + Scr group
ELA-11 attenuates doxorubicin-induced (Figure 3E). And LDH results indicated that ELA-11 inhibited
injury in rat primary cardiomyocytes DOX-induced cardiotoxicity (Figure 3F). Our study revealed that
ELA-11 could attenuate DOX-induced injury in rat primary
To determine the effect of ELA-11 in vitro, we first cultured cardiomyocytes.
rat primary cardiomyocytes with ELA-11, we found that ELA-11
could enter into the cytoplasm (Figure 3A). To figure out the
optimal concentration of DOX, we incubated DOX with different ELA-11 inhibits doxorubicin-induced
concentrations (0.1, 0.5, 1, 2, and 5 uM) for 12 h, CCK-8 results cardiac injury through oxidative stress-
demonstrated that 0.5 uM DOX could reduce cell viability, but 1, induced apoptosis
2, and 5 uM decreased cell viability more significantly and there
were no difference among 1, 2 and 5 uM DOX (Figure 3B). Then Based on the effect of ELA-11 in vivo, we further detected the
CCK-8 results manifested that 1 uM DOX cultured for 12 and apoptosis level by TUNEL, the results revealed that the number
24 h could effectively reduce the cell survival rate, however there of apoptotic cells was increased in the DOX-induced group but

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FIGURE 5
ELA-11 protected cardiomyocytes against oxidative stress-induced apoptosis in CoCl2 model. (A) Cell viability in CoCl2 -induced
cardiomyocyte model. (B) LDH release in CoCl2 -induced cardiomyocyte oxidative stress injury (N = 9 per group). (C,D) The expression of PARP and
caspase-3 in CoCl2 -induced cardiomyocyte oxidative stress injury by western blot. (E) Representative photographs of ROS production in CoCl2
-induced cardiomyocyte oxidative stress injury. (F–H) Detection of MDA production, SOD activity and GSH -PX release in CoCl2 -induced
cardiomyocyte oxidative stress injury (N = 9 per group). ***p < 0.001 and ### p < 0.001, one-way ANOVA.

reduced after ELA-11 co-treatment (Figure 4A). Furthermore, inhibit CoCl2-induced cardiac apoptosis (Figures 5C,D).
DOX reduced mitochondrial membrane potential compared Furthermore, ELA-11 inhibited ROS production with CoCl2
with scr group, but ELA-11 elevated mitochondrial membrane treatment (Figure 5E). And ELA-11 inhibited superoxide
potential compared with DOX treatment (Figure 4B). The production and peroxidase activity (Figures 5F–H). Our
western blot results revealed that cleaved caspase-3 and PARP results further proved ELA-11 protected cardiac injury in
were activated by DOX, but ELA-11 reduced the elevated CoCl2 model mediated by oxidative stress-induced apoptosis.
expression of cleaved caspase-3 and PARP (Figure 4C). Next,
ROS release results indicated that ELA-11 attenuated DOX-
induced free radical production (Figure 4D). Furthermore, we ELA-11 attenuated cardiac injury through
evaluated cellular oxidative stress levels by MDA production, ERK/MAPK and PI3K/AKT signaling
SOD activity and GSH-Px content. Our results demonstrated pathway
that DOX increased MDA production and reduced SOD activity
and GSH-Pxproduction, but ELA-11 inhibited MDA production, We further verified the possible signaling pathway of ELA-11
promoted SOD activity and GSH-Px production (Figure 4E). in rat primary cardiomyocytes by Western blot. We first
Therefore, ELA-11 could inhibit DOX-induced injury by determined the expression of p38, JNK and ERK, however,
attenuating oxidative stress-induced apoptosis. the result showed that there was no expression of JNK and
p38 (data not shown), but phosphorylated ERK was down-
regulated by DOX and ERK protein phosphorylation was
ELA-11 protected cardiomyocytes against activated upon ELA-11 treatment compared with DOX
oxidative stress-induced apoptosis in (Figures 6A,B). Next, we found that DOX also down-regulated
CoCl2 model the expression of phosphorylated AKT and PI3K, but ELA-11
activated phosphorylation of AKT and PI3K protein expression
To validate the role of ELA-11 in another oxidative stress (Figures 6C–E). When CoCl2 was blunted, the phosphorylation
model, we further constructed chemical ischemic model in rat of ERK, AKT and PI3K protein expression was downregulated.
primary cardiomyocytes. Our results indicated that ELA-11 With intervention of ELA-11, ELA-11 activated the expression of
protected cardiomyocytes from CoCl2-induced injury (Figures ERK, AKT and PI3K phosphorylated proteins after CoCl2
5A,B). Then western blot evidence proved that ELA-11 could treatment in rat primary cardiomyocytes (Figures 6F–J). These

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FIGURE 6
ELA-11 attenuated cardiac injury through ERK/MAPK and PI3K/AKT signaling pathway. (A) The expression of ERK in DOX-induced cardiac injury
by western blot. (B) The quantitative data for western blot. (C–E) The expression of PI3K and AKT in DOX-induced cardiac injury by western blot and
its quantitative data. (F,G) Western blot analyzed the expression the expression of ERK in CoCl2 -induced cardiac injury and its quantitative data.
(H–J) Western blot analyzed the expression the expression of PI3K and AKT in CoCl2 -induced cardiac injury and its quantitative data. ***p <
0.001 and ### p < 0.001, one-way ANOVA.

results revealed that ELA-11 protected cardiomyocytes from and western blot results demonstrated that
apoptosis through the ERK/MAPK and PI3K/AKT signaling ML221 significantly decreased the phosphorylation of AKT,
pathways. PI3K, and ERK proteins compared with ELA-11, when ELA-
11 and ML221 co-incubation with DOX, the ability of ELA-11 to
activate phosphorylated ERK, PI3K and AKT was inhibited
ELA-11 protects cardiomyocytes by (Figures 7C–G). Furthermore, we found that ML221 could
binding apelin receptor also inhibit the effect of ELA-11 in CoCl2-induced apoptosi s
(Figures 7H,I). The phosphorylation of AKT, PI3K and ERK
4- oxo-6-((pyrimidin-2-ylthio) methyl)-4H-pyran-3-yl4- proteins was significantly up-regulated after co-treatment with
nitrobenzoate (ML221) is the first reported APJ antagonist ML221 and ELA-11 in CoCl2 model (Figures 7J,K,L–N). These
which exerts antagonistic effect mainly by inhibiting cAMP results suggested that ELA-11 inhibited apoptosis by binding
and recruiting β-arrestin by ELA-11. Report has shown that to APJ. Finally, we drew a pattern diagram of ELA-11 for
ML221 has potential to attenuate the activation and signaling of inhibiting apoptosis and oxidative stress injury in
the APJ receptor and reduce elabela-induced microvascular cardiomyocytes (Figures 8).
endothelial cell proliferation (Godoy-Parejo et al., 2019). To
determine the role of ML221, we used to detect its interaction
with ELA-11 by Western blot. We found that ML221 inhibited Discussion
the effect of ELA-11 on cardiomyocyte apoptosis after DOX
administration (Figures 7A,B). We wondered whether Our current study revealed that ELA-11 has a protective
ML221 was involved in the ERK/MAPK and PI3K/AKT effect on apoptosis and attenuates DOX-induced cardiotoxicity
signaling pathways when the cells were treated with DOX, in vitro and in vivo. Furthermore, we demonstrated that ELA-11

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Wang et al. 10.3389/fphar.2022.873614

FIGURE 7
ELA-11 protects cardiomyocytes by binding APJ. (A,B) Western blot result of PARP and caspase-3 with ML221 treatment in DOX-induced group
and its quantiied data. (C,D) Western blot result of ERK with ML221 treatment in DOX -induced group and its quantiied data. (E–G) Western blot and
quantiied data for PI3K and AKT in DOX-induced group. (H,I) The expression of PARP and casapse-3 in CoCl2 -induced group by western blot and
quantiied. (J,K) The expression of ERK in CoCl2 -induced group by western blot and quantiied. (L–N) The expression of PI3K and AKT in CoCl2
-induced group with quantiied data. ***p < 0.001, and ### p < 0.001, one-way ANOVA.

attenuated oxidative stress-induced apoptosis through ERK/ of disease monitoring (Ishimaru et al., 2017). In addition,
MAPK and PI3K/AKT signaling pathways by targeting endocrine-derived peptides play a dispensable role in different
APJ. ERK/MAPK and PI3K/AKT signaling pathways is pathological processes and engage in organ crosstalk. For
regarded as a promising molecular mechanism in apoptosis, instance, a vasoactive intestinal polypeptide of 28 amino acids
proliferation and differentiation. Our findings provide is released by intestinal neurons (Reginauld et al., 2019). Its level
evidence of the molecular mechanism by which the ELA-11 of variation is related to a variety of human diseases. It is worth
peptide inhibits oxidative stress-induced apoptosis, indicating its observing that some long peptides can be cleaved into smaller
potential use for DOX-induced cardiotoxicity. peptides that have more consequential functions than those of
Many known bioactive substances secreted by heart, longer peptides.
including ANP, have been indicated to be involved in the Because of the cardiac toxicity caused by DOX, its clinical
regulation of cardiovascular disease and sensitive as indicators application is severely limited (Sun et al., 2019). Apoptosis and

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Wang et al. 10.3389/fphar.2022.873614

Oxidative stress irritates the activity of ion exchangers,


including Na+/H+ exchangers (NHE-1) and transient receptor
potentials melastain 2 (TRPM2), resulting in overload of cell
serum and mitochondrial Ca2+. Overloaded Ca2+ leads to
activation of mitochondrial Ca2+ sensitive dehydrogenase and
massive production of respiratory metabolic chain substrate
NADH, which again promotes ROS production (Javadov
et al., 2008). This cycle eventually leads to dyshomeostasis,
apoptosis, inflammatory responses and other pathological
process. Hence, ROS overproduction is the main event of
oxidative stress injury (Schieber and Chandel, 2014).
Mechanically, as an essential signaling pathway of G-protein-
coupled receptors, APJ can activates RAF-1 to phosphorylate
FIGURE 8
threonine and tyrosine through PI3K/AKT, and ultimately
ELA-11 platform for inhibiting apoptosis and oxidative stress
injury in cardiomyocytes. activate ERK1 and ERK2 (i.e., p44MAPK and p42MAPK)
(Chiu et al., 2013). Therefore, AKT and ERK signaling
pathways play a key role in oxidative stress response.
Multiple evidence indicated that mitochondrial
oxidative stress injury induced by DOX came into our insight. permeability conversion pore (mPTP) plays a key role in
CoCl2 is a classic and effective compound used to simulate regulating cardiac apoptosis under pathological conditions
hypoxic and ischemic processes (Ma et al., 2020). In (Hou et al., 2022). Changes in intracellular pH, mitochondrial
consistence with previous studies, our study has demonstrated membrane potential or reactive oxygen species can regulate
that DOX and CoCl2 can induce apoptosis and oxidative stress THE opening of mPTP (Ahmad et al., 2019). Glycogen
injury. APJ receptor is an essential orphan G protein-coupled synthase kinase (GSK)-3α/β is a serine/threonine kinase
receptor super-family. Previous studies have demonstrated that that regulates the opening of mPTP and participates in the
ELA-21 and ELA-32 involved in heart development and stem cell apoptosis process of cardiomyocytes (Ahmad et al., 2014). The
maintenance (Dai et al., 2008). ELA-11 is the shortest peptide in activation of PI3K/AKT signaling pathway may regulate the
ELABELA family and may retain the functional peptide to activation of GSK-3 downstream protein. Knock down the
perform biological functions (Ma et al., 2021). We proved that expression of GSK-3 in adult cardiomyocyte can lead to
ELA-11 can also protect cardiac function by interacting with APJ. mitotic catastrophe which resulting in fatal dilated
As an endogenous ligand of Apelin, APJ plays a cardiomyopathy (Zhou et al., 2016; Ahmad and Woodgett,
cardiovascular protective role by binding to different G 2020). And inhibition of GSK-3αmay be a novel strategy
protein subtypes and trigger multiple signaling pathways, such for limited adverse ventricular remodeling and
as AMPK, PI3K/Akt or MAPK signaling pathways (Li et al., dysfunction after myocardial infarction in future treatment
2021). Elabela was first identified as a novel ligand of APJ (Lal et al., 2015). Whether GSK-3 can play a role in DOX
receptor in zebrafish embryos. The Ela-APJ axis is critical to a induced cardiotoxicity will be further explored in future
variety of biological processes and has been shown to regulate studies.
humoral homeostasis, myocardial contractility, vasodilation, Although recent studies have shown that the short peptide
angiogenesis, cell differentiation, apoptosis, oxidative stress, ELABELA (19–32) can ameliorate DOX-induced
cardiorenal fibrosis and dysfunction (Sato et al., 2017). In cardiotoxicity by promoting autophagic flux through TFEB
human embryonic stem cells, Elabela can activate PI3K/Akt/ pathway, ELA-11, shorter than ELABELA (19–32) reduced
MTORC1 signaling pathway to regulate self-renewal and survival ROS release, thereby inhibiting mitochondrial oxidative stress
of stem cells (Ho et al., 2015). As we all know that DOX induced and further inhibiting apoptosis of cardiomyocytes.
cardiotoxicity involves a variety of molecular mechanisms, Furthermore, In our study, the results supported that ELA-
including energy metabolism, oxidative stress, and 11 resisted cardiotoxicity by inhibiting apoptosis and
programmed cell death (Tocchetti et al., 2014). The excessed oxidative stress by activating PI3K, AKT and ERK
ROS can be effectively eliminated by the antioxidant defense phosphorylation.
system in physiological status (Zhang et al., 2022). However, Our study also has some limitations. First, more clinical
when the antioxidant defense system is unable to consume samples are needed to determine the exact window of time of
excessive levels of ROS, cytotoxic signaling pathways will interactions for clinical application. Second, whether the
activate, leading to DNA damage, mitochondrial dysfunction modification of ELA-11 influences its function in
and abnormal cellular calcium homeostasis. cardiovascular diseases needs further evaluation.

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Wang et al. 10.3389/fphar.2022.873614

Data availability statement Project of School of Medicine, Shanghai Jiaotong University


(ZT202013).
The original contributions presented in the study are
included in the article/Supplementary Materials, further
inquiries can be directed to the corresponding authors. Conflict of interest
The authors declare that the research was conducted in the
Ethics statement absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
The animal study was reviewed and approved by the Peptide
and doxorubicin-induced cardiotoxicity.
Publisher’s note
Author contributions All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their
LQ designed the research. XW, LZ, and MF performed the affiliated organizations, or those of the publisher, the
experiments. ZX analyzed the data. XW and ZC wrote the editors and the reviewers. Any product that may be
manuscript. LQ and ZC supervised the work. evaluated in this article, or claim that may be made by
its manufacturer, is not guaranteed or endorsed by the
publisher.
Funding
This work was supported by the National Natural Science Supplementary material
Foundation of China (Grant No. 81873540), Jiangsu provincial
key research and development program (Grant No. BE2019752), The Supplementary Material for this article can be found
Changning Science and Technology Commission online at: https://www.frontiersin.org/articles/10.3389/fphar.
(CNKW2020Y06) and Technology Transfer and Promotion 2022.873614/full#supplementary-material

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