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PII: S0014-4827(19)30094-1
DOI: https://doi.org/10.1016/j.yexcr.2019.03.006
Reference: YEXCR11342
To appear in: Experimental Cell Research
Received date: 16 October 2018
Revised date: 28 February 2019
Accepted date: 3 March 2019
Cite this article as: Yun-lin Chen, Jinqi Fan, Li Cao, Ting-li Han, Mengying
Zeng, Yanping Xu, Zhiyu Ling and Yuehui Yin, Unique Mechanistic Insights
into the Beneficial Effects of Angiotensin-(1–7) on the Prevention of Cardiac
Fibrosis: A Metabolomic Analysis of Primary Cardiac Fibroblasts, Experimental
Cell Research, https://doi.org/10.1016/j.yexcr.2019.03.006
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Unique Mechanistic Insights into the Beneficial Effects of Angiotensin-(1-7) on the Prevention
1,1 1, 5 ,1 1 2, 3, 4 1
Yun-lin Chen, PhD, Jinqi Fan, PhD, Li Cao, PhD, Ting-li Han, PhD, Mengying Zeng, MD,
1 1 1, *
Yanping Xu, PhD, Zhiyu Ling PhD, Yuehui Yin, MD
1
Department of Cardiology, the 2nd Affiliated Hospital of Chongqing Medical University
2
Department of Obstetrics and Gynecology, the 1st Affiliated Hospital of Chongqing Medical University
3
Liggins Institute, University of Auckland, New Zealand
4
Mass Spectrometry Centre, China-Canada-New Zealand Joint Laboratory of Maternal and Foetal
5
Departments of Biomedical Engineering and Pediatrics, Emory University, Atlanta, GA 30322, USA
*Address correspondence to Prof. Yuehui Yin, MD, Department of Cardiology, the 2nd Affiliated
Hospital of Chongqing Medical University, E-mail: yinyh63@163.com, Tel. & Fax: +86-023-63693
1
Abstract
Background:
Cell metabolic pathways are highly conserved among species and change rapidly in response to drug
stimulation. Therefore, we explore the effects of angiotensin-(1-7) in a primary cell model of cardiac
fibrosis established in angiotensin II-stimulated cardiac fibroblasts via metabolomics analysis and
After exposing cardiac fibroblasts to angiotensin II and/or angiotensin-(1-7), 172 metabolites in these
cells were quantified and identified by gas chromatography-mass spectrometry. The data were
biochemically significant metabolites associated with the antifibrotic action of angiotensin-(1-7). Seven
acid, docosahexaenoic acid (DHA), glutathione, palmitelaidic acid, and pyroglutamic acid. By
metabolic network analysis, we found that these metabolites were involved in six metabolic pathways,
including arachidonic acid metabolism, leukotriene metabolism, and the γ-glutamyl cycle. Since these
metabolic pathways are related to calcium balance and oxidative stress, we further verified that
angiotensin-(1-7) suppressed the abnormal extracellular calcium influx and excessive accumulation of
Furthermore, we found that angiotensin-(1-7) suppressed the abnormal calcium- and ROS-dependent
factor 2 (CTGF), and p-ERK1/2), and excessive collagen deposition in vitro and in vivo.
2
Conclusions: Angiotensin-(1-7) can ameliorate the angiotensin II-stimulated metabolic perturbations
associated with cardiac fibroblast activation. These metabolic changes indicate that modulation of
calcium- and ROS-dependent activation of CaMKIIδ mediates the activity of angiotensin-(1-7) against
cardiac fibrosis. Moreover, pyroglutamic acid and arachidonic acid may be potential biomarkers for
Keywords
2+
Ca /calmodulin-dependent protein kinase II
Introduction
Cardiac fibrosis is an important global health issue associated with nearly all forms of cardiac disease
[1] and is characterized by the uncontrolled activation of cardiac fibroblasts (CFs) (excessive
excitation-contraction coupling, and impaired cardiac architecture [2]. The renin-angiotensin system
(RAS), in which angiotensin II (Ang II) is the predominant effector, can promote the development of
cardiac fibrosis [3]. As a newly identified component of the RAS, angiotensin-(1-7) (Ang-(1-7))
counteracts Ang II. Recent studies, including ours, show that treatment with Ang-(1-7) can attenuate
the cardiac fibrosis induced by Ang II in association with CF activation inhibition [4-8]. Traditional
molecular biology studies have provided useful information regarding the mechanisms of Ang-(1-7)
action. However, the comprehensiveness of investigations into the underlying mechanisms of Ang-(1-7)
comprehensive snapshot of ongoing metabolic changes within a living system [9]. Metabolic changes
3
are the most proximal reporters of alterations in the body in response to a disease process or drug
therapy [10]. Integrating metabolic alterations with prior biological knowledge enables us to rapidly
uncover the mechanisms associated with the pathogenesis of disease or the action of a drug [11]. In
and biomarker discovery. Most of these studies have been performed in in vivo systems, including
human patients and animal disease models [12, 13]. In vitro metabolomic approaches such as cell
culture metabolomics can offer some advantages over in vivo metabolomics, including stricter control
of external variables and greater reproducibility; thus, such approaches are attractive for use in drug
discovery and development [14]. Historically, most drug screening studies have utilized
tumor-originated cell lines or other immortalized mammalian cells. The implementation of primary cells
is more suitable for monitoring drug toxicity and exploring drug action mechanisms because primary
cells retain functions that are more physiologically and clinically relevant to those observed in vivo [15].
However, the application of primary cells in cardiovascular drug research has rarely been reported.
In this study, we aim to systematically explore the mechanism underlying the protective activity of
Ang-(1-7) against cardiac fibrosis. After CFs were treated with Ang II and/or Ang-(1-7), a metabolomic
analysis of the treated CFs and normal CFs was performed to elucidate the mechanisms underlying
the protective activity of angiotensin-(1-7) against cardiac fibrosis. Multivariate analyses, including
orthogonal projections to latent structures discriminant analysis (OPLS-DA) and shared and unique
structures (SUS) plot analysis, were used to identify biochemically significant metabolites linked to the
mechanisms underlying the antifibrotic action of Ang-(1-7). Finally, the biological events associated
with these metabolites were verified by traditional molecular biology experiments in vivo and in vitro.
4
Materials and Methods
This research protocol was approved by the Institutional Animal Care and Use Committee of
Chongqing Medical University and was conducted in compliance with the Guide for the Care and Use
Animal models
A total of 18 male Sprague-Dawley rats weighing approximately 240 g were purchased from
Chongqing Medical University Laboratory Animal Center and fed a chow diet in a 12-hour light/12-hour
dark environment maintained at 25°C. These Sprague-Dawley rats were treated with Ang II (500
ng/kg/min, n = 6), Ang II plus Ang-(1-7) (500 ng/kg/min, n = 6), and physiological saline solution (the
normal group, n = 6) for 28 days using osmotic minipumps (model 2004; Alzet, Cupertino, USA) as
CFs were isolated from neonatal Sprague-Dawley rats (1–3 days old) as previously described [5].
Briefly, rat hearts were rapidly excised and digested with 0.08% collagenase type II (Invitrogen,
California, USA) and 0.125% trypsin (Beyotime, Shanghai, China). After digestion, the cells were
supplemented with 10% fetal bovine serum (Invitrogen, California, USA) plus 1% penicillin–
streptomycin (Beyotime, Shanghai, China) and incubated in a humidified incubator containing 5% CO2
at 37°C. The CFs at passage one or two were used for subsequent experiments.
Total RNA was isolated from the treated CFs using TRIzol reagent (TaKaRa, Dalian, China).
Approximately 1 µg of RNA was used for reverse transcription reactions with PrimeScript RT Master
5
Mix (TaKaRa, Dalian, China) using the standard protocol described in the manufacture user manual,
with a 15-min RT step. Subsequently, qPCR reactions were run on an Applied Biosystems 7500
Real-Time PCR System using TB Green Premix Ex Taq II (TaKaRa) and the probes for
rat calcium/calmodulin-dependent protein kinase II delta (CaMKIIδ), NADPH oxidase 4 (Nox4), cellular
(Supplemental Table 1). The relative expression levels of these genes were assessed by the
delta/delta Ct method.
Rat hearts were fixed in formaldehyde, embedded in paraffin, and sectioned at 5-μm thickness for
Masson’s trichrome staining (Solarbio Life Science, Beijing, China). Fibrotic areas were measured and
Immunohistochemistry
Paraffin-embedded rat ventricular tissues were sliced into 5-μm sections. After deparaffinization,
hydration and antigen retrieval, the sections were washed in PBS. Peroxidase activity was blocked
with 3% H2O2 for 10 minutes, and the sections were then incubated with primary antibodies against
α-SMA (1:400, Sigma-Aldrich, Missouri, USA) overnight at 4°C. Next, the sections were washed and
incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Finally, the
sections were processed with DAB working solution and mounted. Staining area quantification was
Snap-frozen heart tissues and cultured CFs were lysed in RIPA buffer (Beyotime, Shanghai, China)
containing a protease and phosphatase inhibitor cocktail (MedChemExpress, New Jersey, USA).
6
Protein lysates were boiled at 100°C for 5 minutes, and the protein concentration was assessed with a
BCA protein assay. A total of 30 μg of each sample was separated by 10% SDS-PAGE, transferred
onto PVDF membranes (Roche, Basel, Switzerland), and blocked in PBS containing 5% BSA
(Beyotime, Shanghai, China) and 0.1% Tween 20 (Beyotime, Shanghai, China). The membranes were
incubated with primary antibodies against collagen I, collagen III, α-SMA, CTGF, CaMKIIδ,
overnight at 4°C (Abcam, Cambridge, UK; Sigma-Aldrich, Missouri, USA; GeneTex, California, USA).
Then, the membranes were incubated with HRP-conjugated secondary antibodies (1:8000, Beyotime,
Shanghai, China) for 1 hour at room temperature. Protein bands were detected using the
WesternBright ECL HRP substrate (Advansta, CA, USA) and a Bio-Rad ChemiDoc MP system
(Bio-Rad, California, USA). The band intensities were quantified by Image Lab software version 5.1
Immunofluorescence staining
Frozen left ventricular tissue samples for immunofluorescence staining were prepared according to the
reference [18]. To prepare cell samples for immunofluorescence staining, the treated CFs were rinsed
with PBS, fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature, permeabilized
with PBS containing 0.125% Triton X-100, and blocked with PBS containing 10% goat serum. Next,
these samples were incubated with primary antibodies against α-SMA (1:400, Sigma-Aldrich, Missouri,
USA) and ox-Met(281/282)-CaMKIIδ(1:100, Gene Tex, California, USA) overnight at 4°C, followed by
Alexa Fluor 594-conjugated secondary antibodies (1:1000, Gene Tex, California, USA) for 1 hour at
room temperature. Nuclei were stained with DAPI (Beyotime, Shanghai, China). Image acquisition and
analysis were performed using a Leica TCS-SP5 laser scanning confocal microscope and LAS AF Lite
7
software (Hessen, Germany).
CFs were cultured in a 96-well plate (5000 cells per well) and treated with Ang II (10 μM,
MedChemExpress, New Jersey, USA) and/or Ang-(1-7) (10 μM, MedChemExpress, New Jersey, USA)
for 48 hours. To assess cell proliferation, 10 μl of Cell Counting Kit-8 (Dojindo, Tokyo, Japan) solution
was added to each well; the absorbance at 540 nm was measured with a microplate reader
(Bio-Tek, Vermont, USA) after the plate was incubated for 2 hours under the conditions described
above.
2+ 2+
Measurements of the intracellular Ca concentration ([Ca ]i) were performed as described previously
[19]. CFs were plated onto 35-mm glass-bottom dishes (NEST, Jiangsu, China) and grown to a
confluence of approximately 70%. The cells were incubated with Fluo-4 AM (10 μM, Invitrogen,
California, USA) at 37°C for 30 minutes in Hanks’ balanced salt solution (HBSS, 137.93 mM NaCl, 5.33
mM KCl, 4.17 mM NaHCO3, 0.441 mM KH2PO4, 0.338 mM Na2HPO4, and 5.56 mM D-glucose)
2+ 2+
without Ca . The CFs were then stimulated with Ang II or Ang-(1-7) in HBSS with or without Ca (1.26
mM CaCl2). Fluorescence images of a group of cells (≥ 10 cells) were acquired and analyzed with the
Leica TCS-SP5 laser scanning confocal microscope (excitation at 494 nm and emission at 516 nm)
2+
and LAS AF Lite software. Changes in [Ca ]i were reported as the Fluo-4 ratio F/F0, where F indicates
the fluorescence intensity at different time points and F0 is the baseline fluorescence intensity recorded
-
To measure the intracellular H2O2 and O2 levels, the fluorescent dyes dichloro-dihydro-fluorescein
8
diacetate (DCFH-DA, 10 μM, Beyotime, Shanghai, China) and dihydroethidium (DHE, 10 μM,
Sigma-Aldrich, Missouri, USA), respectively, were used as described previously [20, 21]. After
treatment with Ang II and/or Ang-(1-7) for 15 minutes, cells in 6-well culture dishes were washed 3
times with HBSS. Subsequently, fluorescence was detected by an Olympus IX53 fluorescence
microscope (Tokyo, Japan). The mean fluorescence intensity in different groups was quantified using
analysis
The metabolite extraction and derivatization protocols were modified from those of Smart et al. [22, 23].
After 48 hours of treatment with Ang II and/or Ang-(1-7), CFs were quenched by liquid nitrogen.
Metabolites were extracted from these cells using cold methanol/chloroform (9:1 v/v, Sigma-Aldrich,
Missouri, USA), and the internal standard, namely, 2,3,3,3-d4-alanine (20 µl for every 1.5 ml of
extraction solvent), was concurrently introduced into every sample. Next, the cells were removed with
cell scrapers, and the solvent extracts were transferred to Eppendorf tubes. After centrifugation at
17,000 g for 15 minutes at 4°C, the supernatant was collected from each tube, divided into two tubes,
and dried in a SpeedVac (Labconco Corp., Missouri, USA) at room temperature for 4−5 hours. Then,
one tube of each sample was used to measure the protein concentration by a BCA assay for biomass
normalization, while the supernatant in the other tube was chemically derivatized by the methyl
chloroformate (MCF) approach prior to GC-MS analysis. The MCF derivatives were analyzed in an
Agilent GC7890B system coupled to an MSD5977A mass selective detector (EI) set at 70 eV. The GC
column installed for metabolite analysis was a ZB-1701 GC capillary column (30 m × 250 µm id × 0.15
µm with a 5-m guard column, Phenomenex). The GC analysis parameters were implemented
9
according to the protocol of Smart et al. [24]. Chromatogram deconvolution was performed by the
Automated Mass Spectral Deconvolution and Identification System (AMDIS), and compounds were
identified by an in-house MCF-derivatized mass spectral library and the NIST mass spectral library.
The relative abundance levels of the identified metabolites were extracted by an in-house
XCMS-based R script.
For the GC-MS data, the metabolite abundance levels were normalized to the concentration of the
internal standard and the total protein detected in each sample. Batch variation was removed by
median centering, and the metabolite data were log transformed, mean centered, and Pareto scaled.
Then, the data were analyzed by principal component analysis (PCA) and OPLS-DA in SIMCA 14.1
(Umetrics, UMEÅ, Sweden) [25]. Seven-fold cross-validation was employed as a measure of the
robustness of the multivariate models. Loading plots with jackknife confidence intervals, variable
importance in projection (VIP) scores, and S-plots from OPLS-DA were used to identify biochemically
interesting metabolites. The significant metabolites were selected based on the following criteria: VIP
scores > 1, |p (corr)| > 0.5, |p|> 0.1, and jackknife confidence intervals excluding zero [26, 27]. The
terms p (corr) and p indicate the correlation and covariance loading profiles, respectively. An SUS plot
was used to identify the significant metabolites associated with the antifibrotic action of Ang-(1-7) that
were shared and unique between the OPLS-DA models (normal vs Ang II; Ang II + Ang-(1-7) vs Ang II).
Then, a specific metabolic network based on the Kyoto Encyclopedia of Genes and Genomes (KEGG)
database was built by MetScape 2 to unravel the biological implications of these shared metabolites
[28].
The molecular biological data are expressed as the means ± SEMs. The assumptions of normality and
10
variance homogeneity were tested by the Shapiro-Wilk test (p > 0.1) and Levene’s test (p > 0.1),
respectively. Statistical significance across groups was determined by one-way or two-way ANOVA
followed by Tukey’s honest significant difference (HSD) post hoc test using R software; p-values < 0.05
Results
To determine whether Ang-(1-7) attenuates the differentiation of CFs induced by Ang II, we tested the
effects of Ang II and Ang-(1-7) on the expression of α-SMA, collagen I, and collagen III by Western
blotting and/or immunofluorescence staining. As shown in Figure 1a and 1b, Ang II significantly
increased the expression levels of α-SMA, collagen I, and collagen III compared with those of the
normal cells, but pretreatment with Ang-(1-7) prevented these increases. We then assessed the
proliferation of CFs by the CCK-8 assay. The exposure of CFs to Ang II led to a 1.76-fold increase in
cell proliferation; this increase was suppressed by pretreatment with Ang-(1-7) (Figure 1c).
Ang-(1-7) ameliorates the perturbations in arachidonic acid (AA) and glutathione metabolism in
A total of 275 metabolite peaks were detected in the primary cell cultures, 172 of which were identified
by the in-house MCF GC-MS mass spectral library. PCA was performed to obtain an overview of the
metabolic profiles. The PCA score plot in Figure 2 shows a clear separation between the control group
(a cardiac fibrosis cell model established in Ang II-stimulated CFs) and the two treatment groups
(normal CFs and Ang II-stimulated CFs treated with Ang-(1-7)). The principal components t1 and t2
2
explained 64.9% and 12.1% of the total variance, respectively (Q values: t1 = 0.61, t2 = 0.7),
11
suggesting that the cellular metabolic phenotypes were significantly different between the control
group (Ang II group) and the treatment groups (normal and Ang II + Ang-(1-7) groups).
OPLS-DA was then used to screen statistically significant metabolites that contributed to the
classification of each of the treatment groups and the control group. As shown in Figure 3a and Figure
4a, both OPLS-DA models (model 1, normal vs Ang II; model 2, Ang II + Ang-(1-7) vs Ang II) yielded
2 2
good classification under different conditions (R = 0.87, Q = 0.73, 1 predictive component + 2
2 2
orthogonal components; R = 0.98, Q = 0.75, 1 predictive component + 5 orthogonal components).
According to the cross-validated score plots, no sample was misclassified, which further indicated the
In the OPLS-DA models, both loading plots with confidence intervals and VIP scores were used to
assess variable significance (VIP scores > 1 and jackknife confidence intervals excluding zero) [25].
The models identified 30 statistically significant metabolites (Figure 3b) for the normal group and 27 for
the Ang II + Ang-(1-7) group (Figure 4b). An S plot combining the covariance (p (corr), the
concentration of metabolites) and correlation (p, the contribution to class separation) from the
significant metabolites with |p (corr)| > 0.5 and |p| > 0.1) [26, 27]. A total of 17 and 7 metabolites met
the selection criteria for model 1 and model 2, respectively (Figures 3c and 4c). Compared to the Ang II
group, the normal and Ang II + Ang-(1-7) groups exhibited decreased concentrations of all
Furthermore, an SUS plot was used to compare the outcomes of the two established OPLS-DA models
(normal vs Ang II and Ang II + Ang-(1-7) vs Ang II). Seven biochemically interesting metabolites near
the diagonal line ((-1, -1) to (1, 1)) were equally affected in both models (Figure 5). In other words, the
12
changes in these seven metabolites represent the rescue effect of Ang-(1-7) on Ang II-stimulated CFs.
Finally, the metabolomic data were linked to prior knowledge of metabolic pathways (KEGG analysis)
and visualized by MetScape 2, as shown in Figure 6. Among the identified metabolites, 56 were used
to reconstruct a metabolic network associated with the abnormal activation of CFs. The seven
metabolism, the γ-glutamyl cycle, leukotriene metabolism, omega-6 fatty acid metabolism, and
prostaglandin biosynthesis.
Next, we explored whether the antifibrotic effect of Ang-(1-7) is associated with the reestablishment of
calcium hemostasis in CFs. Figures 7a and 7b show that Ang II triggered a lower and transient peak of
2+ 2+ 2+
[Ca ]i in the absence of extracellular Ca but a higher and sustained peak of [Ca ]i in the presence of
2+ 2+
extracellular Ca . Notably, the Ang II-induced increase in [Ca ]i was suppressed by Ang-(1-7)
2+
pretreatment in the presence of extracellular Ca but was unaffected in the absence of extracellular
2+
Ca (Figure 7c and 7d). These results suggested that the extracellular calcium influx contributes to the
2+
increase in [Ca ]i in Ang II-stimulated CFs. This phenomenon can be abolished in the presence of
Furthermore, we investigated the effect of Ang-(1-7) on CaMKIIδ and the associated downstream
signaling pathways by RT-qPCR and Western blot analyses. Supplemental Figure 1 shows that the
mRNA expression levels of CaMKIIδ and CTGF were significantly increased in the Ang II-stimulated
CFs. This effect can be significantly suppressed by Ang-(1-7) treatment. Correspondingly, the protein
expression levels of p-Thr(287)-CaMKIIδ and total CaMKIIδ were significantly higher in the Ang II
group than in the normal group. This increased expression was restored to normal levels by
13
pretreatment with Ang-(1-7) (Figure 5f). The ratio of p-Thr287 expression to total CaMKIIδ expression
was higher in the Ang II group than in the normal group, but pretreatment with Ang-(1-7) restored the
increased ratio to the normal level. In addition, treatment with Ang II significantly increased the protein
levels of p-ERK1/2 and CTGF over the levels observed in the normal group, but pretreatment with
Ang-(1-7) significantly inhibited the Ang II-induced phosphorylation of ERK1/2 and the increased
expression of CTGF (Figure 7f). Ang-(1-7) pretreatment decreased the ratio of p-ERK1/2 to ERK1/2
To explore whether Ang-(1-7) suppresses the activation of CFs induced by Ang II via modulation of
intracellular oxidative stress, the effects of Ang-(1-7) on the level of ROS and the expression of Nox4
and ox-CaMKIIδ were studied in Ang II-stimulated CFs. CFs were stimulated with Ang II, which
resulted in increased production of intercellular ROS, including hydrogen peroxide (Figure 8a) and the
superoxide anion (Figure 8b). These increases were alleviated by pretreatment with Ang-(1-7) (Figure
8a and 8b). Furthermore, we observed a significant increase in the expression levels of Nox4 (mRNA
and protein levels) and ox-CaMKIIδ (protein level) in CFs treated with Ang II, but this increase was
Ang-(1-7) infusion protects against Ang II-induced cardiac fibrosis by suppressing abnormal
To further validate the effect of Ang-(1-7) on cardiac fibrosis induced by Ang II in vivo, Ang II-infused
rats were treated with Ang-(1-7). Masson’s trichrome staining of paraffin sections showed that Ang II
infusion promoted extensive fibrosis in the left ventricular sections of the rats (Normal: 3.28±0.81% vs
Ang II 14.75±5.17%, p < 0.05, Figure 9a). This extensive cardiac fibrosis can be reversed by treatment
14
with Ang-(1-7) (Ang II + Ang-(1-7): 7.48±0.59% vs Ang II: 14.75±5.17%, p < 0.05, Figure 9a).
α-SMA after Ang II infusion, whereas the Ang-(1-7) treatment abolished the increase in the expression
of α-SMA (Figure 9b). In addition, Western blot analysis revealed elevated protein levels of CTGF and
p-ERK1/2 in Ang II-infused rats but not in Ang II-infused rats treated with Ang-(1-7) (Figure 9c). No
change was observed in the protein expression of ERK1/2 after the Ang II and Ang-(1-7) infusion
(Figure 9c).
Furthermore, we explored whether Ang-(1-7) ameliorates Ang II-induced cardiac fibrosis by inhibiting
ox-CaMKIIδ (a marker of CaMKII oxidative activation) and α-SMA (a marker of CF activation) was
observed in ventricular sections of Ang II-infused rats, implying that CF activation is likely accompanied
hearts due to the incompatibility of the antibody with immunofluorescence staining. Western blot
analyses showed that Ang II infusion significantly increased the expression levels of CaMKIIδ,
p-CaMKIIδ, ox-CaMKIIδ and Nox4 in rat ventricles, whereas these protein expression levels were
significantly suppressed by Ang-(1-7) treatment (Figure 10b, 10c, 10d, and 10e).
Discussion
The results of our previous studies suggest that Ang-(1-7) attenuates cardiac fibrotic remodeling by
reducing the Ang II-induced proliferation and differentiation of CFs [4-6]. However, the antifibrotic
mechanism of Ang-(1-7) remains unclear. This study was the first to explore the mechanism underlying
Ang-(1-7) activity in Ang II-stimulated CFs via an untargeted metabolomic analysis. We successfully
15
determined that Ang-(1-7) suppressed the metabolic perturbations induced by Ang II in CFs, including
those affecting AA metabolism and the γ-glutamyl cycle. Considering that metabolic changes in AA
metabolism and the γ-glutamyl cycle are associated with calcium imbalance and oxidative stress,
respectively, we further verified that Ang-(1-7) inhibited Ang II-induced CF activation by suppressing
calcium overload, excessive oxidative stress, and the activity of CaMKIIδ-related signaling pathways in
The change in the AA level in CFs leads us to postulate that Ang-(1-7) exerts its antifibrotic effect by
suppressing the calcium overload induced by Ang II. The results of our metabolomic analysis revealed
that Ang II enhanced AA production in CFs, and subsequent metabolic network analysis showed that
AA was involved in multiple metabolic pathways associated with the intracellular calcium balance, such
reveal that a calcium imbalance can result in abnormal activation of CFs [19, 30-32]. Extensive
2+
evidence demonstrates that AA can increase [Ca ]I via transient receptor potential channels, which
2+
are the main membrane channels mediating Ca influx into CFs [33-37]. Consistent with the results of
previous studies, our study also showed that Ang II increased the endogenous AA level and promoted
2+
the influx of extracellular Ca into CFs [38, 39]. More importantly, Ang-(1-7) not only reduced the
endogenous AA concentration but also profoundly decreased the excessive influx of calcium into Ang
II-stimulated CFs. These novel findings provide direct evidence that Ang-(1-7) can reverse the
deleterious effect of Ang II on CFs by maintaining calcium homeostasis and that AA has potential as a
The alterations in the γ-glutamyl cycle reflect the antioxidant role of Ang-(1-7) against Ang II-triggered
ROS production in CFs. Our metabolic network analysis highlighted the upregulation of the γ-glutamyl
16
cycle in Ang II-stimulated CFs. Dysregulation of this cycle has been reported to be related to
uncontrolled oxidized stress during cardiac fibrotic remodeling [40, 41]. The γ-glutamyl cycle governs
the biosynthesis of glutathione; this process involves the ligation of glutamate with two amino acids
(cysteine and glycine) to form glutathione, the catabolism of glutathione to pyroglutamic acid (also
called 5-oxoproline) and the subsequent degradation of pyroglutamic acid into glutamate [42]. Here,
we reported that glutathione and pyroglutamic acid levels were positively correlated with the ROS level
in Ang II-stimulated CFs. Similarly, Atze et al. demonstrated that accumulation of pyroglutamic acid in
cardiomyocytes elevated the ROS level in mice with heart failure [43]. Intriguingly, Ang-(1-7) can
counteract the accumulation of pyroglutamic acid in CFs treated with Ang II. Furthermore, in our
experiments in vivo and in vitro, the expression of Nox4, which is a major source of ROS in CFs, was
increased after Ang II treatment. This result is consistent with the findings reported by other studies
showing that Nox4-mediated increases in H2O2 levels promote CF differentiation and proliferation and
that Nox4 upregulation in the myocardium causes cardiac fibrosis in rats [17, 44]. Interestingly, our
results showed that treatment with Ang-(1-7) inhibited Nox4 overexpression and excessive generation
of H2O2 in the CFs incubated with Ang II. Ang-(1-7) also markedly reduced excessive
collagen deposition and increased the protein expression levels of α-SMA and Nox4 in Ang II-infused
rats. These results are in line with those reported in a recent animal study published by Wang et al. [45].
Therefore, our findings suggest that Ang-(1-7) can ameliorate cardiac fibrosis by eliminating
Nox4-mediated ROS production in CFs and that this antioxidant outcome can be reflected by the level
of pyroglutamic acid.
CaMKIIδ acts as a central enzymatic node connecting the upstream calcium overload and ROS
perturbations with downstream profibrotic signaling pathways in the heart [46]. Calcium overload and
17
oxidative stress can promote posttranslational activation of CaMKIIδ through autophosphorylation of
respectively [47-51]. Indeed, our cell and animal results revealed that Ang II-induced excessive CF
overload and excessive ROS production. On the other hand, previous studies revealed that CTGF-
activation of CFs [52-54]. Consistently, we also showed that the downstream molecules of CaMKIIδ,
such as CTGF and p-ERK1/2, were upregulated after Ang II treatment. Most profoundly, pretreatment
with Ang-(1-7) can abolish both the abnormal activation of CaMKIIδ and the upregulation of its
downstream cascade observed in the Ang II-induced cardiac fibrosis. The effective inhibition of
CaMKIIδ by Ang-(1-7) may be the key step for protection against cardiac fibrosis triggered by Ang II.
This study has several limitations. Our study focused only on the downstream signaling pathways in
response to the metabolic changes in AA metabolism and the γ-glutamyl cycle in CFs treated with Ang
II and Ang-(1-7), and the upstream regulating mechanisms remain to be investigated. Furthermore,
multiple inbred rodent strains should be implicated to validate our hypothesis in vivo because a recent
study suggested that the heterogeneous nature of CFs is genetically diverse in rodent strains [55].
Conclusions
In this study, the results of our mass spectrometry-based metabolomic analysis support the hypothesis
that Ang-(1-7) exhibits protective activity against cardiac fibrosis via the modulation of AA metabolism
and the γ-glutamyl cycle. These metabolic reprogramming observations guided us to unravel the
18
protective mechanism of Ang-(1-7), which acts by restoring calcium homeostasis, protecting against
ROS damage, and suppressing downstream CaMKIIδ-related signaling pathways in Ang II-stimulated
CFs. This enhanced mechanistic understanding of Ang-(1-7) activity could further promote the
pyroglutamic acid and AA are the biomarkers most likely to be useful for monitoring the protective
Acknowledgments
Chen YL, Cao L, Ling ZY and Zeng MY performed the experiments. Chen YL and Han TL analyzed the
data. Chen YL, Fan JQ, and Yin YH designed the experiments. Chen YL, Fan JQ, and Han TL wrote
the manuscript, and all authors contributed to the preparation of the manuscript.
Funding
This study was supported by grants from the National Natural Science Foundation of China (grant nos.
Disclosures
None.
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Data
Figure 1. Ang-(1-7) inhibited the cardiac fibroblast differentiation and proliferation induced by Ang II. Cardiac
fibroblasts were cultured in microplates and incubated with Ang II (10 μM) and/or Ang-(1-7) (10 μM) for 48 hours. a)
Immunofluorescence images indicating that Ang-(1-7) inhibited the Ang II-induced differentiation of cardiac fibroblasts,
as evidenced by the reduction in α-SMA (red) expression. Nuclei were stained with DAPI (blue). The scale bars
represent 50 μm. b) Western blot analysis of α-SMA, collagen I, and collagen III and quantification of the protein bands.
c). Assessment of cardiac fibroblast proliferation with a CCK-8 assay. These experiments were repeated at least 3
times with reproducible results. The data are expressed as the means ± SEMs. * indicates p < 0.05 compared to the
Ang II group. Significant differences between conditions were determined by one-way ANOVA and Tukey’s multiple
comparisons test.
28
Figure 2. Principal component analysis (PCA). PCA score plot of the normal (red boxes), Ang II (green boxes), and
2 2
Ang II + Ang-(1-7) (blue boxes) groups (R = 0.77; Q = 0.70; 2 cumulative principal components).
26
Figure 3. Identification of biochemically significant metabolites between the normal and Ang II groups by
2 2
OPLS-DA. a) Cross-validated score plots for the OPLS-DA model (R = 0.87; Q = 0.73; 1 predictive component + 2
orthogonal components). The red and green labels indicate the normal and Ang II groups, respectively. The modeled
score (t [1]) and the cross-validated score (tcv [1]) have identical orthogonal scores (to [1]). b) Loading plot with jackknife
confidence intervals. Both the black and the red bars represent metabolites with a VIP score > 1, but the red bars also
indicate metabolites identified as biochemically significant using the S-plot. c) S-plot. Seventeen biochemically
significant metabolites (statistically significant metabolites with a relatively high concentration and reliability) are
indicated by the red circles. The black circles represent metabolites with a VIP > 1. The white circles indicate statistical
nonsignificance.
27
Figure 4. Identification of biochemically significant metabolites between the Ang II + Ang-(1-7) and Ang II
2 2
groups by OPLS-DA. a) Cross-validated score plots for the OPLS-DA model (R = 0.98; Q = 0.75; 1 predictive
component + 5 orthogonal components). The blue and green labels represent the Ang II + Ang-(1-7) and Ang II groups,
respectively. The modeled score (t [1]) and the cross-validated score (tcv [1]) have identical orthogonal scores (to [1]). b)
Loading plot with jackknife confidence intervals. Both the gray and the blue bars represent metabolites with a VIP
score > 1, but the blue bars also indicate metabolites identified as biochemically significant using the S-plot. c) S-plot.
Twenty-seven biochemically significant metabolites (statistically significant metabolites with a relatively high
concentration and reliability) are indicated by the blue circles. The gray circles represent metabolites with a VIP > 1.
The white circles indicate statistical nonsignificance.
28
Figure 5. Identification of biochemically significant metabolites common to the normal and Ang II + Ang-(1-7)
groups using an SUS plot. The red and blue circles indicate the biochemically significant metabolites common to the
two OPLS-DA models (normal vs Ang II and Ang II + Ang-(1-7) vs Ang II). To improve clarity, all nonsignificant
metabolites were eliminated from each model.
29
Figure 6. The global metabolic network of all identified metabolites based on the KEGG database. The larger
circles indicate the identified metabolites. Biochemically significant metabolites for the normal group are shown as red
circles. Biochemically interesting metabolites for the Ang II + Ang-(1-7) group are highlighted by blue halos. Red circles
with a blue halo indicate biochemically significant metabolites common to the normal and Ang II + Ang-(1-7) groups.
The shared metabolites can be explained by their presence in the six metabolic pathways illustrated on the right.
30
Figure 7. Ang-(1-7) suppressed calcium overload and excessive activation of the associated signaling pathway
2+
in Ang II-stimulated CFs. The concentration of intracellular calcium (Ca ) is indicated by the green fluorescence
2+
intensity. a-d) The effect of Ang II (10 µM) on the intracellular Ca concentration under the following conditions: a) in
2+ 2+
the absence of extracellular Ca ; b) in the presence of extracellular Ca (1.8 mM); c) under pretreatment with
2+
Ang-(1-7) (10 µM for 48 h) in the absence of extracellular Ca ; and d) under pretreatment with Ang-(1-7) (10 µM for 48
2+
indicates the peak intracellular Ca concentration. The scale bars represent 50 μm. e) Summary of the results
2+
presented in a-d. The mean peak intracellular Ca values are plotted against each condition (*p < 0.05 vs condition a;
#p < 0.05 vs condition b). Significant differences between different conditions were determined by two-way ANOVA and
Tukey’s multiple comparison test. f) Western blot analysis showing the expression of p-T287-CaMKIIδ, CaMKIIδ, CTGF,
p-ERK1/2, and ERK1/2 in normal CFs, Ang II-stimulated CFs, and Ang II-stimulated CFs treated with Ang-(1-7). These
experiments were repeated at least 3 times with reproducible results. The data are presented as the means ± SEMs. *p
< 0.05 vs the Ang II group. Significant differences between different conditions were determined by one-way ANOVA
with Tukey’s multiple comparison test.
31
Figure 8. Ang-(1-7) suppressed abnormal oxidative stress and excessive activation of the associated signaling
pathway in Ang II-stimulated CFs. a) H2O2 production in normal CFs, Ang II-stimulated CFs, and Ang II-stimulated
CFs treated with Ang-(1-7). The green fluorescence intensity reflects the level of H2O2. The scale bars represent 100
-
μm. b) O2 production in normal CFs, Ang II-stimulated CFs, and Ang II-stimulated CFs treated with Ang-(1-7). The red
-
fluorescence intensity reflects the level of intracellular O2 . The scale bars represent 100 μm. c) Western blot analysis
showing the expression of Nox4, ox-M281/282-CaMKIIδ, and CaMKIIδ in normal CFs, Ang II-stimulated CFs, and Ang
II-stimulated CFs treated with Ang-(1-7). These experiments were repeated at least 3 times with reproducible results.
The data are presented as the means ± SEMs. *p < 0.05 vs the Ang II group. Significant differences between different
conditions were determined by one-way ANOVA with Tukey’s multiple comparison test.
32
Figure 9. Ang-(1-7) treatment protected against Ang II-induced cardiac fibrosis in rats. a) Masson’s trichrome
staining for fibrotic areas in hearts from rats infused with Ang II (control), Ang II plus Ang-(1-7), and physiological saline
solution. The scale bars represent 100 μm. b) Immunohistochemical staining of α-SMA in the left ventricular sections of
the rats treated with Ang II (control), Ang II plus Ang-(1-7), and physiological saline solution. The scale bars represent
200 μm. c) Western blot analyses showing the protein levels of CTGF, p-ERK1/2, and ERK1/2 in hearts from rats
treated with Ang II (control), Ang II plus Ang-(1-7), and physiological saline solution. These experiments were repeated
at least 6 times with reproducible results. The data are presented as the means ± SEMs. *p < 0.05 vs the Ang II group.
Significant differences between different treatments were determined by one-way ANOVA with Tukey’s multiple
comparison test.
33
Figure 10. Ang-(1-7) suppressed the excessive CaMKIIδ activation of CFs in the rat model of Ang II-induced
cardiac fibrosis. a) The immunofluorescence colocalization of positive α-SMA and ox-CaMKIIδ cell markers in
DAPI-stained cells indicates that CaMKIIδ activation of CFs occurred in hearts from Ang II-infused rats. The scale bar
indicates 100 μm. Western blotting (b, c, d) and quantification analysis (e) showing the protein expression levels of
CaMKIIδ, p-T287-CaMKIIδ, ox-M281/282-CaMKIIδ, and Nox4 in the hearts of rats treated with Ang II (control), Ang II
plus Ang-(1-7), and physiological saline solution. These experiments were repeated at least 6 times with reproducible
results. The data are presented as the means ± SEMs. *p < 0.05 vs the Ang II group. Significant differences between
different treatments were determined by one-way ANOVA with Tukey’s multiple comparison test.
34
,
Figure 11. The hypothetical mechanism underlying the protective activity of Ang-(1-7) against cardiac fibrosis.
In Ang II-stimulated CFs, AA induces calcium overload through TRP channels. Nox4 is a membrane protein that
converts oxygen (blue circles) to ROS (red circles). The increased ROS level is reflected by the upregulation of
γ-glutamyl cycle activity, which increases the levels of glutamic acid, glycine, GSH (or GSSH), and pyroglutamic acid.
The accumulation of calcium and ROS activates CaMKIIδ by the posttranslational modifications of phosphorylation at
threonine 287 (p-T287) and oxidation at methionine 281/282 (ox-M281/282). Activated CaMKIIδ upregulates the
downstream CTGF- and ERK1/2-dependent signaling pathways, which leads to cardiac fibroblast proliferation and
differentiation into myofibroblasts. Pretreatment of Ang II-stimulated CFs with Ang-(1-7) can abolish calcium overload,
decrease Nox4-mediated ROS generation, downregulate CaMKIIδ-related pathways, and finally attenuate fibroblast
proliferation as well as reduce the expression of collagen I/II and α-SMA in myofibroblasts. Furthermore, Ang-(1-7)
lowers the levels of AA, GSH (or GSSG), and pyroglutamic acid.
35