Atorvastatin treatment ameliorates cardiac function and remodeling
induced by isoproterenol attack through mitigation of ferroptosis
Dong Ning a, 1
, Xinquan Yang a, f, 1
, Ti Wang c
, Qiaohui Jiang c
, Jiangquan Yu c, d, **,
Daxin Wang b, e, *
a Department of Cardiology, The Second Xiangya Hospital, Central South University, Changsha, Hunan, China
b The Hospital Affiliated to Medical School of Yangzhou University(Taizhou People’ Hospital), Taizhou, Jiangsu, China c School of Medicine, Yangzhou University, Yangzhou, Jiangsu, China
d Clinical Medical College of Yangzhou University(Northern Jiangsu Province Hospital),Yangzhou, Jiangsu, China
e Yangzhou Biomedical Engineering Research Center, China
f The First Affiliated Hospital, School of Public Health, Zhejiang University School of Medicine, Hangzhou, China
article info
Article history:
Received 17 July 2021
Accepted 6 August 2021
Available online 12 August 2021
Keywords:
Atorvastatin
Heart failure
Cardiac remodeling
Ferroptosis
Ferritinophagy
abstract
Ferroptosis has been identified as an important role in damaged heart. Meanwhile, statin therapy has
been reported to be beneficial for the treatment of heart failure(HF) under different conditions. However,
the beneficial effects of statin treatment on regulation of ferroptosis in failing heart is unveiled. The aim
of this study is to explore the protective efficacy of atorvastatin against the ferroptosis related signaling
pathway in isoproterenol(ISO)-induced HF. We found that ATV and ferrostatin-1(Fer-1,as a positive
control) significantly improved ISO-decreased cell viability and cell survival by reducing oxidative stress
and Fe2þ-dependent lipid peroxidation in H9C2 cells. Additionally, ISO triggered marked ferritinophagy
accompanied by up-regulating protein levels of LC3BII,NCOA4 and Beclin1 and down-regulating protein
levels of P62 and FTH1 in damaged cells, which nevertheless was significantly blocked by administration
of ATV and these results were in parallel with the results obtained after 3-methyadenine(3-MA) treatment. Consistently, C57BL/6J mice were used in used in this study and administered 5 mg/kg/day ISO for
2 weeks to simulate cardiac injury. 20 mg/kg/day ATV treatment for 2 weeks simultaneously markedly
improved cardiac dysfunction and remodeling induced by ISO attack. ATV showed significantly protective effects through suppressing the activation of ferroptosis related signaling, as evidenced by
decreasing the mRNA levels of PTGS2(a marker of ferroptosis), contents of malonaldehyde and protein
levels of NOX4 and increasing the contents of glutathione(GSH), the ratio of GSH/GSSG and protein levels
of GPX4 and SLC7A11. Moreover, ISO evidently triggered degradation of FTH1 in failing heart. However,
ATV significantly prevented these changes in damaged heart. Overall, these results reveal atorvastatin
suppresses ferroptosis and exhibits protective effect on failing myocardium of mice after ISO insult
though inhibiting ferritinophagy-mediated ferroptosis, which might be a potential therapeutic strategy
in the prevention of ISO-associated cardiomyopathy.
© 2021 Elsevier Inc. All rights reserved.
1. Introduction
Heart failure(HF) is the major cause of mortality globally and has
been regarded as an important concern of public health system [1].
Cardiac dysfunction and remodeling secondary to irreversible
myocardial damage possibly caused by cardiac ischemia or hypoxia
remains a huge clinical challenge despite of many supplements and
drugs available in the hospital and community. Pathological hypertrophy in response to multiple forms of stress is initially adaptive change and beneficial, however, if these pathologic stimuli is
persistent, enlargement and dilation of cardiac muscles extrude
adjacent arteries and result in myocardial death. Subsequently, the
damaged area can be replaced by abnormal collagen deposition due
* Corresponding author. The Hospital Affiliated to Medical School of Yangzhou
University(Taizhou People’ Hospital), Taizhou, Jiangsu, China.
** Corresponding author. School of Medicine, Yangzhou University, Yangzhou,
Jiangsu, China.
E-mail addresses: [email protected], [email protected] (J. Yu),
[email protected], [email protected] (D. Wang). 1 Equally contributing authors.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
https://doi.org/10.1016/j.bbrc.2021.08.017
0006-291X/© 2021 Elsevier Inc. All rights reserved.
Biochemical and Biophysical Research Communications 574 (2021) 39e47
to uncontrolled activation of cardiac fibroblasts or other nonmuscle cells [2]. Complex signal pathway network imbalance
makes the current clinical treatments unable to effectively prevent
the compensatory cardiac remodeling from turning to decompensated. Therefore, proposing novel therapeutic ideas or promoting the translational medicine from frontier basic studies to
improve the prognosis of such patients will become an urgent task.
Ferroptosis, one novel form of regulated cell death, is characterized by Fe2þ-dependent irreparable lipid peroxidation, along
with impaired cellular reducing capacity induced by iron dysbiosis
[3]. Excessive iron through Fenton reaction on various lipid membranes in cell will form a vicious circle and then promote production of ROS [4]. During this process which has been suggested to be
initiated by multiple reaction, oxygenation of polyunsaturated fatty
acids on membranes leads to accumulation of toxic lipid metabolites and subsequent membrane injury [5].Importantly, neutralizing lipid radicals or detoxifying secondary reactive products
through restoring the function of glutathione peroxidase 4(GPX4)
can turn lipid peroxidation down, improve capacity of resistance to
ferroptosis and subsequently protect against cardiomyopathy
induced by different pathological attack [6,7].
Statins are the first-line treatment for patients with cardiovascular disease. At the same time, there are also many studies
reporting that such drugs have a satisfactory effect on improving
the risk of death in patients with non-fatal HF. After MI, patients
with 50% LDL-C reduction using high-intensity statins at
discharge appear to have a lower incidence of mortality and major
cardiovascular events [8]. In the past, targeting for reduction of
cholesterol synthesis through inhibition of HMG-CoA reductase has
been considered as the main mechanism of statins [9]. In addition,
such drugs can also achieve the purpose of inhibiting adverse
remodeling in damaged heart possibly through improving endothelial function, suppressing systemic or local inflammation,
enhancing the ability to resist oxidative stress. ISO, a synthetic
catecholamine and non-selective b-adrenergic receptor agonist, is
widely used in reproducible and well-standardized preclinical
model of cardiac remodeling and dysfunction mainly due to excess
production of reactive oxygen species(ROS) by persistent badrenergic stimulation [10]. Previously, Liu et al. demonstrated that
ferroptosis-like cell death and disorder of iron metabolism were
observed in ISO-treated cardiomyocytes or murine model, which
was characterized by increased lipid peroxidation and labile iron
pool and promoting degradation of ferritin heavy chain [10].However, little is known about the influence of statins to ferroptosis in
ISO-induced HF. In light of our previous study highlighting the
protective role of long-term 20 mg/kg atorvastatin(ATV) in
amelioration of cardiac dysfunction induced by obesity [11], in this
study, we used ISO to establish the cardiotoxicity and had new
discoveries that administration of atorvastatin could protect heart
against ISO attack through suppressing ferritinophagy-mediated
ferroptosis both in vitro and in vivo, which may offer novel insights into theoretical strategies for ameliorating HF.
2. Materials and methods
2.1. Cell culture and treatment
H9c2 cardiomyocytes were purchased from the Cell Bank of
Shanghai Institute of Biochemistry and Cell Biology(Shanghai,China) and cultured in complete DMEM medium(10% v/v fetal bovine serum,100 U/ml penicillin/streptomycin)
at 37 C and 5%(v/v) CO2 in a humidified incubator. Upon reaching
approximately 60% confluence, the cells were treated with ISO(-
Sigma,USA) or Erastin(MCE,USA) alone, or in combination with
ATV(Sigma,USA) or Ferrostatin-1(Fer-1; MCE,USA) or 3-
Methyladenine(3-MA; MCE,USA).
2.2. Cytotoxicity assays
Cell viability was performed by using CCK-8 assay kit(Beyotime,China) according to the manufacturer’s instructions. 48 h after
cell treatment, 10 mL of sterile CCK-8 was added to each well in 96-
well plates and incubated in the dark for 2h at 37 C. The
absorbance(450 nm) was determined using microplate reader(TECAN, Austria). Cell death was assessed by Calcein/Propidium
Iodide (PI) staining assay(Beyotime,China). Briefly, after treatment,
cell were incubated with 1 mM-working solution for 15min at 37 C
in the incubator. Fluorescence microscope was used to acquire the
images and positive cells were analyzed by ImageJ software.
2.3. Lipid peroxidation assay
D3861-BODIPY™581/591C11(Thermo Fisher Scientific,USA)
was used to evaluate the degree of lipid peroxidation in vitro. According to the manufacturer’s instructions, cells were incubated
with 10 mM-working solution in serum-free medium for 30min at
37 C in the dark environment. Fluorescence microscope was used
to obtain images which then were analyzed by ImageJ software.
2.4. Intracellular Fe2þ detection
FerroOrange(Dojindo,Japan) was used to evaluate the level of
intracellular Fe2þ according to the manufacturer’s protocol. cells
were incubated with 1 mM-working solution for 30min at 37 C in
the dark environment. Images were acquired by using fluorescence
microscope and then analyzed by using ImageJ software.
2.5. Animals study
8-week C57BL/6J male mice purchased from Comparative
Medicine Center of Yangzhou University were retained with unrestricted access to sterilized diet and water at standard bio-clean
laboratory settings (Experimental Animal Center of College of
Veterinary Medicine of Yangzhou University). All animal procedures were approved by Animal Care Committee of the Yangzhou
University and conformed to the Guide for the care and use of
laboratory animals(SYXK-2017-0044). Animals were randomly
divided into four groups(n ¼ 6e8 mice per group): control group or
ISO group: injected with saline or ISO(5 mg/kg) subcutaneously for
14 days and, meanwhile, received vehicle saline via gavage for 14
days respectively; ATV(Pfizer,USA) group or ISO þ ATV group:
injected with saline or 5 mg/kg ISO(Sigma,USA) subcutaneously for
14 days and, meanwhile, received 20 mg/kg ATV via gavage for 14
days respectively. During the whole experiment, all mice were kept
at room temperature with an automatic system of 12/12 h light/
dark cycle. After the final gavage intervention and echocardiography, mice were sacrificed and then parts of heart tissues were snapfrozen in liquid nitrogen and stored at 80 C for subsequent
analysis.
2.6. Echocardiography
Continuous-flow 1.5% isoflurane(MERCK,USA) was used to
anesthetize to experimental mice. An experienced sonographer(-
Animal Core Facility of Nanjing Medical University) who was blinded to study design used VEVO 3100 high-resolution in vivo
imaging system (Visual Sonics,Canada) and 35 MHz linear transducer and ultrasound probe to perform two-dimensionally guided
M-mode in the parasternal short-axis view for evaluating left
ventricular(LV) dimensions and function. All parameters were done
D. Ning, X. Yang, T. Wang et al. Biochemical and Biophysical Research
for three cycles on average. Tissue records and analysis were performed offline by an experienced expert who was blind to our study
design.
2.7. Histological analysis
After mice were sacrificed, parts of heart samples were fixed in
4% paraformaldehyde at room temperature for 24 h and then
embedded in paraffin according to routine procedure. 6 mm-thick
sections were prepared and then stained with HaematoxylinEosin(HE), Masson’s Trichrome and Prussian/Diaminobenzidine
stain following the procedure stain under light microscope for
evaluating the cardiac morphological changes and accumulation of
iron. The cardiomyocyte cross-sectional area, fibrotic area and
content of iron were measured and calculated by using ImageJ
software.
2.8. Assessment of in-vivo biochemical parameters
The amount of MDA and GSH in the hearts was measured using
commercial MDA and GSH assay kits according to the manufacturer’s protocols(Beyotime,China). Briefly,10.0% heart homogenates
were prepared and then centrifuged(12,000g, 10min, 4 C). The
supernatants were mixed with thiobarbituric acid solution reconstituted in glacial acetic acid and then incubated(15min,
95 C).Then, the supernatants were transferred into a 96-well
microplate for evaluating changes in levels of MDA(the absorbance at OD 532 nm). Approximate 30 mg of fresh left ventricle
tissue was homogenized in reagent M solution(protein removal)
and then centrifuged (12,000g, 10min, 4 C). After being diluted by
GSH auxiliary solution, the supernatants were incubated(5min,
room temperature) and then measured in a 96-well microplate for
evaluating changes in levels of GSSG(the absorbance at OD412nm).
The concentration of GSH was calculated by subtracting 2x GSSG
from the total glutathione concentration. Commercial BCA assay
kits(Solarbio, China) were used to determine protein
concentrations.
2.9. Western blotting
Western blotting was performed was performed to assess protein expression. Total protein was extracted in RIPA buffer(Solarbio,
China) containing protease inhibitor. Briefly, 25 mg of protein were
separated by 8e15% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis(80V and 30min during spacer gel; 160V and
30e40min during separation gel) and then to polyvinylidene
difluoride membrane(100V and 60e120min). The membranes
were blocked in 5% skim milk for 2e4 h at room temperature and
then incubated with primary antibodies(Table S1) at 4 C overnight
followed by routing washing procedure and then appropriate
horseradish peroxidase-labelled secondary antibodies at room
temperature for 1e2 h. The densitometric analysis was performed
using ImageJ software. GAPDH was used as internal control for
protein normalization.
2.10. Real-time PCR
Total RNA from intraarticular heart tissues were extracted using
TRIzol kit(Tiangen, Chian). According to standard instruction, cDNA
was synthesized using HiScript Q RT SuperMix kit(Vazyme, China).
Relative gene quantification by real-time PCR was performed by
using SYBR Fast qPCR Mix kit(Abclonal, China) in real time with the
Applied Biosystems(USA). Murine 18S ribosomal RNA was ampli-
fied as a reference standard. The primer sequences(Sangon,China)
were shown in Table S2.
2.11. Statistical analysis
The data were presented as the mean ± standard deviation by
using GraphPad Prism 8.0 software, and statistical analysis was
performed by using one-way univariate analysis of variance and
Mann-Whitney nonparametric test for skewed data that deviate
from normality(SPSS 25.0 software, Chicago, IL, USA). For the
comparisons among multiple groups, after evaluating the homogeneity of variance by the Brown-Forsythe test, One-way ANOVA
was used followed by post hoc analysis using the LSD(Fisher’s least
significance difference) post hoc test for normally distributed data
or Dunnett’s post hoc test for skewed data which was heterogeneity
of variance. Differences at P < 0.05 were considered to be statistically significant.
3. Result
3.1. Atorvastatin treatment inhibited isoproterenol-induced
ferroptosis in H9C2 cardiomyocytes
H9C2 cells were exposed to different concentrations of ISO for
evaluating cell viability and cell death by CCK-8 and Calcein/PI
staining. Exposure to 10, 20, 50 and 100 mM ISO for 24 or 48 h
resulted in a dose-dependent manner of cell viability(Fig. 1A). Then,
we selected 50 mM of ISO as an optimal concentration for the
subsequent experiments in this study. ATV(0.1,0.5,or1mM) coculture with ISO(50 mM) for 48 h or erastin(2.5 mM) for 24 h dosedependently reversed the decrease in cell viability(Fig. 1BeE).
Ferrostatin-1(Fer-1), a ferroptosis-specific inhibitor, was regarded
as a positive control. Fer-1 treatment(1,2.5,5 or 10 mM) for 48 h also
dose-dependently reduced the cytotoxicity of ISO in H9C2
cells(Fig. 1F). As the results reflected by PI staining, the increased
number of positive dead cells induced by ISO treatment was
markedly inhibited by administration of ATV or Fer-1(Fig. 1GeH).
Subsequently, we found that reactive oxygen species (ROS) content
increased in cells exposed to ISO, and both ATV and Fer-1 could
reverse this effect(Fig. 1IeJ). Similarly, in comparison with ISOtreated cells, ATV and Fer-1 reversed the increase in lipid
peroxidation(Fig. 1K-L). Consistently, the analysis of western blot
showed that cells exposed to ISO exhibited lower GPX4 and
SLC7A11 levels and higher NOX4 expression, which however were
reversed after treatment with ATV or Fer-1(Fig. 1MeN). These
findings indicate that inhibition of ferroptosis by ATV alleviates
ISO-induced H9C2 cell injury.
3.2. Atorvastatin treatment inhibited isoproterenol-induced
ferritinophagy in H9C2 cardiomyocytes
Previously, nuclear receptor coactivator 4(NCOA4)-mediated
autophagic degradation of ferritin can induce cardiomyocyte death,
highlighting importance of activation of ferritinophagy in the
development of HF [12]. Therefore, we surmised that atorvastatin
ameliorated cell injury by mediating ferritinophagy. 10 mM of 3-
methyladenine (3-MA), as an inhibitor of autophagy, restored to
the level of cell viability of ATV group and improved cell survival in
the damaged cells(Fig. 2AeC). Given that Fe2þ-dependent lipid
peroxidation plays an important role of ferroptosis, we measured
the level of intracellular Fe2þ by using FerroOrange. ISO treatment
induced excessive content of Fe2þ than that of normal control cells,
while these changes were significantly ameliorated by administration of ATV or 3-MA(Fig. 2DeE). Meanwhile, ISO downregulated
the expression of FTH1 and P62 and upregulated the ratio of conversion from LC3BeI to LC3B-II and expression of NCOA4 and
Beclin1 in H9C2 cells(Fig. 2FeG). However, treatment with ATV or
3-MA reversed the changes in levels of these proteins(Fig. 2FeG).
D. Ning, X. Yang, T. Wang et al. Biochemical and Biophysical Research
Overall, these findings suggest that ATV improves cell ferroptosis in
ISO-challenged H9C2 cells through mediating NCOA4-mediacated
autophagic degradation of ferritin.
3.3. ATV treatment prevents ISO-induced cardiac dysfunction and
remodeling in mice
In order to observe the effects of atorvastatin treatment on
cardiac dysfunction, intermittent subcutaneous injection of ISO was
performed to induce cardiac injury in mice. As expected, compared
with mice in CON group, impaired left ventricular function as
shown as by marked decreases in LVEF%, LVFS% and E/A (Fig. 3AeC)
in ISO group as well as LV Mass, LVEDV, LVIDd, RWT (Figs. S1DeH).
However, treatment with atorvastatin at dose of 20 mg/kg for 14
days could significantly reverse these parameters, indicating that
atorvastatin protects against cardiac dysfunction and pathological
structural alterations caused by ISO.
ISO-induced enlargement of areas of cardiomyocytes along with
Fig. 1. Atorvastatin treatment inhibited isoproterenol-induced ferroptosis in H9C2 cardiomyocytes. The cell viability of H9C2 cells in different groups were analyzed by CCK-8
assay(A-F). Cells were coclutured with 50 mM ISO and ATV(1 mM) or Fer-1(2.5 mM) for 48h and analyzed by PI staining(G-H), ROS staining(I-J) and lipid bodipy staining(K-L)
a magnification of 200.(M N)Representative western blots for NOX4(67 kDa),GPX4(17 kDa) and SLC7A11(40 kDa). Statistical significance: #P < 0.05; ##P < 0.01 and ###P < 0.001
versus the control group; *P < 0.05; **P < 0.01 and ***P < 0.001 versus the ISO group.
D. Ning, X. Yang, T. Wang et al. Biochemical and Biophysical Research Communications 574 (2021) 39e47
42
disorder arrangement, cardiomyocyte necrosis, nuclear dissolution
and more inflammatory cell infiltration also could be effectively
inhibited by administration of atorvastatin as indicated by
morphology of heart tissue stained with H&E(Fig. 3EeF) and hypertrophic markers including ANP and BNP(Fig. 3GeH). To evaluate
the cardiac fibrosis in heart, Masson’s trichrome staining was performed. As shown in Fig(Fig. 3IeJ), there was a significant increase
in cardiac fibrosis in the ISO group in comparison with the CON
group. ISO þ ATV group had a significant reduction in fibrotic areas.
Consistently, the mRNA levels of fibrosis markers including COL I,
COL III, OPN, TGFb, SMAa, MMPs were highly induced by ISO, which
however were clearly reversed by atorvastatin
treatment(Fig. 3K).Western blotting analysis suggested that the
levels of OPN-N and the ratio of CL-OPN to Full-OPN were greatly
enhanced by ISO as well as fibrotic proteins. Obviously, atorvastatin
treatment significantly reversed these parameters in damaged
heart(Fig. 3LeO). These findings indicated that atorvastatin could
significantly suppress cardiac hypertrophy and fibrosis in ISOinduced cardiac injury.
3.4. ATV treatment inhibits ferritinophagy-related ferroptosis
signaling pathways in damaged hearts
As shown as in Fig. 4A-D, results using commercial kits pointed
to that ISO induced reduction of GSH content and ratio of GSH/GSSG
and production of MDA(Fig. 4AeD). Consistently, we also found
that compared with CON group, significantly increased levels of
cardiac Ptgs2 mRNA, one of ferroptosis marker, in ISO
group(Fig. 4E). WB analysis showed that the protein levels of both
GPX4 and SLC7A11 were downregulated, while the NOX4 protein
level was upregulated in damaged tissues in comparison to control
hearts(Fig. 4FeG). Nevertheless, after treatment with atorvastatin,
great increases in the GSH level and ratio of GSH/GSSG and marked
decrease in the MDA level in cardiac tissues could be
observed(Fig. 4AeD). As well, the changes in GPX4, SLC7A11, NOX4
at protein level and PTGS2 at mRNA level were significantly
reversed following administration of such drug in failing
heart(Fig. 4EeG). We also found the accumulation of iron in ISO
group reflected by an increase in the signals of Prussian blue
Fig. 2. Atorvastatin treatment inhibited isoproterenol-induced ferritinophagy in H9C2 cardiomyocytes. Cells were coclutured with 50 mM ISO and ATV(1 mM) or 3-MA(10 mM) for
48h and analyzed by CCK-8 assay(A), PI staining(BeC) and FerroOrange staining(D-E) at a magnification of 200. (F-G)Representative Western blots for
Beclin1(54 kDa),P62(62 kDa),LC3B(16e18 kDa),NCOA4(70 kDa) and FTH1(21 kDa). Statistical significance: #P < 0.05; ##P < 0.01 and ###P < 0.001 versus the control group; *P < 0.05;
**P < 0.01 and ***P < 0.001 versus the ISO group.
D. Ning, X. Yang, T. Wang et al. Biochemical and Biophysical Research
Fig. 3. ATV treatment prevents ISO-induced cardiac dysfunction and remodeling.(A) Representative echocardiograms from four groups. Parameters of echocardiography revealing
cardiac function as shown by changes in LVEF(B),LVFS(C), E/A(D).(E-F)Representative H&E staining of mouse hearts at a magnification of 400(scale ¼ 100 mm) and cross-sectional
area of cardiomyocytes. (GeH) ANP mRNA and BNP mRNA levels in the hearts. (IeJ) Representative photography of Masson’s trichrome staining at a magnification of 200
D. Ning, X. Yang, T. Wang et al. Biochemical and Biophysical Research Communications 574 (2021) 39e47
staining(Fig. 4HeI).Results from WB exhibited that the expression
of vital autophagic markers Beclin1 and LC3B-II was upregulated
and the protein levels of p62, and FTH1 were reduced by ISO
insult(Fig. 4JeK), indicating the activation of ferritinophagy in
failing heart. Consistently, decreases in Beclin1 and LC3B-II and
increases in P62 and FTH1 at protein level as well as marked
reduction of iron in failing hearts could be observed in ISO þ ATV
group(Fig. 4JeK), suggesting the beneficial of atorvastatin on ISOquantitative analysis of fibrotic areas calculated by positive area.(K) RT-PCR for profibrotic genes at mRNA level in the hearts including COLI,COLIII,TGFb,SMAa,OPN and MMPs.(L-M)
Representative western blots for COL I(130 kDa),COL III(139 kDa),TGFb(44 kDa),SMAa(42 kDa) in the heats. (NeO) Representative western blots for cleavage of OPN(Full-
66kda,Fragment-40kDa) in the hearts. Statistical significance: #P < 0.05; ##P < 0.01 and ###P < 0.001 versus the control group; *P < 0.05; **P < 0.01 and ***P < 0.001 versus
the ISO group. n ¼ 3e8/group.
Fig. 4. ATV treatment inhibits ferritinophagy-related ferroptosis signaling pathways. (AeD) The levels of GSH,GSSG, ratio of GSH/GSSG and MDA in heart homogenates (E) PTGS2
mRNA levels in the hearts.(F-G)Representative Western blots for NOX4(67 kDa),GPX4(17 kDa) and SLC7A11(40 kDa) in the hearts.(HeI) Representative photography of Prussian
staining at a magnification of 200 and quantitative analysis of area of iron deposition calculated by positive area.(JeK) Representative Western blots for
Beclin1(54 kDa),LC3B(16e18 kDa),P62(62 kDa) and FTH1(21 kDa)in the hearts. Statistical significance: #P < 0.05; ##P < 0.01 and ###P < 0.001 versus the control group; *P < 0.05;
**P < 0.01 and ***P < 0.001 versus the ISO group. n ¼ 3e8/group.
D. Ning, X. Yang, T. Wang et al. Biochemical and Biophysical Research Communications 574 (2021) 39e47
induced cardiac injury possibly through retarding the progression
of ferritinophagy.
4. Discussion
There is strong evidence supporting satisfactory protective role
of statin treatment in treating HF in different background [9,13].
Nevertheless, the beneficial effects of statin treatment on regulation of ferroptosis in failing heart remains unexplored. In the present study, we showed that ATV attenuated the heart function
injury and pathological changes of HF mice which were treated
with intermittent administration of ISO and exhibited more severe
cardiac dysfunction and fibrotic response referring to elegant work
presented by Ma et al. [14].We demonstrated that ATV improved
ISO-induced cardiac dysfunction by substantially inhibiting cardiac
remodeling, oxidative stress and iron accumulation both in vivo
and in vitro through suppressing ferritinophagy-related ferroptosis
signaling pathways.
Currently, ferroptosis is a newly reported form of regulated cell
death in recent years, which is characterized by the accumulation
of fatal lipid hydroperoxides caused by intracellular free ferrous
iron overload and has become an important hot spot for studying
the progression of HF. Ferritin comprising a total of 24 heavy (FTH1)
and light (FTL1) chain subunits has been identified a central role in
maintaining the iron balance through storing free cellular iron [4].
Owing to its ferroxidase activity to convert ferrous iron to ferric iron
through, FTH1 plays an important role in preventing the process of
undesirable Fenton reactions [15]. In heart, loss of FTH1 can
resulting in mild cardiac injury upon aging and increasing the
susceptibility to iron overload‒associated ferroptosis and cardiomyopathy by increasing oxidative stress, highlighting an essential
role of iron-storage protein ferritin in maintaining iron homeostasis
and cardiac function [16]. At the same time, autophagy(NCOA4)-
dependent degradation of ferritin which promotes ferroptosis by
increasing intracellular ferrous iron levels and then triggering fatal
lipid peroxidation is regarded as mainstream mechanism of ferroptosis in cardiomyocytes, suggesting that targeting for inhibition
of this process to control the balance of intracellular iron level
would become a novel therapeutic mechanism for HF [12,17].
Consistent with the previous research [10], our study observed
that the reduction of FTH1 protein level and increases in labile iron
pool and lipid peroxidation in damaged cardiomyocytes after ISO
attack were accompanied by up-regulation of Beclin1,LC3BII/LC3BI
and NCOA4 and down-regulation of P62, supporting the autophagic
degradation of FTH1. Using the specific inhibitor of ferroptosis (Fer-
1) or inhibitor of autophagy (3-MA) as positive control in vitro
study, we revealed the inhibitory effect of ATV on ferroptosis in ISOinduced cardiac injury as indicated the changes in those parameters
from analysis of our results possibly through mediating ferritinophagy and then regulating Fe2þ dependent lipid peroxidation.
In vivo, our study consistently testified that ATV restored ISOinduced changes in ferritinophagy-related signaling pathways in
falling heart. We considered this part as an important signaling
turning point and consequently, concomitant enhanced antioxidant defence of GPX4-GSH system and reduction in iron accumulation could be observed as well as the improved heart function and
pathological remodeling. At present, reports on the regulation of
autophagy by ATV are still not unified. ATV might exert protective
effects though re-activating the suppressed response of autophagy
[18,19], however, on the other hand, it can rebalance excessive
autophagy and its related biological responses to achieve pharmacological protection [20,21].Hence, further study aiming at how
this drug regulates NCOA4/FTH1 autophagic axis should be investigated in-depth, which might explain the findings we observed
that ATV inhibited ferroptosis in HF induced by ISO challenge.
In summary, this study demonstrated that atorvastatin treatment exhibited cardiac protective effects on cardiac function and
remodeling after cardiac injury caused by ISO attack though
inhibiting ferritinophagy-related ferroptosis, indicating a novel
insight toward uncovering the mechanisms of statins in treatment
of HF.
Declaration of competing interest
The authors declare that there is no conflict of interests
regarding the publication of this paper.
Acknowledgments
This work was supported by grants from the Top-notch Talents of
“Six-one Projects” of Jiangsu Commission of Health(LGY2019036),
Yangzhou social development project (YZ2018075 and YZ2020098)
and Yangzhou Medical Talent(ZDRC201845).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2021.08.017.
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