Protective effect of HDL on NADPH oxidase-derived super oxide anion mediates hypoxia-induced cardiomyocyte apoptosis
Protective effect of HDL on NADPH oxidase- derived super oxide anion mediates hypoxia- induced cardiomyocyte apoptosis
Su-Ying Wen 0 1 2
Shanmugam Tamilselvi 0 2
Chia-Yao Shen 0 2
Cecilia Hsuan Day 0 2
Li- Chin Chun 0 2
Li-Yi Cheng 0 2
Hsiu-Chung Ou 0 2
Ray-Jade Chen 0 2
Vijaya Padma Viswanadha 0 2
Wei-Wen Kuo 0 2
Chih-Yang Huang 0 2
0 Funding: This study is supported in part by Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence MOHW106-TDU-B- 212-113004. There was no additional external
1 Department of Dermatology, Taipei City Hospital , Renai Branch, Taipei, Taiwan , 2 Center for General Education, Mackay Junior College of Medicine , Nursing, and Management, Taipei, Taiwan , 3 Graduate Institute of Basic Medical Science, China Medical University , Taichung, Taiwan , 4 Department of Nursing, MeiHo University , Pingtung, Taiwan , 5 Department of Hospital and Health Care Administration, Chia Nan University of Pharmacy & Science, Tainan County, Taiwan, 6 Department of Biological Science and Technology, Asia University , Taichung, Taiwan , 7 Department of Surgery, School of Medicine, College of Medicine, Taipei Medical University , Taipei, Taiwan , 8 Department of Biotechnology, Bharathiar University , Coimbatore , India , 9 Department of Biological Science and Technology, China Medical University , Taichung, Taiwan , 10 Graduate Institute of Chinese Medical Science, China Medical University , Taichung , Taiwan
2 Editor: Partha Mukhopadhyay, National Institutes of Health , UNITED STATES
Cardiovascular diseases are the leading cause of death of death in Taiwan. Atherosclerosis can lead to serious problems, including heart attack, stroke, or even death. Coronary heart disease (CHD) occurs when plaque builds up in the coronary arteries to cause the ischemic heart disease which will enhance myocardial remodeling and also induce myocardial hypoxia. High density lipoprotein (HDL) has been proposed to have cardio-protective effects. Under hypoxic conditions (1%O2 for 24hr), in H9c2 cells, reactive oxygen species (ROS) is induced which leads to cardiomyocyte apoptosis and cardiac dysfunction. Therefore, the present study described the protective effect of HDL on hypoxia-induced cardiomyocyte damage. We investigated the NADPH oxidase-produced ROS-related signaling pathways and apoptosis in cardiomyocytes under hypoxia conditions. Results showed that the ROS mediated cardiac damage might occur via AT1 and PKC activation. Furthermore, hypoxia downregulated the survival protein (p-AKTser473) and anti-apoptotic protein (BCL2), whereas pro-apoptotic protein, Bax and caspase 3 were upregulated. These detrimental effects by
Data Availability Statement; All relevant data are within the paper
Competing interests: The authors have no
conflicts of interest.
ROS and apoptosis were prevented by HDL pretreatment. Our findings revealed the
underlying molecular mechanism by which HDL suppresses the hypoxia-induced cardiomyocyte
dysfunction. Further, we elucidated the role of HDL on preventing hypoxia induced
cardiomyocyte apoptosis is mediated through the inhibition of NADPH oxidase-derived ROS.
Atherosclerosis is one of the top ten reasons of death in Taiwan caused due to the
accumulation of fatty substances, cholesterol, cellular waste products, calcium and fibrin on the inner
coating of artery[
]. Arteries are the blood vessels which deliver blood from the heart
throughout the body. Formation of plaques in the arteries could cause chest pain on
exertion, and also reduces the supply of oxygen to all the parts of body leading to hypoxic
Under normoxic condition, the working heart produces an abundant supply of ATP
(>95%), mainly from fat oxidation. Under anaerobic conditions, mammalian heart cells
cannot generate enough energy to perform essential cellular functions; therefore, a continuous
supply of oxygen is essential to retain the function and viability of heart [
injury (I/R) is one of the major stresses of heart, leading to detrimental effects through the
activation of fetal cardiac genes and specific signal transduction pathways [
]. Gene expression is
altered by several mechanisms based on the availability of oxygen in the heart which includes
the regulation of gene transcription by hypoxia-inducible factor 1α (HIF-1α) [
mediates the transcription of many important genes, which can control vital cellular processes
like vascular remodeling, metabolism, apoptosis, control of ROS, vasomotor reactivity, and
Oxidative stress is caused by reactive oxygen species (ROS) which produces oxygen free
radicals, that contain unpaired electrons and are highly reactive and short-lived [
normoxic condition, ROS can be formed by different mechanisms which includes the
production through oxidative phosphorylation in the mitochondria as a byproduct of normal cellular
metabolism in heart [
In cardiomyocytes, hypoxia has reported to induce various harmful effects which
include cell proliferation, cell hypertrophy and cell death. As a response to hypoxia,
cardiomyocytes express a number of genes, that induced by a variety of signaling cascades
High density lipoprotein (HDL) is a circulating-complex of lipids, bioactive particles,
containing multiple acute phase response proteins, protease inhibitors, and complement
regulatory proteins. Many reports have documented that HDL-cholesterol levels are
inversely associated with the risk of cardiovascular diseases [
]. The effect of HDL
could be influenced by abundance of several bioactive proteins and lipids, and could exert
anti-inflammatory, anti-oxidative, anti-coagulative and other atheroprotective functions
Therefore, the aim of this study was to explore the underlying protective mechanisms of
HDL against hypoxia-induced oxidative stress in cardiomyocytes. We investigated the ROS
mediated activation and subsequent inflammatory and apoptotic signaling pathways.
Materials and methods
H9c2 cell lines were obtained from American Type Culture Collection (ATCC), cultured in
Dulbecco's modified essential medium (DMEM) supplemented with 10% Cosmic Calf serum
(CCS), 2mM glutamine, 100units/ml penicillin, 100μg/ml streptomycin, and 1mM pyruvate in
humidified air (5% CO2) at 37ÊC. During the treatment, cells were pretreated with HDL (25,
50 and 100μg/ ml) for 2 hours and then incubated in hypoxic chamber (1% O2 for 24 hours).
The specificity of the inhibit ROS and mitochondria complex I inhibitor by adding N-acetly
cysteine (NAC) (500μM).
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Reactive oxygen species and mitochondrial superoxide production
Intracellular generation of ROS was monitored by flow cytometry using peroxide-sensitive
fluorescent probe 20, 70-dichlorofluorescein diacetate (DCFH-DA used as a Molecular Probes).
The fluorescent dichlorofluorescein (DCF) formed by oxidation of DCFH was quantified by
flow cytometry. Furthermore, mitochondrial superoxide (O2radical dot−) was evaluated by
MitoSOX Red mitochondrial superoxide indicator (Invitrogen, Eugene, OR). Cells were
harvested after treatment. Cells were resuspended with MitoSOX (2 μM) and incubated at 37ÊC
for 10±15 min. Samples were analyzed by a BD FACSCantoM II flow cytometer (BectonÐ
Dickinson, Franklin Lakes, NJ) to detect the mean mitochondrial ROS production.
H9c2 cells were washed once with PBS and harvested, then cell suspension was spun down,
and lysed in RIPA buffer (HEPES 20mM, MgCl2 1.5mM, EDTA 2mM, EGTA 5mM,
dithiothreitol 0.1mM, phenylmethylsulfonyl fluoride 0.1mM, pH 7.5) for 4hr at 4ÊC. Total cell lysate
was spun down 12,000 rpm for 20 min at 4ÊC and the supernatant was collected in new
eppendorf tube. Total protein was estimated by Bradford's method. Proteins (30 μg) were separated
by electrophoresis on SDS-polyacrylamide gel. After the protein had been transferred to
polyvinylidene difluoride (PVDF) membrane, the blots were incubated with blocking buffer (1X
PBS and 5% nonfat dry milk) for 1 hour at room temperature and then probed with primary
antibodies (1:1000 dilutions) overnight at 4ÊC, followed by incubation with horseradish
peroxidase-conjugated secondary antibody (1:5000) for 1 hour. To control equal loading of total
protein in all lanes, blots were stained with mouse anti-β-actin antibody at a 1:50000 dilutions.
Antibodies AT 1 (sc-1173), P47 (sc-14015), p-PKCδ (sc-11776), Bcl 2 (sc-7382), Bax (sc-526),
p-P38 (sc-7973), p-ERK (sc-7383), p-JNK (sc-6254), caspase 3 (sc-7148) and β-actin
(sc47778) and their respective secondary antibodies were purchased from Santa Cruz
Biotechnology, CA. Antibodies gp41 (ab 31092) and Rac 1 (ab-33186) were purchased from ab cam, UK
and pAkt (cs# 92755) was purchased from cell signaling Technology, Inc, USA. The bound
immunoproteins were detected by an ECL kit.
DAPI staining and TUNEL assay
After treatment H9c2 cells were grown on 6 mm plates. Then the cells were washed with PBS
and were fixed with 4% paraformaldehyde solution for 30 min at room temperature. After a
wash with PBS, cells were treated with permeation solution (0.1% Triton X-100 in 0.1%
sodium citrate) for 2 min at 4ÊC. Following wash with PBS, samples were first incubated with
TUNEL reagent containing terminal deoxynucleotidyl transferase and fluorescent
isothiocyanate-dUTP. The cells were also stained with 1μg/ml DAPI for 30 min to detect cell nucleus by
UV light microscopic observations (blue). Samples were analyzed in a drop of PBS under a
fluorescence and UV light microscope, respectively. Apoptotic cells were calculated by
Neonatal cardiomyocyte culture
Neonatal cardiomyocytes were isolated and cultured using the commercial Neonatal
Cardiomyocyte Isolation System Kit (Cellutron Life Technologies, Baltimore, MD, USA) according
to manufacturer's directions. Briefly, hearts were quickly removed from one- to two-days-old
Sprague-Dawley decapitated rats, the ventricles were pooled, and the ventricular cells were
dispersed by digestion solution at 37ÊC. Ventricular cardiomyocytes were isolated and cultured
in DMEM containing 10% fetal bovine serum, 100μg/ml penicillin, 100μg/ml streptomycin,
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and 2 mM glutamine. Cells were incubated in serum-free essential medium overnight before
treatment with indicated agents. All experimental procedures were performed according to
the NIH guide for the care and use of laboratory animals. All protocols were approved by the
Institutional Animal Care and Use Committee of China Medical University, Taichung,
Statistical differences were assessed by one way-ANOVA or t-test. P < 0.05 was considered
statistically significant. Data were expressed as the mean ± SEM.
Sustained incubation of H9c2 cells under hypoxia conditions increases reactive oxygen species (ROS) production and decreases the expression of antioxidant enzyme
Cardiovascular injury, one of the most common complications of hypoxia, is linked to the
elevated ROS levels, which subsequently induce cell apoptosis. Therefore, we examined the
cellular ROS levels in cardiomyoblast H9c2 cells incubated in sustained hypoxia conditions. The
effects of hypoxia on the expression of gp91phox, p47phox and Rac-1 were observed by
western blotting in H9c2 cells. We found that the protein levels of NADPH oxidases were increased
in H9c2 cells exposed to hypoxia for 0-24h (Fig 1A).
As shown in Fig 1B, after treatment with hypoxia for different time periods, the protein
expression levels of AT1, p-PKC-α and p-PKC -δ were increased in H9c2 cells. Intracellular
ROS level has been regulated by the balance between ROS generation and the activity of
antioxidant enzymes such as catalase or SOD. Thus, the involved ROS might inactivate
anti-oxidative enzymes that additionally increase the imbalance in favor of oxidative stress. Therefore,
we investigated the expression of its isoforms in H9c2 cells in response to hypoxia. Our results
showed that the antioxidant enzymes SOD2 decreased in H9c2 cells treated with hypoxia
Fig 1. Hypoxia-increased oxidative stress in H9c2 cells. (A) The level of NADPH oxidase (Nox2-gp91 phox, p47phox, and Rac1) protein
expressions. (B) The level of AT1 receptor, p-PKCα and p-PKCδ, and antioxidant enzyme SOD2 protein expressions. `Image J' software
was used to calculate the expression level of protein.
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Hypoxia-induced apoptosis in cardiomyocyte
We examined the survival protein (p-Aktser473), Bcl2, and pro-apoptotic protein (Bax) in
different time of hypoxia condition. The results showed that hypoxia downregulated the
survival protein (Fig 2A) whereas pro-apoptotic protein was upregulated. To analyze
hypoxia induced cell apoptosis in cardiac myocyte, TUNEL assay was performed. After 24h
incubation in hypoxic chamber, we observed a significant increase in apoptotic bodies
Roles of MAPK family proteins in hypoxia induced H9c2 cell apoptosis
To investigate whether MAPK family proteins are involved in the hypoxia-induced H9c2
apoptosis, we examined the expression levels of MAPK family proteins by Western blot. Our
results showed that the expression of p-ERK, and p-P38 were increased after treatment with
hypoxia for 0-24h, whereas pJNK expression was not increased notably (Fig 2B).
Fig 2. Effect of hypoxia-induced apoptosis and activation of MAPK family proteins, and survival proteins in H9c2 cells. (A) Survival
proteins for 0-24h. (B) MAPK family (p-ERK, p-JNK, p-P38). (C)Fluorescence images show the cells stained with 4,
6-diamidino2-phenylindole (DAPI) and stained using terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL) assay. (D)
Graphical representation of TUNEL assay. Data showed the means ± SEM of 3 independent analyses. *P<0.05 vs. hypoxia treatment.
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Effects of HDL on hypoxia-induced NADPH oxidase complex and antioxidant enzyme
The effects of HDL (25, 50 and100μg/ml) on expression levels of p-PKC- α, gp91phox and
Rac-1 were examined by Western blotting in H9c2 cells. We found that hypoxia treatment of
H9c2 cells resulted in the increase of NADPH oxidase activity. However, pretreatment with
HDL (25±100μg/ml) led to a dose-dependent reduction in gp91phox and Rac-1 protein
expression (Fig 3A). As shown in Fig 3A, incubation of H9c2 cells with hypoxia resulted in
significant phosphorylation of protein kinase C (p-PKC-α) and HDL attenuated the expression
of p-PKC-α. We further investigated the effects of HDL on generation of ROS, a potential
factor related to hypoxia-induced H9c2 cells injury, using hydroxyl radical sensitive probe
2',7'dichlorofluorescein acetoxymethyl ester (DCF-AM), superoxide sensitive probe
dihydroethidium (DHE) and MitoSOX™ Red mitochondrial superoxide indicator. The production of ROS
and superoxide generations were examined by flow cytometry. Results revealed a significant
Fig 3. Inhibitory effect of HDL on hypoxia induced ROS production in H9c2 cells. (A) Representative western blots. `Image J' software
was used to calculate the expression level of protein (B). ROS was examined by DCF-AM (10μM), DHE (5μM), and MitoSOX™ (5μM).
Fluorescence intensity of cells was measured by flow cytometry. (C) Neonatal cardiomyocytes were treated with HDL (25±100μg/ml) for 2h,
or NAC (500 μM), roteone (5 μM), and then incubated with 1%hypoxia for an additional 24h, and followed by 1h incubation with DHE (10μM).
Fluorescence intensity of cells was measured by immunofluorescence microscopy (Olympus CKK53). Data showed the means ± SEM of 3
independent analyses.#P<0.05 comparison of control and hypoxia groups, *P<0.05 HDL/ NAC treated groups vs. hypoxia treatment.
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reduction of ROS in H9c2 cells pretreated with HDL (25±100μg/ml) in a dose-dependent
manner (Fig 3B).
Sustain exposure of HDL can reduce NADPH oxidase activity in neonatal cardiomyocytes treated with hypoxia
To explore the effects of HDL on NADPH oxidase, a major source of ROS in H9c2 cells, we
measured the formation of superoxide by using superoxide sensitive probe dihydroethidium
(DHE). As shown in Fig 3C, neonatal cardiomyocytes pretreated with HDL (25±100μg/ml) for
2h before exposure to hypoxia for 24h, we used superoxide sensitive probe dihydroethidium
(DHE) to confirm by microscopic observation. The results showed that hypoxia induced
superoxide generation was reduced in HDL (25±100μg/ml) pretreated cells, similar results
were observed by the treatments with anti-oxidant (NAC, 500 μM) and mitochondrial
superoxide inhibitor (Rotenone, 5 μM).
Effect of HDL on hypoxia-induced MAPK protein phosphorylation
Incubation of H9c2 cells with hypoxia resulted in significant phosphorylation of p38, and
ERK, but less change in the expression of pJNK. Therefore, we examined whether HDL could
inhibit MAPK proteins (p-p38 and pERK) activation. H9c2 cells were treated with HDL (25±
100μg/ml) for 2h before exposure to hypoxia for 24h, our data showed HDL can downregulate
the phosphorylation of p38 only (Fig 4A). Whereas significant reduction was not observed in
the expression of pERK (data not shown). Thus, HDL pretreatment could suppress hypoxia
mediated apoptosis by downregulating p38 MAPK.
HDL attenuated the apoptotic effects of hypoxia by regulating Bcl2 family protein, and the activation of casepase3
Immunoblotting studies demonstrated that hypoxia downregulated the anti-apoptotic and
survival proteins (Bcl2, p-Aktser473), also upregulated the pro-apoptotic protein (Bax), whereas
HDL pretreatment effectively reverted these effects. Since activated caspase 3 is a key factor in
the execution of mitochondrial apoptosis [
], we subsequently determined the expression of
caspase 3, both pro-form and active-form and Bax by immunoblotting (Fig 4A). The data
showed that active caspase 3 was significantly increased in cells that had been treated with
hypoxia whereas suppressed in cells pretreated with HDL. Furthermore, at cellular level, the
antiapoptotic effects of HDL on hypoxia-induced cell death was evaluated by TUNEL and DAPI
staining assays. As shown in Fig 4B & 4C, hypoxia treated cells showed a typical features of
apoptosis, including the formation of condensed nuclei. However, apoptosis was not observed
in the HDL-pretreated H9c2 cells. As described above, both results of cell viability assay
and phenotypic observation of apoptosis under microscopy suggested that HDL is a potent
inhibitor of hypoxia-induced apoptosis in cultured H9c2 cells. NAC (500μM) could suppress
TUNEL-positive cell, induced by hypoxia.
Hypoxic condition is one of the common causes of cell damage, which is implicated in many
pathologic conditions which include stroke, myocardial infarction (MI), diabetes mellitus and
multiple organ failure [
]. Plaques formation in the heart's arteries is known to lead to
myocardial remodeling and myocardial hypoxia . Angiotensin II (Ang II) was reported to
mediate hypoxia-induced caspase-3 activation through ERK pathway in primary cortical
neuronal cultures [
]. However, in the present study, we found that hypoxia induced apoptosis
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Fig 4. Inhibitory effect of HDL on hypoxia induced MAPK and apoptotic protein expression in H9c2 cells. (A). Representative
western blots. `Image J' software was used to calculate the expression level of protein. (B) Fluorescence images show the cells stained with
4,6-diamidino-2-phenylindole (DAPI) (upper panel) and stained using terminal deoxynucleotidyl transferase dUTP-mediated nick-end
labeling (TUNEL) assay (bottom panel), and photomicrographs were from immunofluorescence microscopy (Olympus CKK53). C).
Graphical representation of TUNEL assay. #P<0.05 comparison of control and hypoxia groups, *P<0.05 HDL treated groups vs. hypoxia
was successfully reverted by HDL pretreatment in H9c2 cells. In human umbilical vein
endothelial cells, the mitochondrial pathway of apoptosis was shown to be blocked by HDL [
Tumor necrosis factor-α-induced apoptosis was reported to be inhibited by HDL in
endothelial cells [
Our study was extended to explore the mechanisms of hypoxia-induced cell apoptosis in
heart, focusing on the NADPH oxidase-generated ROS induced signaling and also
concentrated on the therapeutic potential of inhibiting NADPH oxidase in hypoxia-exposed cardiac
cells. The effects of ang II are mediated by two distinct receptors, referred to as the Ang II
type-1 (AT1) and type-2 (AT2) receptor subtypes. AT1 receptor is dependent on the cell and
organ type, binding of Ang II with AT1 receptor leads to cellular contraction, hypertrophy,
proliferation, and/or apoptosis and importantly, the generation and release of ROS [
activation of AT1 receptor, p47phox becomes phosphorylated and translocated to the
membrane, where p47phox forms a complex with gp91phox and p22phox and the assembly
performs as an active oxidase [
]. The effect of hypoxia resulted in increased expression of AT1
and NADPH oxidase proteins (Fig 1). Various protein kinases have been identified to be
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involved in the regulation of NADPH oxidase activity [
], among them the protein kinase
C (PKC) family was found to play a critical role [30±32]. Our results proved that
phosphorylation of PKC α and δ was increased under hypoxia condition. Furthermore, hypoxia activated
NADPH oxidase via AT1 receptor was also evidenced. SOD is known to protect cells against
superoxide-mediated cytotoxicity by rapidly dismutating O2± to H2O2. Treatment of hypoxia
decreased Cu/Zn-SOD and Mn-SOD protein expression level (Fig 1). Whereas HDL
pretreated cells resulted in decreased ROS generation, and subsequently attenuated
hypoxiaimpaired superoxide dismutase (SOD) activity and suppressed ROS-induced intracellular
signaling pathways (Fig 3). HDLs from healthy persons have been found to activate endothelial
nitric oxide (NO) generation which decreases the production of endothelial ROS . Many
studies have reported that MAPK signaling pathway is stimulated by hypoxia. [34±38]. Here
we showed that hypoxia increased the expression of p-P38 and p-ERK, but not p-JNK
Fig 5. Schematic representation of the effect of hypoxia and the preventive role of HDL on H9c2 cells. Hypoxia induces
ROS mediated apoptosis through the overexpression of AT1 and PKC, which is prevented by the treatment of HDL.
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significantly and HDL pretreatment could protect these effects caused by hypoxia only by
downregulating p-P38 MAPK.
Our in vitro studies revealed that HDL could prevent from hypoxia-induced cardiomyocyte
apoptosis and oxidative dysfunction via modulating mitochondria dependent pathway (Fig 5).
Further, the findings provided an insight of underlying molecular mechanism, which leads to
the progression of cardiovascular disease. In H9c2 cells, it was confirmed that hypoxia
triggered AT1 receptor and NADPH oxidase activity. Future direction of the present study would
be whether the protective effect of HDL against hypoxia-induced apoptosis is mediated by the
downregulation of AT1receptor or the involvement of any other receptors and also the specific
component of HDL such as Apo A-1, SR-B1 is responsible for the activity of HDL. The
findings would pave the way to the discovery of accurate drugs for cardiovascular diseases.
Conceptualization: CYH WWK.
Formal analysis: SYW ST.
Funding acquisition: CYH.
Investigation: SYW HCO CYS.
Methodology: SYW VPV RJC.
Project administration: CYH.
Supervision: CYH WWK.
Validation: LCC CHD LYC.
Writing ± original draft: ST VPV.
Writing ± review & editing: ST SYW WWK.
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