Cardioprotective Effects of Quercetin in Cardiomyocyte under Ischemia/Reperfusion Injury

Quercetin, a polyphenolic compound existing in many vegetables, fruits, has antiinflammatory, antiproliferation, and antioxidant effect on mammalian cells. Quercetin was evaluated for protecting cardiomyocytes from ischemia/reperfusion injury, but its protective mechanism remains unclear in the current study. The cardioprotective effects of quercetin are achieved by reducing the activity of Src kinase, signal transducer and activator of transcription 3 (STAT3), caspase 9, Bax, intracellular reactive oxygen species production, and inflammatory factor and inducible MnSOD expression. Fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can reveal the differentially expressed proteins of H9C2 cells treated with H2O2 or quercetin. Although 17 identified proteins were altered in H2O2-induced cells, these proteins such as alpha-soluble NSF attachment protein (α-SNAP), Ena/VASP-like protein (Evl), and isopentenyl-diphosphate delta-isomerase 1 (Idi-1) were reverted by pretreatment with quercetin, which correlates with kinase activation, DNA repair, lipid, and protein metabolism. Quercetin dephosphorylates Src kinase in H2O2-induced H9C2 cells and likely blocks the H2O2-induced inflammatory response through STAT3 kinase modulation. This probably contributes to prevent ischemia/reperfusion injury in cardiomyocytes.


Introduction
Because of their high incidence and mortality rate, cardiovascular diseases have recently become a primary health concern worldwide. Myocardial ischemia/reperfusion injury, which causes excess reactive oxygen species (ROS) production that can lead to cardiac hypertrophy or dysfunction, is the most acute cardiovascular disease [1,2]. In 2010, Chou et al. showed that ROS may affect intercellular connections and cytoskeleton resulting in cell detachment, morphology change, or death. Src kinase also plays a key role in ROS-induced phosphorylation and cell damage in cardiomyocytes [2].
The ROS in this study includes hydrogen peroxide (H 2 O 2 ), singlet oxygen (O • ), superoxide (O 2− ), and the hydroxyl radical (OH • ). Among these ROS species, H 2 O 2 is the most stable and the most abundant in human cells. Although the optimal amount of ROS plays an important role in signal transduction, excess ROS causes cell damage [3]. H 2 O 2 regulates signal transduction-related proteins by phosphorylating or modifying the active sites of proteins but also inhibits phosphatase activity [4].
Quercetin, a type of polyphenolic compound, has antiinflammatory, antiproliferation, anti-histamine, and antioxidant effects. Quercetin exists in many types of vegetables and fruits. Several reports have shown that quercetin has protective effects on different types of cells, including myocytes, testis, renal cells, and liver cells in ischemia/reperfusion injury [5]. A study conducted in 1992 showed that quercetin reduces the oxidative stress caused by ischemia/reperfusion in cardiomyocytes by inhibiting the xanthine dehydrogenase/xanthine oxidase system [6]. Several reports have also indicated that quercetin and isorhamnetin can scavenge ROS and inhibit the activation of ERK or MAP kinase in ROS-induced cardiomyopathy [7,8]. In cancer therapy, combining quercetin with doxorubicin augmented the effects of doxorubicin in highly invasive breast cancer cells [9] and can protect cardiomyocytes from doxorubicin-induced toxicity by chelating iron, inducing antioxidant activity, and inhibiting carbonyl reductase [10]. Although quercetin has been reported to play a role in protecting myocardial cells from ischemia/reperfusion injury, its protective mechanism remains unclear in current knowledge. Ischemia/reperfusion injury in cardiomyocytes is the result of myocardial inflammation [11]. Muthian and Bright showed that quercetin blocks the IL-12-induced inflammatory response through a signal transducer and activator of transcription 3 (STAT3) activation in T lymphocytes [12]. However, previous research failed to show a direct relationship between quercetin and STAT3-activated inflammation in cardiomyocytes. STAT3 is a transcription factor that plays an important role in numerous cytokine signaling transductions including cell survival, proliferation, cell cycle progression, and cell growth. STAT3 has two important phosphorylated and activate sites: Tyr705 and Ser727. STAT3 activation was phosphorylated at tyrosine 705 induced by various factors, including cardiotrophin-1, IL-6, tumor necrosis factor-alpha (TNF-), and interferon-gamma (IFN-) [5,13]. pY705-STAT3 is also essential for the dimerization of STAT3 and the translocation of STAT3 into the nucleus. In addition, STAT3 has been observed to be phosphorylated at serine 727 under oxidative stress to enhance the transcription activity of STAT3 in previous cerebral ischemia preconditioning study [14]. Moreover, The JAK2/STAT3 signaling pathways participate in an oxidative stress-induced immune response [3,15].
Two-dimensional gel electrophoresis (2-DE) is a common tool for analyzing thousands of proteins in different biological samples and is complementary to LC-MS results. However, varying quantification between gels remains the primary challenge in 2-DE. Therefore, 2D-DIGE reduces the variation between gels and gels, which codetected the sample abundances on the same gel by using differential fluorescent labeling [2].
This study investigates the potential protective role of quercetin in H 2 O 2 -induced H9C2 cell injury. We focus on the correlation between quercetin in cardiomyocytes and the cardioprotective role of Src kinase inhibition and inflammatory response of STAT3 using 2D-DIGE combined with MALDI-TOF MS and immunoblotting.  phopho-FAK, Bax, caspase9, Bcl-2, GAPDH, and STIP1 were purchased from Genetex (Hsinchu, Taiwan). Horseradish peroxidase and fluorescence conjugated secondary antibodies against mouse and rabbit were purchased from Sigma-Aldrich (St. Louis, USA). Annexin V-FITC and a propidium iodide (PI) labeling kit were purchased from Invitrogen. We purchased 2,7-dichlorofluorescein diacetate (DCFH-DA) from Molecular Probes. The chemicals and reagents of 2D-DIGE were purchased from GE Healthcare (Uppsala, Sweden).

Cell
Lines, Cell Culture, and Cell Treatment. The H9C2 rat cardiomyocyte cell line purchased from American Type Culture Collection (Manassas, VA, USA) was chosen as a cellular model for this study as this cell line retains the characteristics of isolated primary cardiomyocytes and has been used as a model in ischemia and reperfusion studies [2]. The H9C2 was cultured in Dulbecco's modified Eagle medium (DMEM) (Invitrogen) containing 10% fetal bovine serum (FBS) at 37 ∘ C. Cells cultured in normal growth medium were treated with various concentrations of H 2 O 2 for 20 min. H9C2 cells were pretreated with quercetin (Sigma) for 1 h followed by treatment with H 2 O 2 for 20 min.

Immunostaining and Fluorescence
Microscopy. For completing immunofluorescence staining, H9C2 cells grown on coverslips (12 mm) were treated with 5 mM H 2 O 2 for 20 min alone, 1 mM quercetin for 1 h prior to treatment with 5 mM H 2 O 2 for 20 min, or left untreated [2]. The cell fixing, immunostaining, and fluorescence image analysis methods in this study were similar to our previous paper [2].

Wound
Healing Assay. H9C2 cells (10 5 cells/well) were incubated in 24-well plate at 37 ∘ C for 12 h and then scraped with a 10 L tip and treated with H 2 O 2 for 20 min, pretreated with quercetin, or left untreated. H9C2 cells were incubated with medium containing 10% FBS, and a fluorescence microscope captured images at different incubation times (0 h, 6 h, 24 h, 30 h, and 42 h).

Adhesion
Assays. H9C2 cells (8 × 10 4 cells/well) were incubated in a 3 cm dish containing DMEM containing 10% FBS and treated with 1 mM quercetin for 1 h followed by 5 mM H 2 O 2 for 20 min. After treatment, H9C2 cells were incubated with serum-free medium for 1 h and 4 h and then were counted. The cell culture environment and cell counting were similar to our previous study [2]. All conditions have been performed in duplicate-independent experiments.    an important signal mediator, induces large scale of protein phosphorylation and protein modification resulting in cellular physiology alteration including cell morphology, adhesion, and viability. Because heart ischemia/reperfusion injury stimulates H 2 O 2 production, H9C2 cells were treated with varying H 2 O 2 doses to find the optimal phosphotyrosine response. The optimal response represents the maximal ratio of phosphotyrosine intensity to H 2 O 2 concentration by immunoblotting (Figure 1(a)). Results show that 5 mM H 2 O 2 treatment led to a robust phosphotyrosine response, but the phosphotyrosine response decreased in 10 mM H 2 O 2 cells. Quercetin may also play an important role in oxidative stressdamaged cells, and phosphotyrosine signals were detected with a range of quercetin followed by treatment with 5 mM H 2 O 2 (Figure 1(b)). These results reveal that H9C2 pretreated with 1 mM quercetin and subsequently treated with 5 mM H 2 O 2 induced a lesser phosphotyrosine response than that of H9C2 cells treated with 5 mM H 2 O 2 . Subsequent experiments were carried out based on these H 2 O 2 and quercetin treatment concentrations.

Quercetin Inhibits Hydrogen Peroxide-Induced Changes in
Cell Morphology and Loss of Cell Adhesion. H 2 O 2 stimulates the activation of Src kinase that regulates cytoskeleton, cell adhesion, and cell motility. Previous report indicated that PP1, a Src kinase inhibitor, inhibits H 2 O 2 -induced Src kinase activation [2]. In this study, quercetin pretreatment reduces the tyrosine phosphorylation of Src kinase and FAK in H 2 O 2treated H9C2 cells (Figure 2(a)). The immunostained images represent the H9C2 proteins against specific antibodies, including cytoskeleton protein (F-actin and -tubulin) and cell-cell interaction protein (ZO-2). Oxidative damage affects cytoskeleton proteins and ZO-2, effectively altering cell morphology (Figure 2(b)). Quercetin pretreatment improved changes in ROS-induced cell morphology.
In the wound healing assay, H9C2 cell images were captured at different time points (0 h, 6 h, 24 h, 30 h, and 42 h) using a microscope (Zeiss). Cells were untreated, H 2 O 2 treated, and quercetin pretreated followed by hydrogen peroxide treatment (Figure 2(c)). After incubation, the closure areas of H 2 O 2 -treated H9C2 cells were larger than those   of untreated and quercetin pretreatment followed by H 2 O 2 treatment. An adhesion assay was also performed to analyze the effects of quercetin on ROS-damaged cardiomyocytes. H9C2 cells untreated, treated with H 2 O 2 alone, or pretreated with quercetin were followed by treatment with H 2 O 2 . Cells were then incubated in a serum-free medium for 1 h or 4 h. The adherent cells were counted after incubation. Results show that H 2 O 2 -treated cells had reduced adhesive ability; yet, this could be significantly improved by pretreatment with quercetin ( Figure 2(d)). Thus, quercetin can stimulate cell migration and maintain cell adhesion in H 2 O 2 -damaged H9C2 cell.

Quercetin Reduces Hydrogen Peroxide-Induced H9C2
Cell Apoptosis. Excess ROS production from ischemia/ reperfusion-injured cardiomyocyte [18] alters redox homeostasis and induces cell apoptosis. During cell apoptosis, the asymmetric distribution of phospholipids of the plasma membrane gets lost and phosphatidylserine is translocated to the outer surface of the plasma membrane which has a high affinity to annexin V-FITC. PI can penetrate the cell nucleus when cells undergo apoptosis. Cell apoptosis was detected using FACS. The dot plots of annexin V and PI staining are analyzed using FACS, appearing in Figures 5(a), 5(b), and 5(c). The cell apoptosis rate increased from 5% to 12.5% upon H 2 O 2 treatment, whereas the cell apoptosis rate decreased to 5.5% after H9C2 cells were pretreated with quercetin before H 2 O 2 treatment. In addition, the PI staining signal of H 2 O 2 -treated H9C2 shifted forward, compared to that of untreated cells and cells pretreated with quercetin followed by H 2 O 2 ( Figure 5(d)). The levels of Bax, Caspase 9, and Bcl-2 were detected using immunoblotting for untreated H9C2 cells, treated H 2 O 2 , and pretreated quercetin followed by H 2 O 2 treatment. ROS increased the expression level of apoptosis factors caspase 9 and Bax and reduced antiapoptosis marker Bcl-2 expression ( Figure 5(e)). According to the data, quercetin can protect and stabilize the total chromosome of DNA in H9C2 cells from oxidative damage by inhibiting cell apoptosis and chromosome attrition.

2D-DIGE Analysis of Untreated and H 2 O 2 -Treated H9C2 Cells and Quercetin Pretreatment Followed by Treatment
Three types of cell lysates were analyzed using 2D-DIGE. The results of 2D-DIGE analysis and DeCyder processing identified 1535 proteins spots, and 44 proteins showed differential expression (≧1.5-fold or ≦ −1.5-fold; < 0.05) among these 3 conditions ( Figure 6).  Figure 7 shows the 2D-gel images, 3D images, and protein abundances from untreated, H 2 O 2 treated, and quercetin pretreated followed by H 2 O 2 cells. Figure 8 shows the functional distribution of identified proteins from 2D-DIGE results. Most of proteins identified using MALDI-TOF MS are related to the cytoskeleton (9%), redox regulation (9%), and protein degradation (14%), implying that quercetin can reverse ROS damage to the cytoskeleton and redox homeostasis in cardiomyocytes.

Verification by Immunoblotting and
Immunostaining. The levels of the alpha-soluble NSF attachment protein ( -SNAP) and cell division protein kinase 4 (CDK4) were examined by immunoblotting or immunostaining to validate the results of 2D-DIGE analysis. These results indicate that -SNAP and CDK4 were overexpressed in response to H 2 O 2 . However, quercetin suppressed ROS-induced -SNAP and CDK4 protein expression in H9C2 cells (Figures 9(a) and 9(b)). These data are consistent with 2D-DIGE results.

Discussions
Cardiovascular diseases have become a primary health concern worldwide in recent years. Ischemia/reperfusion injury in cardiomyocytes, which leads to excess ROS generation, is a particularly serious result of cardiovascular diseases. Many studies have focused on how to alleviate ischemia reperfusion-induced ROS in cardiomyocytes. For example, many plant molecules, including resveratrol, quercetin, sasanquasaponin, proanthocyanidin, safflower, and orientin, function as protectors in ischemia/reperfusion-damaged cardiomyocytes [19][20][21][22][23]. However, the role of quercetin in the ischemia/reperfusion injury of cardiomyocytes remains unclear.
According to previous reports, Src kinase regulates many cell signals, including cell adhesion, migration, proliferation, and apoptosis [24,25]. During oxidative stress, Src kinase induces cell death by inactivating PI-3 K, cell migration, and spreading [26]. PP1, a Src kinase inhibitor, can rescue ROS-damaged H9C2 cells by inhibiting cell apoptosis and enhancing cell adhesion/viability [2]. However, the inhibition of Src kinase activity with PP1 is generally unsuitable for mammalian cells. Alternatively, in our findings, H9C2 cells pretreated with quercetin for 1 h are protected against H 2 O 2induced apoptosis in this study. The role of quercetin in H 2 O 2 -treated cardiomyocytes is to inhibit inflammatory response and maintain cell physiology, including morphology, redox status, and metabolism, by regulating Src kinase, FAK, and STAT3.
The results of this study indicate that H 2 O 2 stimulates the tyrosine phosphorylation of Src kinase and FAK, which affect cell morphology and tight junction proteins, leading to cell detachment [27]. Quercetin, however, inhibits the tyrosine phosphorylation of Src kinase and FAK which maintain cellcell interaction and morphology. Many studies have shown that quercetin protects retina, testis, neuron, cerebral, and cardiovascular cells from ischemia/reperfusion injury [28][29][30][31]. This study further demonstrates that quercetin increases migration and survival in H 2 O 2 -treated cardiomyocytes ( Figure 2).
-SNAP is a component of the soluble N-ethylmaleimidesensitive fusion factor attachment protein receptors (SNAREs) complex required for vesicular transport between the endoplasmic reticulum and the Golgi apparatus. The major function of -SNAP is to recycle the SNARE complex. Several reports have shown that SNAREdependent trafficking is required for integrin signaling through a FAK/Src/PI3 K-dependent pathway [32], and the inhibition of SNARE-mediated exocytosis attenuates ischemia/reperfusion injury [33]. -SNAP may play a critical role in regulating Src kinase signaling and inducing ischemia/reperfusion injury in cardiomyocytes. This study shows that -SNAP was robust to overexpression (9.85-fold) in 5 mM H 2 O 2 -treated H9C2 cells. However, pretreatment with quercetin reduced H 2 O 2 -induced -SNAP expression. Quercetin inhibits ROS-induced -SNAP overexpression in cardiomyocytes, which could be effectively applied for protecting cardiomyocytes from oxidative stress ( Figure 10).
The major functions of the Ena/VASP-like (Evl) protein include the regulation of cytoskeletal dynamics and organization axon guidance, platelet aggregation, cell motility, and cell adhesion [34,35]. However, several studies have shown that the Evl protein has another function in homologous pairing and strand exchange through interaction with RAD51 and RAD51B [36]. Because H 2 O 2 targets DNA, oxidative stress causes base damage such as strand breaking in DNA. At this moment, the repaired mechanisms, including base excision repair (BER), transcription-coupled repair (TCR), mismatch repair (MMR), nonhomologous end-joining (NHEJ), translesion synthesis (TLS), global genome repair (GGR), and homologous recombination (HR), will been turned on [37]. ROS-treated cells exhibited DNA damage, stimulating homologous recombination. In this case, Evl expression increased in cardiomyocytes, but quercetin pretreatment reduced the expression of ROS-induced Evl ( Figure 10). This suggests that quercetin may stabilize the DNA structure of ROS-damaged cardiomyocytes.
Isopentenyl-diphosphate delta-isomerase 1, which is located in peroxisomes, catalyzes the isomerization of 1,3allylic rearrangement of the homoallylic substrate isopentenyl (IPP) to dimethylallyl diphosphate (DMAPP), which is a strong electrophile allylic isomer. DMAPP is also an important product in the synthesis of many lipophilic molecules such as sterols, ubiquinones, and terpenoids. Yochem et al. demonstrated that losing idi-1 gene is lethal in Caenorhabditis elegans, leading to accumulated and enlarged lysosomes and autophagosomes [38]. This study shows that ROS-treated block isopentenyl-diphosphate delta-isomerase 1 expression may induce cell death; however, quercetin pretreatment reversed isopentenyl-diphosphate delta-isomerase 1 expression in H9C2 cell ( Figure 10).
Elongation factor 1-alpha (EF-1 alpha) is a multifunctions protein that promotes peptide synthesis through GTPdependent binding of aminoacyl-tRNA to the A-site of ribosomes and binds to filamentous actin [39] and severs microtubules, leading to abnormal tetraploid cells and cell death [40]. In 1996, GaŁasiński demonstrated that quercetin prevents the peptide elongation by interacting with EF-1 alpha in plant [41]. The present data show that H 2 O 2 downregulates the expression of EF-1 alpha in H9C2 cells, whereas quercetin pretreatment reverses the expression of EF-1 alpha. Quercetin can prevent ROS-induced cytoskeleton damage and promote protein synthesis in cardiomyocytes ( Figure 10). Cellular antioxidant enzymes including superoxide dismutases (Mn-SOD and CuZn-SOD), catalase (CAT), peroxidases, and glutathione S-transferases regulate redox homeostasis in mammalian cells. Catalase and peroxidases scavenge H 2 O 2 or convert it to hydroxyl radicals. Superoxide dismutases convert superoxide anions (O 2− ) to H 2 O 2 . The observation of the oxidative state in this study demonstrates that ROS inhibits the MnSOD expression that leads to O 2− accumulation in cell. However, quercetin pretreatment not only reduces ROS production, but also prevents MnSOD expression in H 2 O 2 -treated H9C2 cells (Figure 3).
Inflammation contributes to the pathophysiology of cardiac ischemia/reperfusion injury. Myocardial ischemia and reperfusion, sepsis, viral myocarditis, and immune rejection induce the inflammatory response [11]. Cardiac ischemia/reperfusion is an acute inflammatory response that may activate phospholipase A2, metabolizing arachidonic acid into inflammatory factors by cyclooxygenases (COX-1 and COX-2), cytochrome P450, and lipoxygenase. These enzymes increase ROS production in the mitochondria. Xanthine oxidase and NADPH oxidase, which produce ROS in cells, result in inflammatory gene expression. These results suggest that quercetin blocks ROS-induced inflammatory responses such as COX-2, which converts arachidonic acid to prostaglandin ( Figure 10). STAT3, which belongs to the STAT protein family, is a protein transcription factor regulating many downstream signals for cell survival, apoptosis, proliferation, angiogenesis, and metabolic and anti-oxidative pathways [42]. Previous reports mentioned that oxidative stress activates the JAK2/STAT3/IL6 signal pathway in obese Zucker rats' fatty livers [15]. Furthermore, ROS production in hepatoma cells infected with Hepatitis C virus (HCV) activated STAT3 through JAK, Src kinase, and p38 MAP kinase pathways [43], and the decreased phosphorylation of p38 MAPK blocks the oxidative stress-induced senescence of myeloid leukemic cells [44]. PI-3K/AKT pathways, playing an important role in cell survival, proliferation, and growth, were activated by IL-1, leading to the proinflammatory gene activation of NF-B regulation. This study shows that H 2 O 2 induced the phosphorylation of Src kinase, AKT, p38, and STAT3 (pY-705 and pS-727) in cardiomyocytes inhibited by pretreatment with quercetin. Quercetin protects H9C2 cells from ROS-induced hyperinflammatory responses that inhibit the activation of Src, p38, and STAT3 ( Figure 10).
In summary, this study shows that quercetin inhibits Src kinase, a potential therapeutic target in vitro, and kinases such as FAK, p38, and STAT3. Thus, quercetin has comprehensive effects on cardiomyocyte. The inhibition of an inflammatory response through STAT3 inactivation in cardiomyocyte may be beneficial for an ischemia/reperfusion injury model. Hence, quercetin should be tested in an animal model to verify its therapeutic role.