Epigallocatechin-3-gallate-mediated cardioprotection by Akt/GSK-3β/caveolin signalling in H9c2 rat cardiomyoblasts
© Hsieh et al.; licensee BioMed Central Ltd. 2013
Received: 22 August 2013
Accepted: 18 November 2013
Published: 19 November 2013
Epigallocatechin-3-gallate (EGCg) with its potent anti-oxidative capabilities is known for its beneficial effects ameliorating oxidative injury to cardiac cells. Although studies have provided convincing evidence to support the cardioprotective effects of EGCg, it remains unclear whether EGCg affect trans-membrane signalling in cardiac cells. Here, we have demonstrated the potential mechanism for cardioprotection of EGCg against H2O2-induced oxidative stress in H9c2 cardiomyoblasts.
Exposing H9c2 cells to H2O2 suppressed cell viability and altered the expression of adherens and gap junction proteins with increased levels of intracellular reactive oxygen species and cytosolic Ca2+. These detrimental effects were attenuated by pre-treating cells with EGCg for 30 min. EGCg also attenuated H2O2-mediated cell cycle arrest at the G1-S phase through the glycogen synthase kinase-3β (GSK-3β)/β-catenin/cyclin D1 signalling pathway. To determine how EGCg targets H9c2 cells, enhanced green fluorescence protein (EGFP) was ectopically expressed in these cells. EGFP-emission fluorescence spectroscopy revealed that EGCg induced dose-dependent fluorescence changes in EGFP expressing cells, suggesting that EGCg signalling events might trigger proximity changes of EGFP expressed in these cells.
Proteomics studies showed that EGFP formed complexes with the 67 kD laminin receptor, caveolin-1 and -3, β-actin, myosin 9, vimentin in EGFP expressing cells. Using in vitro oxidative stress and in vivo myocardial ischemia models, we also demonstrated the involvement of caveolin in EGCg-mediated cardioprotection. In addition, EGCg-mediated caveolin-1 activation was found to be modulated by Akt/GSK-3β signalling in H2O2-induced H9c2 cell injury.
Our data suggest that caveolin serves as a membrane raft that may help mediate cardioprotective EGCg transmembrane signalling.
Green tea polyphenols (GTPs) have potent antioxidant and radical-scavenging properties, which may partially account for their cardioprotective effects . The major catechins in GTPs include epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epigallocatechin-3-gallate (EGCg) [1, 2]. EGCg is the most physiologically potent compound, and primarily accounts for the biological effects of green tea. Two recent reports using two different rat myocardial ischemic models of MI (myocardial infarction)  and IR (ischemia reperfusion)  associated with left anterior descending (LAD) coronary artery ligation have demonstrated that GTPs can efficiently improve cell viability during myocardial ischemic injury. Other studies of myocardial injury have also suggested that the cardioprotective effect of GTPs is associated with the scavenging of active-oxygen radicals, the modulation of redox-sensitive transcription factors (e.g., NFκB, AP-1), the reduction of STAT-1 activation and Fas receptor expression, an increase in NO production, and the exertion of positive inotropic effects [5–9]. Although studies have provided convincing evidence to support the cardioprotective effects of GTPs, it remains unclear whether GTPs affect trans-membrane signalling in cardiac cells.
A growing body of evidence has demonstrated that multiple signal transduction events for cardioprotection are mediated via signalling microdomains, such as lipid rafts or caveolae, on the plasma membrane of cardiac cells [10, 11]. Caveolae are a subset of lipid rafts enriched in the protein caveolin (Cav) . There are three isoforms of Cav, Cav-1, Cav-2 and Cav-3 , each of which functions as a scaffolding protein to organize and regulate membrane receptors and lipid-modified signalling molecules [14–16]. Cav-3 is the muscle-specific isoform in cardiac myocytes, whereas Cav-1 and Cav-2 are present in other cell types in the heart . A study using in vitro and in vivo models of myocardial injury demonstrated that modification of the membrane structure and composition triggers Src activation and Cav-1 phosphorylation, resulting in cardioprotection . More recently, another study with Cav-3 knock-out mice subjected to IR injury has shown that the expression of Cav-3 in cardiac myocytes is essential for isoflurane-induced cardioprotection from myocardial ischemic injury . These data also suggested that Cav may mediate the beneficial actions of a variety of cardioprotective agents .
In this study, we examined the potential mechanism for EGCg-mediated cardioprotection in an H2O2-induced oxidative stress model of myocardial ischemia injury using H9c2 rat cardiomyoblasts. We first verified that the cardioprotection of EGCg is mediated by decreasing reactive oxygen species (ROS) and cytosolic Ca2+ and by preventing alterations in the protein expression of the adherens molecules β-catenin and N-cadherin and the gap junction protein connexin 43 (Cx43) in cardiac cells. In addition, EGCg was found to prevent H2O2-induced cell cycle arrest at G1-S phase via the glycogen synthase kinase-3β/β-catenin/cyclin D1 signalling pathway. To further clarify the putative mechanism underlying EGCg transmembrane signalling in cardiac cells, enhanced green fluorescence protein (EGFP) was ectopically expressed in H9c2 cells. EGFP-emission fluorescence spectroscopy indicated that Triton X-100-resistant microdomains (i.e., lipid rafts) on the cell membrane may take part in the transmission of EGCg signalling to protect cardiac cells from oxidative stress. Using an in vitro H2O2-induced oxidative stress model in H9c2 cells and an in vivo rat model of myocardial ischemia, we demonstrated the involvement of Cav in GTPs-mediated cardioprotection. In addition, we showed that EGCg-mediated Cav-1 activation could be modulated by Akt/GSK-3β signalling in H2O2-induced H9c2 cell injury. Taken together, our data suggest that EGCg may act to protect cardiac cells from H2O2-induced oxidative stress through Akt/GSK-3β dependent Cav signalling pathway.
Chemicals and reagents
H9c2 cell lines were purchased from American Type Culture Collection (ATCC, CRL-1446) (Rockville, MD). All reagents used were ACS or MB grade. EGCg, purchased from Sigma, was prepared as a stock solution of 10 mM by dissolving the compound in deionized water.
Cell culture, EGCg and/or H2O2 treatments, MTT assay
H9c2 cells were cultured in Dulbecco’s modified essential medium (DMEM, Gibco, Invitrogen Taiwan Ltd., Taipei, Taiwan) containing 10% fetal bovine serum (FBS) (Gibco), 25 mM D-glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% streptomycin (100 μg/ml) (Gibco), and 1% penicillin (100 U/ml) (Gibco) at pH 7.4 in a 5% CO2 incubator at 37°C. Cell viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay (ATCC, Manassas, VA, USA). Cells (105) were seeded onto 6-cm plates in DMEM-10% FBS. After adhering overnight, the cells were changed to serum-free medium with or without EGCg for 30 min in a 5% CO2 incubator at 37°C and then washed with phosphate buffer solution (PBS). The washed cells were treated with different concentrations of H2O2 in serum-free DMEM for 30 min in a 5% CO2 incubator at 37°C. After washing with PBS, the cells were incubated in serum-free DMEM for 24 h in a 5% CO2 incubator at 37°C. After 24 h incubation, MTT was then added to the cells at a final concentration of 0.5 mg/ml and the mixture was incubated at 37°C for 4 h. The optical density of the purple MTT formazan product was measured at 570 nm using a microplate reader (Anthos 2000, Austria).
Determination of cellular Ca2+ levels
where Rmin and Rmax are the ratios measured by the release of intracellular dye with 2 mM EGTA in 0.1% Triton X-100 (Rmin) followed by the addition of 2.1 mM Ca2+ (Rmax), whereas Sf2/Sb2 is the ratio of the 380 nm signals in Ca2+-free and Ca2+-replete solutions, respectively.
Measurement of intracellular ROS generation by fluorescence spectrophotometry
Intracellular ROS levels were assessed using 2′,7′-dichlorofluorescein diacetate (DCF-DA) . Cells (1.2 × 106) loaded with DCF-DA in 3 ml PBS at a final concentration of 10 μM were incubated at 37°C for 1 h. After incubation, the cells were then washed three times with PBS by centrifugation at 300 × g at 4°C for 5 min. The cells re-suspended with PBS and brought to a density of 105 cells/ml were measured for DCF-DA fluorescence changes every 10 min after the addition of H2O2 or EGCg by fluorescence spectrophotometry. The fluorescence excitation maximum for DCF-DA was 495 nm, and the corresponding emission maximum was 527 nm.
Cell cycle phase determination
H9c2 cells (107) were seeded in a 10-cm dish in DMEM-0.2% FBS and cultured in a CO2 incubator at 37°C for 24 hr. The cells were then changed to fresh medium, trypsinized, and centrifuged. The pellet was washed and re-suspended in 1 ml of pre-chilled PBS, fixed by the gradual addition of 3 ml of 95% ethanol, and stored in a deep freezer (-20°C) overnight. The cells were then washed three times by centrifugation and re-suspended in pre-chilled PBS. To stain the cells with propidium iodide (PI), the cells were re-suspended in PBS containing 0.1% Triton X-100, 20 μg/ml of PI, and 0.2 mg/ml of RNase A and incubated for 30 min at room temperature in the dark. Samples were analyzed on a flow cytometer (FC500 Flow Cytometry System, Beckman Coulter, Inc.) with a 488 nm excitation laser. The cell cycle phases were determined using the software provided with the instrument (CXP Software, Beckman Coulter, Inc.) .
The sample preparation for SDS-PAGE and electro-transfer was as described previously [23, 24]. The primary antibodies used were mouse monoclonal antibodies against β-actin (C4) (sc-47778), human N-cadherin (H-63) (sc-7939), human-β-catenin (9 F2) (sc-47752), human GSK-3β (H-76) (sc-9166), human pGSK-3β (pY-216) (sc-135653), human cyclin D1 (DSC-6) (sc-20044), Cav-3 (A-3) (sc-5310), rat-nCx43 (D-7) (sc-13558), and rabbit GAPDH (6C5) (sc-32233) (all from Santa Cruz and all 1: 1000 dilution); goat polyclonal anti-human Laminin-R antibody (F-18) (sc-21534) (Santa Cruz; 1: 1000 dilution); and rabbit polyclonal antibodies raised against human Cav-1 (N-20) (sc-894), human Akt1 (H-136) (sc-8312), human Ser 9 phosphorylated GSK-3β (Ser 9) (sc-11757), pCav-1 (Tyr 14) (sc-101653) (Santa Cruz; 1: 1000 dilution), human Ser 473 phosphorylated Akt1 (SAB4504331) (Sigma, 1: 500 dilution), and rat Cx43 (71-0700) (Zymed, Invitrogen; 1: 1000 dilution). After 3 × 10 min washes with PBS containing 0.05% Tween-20, the membrane was incubated for 2 h at 4°C with alkaline phosphatase-conjugated goat anti-rabbit, donkey-anti-goat, or rabbit-anti-mouse IgG antibodies (Santa Cruz; 1:5000 dilution), and the bound antibody was detected using 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium.
EGFP-expressing H9c2 and fluorescence measurements
EGFP-expressing H9c2 cells were generated by co-transfecting pEGFP-N1 (PT3027-5) vector with Lipofectamine 2000 (Invitrogen) into H9c2 cells. The fluorescence changes in transformed cells were measured in a Perkin-Elmer LS 50B spectrofluorimeter (Beaconsfield, Buckinghamshire, England). The fluorescence excitation maximum for EGFP was 488 nm, and the corresponding emission maximum was 507 nm .
Immunoprecipitation and immunoblotting
EGFP expressed H9c2 cells were lysed with pre-chilled RIPA buffer containing 50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 5 mM EDTA, 0.02 mM EGTA, 1% phenylmethanesulfonyl fluoride, and a cocktail of protease inhibitors. The cell lysates were diluted with pre-chilled PBS to a volume of 500 μl and a concentration of 5 mg/ml and incubated overnight at 4°C with 25 μg of rabbit anti-EGFP (PG-10013, Genesis Biotech Inc., Taipei, Taiwan). 50 μl protein G Sepharose 4 Fast flow (GE Healthcare UK Ltd., England) was then added, and the mixture was incubated for 1 h at 4°C. After centrifugation, the pellet was washed with RIPA buffer followed by Tris-OH buffer (50 mM, pH 8.0). The samples dissolved in reducing buffer containing 1% SDS, 100 mM dithiothreitol, 50 mM Tris-OH, pH 7.5 were used for molecular identification of the protein complexes that formed with EGFP in the overexpressed cells by SDS-PAGE, followed by immunoblotting, as described above. In addition, protein bands on the SDS-PAGE gels were cut out for molecular identification by acquiring MALDI-MS spectra at the Proteomics center at National Chung-Hsing University (Taichung, Taiwan) (Additional file 1: Figure S1).
Protein separation by 2-DE and isoelectric focusing (IEF)
After co-immunoprecipitation, the protein complexes conjugated with EGFP were separated by two-dimensional electrophoresis (2-DE) and IEF. Immobilized pH gradient strips (pH 3-10, 13 cm) were rehydrated with 450 μg protein at room temperature overnight (at least 12 h). IEF was performed using an IPGphor 3 apparatus (GE healthcare) for a total of 17 kVh at 20°C. After IEF, strips were equilibrated in 6 M urea, 75 mM Tris–HCl (pH 8.8), 29.3% (v/v) glycerol, 2% (w/v) SDS and 0.002% (w/v) bromophenol blue with 65 mM DTT for 15 min and in the same buffer with 240 mM iodoacetamide for next 15 min. Strips were then transferred onto 10% polyacrylamide gels and sealed with 0.5% (w/v) low-melting-point agarose in SDS running buffer containing 0.02% (w/v) bromophenol blue. The gels were run in a PROTEAN® II xi gel tank (Bio-Rad) at 35 mA per gel at 20°C until the dye reached the bottom of the gels. Gels were stained with Bio-safe™ Coomassie G-250 Stain (Bio-Rad) according to the manufacturer’s protocol. Stained gels were scanned using Scanmaker 9800XL (Microtek) and analyzed using ImageMaster™ 2D Platinum 7.0 (GE healthcare). The proteins of interest were cut out for molecular identification by acquiring MALDI-MS spectra.
RNA extraction and semi-quantitative RT-PCR
The procedures for RNA extraction and semi-quantitative reverse transcription polymerization chain reaction (semi-quantitative RT-PCR) have been described previously . The primers used were GAPDH, (forward) 5-ACC ACA GTC CAT GCC ATC AC-3, (reverse) 5-TCC ACC ACC CTG TTG CTG TA-3, product size 452 bp; Cav-1, (forward) 5-CTA CAA GCC CAA CAA CAA GGC-3, (reverse) 5-AGG AAG CTC TTG ATG CAC GGT-3, product size 342 bp; Cav-2, (forward) 5-GCT CAA CTC GCA TCT CAA GCT-3, (reverse) 5-TCT GTC ACA CTC TTC CAT ATT-3, product size 260 bp; Cav-3, (forward) 5-GGA CAT TGT GAA GGT GGA TTT-3, (reverse) 5-GCA CTG GAT CTC AAT CAG GTA-3, product size 247 bp. The correct sequences for all genes were confirmed by alignment with the reported sequence for each gene.
A rat model of myocardial ischemia involving LAD ligation
Male Sprague–Dawley rats (200-250 g), aged 8-9 weeks, were randomly divided into three groups: a sham control group, a group that underwent LAD ligation without GTP supplementation, and a group that underwent LAD ligation with GTP supplementation (400 mg/Kg/day) for 2 weeks, with 5 animals per group. Once the rat was anesthetized, the heart was exposed via a left thoracotomy, and a 6-0 polypropylene suture was tied onto the LAD coronary artery 3 mm distal to the inferior margin of the left atrium, and the chest wall was closed in layers. Regional myocardial ischemia was confirmed by the observation of a rapid change from reddish to a dark red color on the anterior surface of the LV and rapid development of akinesia and dilatation in the ligated area. All experimental procedures conformed to the “Guidelines for Proper Conduct of Animal Experiments” approved by the Animal Care and Use Committee of Taichung Veterans General Hospital and National Chung-Hsing University.
After surgery, the rats were fed intragastrically with GTPs (400 mg/kg) every day for two weeks. After the rats were sacrificed, the hearts were cut along the long cross-sectional axis of the left ventricle, and cardiac tissues at both the infarcted area and a remote site of myocardium were isolated to determine protein levels of the 67 kD laminin receptor and Cav-1 and-3 by immunoblotting, as described above.
Quantitative values are presented as the mean and standard error of the mean (mean ± SEM). A difference was considered to be statistically significant when the P value was less than 0.05.
EGCg cardioprotective effects on cell viability, ROS formation, and cytosolic Ca2+ overload in H2O2-treated H9c2 cells
Effects of EGCg and H2O2 on the protein levels of N-cadherin, β-catenin, and phosphorylated and non-phosphorylated Cx43 in H9c2 cells
Effects of EGCg and H2O2 on the cell cycle and phosphorylated and GSK-3β, β-catenin, and cyclin D1 protein levels in H9c2 cells
Glycogen synthase kinase 3β (GSK-3β) is a key component of multiple signalling pathways involved in the regulation of cell fate, protein synthesis, glycogen metabolism, cell mobility, proliferation, and survival [26–28]. By preventing cells from entering the cell cycle, GSK-3β participates in the regulation of the β-catenin signalling pathway by modulating cyclin D1 expression levels . For cells treated with 400 μM H2O2, phosphorylation of GSK-3β (Tyr216) was increased by 20%, whereas EGCg pre-treatment with or without H2O2 attenuated both total and phosphorylated GSK-3β levels in cells (Figure 3b). In addition, H2O2 decreased β-catenin (42%) and cyclin D1 (26%) expression levels (Figure 3c), which may cause the subsequent cell cycle arrest at the G1-S phase (Figure 3a). This H2O2-induced inhibition of the β-catenin/cyclin D1 signalling pathway could be efficiently prevented by pre-treatment with 20 μM of EGCg (Figure 3c) or ~10 μM of SB 216763, an inhibitor of GSK-3α/3β (Figure 3d).
EGCg-induced fluorescence changes in intact Triton X-100-soluble and insoluble fractions of EGFP-expressing H9c2 cells
To identify the protein complexes conjugated to EGFP in EGFP-expressing cells, a co-immunoprecipitation assay using a specific antibody against EGFP was used for molecular identification, followed by immunoblotting or MS spectra. The EGFP co-immunoprecipitated proteins separated by one-dimensional SDS-PAGE (Figure 4c, Additional file 1: Figure S1) or 2-D electrophoresis (Figure 4d) were myosin IX [myh 9, 200 kD, EDM15905], 67 kD laminin receptor [LR, 67 kD, 48 kD], vimentin [53.7 kD, NP112402], β-actin [43 kD, ABM16832)], GAPDH (36 kD), Cav-3 (24 kD) and Cav-1 (21 kD). LR has been identified as a receptor for EGCg . Cav-1 and -3 are known to stabilize lipid raft microdomains (Triton X-100-resistant compartment) on cell membranes [31, 32]. Cytoskeletal proteins including β-actin, myosin IX, and vimentin may form complexes with other protein in cellular Triton X-100-resistant microdomains, which may play a role in EGCg transmembrane signalling in cardiac cells.
Effects of H2O2 and EGCg on the expression of Cavs in H9c2 cells
Effects of LAD ligation and GTP treatment on the protein content of LR and Cav-1 and -3 in rat myocardium
Effects of H2O2 and EGCg on the Akt/GSK-3β survival pathway in H9c2 cells
To further establish the relationship between Cav and GSK-3β signalling pathway, we determined the effects of GSK-3β inhibition on the phosphorylation of Cav-1 in H2O2-induced H9c2 cells (Figure 7b). For cells exposed to 400 μM H2O2, phosphorylation of pGSK-3β (S9) and pCav-1(Y14) was decreased, whereas EGCg or GSK-3β inhibitor, SB 216763 pre-treatment increased phosphorylation of both pGSK-3β (S9) and pCav-1(Y14) in H2O2-exposed cells (Figure 7b). Concomitantly, the H2O2,-suppressed cell viability of H9c2 was improved by EGCg and/or GSK-3β inhibitor, SB 216763 pre-treatment (Figure 7c) Apparently, EGCg mediated Cav-1 signalling through activation on Akt/GSK-3β might act to protect cardiac cells against the H2O2-induced oxidative stress in H9c2 cells.
Oxidative stress describing an imbalance between the generation and clearance of reactive oxygen species (ROS) in cells has the causative effect on the development and progression of heart disease [34, 35]. A cell line of H9c2 rat cardiomyoblasts has been used as an in vitro cellular model for cardiac tissues in response to oxidative stress conditions . In addition, H9c2 cells associated with H2O2-induced oxidative stress have been widely used to evaluate the protective role of EGCg against oxidative injury and cell death caused by ROS in cardiac cells . In the present study, we demonstrated the cardioprotection effects of EGCg against H2O2-induced oxidative stress in H9c2 cells by preventing ROS formation and cytosolic Ca2+ overload (Figure 1). This is consistent with the finding by Dreger et al. , which demonstrated that EGCg treatment for 30 min significantly reduced intracellular levels of ROS in a model of H2O2-induced oxidative stress in neonatal rat cardiomyocytes.
Using the H9c2 cell model of H2O2-induced oxidative stress for a proteomics study, Chou et al.  showed that oxidative stress triggers tyrosine phosphorylation on target proteins associated with cell-cell junctions, the actin cytoskeleton, and cell adhesion in cardiac cells. Previously utilizing a surgical model of IR involving a temporary LAD ligation in rats, we demonstrated that green tea polyphenol (GTP) pre-treatment protects cardiomyocytes from IR injury by altering the expression and distribution of adherens and gap junction proteins . In agreement with previous findings, the present study validated that EGCg has a protective effect on H2O2-induced changes in protein expression for the adherens molecules of β-catenin and N-cadherin and the gap junction protein Cx43 in H9c2 cells (Figure 2).
GSK-3β relevant to mitochondrial signalling has emerged as a key end-effector of multiple signalling pathways for cardioprotection . Here, we demonstrated that EGCg pre-treatment could protect the H2O2-induced cell cycle arrest at the G1-S phase by decreasing tyr216 phosphorylation of GSK-3β, leading to the subsequent increase in β-catenin and cyclin D1 protein expression in H9c2 cells (Figure 3). β-catenin is a transcriptional activator of target genes in the nucleus [38, 39]. Cyclin D1 is one of target genes that may be activated by β-catenin for cell proliferation . EGCg modulation of the GSK-3β/β-catenin/cyclin D1 signalling pathway would therefore promote the cardiac cell cycle progression into S phase.
Many of the properties of lipid rafts have been inferred from detergent-resistant membranes that occur in non-ionic detergent (e.g., Triton X-100) lysates of cells . In the present study, we determined the EGCg-induced fluorescence changes in intact, Triton X-100-soluble and insoluble fractions of these cells (Figure 4). Along with the molecular identification for the protein complexes with EGFP in these cells, these data suggested that the lipid raft microdomain-associated proteins (i.e., LR, Cav-1 and Cav-3) as well as cytoskeletal proteins (i.e., β-actin, myosin IX, and vimentin) may play a role in EGCg transmembrane signalling in cardiac cells. Both intact microtubules and actin filaments have been shown to be the primary interacting partners of lipid rafts [15, 31, 32]. There is increasing evidence that lipid rafts in the cell membrane are clustered in response to different stimuli to form signalling platforms for transmembrane transduction . Among these signalling platforms, Zhang et al.  reported that some large redox signalling molecules are recruited into lipid raft microdomains and subsequently produce ROS in bovine coronary arterial endothelial cells. The present study comparing the binding of EGCg to EGFP-expressing cells in conditions with or without H2O2-induced oxidative stress indicated that the strength of EGCg binding to cells exposed to H2O2-induced oxidative stress conditions doubled compared to controls without H2O2 exposure (Figure 5). It appears that oxidative stress-induced cardiac cells increase lipid-raft signalling for the binding of EGCg. Accordingly, these rafts could function as platforms to mediate the EGCg intracellular signalling for cardioprotection against oxidative stress.
Increasing evidence indicates that multiple signal transduction events in the heart occur via caveolae and caveolins (Cavs) to localize signalling molecules and receptors in the membrane for cardioprotection [10, 11, 18, 19]. Both Cav-1 and Cav-3, functioning as scaffolding proteins, can provide direct temporal and spatial regulation with signalling molecules activated by a wide spectrum of cardioprotective agents including the volatile anesthetic isoflurane [17, 18]. Cav-1 has been shown to play a signalling role in cardiomyocytes . In contrast, Cav-3, the muscle-specific isoform, mediates interactions with cytoskeletal elements and is responsible for caveolae formation in cardiac cells . Several myocardial pathologies have been shown to be associated with alterations in Cav expression: Cav-1 and Cav-3 levels are elevated in pressure-overloaded and failing hearts [43, 44], whereas reduced cardiac Cav-1 and Cav-3 expression has been reported in cases of myocardial infarction , cardiac hypertrophy , heart failure , and chronic hypoxia . Cav-1 levels are also altered in renal failure  and pulmonary hypertension . In the present study, using in vitro H2O2-induced oxidative stress in H9c2 cells, we demonstrated that H2O2 caused a 30% decrease in the levels of Cav-1 concomitant with a 20% decrease in phosphorylated Cav-1, and these reductions were counteracted by 10 or 20 μM EGCg pre-treatment for 30 min (Figure 5b). Since pre-treatment with GSK-3β inhibitor, SB 216763, also blunted the effects of H2O2 induced oxidative stress on Cav-1 inhibition (Figure 7b, c), it is very likely that EGCg could act through GSK-3β to affect Cav-1 signalling in H2O2-induced cells. The link between Cav-1 activation and GSK-3β signalling pathway could be achieved by Akt activation (Figure 7a). Thus, during oxidative stress by myocardial ischemia assault Cavs can modulate intracellular signalling for EGCg-medicated cardioprotection via Akt/GSK-3β pathway. In addition, using a rat model of myocardial ischemia involving LAD ligation, we demonstrated that GTPs treatment for 2 weeks efficiently protected infarcted myocardium of LAD-ligated rats from reduced Cav-3 protein levels (Figure 6). It appears that during oxidative injury or myocardial ischemia, Cavs can modulate intracellular signalling for GTP-medicated cardioprotection.
In summary, the results reported here suggested that GTPs may mediate cardioprotection against oxidative stress and ischemic injury through caveolae trafficking via Akt/GSK-3β pathway.
This work was supported by the National Science Council of Taiwan government (to Y-M L., Grants: NSC 100-2320-B-005-001, NSC 101-2320-B-005-001).
- Stangl V, Dreger H, Stangl K, Lorenz M: Molecular targets of tea polyphenols in the cardiovascular system. Cardiovasc Res. 2007, 73: 348-358. 10.1016/j.cardiores.2006.08.022.View ArticlePubMedGoogle Scholar
- Mak JC: Potential role of green tea catechins in various disease therapies: Progress and promise. Clin Exp Pharmacol Physiol. 2012, 39: 265-273. 10.1111/j.1440-1681.2012.05673.x.View ArticlePubMedGoogle Scholar
- Shieh SR, Tsai DC, Chen JY, Tsai SW, Liou YM: Green tea extract protects rats against myocardial infarction associated with left anterior descending coronary artery ligation. Pflugers Arch. 2009, 458: 631-642. 10.1007/s00424-009-0655-1.View ArticleGoogle Scholar
- Liou YM, Hsieh SR, Wu TJ, Chen JY: Green tea extract given before regional myocardial ischemia-reperfusion in rats improves myocardial contractility by attenuating calcium overload. Pflugers Arch. 2010, 460: 1003-1014. 10.1007/s00424-010-0881-6.View ArticlePubMedGoogle Scholar
- Dreger H, Lorenz M, Kehrer A, Baumann G, Stangl K, Stangl V: Characteristics of catechin- and theaflavin-mediated cardioprotection. Exp Biol Med (Maywood). 2008, 233: 427-433. 10.3181/0710-RM-292.View ArticleGoogle Scholar
- Li D, Yang C, Chen Y: Identification of a PKCϵ-dependent regulation of myocardial contraction by epicatechin-3-gallate. Am J Physiol Heart Circ Physiol. 2008, 294: H345-H353.View ArticlePubMedGoogle Scholar
- Lorenz M, Hellige N, Rieder P: Positive inotropic effects of epigallocatechin-3-gallate (EGCG) involve activation of Na+/H+ and Na+/Ca2+ exchangers. Eur J Heart Fail. 2008, 10: 439-445. 10.1016/j.ejheart.2008.03.004.View ArticlePubMedGoogle Scholar
- Hirai M, Hotta Y, Ishikawa N, Wakida Y, Fukuzawa Y, Isobe F, Nakano A, Chiba T, Kawamura N: Protective effects of EGCg or GCg, a green tea catechin epimer, against postischemic myocardial dysfunction in guinea-pig hearts. Life Sci. 2007, 80: 1020-1032. 10.1016/j.lfs.2006.11.032.View ArticlePubMedGoogle Scholar
- Townsend PA, Scarabelli TM, Pasini E, Gitti G, Menegazzi M, Suzuki H, Knight RA, Latchman DS, Stephanou A: Epigallocatechin-3-gallate inhibits STAT-1 activation and protects cardiac myocytes from ischemia/reperfusion-induced apoptosis. FASEB J. 2004, 18: 1621-1623.PubMedGoogle Scholar
- Jin S, Zhou F, Katirai F, Li PL: Lipid raft redox signaling: molecular mechanisms in health and disease. Antioxid Redox Signal. 2011, 15: 1043-1083. 10.1089/ars.2010.3619.PubMed CentralView ArticlePubMedGoogle Scholar
- Das M, Das DK: Lipid raft in cardiac health and disease. Curr Cardiol Rev. 2009, 5: 105-111. 10.2174/157340309788166660.PubMed CentralView ArticlePubMedGoogle Scholar
- Kurzchalia TV, Parton RG: Membrane microdomains and caveolae. Curr Opin Cell Biol. 1999, 11: 424-431. 10.1016/S0955-0674(99)80061-1.View ArticlePubMedGoogle Scholar
- Williams TM, Lisanti MP: The Caveolin genes: from cell biology to medicine. Ann Med. 2004, 36: 584-595. 10.1080/07853890410018899.View ArticlePubMedGoogle Scholar
- Kukkonen JP: A ménage à trois made in heaven: G-protein-coupled receptors, lipids and TRP channels. Cell Calcium. 2011, 50: 9-26. 10.1016/j.ceca.2011.04.005.View ArticlePubMedGoogle Scholar
- Head BP, Patel HH, Roth DM, Murray F, Swaney JS, Niesman IR, Farquhar MG, Insel PA: Microtubules and actin microfilaments regulate lipid raft/caveolae localization of adenylyl cyclase signaling components. J Biol Chem. 2006, 281: 26391-26399. 10.1074/jbc.M602577200.View ArticlePubMedGoogle Scholar
- Everson WV, Smart EJ: Influence of caveolin, cholesterol, and lipoproteins on nitric oxide synthase implications for vascular disease. Trends Cardiovasc Med. 2001, 11: 246-250. 10.1016/S1050-1738(01)00119-0.View ArticlePubMedGoogle Scholar
- Hagiwara Y, Nishina Y, Yorifuji H, Kikuchi T: Immunolocalization of caveolin-1 and caveolin-3 in monkey skeletal, cardiac and uterine smooth muscles. Cell Struct Funct. 2002, 27: 375-382. 10.1247/csf.27.375.View ArticlePubMedGoogle Scholar
- Patel HH, Tsutsumi YM, Head BP, Niesman IR, Jennings M, Horikawa Y, Huang D, Moreno AL, Patel PM, Insel PA, Roth DM: Mechanisms of cardiac protection from ischemia/reperfusion injury: a role for caveolae and caveolin-1. FASEB J. 2007, 21: 1565-1574. 10.1096/fj.06-7719com.View ArticlePubMedGoogle Scholar
- Horikawa YT, Patel HH, Tsutsumi YM, Jennings MM, Kidd MW, Hagiwara Y, Ishikawa Y, Insel PA, Roth DM: Caveolin-3 expression and caveolae are required for isoflurane induced cardiac protection from hypoxia and ischemia/reperfusion injury. J Mol Cell Cardiol. 2008, 44: 123-130. 10.1016/j.yjmcc.2007.10.003.PubMed CentralView ArticlePubMedGoogle Scholar
- Hsu YC, Liou YM: The anti-cancer effects of (-)-epigalocathine-3-gallate on the signaling pathways associated with membrane receptors in MCF-7 cells. J Cell Physiol. 2011, 226: 2721-2730. 10.1002/jcp.22623.View ArticlePubMedGoogle Scholar
- Grynkiewicz G, Poenie M, Tsien RY: A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985, 260: 3440-3450.PubMedGoogle Scholar
- Shieh DB, Li RY, Liao JM, Chen GD, Liou YM: Effects of genistein on β-catenin signaling and subcellular distribution of actin-binding proteins in human umbilical CD105-positive stromal cells. J Cell Physiol. 2010, 223: 423-434.PubMedGoogle Scholar
- Peng KW, Liou YM: Differential role of actin-binding proteins in controlling the adipogenic differentiation of human CD105-positive Wharton’s jelly cells. Biochim Biophys Acta. 1820, 2012: 469-481.Google Scholar
- Liou YM, Kuo SC, Hsieh SR: Differential effects of a green tea-derived polyphenol (-)-epigallocatechin-3-gallate on the acidosis-induced decrease in the Ca2+ sensitivity of cardiac and skeletal muscle. Pflugers Arch. 2008, 456: 787-800. 10.1007/s00424-008-0456-y.View ArticlePubMedGoogle Scholar
- Lindsey ML, Escobar GP, Mukherjee R, Goshorn DK, Sheats NJ, Bruce JA, Mains IM, Hendrick JK, Hewett KW, Gourdie RG, Matrisian LM, Spinale FG: Matrix metalloproteinase-7 affects Connexin-43 levels, electrical conduction, and survival after myocardial infarction. Circulation. 2006, 113: 2919-2928. 10.1161/CIRCULATIONAHA.106.612960.View ArticlePubMedGoogle Scholar
- Vigneron F, Dos Santos P, Lemoine S, Bonnet M, Tariosse L, Couffinhal T, Duplaà C, Jaspard-Vinassa B: GSK-3β at the crossroads in the signalling of heart preconditioning: implication of mTOR and Wnt pathways. Cardiovasc Res. 2011, 90: 49-56. 10.1093/cvr/cvr002.View ArticlePubMedGoogle Scholar
- Omar MA, Wang L, Clanachan AS: Cardioprotection by GSK-3 inhibition: role of enhanced glycogen synthesis and attenuation of calcium overload. Cardiovasc Res. 2010, 86: 478-486. 10.1093/cvr/cvp421.View ArticlePubMedGoogle Scholar
- Juhaszova M, Zorov DB, Yaniv Y, Nuss HB, Wang S, Sollott SJ: Role of glycogen synthase kinase-3beta in cardioprotection. Circ Res. 2009, 104: 1240-1252. 10.1161/CIRCRESAHA.109.197996.PubMed CentralView ArticlePubMedGoogle Scholar
- Takahashi-Yanaga F, Sasaguri T: GSK-3beta regulates cyclin D1 expression: a new target for chemotherapy. Cell Signal. 2008, 20: 581-589. 10.1016/j.cellsig.2007.10.018.View ArticlePubMedGoogle Scholar
- Tachibana H, Koga K, Fujimura Y, Yamada K: A receptor for green tea polyphenol EGCG. Nat Struct Mol Biol. 2004, 11: 380-381. 10.1038/nsmb743.View ArticlePubMedGoogle Scholar
- Chichili G, Rodgers W: Cytoskeleton–membrane interactions in membrane raft structure. Cell Mol Life Sci. 2009, 66: 2319-2328. 10.1007/s00018-009-0022-6.PubMed CentralView ArticlePubMedGoogle Scholar
- Allen JA, Halverson-Tamboli RA, Rasenick MM: Lipid raft microdomains and neurotransmitter signaling. Nat Rev Neurosci. 2007, 8: 128-140. 10.1038/nrn2059.View ArticlePubMedGoogle Scholar
- Sussman MA, Völkers M, Fischer K, Bailey B, Cottage CT, Din S, Gude N, Avitabile D, Alvarez R, Sundararaman B, Quijada P, Mason M, Konstandin MH, Malhowski A, Cheng Z, Khan M, McGregor M: Myocardial AKT: the omnipresent nexus. Physiol Rev. 2011, 91: 1023-1070. 10.1152/physrev.00024.2010.PubMed CentralView ArticlePubMedGoogle Scholar
- Baines CP: The cardiac mitochondrion: nexus of stress. Annu Rev Physiol. 2010, 72: 61-80. 10.1146/annurev-physiol-021909-135929.View ArticlePubMedGoogle Scholar
- Whelan RS, Kaplinskiy V, Kitsis RN: Cell death in the pathogenesis of heart disease: mechanisms and significance. Annu Rev Physiol. 2010, 72: 19-44. 10.1146/annurev.physiol.010908.163111.View ArticlePubMedGoogle Scholar
- Chou HC, Chen YW, Lee TR, Wu FS, Chan HT, Lyu PC, Timms JF, Chan HL: Proteomics study of oxidative stress and Src kinase inhibition in H9c2 cardiomyocytes: a cell model of heart ischemia-reperfusion injury and treatment. Free Radic Biol Med. 2010, 49: 96-108. 10.1016/j.freeradbiomed.2010.04.001.View ArticlePubMedGoogle Scholar
- Sheng R, Gu ZL, Xie ML, Zhou WX, Guo CY: Epigallocatechin gallate protects H9c2 cardiomyoblasts against hydrogen dioxides-induced apoptosis and telomere attrition. Eur J Pharmacol. 2010, 641: 199-206. 10.1016/j.ejphar.2010.05.054.View ArticlePubMedGoogle Scholar
- Daugherty RL, Gottardi CJ: Phospho-regulation of Beta-catenin adhesion and signaling functions. Physiology. 2007, 22: 303-309. 10.1152/physiol.00020.2007.PubMed CentralView ArticlePubMedGoogle Scholar
- Dashwood WM, Carter O, Al-Fageeh M, Li Q, Dashwood RH: Lysosomal trafficking of beta-catenin induced by the tea polyphenol epigallocatechin-3-gallate. Mutat Res. 2005, 591: 161-172. 10.1016/j.mrfmmm.2005.03.029.PubMed CentralView ArticlePubMedGoogle Scholar
- Li PL, Zhang Y, Yi F: Lipid raft redox signaling platforms in endothelial dysfunction. Antioxid Redox Signal. 2007, 9: 1457-1470. 10.1089/ars.2007.1667.View ArticlePubMedGoogle Scholar
- Zhang AY, Yi F, Zhang G, Gulbins E, Li PL: Lipid raft clustering and redox signaling platform formation in coronary arterial endothelial cells. Hypertension. 2006, 47: 74-80. 10.1161/10.1161/01.HYP.0000196727.53300.62.View ArticlePubMedGoogle Scholar
- Tsutsumi YM, Horikawa YT, Jennings MM, Kidd MW, Niesman IR, Yokoyama U, Head BP, Hagiwara Y, Ishikawa Y, Miyanohara A, Patel PM, Insel PA, Patel HH, Roth DM: Cardiac-specific overexpression of caveolin-3 induces endogenous cardiac protection by mimicking ischemic preconditioning. Circulation. 2008, 118: 1979-1988. 10.1161/CIRCULATIONAHA.108.788331.PubMed CentralView ArticlePubMedGoogle Scholar
- Kikuchi T, Oka N, Koga A, Miyazaki H, Ohmura H, Imaizumi T: Behavior of caveolae and caveolin-3 during the development of myocyte hypertrophy. J Cardiovasc Pharmacol. 2005, 45: 204-210. 10.1097/01.fjc.0000152029.53997.57.View ArticlePubMedGoogle Scholar
- Uray IP, Connelly JH, Frazier OH, Taegtmeyer H, Davies PJA: Mechanical unloading increases caveolin expression in the failing human heart. Cardiovasc Res. 2003, 59: 57-66. 10.1016/S0008-6363(03)00352-3.View ArticlePubMedGoogle Scholar
- Jasmin JF, Mercier I, Hnasko R, Cheung MW, Tanowitz HB, Dupuis J, Lisanti MP: Lung remodeling and pulmonary hypertension after myocardial infarction: pathogenic role of reduced caveolin expression. Cardiovasc Res. 2004, 63: 747-755. 10.1016/j.cardiores.2004.05.018.View ArticlePubMedGoogle Scholar
- Piech A, Massart PE, Dessy C, Feron O, Havaux X, Morel N, Vanoverschelde JL, Donckier J, Balligand JL: Decreased expression of myocardial eNOS and caveolin in dogs with hypertrophic cardiomyopathy. Am J Physiol Heart Circ Physiol. 2002, 282: H219-H231.PubMedGoogle Scholar
- Hare JM, Lofthouse RA, Juang GJ, Colman L, Ricker KM, Kim B, Senzaki H, Cao S, Tunin RS, Kass DA: Contribution of caveolin protein abundance to augmented nitric oxide signaling in conscious dogs with pacing-induced heart failure. Circ Res. 2000, 86: 1085-1092. 10.1161/01.RES.86.10.1085.View ArticlePubMedGoogle Scholar
- Shi Y, Pritchard KA, Holman P, Rafiee P, Griffith OW, Kalyanaraman B, Baker JE: Chronic myocardial hypoxia increases nitric oxide synthase and decreases caveolin-3. Free Radic Biol Med. 2000, 29: 695-703. 10.1016/S0891-5849(00)00364-6.View ArticlePubMedGoogle Scholar
- Zager RA, Johnson A, Hanson S, Dela Rosa V: Altered cholesterol localization and caveolin expression during the evolution of acute renal failure. Kidney Int. 2002, 61: 1674-1683. 10.1046/j.1523-1755.2002.00316.x.View ArticlePubMedGoogle Scholar
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