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
Fura 2-AM (fura 2-tetra-acetoxymethyl ester; Molecular Probes, Eugene, OR) was used as the fluorescent indicator. H9c2 cells were dissolved in PBS containing 2 mM fura 2-AM and incubated for 45 min at room temperature and then for 30 min at 37°C, during which time the fura 2-AM was trapped inside by esterase cleavage. The cells were then washed twice with PBS and diluted to a density of 2 × 106
cells/ml in PBS. Recordings were made in a Perkin-Elmer LS 50B spectrofluorimeter equipped with an accessory to measure Ca2+
(Beaconsfield, Buckinghamshire, England). The dye trapped inside the cells was excited every second by exposure to alternating 340 and 380 nm light beams and the intensity of light emission at 510 nm was measured, allowing the monitoring of both the light intensity and the 340 nm fluorescence/380 nm ratio (F340/F380). The 340/380 ratio (R) was calculated and converted to the corresponding levels of [Ca2+
as described previously [20
], using a Kd of 0.14 μM [21
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.