Phlorizin was purchased from Fluka Chemie AG (Switzerland). Insulin (Monotard® HM) was obtained from Novo Nordisk A/S (Bagsvaerd, Denmark). Tiron, PD98059 and SB203580 were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Opti-MEM® I Reduced Serum Medium, Stealth™ Select RNAi (siRNA-GATA-4), scrambled siRNA (siRNA-control), and Lipofectamine 2000™ were obtained from Invitrogen (Carlsbad, CA, USA). Antibodies of phospho-GATA-4 (serine105) (AB5245) and laminin (AB11575) were purchased from Abcam (Cambridge, UK). Antibodies for cardiac TnI (No. 4002), Phospho-GSK3β (Ser 9), GSK3β and Anti-rabbit IgG, HRP-linked were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies to GATA-4, Histone H3 and goat anti-rabbit IgG-HRP-linked were obtained from Santa Cruz biotechnology, Inc. (Santa Cruz, CA, USA). Anti-Actin Monoclonal Antibody was purchased from Chemicon International, Inc. (Billerica, MA, USA). Fluorescent conjugated secondary DyLight™ 549-conjugated AffiniPure Donkey Anti-Goat IgG was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). 4', 6-diamidino-2-phenylindole (DAPI) was purchased from Thermo Fisher Scientific, Inc. (Rockford, IL, USA).
Eight-week-old male Wistar rats, weighing from 200 to 250 g, were obtained from the Animal Centre of National Cheng Kung University Medical College. Diabetes was induced by intravenous injection of 60 mg/kg streptozotocin as described previously. Animals were considered hyperglycemic when their plasma glucose concentration was 20 mmol/L or greater in addition to polyuria and other hyperglycemic features. The levels of plasma glucose were measured in blood samples collected from the femoral vein of anesthetized rats (pentobarbital, 30 mg/kg, i.p.) one hour before treatment. Blood samples were centrifuged at 12,000 g for 3 min then analyzed using glucose kit reagents (AppliedBio assay kits; Hercules, CA, USA). The levels of plasma glucose were then estimated by an auto-analyzer (Quik-Lab, Ames, Miles, Inc., Elkhart, IN, USA), run in triplicate. All studies were carried out 10 weeks after induction of diabetes.
Then, STZ-diabetic rats were anesthetized with an intraperitoneal injection of urethane (1.4 g/kg) and cannulated in the right femoral artery with polyethylene catheters (PE-50). Mean arterial pressure (MAP) and heart rate (HR) were recorded using a polygraph (MP35, BIOPAC, CA, USA). The trachea was intubated for artificial ventilation (Small Animal Ventilator Model 683, Harvard Apparatus, Holliston, MA, USA) at 50 breaths/min with a tidal volume of 8 mL/kg and a positive end expiratory pressure of 5 cm H2O. After making an incision in the chest of the rat at the third intercostal space to expose the heart, a small section (1 cm long) of the ascending aorta was freed from the connective tissue. A Transonic Flowprobe (2.5PSB923, Transonic System Inc., Ithaca, NY, USA) was implanted around the root of the ascending aorta and connected to a transonic transit-time blood flowmeter (T403, Transonic System Inc.). The cardiac output (CO) was calculated from the aortic blood flow; the difference in CO between STZ-diabetic rats and normal rats was found.
Otherwise, 4 groups (n = 6 in each of groups) of age-matched rats were used for investigation: group I, vehicle-treated normal rats; group II, vehicle-treated STZ-diabetic rats; group III, insulin-treated STZ-diabetic rats; group IV, phlorizin- treated STZ-diabetic rats. The STZ-diabetic rats of group III received intraperitoneal (i.p.) injections of long-lasting human insulin (Monotards HM) at 22.75 μg/kg every 8 hr, three times per day, as described previously [27
]. The STZ-diabetic rats of group IV received phlorizin by i.p. injection at a dose of 1 mg/kg every 8 hr according to the method described previously [27
]. Phlorizin was dissolved in a 20% solution of propylene glycol and diluted with saline to the desired concentration for injection. Fluctuations in glycemia were not observed in rats that received repeated injections of insulin or phlorizin. Changes in protein expression were determined from samples collected after a 4-day treatment period. All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996), as well as the guidelines of the Animal Welfare Act.
Cell culture and treatment
Primary cultures of cardiomyocytes from neonatal rats were prepared with some modifications as described previously [1
]. Briefly, heart tissue from a 1- to 2-day-old Wistar rat was cut into 1- to 2-mm pieces and predigested with trypsin to remove red blood cells. The heart tissue was then digested with 0.25% trypsin and 0.05% collagenase. The dissociated cells were placed in uncoated 10-cm dishes and incubated at 37°C in a 5% CO2
incubator for at least 1 hr to remove the nonmyocytic cells. This procedure caused most of the fibroblasts to attach to the dish, while most of the cardiomyocytes remained unattached. The population of cells enriched in cardiomyocytes was then collected and counted. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) purchased from GIBCO-BRL Life Technologies (GIBCO BRL, Gaithersburg, MD, USA) with 1 mmol/L pyruvate, 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 units/mL streptomycin. On the second day after plating, the medium was replaced. Three days after plating, the cells were exposed to hyperglycemic conditions, as described in detail later. Animal handling and disposal were performed in accordance with the NIH guidelines. Embryonic rat-heart-derived H9c2 cells (BCRC No. 60096) were obtained from the Culture Collection and Research Center of the Food Industry Institute (Hsin-Chiu City, Taiwan). The H9c2 cells were maintained in growth medium composed of DMEM supplemented with 10% FBS. The H9c2 cells were plated at a density of 6000 cells/cm2
and allowed to proliferate in growth medium. The medium was changed every 48 hr. To induce differentiation of the H9c2 myoblasts into myotubes, the growth medium was replaced by differentiation medium containing DMEM plus 2% horse serum when the cells neared confluence[28
Hyperglycemia-treated cardiomyocytes were generated by treating the cells with 30 mmol/L glucose for 24 hr [1
]. In brief, cells were supplemented with 10% FBS and antibiotics. After reaching approximately 60% confluence, serum-free cell media with various final concentrations of D-glucose (5.5, 10, 20 or 30 mM) were dispersed and cells were cultured for a further 24 hr [25
]. We used H9c2 cells exposed to 5.5 mM D-glucose as a control. To rule out the effect of osmolarity, we added 24.5 mM mannitol to the control cultures. High glucose (HG)-treated cardiomyocytes were generated by incubating cells with 30 mmol/L glucose for 24 hr [25
]. The cells were treated with tiron (100 nmol/L), PD98059 (20 μmol/L) and SB203580 (25 μmol/L) for 30 min before high-glucose treatment. After treating with a high glucose concentration for 24 hr, the cells were washed twice with PBS and removed by trypsinization. Cells were then collected for Western blot analysis.
Measurement of superoxide generation
Superoxide production was analyzed by the lucigenin method with modification [29
]. H9c2 cells (106
cells) were incubated in either control or HG medium for 24 hr in the presence or absence of the various treatments described above. We also treated cells with rotenone, a mitochondrial complex I inhibitor, as the positive control of superoxide generation [28
]. H9c2 cells were then trypsinized, collected by centrifugation and washed. The resulting pellet was then re-suspended in 900 μL of Krebs buffer containing NaCl (130 mmol), KCl (5 mmol), MgCl2
(1 mmol), CaCl2
(1.5 mmol), K2
(1 mmol), and HEPES (20 mmol, pH 7.4, with 1 mg/mL bovine serum albumin (BSA). To measure ROS production, the suspensions were transferred into a measuring chamber and assessed using a Chemiluminescence Analyzer (Tohoku Electronic Industrial Co., Ltd., Japan). Suspensions were injected with 100 μL lucigenin (final concentration, 4 × 10-4
mmol/L) and photon emissions were counted every 10 sec for up to 10 min.
For intracellular ROS detection, H9c2 cells (about 5,000) were seeded onto 12-well cell culture plates. After 24 hr of growth, cultures were placed in either control or HG medium, treated as described above for 24 hr, and then harvested. Also, cells treated with rotenone were used as a positive control of superoxide generation [28
]. The H9c2 cells were fixed with 4% paraformaldehyde for 30 min. After fixation, the cells were washed three times with PBS, and 5 μmol/L of dihydroethidium (DHE; Invitrogen) was added as a fluorescent indicator of ROS. Images were collected using an Olympus IX70 fluorescence microscope fitted with an Olympus America camera and MagnaFire 2.1 software.
Immunofluorescence cell staining
H9c2 cells (about 10,000) were seeded onto 6-well cell culture plates. After 24 hr of growth, cultures were placed in either control or HG medium, treated as described above for 24 hr, and then harvested. The H9c2 cells were fixed with 4% paraformaldehyde for 30 min. After fixation, the cells were washed three times with phosphate-buffered saline (PBS). The tissue was blocked with 5% BSA for 30 min at room temperature. Primary antibodies diluted in 5% BSA (anti-phospho-GATA-4 1:100) were incubated at 4°C overnight. Thereafter, cells were washed in PBS and secondary antibodies (DyLight™ 549-conjugated AffiniPure Donkey Anti-Goat IgG 1:500) and were incubated for 30 min at room temperature. Cells were then washed with PBS and counterstained with 4', 6-diamidino-2-phenylindole (DAPI). Images were collected using an Olympus IX70 fluorescence microscope fitted with an Olympus America camera and MagnaFire 2.1 software.
Nuclear protein extraction
For nuclear protein extraction, the CNMCS Compartmental Protein Extraction Kit was purchased from BioChain Institute, Inc. CA, USA. In briefly, Cells were collected using routine cell culture techniques. Count cells and add ice cold lysis buffer at 2.0 ml per 20 million cells. Mix the cells with buffer well and rotate the mixture at 4°C for 20 min. Syringe with a needle gauged between 26.5 and 30. Remove the needle tip by bending the needle several times and only leave the needle base on the syringe. Pass the cell mixture through needle base 50-90 times to disrupt the cell membrane and release the nuclei from the cells. The degree of cell membrane disruption and nucleus release can be monitored under microscope. Then spin the cells mixture at 15,000 g at 4°C for 20 min. The cytoplasmic proteins are in the supernatant, take out and save in another tube. Resuspend the pellet with ice cold wash buffer at 4.0 ml per 20 million cells; rotate at 4°C for 5 min. Spin at 15,000 g at 4°C for 20 min. Drain the supernatant. Add ice cold nuclear extraction buffer at 1.0 ml per 20 million cells to resuspend nuclear pellet, rotate at 4°C for 20 min then spin at 15,000 g at 4°C for 20 min. The nuclear proteins are in the supernatant, take out and save for further detections.
Western blot analysis
Specific protein expression levels in rat hearts or H9c2 cells were determined by Western blot analysis. Protein was extracted from tissue homogenates and cell lysates using ice-cold radio-immuno-precipitation assay (RIPA) buffer supplemented with phosphatase and protease inhibitors (50 mmol/L sodium vanadate, 0.5 mM phenylmethylsulphonyl fluoride, 2 mg/mL aprotinin, and 0.5 mg/mL leupeptin). Proteins extracted using RIPA buffer were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransfered and immobilized on a nitrocellulose membrane. The membrane was blocked with 5% non-fat milk in PBS containing 0.1% Tween 20 (PBS-T) and incubated for 2 hr. The membrane was then washed in PBS-T and hybridized with primary antibodies, which were diluted to a suitable concentration in PBS-T, for 16 hr. Incubation with secondary antibodies and detection of the antigen-antibody complex were performed using an Enzymatic Chemiluminescence (ECL) kit (Amersham Biosciences, UK). Immunoblot densities were quantified using a laser densitometer.
Small Interfering RNA (siRNA)
Duplexed RNA oligonucleotides for rat GATA-4 (Stealth RNAi ™) were synthesized by Invitrogen. H9c2 cells were transfected with 40 pmol of GATA-4-specific siRNAs (siRNA-GATA-4) or scramble siRNA (siRNA-control) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocols that cells were treated with siRNA for 48 hr.
Cell contractility assay
In the study of cell contraction, H9c2 cells were measured by the change in the planar surface area, as described previously [31
]. H9c2 cells were cultured in 6-mm flat-bottomed plates approximately 5x105
cells/ plate. Culture plates were then mounted on the heated stage of an inverted light microscope for the contractility assay. Changes in the planar surface areas in response to treatment were observed using a video camera, and images of the cells were captured serially at 0, 20, 40, and 60 min. Cells of the same group (n
= 6-10) were collected at the same time points. The perimeters of individual cells with clearly-defined borders were outlined, and the planar surface area was calculated as a percentage change. Control groups were monitored in cultures with vehicle alone.
Data are expressed as the mean ± SEM for the number (n) of animals in one group as indicated. Statistical analysis was carried out using one-way ANOVA analysis and Newman-Keuls Post-hoc analysis. A P-value of 0.05 or less was considered significant.