Cell culture and reagents
H9c2 cells, which are myoblasts cells from rat myocardium, were purchased from the American Type Culture Collection. H9c2 cells were cultured with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and penicillin (50 IU/mL)/streptomycin (50 μg/mL). A 0.25% (w/v) Trypsin-0.53 mM ethylenediaminetetraacetic acid (EDTA) solution was used to passage cells. Cells were cultured in humidified air with 5% CO2 at 37°C. FBS and EDTA were purchased from Gibco (NY, USA). The 5,58,6,68‐tetraethylbenzimidazolcarbocyanine iodide (JC-1), Ro-32-0432, and diphenyleneiodonium chloride (DPI) were purchased from Sigma (MO, USA). Dihydroethidium (DHE) was obtained from Thermo Scientific (MA, USA). Anti-β-actin, anti-phospho-AMPK, anti-AMPK, anti-Nox-2, anti-Rac-1, anti-phospho‐PKC, anti-PKC, anti-phospho-p53, anti-Bax, anti-Bcl2, anti-cytochrome c, anti-Na/K ATPase, and anti‐PGC-1a were all obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Horseradish peroxidase (HRP)-conjugated anti‐rabbit and anti‐mouse secondary antibodies were purchased from Transduction Laboratories (CA, USA).
Hypoxia and ischemia induction
H9c2 cells were washed two times with PBS to remove glucose and serum in the culture medium. In control cells, the medium was replaced with glucose-free DMEM; in DAPA-treated cells, the medium was replaced with DAPA containing glucose-free DMEM. The cells were transferred to a hypoxia chamber containing of 95% N2 and 5% CO2 for 1 hr. After hypoxia exposure, the cells were placed in a 5% CO2 and 95% O2 incubator for reoxygenation with high-glucose DMEM containing of 10% FBS; in DAPA treated cells, DAPA was added to high-glucose DMEM. In animal study, vehicle or DAPA (0.1 mg/kg per day) was administered by the oral route. DAPA was dissolved in 60% propylene glycol and was given daily by gavage for 5 days before and 4 days after operation till sacrifice. For ischemia induction in animals, the heart was accessed by left thoracotomy, and the pericardium was removed in Sprague Dawley (SD) rats. Ischemia was induced via the ligation of the left anterior descending coronary artery (LAD) with a 6-0 silk suture. After 1 hr, ligation of LAD was released to allow reperfusion. The animals in the control group underwent the same surgical procedures but without LAD ligation. Four days after surgery, the animals were sacrificed for further experiments.
Protein isolation, tissue extraction and Western blotting assay
Protein expression levels were investigated by Western blotting. Total protein was isolated from cells of the left ventricle. After sacrifice, the hearts of all the animals were collected. The tissue of left ventricle was washed 2 times with PBS buffer, and then 100 mg of tissue was cut for homogenization with radioimmunoprecipitation assay (RIPA) lysis buffer. The homogenates were centrifuged at 13,000×g for 30 min, and the supernatant was collected and placed at −80 °C until use. The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane after the proteins were separated by electrophoresis on sodium dodecyl sulfate polyacrylamide gels. The membranes were blocked by buffer for 1 hr at 37 °C. Then, the membranes were incubated with primary antibodies for 18 hr at 4 °C followed by hybridization with HRP-conjugated secondary antibodies for 1 hr. The intensities were quantified by densitometric analysis. Plasma was obtained through blood collection for lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) assay.
Determination of cardiac functional parameters
Four days after operation, echocardiography was performed to assess cardiac function. Isoflurane-anesthetized animals were placed in a supine position. Echocardiographic data were collected by a Vevo 770 microimaging system with a 25-MHz probe (VisualSonics, Toronto, ON, Canada). Parameters were collected based on the M-mode and two-dimensional images obtained in the parasternal long and short axes at the level of the papillary muscles.
Apoptotic cells were analyzed by the ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Calbiochem). After H/R treatment, cells were rinsed twice in PBS before fixation for 30 min at room temperature with 4% paraformaldehyde. Next, cells were washed in PBS before incubation in the prepared solution (0.1% Triton X-100, 0.1% sodium citrate) for 5 min. Cells were then incubated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction mixtures in a humidified atmosphere for 1 hr at 37 °C in the dark, washed in PBS, and analyzed by flow cytometry. The BioVision CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit (Milpitas, CA, USA) was used for detection of active caspase 3. For investigating apoptosis in animal cardiac tissues, tissues were soaked in 4% paraformaldehyde. Paraffin-embedded heart was cut into 2-μm-thick sections. TUNEL staining was performed for apoptosis. In brief, the tissue sections were deparaffinized in xylene, rehydrated through a graded alcohol series (100%, 90%, 85%, and 75%), and then rinsed in PBS (pH 7.2). A DNA fragmentation detection kit (FragEL; Calbiochem) was used to detect apoptotic cells in cardiac tissue sections using TUNEL assay.
Investigation of mitochondrial membrane potential
The JC-1 is widely used to study mitochondrial membrane potential. In healthy cells, JC-1 concentrates in the mitochondrial matrix where it forms red fluorescent aggregates. In apoptotic and necrotic cells, JC-1 exists in monomeric form and stains cells green. After stimulation of H/R, cells were rinsed with DMEM and then loaded with JC-1 (5 μM). After 30-min incubation at room temperature, cells were assayed by flow cytometry.
Real-time polymerase chain reaction (PCR) assay was performed to investigate mitochondrial deoxyribonucleic acid (mtDNA) content. The primers for mitochondrial complex II were sense primer 5’-CAAACCTACGCCAAAATCCA-3’ and antisense primer 5’-GAAATGAATGAGCCTACAGA-3’. The primers forβ-actin were sense primer 5’-AGGTCATCACTATTGGCAACGA-3’ and antisense primer 5’-CACTTCATGATGGAATTGAATGTAGTT-3’. The result of real-time PCR was assayed by SYBR Green on an ABI 7000 sequence detection system according to the protocol.
NADPH oxidase activity assay
The lucigenin method was used to determine the nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase activity in cells. The crude membrane fraction of cells was prepared by a kit. The total protein concentration was adjusted to 1 mg/mL. An aliquot 200 µL of protein was incubated in the presence of 5 µm lucigenin and 100 µm NADPH. The luminescence was measured after 10 min by a plate reader (VICTOR3; PerkinElmer) to determine the relative changes in NADPH oxidase activity.
Measurement of ROS formation
ROS generation in H9c2 cells was tested using DHE. Cells were pre-treated with or without DAPA for 2 hr. Next, cells were exposed to H/R. After removing the medium from the wells, cells were incubated with 10 µm DHE for 30 min. The fluorescence intensity was calculated by a flow cytometry.
Transfection with small interfering RNA
AMPK siRNA, PGC-1-a siRNA (AMPK ON‐TARGET plus SMART pool and PGC-1-a ON-TARGET plus SMART pool) and negative control siRNA (ON-TARGET plus non-targeting pool) were purchased from Dharmacon. Two days after transfection, cells were treated with a reagent as indicated for further experiments.
Results are expressed as the means ± standard deviation (SD). Statistical analyses were performed using analysis of variance, followed by Turkey’s test. A p value < 0.05 was considered statistically significant.