2.1 Animal care
Animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (Publication 85 − 23, National Institutes of Health, Bethesda, MD), and all procedures were approved by the First Affiliated Hospital of Nanchang University (Nanchang, Jiangxi, China).
2.8 Immunofluorescence analysis
Immunofluorescence assay was carried out as described above. The muscle cells fixed with paraformaldehyde were treated with 0.5% Triton X-100 PBS for 15 minutes. The myocytes were then immunostained with anti-PTEN (1:100) antibody. After washing with PBS, FITC-conjugated anti-rabbit IgG (1:2000, Jackson, USA) were stained for 2 h, respectively. The stained cells were observed under a Zeiss LSM800 confocal microscope (Zeiss, Heidelberg, Germany).
2.10 TUNEL staining
Apoptosis rates in cultured neonatal cardiomyocytes and mouse heart tissue sections were analyzed by TUNEL staining using the in-situ cell death detection kit (Roche Applied Science) according to the manufacturer's protocol. In summary, the slides were incubated with TUNEL reaction mixture, apoptotic cells were labeled, and the total number of cells was determined by DAPI staining. The slides were viewed under a Zeiss LSM800 laser scanning confocal microscope (Zeiss, Wetzlar, Germany). Apoptosis rate was calculated as the percentage of TUNEL positive cells in the total number of DAPI stained cells. All the experiments were repeated three times independently.
2.11 Measurement of cTnI, lactate dehydrogenase (LDH), superoxide dismutase (SOD) and CK levels creatine kinase-MB (CK)
The coronary effluent and culture medium of cardiomyocytes after reperfusion were placed in a thermostatic chamber. Electrochemiluminescence immunoassay was used to detect the level of cTnI, LDH and CK according to the instructions of the kit (Roche, Germany). The measurement of SOD, activity is performed according to the test kit instructions (Nanjing Kaiji Bio, Nanjing, China). All the experiments were repeated three times independently.
2.12 Seahorse analysis
As previously described, mitochondrial metabolic flux was tested in adult cardiomyocytes. Briefly, cardiomyocytes were inoculated into XF24 hippocampal plate coated with laminin at a density of 104 cells per well and cultured overnight in BCAA-free substrate-restricted medium, then analyzed. OCR and ECAR were determined using the Seahorse XF24 extracellular flux analyzer (Seahorse Bioscience, North Billerica, MA, USA). All the experiments were repeated three times independently.
2.13 Mitochondrial fusion/fission detection
The mitochondrial fusion/fission detection of myocardiocytes was detected as described previously[19].
2.14 I/R injury model in Langendorff-perfused rat hearts
I/R injury model in Langendorff-perfused rat hearts was performed as previously[19]. In brief, the rats were anesthetized with sodium pentobarbital (45 mg/kg I.P.), the hearts were rapidly resected-and the Krebs-Henselite (K-H) solution was infused at 37 ° C using a Langendorff apparatus at a constant pressure of 80 mm Hg as described in the previous paper. A water-filled latex balloon was connected to a pressure sensor (Gould P23DB, AD instrument) and inserted into the left ventricular cavity to achieve a stable LVEDP of 5-10mmHg during the initial equilibrium. After balanced perfusion, the heart was ischemia-free for 30 minutes, followed by another 45 minutes of perfusion. LVDP and ± dp/dt Max were evaluated using PowerLab system (AD instrument).
2.15 In vivo adenoviral gene delivery
The surgical procedures and adenoviral delivery were carried out as described[19].
2.16 Statistical analysis
Data are expressed as mean ± SEM. Statistical significance was determined by multiple comparisons or repeated measures using analysis of variance or repeated analysis of variance. The student t test was used to estimate the significant difference between the two averages. P < 0.05 was considered statistically significant.