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).
2.2 Isolation and culture of rat neonatal ventricular myocytes
Rat neonatal left ventricular myocytes were isolated from adult rat hearts using a standard enzymatic method as previously described.
2.3 Simulated H/R in isolated cardiomyocytes
A cellular model of simulated H/R (20-min/30-min) in neonatal left ventricular myocytes was used as previously described. Briefly, the primary neonatal left ventricular myocytes were cultured in anoxic solution in an anoxic incubator (95% N2/5% CO2) for 3 hours. The hypoxia solution was then replaced by reoxygenation solution and cultured in a high-oxygen incubator (95%O2/5%-CO2) for 3 hours.
2.4 Construction and infection of recombinant adenoviruses
Recombinant adenoviruses expressing Rat N1ICD/PTEN/Pink1 complementary DNA (cDNA) were prepared using the pAdEasyTM vector system (Qbiogene) as described previously. Primary cardiac myocytes were infected with adenoviral particles at the multiplicity of infection of 100.
2.5. Cell viability assay
The cell viability of neonatal cardiomyocytes was detected with CCK-8 assay (Dojindo) as described previously.
2.6 Real-time PCR
Total RNA was extracted from frozen heart tissues or cultured cells and RNA reverse-transcription were performed as we previously described. RT-PCR was conducted using a SYBR Green Master Mix (Cowin, Beijing, China). All the experiments were repeated three times independently.
2.7 Western blot analysis
The adult cardiomyocytes were lysed in cell lysis buffer (Beyotime Institute of Biotechnology) at 4°C. Protein samples were separated by 8%‐10% SDS‐PAGE, then transferred to nitrocellulose membranes (Millipore). Membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies at room temperature for 1 hour. The fluorescent signals were detected using enhanced chemiluminescence by ImageQuant LAS4000 (GE). All the experiments were repeated three times independently.
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).
2.9 Hes1 and PTEN promoter luciferase reporter assay
Commercial PTEN luciferase reporter kit from SABiosciences Qiagen was used to determine the effect of HES1 over-expression on transcriptional activity of PTEN. Neonatal cardiomyocytes cells were transfected with the reporter construct using Lipofectamine 3000 (Thermo Fisher). Twenty-four hours after transfection, cells were harvested using lysis buffer. Samples were centrifuged, and 20 mL aliquot was used for measurement of dual luciferase activity using a luminometer.
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.
2.14 I/R injury model in Langendorff-perfused rat hearts
I/R injury model in Langendorff-perfused rat hearts was performed as previously. 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.
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.