2.1. Animals
All animals were treated in compliance with the guidelines for proper conduct of animal experiments and related activities (Ministry of Education, Culture, Sports, Science, and Technology of Japan). Additionally, the protocols, which follow the ARRIVE guidelines [14], were approved by the Animal Care and Use Committee at the University of Tokushima. Adult male Wistar rats and male C57BL/6 mice at 4 weeks of age were purchased from Japan SLC, Inc. (Shizuoka, Japan). mTOR+/− mice were used as previously described. [15] Mice were housed under temperature- (23 ± 3 ℃) and humidity-controlled conditions with a 12 h light/12 h dark cycle. Mice had free access to water and a control diet (14% of calories from fat; Oriental Yeast Co., Ltd., Tokyo, Japan) or an HFD (60% of calories from fat; Oriental Yeast Co., Ltd.). Mice were allowed to adapt to these conditions for 6 weeks before performing the experiments on them at 10 weeks of age.
2.2. In vivo myocardial I/R experiments
The surgical methods used were similar to those described previously. [16] [17] [18] Mice were anesthetized with sodium pentobarbital (80 mg/kg, i.p.) and mechanically ventilated with 100% oxygen using a pressure-controlled ventilator (TOPO ventilator, Kent Scientific Co., Torrington, CT, USA). The core body temperature was maintained with a heating pad, and electrocardiogram leads were placed to record the heart rate. Ischemia was induced by occluding the left coronary artery (LAD) with a 7–0 silk suture for 30 min, after which the ligature was released and the heart was perfused for 2 h. Mice were randomly assigned to each experimental group. Saline (0.9%) or Leu (200 mg/kg, IV) was administered 20 min before the occlusion. In the ischemic preconditioning (IPC) group, IPC was induced by occluding the LAD for 5 min, followed by 15 min of reperfusion immediately before the I/R procedure.
2.3. Western blotting
Lysates were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% polyacrylamide precast gels (Invitrogen, Carlsbad, CA, USA) and transferred to polyvinylidene difluoride membranes through electroelution. Membranes were blocked in 20 mM Tris-buffered saline with 1% Tween containing 5% skimmed milk and incubated with primary antibodies overnight at 4 ℃. Immunolabeled blots were visualized using horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and enhanced chemiluminescence reagent (GE Healthcare, Waukesha, WI, USA). [19]
2.4. Reactive oxygen species (ROS)
To measure ROS production in the myocardium, the OxiSelect™ in vitro ROS/RNS Assay kit (Cell Biolabs, San Diego, CA, USA) was used. Before performing the I/R procedure, mice were injected with 200 µL of saline, Leu, or Leu and rapamycin (Rap) into the aorta. All experimental procedures were performed according to the manufacturer’s instructions. The ROS content was determined using the predetermined dichlorodihydrofluorescein standard curve, and mean fluorescence units were recorded.
2.5. Isolation and maintenance of rat cardiomyocytes
Cardiomyocytes were isolated from adult male Wistar rats. Rats were heparinized (1.0 IU/g, i.p.) 30 min before anesthetizing them with pentobarbital (80 mg/kg, i.p.). Myocytes were obtained via enzymatic (210 U/mg collagenase II; Worthington, Lakewood, NJ, USA) digestion of the heart using a Langendorff apparatus. Enzymatic digestion was performed as previously described. [20] [21] Isolated myocytes were then cultured in 4% fetal bovine serum on laminin (2 µg/cm2)-coated plates for 1 h. Culturing/maintenance media were changed to serum-free media [1% bovine serum albumin + 0.1% penicillin/streptomycin M199 media (Invitrogen, Carlsbad, CA, USA)] to eliminate all non-myocytes, and cardiac myocytes were incubated at 37 ℃ in 5% CO2 for 24 h.
2.6. Mitochondrial dynamics analysis in isolated rat cardiomyocytes
Six hours prior to the pretreatment, all media were replaced with amino acid-free Dulbecco’s Modified Eagle’s medium to wash out any amino acids in M199 media. L-Leu (2.3 g; Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 100 mL distilled water, creating a 175 mM stock solution. Cells were pretreated with Leu (160 µM) or control [phosphate-buffered saline (PBS)] for 2 h. We evaluated the changes in mitochondrial dynamics of each group for 4 h at 30 min intervals after the administration of Leu or PBS.
After pretreatment with Leu or PBS, simulated ischemia/reperfusion was performed. Simulated ischemia was induced by replacing the air content with a 95% N2 and 5% CO2 gas mixture at 2 L/min in a chamber and the media with glucose-free media. This was performed for 60 min, followed by 60 min of “reperfusion” by replacing the media with normal maintenance media and by incubating the cells with 21% O2 and 5% CO2. Finally, the cells were maintained in Krebs solution and fixed with 4% paraformaldehyde after incubating them for 30 min with MitoTracker Green FM (400 nmol/L; Molecular Probes, Invitrogen). Confocal image stacks were captured using a Leica laser microscope (Leica, Tokyo, Japan), as described previously. [22] Mitochondrial density was quantified using the ImageJ software (NIH, Bethesda, MD, USA). The number and volume of each mitochondria were quantified using the Image J 3D Object Counter plug-in. The percentage of cells with a fusion pattern was determined based on the criteria that evaluated mitochondrial fusion with mitochondrial volume and a decrease in the number of mitochondria. [23] [24]
2.7. Electron microscopy
Whole hearts or cardiomyocytes were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 h at room temperature, post-fixed with 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h at room temperature, and embedded as monolayers using LX-112 embedding kits (Ladd Research, Williston, VT, USA). Sections were stained with uranyl acetate and lead citrate, and observed under an electron microscope (EM). Random sections were taken by an EM technician who was blinded to the treatments. [25]
2.8. Statistical Analyses
All results were analyzed by observers who were blinded to the animal treatment history. Data are presented as mean ± standard deviation. Differences between the treatment groups were tested for statistical significance by one-way analysis of variance, followed by Bonferroni’s post hoc test. Differences were considered significant at P < 0.05.