2.1 Chemicals and reagents
Isoflurane (2%, Henry Schein Animal Health, Fort Worth, TX, USA), 8–0 polyprolene suture (Prolene; Ethicon, Somerville, N.J., USA), buprenorphine (0.01 mg/kg, Prolene; Ethicon, Somerville, N.J., USA), glucose (Carson-Millonig formulation, RI31911; Ricca Chemicals, Arlington, TX, USA), 10% formaldehyde buffer (4℃, pH 7.4, Carson-Millonig formulation, RI31911; Ricca Chemicals, Arlington, TX, USA), transmission electron microscope (TEM, JEM-1400, JEOL, US).
2.2 Animals and treatments
Animal experiments were carried out with the support of the University of Mississippi and the UMMC Health Science animal welfare committee. The IACUC was approved under the protocol 1449[33]. Young (3 months, 25-30g) and old (18 months, 32-38g) C57BL/6J mice were purchased from Charles River Laboratory (Wilmington, Massachusetts, USA). After one week of adaptation, the aged mice were randomly divided into two groups: AL group and CR group. The first week after the adaptation period was the zero week of intervention. In the following seven weeks, AL group had a normal diet, CR group had a normal diet in the zero week. In the first to second weeks, CR group had a calorie restriction of 10%, 20% in the third to fourth weeks and 30% in the fifth to sixth weeks, and the weight loss of mice was not more than 20% (Fig. 1).
2.3 Ischemia reperfusion in vivo
Ischemia reperfusion referred to the method established by Hanying et al. for the I/R model and made adjustments according to experimental requirements[34]. Animals were anaesthetized with isoflurane (2%) and kept body temperature at 37℃. A left thoracotomy was performed and the ribs were spread apart to expose the left ventricle (LV). Ischemia was induced by ligating the left anterior descending (LAD) branch of coronary artery with an 8–0 polyprolene suture. An electrocardiogram (ST-segment elevation) and blanching of LV confirmed ischemic repolarization changes. After 45 mins of ischemia, the suture was released to induce reperfusion. Once the skin was sutured back together, intraperitoneal injection of buprenorphine (0.01 mg/kg) was provided for analgesia. The animal was allowed to recover for 24 hrs as reperfusion.
2.4 Measurement of body composition
Whole body composition was determined in live, unanesthetized mice by use of quantitative magnetic resonance (QMR) system, which relies on nuclear magnetic resonance (NMR) technology (EchoMRI, USA). Duplicate QMR scans with accumulation times of 2 min were performed by placing previously-weighed mice into a well-ventilated plastic cylinder (1.5 mm thick, 4.7 cm inner diameter), with a cylindrical plastic insert added to limit movement. While in the tube, animals were briefly subjected to a low-intensity (0.05 Tesla) electromagnetic field. Using various pulse sequences, the QMR system provides estimates of fat and lean tissue mass, which were expressed at % body weight[35].
2.5 Detection of serum adiponectin and leptin levels
Before Ischemia reperfusion, 10mL of ether-moistened skimmed cotton wool was spread on the bottom of the glass container, and then we put the animal in the bottle and closed the lid. The animals entered the anesthesia state within 20–30 seconds. Mouse blood was collected from the eyes after successful anesthesia with ether. The mice were sacrificed by cervical dislocation after blood collection. The whole blood of the mice was separated to obtain the serum, which was stored in the refrigerator at -80℃ for analysis. The serum levels of serum adiponectin and leptin in mice were determined using the corresponding enzyme-linked immunoassay (ELISA) kit (AndyGene, Beijing, China). The test steps are performed in strict accordance with the kit instructions.
2.6 Ip-GTT and blood biochemical items
Basal blood glucose was measured after 5 hrs fasting. The dose of injection of peritoneal (i.p.) glucose based on the fat content measured by ECHO-MRI equipment. Repeat test blood glucose and gather blood sample from the tail at 0, 15, 30, 60, 90 and 120 mins after injection respectively. There were four calculated marks set out, fasting glucose as G and fasting insulin as I in the formula below: QUICKI: quantitative insulin sensitivity check index; QUICKI = 1/ (logI + logG); HOMA-IS: homeostasis model assessment of insulin sensitivity; HOMA-IS = 10000/I× G; HOMA-IR: homeostasis model assessment of insulin resistance; HOMA-IR = I× G/22.5; HOMA-B%: homeostasis model assessment of β-cell function; HOMA-Β%= I× 20/(G-3.5)[36].
2.7 Transmission electron microscopy
The heart will be quickly removed and cut into halves from the center of the left ventricular along the long axis for the electron microscope. In brief, fresh heart tissue of 1mm3 size fixed in 10% formaldehyde buffer (4℃ pH 7.4, Carson-Millonig formulation, RI31911; Ricca Chemicals, Arlington, TX, USA) for at least 8 hrs, stained with OsO4 for 1 hr. Dehydrated in a graded ethanol series (35, 50, 70, 95,100 and 100%, 10 mins per step), then washed with acetone (2×15 mins) and acetone:Epon(1:1, 1 hr), embedded in 100% Epon (Ted Pella, Redding, CA,USA) for 30 mins and 60℃ overnight finally. Semithin sectioned (1 mm) stained with 1% toluidine were determined interested areas, then ultrathin sections (70 nm) were applied on copper grids, dried, stained with uranyl acetate (2%, 3 mins), calcinated lead citrate (30 s), and rinsed in ddH2O for 20 s. The areas of interest were photographed at magnification of 3,000× to 10,000× using transmission electron microscope (TEM, JEM-1400, JEOL, US).
2.8 Western blotting
Cardiac LV tissue was homogenized (20 mg) with RIPA lysis buffer containing protease and phosphatase inhibitors. After centrifugation at 12,000 × g for 10 mins, 4℃, extract and collect the supernatant (total protein). Protein concentrations were determined using the BCA protein assay kit (Beyotime Institute of Biotechnology, China). Protein/lane of each sample was separated by 8% SDS polyacrylamide gel electrophoresis. The proteins were then transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% (w/v) BSA in TBS containing 0.1% Tween-20 for 2 hrs at room temperature, the membrane was incubated with primary antibody overnight at 4℃ and secondary antibody incubated at room temperature 1 hr. Rabbit antibodies p-Akt (SER473,13038), Akt (4691s), p-FoxO1 (SER256,9461), FoxO1 (2880s), p-Raptor (SER792,2083s), Raptor (2280s, 244871), mTOR (2983), p-ULK1 (Ser555,5869s), ULK1 (8054s). Finally, the target protein was detected using ECL luminescence reagent (Boster, Wuhan, China).
2.9 Statistical analyses
Statistical analysis was performed using SPSS 26.0 software (IBM, Chicago, IL, USA). All values are presented as mean ± SEM (n observations). The signifcance of diferences was determined by the use of a two-tailed Student’s t test and analysis of variance (ANOVA). Signifcant diferences were denoted P < 0.05.