Animals

Sprague-Dawley (SD) rats were obtained from Jinan Peng Yue Experimental Animal Breeding Co. Ltd. The animals (SCXK (Lu) - 20,180,030) were housed in chambers with a temperature of 23 °C and a 12 h light/ 12 h dark cycle. They had unrestricted access to water and were given a standard diet for rodents. The Ethics Committee of Yantaishan Hospital (YSLZ2023152) approved all the procedures involving animals, and the animal experiments were conducted according to the ARRIVE guidelines (https://arriveguidelines.org).

Generation and transfection of recombinant lentivirus

HIF-1αcomplete cDNAs were cloned and recombined with a lentivirus expression vector acquired from GeneChem in Shanghai, China. An empty lentivirus was employed as a control. Lentiviruses acquired from GeneChem (Shanghai, China) were employed to encode and combine Sestrin2 siRNA and its non-targeting siRNA.

Animal model and grouping

The AMI model was created by ligating the left anterior descending coronary artery [15]. Following anesthesia administration (1%, 40 mg/kg pentobarbital sodium with intraperitoneal injection) in rats, a neck incision was performed to establish a connection with the ventilator. Subsequently, thoracotomy was conducted to expose the heart, and the left anterior descending branch of the coronary artery was ligated. Successful construction of the AMI model was indicated by the elevation of the ST segment in the electrocardiogram, deceleration of the heart rate, and whitening of the left ventricle. Left Ventricular End-Diastolic Diameter (LVEDD), left ventricular ejection fraction (LVEF), LV end-systolic Diameter (LVESD), LV fractional shortening (LVFS) [16]. The successfully modeled rats were randomly allocated into four groups (n = 6), namely the model group, NC group, HIF-1α overexpression (HIF-1α-OE) group, HIF-1α overexpression + si-sestrin2 (HIF-1α-OE + si-sestrin2) groups, each consisting of nine animals. Additionally, a sham surgery group comprising nine SD rats that underwent thoracotomy without ligation of the left anterior descending coronary artery. Prior to surgery, a negative control, a recombinant plasmid overexpressing HIF-1 α, and si-sestrin2 lentivirus were administered via the tail vein to the NC group, HIF-1α-OE group, and HIF-1α-OE + si-sestrin2 group, respectively. The model group and sham group received an equivalent volume of normal saline. Subsequently, injections were administered weekly, and rats were euthanized 28 days post-surgery to obtain myocardial tissue, which was stored at − 80 °C for future use.

2,3,5-triphenyl-2H-tetrazolium chloride (TTC)

After AMI, the heart was immediately removed and frozen at − 80 °C for later evaluation of the myocardial infarction’s severity using TTC (Solarbio, Beijing, China) staining. In order to achieve consistency, every heart was divided into five slices of the same thickness. These slices were subsequently immersed in a 1% TTC solution and incubated at a temperature of 37 °C for a duration of 25 minutes. After being exposed to 10% formaldehyde for a duration of 24 hours, the slices were organized based on their decreasing size and captured using an Olympus camera. Image J software was utilized to determine the region affected by infarction. Myocardial infarction area (%) = myocardial infarction area/total myocardial area × 100% [17].

Haematoxylin-eosin (H&E) staining

Myocardial tissue of rats was fixed in a 4% paraformaldehyde solution and subsequently embedded in paraffin slices with a thickness of 4 μm. Then, H&E staining (Solarbio, Beijing, China) procedure was carried out. A light microscope (Leica, Germany) was used to observe myocardial tissue damage and inflammatory infiltration.

Detection of LDH, CK-MB, TNF-α, IL-6, SOD, and MDA

After 24 hours of effective modeling, blood was collected from the abdominal aorta and left to sit at room temperature for a period of 2 hours. Afterward, the blood underwent centrifugation at 2500×g for 15 minutes to collect serum. The serum levels of LDH (BC0680, Solarbio, Beijing, China), CK-MB (SEKR-0059, Solarbio, Beijing, China), TNF-α (PT516, Beyotime, Shanghai, China), IL-6 (PI328, Beyotime, Shanghai, China), SOD (A001–3-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and MDA (A003–1-2, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were assessed using corresponding kits, following the manufacturers’ instructions.

TdT-catalyzed dUTP Nick-end labeling (TUNEL) assay

Myocardial tissue section of rats underwent dewaxing and repair, then TdT enzyme (Beyotime, Shanghai, China) was added for a 60-minute reaction at 37 °C. Afterward, streptavidin labeled with horseradish peroxidase (HRP) was introduced and left to interact for 30 minutes at 37 °C. After 3,3′ Diaminobenzidine (DAB) staining (Beyotime, Shanghai, China) and hematoxylin staining for a duration of 10 minutes, the cells undergoing apoptosis displayed brown particles.

Immunohistochemistry (IHC) staining

After dewaxing and repairing the antigen, the paraffin tissue section was diluted with a solution of 1:1000. Then, the rabbit anti- α-SMA (ab7817, Abcam, UK), HIF-1α (ab16897, Abcam, UK), and sestrin2 (ab178518, Abcam, UK) antibody diluent was added. The resulting mixture was incubated at 4 °C overnight, after which a second antibody (1:1500 dilution) was added for further incubation. The resulting mixture was subjected to DAB coloration, hematoxylin re-staining, and microscopic observation of a-SMA, HIF-1α, and Sestrin2. Positive cells were identified by their brown-yellow coloration with a microscope (Leica, Germany).

Western blot

Total protein from myocardial tissue was lysed by RIPA lysis buffer from Beyotime (Shanghai, China). The protein content was assessed using the BCA kit (Solarbio, Beijing, China), the sample was subsequently combined with 2 × SDS loading buffer. The membrane transfer was conducted using SDS polyacrylamide gel electrophoresis. Subsequently, the PVDF membrane (Millipore, USA) was detached to indicate its orientation and then sealed with 5% TBST skimmed milk powder. The primary antibodies HIF-1α (ab16897, Abcam, UK), sestrin2 (ab178518, Abcam, UK), p-AMPK (Cell Signaling Technology, USA), and AMPK (Cell Signaling Technology, USA) were incubated overnight at 4 °C. The membranes were washed with TBST, followed by the addition of the second antibody, which was labeled with HRP and incubated at 37 °C for 60 minutes, then washed the membrane with TBST. The color development process was facilitated by the utilization of the enhanced chemiluminescence solution (ECL, Solarbio, Beijing, China), and the resulting image was captured. The densitometry analysis of the western blots was performed using the Image J software (NIH, USA).

Statistical analyses

The statistical analysis of the data was conducted using SPSS 23.0 software (IBM, USA), and the drawing was completed with GraphPad Prism 8 software. The mean ± SD was used to present the experimental results. Statistical significance was determined using a one-way ANOVA followed by Turkey’s post hoc test for comparing multiple groups for data followed a normal distribution, with a significance level of P < 0.05.