Protective Effects of High-Intensity Interval Training on Myocardial Ischemia-Reperfusion in Rats and Its Regulatory Mechanisms

Determination of the area at risk and MI sizeThe pigmentation techniques of Zhao et al. (2021) were applied. Once the experiment was concluded, each group consisted of six rats, and their hearts were collected. Then, an intravenous injection of heparin (1,000 U) was administered, followed by the resection of the heart and re-ligation of the previous myocardial infarction (MI) site. Afterward, we injected 1% Evans blue dye (MilliporeSigma) through the ascending aorta, and upon completion of the perfusion, we applied five transverse sections through the left ventricle and interventricular septum. Then, we submerged the slices in a solution containing 1% triphenyl tetrazolium chloride and then subjected to incubation in a thermostatic water bath set to 37°C for a duration of 20 min. After incubation, the portions were weighed and treated with a 10% formalin solution at room temperature for a period of 24 h in order to ensure fixation. The colors blue, pale, and red respectively corresponded to normal, infarcted, and ischemic tissue. By adding the weight of the myocardial tissue within the vascular territory defined by AAR, which is located distal to the culprit lesion of the infarct-related artery, the total weight of the AAR and MI was calculated. AAR and MI are expressed as percentages, with AAR being calculated as the ratio of the weights of the red area over the left ventricle, and MI being calculated as the weights of the white area over the red area (El-Shitany et al., 2012).TUNEL staining analysis of the heartsFollowing completion of the experiments, four rats from each experimental group were taken, and their hearts were sectioned into 4-μm thick slices and subsequently fixed with acetone at ambient temperature for a duration of 24 h. Subsequently, the samples were transferred to a solution comprising a conjugate of horseradish peroxidase and terminal deoxynucleotidyl transferase along with detection buffer (Roche Diagnostics GmbH), and then incubated at a temperature of 37°C for a duration of 60 min. A diamino‑benzidine‑chromogen (Boehringer) was used. To determine the final number of TUNEL-positive nuclei, several additional steps were carried out. Initially, the samples were evaluated through an inverted light microscope (CKX53; Olympus Corporation) at a magnification of ×400. Then, we selected a random area and counted the nuclei within that area. The value was subsequently converted to a percentage by dividing it by the total number of cell nuclei.Calculation of serum levels of TNF-α, IL-1β, IL-6, and IL-10The blood samples were collected from six rats in each group at the end of the experiments. An ELISA was utilized to measure the serum levels of TNF-α (RTA00), IL-1β (RLB00), IL-6 (R6000) and IL-10 (R1000). The assay was carried out using a commercially available ELISA kit (R&D Systems, Inc., Minneapolis, Minnesota, USA) and performed in accordance with the manufacturer's instructions. Briefly, serum was pipetted in wells precoated with specific antibody for rat TNF-α, IL-1β, IL-6, or IL-10 and allowed to incubate for 2 h.After wells were rinsed to remove all unbound substance, an enzyme-linked antibody specific for rat TNF-α, IL-1β, IL-6, and IL-10 was added to wells for 2 h.Following the removal of unbound enzyme-linked antibody, the wells were thoroughly washed. A substrate solution was subsequently introduced into the wells and left to react for 30 min, which resulted in the development of a colored product. Optical density readings at 450 nm were then obtained to quantify the intensity of the color. To calculate the concentration of IL-1, IL-6, IL-10, or TNF-α in each sample, known standards provided with the kit were employed in each assay. The concentration of each cytokine was measured in picograms per milliliter. For all kits utilized, the minimum detectable dose was 10 pg/ml, with both intra-assay and inter-assay variations of 10%.Western blot analysis of TNF-ɑ, activated Caspase-3, Bax, and Bcl-2 in the heartAfter completing the experiments, we submerged the hearts of four rats from each group in tissue protein extraction reagent (T-PER; Thermo Fisher Scientific, Inc.) at 4°C to enable homogenization. We prepared the myocardial cell lysate by treating the homogenized tissue with a cold lysis buffer comprising 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS. After being centrifuged at 10,000 × g for 10 min at 4°C, protein concentrations in the samples were assessed using the Bradford assay method. After loading 60 μg of protein onto a 15% SDS-PAGE gel, the proteins were separated and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% powdered skimmed milk in Tris-buffered saline comprising 0.1% Tween-20 (TBST) at 37°C for 30 min, and then incubated with anti-caspase-3 (cat. no. IMG-144A; Imgenex), anti-TNF-α (cat. no. sc-52746; Santa Cruz Biotechnology), anti-BAX (cat. no. NBP1-28566; Novus Biologicals), and anti-Bcl-2 antibodies (cat. no. 633502; BioLegend), all at a dilution of 1:1,000 in 5% powdered skimmed milk, at 4°C for 24 h. The nitrocellulose membranes were subsequently immersed in a solution containing a secondary antibody (m-IgGκ BP-HRP; cat. no. sc-516102; Santa Cruz Biotechnology; 1:1,000) that was conjugated to horseradish peroxidase, after which the membranes were washed with TBST. An enhanced chemiluminescence substrate system was utilized to visualize the peroxidase activity, and the membranes were then subjected to exposure on hyperfilms. β-actin (cat. no. 643802; BioLegend; 1:1,000) was included as a loading control and incubated at room temperature for 2 h. Densitometry analysis was performed using ImageJ version 1.6 (National Institutes of Health), and normalization against the background density was performed for each gel.