Thirty 10-week-old Sprague Dawley male rats, weighing 200 $\pm$ 10 g, were acquired from Beijing Vital River Laboratory Animal Technology (Beijing, China). The rats were provided with free access to food and water. A 12-h light-dark cycle was applied. The Ethics Committee of Affiliated Hospital of Jiangnan University granted approval for the study (JN. No20240630), in alignment with the “Guide for the Care and Use of Laboratory Animals” issued by the US National Institutes of Health.

The following five groups were randomly divided to all rats: (1) Sham group: The rats underwent a sham operation. (2) Ischemia-reperfusion (I/R) group (I/R model rats): The rats underwent an ischemia-reperfusion operation. (3) I/R+saline group: I/R model rats were intraperitoneally administrated with saline (1:10, HBPT033, Qingdao Haibo Biological, Qingdao, China). (4) I/R+PF-4708671 group: I/R model rats were intraperitoneally administrated with 75 mg/kg PF-4708671 (ZY6M2271, Shanghai Zeye Biotechnology Co., Ltd., Shanghai, China) (PF-4708671 was injected intraperitoneally for 1 day before ischemia). (5) I/R+PF-4708671+RIP3 group: Administer an intraperitoneal injection of 50 mg/kg RIP3 activator to the I/R+PF-4708671 treatment group of rats (HY-144828, MedChemExpress, Middlesex County, NJ, USA) (RIP3 activator was injected intraperitoneally at the beginning of the ischemia–reperfusion operation). PF-4708671 was dissolved in a saline solution as previously described [16].

The model for MI/R injury was developed following the previously outlined methodology [23]. Briefly, the rats were anaesthetized with 3% isoflurane (132220, Klamar, Shanghai, China) using a gas inhalation anesthesia machine (ABS-6A, YUYAN INSTRUMENTS, Shanghai, China). Following tracheotomy, a 7-0 nylon suture was used to ligate the left anterior descending artery (LAD), and a silicone tube (outer diameter = 86 mm) was inserted 1 mm below the ligation to induce myocardial ischemia. The success of the occlusion was confirmed by ligating the distal ventricle until it appeared white. After 30 minutes of occlusion, the silicone tube was removed to facilitate blood reperfusion for a duration of 2 hours, the restoration of myocardial color after reperfusion indicates that the I/R model has been successfully established [24, 25]. Then, 2 hours after surgery, anesthesia was provided by intraperitoneal injection of 1% pentobarbital sodium (40 mg/kg) (20090512, SINOPHARM, Beijing, China). Cervical dislocation: cervical dislocation is performed manually and euthanasia is achieved in approximately 10–15 seconds. The heart tissue and blood samples were collected for pathological evaluation and biochemical analysis, respectively.

Heart tissues were fixed in 4% formaldehyde (P6148, Sigma-Aldrich, St. Louis, MO, USA) for 48 h, dehydrated in an alcohol gradient series, and paraffin-embedded. Tissues were cut into approximately (~5-µm slices) and subsequently stained with hematoxylin (ST2023, Saint-Bio, Shanghai, China) for 3 minutes, followed by eosin for 30 seconds. Stained tissue sections were sealed with neutral resin and stored at room temperature. Heart tissue pathological assessments were performed under a light microscope (BX43, Olympus, Tokyo, Japan).

Myocardial infarction (MI) areas were measured by 2,3,5-triphenyltetrazolium chloride (TTC, BB-44549, Bestbio, Shanghai, China) staining. The collected hearts were quickly sectioned into 5 to 6 thin transverse slices and incubated in 1% TTC for 30 min at 37 °C to delineate the infarct area from viable myocardium. The MI region was imaged by a digital camera (v30, Epson, Suwa city, Japan) and quantified using Image J v1.8.0 software (NIH, Madison, WI, USA). Infarct size (%) was calculated using the following equation: Infarct myocardium area/(infarct myocardium area + normal myocardium area) $\times$ 100%.

After 30 minutes of occlusion, two hours of reperfusion were performed. Echocardiography was performed in mice 3 days after I/R to detect cardiac function. Isoflurane (0.5–1.5% in oxygen) anesthesia lasts 10–15 min and positioned on an orbital system to ensure that their body temperature was maintained at 37 °C $\pm$ 0.5 °C. Echocardiographic assessments were conducted with a high-resolution ultrasound system designed for small animals, featuring a 40 MHz single crystal transducer (Prospect, S-Sharp, Taipei, Taiwan). An M-mode image of the left ventricular papillary muscle level was obtained from the long axis view and used to evaluate the left ventricular systolic pressure (LVSP), the left ventricular end-diastolic pressure (LVEDP), the maximum rate of increase in the left ventricular pressure (+dp/dtmax), and the maximum rate of the decrease in left ventricular pressure (-dp/dtmax).

Blood were taken and spun at 5000 rpm for 10 minutes. Plasma samples were collected, and 20 µL of each sample was reacted with 200 µL of the working solution to identify cardiac troponin-1 (cTn-I), lactate dehydrogenase (LDH), creatine kinase-MB (CK-MB), and aspartate aminotransferase (AST) were measured using ELISA kits (cTn-I, yb-E12746, Shanghai Yubo Biotechnology Co., Ltd., China; LDH, SP12758; CK-MB, SP12933; AST, SP12839, Wuhan Saipei Biotechnology Co., Ltd., Wuhan, China).

APO-BrdU™ TUNEL assay kit (A23210, Thermo Fisher, Mountain View, CA, USA) was utilized to conduct TUNEL staining for the identification of apoptotic cells. Tissue sections (~5 µm) were permeabilized in 10% Triton for 10 min and stained with TUNEL staining solution for 1 h. 4,6-dianmidino-2-phenylindole (DAPI) staining (PM11055, PERFEMIKER, Shanghai, China) was performed on each section for a duration of 15 minutes at room temperature. Subsequently, the slices were examined and imaged using a fluorescence microscope (Ti2-U, Nikon, Tokyo, Japan).

Extracted proteins were subjected to denaturation in a loading buffer and boiled. The samples (~40 µL) were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (1620177, Bio-rad, Hercules, CA, USA). The membranes were probed with antibodies against RIP3 (ab226297, 1:500, Abcam, Cambridge, UK), p-RIP3 (phospho T231 + S232) (ab222320, 1:500, Abcam, Cambridge, UK), S6K1 (ab32529, 1:500, Abcam, Cambridge, UK), p-S6K1 (phospho T229) (ab59208, 1:500, Abcam, Cambridge, UK), Bcl-2 (ab32124, 1:500, Abcam, Cambridge, UK), Bax (ab32503, 1:500, Abcam, Cambridge, UK), cleaved-caspase3 (ab32042, 1:500, Abcam, Cambridge, UK), caspase3 (ab32351, 1:500, Abcam, Cambridge, UK), RIP1 (ab202985, 1:500, Abcam, Cambridge, UK), P-MLKL (ab196436, 1:500, Abcam, Cambridge, UK), MLKL (ab184718, 1:500, Abcam, Cambridge, UK) and $\beta$-actin (ab8227, 1:500, Abcam, Cambridge, UK) at 4 °C overnight. Subsequently, goat anti-rabbit immunoglobulin G (IgG) H&L (HRP) Secondary antibodies (ab6721, 1:1000, Abcam, Cambridge, UK) were applied to the membrane. The blots were developed with Immobilon ECL Ultra Western HRP Substrate (cat. no. WBULS0500, Merck Millipore, USA) and imaged on a ChemiDoc XRS Imaging System (1708265, Bio-Rad, California, USA). Finally, ImageJ software was used for greyscale analysis.

Following the blood collection, the hearts were preserved in 4% paraformaldehyde (P6148, Sigma-Aldrich, USA), buried in paraffin, and then cut into measuring 3–4 µm, which were placed on slides. The resulting sections were subsequently stained with DAPI (PM11055, PERFEMIKER, Shanghai, China)/propidium iodide (PI) (HY-D0815, MedChemExpress, NJ, USA) in accordance with the provided protocol. Observations and photographic documentation were conducted using a fluorescent microscope (Ti2-U, Nikon, Tokyo, Japan).

Frozen heart tissues were lysed in non-denaturing lysis buffer (20 mM, pH = 8.0, Tris HCl, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40) supplemented with Calbiochem® Protease and Phosphatase Inhibitor Cocktails (Calbiochem, Merck, Darmstadt, Germany). Then the samples were pre-cleared with protein G-immobilized agarose for 1 h at 4 °C, followed by incubation with S6K1 or RIP3 antibodies at 4 °C overnight. The samples were then subjected to SDS-PAGE/immunoblotting. The primary antibodies against S6K1 (1:1000, AB 32529, Abcam, Cambridge, UK), RIP3 (1:1000, AB 226297, Abcam, Cambridge, UK), and rabbit IgG, Isotype Control (1:1000, Ab172730, Abcam, Cambridge, UK) were added to the experiment. Goat anti-rabbit IgG H&L (HRP) (1:1000, ab6721, Abcam, Cambridge, UK) was used as the secondary antibody.

Each experiment was repeated three times in this study, and the results were expressed as means and standard deviations (SD). Statistical analysis of the data was conducted using GraphPad Prism 7 software (Dotmatics, San Diego, CA, USA). Multigroup comparisons were conducted using one-way analysis of variance (ANOVA) with Tukey’s post hoc test, while comparisons between two groups were performed using the t-test. Normal distribution data were subsequently analyzed using the Bonferroni test. A p-value of less than 0.05 was regarded as statistically significant. Unless indicated otherwise, all n values pertain to mice and are specified in the figure legends.

First, a western blot experiment showed that p-S6K1 (Thr389/Thr412) in the I/R+PF-4708671 group was markedly down-regulated compared with the other three groups. The results showed that PF-470867 inhibited S6K1 activity (Fig. 1A) (p 0.05). Then, H&E staining showed that the heart from the sham rat had viable myocardial tissue with a low level of tissue damage. In the I/R and I/R+ saline groups, there were apparent interstitial edema, the disappearance of myocardial fiber transverse striations, decreased cytoplasm level, and inflammatory infiltration (red arrows) in the heart tissues of the rats. Interstitial edema and inflammatory infiltration (red arrows) indicated a milder condition in rats in the I/R+PF-4708671 group compared with the I/R group (Fig. 1B). Finally, the MI region was measured by TTC staining, which showed that compared with the sham group, the I/R-induced infarct size was significantly increased, while the infarct size decreased after treatment with PF-4708671 (Fig. 1C) (p 0.05). These findings indicated that inhibiting S6K1 activity could reduce I/R-induced myocardial injury.

Fig. 1.

Inhibition of S6K1 mitigated I/R-induced myocardial injury. (A) Expression of p-S6K1; S6K1 was detected by western blotting (n = 6 rats each). (B) H&E staining of myocardial tissue (n = 6 rats each). Red arrows, interstitial edema and inflammatory infiltration. Scale bars, 50 µm. (C) Myocardial infarct size was assessed by TTC staining (n = 6 rats each) (*p 0.05 vs. the sham group; ^#p 0.05 vs. the I/R group; ^%p 0.05 vs. I/R + the saline solution group). S6K1, p70 ribosomal S6 kinase; TTC, 2,3,5-triphenyltetrazolium chloride; H&E, Hematoxylin-eosin; I/R, Ischemia-reperfusion.

Echocardiography was used to determine LVSP, -dp/dtmax, LVEDP, and +dp/dtmax levels to evaluate the effect of S6K1 inhibition on myocardial function in I/R rats. As shown in Fig. 2A, compared with the sham group, the expression of LVSP and $\pm$dp/dtmax induced by I/R was significantly lower, while the expression of LVEDP was markedly higher, which were reversed after PF-4708671 treatment (Fig. 2A) (p 0.05). Subsequently, the expression of cTn-I, LDH, CK-MB, and AST was determined by ELISA, with the results showing significantly increased expression of cTn-I, LDH, CK-MB, and AST induced by I/R compared with the sham group; however, PF-4708671 reversed the above effects (Fig. 2B) (p 0.05). These findings suggest that inhibiting S6K1 improved myocardial function in rats receiving myocardial I/R.

Fig. 2.

Inhibition of S6K1 improved myocardial function. (A) The LVSP, LVEDP, +dp/dtmax, and -dp/dtmax levels of the sham, I/R, I/R + saline solution, and I/R + PF-4708671 groups were measured (n = 6 rats each). (B) cTn-I, AST, LDH, CK-MB levels of the above four groups were measured (n = 6 rats each) (*p 0.05 vs. the sham group; ^#p 0.05 vs. the I/R group; ^%p 0.05 vs. the I/R + saline solution group). cTn-I, cardiac troponin-1; LDH, lactate dehydrogenase; CK-MB, creatine kinase-MB; AST, aspartate aminotransferase; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; +dp/dtmax, maximum rate of increase in the left ventricular pressure; -dp/dtmax, maximum rate of the decrease in left ventricular pressure.

Cardiomyocyte apoptosis was assessed by TUNEL staining, which showed a pro-apoptosis effect following I/R induction compared to the sham group, which was reversed by PF-4708671 treatment (Fig. 3A) (p 0.05). Of note, compared to the Sham group, the expression of Bcl-2 was decreased and the expression of cleaved-caspase-3/caspase-3 and Bax was increased after I/R induction; however, these effects were reversed after treatment with PF-4708671 (Fig. 3B) (p 0.05). The results indicate that inhibiting S6K1 activity reduces apoptosis in cardiomyocytes, with the apoptotic pathway being affected by PF-4708671.

Fig. 3.

Inhibition of S6K1 activity ameliorated myocardial apoptosis and necrosis in I/R rats. (A) Myocardial apoptosis was detected by the TUNEL staining (n = 6 rats each). Scale bars, 200 µm. (B) Bcl-2, Bax, cleaved-Caspase-3, and Caspase-3 in the myocardial tissue were detected by western blotting (n = 6 rats each). (C) The necrosis of I/R rat cardiomyocytes assessed by DAPI–PI staining (n = 6 rats each). Scale bars, 200 µm. (D) Western blot analysis was used to assess the expression of key molecules for necrosis in myocardial tissue (n = 6 rats each) (*p 0.05 vs. the sham group; ^#p 0.05 vs. the I/R group; ^%p 0.05 vs. the I/R + saline solution group). MLKL, mixed lineage kinase domain-like protein; RIP1, receptor-interacting protein kinase 1; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; DAPI, 4,6-dianmidino-2-phenylindole; PI, propidium iodide; RIP3, receptor-interacting protein kinase 3.

To determine whether PF-4708671 improves necrosis in cardiomyocytes of I/R rats, we conducted DAPI-PI staining and assessed necrosis markers. The DAPI–PI staining results showed that compared with the sham group, the number of necrosis cells in the I/R group was markedly increased, while the number of necrosis cells decreased after treatment with PF-4708671 (Fig. 3C) (p 0.05). The western blot analysis results suggested that the levels of RIP3, RIP1, and p-MLKL/MLKL proteins in the I/R+PF-4708671 group were lower than those in the I/R and I/R+ saline groups (Fig. 3D) (p 0.05). These findings indicated that inhibiting S6K1 activity improved the necrosis of cardiomyocytes in I/R rats.

Concerning the relationship between RIP3 and S6K1, protein blot analysis showed that after I/R induction, the expression of phosphorylation-S6K1/S6K1 and phosphorylation-RIP3/RIP3 increased significantly compared to the sham group; however, PF-4708671 treatment reversed the effects (Fig. 4A) (p 0.05). These findings suggested that I/R injury-induced RIP3 phosphorylation could be downregulated by the S6K1 inhibitor, i.e., PF-4708671-treatment. Furthermore, the co-immunoprecipitation assay was conducted to determine whether activated RIP3 directly. As shown in Fig. 4B,C, S6K1 was detected after immunoprecipitation with RIP3 antibody and RIP3 after immunoprecipitation with S6K1 antibody, but the expression of both was not detected in the control IgG group, respectively (Fig. 4B,C), indicating that there is a reciprocal binding effect of S6K1 and RIP3.

Fig. 4.

RIP3 was a direct target of S6K1. (A) p-S6K1, S6K1, p-RIP3, and RIP3 were detected in the myocardial tissue by western blotting (n = 6 rats each). (B,C) Co-immunoprecipitation was performed in the myocardial tissue. The protein complex was downregulated by S6K1 (B) or RIP3 (C) or immunoglobulin G (IgG), followed by immunoblotting with S6K1 and RIP3 antibodies, respectively. IP, Immunoprecipitation. (*p 0.05 vs. the sham group; ^#p 0.05 vs. the I/R group; ^%p 0.05 vs. the I/R + saline solution group).

To clarify whether the activation of RIP3 can block the protective effect of S6K1 inhibition on myocardium I/R injury, we treated I/R-induced myocardial tissue with a RIP3 activator for subsequent experiments. H&E staining showed that the rats in the I/R + PF-4708671 + RIP3 activator 1 group exhibited significantly aggravated interstitial edema and inflammatory infiltration compared with the I/R+PF-4708671 group (Fig. 5A). Subsequently, we carried out TTC staining on I/R-induced myocardial tissue after treatment with RIP3 activator. The results showed that compared with the I/R+PF-4708671 group, the infarct size increased after treatment with RIP3 activator (Fig. 5B) (p 0.05). In addition, RIP3 activator blocked the protective effect of PF-4708671 on cells (Fig. 5C) (p 0.05). The results from the western blot analysis indicated that RIP3 activator blocked the alleviated effect of PF-470867 on the I/R-induced downregulation of RIP1, RIP3, and p-MLKL/MLKL (Fig. 5D) (p 0.05). These data indicated that activation of RIP3 blocked the protective effect of S6K1 inhibition on against myocardium I/R injury.

Fig. 5.

Activation of RIP3 blocked inhibition of S6K1 effects on myocardium ischemia/reperfusion injury. (A) Myocardial tissue was detected by Hematoxylin-Eosin staining (n = 6 rats each). Red arrows, interstitial edema and inflammatory infiltration. Scale bars, 100 µm. (B) The size of the myocardial infarction was assessed by TTC staining (n = 6 rats each). (C) I/R rat cardiomyocyte necrosis assessed by DAPI-PI staining (n = 6 rats each). Scale bars, 200 µm. (D) The expression of key molecules for necrosis in myocardial tissue assessed by Western blot analysis (n = 6 rats each) (*p 0.05 vs. the I/R group; ^#p 0.05 vs. the I/R + saline solution group; ^%p 0.05 vs. the I/R + PF-4708671 group).