Male Wister rats weighing 220 ± 10 g were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All rats were kept in the Animal Experiments Committee of Zhengzhou University (Ethics No. ZZULAC20211112[06]) at 24°C with a 12-h light/dark cycle and free access to food and water. This study conformed to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).

To induce cardiotoxicity, the rats were injected with a cumulative dose of 15 mg/kg Dox (HY-15142, Med Chem Express, Monmouth Junction, NJ, USA) or vehicle via three weekly injections (2.5 mg/kg i.p. once every 3 days). To investigate the role of ticagrelor in vivo, 1 h before each Dox injection, the rats were injected with 7.5 mg/kg ticagrelor (i.g., HY-10064, Med Chem Express). There were 12 rats in each group, 36 rats in total. Subsequent analyses were performed 8 weeks after the last injection. At the end of the experiment, six rats per group were sacrificed to make heart slices and other rats heart and blood samples were collected.

Rat echocardiography was performed under isoflurane anesthesia using a Visualsonics imaging system under M-mode (Vivo 2100, Toronto, Canada). General anesthesia was initially induced with 5% isoflurane oxygen and then reduced to 1.5% to maintain anesthesia. Left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF), left ventricular end-systolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD) were measured using software included in the Visualsonics system.

Rats heart were washed with cold saline and placed in 4% paraformaldehyde at 25°C for 24 h. Four micrometers sections were collected and subjected to hematoxylin and eosin (HE), Masson’s trichrome, and wheat germ agglutinin (WGA) (#L4895; Sigma Aldrich, USA) staining. An optical microscope (BX60; Olympus Corporation, Tokyo, Japan) was used to observe stained sections. Images were analyzed using ImageJ Launcher 1.8.0 (National Institutes of Health, Bethesda, MD, USA).

Heart sections (4 μm) were fixed with 4% paraformaldehyde, blocked with 10% fetal bovine serum (FBS, #35-081-CV, Corning Inc., Corning, NY, USA), and permeabilized with Triton X-100. Anti-p-Ser9-GSK-3β (1:200, #93235, Cell Signaling Technology, Danvers, MA, USA), anti-cleaved-capsase-1 (1:200, #PA5-99390, Invitrogen, Carlsbad, CA, USA), anti-GSDMD-NT (1:200, #PA5-115330, Invitrogen) and anti-ASC (1:200, #6741R-A5555, Bioss) antibodies were incubated overnight at 4°C, followed by secondary antibodies conjugated to Alexa Fluor 680 (1:500, #A10043, Invitrogen) and 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (#S2110, Solarbio, China). Anti-α-actinin was purchased from Abcam (Cambridge, MA, USA, 1:200, #ab68194). All slices were analyzed using the EVOS M7000 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).

After heart sections (4 μm) were fixed, blocked and permeabilized, anti-IL-1β (1:200, #PA5-105048, Invitrogen) and anti-IL-18 (1:200, #ab191860, Abcam) antibodies were used to incubate the slices for 2 h, followed by incubation with secondary antibodies conjugated to AlexaFluor 488 (1:200; Invitrogen) or AlexaFluor 594 (1:200; Invitrogen) at 37°C for 1 h. All slices were analyzed using the EVOS M7000 Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).

Serum levels of LDH, CK-MB, cTnI, and BNP levels were assessed using standardized commercially available kits (Jian Cheng, Nanjing, China). Serum IL-1β and IL-18 levels were assessed using rat enzyme-linked immunoassay (ELISA) kits (Abcam) according to the manufacturer’s instructions. The information of the above kits were shown in Table 1.

Both primary rat cardiomyocytes (RCMs) and rat embryonic cardiac-derived H9c2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS and 1% penicillin/streptomycin (#P1400, Solarbio, Beijing, China).

Clean heart ventricles were digested with 0.1 mg/ml trypsin (#T1300, Solarbio, Beijing, China) and 0.1 mg/ml collagenase II (#LS004176, Worthington, Lakewood, NJ, USA) from 1-day-old rat hearts for RCMs. RCMs were seeded in 6-well plates at 70–90% confluency and incubated for 24 h in medium containing various combinations of the following reagents: 1/10/20 μm ticagrelor and 0.1/1/10 μm Dox. All inhibitors were dissolved in 0.1% DMSO (#D8370, Solarbio) and an equivalent quantity of DMSO was used as a control.

H9c2 cells were purchased from ATCC (Manassas, VA, USA). When the cells reached 70–90% confluence, they were incubated for 24 h in medium containing the following reagents: 10 μm MCC950 (inhibitor of NLRP3, #HY-12815, MedChemExpress), 10 μm MK2206 (inhibitor of Akt, #HY-10358, MedChemExpress), 20 μm TDZD-8 (inhibitor of GSK-3β, #HY-11012, MedChemExpress), 25 μm VX765 (inhibitor of caspase-1, #HY-13205, MedChemExpress), 10 μm ticagrelor, and 1 μm Dox. All the inhibitors were dissolved in 0.1% DMSO, and an equivalent quantity of DMSO was used as a control.

RCMs were seeded in a 96-well plate. After culturing for 24 h with different pharmaceutical ingredients, 10 μl MTT (0.5 mg/ml, #HY-15924, MedChemExpress) was added and the cells were incubated at 37°C for 4 h. The culture medium was removed and DMSO was added. The absorbance was measured at 570 nm using a microplate reader (Thermo Fisher Scientific).

RCMs and H9c2 cells were seeded in 24-well plates and PI dissolved in 0.1% DMSO (2 μg/ml, MedChemExpress) was added. Images of the pyroptotic cells were captured using a Nikon Ti2 microscope (Nikon, Minato Ward, Tokyo, Japan) after 2 h.

The release of LDH into the supernatants was measured using the CyQUANT LDH Cytotoxicity Assay Kit (#C20301, Invitrogen) according to the manufacturer’s instructions.

H9c2 cells were seeded in 35 mm confocal dishes. After incubation with the ingredients, H9c2 cells were fixed with 4% paraformaldehyde for 2 h. Next, 2 mg/ml glycine was added to neutralize residual paraformaldehyde. The culture medium was discarded and the cells were washed with PBS. 0.5 μm rhodamine-labeled phalloidin incubated the dishes for 30 min. The cells were incubated with DAPI (0.5 M DAPI incubated them for 10 min. The cells were examined under an Eclipse E100 fluorescence microscope (Nikon, Japan).

Rat GSK-3β shRNA and negative control shRNA were constructed by Hanbio (Wuhan, China). H9c2 cells were seeded in 6-well plates and transfected with 2.5 μg of shRNAs using 5 μl Lipofectamine 3000 (Invitrogen). The sequences of the specific shRNAs are listed in Table 2.

The AAV9-shGSK-3β and AAV9-shNC constructs were constructed by Hanbio, using shRNAs (Table 2). The viral vector was injected into the heart after opening the thoracic cavity of rats. Two weeks before drug administration, AAV9 vectors (50 μl) at a dose of 1 × 10¹¹ viral genome particles were randomly injected into the left ventricle of the rats. These virus solutions were distributed to five sites. A single injection was administered to the left ventricular apex. Double injections at a spacing of 5 mm were performed in the anterior and lateral walls of the left ventricle. There were 12 rats in each group, 36 rats in total. At the end of the experiment, six rats per group were sacrificed to make heart slices and other rats heart and blood samples were collected.

Proteins from rat heart tissues and cell lysates were collected. Western blotting was performed using the standard methods. Membranes were incubated with specific antibodies against GSK-3β (1:1,000, #12456, Cell Signaling Technology), p-Ser9-GSK-3β (1:1,000), cleaved-capsase-1 (1:1,000), GSDMD-NT (1:1,000), NLRP3 (1:1,000, #ab263899, Abcam), ASC (1:1,000), p-Ser473-Akt (1:1,000, #4060, Cell Signaling Technology), Akt (1:1,000, #9272, Cell Signaling Technology), PARP (1:1,000, #191217, Abcam), caspase-3 (1:1,000, #184787, Abcam) and GAPDH (1:10,000, # 60004-1, Proteintech). The band intensities were analyzed using ImageJ.

Data are presented as the mean ± SD and were analyzed using SPSS (version 21.0; SPSS Inc., Chicago, IL, USA). Kolmogorov-Smirnov test was used to check whether the samples conform to the normal distribution. One-way analysis of variance followed by the Bonferroni post-hoc test was used to compare multiple groups. P-values less than 0.05 were considered statistically significant.

We used Dox to induce cardiotoxicity in rats and observed the effects of ticagrelor (Figure 1A). Echocardiographic analysis (Figure 1B) showed that Dox administration decreased LVEF and LVFS in rats, along with increased LVESD and LVEDD (Figure 1C) at the end of the experiments. Ticagrelor treatment significantly attenuated the LV dysfunction. Dox-induced heart lesions were confirmed by HE and Masson’s trichrome staining (Figure 1B). The morphology and arrangement of cardiac cells in the Con group were normal, while the cardiac structure in Dox group was damaged with obvious fiber breakage. Ticagrelor pretreatment reduced the area of fibrosis induced by Dox from 30.9 to 14.1% (Figures 1B, D). In addition, WGA staining (Figures 1B, E), the heart weight/body weight ratio (HW/BW) (Supplementary Table 1 and Figure 1F) and heart weight per tibia length (HW/TL) (Figure 1G) in rats confirmed that Dox induced cardiac hypertrophy, which could be improved by ticagrelor. As for cardiac-specific parameters, Dox significantly increased the serum level of LDH, CK-MB, cTnI, and BNP and ticagrelor suppressed their abnormality (Figure 1H).

Ticagrelor effectively prevented Dox-induced cardiac function damage in rats. Is this effect related to pyroptosis? How can pyroptosis be regulated? We observed pyroptosis in rat heart tissues. We performed immunofluorescence staining on rat heart sections and found that ticagrelor reduced the number of GSDMD-NT-positive cardiomyocytes induced by Dox (Figures 2A, D). Moreover, the expression of GSDMD-NT in the myocardial tissue protein extract showed a consistent trend (Figures 2H, I).

Pyroptosis mainly occurs via the canonical caspase-1-dependent pathway, and we examined whether Dox caused pyroptosis in rat cardiomyocytes through the activation of caspase-1. GSK-3β, has been proved to play a role in Dox induced cardiotoxicity (20, 21). Our results showed that after Dox administration, the proportion of p-GSK-3β positive heart slices decreased (Figures 2A, B) and cleaved-caspase-1 increased (Figures 2A, C). Western blot analysis showed that Dox decreased the p-GSK-3β/GSK-3β ratio (Figures 2E, F) and increase cleaved-caspase-1 expression (Figures 2E, G) at the protein level. Importantly, we found that ticagrelor increased the p-GSK-3β/GSK-3β ratio and cleaved-caspase-1 by Dox.

After GSDMD-NT forms plasma membrane pores, cells secrete IL-1β and IL-18 (15). In rat heart slices, Dox increased the expression of IL-1β and IL-18, and the addition of ticagrelor inhibited the induction of Dox (Figure 3A). Quantitative results also showed that ticagrelor reduced the Dox-induced high expression of IL-1β from 18.9 to 5.2% (Figure 3B) and IL-18 from 22.3 to 4.5% (Figure 3C). ELISA was used to determine the levels of IL-1β and IL-18 in rat serum, which showed that ticagrelor reduced the excessive secretion of IL-1β (Figure 3D) and IL-18 (Figure 3E) induced by Dox. Similarly, the expression of IL-1β (Figures 3F, G) and IL-18 (Figures 3F, H) proteins in the rat heart tissue was promoted by Dox and decreased by ticagrelor. The release of IL-1β and IL-18 is closely related to the activity of NLRP3 inflammasome. We detected the ASC sparks in the rats heart tissue slices, which indicates the activation of NLRP3 inflammasome. The results showed that Dox increase the number of ASC sparks and ticagrelor inhibit the formation of ASC sparks (Figure 3I). Similarly, Dox increased the protein expression of NLRP3 inflammasome components in rats heart, while ticagrelor preconditioning decreased their abnormal increase (Figures 3J–L).

We verified the effect of ticagrelor on Dox-induced pyroptosis in RCMs and H9c2 cells. Viability was evaluated using the MTT assay. Under normal conditions, ticagrelor at a concentration of 20 μm did not reduce cell viability over 24 h. A dose-dependent increase in cell viability was observed following 1 h pretreatment with ticagrelor (1, 10, and 20 μm) and Dox (0.1, 1, and 10 μm) (Figure 4A). A concentration of 10 μm ticagrelor and a concentration of Dox (1 μm) were used for the subsequent in vitro experiments. The immunofluorescence results for α-actinin showed that Dox significantly increased the surface area of RCMs and could be inhibited by ticagrelor (Figures 4B, C). Western blot analysis showed that the p-GSK-3β/GSK-3β ratio was decreased (Figures 4D, E), whereas cleaved-caspase-1 expression (Figures 4D, F) and GSDMD-NT were increased (Figures 4G, H) in the Dox group. Ticagrelor inhibited changes in these proteins. Moreover, ticagrelor reduced the number of PI-positive (Figure 4I) and LDH-releasing cells (Figure 4J) upon Dox stimulation.

Similar to the above results in RCMs, ticagrelor countered the cell surface area of H9c2 cells (Figures 5A, B), the p-GSK-3β/GSK-3β ratio (Figures 5C, D), and the expression of cleaved-caspase-1 expression (Figures 5C, E) and GSDMD-NT (Figures 5F, G) proteins induced by Dox in H9c2 cells. Ticagrelor also reduced the number of PI-positive (Figure 5H) and LDH-releasing cells (Figure 5I) after Dox stimulation. Although the effect of Dox on pyroptosis is increasingly recognized, DOX-induced apoptosis is still the main mechanism of cell death. We measured cleaved caspase-3 and cleaved PARP in rats heart, RCMs and H9c2 cells to confirm this (Supplementary Figure 1). Ten micrometers MCC950 showed no effects on cell viability and cytotoxicity (Supplementary Figure 2). We added MCC950 and ticagrelor, respectively, before adding Dox, and finally detected the expression changes of NLRP3 and ASC (Figures 5J–L). The results showed that both MCC950 and ticagrelor prevent the deleterious action of Dox.

The in vivo and in vitro results of this study indicate that Dox decreases the content of phosphorylated GSK-3β at serine 9, which seems to be related to pyroptosis in H9c2 cells. Akt inactivates GSK-3β by phosphorylating it at serine 9 (22). The effect of Dox on Akt expression was also investigated. As shown in Figure 6, the ratio of p-Akt (Ser473)/Akt in rat heart tissue (Figures 6A, B), RCMs (Figures 6A, C), and H9c2 cells (Figures 6A, D) was suppressed by Dox stimulation, and ticagrelor increased the phosphorylated level of Akt. To further verify the role of the GSK-3β/caspase-1/GSDMD pathway in Dox-induced pyroptosis of H9c2 cells, the Akt inhibitor MK2206 (10 μm), GSK-3β inhibitor TDZD-8 (20 μm) and caspase-1 inhibitor VX-765 (25 μm) were applied to H9c2 cells. According to the quantitative analysis of proteins in Figure 6E, the results showed that inhibition of Akt, GSK-3β and caspase-1 effectively alleviated the Dox-induced increase in GSDMD-NT in H9c2 cells, that is, it alleviated pyroptosis in H9c2 cells. Compared with the DOX group, MK2206 affected not only the p-Akt/Akt ratio but also the p-GSK-3β/GSK-3β ratio and cleaved-caspase-1. TDZD-8 altered the p-GSK-3β/GSK-3β ratio and cleaved-caspase-1. However, VX-765 did not interfere with the ratios of p-Akt/Akt and p-GSK-3β/GSK-3β (Figures 6F–I). Therefore, in Dox-induced cardiotoxicity, we speculate that GSK-3β plays a regulatory role upstream of caspase-1 and that the pathway could be GSK-3β/caspase-1/GSDMD.

If the effect of ticagrelor in alleviating Dox-induced pyroptosis is mediated by GSK-3β, how will knockdown of GSK-3β influence the effect of ticagrelor? We used two significant shRNAs to knock down GSK-3β in H9c2 cells (Figures 7A, B), after which ticagrelor was added for pretreatment and DOX was incubated for 24 h. The expressions of cleaved-caspase-1 and GSDMD-NT were detected using western blotting (Figure 7C). The results showed that shGSK-3β 1/2 weakened the inhibitory effect of ticagrelor on cleaved-caspase-1 (Figure 7D) and GSDMD-NT (Figure 7E).

In vivo, shGSK-3β/1 and shGSK-3β/2 were injected into rat hearts using the AAV9 vector (Figure 8A). After verification by western blotting, the shGSK-3β/2 sequence was selected (Figures 8B, C). Consistent with the results of the cell experiments, knockdown of GSK-3β in the rat heart reduced the potency of ticagrelor to reduce cleaved-caspase-1 and GSDMD-NT protein (Figures 8D–F). In addition, we evaluated cardiac function (Figures 8G, H), pathological characteristics (Figure 8I), cardiomyocytes area (Figure 8J), the ratios of HW/BW (Figure 8K) and HW/TL (Figure 8L), and serum cardiac-specific parameters (Figure 8M) of the rats in each group. The efficacy of ticagrelor against Dox-induced cardiac injury has been evaluated previously. These results showed that knockdown of GSK-3β reduces the effect of ticagrelor, which is specifically manifested by the reduction of LVEF and LVFS, and the recovery of LVEDD, LVESD, fibrotic area, cardiomyocytes area, LDH, CK-MB, cTnI, BNP, HW/BW, and HW/TL.