C57BL/6J mice aged 6–8 weeks were obtained from the HYcell Biotechnology (Wuhan, China) and allocated into three distinct groups: a sham group (which underwent surgery without ligation), an HCM group, and an HCM group subjected to 30 Gy radiation treatment group (n = 5 per group). To create a mouse model of pressure overload-induced HCM, TAC surgery was performed in accordance with established protocols [30]. In brief, the mice were anesthetized with a combination of 2% isoflurane (AErrane, Baxter, Deerfiel, IL, USA) and 1.0 L/min of 100% oxygen within an induction chamber. A longitudinal skin incision measuring 1.0 cm was made at the suprasternal notch to expose the thymus and aorta. Following this, a 2 mm longitudinal incision was made in the sternum. A bent 26 G needle and a 6-0 silk suture were then employed to ligate the aorta between the right innominate and left common carotid arteries. In the sham control group, identical procedures were executed, with the sole exception that the artery was not subjected to ligation.

One week following the successful establishment of the HCM mouse model, a radiation dose of 30 Gy was administered over a two-week period. The body weights of the mice across all three experimental groups were recorded initially at baseline and subsequently at regular intervals throughout the duration of the study. Observations were conducted regarding mental health status, dietary and water intake, urination and defecation patterns, as well as the condition of the skin in the irradiated area for each group. Any alterations or abnormalities were meticulously documented and monitored. All mice were euthanized via cervical dislocation, and cardiac tissues were subsequently harvested. The mice were provided with humane care and were fed in accordance with the standards set forth in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. All animal maintenance and experimental procedures were conducted in compliance with the approval of the Ethics Committee of the Huashan Hospital Institutional Review Board (No 2020 Huashan Hospital JS-590).

To assess cardiac morphology and function, echocardiographic analysis was conducted utilizing an ultrasound device (Mylab X5 Vet, Esaote, Genova, Italy), as outlined in a prior study [31]. In summary, mice were subjected to imaging under light anesthesia with a combination of isoflurane (1–2%, AErrane, Baxter, Deerfiel, IL, USA) and oxygen (1 L/min). Systolic and diastolic functions were evaluated through conventional M-mode and two-dimensional echocardiography, augmented by pulse-wave Doppler techniques. A range of parameters was documented, including fractional shortening (FS), left ventricular (LV) ejection fraction (LVEF), systolic posterior wall (SPW) thickness, LV posterior wall (LVPW) thickness, LV internal diastolic (LVID) diameter, LV internal systolic (LVIS) diameter, interventricular septal systolic (IVSS) thickness, interventricular septal diastolic (IVSD) thickness, LV end-diastolic volume, LV end-systolic volume, relative wall thickness, and heart rate. LV volumes were determined by directly tracing of the LV wall contour in the long-axis view to compute volumes at end-diastole and end-systole. For each parameter, three measurements were taken per subject.

H&E (Hematoxylin, H9627-25G, Sigma-Aldrich, St Louis, MO, USA; Eosin Y, D12621, Xiya Reagent, Linshu, Shangdong, China) and WGA (L4895-2M, Sigma-Aldrich, St Louis, MO, USA.) staining was performed to evaluate the cross-sectional area of cardiomyocytes and the degree of LV hypertrophy, respectively. Cardiac tissues from the murine subjects were harvested and subsequently fixed in formalin. Following standard protocols for dehydration utilizing ethanol and achieving transparency with xylene, the samples were embedded in paraffin for sectioning. Subsequently, 5 µm sections were then stained with H&E and WGA. Imaging was conducted using an Olympus fluorescence microscope (CX-21, Tokyo, Japan), and the cross-sectional area of the cardiomyocytes was quantified employing the ImageJ software1.53 (National Institutes of Health, Bethesda, MD, USA).

The HL-1 mouse cardiac muscle cell line (SCC065, acquired from Sigma-Aldrich, St.Louis, MO, USA) was cultured in Dulbecco’s Modified Eagle Medium (DMEM, 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin). DMEM and penicillin/streptomycin were obtained from Hyclone (SH30023, SH30022, SH30021, SH30265.01B, SH30809, SV30010, Logan, UT, USA), and FBS was purchased from Tianhang (141215, Huzhou, Zhejiang, China). All cell lines underwent validation through short tandem repeat (STR) profiling. All cell lines underwent validation using a MycoProbe Mycoplasma Detection kit (CULoo1B, R&D systems, Minneapolis, MN, USA) and were confirmed to be free of mycoplasma contamination. The cells were maintained in an incubator under a 5% CO₂ atmosphere at 37 °C until they reached the logarithmic growth phase. At this stage, the HL-1 cells were allocated into several experimental groups: a control group, a radiation group, a NLRP3 inhibitor group, a NLRP3 inhibitor (MCC950, S8930, Selleck, Houston, TX, USA) combined with radiation group, a cGAS overexpression group, and a cGAS overexpression combined with MCC950 and radiation group. The dose-response relationship regarding cell viability post-irradiation was assessed, revealing a decline in cell viability with increasing radiation doses. At a dose of 40 Gy, cell viability approximated the half-maximal inhibitory concentration (IC50), while at 50 Gy, viability fell below the IC50 threshold (refer to Supplementary Fig. 1). Consequently, a radiation dose of 40 Gy was selected for subsequent in vitro experiments with cells in the radiation group receiving a single exposure of 40 Gy via an X-ray irradiator (RS-2000, Radsource, Suwanee, GA, USA.).

For the transfection procedure, HL-1 cells were seeded at a density of 1.0$\times$10⁵ cells per well in 6-well plates and incubated overnight to allow for adherence. The transfection of the pCDNA3.1-FLAG-cGAS plasmid, which was synthesized by ELK Biotechnology (Wuhan, Hubei, China, refer to Supplementary Fig. 2), was performed utilizing Lipofectamine 3000 transfection reagent (11668019, Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s guidelines. Control groups were transfected with blank pCDNA3.1 vector. Additionally, to inhibit NLRP3, HL-1 cells were treated with 20 µM of MCC950.

To evaluate cell viability, a CCK-8 assay (C0038, Beyotime, Shanghai, China) was conducted in accordance with the manufacturer’s guidelines. A total of 5000 HL-1 cells were plated in each well of a 96-well plate, subjected to various experimental treatments, and incubated overnight. Subsequently, the cells were treated with 10 µL of CCK-8 per well and incubated for a duration of 1–4 hours at 37 °C in an incubator. The optical density at 450 nm (OD450) was then measured using a microplate reader (DR-200BS, Hiwell-Diatek, Wuxi, Jiangsu, China).

Whole heart tissue samples from the mice or HL-1 cells were subjected to lysis using a cell lysis buffer (AS1004, Aspenbio, Wuhan, Hubei, China) supplemented with a protease inhibitor cocktail (04693159001, Roche Diagnostics, Mannheim, Germany) and phenylmethylsulfonyl fluoride (PMSF, AS1006, Aspenbio, Wuhan, Hubei, China). The protein concentrations were quantified utilizing the BCA method (AS1006, Aspenbio, Wuhan, Hubei, China). For each well, 10 µg of protein was applied for separation via SDS-PAGE. Following this, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) employing a blot transfer apparatus (AS1015, Aspenbio, Wuhan, Hubei, China). After blocking with a blocking solution, the membranes were incubated overnight with primary antibodies, including anti-cGAS (1:1000, 26416-1-AP, Proteintech, Wuhan, Hubei, China), anti-cAMP (1:2000, ab134901, Abcam, Cambridge, UK), anti-STING (1:1000, 19851-1-AP, Proteintech, Wuhan, Hubei, China), anti-NLRP3 (1:1000, 15101, CST, Boston, MA, USA), anti-caspase-1 (1:500, AF5418, Affbiotech, Cincinnati, OH, USA), anti-cleaved caspase-1 (1:500, 89332, CST, Boston, MA, USA), anti-GSDMD (1:1000, ab219800, Abcam, Cambridge, UK), anti-GSDMD-N (1:1000, 10137, CST, Boston, MA, USA), and anti-$\beta$-Actin (1:5000, TDY051, Tdybio, Beijing, China). The following day, after washing the membranes five times, they were incubated with the appropriate secondary antibodies for 30 minutes at room temperature (RT). The secondary antibodies utilized in this study included goat anti-rabbit IgG-HRP, goat anti-mouse IgG-HRP, goat anti-rat IgG-HRP, rabbit anti-goat IgG-HRP, and rabbit anti-sheep IgG-HRP (1:10,000 dilution, BA1055, BA1075, BA1060, Boster, Wuhan, Hubei, China; ab97057, ab6747, Abcam, Cambridge, UK). Detection was performed using ECL substrate (AS1059, Aspenbio, Wuhan, Hubei, China) in accordance with the manufacturer’s instructions.

To assess the concentrations of Interleukin (IL)-1$\beta$, IL-18, and cGAMP in the supernatants, we employed Mouse IL-1 and Mouse IL-18 ELISA kits (ELK1271, ELK2269, ELK Biotechnology, Wuhan, Hubei, China), as well as a cGAMP ELISA kit (501700, Cayman, Ann Arbor, MI, USA), following the protocols provided by the manufacturers.

To evaluate the reduction in mitochondrial membrane potential, the JC-1 probe (C2006, Beyotime, Shanghai, China) was employed in accordance with the manufacturer’s guidelines. Cells were treated with the JC-1 solution at 37 °C for 30 minutes, ensuring protection from light exposure. Subsequent to the incubation period, the cells were rinsed three times with phosphate-buffered saline (PBS), and the cell nuclei were stained with DAPI (D8417-1MG, Sigma-Aldrich, St Louis, MO, USA) at RT for 20–30 minutes. Following the application of an anti-fade mounting medium, fluorescence microscopy (CX-21, Olympus, Tokyo, Japan) was employed to capture the resulting images.

The morphology of cell membranes was analyzed utilizing scanning electron microscopy (SEM) in accordance with established protocols [32]. Briefly, HL-1 cells were cultured on 10 mm glass coverslips and subsequently fixed for 2 h at RT in PBS buffer containing 2% paraformaldehyde and 3% glutaraldehyde. Following fixation, the cells were rinsed with PBS buffer and post-fixed in 1% osmium tetroxide (OsO₄) for 30 minutes. The cells were then dehydrated through a graded series of ethanol solutions. Following ethanol dehydration, the cells underwent critical point drying, were mounted onto stubs, and coated with a thin layer of conductive metal, specifically gold and palladium, via a sputter coating technique. SEM images were captured using the FEI Quanta 650 FEG SEM system (JEOL, Tokyo, Japan), and representative images were selected for inclusion in the results section.

To evaluate mitochondrial morphology, HL-1 cells were subjected to dual-labeling with MitoTracker^® Red (C1035, Beyotime, Shanghai, China) and PicoGreen^TM double-stranded DNA stain (P2023, UElandy, Hangzhou, Zhejiang, China). Briefly, HL-1 cells were incubated with PicoGreen^TM stain at RT in the dark for 3 minutes. Following this, the cells were washed three times with PBS and subsequently stained with MitoTracker^® Red (100 nM) for 20 minutes at 37 °C, while avoiding exposure to light. The cells were then fixed using 4% paraformaldehyde and counterstained with the nuclear dye DAPI. Imaging was captured by a confocal microscope (TCS SP5, Leica Microsystems, Wetzla, Germany), and the resulting images were analyzed using ImageJ software (version 1.6.0, National Institutes of Health (NIH), Bethesda, MD, USA).to quantify signal intensity.

Data were analyzed and profiled by GraphPad Prism 9.0 (La Jolla, CA, USA). Prior to statistical analysis, the normality of data distribution was assessed using a combination of the Shapiro-Wilk test and visual inspection methods, such as Q-Q plots and histograms. The results are presented as mean values for each experimental repetition, with the variability within each repetition represented by the standard deviation ($\pm$ SD). For datasets with a normal distribution, comparisons between two groups were conducted using a two-tailed Student’s t-test, while comparisons among multiple groups were assessed using a one-way analysis of variance (ANOVA) followed by Tukey’s post hoc post-test. For datasets that did not follow a normal distribution or when normality could not be reasonably assumed, non-parametric tests such as the Mann-Whitney U test or Kruskal-Wallis test with Dunn’s post hoc test were applied as appropriate. A p-value of less than 0.05 was considered statistically significant.

Echocardiographic assessments were conducted to evaluate alterations in cardiac morphology and function across three groups of experimental mice, which demonstrated notable differences in several cardiac parameters among the groups (Fig. 1A–C).

Fig. 1.

Radiation attenuates cardiac hypertrophy and improves cardiac function in a pressure overload-induced hypertrophic cardiomyopathy (HCM) mouse model. (A–C) M-mode echocardiography photos for mouse hearts from sham-operated (A), HCM (B), and HCM + radiation mouse groups (C). Scale bars represent 5 mm (vertical axis) and 250 ms (horizontal axis). Quantitative analysis of left ventricular ejection fraction (LVEF) (D), fractional shortening (FS) (E), LV posterior wall (LVPW) thickness (F), systolic posterior wall (SPW) thickness (G), LV internal diastolic (LVID) diameter (H), LV internal systolic (LVIS) diameter (I), LV end-diastolic volume (J), LV end-systolic volume (K), and relative wall thickness (L). Mean $\pm$ SD, n = 5 per group. **p 0.01, ***p 0.001, and ****p 0.0001.

In comparison to the sham-operated mice, the mice subjected to pressure overload-induced HCM mice exhibited a significant reduction in LVEF and FS (p 0.001, Fig. 1D,E), which signifies compromised cardiac function. Additionally, the HCM mouse model displayed an increase in LVPW thickness and SPW thickness (p 0.001, Fig. 1F,G), indicating the presence of hypertrophic alterations in the myocardium. Furthermore, the HCM mice showed an increase in LVID diameter and LVIS diameter (p 0.01, Fig. 1H,I), alongside a decrease in IVSD thickness (p 0.01) and IVSS thickness (p 0.05, Supplementary Fig. 3A,B), which collectively suggest an expansion of cardiac chamber dimensions.

Notably, the HCM mice that received a radiation dose of 30 Gy exhibited marked enhancements in cardiac function, as indicated by significantly elevated values of EF, FS, LVPW diameter, and systolic posterior wall thickness when compared to the HCM mouse model (Fig. 1D–G). While the LVID and LVIS diameter measurements in the HCM + 30 Gy group remained higher than those observed in the sham-operated mice, they were nonetheless reduced in comparison to the HCM mice (Fig. 1H,I).

The LV end-diastolic and end-systolic volumes were significantly reduced in the HCM + 30 Gy group in comparison to the HCM group (Fig. 1J,K), indicating a potential enhancement in cardiac remodeling. However, there was no observed alteration in the relative wall thickness, which represents the ratio of wall thickness to chamber diameter, between the HCM + 30 Gy radiation group and the HCM group (Fig. 1L). Additionally, no notable changes in heart rate were detected across all three experimental groups (Supplementary Fig. 3C).

The results of this study confirm the effective creation of a mouse model of HCM induced by pressure overload through the use of TAC surgery. Furthermore, the findings indicate that radiotherapy exerts a positive influence on both cardiac morphology and function in the context of HCM.

Pathological myocardial hypertrophy often leads to the enlargement of cardiomyocytes, which serves as a significant marker for HCM. Therefore, we performed WGA staining to assess the cross-sectional area of cardiomyocytes, as this measurement is a dependable indicator of cellular hypertrophy and other pathological alterations [33, 34]. The findings from H&E and WGA staining of paraffin-embedded heart tissue sections from three experimental mouse groups demonstrated significant variations in both the cross-sectional area of cardiomyocytes.

In sham-operated mice, the cross-sectional area of cardiomyocytes was observed to be within normal limits (mean = 217 µm²), with no significant hypertrophy detected (Fig. 2A,B,G). Conversely, mice with HCM exhibited a marked increase in cardiomyocyte size (mean = 356 µm²) when compared to the sham-operated group (p 0.001, Fig. 2G), which is indicative of cardiac hypertrophy (Fig. 2C,D). Notably, the mean cardiomyocyte size in the HCM + 30 Gy group (mean = 292 µm²) remained significantly larger than that of the sham group (p 0.01); however, radiation treatment resulted in a significant reduction in cardiomyocyte size relative to the HCM group (p 0.05, Fig. 2G), suggesting a potential attenuation of cardiac hypertrophy (Fig. 2E,F). Furthermore, the degree of LV hypertrophy appeared to be diminished in HCM mice subjected to radiation compared to their untreated counterparts (Fig. 2F vs. Fig. 2D), indicating a possible beneficial effect of radiation therapy on myocardial pathological changes. These results imply that radiation treatment may have a modulatory influence on myocardial cell hypertrophy in the context of HCM.

Fig. 2.

Radiation improves cardiomyocyte hypertrophy. Light microscope images showing the cross-sectional area of cardiomyocytes of the LV in three experimental mouse groups. Heart tissue sections were immuno-stained with hematoxylin & eosin (H&E) and Wheat Germ Agglutinin (WGA) (green) in sham-operated mice (A,B), HCM mice (C,D), and HCM + radiation mice (E,F). WGA-stained (green) images show myocardial hypertrophy. (A,C,E) the scale bar = 50 µm. (B,D,F) the scale bar = 20 µm. (G) Quantification of cross-sectional myocyte area in sham-operated, HCM, and HCM + radiation mice. *p 0.05, **p 0.01, and ***p 0.001.

Considering the role of NLRP3 and the cGAS-STING pathway in the modulation of pyroptotic cell death within the context of cardiovascular disease [35], we proceeded to examine the impact of NLRP3 inhibition and cGAS overexpression on cell viability. HL-1 cardiomyocyte cells were divided into six experimental groups: a control group, a radiation group, a NLRP3 inhibitor group, a NLRP3 inhibitor (MCC950) + radiation group, a cGAS overexpression group, and a cGAS overexpression + MCC950 + radiation group. Initially, the effectiveness of radiation on HL-1 cells was assessed using SEM. As shown in Fig. 3B, X-ray irradiated HL-1 cells exhibited signs of plasma membrane rupture (indicated by red arrows) and a flattened cytoplasm, which are characteristic features of pyroptotic cells, in contrast to the control cells depicted in Fig. 3A. This observation confirms that the cells experienced pyroptosis as a result of radiation exposure.

Fig. 3.

NLR family pyrin domain containing 3 (NLRP3) inhibition rescues radiation-induced cell cytotoxicity. (A,B) Representative scanning electron microscopy (SEM) images of control HL-1 cells (A) and X-ray irradiated HL-1 cells (B). Red arrows indicate ruptured plasma membrane. The scale bar = 20 µm. (C) Quantitative analysis of cell viability in HL-1 cells under different conditions. Cell viability was assessed by Cell Counting Kit-8 (CCK8) assay. Mean $\pm$ SD, **p 0.01, and ****p 0.0001, compared to untreated HL-1 cells.

The results obtained from the CCK-8 assay confirmed that radiation significantly reduced cell viability in comparison to control groups, thereby illustrating the cytotoxic effects of radiation on HL-1 cells (p 0.0001, Fig. 3C). The NLRP3 inhibitor (MCC950) did not exhibit any significant impact on cell viability when assessed alone against control cells. However, in HL-1 cells subjected to radiation, the administration of MCC950 markedly enhanced cell proliferation relative to the radiation only group (p 0.0001), indicating a protective role of the NLRP3 inhibitor against radiation-induced cytotoxicity. Conversely, the overexpression of cGAS in cells exposed to radiation resulted in a further decline in cell viability compared to the radiation group, suggesting a harmful effect associated with cGAS overexpression. Notably, this detrimental effect was mitigated by the inhibition of NLRP3, as evidenced in the pcDNA3.1-FLAG-cGAS+ MCC950+radiation group (Fig. 3C). These findings highlight the potential protective benefits of NLRP3 inhibition on cell viability following radiation exposure, while also emphasizing the adverse consequences of cGAS overexpression.

Mitochondrial homeostasis plays a vital role in the regulation of pyroptosis. An overproduction of mitochondrial ROS can lead to the formation of macropore in the outer mitochondrial membrane of mitochondria and subsequent damage to mitochondrial DNA. This damage facilitates the release of mtDNA into the cytoplasm, which activates the cGAS-STING pathway, thereby promoting pyroptosis [21]. Therefore, our study sought to examine the impacts of radiation exposure, NLRP3 inhibition, and cGAS overexpression on mitochondrial membrane potential and morphology.

Staining with the JC-1 probe demonstrated that exposure to radiation resulted in a substantial reduction of mitochondrial membrane potential in HL-1 cells, as indicated by a significant decrease in the red/green fluorescence ratio (p 0.0001) when compared to control cells (Fig. 4A). The JC-1 aggregates emitted red fluorescence, while the presence of green fluorescence signified the existence of JC-1 monomers. In contrast, the group treated with the NLRP3 inhibitor (MCC950) in conjunction with radiation exhibited a notable preservation of mitochondrial membrane potential relative to the radiation-only group (p 0.001), implying a potential protective role of NLRP3 inhibition against radiation-induced mitochondrial impairment. Conversely, the overexpression of cGAS was found to further intensify the loss of mitochondrial membrane potential in cells subjected to X-ray irradiation compared to those exposed to radiation alone. Notably, NLRP3 inhibition significantly mitigated the detrimental effects of cGAS overexpression in X-ray irradiated cells (p 0.0001). It is important to note that treatment of HL-1 cells with MCC950 alone did not influence mitochondrial membrane potential.

Fig. 4.

Radiation-induced loss of mitochondrial membrane potential and mitochondrial DNA (mtDNA) leakage into the cytoplasm through activation of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)/STING/NLRP3. (A) Quantitative analysis of mitochondrial membrane potential using JC-1 probe red/green fluorescence ratios in HL-1 cells. (B) The expression levels of mtDNA and MitoTracker under different conditions. Mean $\pm$ SD, *p 0.05, **p 0.01, ***p 0.001, and ****p 0.0001, compared to untreated control HL-1 cells. (C–H) Representative images showing mitochondrial DNA (green) and mitochondria (red) in HL-1 cells under different conditions. The scale bar = 10 µm. Nuclei were, DAPI (blue).

The labeling of biologically active mitochondria (indicated in red) and mtDNA (indicated in green) provided evidence that radiation exposure significantly enhanced both the quantity of mitochondria and the release of mtDNA into the cytoplasm (Fig. 4B,D), as illustrated in Fig. 4B,C (p 0.0001). Notably, the inhibition of NLRP3 using MCC950 in X-ray irradiated HL-1 cells resulted in a significant reduction in mitochondria quantity compared to the radiation-only group (p 0.05, Fig. 4B,D,E,F). Furthermore, the overexpression of cGAS further intensified the increase in mitochondrial numbers relative to the radiation only group (Fig. 4B,D,G), while the application of MCC950 significantly alleviated this effect (p 0.05, Fig. 4B,G,H). Collectively, these results suggest that radiation-induced oxidative stress disrupts mitochondrial membrane potential, facilitating the leakage of mtDNA, a process that is mediated by the cGAS/STING/NLRP3 signaling pathway.

The cGAS-STING signaling pathway plays a crucial role in the regulation of pyroptosis via NLRP3 inflammasomes [36]. In summary, the detection of cytosolic DNA by cGAS leads to the production of cGAMP, which initiates a cascade reaction within the cGAS-STING pathway, a process that is mediated by the NLRP3 inflammasome. The activation of NLRP3 subsequently recruits and activates caspase-1, which in turn activates the pro-inflammatory cytokines IL-1$\beta$ and IL-18, as well as GSDMD, a protein that forms membrane pores upon cleavage, ultimately resulting in pyroptotic cell death. The N-terminal fragment of GSDMD, referred to as GSDMD-N, is specifically responsible for the formation of these membrane pores, thereby compromising cellular integrity [5, 37, 38, 39]. Additionally, we demonstrated that the overexpression of cGAS significantly elevated the levels of NLRP3 in normal HL-1 cells, thereby confirming the signaling cascades associated with the cGAS/STING/NLRP3 signaling axis (Supplementary Fig. 4A,B).

In light of the involvement of these proteins in cGAS/STING/NLRP3-mediated pyroptosis, an analysis of their levels was conducted in HL-1 cells through western blotting techniques. The findings from the western blot analysis revealed that the irradiation of HL-1 cells resulted in a marked upregulation of cGAS, STING, NLRP3, caspase-1, cleaved caspase-1, GSDMD, and GSDMD-N when compared to the control HL-1 cells (Fig. 5A–J).

Fig. 5.

Radiation induces pyroptosis through activation of cGAS/STING/NLRP3 axis in HL-1 cells. Immunoblots and histograms show the protein levels of cGAS, STING, NLRP3 (A–D), caspase-1, cleaved caspase-1 (E–G), GSDMD, and GSDMD-N (H–J) in different HL-1 cell treatment groups. A1: control HL-1 cells; B1: Irradiated HL-1 cells; C1: MCC950-treated HL-1 cells; D1: MCC950 + irradiated HL-1 cells; E1: cGAS overexpression + irradiated HL-1 cells; and F1: cGAS overexpression + MCC950 + irradiated HL-1 cells. Mean $\pm$ SD, **p 0.01, ***p 0.001, and ****p 0.0001.

Inhibition of NLRP3 in irradiated HL-1 cells led to a marked decrease in the levels of cleaved caspase-1 (p 0.01, Fig. 5G) and GSDMD-N (p 0.0001, Fig. 5J). Furthermore, the protein expression of NLRP3 was reduced in cells treated with MCC950, thereby confirming the efficacy of NLRP3 inhibition (p 0.0001, Fig. 5D). Conversely, overexpression of cGAS in X-ray irradiated cells resulted in an exacerbation of all pyroptotic protein levels compared to radiation treatment alone, with significant increases observed in STING (p 0.01), NLRP3 (p 0.001), caspase-1 (p 0.001), cleaved caspase-1 (p 0.01), GSDMD (p 0.01), and GSDMD-N (p 0.001, Fig. 5A–J). Notably, the inhibition of NLRP3 reversed this trend, as indicated by the reduced levels of caspase-1, cleaved caspase-1, GSDMD, and GSDMD-N in the cGAS overexpression + radiation + MCC950 group compared to the cGAS overexpression and X-ray irradiation group (p 0.0001, Fig. 5F–J). The levels of cGAS and STING remained constant across these two groups, reinforcing the hypothesis that NLRP3 functions downstream of the cGAS-STING signaling pathway (Fig. 5A–C). Collectively, these findings suggest that radiation-induced pyroptosis is mediated through the cGAS/STING/NLRP3 axis, and that NLRP3 inhibition can mitigate pyroptosis activated by the cGAS-STING pathway.

In alignment with these findings, the experiment involving HCM mice demonstrated comparable outcomes, as the group subjected to HCM and radiation exhibited significantly elevated protein levels of cGAS, STING, NLRP3, caspase-1, cleaved caspase-1, GSDMD, and GSDMD-N in comparison to the HCM-only group (Fig. 6A–J). This observation indicates the activation of the cGAS-STING signaling pathway in response to cellular stress induced by radiation. Conversely, it was unexpectedly noted that in the HCM mouse model, the performance of TAC surgery led to a reduction in the levels of pyroptotic proteins when compared to the sham-operated group (Fig. 6A–J).

Fig. 6.

Radiation induces pyroptosis through activation of cGAS/STING/NLRP3 axis in HCM mouse model. Immunoblots and histograms show the protein levels of cGAS, STING, NLRP3 (A–D), caspase-1, cleaved caspase-1 (E–G), GSDMD, and GSDMD-N (H–J) in three mouse experimental groups, n = 5 per group. A2: sham-operated mice; B2: pressure overload-induced HCM mice; C2: HCM + radiation mice. (K,L) Quantitative analysis of cGAMP levels in three experimental mouse groups (K), and HL-1 cells under different conditions (L). (M,N) Quantitative analysis of IL-18 (M) and IL-1$\beta$ (N) levels in HL-1 cells under different conditions. Mean $\pm$ SD, *p 0.05, **p 0.01, ***p 0.001, and ****p 0.0001.

The concentration of cGAMP, the second messenger that binds to STING, was assessed using ELISA assays. In alignment with the observed trends in other pyroptotic proteins previously discussed, a notable increase in cGAMP levels was detected in mice subjected to HCM and radiation when compared to those with HCM alone (p 0.05, Fig. 6K). In HL-1 cells, exposure to radiation resulted in elevated cGAMP levels relative to the control cells; however, this increase did not reach statistical significance. Additionally, NLRP3 did not appear to influence cGAMP levels in cells exposed to X-ray irradiation. While overexpression of cGAS seemed to elevate cGAMP levels, this increase was not statistically significant when compared to the group exposed solely to radiation (Fig. 6L).

As previously mentioned, IL-18 and IL-1$\beta$ serve as critical downstream mediators in the process of pyroptosis. As shown in Fig. 6M,N, the exposure to radiation resulted in a significant elevation of both IL-18 and IL-1$\beta$ levels in HL-1 cells (p 0.001 and p 0.01, respectively), thereby indicating the activation of the NLRP3 inflammasome in response to radiation-induced cellular stress. Conversely, the application of MCC950, an inhibitor of NLRP3, resulted in a marked reduction in the expression of IL-1$\beta$ and IL-18 in the presence of radiation. Furthermore, the overexpression of cGAS led to an increase in IL-1$\beta$ and IL-18 levels; however, this increase was mitigated by the inhibition of NLRP3, as evidenced in group subjected to cGAS overexpression, MCC950 treatment, and radiation exposure (Fig. 6M,N). Collectively, these findings underscore the protective role of NLRP3 inhibition in mitigating pyroptosis through the downregulation of pro-inflammatory cytokine production.