GSDMD-mediated pyroptosis promotes cardiac remodeling in pressure overload

ABSTRACT

Background

Gasdermin D (GSDMD) forms membrane pores to execute pyroptosis. But the mechanism of how cardiomyocyte pyroptosis induces cardiac remodeling in pressure overload remains unclear. We investigated the role of GSDMD-mediated pyroptosis in the pathogenesis of cardiac remodeling in pressure overload.

Methods

Wild-type (WT) and cardiomyocyte-specific GSDMD-deficient (GSDMD-CKO) mice were subjected to transverse aortic constriction (TAC) to induce pressure overload. Four weeks after surgery, left ventricular structure and function were evaluated by echocardiographic, invasive hemodynamic and histological analysis. Pertinent signaling pathways related to pyroptosis, hypertrophy and fibrosis were investigated by histochemistry, RT-PCR and western blotting. The serum levels of GSDMD and IL-18 collected from healthy volunteers or hypertensive patients were measured by ELISA.

Results

We found TAC induced cardiomyocyte pyroptosis and release of pro-inflammatory cytokines IL-18. The serum GSDMD level was significantly higher in hypertensive patients than in healthy volunteers, and induced more dramatic release of mature IL-18. GSDMD deletion remarkably mitigated TAC-induced cardiomyocyte pyroptosis. Furthermore, GSDMD deficiency in cardiomyocytes significantly reduced myocardial hypertrophy and fibrosis. The deterioration of cardiac remodeling by GSDMD-mediated pyroptosis was associated with activating JNK and p38 signaling pathways, but not ERK or Akt signaling pathway.

Conclusion

In conclusion, our results demonstrate that GSDMD serves as a key executioner of pyroptosis in cardiac remodeling induced by pressure overload. GSDMD-mediated pyroptosis activates JNK and p38 signaling pathways, and this may provide a new therapeutic target for cardiac remodeling induced by pressure overload.

KEYWORDS:

• Gasdermin D (GSDMD)
• pyroptosis
• pressure overload
• transverse aortic constriction (TAC)
• cardiac remodeling

Introduction

Although various antihypertensive drugs have been developed to reduce blood pressure, such complications as cardiac remodeling remain intractable to clinical therapy. Cardiac remodeling induced by pressure overload is one of the most important pathological factors of heart failure and contributes to morbidity and mortality worldwide (1). The underlying pathogenic mechanisms remain enigmatic.

Pyroptosis is a lytic program of cell death induced by robust inflammatory cascades upon activation of inflammatory caspases. It plays a crucial role in the pathogenesis of various diseases (2,3). Pyroptosis is distinct from other types of cell death that have been proved. It has typical morphological features, such as prominent bubble-like formations, pore formation in the plasma membrane, cell swelling, membrane rupture and pro-inflammatory cytokines secretion (2–8). Previous studies reported that inflammation is associated with cardiac remodeling and heart failure (6). However, the underlying mechanism of how pyroptosis triggers cardiac remodeling is unknown. Investigating the potential of anti-inflammatory treatment in cardiovascular diseases may be one focus of future research.

Gasdermin D (GSDMD) belongs to the gasdermin family and is highly conserved in mammals, which can be cleaved into N-terminal (GSDMD-N) to initiate the progression of pyroptosis after inflammasome activation by perforating the plasma membrane. The pro-inflammatory cytokines IL-18 are released from the GSDMD pores, and lead to pyroptotic cell death (9). It has been demonstrated that GSDMD serves as the final and direct executor of pyroptosis, and thus inhibiting GSDMD is an attractive strategy to curb inflammation (9,10). Accumulating evidence has suggested that excessive activation of inflammation is implicated in the pathogenesis of cardiac remodeling (6). However, the potential effects of GSDMD and its underlying mechanisms in cardiac remodeling are still elusive. The current study aimed to elucidate the pivotal role of GSDMD-mediated pyroptosis in cardiac remodeling induced by pressure overload. The results may provide a new therapeutic target for cardiac remodeling.

Methods

Animals

Wild-type C57BL/6J male mice (8–10 weeks old, 20–25 g) were purchased from Shanghai SLAC Laboratory Animal Company. Cardiomyocyte-specific GSDMD-knockout mice were bred by crossing the GSDMD^F/F strain with the Myh6-Cre strain (Cyagen Biosciences Inc. Guangzhou, China) as we described in our previous study (2). The mice were housed in a quiet environment that was maintained at 18–22°C, under 12-hr light/dark cycles, with stable humidity, and free access to food and water.

Transverse aortic constriction (TAC)

Pressure overload was induced by TAC (n = 10) according to the methods we described previously (1,11). Briefly, mice were anesthetized by intraperitoneal injection of a mixture of ketamine (150 mg/kg) and xylazine (10 mg/kg). The thoracic cavity was opened and a mouse ventilator (Type 7025; Harvard Apparatus, March-Hugstetten, Germany) was connected. The aortic arch was ligated with a 27-gauge needle using 6–0 silk surgical thread between the origins of the innominate artery and left common carotid artery, and then the needle was removed to yield a constriction. Finally, the thoracic cavity was closed with a 4–0 silk suture. Meloxicam (0.13 mg each) was administered subcutaneously for analgesia. The corresponding sham-operated mice (n = 10) underwent the same surgical procedure but without ligation of the aorta. Samples were harvested four weeks after surgery.

Echocardiography

The echocardiography was evaluated four weeks after surgery by a high-frequency ultrasound system Vevo 2100 (VisualSonics, Toronto, ON, Canada) as reported in our previous studies (1,11). The center frequencies of the transducers were 30-MHz, providing resolutions of 115 μm (lateral) × 55 μm (axial). During the echocardiographic study, the mouse was positioned on a heating pad to maintain normothermia and anesthetized with 1.5% isoflurane. We assessed the following parameters: peak systolic velocity of aortic arch flow (PSVa), left ventricular posterior wall end-diastolic and end-systolic thickness (LVPWTd and LVPWTs), left ventricular end-diastolic and end-systolic dimensions (LVEDD and LVESD), and left ventricular ejection fraction and fractional shortening (LVEF and LVFS).

Invasive hemodynamics

Left ventricular hemodynamics were evaluated as we described previously (1,11). Briefly, a micromanometric catheter (Millar 1.4F, SPR 835; Millar Instruments, Houston, TX) was inserted into the right common carotid artery and carefully advanced into the left ventricle. The micromanometer was connected to a Power Laboratory system (AD Instruments, Castle Hill, NSW, Australia) to record left ventricular end-systolic and end-diastolic pressure (LVESP and LVEDP), the maximal contraction and relaxation velocity (Max dP/dt and Min dP/dt), and contractility index.

Histological analysis

Hearts from anesthetized mice were excised, rinsed in saline, fixed in 10% formalin, embedded in paraffin, and cut into 4 μm-thick sections in the short axis at the papillary muscle level. Tissue sections were stained with hematoxylin and eosin for cardiomyocyte size and Masson trichrome for interstitial fibrosis as we previously described (1,11). Digital photographs were taken and quantified by image analysis systems (Qwin V3, Leica, Wetzlar, Germany; ImageJ 1.52 v National Institutes of Health, Bethesda, United States).

Real-time quantitative polymerase chain reaction (RT-qPCR)

Total RNA was prepared from fresh-frozen left ventricular myocardium using Trizol reagent (Invitrogen, Carlsbad, CA). The reverse transcription of PCR was performed using Toyobo ReverTra Ace-a-real-time – PCR kit (Toyobo Co, Ltd, Osaka, Japan) according to the manufacturer’s instructions. The real-time PCR was performed with ABI PRISM 7900 System (Applied Biosystems, Carlsbad, CA). The genes were amplified using their specific primers as follows: 1) ANP, 5”-GGTGTCCAACACAGATCTGA-3‘and 5’-CCACTAGACCACTCATCTAC-3;‘ 2) BNP, 5’-AGTCCTTCGGTCTCAAGGCA-3‘and 5’-CCGATCCGGTCTATCTTGTGC-3;‘ 3) Col1a1, 5’-GGACGCCATCAAGGTCTACTGC-3‘and 5’-GAACGGGAATCCATCGGTCAT-3;‘ 4) Col3a1, 5’-CTCAAGAGTGGAGAATACTGGGTT-3‘ and GGTATGTAATGTTCTGGGAGGC-3;’ 5) GAPDH, 5”-ACCACAGTCCATGCCATCAC-3“and 5”-TCCACCACCCTGTTGCTGTA-3.’ Expression levels of hypertrophic and fibrotic-related genes were shown as 2^−ΔΔCt of the target gene relative to GAPDH. All real-time PCR reactions were performed in triplicate.

Western blot (WB)

Western blot was performed as previously described (1,11). Briefly, total protein extracted from homogenized left ventricular tissues was electrophoresed in 12% polyacrylamide gel and transferred to PVDF membrane (Millipore, Billerica, MA, USA). The blotted membranes were incubated with primary antibodies against GSDMD (1:1000; CST, Danvers, MA, USA), cleaved IL-18 (1:1000; CST, Danvers, MA, USA), IL-18 (1:1000; CST, Danvers, MA, USA), phospho-JNK (1:1000; CST, Danvers, MA, USA), JNK (1:1000; CST, Danvers, MA, USA), phospho-p38 (1:1000; CST, Danvers, MA, USA), p38 (1:1,000; CST, Danvers, MA, USA), phospho-ERK1/2 (1:1000; CST, Danvers, MA, USA), ERK1/2 (1:1000; CST, Danvers, MA, USA), phospho-Akt (1:1000; CST, Danvers, MA, USA), and Akt (1:1000; CST, Danvers, MA, USA). After three washes, the blotted membranes were then incubated with horseradish peroxidase-conjugated rabbit secondary antibody (1:5000, Kang-Chen Biotechnology, Shanghai, China). GAPDH was used as the internal control that was detected by horseradish peroxidase-conjugated monoclonal mouse GAPDH (1:5000; Kang-Chen Biotechnology, Shanghai, China). The antigen-antibody complexes were detected using ECL chemiluminescence reagents (GE Healthcare, Piscataway, NJ) and visualized by densitometry using LAS-3000 Image software (FUJIFILM, Kanagawa, Japan).

Enzyme-linked immunosorbent assay (ELISA)

The data of 68 consecutive participants who took 24-hour ambulatory blood pressure monitoring in our Medical Examination Center were prospectively collected and analyzed by propensity score matching according to age, sex, and body mass index. Finally, peripheral blood samples were collected from 20 healthy volunteers and 20 hypertensive patients. The serum levels of GSDMD and IL-18 were determined by ELISA kits (Human GSDMD ELISA kit: TSZ Biosciences, USA; Human IL-18/IL-1F4 ELISA kits: R&D Systems, USA) according to the manufacturer’s protocols as we previously described (2,11). Blood samples were placed at room temperature for 30 min prior to centrifugation for 15 minutes at 1000 g, 4°C. Serum samples were aliquoted and stored at −80°C. Absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA). The results were determined by comparing the samples to the standard curve generated by the kit. All samples and standards were assayed in triplicate.

Primary adult mouse ventricular myocytes (AMVMs) culture

Adult mouse ventricular myocytes (AMVMs) were isolated from WT mice (C57BL/6J) and GSDMD-CKO male mice as described previously (12). Briefly, the mouse’s heart was perfused with EDTA buffer, Perfusion buffer, followed by collagenase buffer, then dissociated. After that, the enzyme reaction was neutralized with the Stop solution and the cell suspension was filtered with a 100 μm filter. Cardiomyocytes were collected by gravity settlement and used for culture after re-introduction of calcium. Pure cardiomyocytes were resuspended in plating medium (Medium 199, supplemented with 5% FBS, 2,3-Butanedione monoxime and 1% Penicillin-Streptomycin), and then plated on culture dishes pre-coated with laminin (5 μg/ml, Thermo Scientific). One hour later, the plating medium was changed to the culture medium (Medium 199, supplemented with 5% (w/v) bovine serum albumin, ITS supplement, 2,3-Butanedione monoxime, chemically defined lipid concentrated and 1% Penicillin-Streptomycin) and cardiomyocytes were incubated with Ang II (1 μmol/L) for 24 hours.

Lactate dehydrogenase (LDH) measurement

Cardiomyocyte supernatants were collected and examined for LDH level with an LDH Cytotoxicity Assay Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions.

Propidium iodide (PI) staining

Cultured adult cardiomyocytes were stained with Hoechst 33 342 (Beyotime, Shanghai, China) and PI (Sangon Biotech, Shanghai, China) to examine cardiomyocyte necrosis. Fluorescent images were obtained with a fluorescence microscope (Olympus, Tokyo, Japan). Five random fields for PI-positive and Hoechst stained-cells were counted with ImageJ.

Statistical analysis

All parameters obtained from at least three independent experiments were presented as the means ± standard error and analyzed using the two-tailed indirect Student’s t-test or one-way analysis of variance (ANOVA) followed by the Student – Newman–Keuls (S-N-K) test for multiple comparisons. Statistical analysis was performed by GraphPad Prism 8 (GraphPad Software, Version 8.01, San Diego, CA, USA). Statistical significance was defined as P < .05.

Ethics statement

The experimental protocol was approved by the Institutional Review Board of Shanghai East Hospital, and was in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (No. 85–23, revised 1996; National Institutes of Health, Bethesda, MD, USA). Written informed consent was obtained from all participants included in the study.

Results

Pressure overload activated pyroptosis in a GSDMD-dependent manner

The changes of GSDMD and other pyroptosis related proteins were verified in pressure overload hearts. GSDMD was significantly upregulated in the TAC mice and GSDMD-NT was cleaved to provoke pyroptosis. Cleaved IL-18 was remarkably upregulated in the TAC mice. Moreover, the serum levels of GSDMD and IL-18 were significantly higher in hypertensive patients than in healthy volunteers (Figure 1).

Figure 1. Changes of signaling pathways related to cardiomyocyte pyroptosis, hypertrophy and fibrosis. a, Western blot analysis for GSDMD, IL-18, JNK, p38, Akt, and ERK1/2 in all groups. b, Quantitative real-time PCR analysis of ANP, BNP, Col1a1, and Col3a1 gene expression in all groups. c, the serum levels of GSDMD and IL-18 in healthy volunteers or hypertensive patients. WT, wild-type mice; KO, cardiomyocyte-specific GSDMD-knockout mice; TAC, transverse aortic constriction; HTN, hypertension. †P < .05 versus the corresponding values of the sham or control group; *P < .05 versus the corresponding values of the WT group.

To fully verify the key role of GSDMD in cardiomyocyte pyroptosis, we generated cardiac-specific knockout (GSDMD^flox/flox; CreαMHC (GSDMD-CKO)) mice. The depletion of GSDMD protein in cardiomyocytes was verified by western blotting. We found that GSDMD deletion abolished TAC-induced cardiomyocyte pyroptosis. The deletion of the GSDMD gene decreased IL-18 release (Figure 1).

In addition, Ang II treatment caused notable cell membranes damage as assessed by LDH level and PI staining, while GSDMD deletion reduced the LDH level in the supernatant of Ang II-treated AMVMs and decreased the PI-positive cardiomyocytes ratio (Figure 2).

Figure 2. GSDMD deletion reduces Ang II-induced adult mouse ventricular myocytes (AMVMs) cardiomyocytes pyroptosis. a, Lactate dehydrogenase (LDH) levels of cardiomyocyte supernatants. b, PI-positive cardiomyocytes ratio. c, Cardiomyocytes necrosis by propidium iodide (PI) staining, white arrows indicate necrotic cardiomyocytes. WT, wild-type mice; KO, cardiomyocyte-specific GSDMD-knockout mice. *P < .05 versus the corresponding values of the PBS group; †P < .05 versus the corresponding values of the PBS WT group.

Thus, both in vivo and in vitro results indicated that GSDMD plays a key role in TAC-induced cardiomyocyte pyroptosis.

The deterioration of cardiac remodeling by GSDMD-Mediated pyroptosis was associated with specific signaling networks of hypertrophy and fibrosis

Pressure overload caused cardiac dysfunction in the TAC group four weeks after surgery, as demonstrated by reduced LVEF, LVFS, Max dP/dt, Min dP/dt and contractility index, in line with elevated PSVa, LVEDP and LVESP. On the contrary, GSDMD deletion improved cardiac function as evidenced by the aforementioned indices. Coherently, TAC operation induced myocardial hypertrophy and fibrosis in the WT mice. GSDMD deletion repressed LVPWTs and LVESD. Hematoxylin and eosin staining showed that the cardiomyocyte size was significantly decreased after GSDMD deletion. Consistently, GSDMD deletion ameliorated interstitial fibrosis compared to the control group, as evaluated by Masson trichrome staining (Figure 3). The results indicated that GSDMD aggravated cardiac remodeling induced by pressure overload and represented a potential biomarker for the diagnosis of cardiac remodeling.

Figure 3. The results of echocardiography, invasive hemodynamics, and histological analysis in all groups four weeks after surgery. a, the color Doppler images of aortic arch flow. b, the Doppler flow spectra at the aortic arch. c, M-mode of left ventricle. d, Left ventricular pressure curves, the contraction and relaxation velocity. e, Tissue sections stained by hematoxylin and eosin for cardiomyocyte size. f, Tissue sections stained by Masson trichrome for interstitial fibrosis. WT, wild-type mice; KO, cardiomyocyte-specific GSDMD-knockout mice; TAC, transverse aortic constriction; HW, heart weight; BW, body weight; PSVa, peak systolic velocity of aortic arch flow; LVPWTd, left ventricular posterior wall end-diastolic thickness; LVPWTs, left ventricular posterior wall end-systolic thickness; LVEDD, left ventricular end-diastolic dimension; LVESD, left ventricular end-systolic dimension; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVESP, left ventricular end-systolic pressure; LVEDP, left ventricular end-diastolic pressure; Max dp/dt, maximal contraction velocity; Min dp/dt, maximal relaxation velocity; CSA, cross-sectional area. Red bar = 20 μm for HE staining, blue bar = 50μm for Masson trichrome staining. †P < .05 versus the corresponding values of the sham group; *P < .05 versus the corresponding values of the WT group.

In our study, the MAPK kinases (including JNK, p38, ERK1/2) and Akt signaling pathway were prominently activated in the TAC group. GSDMD deletion inhibited JNK and p38 signaling pathways. But ERK1/2 or Akt levels of the GSDMD-CKO group were comparable with the WT group (Figure 1). The data showed that the deterioration of cardiac remodeling by GSDMD-mediated pyroptosis was associated with activating JNK and p38 signaling pathways, but not ERK or Akt signaling pathway. In addition, the gene levels of ANP, BNP, SAA, Col1a1, and Col3a1 were dramatically upregulated in the TAC group, while the activation was abrogated by GSDMD deletion (Figure 1).

Discussion

Pyroptosis causes extracellular spilling of the intracellular contents, such as pro-inflammatory cytokines, which is characterized by a breach of plasma membrane integrity (2–8). Previous studies found that pyroptosis participates in a range of pathophysiological processes (2,3). It has been reported that inflammasome is activated after pressure overload and pyroptosis might play a critical role in cardiac remodeling (6). However, the role of GSDMD-mediated pyroptosis in cardiac remodeling and possible underlying mechanisms has been little revealed. Our results identified GSDMD as the executor of pyroptosis in pressure overload heart, and its cardiomyocyte-specific deficiency markedly attenuated hypertrophy and fibrosis, and improved cardiac function.

We have found that GSDMD-NT was increased for GSDMD pores assembling (by PI uptake), cell death, and cell lysis (by LDH release) in the TAC group, followed by IL-18 release and stimulation of pyroptosis. Coincidently, a recent study also indicated GSDMD deficiency reduced pressure overload-induced cardiac hypertrophy, dysfunction, and associated cardiomyocyte pyroptosis in mice (13). In the present study, we further demonstrated that GSDMD deletion not only significantly suppressed TAC-induced cardiomyocyte pyroptosis and alleviated cardiac remodeling, but also attenuated JNK and p38 activation. These findings suggested that GSDMD-mediated pyroptosis might be a determinant of cardiac remodeling in pressure overload. Our results provide the first demonstration that GSDMD triggers cardiac remodeling at least in part related to the regulation of JNK and p38 signaling pathway activation.

Previous studies found that GSDMD could be released from pyroptotic cardiomyocytes into the periphery, and GSDMD levels were also increased in the serum of patients after reperfusion by percutaneous coronary intervention (2). Our results also confirmed that GSDMD can used for a potential biomarker for cardiac remodeling, and it might be associated with pyroptosis. However, considering inflammatory cytokines are capable to be released through GSDMD pores without concomitant cell death (9), further studies are warranted to investigate the details and elucidate the underlying associated mechanisms.

In conclusion, our data demonstrate that GSDMD-mediated pyroptosis plays a pivotal role in cardiac remodeling induced by pressure overload, which is associated with JNK and p38 signaling pathway. These findings provide new insights into the treatment of cardiac remodeling. In addition, GSDMD may be a potential biomarker and a therapeutic target for cardiac remodeling induced by pressure overload.

Limitations

Although our data have indicated that the deterioration of cardiac remodeling by GSDMD-mediated pyroptosis was associated with activating JNK and p38 signaling pathways, the underlying regulatory mechanisms remain to be elucidated. Rescue assay targeting the aforementioned signaling effectors would help to recapitulate the role of related signaling pathways in GSDMD-mediated cardiomyocyte pyroptosis.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Health Science and Technology Project of Shanghai Pudong New Area Health Commission (PW2019A-13), the National Natural Science Foundation of China (81500191), the Health Science and Technology Project of Jiangxi Province (SKJP220210327), and Top-level Clinical Discipline Project of Shanghai Pudong District Grant (PWYgf2021-01). Shanghai Key Clinical Specialty Project (shslczdzk06202)