TRADD-mediated pyroptosis contributes to diabetic cardiomyopathy

Abstract

Regulated cell death like pyroptosis is one vital cause of diabetic cardiomyopathy (DCM), which eventually leads to heart failure. Tumor necrosis factor (TNF) receptor-associated death domain protein (TRADD) is an adapter protein with multiple functions that participates in the pathophysiological progress of different cardiovascular disorders via regulating regulated cell death. Studies have shown that TRADD combines with receptor-interacting protein kinase 3 (RIPK3) and facilitates its activation, thereby mediating TNF-induced necroptosis. However, no direct relationship between TRADD and pyroptosis has been identified. In this study, we investigated the role and mechanisms of TRADD in pyroptosis during DCM. We established a streptozotocin (STZ)-induced diabetic mouse model and high glucose (HG)-treated cardiomyocytes model. We showed that the expression levels of TRADD were significantly increased in the hearts of diabetic mice and HG-treated cardiomyocytes. Knockdown of TRADD did not affect blood glucose and triglyceride levels, but significantly improved cardiac function, and attenuated myocardial hypertrophy, fibrosis, and pyroptosis in the heart of diabetic mice. Furthermore, both knockdown of TRADD and application of TRADD inhibitor apostatin-1 (Apt-1, 10 μM) significantly ameliorated cell injury and pyroptosis in HG-treated cardiomyocytes. We demonstrated that HG treatment increased the expression of X-box binding protein 1 (XBP1) and enhanced the binding of XBP1 to the TRADD promoter to elevate TRADD expression in the cardiomyocytes. Collectively, this study provides evidence that TRADD-mediated pyroptosis contributes to DCM, suggesting that strategies to inhibit TRADD activity may be a novel approach for DCM treatment.

Introduction

Diabetes triggers the pathophysiological condition of diabetic cardiomyopathy (DCM). The primary manifestations of diabetic cardiomyopathy are cardiac systolic and diastolic dysfunctions that can ultimately cause heart failure [1, 2]. It is believed that insulin resistance, endothelial dysfunction, regulated cardiomyocyte apoptosis, autophagy, myocardial remodeling, and myocardial fibrosis may have a part in the development and occurrence of DCM [3]. Nonetheless, precise molecular mechanisms are not completely understood, which hinders the development of new prevention and treatment strategies for DCM. Therefore, there is an urgent need to explore new ideas and biological targets.

Pyroptosis being the regulated cell death form is accompanied by inflammation [4]. Major characteristics of pyroptosis include nuclear shrinkage, chromatin DNA fragmentation and degradation, holes formation in the cell membrane, increased permeability, and subsequent pro-inflammatory cytokines release, lactate dehydrogenase, and other intracellular substances, eventually leading to cell lysis [5, 6]. Extracellular and intracellular harmful stimuli activate pattern-recognition receptors (PRRs), recruiting the precursor Caspase 1, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), and apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) to form inflammasomes. Inflammasome activation promotes cleavage of precursor Caspase 1, and activation of interleukin-1β (IL-1β) and interleukin-18 (IL-18). Then, cleavage of downstream target protein gasdermin D (GSDMD) occurs at the GSDMD-N terminus, leading to cell rupture and cell contents release, ultimately resulting in pyroptosis [7–9]. Increasing evidence suggests that pyroptosis is linked to the progression of cardiovascular disorders [10–12]. Studies reported that pyroptosis mediated by GSDMD promoted myocardial ischemia-reperfusion injury [8]. Furthermore, NLRP3 inflammasome level was enhanced in the mice myocardium through dilated cardiomyopathy induced by doxorubicin (DOX), and the activities of GSDMD, IL-1β, IL-18, and Caspase 1 were enhanced. Knockout of Caspase 1 or NLRP3 gene inhibited the activation of the inflammasome and cell pyroptosis, thereby alleviating DOX-induced myocardial injury and cardiac dysfunction [13]. Although some studies have found that pyroptosis may be related to DCM [14, 15], the detailed mechanism is still unclear.

TRADD, a 34 kDa adapter protein, is a signal transduction molecule associated with tumor necrosis factor receptors. Structurally, TRADD has a C-terminal death domain (DD) and an N-terminal tumor necrosis factor receptor-associated factor 2 (TRAF2) binding domain [16–21]. Functionally, TRADD is a multifunctional adapter protein that has vital functions in regulating the balance between cell survival and cell death [22]. Studies have shown that TRADD combined with receptor-interacting protein kinase 3 (RIPK3) facilitated its activation, thereby mediating tumor necrosis factor (TNF)-induced necroptosis [23]. However, no direct relationship between TRADD and pyroptosis has been identified. TRADD participates in the pathophysiological progression of cardiovascular disorders. The research found that TRADD knocking out alleviated myocardial fibrosis, improved left ventricular ejection fraction and remodeling, and inhibited myocardial hypertrophy in mice with transverse aortic constriction (TAC) surgery [24]. TRADD mRNA expression in umbilical vein endothelial cells of humans significantly increased by the stimulus of oxidized low-density lipoprotein. TRADD downregulating inhibited apoptosis and the expression of inflammatory factors, thereby alleviating endothelial cell dysfunction induced by oxidized low-density lipoprotein [25]. However, it is unclear whether TRADD is a potential target for DCM. Therefore, the TRADD knockout mice injected with streptozotocin (STZ) and primary cardiomyocytes stimulated with high glucose were adopted for exploring the mechanism and function of TRADD in DCM, aiming to provide evidence in clinical treatment and prevention of DCM.

Materials and methods

Animal treatment

A substantial portion (exons 3–5) of TRADD protein (amino acids 51–310), including the RIP- and TRAF2-interacting domains were deleted. Then the TRADD^+/− mice were bred to produce TRADD^−/− mice. Successful deletion of TRADD was demonstrated by PCR analysis of DNA obtained from mouse tail tissue. TRADD knockout (TRADD^−/−) mice were born at expected Mendelian ratios, which indicated no embryonic lethality. TRADD^−/− mice (Cyagen Biosciences Inc, Suzhou, China) and male C57BL/6 mice (wild type, WT) were grouped randomly into control and experimental groups. The same amounts of citrate buffer or STZ (60 mg·kg⁻¹·d⁻¹, Sigma–Aldrich, St. Louis, MO, USA) respectively were intraperitoneally injected for 5 consecutive days. The fasting blood glucose (FBG) was measured in the blood collected from the tail vein. Mice having persistently elevated and stable FBG levels above 16.7 mmol/L are considered to have diabetes.

The animal experiments were approved by the Nantong University Committee, Nantong, China (approval No. S-20180323-012), performed in compliance with the ARRIVE recommendations and conducted as per the instructions for Care and Use of Laboratory Animals published by the National Institutes of Health. Operators were blinded, regarding these groups during experiments.

Glycosylated hemoglobin (HbA1c) and triglyceride (TG) measurement

The measurement of HbA1c in plasma and TG in serum was performed respectively by HbA1c Assay Kit (A056-1-1, Jiancheng, Nanjing, China) and TG Assay Kit (A110-1, Jiancheng, Nanjing, China) according to the manufacturer’s instructions.

Cardiomyocyte culture and treatment

The neonatal Sprague-Dawley (SD) rats’ hearts were harvested and rinsed with precooled PBS. The cleaned myocardium was minced, and 0.25% trypsin solution (Beyotime, Shanghai, China) was used for its digestion at a 37 °C water bath until the tissue chunks completely disappeared, which typically required about 10 rounds of digestion. The supernatant was obtained and filtered after digestion. The collected supernatant was centrifuged to obtain the cardiomyocyte precipitate, which was then cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Wisent Inc, Montreal, QC, Canada) having 12% fetal bovine serum (FBS, Wisent Inc, Montreal, QC, Canada). Four hours later, the unattached cardiomyocytes were transferred to a new medium and cultured in normal glucose (NG, 5.5 mmol/L) or high glucose (HG, 33.3 mmol/L) medium for an additional 48 h.

Furthermore, the cardiomyocyte was transfected with TRADD siRNA #1 5′-GACGTACTGCAGATACTCA-3′; #2, 5′-GAGCGCTGTTTGAATTACA-3′; #3, 5′-GCTGCGCCGTTTCATACAA-3′, and non-specific control RNA (NC) siRNA (RiboBio, Guangzhou, China); X-box binding protein 1 (XBP1) siRNA 745 5′-CCAAGCUGGAAGCCAUUAATT-3′, XBP1 siRNA 561 5′-GCUGUUGCCUCUUCAGAUUTT-3′, XBP1 siRNA 279 5′-GCAAGUGGUGGAUUUGGAATT-3′, and NC siRNA 5′-UUCUCCGAACGUGUCACGUTT-3′ or treated by the TRADD inhibitor apostatin-1 (Apt-1, 10 μmol/L, Selleck, Houston, TX, USA).

Adenosine triphosphate (ATP) and lactate dehydrogenase (LDH) assessment

ATP in cardiomyocytes and LDH levels in the cell culture medium were assessed via the assay kits (S0026 and P0395S, Beyotime, Shanghai, China) according to the kits’ instructions.

IL-1β and IL-18 measurement

IL-1β and IL-18 levels in the serum and cardiomyocytes were measured with an enzyme-linked immunosorbent assay (ELISA, PI301, PI553). The procedure was performed according to the manufacturer’s instructions (Beyotime, Shanghai, China).

Luciferase reporter assay

After transfecting with the TRADD promoter (−798/+105) fusion plasmid (1 μg) or pRL-TK reporter plasmid (0.1 μg, Zoonbio Biotechnology, Nanjing, China), the cells were stimulated by NG or HG for 48 h. Then, cells were lysed with 360 μL of luciferase assay buffer, and luciferase assay was conducted via a dual-luciferase reporter gene assay system (E1910, Promega, Madison, WI, USA).

Echocardiography

Isoflurane (2%) was used as the mice anesthesia, and isoflurane (1.5%) for maintaining it. An echocardiography (ECG) system with an imaging sensor was used to observe the heart structure. Two-dimensional M-mode ECG (Visual Sonic Vevo 2100, Canada) was utilized to record and calculate the ejection fraction (EF), fraction shortening (FS), and the ratio of the early peak velocity (E) to the late peak velocity (A) at mitral valve (E/A).

Myocardium staining

Paraformaldehyde was used to fix the freshly extracted mouse hearts and then processed into 5 μm paraffin sections. Hematoxylin and eosin (H&E, C0105S Beyotime, Shanghai, China) staining, as well as wheat germ agglutinin (WGA, L4895, Sigma–Aldrich, Milwaukee, WI, USA) staining were utilized. The cross-sectional area of cardiomyocytes was evaluated through morphometric analysis to quantify myocardial hypertrophy. Masson’s trichrome (G1006, Servicebio, Wuhan, China) and Sirius Red (G1078, Servicebio, Wuhan, China) staining were made for assessing the myocardial fibrosis. Collagen volume fraction (CVF) was then calculated.

Quantitative real-time PCR

TRIzol reagent (9108, Takara, Kyoto, Japan) was used to extract RNA, and its concentration was measured. After performing reverse transcription, the cDNA was amplified in real-time PCR (ABI 7500, Carlsbad, CA, USA) using SYBR Green qPCR Mix (Takara). The utilized primer sequences (Sangon Biotech, Shanghai, China) were: for mouse atrial natriuretic peptide (ANP), forward 5′-GAGAAAGATGCCGGTAGAAGA-3′ and reverse 5′-AAGCACTGCCGTCTCTCAGA-3′; for mouse brain natriuretic peptide (BNP), forward 5′-CTGCTGGAGCTGATAAGAGA-3′ and reverse 5′-TGCCCAAAGCAGCTTGAGAT-3′; and for 18S, forward 5′-AGTCCCTGCCCTTTGTACACA-3′ and reverse 5′-CGATCCGAGGGCCTCACTA-3′. Ct values from experiments were normalized to 18S, and the relative expression of mRNA was determined and compared with the reference.

Hydroxyproline content detection

Myocardial samples were immersed in a digestive solution and kept for 3 h in a water bath at 37 °C. After sufficient digestion, samples were centrifuged for 10 min at 3500 rpm. The supernatant obtained was measured for absorbance at 550 nm using a 1 cm path length to determine the concentration.

Western blotting

Extraction of proteins was made from cardiomyocytes and myocardium and separated using SDS-PAGE. The proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), which were blocked with 5% non-fat milk for 2 h and then incubated with antibodies against anti-TRADD (DF6279), anti-Cleaved Caspase 1 (AF4005, 1:1000, Affinity, USA); anti-Caspase 1 (22915-1-AP, 1:2000, Proteintech, USA); anti-GSDMD (NBP2-33422), anti-NIMA related kinase 7 (NEK7, NBP2-80470, 1:1000, Novus, USA); anti-ASC (A1170, 1:1000, Abclonal, USA); anti-NLRP3 (ab263899), anti-XBP1 (ab37152) (1:1000, Abcam, UK); anti-GAPDH (GB12002, 1:5000, Servicebio, Wuhan, China) or anti-β-tubulin (AT0003, 1:3000, CMCTAG, Milwaukee, WI, USA) severally at 4 °C for more than 12 h. Membranes were then treated at room temperature with HRP-conjugated IgG (A0192, A0208, A0216, Beyotime, Shanghai, China) for 2 h. Protein bands were visualized by enhanced chemiluminescence (WP20005, Thermo Fisher Scientific Inc, Rock-Ford, IL, USA).

Immunofluorescence

Cardiomyocytes were incubated with either antibodies against anti-TRADD (1:500) or GSDMD (1:500) at 4 °C for more than 12 h after fixation. Samples were subsequently treated with Alexa Fluor 488-conjugated IgG (Beyotime, Shanghai, China) for another 2 h. Cardiomyocytes were observed and photographed using a laser scanning confocal microscope (Leica, Wetzlar, Germany).

Propidium iodide (PI) and Hoechst 33342 staining

Cardiomyocytes were incubated with PI and Hoechst 33342 solution (P0137, Beyotime, Shanghai, China) at room temperature for 3–5 min. Cardiomyocytes were observed and photographed using a laser scanning confocal microscope (Leica, TCS SP8, Wetzlar, Germany).

Chromatin immunoprecipitation (CHIP)

CHIP was carried out according to the instructions of Pierce Agarose ChIP Kit (25156, Thermo Fisher Scientific Inc, MA, USA). The primers for XBP1-binding with TRADD promoter were 5′-GTGGTCCACCCAGCAATACA-3′ and 5′-ACCACTCTTCAGGACCCAGT-3′.

Statistical analysis

The data were presented as mean ± standard deviation and analyzed using one-way ANOVA and Bonferroni post-hoc test through Stata 15.0. The statistical significance was set as P < 0.05.

Results

TRADD expression was increased in the hearts of diabetic mice and HG-treated cardiomyocytes

For assessing the role of TRADD in DCM, we first measured the expression of TRADD in the hearts of STZ-induced diabetic mice and HG-treated cardiomyocytes. TRADD expression was enhanced in the hearts of mice after STZ injecting in comparison to those in control (Fig. 1a). Consistently, compared to NG, HG treatment for 24 h, 48 h, or 72 h markedly increased the TRADD expression. Among them, the expression level of TRADD reached the highest level after HG treatment for 48 h (Fig. 1b). Given the increased levels of TRADD in vivo and in vitro, the involvement of TRADD in DCM was strongly suggested.

Fig. 1. TRADD expression was increased in the hearts of diabetic mice and HG-treated cardiomyocytes.

a Male C57BL/6 mice were injected intraperitoneally with STZ (60 mg·kg⁻¹·d⁻¹, DCM) or the same amount of citrate buffer (control) respectively for 5 consecutive days. The mice were continued to be fed for 12 weeks. Protein levels of TRADD were measured in the myocardium. ^**P < 0.01 versus control, n = 6. b Cardiomyocytes were cultured with normal glucose (NG, 5.5 mmol/L) or high glucose (HG, 33.3 mmol/L) medium for 8, 12, 24, 48 or 72 h. Protein levels of TRADD were measured in the cardiomyocytes. ^&&P < 0.01 versus NG, n = 6.

TRADD deficiency did not affect blood glucose and triglyceride levels in diabetic mice

TRADD^−/− mice were used to investigate a relationship between TRADD and DCM. Our results showed that STZ injection elevated FBG, HbA1c, and TG levels of both WT mice and TRADD^−/− mice, suggesting the establishment of a diabetic mouse model. Nonetheless, no statistical difference was found in FBG, HbA1c, or TG levels of mice in the WT-DCM group and TRADD^−/−-DCM group (Fig. 2), indicating that TRADD deficiency did not affect blood glucose and TG levels in diabetic mice.

Fig. 2. TRADD deficiency did not affect blood glucose and triglyceride levels in diabetic mice.

Male C57BL/6 mice were injected intraperitoneally with STZ (60 mg·kg⁻¹·d⁻¹, DCM) or the same amount of citrate buffer (control) respectively for 5 consecutive days. a, b Fast blood glucose was measured periodically from the control and mice with DCM before (0 week) and over the course of 12 weeks after STZ injection. c HbA1c level in the plasma after 12 weeks. d TG level in the serum after 12 weeks. ^**P < 0.01 vs WT; ^&&P < 0.01 vs TRADD^−/−, n = 6.

TRADD deficiency improved cardiac function and attenuated myocardial hypertrophy and fibrosis in diabetic mice

Cardiac function, myocardial hypertrophy, and fibrosis were further detected. Cardiac function assessed using echocardiography showed that both WT mice and TRADD^−/− mice exhibited decreased EF, FS, and E/A ratio following STZ injection, suggesting compromised cardiac function in diabetic mice (Fig. 3a–c). H&E and WGA staining of the myocardium, along with ANP and BNP mRNA expression revealed exacerbated cardiac hypertrophy in mice after STZ injection (Fig. 3d–g). Furthermore, Masson staining, Sirius Red staining, and CVF quantification as well as hydroxyproline measurement revealed typical myocardial fibrosis in mice following STZ injection (Fig. 3h–k). Moreover, TRADD deficiency improved cardiac dysfunction, and attenuated cardiac hypertrophy and fibrosis in diabetic mice (Fig. 3).

Fig. 3. TRADD deficiency improved cardiac function and attenuated myocardial hypertrophy and fibrosis in diabetic mice.

Male C57BL/6 mice or TRADD knockout mice (TRADD^−/−) were injected intraperitoneally with STZ (60 mg·kg⁻¹·d⁻¹, DCM) or the same amount of citrate buffer (control) respectively for 5 consecutive days. The mice were continued to be fed for 12 weeks. a Images of typical two-dimensional M-mode echocardiography and pulse Doppler ultrasound. b, c Ejection fraction (EF), fractional shortening (FS), and the ratio (E/A) of early mitral valve peak blood flow velocity (E) to late mitral valve peak blood flow velocity (A) in mice. d, e Representative images of H&E staining and WGA staining of the myocardium. f Cardiomyocytes areas quantification under WGA staining. g ANP and BNP mRNA levels in the myocardium. h Representative images of Masson staining. i, j Representative images of Sirus-red staining and CVF quantification. k Hydroxyproline content in the myocardium. ^**P < 0.01 vs WT; ^&P < 0.05, ^&&P < 0.01 vs TRADD^−/−; ^#P < 0.05, ^##P < 0.01 vs WT-DCM, n = 6.

TRADD deficiency attenuated pyroptosis in the hearts of diabetic mice

The protein expression of NLPR3, ASC, Cleaved Caspase 1, and GSDMD in the myocardium and inflammatory factors of IL-1β and IL-18 in the serum were examined to further confirm the role of TRADD in pyroptosis of diabetic mice hearts. It was found that NLPR3, ASC, Cleaved Caspase 1, IL-1β, IL-18, and GSDMD were significantly enhanced in both WT and TRADD^−/− mice following STZ injection, suggesting increased pyroptosis in the hearts of diabetic mice. We also found that the above indexes were all lower in the mice of the TRADD^−/−-DCM group than those in the WT-DCM group (Fig. 4a–f).

Fig. 4. TRADD deficiency attenuated pyroptosis in the hearts of diabetic mice.

Male C57BL/6 mice or TRADD knockout mice (TRADD^−/−) were injected intraperitoneally with STZ (60 mg·kg⁻¹·d⁻¹, DCM) or the same amount of citrate buffer (control) respectively for 5 consecutive days. The mice were continued to be fed for 12 weeks. a–c Protein levels of NLPR3, ASC, and Cleaved Caspase 1 in the myocardium. d, e IL-1β and IL-18 levels in the serum. f, g Protein levels of GSDMD and NEK7 in the myocardium. ^**P < 0.01 vs WT; ^&&P < 0.01 vs TRADD^−/−; ^#P < 0.05, ^##P < 0.01 vs WT-DCM, n = 6.

It has been reported that NEK7 is involved in the NLRP3 inflammasome activation and formation to contribute to diabetic complications. Our results showed increased NEK7 expression in both WT and TRADD^−/− mice hearts following STZ injection, which was lower in the mice of the TRADD^−/−-DCM group than those in the WT-DCM group (Fig. 4g). All the results confirmed that TRADD deficiency attenuated pyroptosis in the hearts of diabetic mice.

TRADD downregulation ameliorated cell injury and pyroptosis in HG-treated cardiomyocytes

Cardiomyocytes in vitro were used to examine the role of TRADD in HG-treated cardiomyocytes. HG treatment enhanced the release of LDH but reduced ATP levels in the cardiomyocytes, while TRADD downregulation by TRADD siRNA transfection remarkedly decreased LDH release but increased ATP levels (Fig. 5a, b). HG treatment increased the number of positive PI staining cardiomyocytes, which was reversed by TRADD downregulation (Fig. 5c). Besides, HG treatment enhanced IL-1β and IL-18 levels, which were alleviated by TRADD downregulation (Fig. 5d, e). Furthermore, NLPR3, ASC, Cleaved Caspase 1, GSDMD, and NEK7 expression were considerably elevated in HG-treated cardiomyocytes, which were all attenuated by TRADD downregulation (Fig. 5f–j). These results suggested that TRADD downregulation ameliorated cell injury and pyroptosis in HG-treated cardiomyocytes.

Fig. 5. TRADD downregulation ameliorated cell injury and pyroptosis in HG-treated cardiomyocytes.

Cardiomyocytes were transfected with TRADD siRNA or non-specific control RNA (NC) siRNA before culturing with normal glucose (NG, 5.5 mmol/L) or high glucose (HG, 33.3 mmol/L) medium for 48 h. a LDH content in cell culture medium. b ATP levels in the cardiomyocytes. c PI (red) and Hoechst 33342 (blue) staining of cardiomyocytes, bar = 200 μm. d, e IL-1β and IL-18 levels in the cardiomyocytes. f–h Protein levels of NLPR3, ASC, and Cleaved Caspase 1 in the cardiomyocytes. i Immunofluorescence staining of GSDMD (green) and DAPI (blue), bar = 50 μm. j Protein levels of NEK7 in the cardiomyocytes. ^**P < 0.01 vs NC siRNA; ^&P < 0.05, ^&&P < 0.01 vs TRADD siRNA; ^#P < 0.05, ^##P < 0.01 vs NC siRNA+HG, n = 6.

TRADD inhibitor Apt-1 ameliorated cell injury and pyroptosis in HG-treated cardiomyocytes

Apt-1, a potent TRADD inhibitor, was used to further elucidate the role of TRADD in HG-treated cardiomyocytes. Our results showed that Apt-1 treatment markedly reduced LDH release but increased ATP content in HG-treated cardiomyocytes (Fig. 6a, b). Apt-1 treatment reduced the number of positive PI staining cardiomyocytes (Fig. 6c). Moreover, Apt-1 treatment significantly decreased IL-1β and IL-18 levels, and dramatically suppressed NLPR3, ASC, Cleaved Caspase 1, GSDMD, and NEK7 expression in HG-treated cardiomyocytes (Fig. 6d–j). These results suggested that TRADD inhibitor Apt-1 ameliorated cell injury and pyroptosis in HG-treated cardiomyocytes.

Fig. 6. TRADD inhibitor Apt-1 ameliorated cell injury and pyroptosis in HG-treated cardiomyocytes.

Cardiomyocytes were treated with the TRADD inhibitor apostatin-1 (Apt-1, 10 μmol/L) before culturing with normal glucose (NG, 5.5 mmol/L) or high glucose (HG, 33.3 mmol/L) medium for 48 h. a LDH content in cell culture medium. b ATP levels in the cardiomyocytes. c PI (red) and Hoechst 33342 (blue) staining of cardiomyocytes, bar = 200 μm. d, e IL-1β and IL-18 levels in the cardiomyocytes. f–j Protein levels of NLPR3, ASC, Cleaved Caspase 1, GSDMD, and NEK7 in the cardiomyocytes. ^**P < 0.01 vs NG, ^##P < 0.01 vs HG, n = 6.

High glucose increased XBP1 expression and enhanced the bind of XBP1 to TRADD promoter to elevate TRADD expression in the cardiomyocytes

A series of studies were then conducted to determine the TRADD upregulation mechanism in HG treatment cardiomyocytes. Luciferase (Luc) reporter assays showed HG treatment increased TRADD promoter activity with −798/+115 Luc, −325/+115 Luc, and −152/+115 Luc, but not with the −92/+115 Luc (Fig. 7a). Bioinformatic analyses revealed XBP1-binding site in the upstream region of 120 bp to 115 bp (Fig. 7b). CHIP assay confirmed that HG enhanced the bind of XBP1 to TRADD promoter in the cardiomyocytes (Fig. 7c).

Fig. 7. High glucose increased XBP1 expression and enhanced the bind of XBP1 to TRADD promoter to elevate TRADD expression in the cardiomyocytes.

a After TRADD promoter–luciferase fusion plasmid transfection for 24 h, the cardiomyocytes were cultured with normal glucose (NG, 5.5 mmol/L) or high glucose (HG, 33.3 mmol/L) medium for 48 h. The promoter activity of TRADD was determined. ^**P < 0.01 vs NG, n = 6. b The bind site of XBP1 in the upstream region of TRADD promoter. c The cardiomyocytes were cultured with NG or HG medium for 48 h. The binding activity of XBP1 with TRADD promoter was measured by CHIP. ^**P < 0.01 vs NG, n = 6. d Cardiomyocytes were transfected with TRADD siRNA or non-specific control RNA (NC) siRNA before culturing with NG or HG medium for 48 h. Protein levels of XBP1 in the cardiomyocytes were measured. ^**P < 0.01 vs NC siRNA, ^&&P < 0.01 vs TRADD siRNA. e Cardiomyocytes were transfected with XBP1 siRNA 745, XBP1 siRNA 561, XBP1 siRNA 279 or NC siRNA. Protein levels of XBP1 in the cardiomyocytes were measured. ^**P < 0.01 vs NC siRNA. f Cardiomyocytes were transfected with XBP1 siRNA or NC siRNA before culturing with NG or HG medium for 48 h. Immunofluorescence staining of TRADD (green) and DAPI (blue), bar = 25 μm.

XBP1 is an important transcription factor that influences pathological and physiological functions like cell survival, inflammation, and proliferation. Western blotting indicated that HG treatment increased XBP1 expression in cardiomyocytes, which was not affected by TRADD downregulation (Fig. 7d). Moreover, immunofluorescence staining demonstrated that the enhancement of TRADD expression in HG-treated cardiomyocytes was attenuated by XBP1 downregulation with siRNA (Fig. 7e, f). The above results implied that high glucose increased XBP1 expression and enhanced the bind of XBP1 to TRADD promoter to elevate TRADD expression in the cardiomyocytes.

Discussion

STZ is a toxic glucose analog that impairs the signaling function of β-cell mitochondrial metabolism, leading to insulin-dependent diabetes [26]. In the present study, a large dose of STZ was administered to establish a diabetes model. DCM is a major complication of diabetes with glucotoxicity and lipotoxicity, leading to severe cardiac dysfunction, myocardial injury, myocardial hypertrophy, and fibrosis, greatly reducing the quality of life of patients. Moreover, compared to other complications, the mechanism of DCM is more complex [27]. Previous research has confirmed that TRADD is a vital signal transduction protein involved in controlling the balance between cell death and cell survival [22]. Here we found that TRADD expression was elevated in cardiomyocytes following HG treatment and in the hearts of STZ-induced mice, suggesting that TRADD may be related to the progression of DCM. So, TRADD knockout mice were utilized to validate our hypothesis. It was revealed that STZ-induced cardiac dysfunction, myocardial hypertrophy, and fibrosis in WT diabetic mice were alleviated when the TRADD gene was knocked out. It is reported both Apt-1 and ICCB-19 specifically bind to the N-terminal of TRADD, thereby inhibiting the function of TRADD and the formation of complex I. They have similar effects, and Apt-1 was chosen in the present study. The study in vitro showed that TRADD downregulation or Apt-1 administration attenuated cardiomyocyte injury under HG stimulation. These findings suggested that TRADD could negatively regulate DCM. However, the specific mechanism of TRADD involvement in DCM remains unclear.

Pyroptosis is a typical condition of programmed and inflammatory cell death. It triggers a strong inflammatory response through NLRP3 inflammasome activation, which, in conjunction with ASC, promotes hydrolysis of Caspase 1 precursor. It then facilitates IL-1β and IL-18 activation and finally cleaves downstream effector GSDMD [7–9]. Most of the characteristics of pyroptosis are consistent with the pathological changes of cardiomyocytes in DCM. Therefore, we focused on the regulation of TRADD in pyroptosis, aiming to discover a novel target and mechanism of DCM. Studies have reported that TRADD serves vital roles in regulated cell death. MiR-1184 has an inhibitory impact on apoptosis and inflammatory responses in sepsis via targeting TRADD [28]. Downregulation of TRADD protected against TNF-α-mediated necroptosis and impaired autophagy in chondrocytes [29]. Herein, TRADD knockdown reduced the expression of pyroptotic proteins, such as NLRP3, ASC, Cleaved Caspase 1, GSDMD, and inflammatory factors such as IL-1β and IL-18 in STZ-treated mice hearts. In cardiomyocytes, downregulation of TRADD by either siRNA transfection or the inhibitor Apt-1 abolished HG-induced pyroptosis, as seen by reduced expression of NLRP3, ASC, Cleaved Caspase 1, IL-1β, IL-18, and GSDMD levels. These results suggested that TRADD acted as a negative regulator of DCM by mediating cardiomyocyte pyroptosis.

Previous positive genetic analysis on the inflammasome activation in mice found that NEK7 was a key component of the NLRP3 inflammasome. NEK7 is a serine/threonine kinase, which is crucial for maintaining homeostasis, cell cycle progression, and cell division. Recently conducted studies have shown that parameters like ROS and potassium, activate Capase-1 through regulating the interaction of NEK7 and NLRP3 inflammasome, eventually leading to pyroptosis [30]. Consistently, we observed that the augment of NEK7 expression in STZ-induced mice heart and in HG-stimulated cardiomyocytes was attenuated by TRADD knockout or TRADD downregulation. Therefore, NEK7 served as a downstream regulator of TRADD in pyroptosis, contributing to DCM.

Reportedly, the E74-like ETS transcription factor 3 (ELF3) regulates the transcriptional activity of microtubule affinity regulating kinase 4 (MARK4), participating in HG-induced endothelial cell inflammation [31]. HG stimulation activates the transcription factor Forkhead O1 (FoxO1) to upregulate the transcription and expression of macrophage osteopontin [32], suggesting that HG may regulate the target protein transcription and expression by influencing transcription factors. Recent studies have shown that HG increased the expression of XBP1 in primary mouse Müller cells and rat pancreatic β cells [33, 34]. Activated XBP1 transfers to the nucleus, attaches to the promoter of the target gene, and subsequently initiates target gene transcription, thereby regulating cell growth, apoptosis, and oxidative stress [35–37]. There have been studies reporting the relationship between XBP1 and pyroptosis. Previous research confirmed that XBP1 inhibited the IRE-1α/XBP1s branch and attenuated Cd-induced NLRP3 inflammasome activation and pyroptosis in HK-2 cells [38]. We found that HG enhanced the bind of XBP1 to the TRADD promoter of cardiomyocytes. And our experiments revealed the protein expression of XBP1 in cardiomyocytes increased following high glucose stimulation, which is not affected by TRADD siRNA transfection. Moreover, the increase of TRADD expression in high glucose-stimulated cardiomyocytes was attenuated by XBP1 knockdown. The findings herein demonstrate that XBP1 can serve as an upstream regulator of TRADD transcription, then mediate pyroptosis, and finally contribute to DCM.

However, there are several limitations in our present study. First, primary cardiomyocytes from diabetic TRADD^−/− mice will be beneficial to elucidate the real roles of TRADD in the pathological process of diabetic cardiomyopathy. Second, dynamic changes including cardiac function during diabetes might be an ideal strategy in further studies.

In summary, high glucose increased XBP1 expression and enhanced the bind of XBP1 to TRADD promoter to elevate TRADD expression. Then TRADD augmented the activation of NEK7 and NLRP3 inflammasome, inducing pyroptosis and eventually leading to DCM. TRADD deficiency improved cardiac dysfunction and attenuated myocardial, fibrosis and pyroptosis to alleviate DCM (Fig. 8). These findings offer a better understanding and have a potential guiding significance for clinical treatment of DCM.

Fig. 8. TRADD-mediated pyroptosis contributes to diabetic cardiomyopathy.

High glucose increased XBP1 expression and enhanced the bind of XBP1 to the TRADD promoter to elevate TRADD expression in the cardiomyocytes. Then TRADD augmented the activation of NEK7 and the NLRP3 inflammasome, inducing pyroptosis and eventually leading to DCM. TRADD deficiency improved cardiac dysfunction and attenuated myocardial hypertrophy, fibrosis, and pyroptosis to alleviate DCM.

Author contributions

GLM, JHS, and YC designed research; YYZ, DNS, XLP, WQS, and SJY performed research; QYZ analyzed data; YYZ wrote the original paper; GLM, JHS, and YC reviewed and edited the paper.

Competing interests

The authors declare no competing interests.