Cell culture

Human cardiomyocyte cell line AC16 was cultured in a cell incubator with 5% CO2 at 37 °C. Dulbecco’s Modified Eagle’s medium (DMEM, Hyclone, SH30243.01; Logan, UT, US) was supplemented with 10% fetal bovine serum (GIBCO, 16000e044; Carlsbad, CA, USA) to culture AC16 cells. Additionally, to avoid bacterial contamination, 1% penicillin with streptomycin (Solarbio, Beijing, China) was added in the DMEM medium. For heart failure model, AC16 cells were treated with DOX (Sigma-Aldrich, 25316-40-9) at 2 µmol/L for 12, 24, and 48 h. The mRNA level of DUOX1 was quantified using quantitative real-time polymerase chain reaction (qRT-PCR), and the protein levels of DUOX1 were assessed by western blot assay.

Construction of vectors with DUOX1 overexpression

The primers for DUOX1 (NM_017434.5) containing EcoR I and BamH I were designed, and DUOX1 was synthesized based on the designed primer with two kinds of restriction enzyme. Then, to construct the DUOX1 overexpression plasmids, the DUOX1 after amplification was implanted into the vector of pLVX-Puro (Clontech).

After the cells grew to 90% confluency, transfection was conducted. Based on the manufacturer’s instruction, pLVX-Puro-DUOX1, psPAX2, and pMD2G (Addgen) were co-transfected into 293T cells using Lipofectamine™ 2000 (Invitrogen, CA, USA).

The primers for DUOX1 were designed as follows:

DUOX1-F: 5′-CGGAATTCATGGGCTTCTGCCTGGC-3′ (EcoR I).

DUOX1-R: 5′-CGGGATCCCTAGAAGTTCTCATAATGGTGGGAG-3′ (BamH I).

Construction and transfection of DUOX1 silencing vectors

A siRNA sequence targeting DUOX1 was synthesized and inserted into the PLKO.1 vector. The lentiviral production system included the expression vectors psPAX2, pMD2G, and pLKO.1-shDUOX1, which were all co-transfected into 293T cells (ATCC) and packaged. Meanwhile, the plasmid of the scrambled shRNA was constructed as the negative control. RNA interference is a powerful tool to induce loss-of-function phenotypes by inhibiting gene expression at the post-transcriptional level. One is that dsRNA is cut into siRNA, which allows a specific mRNA to be degraded; the other is that as long as there is a long dsRNA, it can degrade all RNA and inhibit the synthesis of all proteins. The pLKO.1-puro plasmid inserted the following sequences of DUOX1 interference: DUOX1 site 1 (1842–1860): CCATCGTCCTTGAACAATT; DUOX1 site 2 (3085–3103): GGAACTGACATGGGAAGAT; and DUOX1 site 3 (4572–4590): GCTGCCAAGTGTTCTGTAA.

qRT-PCR

To quantify the mRNA levels of DUOX1, the total RNA in cells was extracted using a TRIzol reagent (Invitrogen, 1596-026). After quantifying and confirming the RNA integrity, the RNA samples were reverse-transcribed to cDNA; then, cDNA was amplified using SYBR Green qPCR Master Mixes (Thermo Fisher, Rockford, IL, USA). The Ct value was defined as the number of cycles that the fluorescent signal in the reaction tube reaches the set threshold. The relative DUOX1 mRNA levels normalized to GAPDH were calculated using the 2−△△Ct method. The primer sequences for DUOX1 were designed as follows: Primer F: 5′ AGTGCCTGGATTGTTGCC 3′ and Primer R: 5′ TTGCTGGACAGGACGAAG 3′. The primer sequences for GAPDH were designed as follows: Primer F: 5′ GGATTGTCTGGCAGTAGCC 3′ and Primer R: 5′ ATTGTGAAAGGCAGGGAG 3′.

Western blot analysis

The total protein from AC16 cells was extracted using a RIPA buffer (Solarbio, R0010, Beijing, China), and the protein level was determined by BCA protein assay kit (Thermo Fisher Scientific, PICPI23223). The sample was boiled at 95 °C for 10 min and separated through the gels with 10% concentration of SDS-PAGE gel (JRDUN Biotechnology Co., Ltd, Shanghai, China). Then, the separated proteins were transferred to polyvinylidene fluoride membrane and blocked with 5% nonfat milk. After blocking, the membrane was further incubated with anti-DUOX1, anti-active caspase-1, anti-GAPDH (67226-1-lg, 22915-1-AP, 60004-1-1G, Proteintech), anti-apoptosis-associated speck-like protein containing a CARD (ASC), anti-NLRP3, anti-pro-caspase-1, and anti-Gasdermin D-N domain (GSDMD-N) (Ab155970, Ab263899, Ab179515, Ab215203, Abcam) overnight at 4 °C with gentle shaking. After washing thrice by TBST(TBS + Tween20), the polyvinylidene fluoride membrane was incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000, Beyotime, Shanghai, China) for 1 h at 25 °C. Finally, the expression levels of proteins were measured using a chemiluminescent imaging system (Tanon 5200, Shanghai, China).

ROS detection

To detect the ROS production, Active Oxygen Detection Kit (S0033, Beyotime) was employed. First, the cell pellets were obtained after centrifugation and resuspended using 1 mL of cooled PBS. Following the manufacturer’s instruction, the DCFH-DA staining working solution was diluted to 10 µM using a serum-free medium. Then, the DCFH-DA probe solution was added to the cell suspension in the dark for 20 min at 37 °C. To make the probe in full contact with the cells, the samples were mixed every 3–5 min for 20 min. Then, the cells were washed thrice using a serum-free medium to remove the DCFH-DA probe that did not enter the cells. Finally, the samples were detected by flow cytometry (BD Biosciences, Franklin Lake, NJ, USA).

Pyroptosis assay

To detect the level of pyroptosis, the cells were resuspended in PBS. Then, active caspase-1 (1:30; EL900443, EterLife) was added in the cell incubation for 1 h at 25 °C. To remove non-combined caspase-1, the cells were washed with PBS for three times and incubated with propidium iodide solution (PI, P3566; Invitrogen) at 3 µM for 15 min in the dark. Finally, the rates of cell pyroptosis were estimated based on flow cytometry.

Enzyme-linked immunosorbent assay (ELISA)

The secreted IL-1β and IL-18 in the supernatants were detected using ELISA. The cells were collected by centrifuging at 3000 rpm/min for 20 min. After removing the cell pellets, the secreted IL-1β and IL-18 were quantified using Human IL-1β and IL-18 ELISA kits (NeoBioscience, Shenzhen, Guangdong, China), following the manufacturer’s instructions. The amount of IL-1β and IL-18 was detected at 450 nm by a microplate reader (Bio-Rad, Hercules, CA, USA).

Biochemical detection

The lactate dehydrogenase (LDH) in supernatant and cellular protein level was detected using the LDH (A020-1) Kit and BCA (A045-3) Kit (Nanjing, Jiancheng Biotechnology Research Institute, Jiangsu, China). The supernatant and kit solution were mixed for 15 min at 37 °C in a water bath. Then, the LDH and cellular protein were measured at 440 nm (LDH) and 562 nm (BCA).

Statistical analysis

Each experiment was performed in triplicate. The data were presented as mean value ± standard deviation of three independent biological replicates. To compare the difference in mean values among the groups, one-way analysis of variance with Tukey’s post hoc tests was applied. P < 0.05 was regarded as statistically significant. All statistical analyses in the study were performed using the GraphPad Prism 7.0 software (San Diego, CA, USA).