Totally, 50 C57BL/6 mice (Nanjing Junke Biological Engineering Co., Ltd.) were fed in a specific pathogen-free environment under a 12 h light/12 h dark cycle. Each animal experiment strictly followed the Guide for the Care and Use of Laboratory Animal by International Committees. The mice were randomly separated into the following five groups before surgery (10 mice/group): control group; sham operation group; I/R group; I/R + short hairpin RNA (shRNA) against negative control (sh-NC) group; I/R + shRNA against PVT1 (sh-PVT1) group. This study gained the approval of the Institutional Animal Care of Guangdong Provincial People's Hospital (GDREC2016255H).

After the mice were adaptively reared for 1 week, they were anesthetized by intraperitoneal injection of sodium pentobarbital (45 mg/kg). After anesthesia, there was no response in the clip toe test and the trachea was intubated. The ventilator parameters were set to synchronize with the respiratory rate of the anesthetized mice. Then, the endotracheal tube was connected to the ventilator. Before the operation, a physiological signal collection and processing system was used to record the rat's electrocardiogram. The chest was opened through the third intercostal space on the left edge of the sternum, and the left anterior descending (LAD) coronary artery was ligated with 7-0 silk thread. After 45 min, the ligature was loosened for reperfusion. No treatment was done for mice in the control group. This study only opened the chest of mice in the sham operation group, but threaded the corresponding part without ligating the LAD. After the operation, mice were continuously monitored by ECG for 15 min and placed on a heating pad at 30°C until they were awakened. 48 h before modeling, mice in the I/R + sh-NC and I/R + sh-PVT1 groups were injected by 10 μL sh-NC or sh-PVT1 (GenePharma, Shanghai, China) that was premixed with Lipofectamine 2000 reagent (Thermo Fisher Scientific, USA). The sequence of sh-PVT1 was as follows: 5′-CCUGCAUAACUAUCUGCUUTT-3′.

Under anesthesia, abdominal aortic blood was firstly collected. Then, the limbs were fixed, the chest was opened and the mouse's heart was obtained. After removing the auricle and vascular tissue, the heart was transected into two parts. Half of the tissue at the bottom of the heart was quick-frozen by liquid nitrogen, and then transferred to a −80°C refrigerator for subsequent experiments. Apical 1/2 tissue was fixed in 40 g/L paraformaldehyde and transferred to 70% ethanol solution on the second day. Then, through the Excelsior™ AS tissue processing system, the tissue was dehydrated by gradient ethanol, transparent by xylene and waxed in liquid paraffin. Subsequently, the HistoCore Arcadia paraffin embedding system was used for pathological examination after embedding and retention.

Total RNA was extracted from myocardial tissues and cells utilizing TRIzol (Beyotime, Shanghai, China) and cDNA was reverse transcribed with PrimeScript RT Master Mix (Servicebio, Wuhan, China). These primer sequences were as follows: PVT1: 5′-TGAGAACTGTCCTTACGTGACC-3′ (F), 5′-AGAGCACCAAGACTGGCTCT-3′ (R); IL-1β: 5′-CACCTCTCAAGCAGAGCACAG-3′ (F), 5′-GGGTTCCATGGTGAAGTCAAC-3′ (R); IL-6: 5′-GCTACAGCACAAAGCACCTG-3′ (F), 5′-GACTTCAGATTGGCGAGGAG-3′ (R); TNF-α: 5′-GGCAGCCTTGTCCCTTGAAGAG-3′ (F), 5′-GTAGCCCACGTCGTAGCAAACC-3′ (R); GSDMD: 5′-CCAACATCTCAGGGCCCCAT-3′ (F), 5′-TGGCAAGTTTCTGCCCTGGA-3′ (R); GAPDH: 5′-CTGGGCTACACTGAGCACC-3′ (F), 5′-AAGTGGTCGTTGAGGGCAATG-3′ (R). The expression of above targets was quantified with the 2^−ΔΔCt method, while GAPDH served as an internal control.

Abdominal aortic blood samples were centrifuged at 3,000 g. Then, the serum samples were harvested. Following the instructions of ELISA kits, brain natriuretic peptide (BNP; Biocompare, USA), cardiac troponin I (cTnI; Biocompare, USA) and IL-1β (Biocompare, USA) levels were tested in serum or cell supernatant. Through automatic biochemical analyzer, lactate dehydrogenase (LDH) and creative kinase isoenzyme MB (CK-MB) levels were examined in serum or cell supernatant.

The paraffin-embedded mouse myocardial tissue was sectioned to 5 μm. H&E staining and Masson staining were carried out following the kit instructions (Servicebio, Wuhan, China). The paraffin sections were deparaffinized to water. Then, H&E staining was successively performed. The sections were stained with hematoxylin for 5 min and rinsed by tap water, followed by being differentiated by differentiation solution for 3 s. After being rinsed by tap water, the sections returned to blue for about 3 s, rinsed by tap water and dehydrated by 85 and 95% ethanol in turn for 4 min. Afterwards, the sections were stained by eosin dye solution for 5 min and dehydrated with anhydrous ethanol for 3 times (5 min each time) and transparent by xylene twice for 2 min. Then, the sections were mounted with neutral gum. Masson staining was carried out according to the following procedures. The sections were stained through Masson A solution lasting 15 h as well as heated in a 65°C oven lasting 30 min. After being washed using tap water, the sections were stained by Masson B solution and C solution mixed dyeing for 1 min and differentiated with 1% hydrochloric acid alcohol lasting 1 min. Then, the sections were washed by tap water as well as stained utilizing Masson D solution lasting 6 min and immersed by Masson E solution for 1 min and Masson F solution for 15 s. Afterwards, the sections were differentiated by 1% glacial acetic acid for 3 times (8 s each time) and dehydrated with absolute ethanol for 3 times (5 min each time). After being transparent, the slides were mounted with neutral gum. All slides were scanned at 200 × by TissueFAXS PLUS scanning system (Taborstrasse, Austria).

According to the instructions of the reagents, protein specimens were extracted from myocardial tissues, serum samples and cells through RIPA lysate. After centrifugation at 14,000 g, the supernatant was collected and mixed with the loading buffer. The protein was denatured by boiling at 100°C for 10 min. About 50 μg protein was electrophoresed in 10% SDS-PAGE (Beyotime, Shanghai, China), then transferred to 0.22 μm PVDF membrane (Merck KGaA, Germany). Afterwards, the membrane was incubated with primary antibodies against α-myosin heavy chain (α-MHC; 1/1,000; ab174640; Abcam, USA), β-myosin heavy chain (β-MHC; 1/1,000; ab170867; Abcam, USA), TNF-α (1/1,000; ab215188; Abcam, USA), IL-1β (1/1,000; ab254360; Abcam, USA), IL-6 (1/1,000; ab259341; Abcam, USA), GSDMD (1/200; ab219800; Abcam, USA), TLR4 (1/50; ab1355; Abcam, USA), MyD88 (1/1,000; ab219413; Abcam, USA), NLRP3 (1/1,000; ab263899; Abcam, USA), NF-κB phosphorylation p-p65 (1/1,000; ab76302, USA), NF-κB p65 (#8242, 1:1,000; Cell Signaling Technology, USA), Cleaved caspase-3 (1/5,000; ab21443; Abcam, USA), Bax (1/1,000; ab23247; Abcam, USA), Bcl-2 (1/1,000; ab259833; Abcam, USA), Caspase1 (1/1,000; ab207802; Abcam, USA), and GAPDH (1/1,000; ab9484; Abcam, USA) overnight at 4°C. The next day, the membrane was washed utilizing TBST as well as incubated with HRP-conjugated secondary antibodies (1/10,000; ab7090; Abcam, USA) lasting 1 h at room temperature. Then, the ECL substrate (Thermo Scientific, USA) was dropped on the membrane, followed by being exposed in the FluorChem™E chemiluminescence imaging system. The integrated optical density of the protein band was measured using ImageJ software (version 1.8.0).

Paraffin-embedded sections of myocardial tissue were baked in a 60°C incubator for 2 h. The sections were deparaffinized by xylene and hydrated with ethanol gradient. 3% hydrogen peroxide was added to eliminate endogenous peroxidase activity. After washing, antigen retrieval was carried out in PBS buffer. Then, the sections were blocked by goat serum for 15 min. The primary antibody against GSDMD (1/200; sc-393581; Santa Cruz Biotechnology, USA) and Caspase1 (1/1,000; ab207802; Abcam, USA) was added dropwise to the sections and incubated overnight at 4°C. After washing with PBS, the sections were incubated with secondary antibody (1/10,000; ab7090; Abcam, USA) lasting 2 h. Then, the color was developed with DAB color developing solution. The sections were counterstained by hematoxylin, dehydrated, transparent, and mounted. The results were investigated under a microscope, which were analyzed by Image Plus image analysis software.

H9C2 cells (ATCC, USA) were grown in DMEM cell culture medium containing 1.5 g/L NaHCO₃, 10% FBS, 1% glutamine, and 1% penicillin in a constant temperature incubator of 5% CO₂ at 37°C. For H/R, the cells were cultured in an environment of 95% N₂ and 5% CO₂ at 37°C lasting 4 h. After changing the medium, the cells were reoxygenated with 5% CO₂ at 37°C lasting 3 h. PVT1 sequence was designed and synthesized by GenePharma company (Shanghai, China), which was subcloned into pcDNA3.1 (Invitrogen, USA). The pcDNA3.1 vector served as a control. Lipofectamine 2000 (Invitrogen, USA) was utilized for transfection of plasmids (500 ng pcDNA3.1 vector (empty vector), 500 ng PVT1) into H9C2 cells for 48 h. The sense and antisense oligonucleotides of the sh-PVT1 (5′-CCUGCAUAACUAUCUGCUUTT-3′) were synthesized and cloned into the pENTR™/U6 vector (Invitrogen, USA). For silencing PVT1, H9C2 cells (5 × 10⁴) were transfected by 1 μg sh-NC or 1 μg sh-PVT1 for 48 h via Lipofectamine 2000 reagent before H/R. After transfection for 48 h, above transfected cells were used for the subsequent experiments. Furthermore, the cells were treated with 25 nM Necrosulfonamide (NSA; abs814352; Absin, Shanghai, China), followed by PVT1 overexpression vector transfection and H/R treatment.

H9C2 cells in each group were planted onto a 96-well plate (1 × 10⁴/well). Each group set eight multiple holes. A blank group was set, which was only added by culture medium without cells. Each well was incubated with 10 μL of CCK-8 solution (Dojindo, Japan) lasting 1 h at 37°C. After incubation, a microplate reader was utilized for measuring the absorbance at 450 nm wavelength.

Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) cell apoptosis kit (Beyotime, Shanghai, China) was utilized for detecting cellular apoptosis. H9C2 cells were collected and washed utilizing PBS. The cells were resuspended by 250 μL prepared binding buffer. The density of the cells was adjusted to 1 × 10⁹/L. Then, 100 μL cell suspension was inoculated into a 5 mL flow tube. The cells were incubated with 5 μL Annexin V/FITC and 10 μL PI in the dark at room temperature. After 15 min, the apoptosis rate was detected by flow cytometry.

H9C2 cells were seeded and climbed on the 6-well plate. When the cell fusion was about 80%, the cells were treated with H/R, transfection and drug treatment. Then, the cells were fixed by paraformaldehyde, permeabilized by PBS containing 0.1% TritonX-100, and blocked by BSA. Then, the sections were incubated with primary antibody against GSDMD (1/200; ab219800; Abcam, USA), followed by FITC-labeled fluorescent secondary antibody (1/200; ab7064; Abcam, USA). After the nucleus was stained with 1 μg/ml DAPI for 10 min, images were investigated under a confocal laser microscope.

All analyses were presented utilizing Graphpad Prism software (version 7.0). Statistical differences between multiple groups were compared through one-way analysis of variance (ANOVA) followed by Tukey's test. The results were displayed as the mean ± standard deviation. Each experiment was carried out in triplicate. P < 0.05 was indicative of statistical significance.

Here, this study constructed a myocardial I/R mouse model. Our results showed that lncRNA PVT1 displayed significant up-regulation in myocardial I/R tissues compared with sham myocardial tissues (Figure 1A). This indicated that PVT1 might participate in myocardial I/R progression. For investigating this influence of PVT1 on myocardial I/R damage, PVT1 was silenced by its shRNA. In Figure 1A, the expression of PVT1 was markedly inhibited by sh-PVT1 in myocardial I/R tissues compared with sh-NC. Indicators of myocardial injury including cTnI, LDH, BNP and CK-MB were examined by ELISA. As demonstrated by our data, serum levels of cTnI (Figure 1B), LDH (Figure 1C), BNP (Figure 1D), and CK-MB (Figure 1E) were markedly elevated in myocardial I/R mice compared with sham mice. But PVT1 knockdown significantly decreased serum cTnI, LDH, BNP and CK-MB levels in myocardial I/R models. To evaluate the effect of PVT1 knockdown on the morphology of I/R heart tissue, H&E and Masson staining of heart tissue was performed at the end of the intervention. The results of H&E staining showed that the myocardial fibers of the control group and the sham group were arranged regularly; there was no breakage or necrotic gap; and the myocardial cell nucleus was fusiform or oval (Figure 1F). For I/R and I/R + sh-NC groups, the myocardial fiber structure was damaged; the myocardial fiber was broken and dissolved; the intermuscular space was enlarged; there were inflammatory cellular infiltrations in the infarct. The destruction of myocardial fiber structure and infiltrations of inflammatory cells were improved in I/R + sh-BCRT1 group. As shown in Masson staining, the myocardial tissue fibers were neatly arranged, evenly stained, and there was no collagen in the control group and the sham group (Figure 1G). For I/R and I/R + sh-NC groups, the degree of myocardial fibrosis was obvious; myocardial cells were significantly reduced; and there was increased collagen. For I/R + sh-BCRT1 mice, myocardial fibrosis was markedly ameliorated; the reduction of myocardial cells was improved; and the collagen was decreased. These findings demonstrated that PVT1 knockdown could alleviate myocardial I/R injury.

Western blot was applied for testing the expression of cardiac function markers including α-MHC and β-MHC in myocardial tissues. Our data showed that α-MHC expression was significantly decreased while β-MHC expression was significantly increased in I/R myocardial tissues compared with sham tissues (Figures 2A–C). However, silencing PVT1 markedly alleviated I/R-induced the decrease in α-MHC and the increase in β-MHC. This indicated that PVT1 knockdown could improve cardiac function in myocardial I/R mice. Serum TNF-α, IL-1β, and IL-6 levels were examined by RT-qPCR. In comparison to sham mice, there were increased serum levels of TNF-α (Figure 2D), IL-1β (Figure 2E), and IL-6 (Figure 2F) mRNAs in myocardial I/R mice. Nevertheless, silencing PVT1 distinctly decreased their serum levels in myocardial I/R mice. Western blot was also carried out to test the expression of TNF-α, IL-1β, and IL-6 in serum samples (Figure 2G). Consistently, the expression TNF-α (Figure 2H), IL-1β (Figure 2I), and IL-6 (Figure 2J) proteins was markedly up-regulated in serum of myocardial I/R mice compared with sham mice. Their expression was significantly ameliorated by PVT1 knockdown. Therefore, silencing PVT1 could alleviate I/R-induced cytokine release.

Here, we detected the expression of GSDMD mRNA in myocardial tissues by RT-qPCR. In Figure 3A, GSDMD exhibited the up-regulated mRNA expression in myocardial I/R tissues than sham specimens. But silencing PVT1 markedly decreased the expression of GSDMD mRNA in myocardial I/R tissues. Immunohistochemistry was carried out to examine the expression of GSDMD protein. In comparison to sham mice, increased GSDMD expression was found in myocardial I/R tissues, which was distinctly lowered by PVT1 knockdown (Figures 3B,C). Western blot showed that I/R or PVT1 knockdown did not affect GSDMD-FL expression but affected GSDMD-N expression (Figures 3D–F). GSDMD-N expression was distinctly up-regulated in myocardial I/R tissues, which was lowered by PVT1 knockdown. Above data suggested that GSDMD was required for myocardial I/R-induced pyroptosis and silencing PVT1 alleviated GSDMD-induced pyroptosis in myocardial I/R mice.

Increasing evidence suggests that inhibition of the TLR4/MyD88/NF-κB/NLRP3 inflammasome pathway may reduce myocardial infarction-induced pyroptosis (9, 21). The activation of TLR4/MyD88/NF-κB/NLRP3 inflammasome pathway was examined in myocardial tissues by western blot (Figure 4A) (22). Our western blot showed that the expression of TLR4 (Figure 4B), MyD88 (Figure 4C), NLRP3 (Figure 4D), and p-p65/p65 (Figure 4E) was distinctly elevated in myocardial I/R tissues than sham specimens. However, their expression was markedly suppressed by PVT1 knockdown. These data suggested that suppression of pyroptosis through PVT1 knockdown involved TLR4/MyD88/NF-κB/NLRP3 inflammasome pathway in myocardial I/R tissues.

We also investigated whether PVT1 knockdown affected cardiomyocyte apoptosis. Western blot was presented for examining Cleaved caspase-3, Bax, and Bcl-2 expression in myocardial tissues (Figure 5A). We found that the expression of Cleaved caspase-3 (Figure 5B) and Bax proteins (Figure 5C) was distinctly increased as well as Bcl-2 expression (Figure 5D) was markedly decreased in myocardial I/R tissues than sham tissues. Moreover, silencing PVT1 markedly alleviated I/R-induced the increase in Cleaved caspase-3 and Bax proteins as well as the decrease in Bcl-2 protein in myocardial tissues. Pyrolysis is dependent on Caspase1. Here, Caspase1 expression was examined by immunohistochemistry. The up-regulation of Caspase1 was found in myocardial I/R tissues than sham tissues, which was suppressed when silencing PVT1 expression (Figures 5E,F).

This study established H/R-induced cellular models. The up-regulation of PVT1 was detected in H/R-induced H9C2 cells than controls (Figure 6A). After transfection with sh-PVT1, PVT1 expression was markedly suppressed. CCK-8 results showed that H/R markedly lowered cellular viability of H9C2 cells (Figure 6B). But PVT1 knockdown ameliorated the decrease in cell viability induced by H/R. In Figures 6C,D, H/R treatment markedly elevated apoptotic levels of H/R-treated H9C2 cells. Nevertheless, silencing PVT1 markedly lowered the apoptotic levels of H/R-induced H9C2 cells.

ELISA was presented for testing the levels of CK-MB and LDH in the supernatant of H9C2 cells. Our data showed that H/R markedly elevated CK-MB (Figure 6E) and LDH (Figure 6F) production in the supernatant, which was alleviated by PVT1 knockdown. Furthermore, we tested IL-1β production in the cell supernatant via ELISA. IL-1β was markedly up-regulated in H/R-induced H9C2 cells than controls (Figure 7A). Meanwhile, its production was significantly inhibited under silencing PVT1 in H9C2 cells. As shown in western blot, H/R or PVT1 knockdown did not alter GSDMD-FL expression in H9C2 cells (Figures 7B,C). But H/R treatment distinctly elevated the expression of GSDMD-N, which was markedly alleviated by PVT1 knockdown (Figure 7D). Moreover, Caspase1 expression was significantly up-regulated in H/R-induced H9C2 cells than controls (Figure 7E). Nevertheless, sh-PVT1 transfection significantly lowered its level H/R-treated cardiomyocytes. Immunofluorescence assays were also carried out to verify GSDMD expression in H9C2 cells. Consistently, the up-regulation of GSDMD was mediated by H/R treatment, which was lowered through PVT1 knockdown in H9C2 cells (Figures 7F,G).

For further investigating the influence of PVT1 on H/R-induced pyroptosis, H9C2 cells were transfected by PVT1 overexpression vector. In Figure 8A, RT-qPCR demonstrated that H/R-induced PVT1 up-regulation was enhanced by PVT1 overexpression vector transfection in H9C2 cells. CCK-8 showed that PVT1 overexpression promoted the decrease in cell viability induced by H/R (Figure 8B). Furthermore, its overexpression strengthened the increase in IL-1β production induced by H/R treatment in H9C2 cells (Figure 8C). Our western blot showed that pyroptosis inhibitor NSA did not affect GSDMD-FL expression but significantly suppressed the expression of GSDMD-N in H/R-induced H9C2 cells (Figures 8D–F). Nevertheless, PVT1 overexpression significantly alleviated the inhibitory effect of NSA on GSDMD-N expression in H9C2 cells.