2.1 Cell cultivation and PA treatment

Rat myocardial cell line (H9c2) obtained from American Type Culture Collection (ATCC) was cultured with Dulbecco's modified eagle medium (DMEM) containing 10% fetal bovine serum at constant 5% CO₂ level and 37°C. According to previous study,²² cells were treated with 400 μM PA for 24 h to establish obesity-induced myocardial injury in vitro model.

2.2 Cell transfection

Small interfering RNA (siRNA) specifically targeting OIP5-AS1 (si-OIP5-AS1), scrambled siRNA (negative control, NC), miR-22 mimics, mimics NC, miR-22 inhibitor, and inhibitor NC were obtained from Riobio Co. Ltd. They were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Twenty-four hours or 48 h after transfection, cells were processed for next treatment and detection.

2.3 Oil red O staining

Cells were fixed with 10% formalin for 30 min, and then washed twice with phosphate-buffered saline (PBS), followed by dipping in 60% isopropanol. Subsequently, cells were stained with Oil red O solution (Solarbio) for 10 min, washed twice with PBS, and observed under microscope.

2.4 Evaluation of membrane pores formation

Pyroptosis is characterized by formation of cell membrane pores. Thus, in the setting of pyropstosis, propidium iodide (PI) can penetrate through the membrane pores and stain the cell nucleus.²³ Therefore, to observe membrane pores formation, cells were treated with PI (10 μM) for 30 min at room temperature, and then restained with 4′,6-diamino-2-phenyl indole (DAPI). The percentage of PI-positive cells was used to quantify cell pyroptosis.

2.5 Lactic dehydrogenase (LDH) release assay

Structural impairment of the cell membrane leads to release of LDH into the cell culture medium. Thus, LDH release is considered as a sensitive marker of cell pyroptosis.²⁴ The level of LDH release was measured using LDH release assay Kit (Jiancheng Biological Engineering Research Institute, Nanjing, China) based on manufacturer's protocol.

2.6 Caspase-1 activity assay

For this experiment, cells were digested by trypsase, centrifuged at 4°C, and collected after carefully sucking out the supernatant. The collected cells were mixed with lysis buffer, and then incubated in ice for 15 min. Following 16,000–20,000 g centrifugation at 4°C for 10–15 min, the supernatant was transferred to the centrifuge tube to detect caspase-1 activity using Caspase 1 Activity Assay Kit (Beyotime).

2.7 IL-1β and IL-18 activity assay

The activity of IL-1β and IL-18 in culture medium or serum was detected using rat IL-1β and IL-18 enzyme linked immunosorbent assay (ELISA) Kit (Multi Science), based on the manufacturer's protocol.

2.8 RNA extraction and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted using TRIzol reagent. Cytoplasmic and nuclear RNA was purified from the cytoplasmic and nuclear RNA fractions using Cytoplasmic and Nuclear RNA Purification Kit (Cat. 21000; Norgen). The isolated RNA was reverse-transcribed into cDNA using a PrimeScript RT Reagent Kit (TaKaRa) and cDNA was subjected to real-time qPCR amplification with the following specific primers:

OIP5-AS1 forward: 5'-GTGTTGTGGAGATTGAGGCAGGAG-3'; OIP5-AS1 reverse: 5'-GGCAAGGTGAAGGACAGACAGC-3'; GAPDH forward: (ssD1021-1-5, RIOBIO); GAPDH reverse: (ssD1022-1-5, RIOBIO).

qPCR was conducted by 7500 Real-Time PCR Systems (ThermoFish) with SYBR Premix Ex Taq II (TaKaRa). Gene relative expression was analyzed using the $${2}^{-\unicode{x02206}\unicode{x02206}{C}_{{\rm{t}}}}$$ method.²⁵

2.9 RNA pull-down

Biotin-labeled RNA probe was designed and synthesized by Riobio Co. Ltd. RNA pull-down assay was performed as previously described²⁶ using a Magnetic RNA-Protein Pull-Down kit (Pierce).

2.10 Luciferase assay

Wild-type vector containing the binding region of OIP5-AS1 with miR-22 (pmirGLO-OIP5-AS1-WT) and its match mutant vector (pmirGLO-OIP5-AS1-Mut) were designed and synthesized by GenePharma Co. Ltd. miR-22 mimics and the corresponding NC were cotransfected with pmirGLO-OIP5-AS1-WT or pmirGLO-OIP5-AS1-Mut, respectively. At 48 h posttransfection, luciferase activity was detected using a Dual-Glo Luciferase Assay System (Promega).

2.11 Animal model and gene therapy

A total of 10 male Wistar rats (mean weight 120 ± 20 g) obtained from Beijing HFK Bioscience Co. Ltd. were fed with fat diet with 60 kcal% for 20 weeks to establish rat obesity model based on previous study.²⁷ Adeno-associated virus 9 (AAV9) vector containing short Hairpin RNA (shRNA) targeting OIP5-AS1 (HBAAV9-CTNT-shOIP5-AS1) and corresponding NC (HBAAV9-CTNT-sh-NC) were constructed by Hanbio. Each rat was administered intravenous injection of 5 × 10¹⁰ particles AAV9 through the tail vein at 13th week of feeding period. To monitor the changes in cardiac function, rats were anesthetized with intravenous injection of pentobarbital sodium (30 mg/kg), and a rat Millar catheter was inserted into the left ventricle (LV) via the left common carotid artery to record the changes in heart rate, LV developed pressure (LVDP), and LV positive/negative first-order derivative of ventricular pressure (±dp/dt) using a homodynamic system (MP150; BIOPAC Systems Inc.).

All animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals (NIH) and were approved by the Institutional Animal Care and Use Committee of the China Medical University (KT2018018).

2.12 Histological analysis

LV tissues were fixed with paraformaldehyde, dehydrated by passage through graded ethanol series, paraffin-embedded, and cut into 5-µm-thick sections. The myocardial cross-sectional area was measured using hematoxylin–eosin (HE) stained sections. Masson and Sirius red staining were employed to evaluate myocardial fibrosis and collagen deposition, respectively. The expressions of NLRP3 and cleaved caspase-1 in the myocardium were detected using immunohistochemistry with primary antibodies against NLRP3 and cleaved caspase-1 (NLRP3: 1:200, Proteintech Group; cleaved caspase-1: 1:200, Proteintech).

2.13 Western blot analysis

Proteins were extracted from cells using RIPA lysis Buffer. The extracted proteins were quantified using the Enhanced BCA Protein Assay Kit (Beyotime), denatured by heat, isolated by SDS-PAGE electrophoresis, and transferred onto PVDF membranes. After blocking with 1% bovine serum albumin solution for 1 h, the membranes were incubated overnight with primary antibodies (NLRP3: 1:1000, Proteintech; cleaved caspase-1: 1:1000, Proteintech) at 4°C, followed by incubation with HRP-conjugated IgG (1:5000; Proteintech). The membranes were exposed using FluorChem M Imaging systems. Relative densitometry was analyzed using Image J2x analysis software (NIH).

2.14 Statistical analysis

Data were presented as mean ± standard deviation (SD). Between-group differences were assessed using the Student's t test. Multigroup comparisons were performed using one-way analysis of variance followed by Fisher's least significant difference test. p Values < 0.05 were considered indicative of statistical significance. All statistical analyses were performed using SPSS version 17.0 software (SPSS Inc.).

3.1 PA activated pyroptosis and induced OIP5-AS1 expression in cardiomyocytes

Fat deposition was observed in H9c2 cells 24 h after 400 μM PA treatment (Figure 1A). Simultaneously, pyroptotic phenotype in PA-treated H9c2 cells was also detected. PA-treated cardiomyocytes showed membrane pore formation in H9c2 cells as evidenced by a significant increase in the rate of cells stained with PI and release of LDH (Figure 1B–D). The expression of NLRP3 and cleaved caspase-1 protein, and the activity of caspase-1, IL-1β and IL-18 were also remarkably increased after PA treatment (Figure 1E–I), suggesting that NLRP3 inflammasome-mediated pyroptosis pathway was activated by PA. Correspondingly, the level of OIP5-AS1 in the PA group was 8.29-fold higher than that in the control group (Figure 1J). Collectively, these results suggested that saturated fatty acid activated pyroptosis and induced OIP5-AS1 expression in cardiomyocytes.

3.2 Downregulation of OIP5-AS1 inhibited PA-induced pyroptosis in cardiomyocytes

To examine the role of OIP5-AS1 in saturated fatty acid-induced pyroptosis, endogenous OIP5-AS1 expression was notably repressed by transfection with si-OIP5-AS1 (Figure 2A). Furthermore, the percentage of cells affected by pyroptosis, release of LDH, the level of NLRP3 and cleaved caspase-1 protein, and the activity of caspase-1, IL-1β, and IL-18 were also significantly decreased by transfection with sh-OIP5-AS1 (Figure 2B–I). Collectively, these results suggested that downregulation of OIP5-AS1 inhibited saturated fatty acid-induced pyroptosis in cardiomyocytes.

3.3 OIP5-AS1 bond to miR-22

Considering that the mechanism for gene regulation by LncRNA is related to its cellular localization,²⁸ the location of OIP5-AS1 in cardiomyocytes was detected. The result of nucleocytoplasmic separation assays indicated that most of OIP5-AS1 (nearly 90%) was located in the cytoplasm (Figure 3A). Therefore, microRNAs (miRNAs) binding to OIP5-AS1 were predicted using miRcode, starBase, and LncBase databases. As shown in Figure 3B, 17 putative miRNAs binding to OIP5-AS1 were predicted. Except for 10 miRNAs (miR-29,¹⁹ miR-26,²⁹ miR-223,³⁰ miR-218,³¹ miR-153,³² miR-141,³³ miR-140,³⁴ miR-137,³⁵ miR-129,³⁶ and miR-128³⁷) that have already been reported to bind to OIP5-AS1, the rest of 7 predicted miRNAs (miR-30, miR-22, miR-216, miR-183, miR-18, miR-155, and miR-148) were detected in the pulled down product of OIP5-AS1 probe. The results of RT-qPCR indicated remarkable enrichment of miR-22 expression (Figure 3C). Furthermore, the results of luciferase assay showed that cotransfection with miR-22 mimics and wild-type OIP5-AS1 luciferase plasmid induced a significant decrease in luciferase activity. However, no significant difference was observed in luciferase activity by co-transfecting miR-22 mimics with mutated-type OIP5-AS1 luciferase plasmid (Figure 3D,E). Collectively, these findings demonstrated the specific binding of OIP5-AS1 with miR-22.

3.4 OIP5-AS1 regulated NLRP3-mediated pyroptosis via miR-22

Because NLRP3 is a target gene of miR-22,³⁸ we investigated whether OIP5-AS1 regulated NLRP3-mediated pyroptosis via miR-22. Treatment with miR-22 inhibitor completely rescued the inhibition of NLRP3 by sh-OIP5-AS1 (Figure 4A). Moreover, suppression of cleaved caspase-1 expression and caspase-1 activity by sh-OIP5-AS1 were completely rescued by miR-22 inhibitor (Figure 4B,C). Additionally, the repression of other pyroptotic phenotype by OIP5-AS1 knockdown, including the release of IL-1β, IL-18, LDH, and the percentage of pyroptotic cells, were partially reversed by miR-22 inhibitor (Figure 4D–G). These findings indicated that OIP5-AS1 regulated NLRP3-mediated pyroptosis via miR-22.

3.5 Downregulation of OIP5-AS1 ameliorated cardiac dysfunction in HFD rats

To evaluate the impact of OIP5-AS1 on obesity-induced cardiac dysfunction in vivo, we examined the effect of inhibition of OIP5-AS1 on cardiac function in HFD rats. As shown in Figure 5A, OIP5-AS1 expression was significantly downregulated in heart by injection of HBAAV9-CTNT-shOIP5-AS1, but no significant change in liver, spleen, lung, and kidney. Meanwhile, miR-22 expression was remarkably upregulated in heart with injection of HBAAV9-CTNT-shOIP5-AS1 (Figure 5B). Furthermore, OIP5-AS1 repression led to a significant increase in the values of LVDP and ±dp/dt (Figure 5D–F), but there was no significant change in the value of HR (Figure 5C), which suggesting that downregulation of OIP5-AS1 ameliorated HFD-induced cardiac dysfunction.

3.6 Downregulation of OIP5-AS1 ameliorated myocardial hypertrophy, fibrosis, remodeling, and pyroptosis in HFD rats

To evaluate the impact of OIP5-AS1 on obesity-induced alteration of myocardial structure in vivo, we examined the effect of inhibition of OIP5-AS1 on HFD-induced myocardial hypertrophy, fibrosis, and remodeling. As shown in Figure 6, the cross-sectional area, fibrosis area fraction, and collagen volume fraction in the myocardium of HFD-rats were found to have significantly reduced by OIP5-AS1 knockdown. In addition, the markers of pyroptosis including the expressions of NLRP3 and cleaved caspase-1 in myocardium (Figure 7A,B) and the serum concentrations of IL-1β and IL-18 (Figure 7C,D) were notably decreased by OIP5-AS1 knockdown in HFD rats. The above findings suggested that downregulation of OIP5-AS1 ameliorated myocardial hypertrophy, fibrosis, remodeling, and pyroptosis in HFD rats.