Introduction

Heart failure (HF) is a major cause of mortality, hospitalizations, and reduced quality of life and a major burden for the healthcare system1. Despite great clinical advances and strategies during the past decades, heart failure continues to pose a major health concern worldwide. Cardiac remodeling, contributing to the pathophysiology of heart failure, is a chronic maladaptive process characterized by vascular dysfunction, myocardial hypertrophy, apoptosis, necrosis, ventricular dilatation, and myocardial fibrosis2,3. Cardiac fibrosis is characterized by increased collagen deposition and/or its altered composition in the myocardium4,5. It is manifested by the replacement of normal myocardium by non-functional fibrotic tissues6. The administration of renin–angiotensin–aldosterone system blockers and other drugs can only delay the development of myocardial fibrosis7. At present, there are no other effective means to reverse or prevent myocardial fibrosis.

Sirtuin 1 (SIRT1), also known as nicotine adenine dinucleotide-dependent deacetylase, has shown a strong protective role in cardiovascular diseases. SIRT1 may attenuate isoproterenol-induced cardiac fibrosis by regulating EndMT via the TGF-β/Smad2/3 pathway8. Drugs or interventions targeted at SIRT1 may become an important therapeutic scheme for preventing cardiac fibrosis9. A few studies have shown that a variety of miRNAs are involved in cardiovascular diseases via targeting SIRT110,11.

MicroRNAs (miRNAs) are a class of highly conserved non-coding single-stranded RNAs (20–26 nucleotides in length) that regulate gene expression in eukaryotes. Bioinformatics analysis showed that SIRT1 has multiple microRNAs (mir-217, mir-140, mir-138, mir-204) binding sites. mir-217 is involved in a variety of cardiovascular diseases, i.e. aggravating hypoxia-induced myocardial cell injury via targeting SIRT112,29. Inflammasome is a group of cytosolic multiprotein complexes including NLRP1, NLRP2, NLRP3, AIM2, and NLRC4. Among them, the NLRP3 inflammasome has been widely reported in studies of tumor and neurological diseases. The NLRP3 inflammasome can be activated by a variety of stresses with multiple activation pathways including microRNAs, long-chain non-coding RNA (lncRNAs)30,31,32. And it has been shown that GAS5 is involved in renal tubular cell pyroptosis33, ovarian cancer34 and diabetic cardiomyopathy (DCM)35 induced by NLRP3 inflammasome activation. Previous studies have shown that pyroptosis activation is associated with Cardiac Dysfunction36. In the current study, we tested the expression of NLRP3, Caspase-1, IL-1β and GSDMD which are pyroptosis markers. The results showed that the expression of these markers was significantly increased in the mice hearts treated with ISO, while the overexpression of GAS5 inhibited this increase of these markers expression induced by ISO.

The evidences mentioned above imply that GAS5 may inhibit myocardial fibrosis via regulating NLRP3 inflammasome activation, but the exact mechanism requires further investigations. The regulatory mechanisms of GAS5 involved in the pathophysiological process of cardiovascular diseases are very complex, and one of them is to regulate various microRNAs and their downstream target genes37,38,39. Increasing studies have reported that lncRNAs play crucial functions in numerous biological processes by interacting with the 3′-untranslated region (UTR) of target mRNAs to silence their expression40. It is well-known that SIRT1 probably provides a new therapeutic regimen for preventing heart failure and plays an important role in the occurrence and development of myocardial fibrosis. Study showed that therapeutic strategies to restore appropriate SIRT1 signaling could mitigate ongoing fibrogenesis41. Previous studies have shown that microRNA targets SIRT to regulate pyroptosis of diseases42,43 and mir-217 could negatively regulate expression of SIRT144. Our study in mice found that the overexpression of GAS5 inhibits the expression of mir-217 and promotes the expression of SIRT1. Therefore we hypothesized that GAS5 could regulate the downstream target gene SIRT1 via mir-217 and subsequently inhibit myocardial fibrosis.

Transforming Growth factor β (TGF-β) is a central trigger of myocardial fibroblast activation that regulates a wide variety of biological processes such as extracellular matrix (ECM) production, cell growth, differentiation, cell homeostasis and migration45,46,47. And it can directly enhance collagen expression to induce fibrotic diseases48. In order to prove our hypothesis, we treated the MCFs with TGF-β1 to induce fibrosis after co-transfection of GAS5 overexpression plasmid and mir-217 mimics or mir-217 inhibitor. The results showed that mir-217 negatively regulated SIRT1 and aggravated the myocardial fibrosis induced by TGF-β1 via NLRP3 inflammasome activation. And the overexpression of GAS5 inhibited the myocardial fibrosis and the NLRP3 inflammasome activation. The mir-217 mimics offset this effect of GAS5 on myocardial fibrosis, pyroptosis and SIRT1, and the mir-217 inhibitor enhanced all these effects of GAS5. It can be concluded that GAS5 inhibits myocardial fibrosis induced by NLRP3 inflammasome activation through the mir-217/SIRT1 pathway. Our study is the first to demonstrate that mir-217 played a vital role in develo** myocardial fibrosis through regulating pyroptosis.

Our study and some recent articles have demonstrated that there is a functional connection between GAS5 and mir-21720,49. We constructed the wild type and variant type vectors of GAS5 based on a highly bound sequence between GAS5 and mir-217, and performed dual luciferase gene assay. The experimental results were negative (RiboBio, Guangzhou). However, target prediction between GAS5 and mir-217 revealed the existence of three highly bound sequences between GAS5 and mir-217, and GAS5 is a long-chain non-coding RNA. So, the negative result may be caused by the diversity of transcripts. Therefore, it cannot be entirely concluded that there is no structural interaction between GAS5 and mir-217. New vectors for dual luciferase gene assay are required to be constructed to verify the structural interaction between GAS5 and mir-217 in future studies.

Materials and methods

Reagents

Isoprenaline (ISO) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in phosphate-buffered saline (PBS). TGF-β1 (100-21) was obtained from Peprotech (Rocky Hill, NJ, USA). mir-217 mimics and mir-217 inhibitor were purchased from Hanbio Biotechnology Co., Ltd. (Shanghai, China). Antibody against α-SMA was purchased from Abcam (Cambridge, MA, USA). The following antibodies were purchased from Proteintech (Chicago, IL, USA): SIRT1, collagen I, collagen III, GSDMD, Caspase-1, IL-1β, NLRP3 and β-actin. AAV9-GAS5 (recombinant adeno-associated virus expressing growth arrest specific transcript-5) and AAV9-NC were constructed by Genechem (Shanghai, China).

Animals and treatment

The study is reported in accordance with ARRIVE guidelines. Adult male C57BL/6 mice, aged 8 ~ 10 weeks, were purchased from the Wenzhou Medical University Laboratory Animal Centre (Wenzhou, China). The Animal Certificate NO is SYXK (浙) 2010-0044. All animal-procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The animals were housed individually in cages under hygienic conditions in a 12/12-h light/dark cycle at 22 ± 3 °C and 45 ± 10% humidity for seven days before the experiments. The study was evaluated and approved by the Wenzhou Medical University Animal Care and Use Ethics Committee (permission number wydw2013-0054). The animals were allowed free access to a standard commercial diet and tap water. Before and after the experiment, all of the mice were treated humanely. Then the mice were randomly divided into four groups (10 per group) for treatment: Control, ISO, AAV9-NC + GAS5, and AAV9-GAS5 + ISO. In the ISO model mice, ISO was injected subcutaneously at 15 mg/kg/day for three days and then at 10 mg/kg/day for 11 days8,50. In AAV9-NC group and AAV9-GAS5 group, AAV9 vectors at a dose of 1 × 1011 viral genome particles were randomly injected into the left ventricle of each mouse. Less than 20 μL AAV9 vectors were injected into the heart at 5 sites. The RT-qPCR results verified the successful mouse cardiac GAS5 overexpression after two weeks, followed by ISO treatment, which is the same as ISO group. In control group, the mice were injected subcutaneously with the same dose of sterile saline.

Echocardiographic analysis of cardiac function

The mice were lightly anesthetized with 1.5% isoflurane in a medical air/oxygen mixture during examinations and placed supine on a heated platform with electrocardiography leads. Heart images were acquired using a Vevo1100 system (VisualSonics System, Toronto, Ontario, Canada). Measurements were made three consecutive times, over at least 10 cardiac cycles each time. The following parameters were recorded: ejection fraction (EF), fractional shortening (FS), and left ventricular mass (LV mass).

Histological quantification of cardiac fibrosis

After the 14-day treatment described above, all mice were sacrificed and the hearts were quickly excised, arrested in diastole with 1.34 M KCl, weighed, placed in 4% paraformaldehyde, and embedded in paraffin. The hearts were cut transversely close to the apex. Sections of the heart (4 ~ 5 μm) were mounted onto slides and stained with hematoxylin and eosin (H&E, Solarbio, Bei**g, China) for histopathological analysis. The tissue sections were also subjected to Masson’s trichrome staining (Solarbio, Bei**g, China) and visualized by light microscopy (ECLIPSE 80i, Nikon Corporation) to determine collagen deposition. The photographs were analyzed using digital analysis software (ImageJ) in a blinded manner.

Histological analysis of NLRP3 inflammasome activation-mediated pyroptosis

For immunohistochemical analysis, cardiac sections were deparaffinized in xylene and rehydrated in a graded ethanol series. Endogenous peroxidase activity was quenched in 3% methanol-H2O2 for 10 min. Antigen retrieval was performed by heating the sections in 10 mM citrate buffer (pH 6.0) in a microwave oven. Non-specific binding was blocked in 1% bovine serum albumin. Next, the sections were incubated with primary antibodies against Caspase-1, IL-1β, and NLRP3 at 4 °C overnight and washed three with PBS for 5 min per wash. The sections were then incubated with second antibodies for 30 min before nuclear staining. The results were analyzed by fluorescence microscopy (ECLIPSE Ti-S; Nikon Corporation).

Cell culture and treatments

Primary myocardial fibroblasts (MCFs) were isolated from the hearts of 3-day-old neonatal male C57BL/6 mice as previously described51, which were obtained from the laboratory animal center of Wenzhou Medical University. MCFs were cultured in 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin (Sigma, Milan, Italy) at 37 °C in a humidified atmosphere containing 5% CO2. After overnight serum starvation, MCFs were randomly grouped into: Control, TGF-β1, TGF-β1 + AAV9-NC and TGF-β1 + AAV9-GAS5.

Adeno-associated virus infection

MCFs in the logarithmic growth phase were selected and detached with trypsin. Then the cells were cultured on 96-well plates in complete culture medium (1640 + 10%FBS) at 37 °C in a humidified atmosphere containing 5% CO2 for 24 h. The adeno-associated virus was added and infected cells in 1/2 volume of the same medium (1640 + 10%FBS). The number of viruses was calculated according to the optimal MOI (106). 1/2 volume of fresh culture fluid was added 4 h later to continue infect at 37 °C for 48 h. The treated cells were divided into two groups: Group A was AAV9-NC and Group B was AAV9-GAS5. According to the optimum MOI, Adeno-associated virus were added to complete medium for culture before cells were infected with virus. Cell passage and plating were initiated when cell density developed to 80%. Then the cells were divided into four groups: Control, TGF-β1, AAV9-NC + TGF-β1, AAV9-GAS5 + TGF-β1. The cells were collected with Trizol and RT-qPCR was performed to verify the viral infection.

Co-transfection cell experiment

DEPC water was added into the GAS5 overexpressed plasmid or NC so that its final concentration is 100 nM, then use 1640 medium to prepare 20 mM for reserve. 3 ~ 12 × 1015 MCFs were seeded into 150 μL sterile medium 24 h before transfection. When the cell fusion rate reached 30% ~ 50%, the transfection was initiated. 5 μL GAS5 plasmid was diluted with 250 μL 1640 medium to make 50 nM transfection concentration. Transfection reagent Lipofectanine TM2000 was diluted and mixed with the diluents of GAS5 overexpression or NC plasmid. The mixed reagent was added to the 6-well plate containing the cell solution for culture. The transfection effect was verified by RT-qPCR and then successfully transfected MCFs were subcultured. Cell passage and plating were initiated when cell density was 80%. 3 ~ 12 × 1015 GAS5 overexpressed MCFs were seeded into 150 μL sterile medium 24 h before the transfection. When the cell fusion rate reached 30% ~ 50%, transfection was initiated. 100 nM mir-217 mimics and inhibitor were diluted with 1640 medium to 20 mM for reserve. Transfection reagent was diluted and mixed with the diluents of mir-217 mimics and mir-217 inhibitor. The mixed reagent was added to the 6-well plate containing the cell solution for culture. Then the cells were divided into six groups: Control, TGF-β1, NC + TGF-β1, GAS5 + TGF-β1, mir-217 mimics + GAS5 + TGF-β1, mir-217 inhibitor + GAS5 + TGF-β1.

Western blot

Heart tissue and MCFs were lysed in RIPA protein extraction buffer (Applygen, Bei**g, China) and centrifuged at 4 °C at 12,000 rpm for 15 min. The supernatant was collected and protein quantification was carried out by a bicinchoninic acid protein assay (Beyotime, Shanghai, China). The extracted protein was separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes. After being blocked with 5% skimmed milk in TBST at room temperature for 1 h, the membranes were incubated with primary antibodies at 4 °C overnight, anti-collagen I (1:1000, Proteintech), anti-collagen III (1:1000, Proteintech), anti-SIRT1 (1:1000, Proteintech), anti-GSDMD (1:1000, Proteintech), anti-caspase-1 (1:1000, Proteintech), anti-β-actin (1:5000, Proteintech), anti-IL-1β (1:1000, HuaAn), anti-NLRP3 (1:1000, HuaAn), anti-α-SMA (1:1000, Abcam). Following washing three times with TBST, the membranes were incubated with a secondary antibody for 1 h at room temperature, then washed three more times with TBST. Protein bands were visualized in ECL and the targeted proteins were scanned with the Molecular Imager ChemiDoc XRS Imaging System (Bio-Rad, Hercules, CA, USA) (Supplementary figure).

Quantitative real-time polymerase chain reaction (RT-qPCR)

The mRNA levels of collagen I, collagen III, α-SMA, NLRP3, IL-1β, and GSDMD were analyzed by RT-qPCR. RNA was extracted from heart tissues and MCFs using Trizol reagent following the manufacturer’s instructions. The RNA purity was evaluated based on the OD260/OD280 ratios with micronuclei acid instrument. The RNA was reverse-transcribed into cDNA using the Takara Kit, Gene CopoeiaTM Kit. Diluted primers, samples, and RNA Free ddH2O were dissolved on ice and fully mixed and centrifuged. The PCR was performed via TIANGEN Kit and Gene CopoeiaTM Kit. The primers used in the study are shown in the Table 1.

Table 1 Gene-specific primers used in quantitative real-time polymerase chain reaction (RT-qPCR).
Full size table

Statistical analysis

The results are expressed as mean ± SEM and were analyzed by one-way analysis of variance (ANOVA) and Tukey's post-hoc test (*p < 0.05 and **p < 0.01) with GraphPad Prism 5.0 software.

Ethics approval

All animal experimental protocols in this study were approved by the Animal Care and Use Committee of Laboratory Animal Centre of Wenzhou Medical University.

Consent to participate

Yes, my co-authors and I would like to participate.