1. Introduction
Myocardial infarction (MI) is a major cardiovascular event and a leading cause of mortality worldwide [
1], [
2]. Following MI, an inflammatory response is crucial to activate the repair mechanisms of the heart [
3], [
4], [
5]. However, prolonged activation of this response can lead to adverse cardiac remodeling, including fibrosis and scarring, ultimately resulting in the deterioration of cardiac function and heart failure (HF) [
6], [
7], [
8], [
9]. Although previous research has highlighted the temporal and stage-specific nature of the inflammatory response after MI [
10], [
11], the complex dynamics of how inflammation contributes to cardiovascular fibrosis are not fully understood.
Recent investigations have highlighted the critical role of NOD-like receptor family pyrin domain containing 3 (NLRP3), a central component of the innate immune system, in driving the inflammatory response after MI [
12], [
13], [
14]. After MI, necrotic cells release cytokines that activate this innate immune pathway, exacerbating the inflammatory response [
15], [
16]. The recognition of pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs) triggers the NLRP3 inflammasome [
17], [
18]. This activation leads to the assembly of the adaptor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and the activation of caspase-1, which processes interleukin (IL)-1β/18 and gasdermin D (GSDMD), culminating in pyroptosis [
19], [
20]. This process underscores the role of the NLRP3 inflammasome in sterile inflammation during acute MI events [
18], [
21], [
22], [
23]. Notably, the expression and activation of NLRP3 inflammasome components, including ASC, cryopyrin, and caspase-1, have been observed in various cardiac cell types post-MI [
6], [
24], [
25], [
26], yet the precise timing of inflammasome activation by these cells and its relationship with myocardial fibrosis remain to be elucidated.
In our investigation, we utilized an MI model induced by occlusion of the left anterior descending (LAD) coronary artery to investigate the pattern of NLRP3 inflammasome activation following MI. Our observations revealed an increase in the activity of the NLRP3 inflammasome within macrophages on the third day post-MI, which subsequently extended to fibroblasts. Interventions aimed at deactivating or inhibiting NLRP3 expression, both in vivo and in vitro, reduced myocardial fibrosis. Additionally, our data suggest that macrophage-associated NLRP3 inflammasome activation may significantly contribute to the transformation of fibroblasts into myofibroblasts, primarily through the 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) signaling pathway.
2. Materials and methods
2.1. Human study
Ethical approval for this investigation was granted by the Ethics Committee of Beijing Anzhen Hospital, China, in alignment with the tenets of the Declaration of Helsinki (2023110X, 2022128X, 2017005). From January 2017 to October 2022, patients with acute coronary syndrome (ACS) and healthy individuals were recruited at the hospital. The inclusion criteria were participants aged > 18 years with ST-segment elevation MI and unstable angina. The exclusion criteria for the study were current or historical diseases/conditions of serious cardiovascular and cerebrovascular diseases (MI, stroke, HF, etc.), valve diseases, thyroid diseases, respiratory diseases, infectious diseases, kidney diseases, pregnancy, and malignancies. Venous blood samples were collected after overnight fasting prior to percutaneous coronary intervention (Table S1 in Appendix A). Subsequently, the serum was separated and preserved at -80 °C for subsequent studies. Additionally, paraffin-embedded heart tissues were obtained from MI patients who underwent surgical resection of ventricular aneurysms. All procedures were preceded by the acquisition of informed consent from the participants.
2.2. Desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) instrumentation and data processing
Analysis and localization of specific biomarkers within cardiac tissue were conducted using a Xevo-G2 XS Q-TOF mass spectrometer (Waters, USA) equipped with a two-dimensional DESI stage from Prosolia (USA). Prior to analysis, heart tissue specimens were preserved at -80 °C and then cryogenically sectioned at a thickness of 10 μm. These sections were then rewarmed and affixed to glass slides for subsequent DESI-MSI examination. The DESI-Q-TOF settings were meticulously adjusted to enhance signal clarity, with adjustments made to spatial resolution (100 μm), spray solvent composition (MeOH:H2O in a 9:1 ratio, augmented with 0.1 mmol∙L−1 NH4Cl and 0.1 mmol∙L−1 leucine-enkephalin (LE)), solvent flow rate (1.5 μL∙min−1), raster velocity (400 μm∙s−1), sprayer orientation angle (70°), distances from sprayer to inlet (5 mm) and sprayer to specimen (1.5 mm), source heat (140 °C), capillary voltages in both positive (3.6 kV) and negative (4.5 kV) modes, cone voltage (80 V), mass detection range (m/z 100-1200), and collision energy (CE) spanning 10-40 eV. The MS data were processed, and two-dimensional ion maps were created using high-definition imaging (HDI) using MassLynx software (Waters, UK).
2.3. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitative analysis of oxylipin
Blood oxylipin concentrations were measured using a QTRAP 6500 LC-MS/MS system, with analytical processing provided by MetWare†.
2.4. Animals and ethics statement
Ethical approval for our animal-based research was obtained from the Animal Care Committee of Beijing University of Chinese Medicine (authorization code: BUCM-4-2018101504-4068), ensuring adherence to the National Institutes of Health (NIH)’s Institutional Animal Care and Use Committee (IACUC) standards (eighth edition). We sourced adult male C57BL/6N mice aged between 7 and 8 weeks and weighing 24-28 g from SPF (Beijing) Biotechnology Co., Ltd. (China). These experiments also included
Nlrp3 gene knockout mice, as detailed in Refs. [
27], [
28]. We established an MI model using LAD ligation [
29], [
30] and subsequently categorized mice into control and various postligation timepoint groups (LAD ligation for 1, 3, 7, or 14 days), as well as a cohort treated with an NLRP3 inhibitor MCC950 (10 mg∙kg
−1; AbMole BioScience, USA).
2.5. Ultrasound echocardiography
In accordance with prior investigations [
30], [
31], [
32], transthoracic echocardiography of sedentary mice in a resting state was conducted using a Vevo 3100 high-resolution imaging apparatus (Vevo TM 2100; Visual Sonics, Canada) under 1.5%-2.0% isoflurane anesthesia. From the left parasternal short-axis view, two-dimensional M-mode representations of the left ventricle were captured. At each measurement site within these cross-sectional views, recordings of three cardiac cycles were taken. Images along the parasternal long and short axes were also acquired. Measurements of left ventricular (LV) dimensions and wall thickness were obtained from M-mode images in the parasternal short-axis view.
2.6. Histological and immunofluorescence staining
Following echocardiographic assessments, the mice were euthanized under deep isoflurane anesthesia. Subsequently, their hearts were excised, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5 μm thick slices. Cardiac structure and fibrosis were assessed using hematoxylin and eosin (H&E) and Masson’s trichrome staining, respectively.
For the detection of α-actinin, cluster of differentiation 31 (CD31), CD86, vimentin, and ASC expression, dual immunofluorescence staining was conducted. The paraffin-embedded heart slices were deparaffinized, rehydrated, subjected to antigen retrieval, and then permeabilized with 0.1% Triton X-100 plus 1% bovine serum albumin. Overnight incubation at 4 °C was performed with primary antibodies against α-actinin, CD31, CD86, vimentin, and ASC. Nuclear staining was performed using 4′,6-diamidino-2-phenylindole (DAPI), and an Olympus IX51 fluorescence microscope (Japan) was used for image acquisition.
2.7. Cardiac biomarker quantification
Serum levels of lactate dehydrogenase (LDH), creatine kinase (CK), and its isoenzyme CK-MB, along with cardiac troponin I (cTn-I), were meticulously quantified. LDH and CK concentrations were measured via an automated Hitachi 7080 biochemical analyzer (Hitachi High-Technologies Corporation, Japan). Moreover, the CK-MB and cTn-I levels were evaluated using specific enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer’s protocols.
2.8. Analysis of single-cell RNA sequencing (scRNA-seq) data
For the scRNA-seq data analysis, datasets pertaining to the cardiac interstitial cell population from scRNA-seq were acquired from the ArrayExpress repository
†, including a comprehensive expression matrix and annotated ensemble genes. Initial quality control measures were implemented to exclude cells of inferior quality and infrequently expressed genes. According to the criteria outlined in Ref. [
33], this single-cell dataset was subjected to preprocessing prior to subsequent analyses. Using Seurat version 4.3.0 (PMID: 29608179, PMID: 31178118) for detailed examination, cell populations were delineated based on marker gene expression using the R package Seurat. Notably, normalization of counts was executed using SCTransform, and principal component analysis (PCA) was applied to categorize the variable genes into 50 principal components (PCs), followed by two-dimensional visualization through
t-distributed stochastic neighbor embedding (
t-SNE) using RunPCA and RunTSNE functions with all 50 PCs, as recommended by the findings from Seurat’s JackStraw. Furthermore, transcriptionally unique clusters were identified employing Seurat’s FindNeighbors and FindClusters functions using all 50 PCs. The Cell ID R package, a clustering-independent multivariate statistical approach for deriving robust gene signatures from scRNA-seq data per cell (PMID: 33927417), was utilized for cell type annotation.
2.9. Cell culture and treatment protocol
Bone marrow-derived macrophages (BMDMs) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. BMDMs were seeded into a six-well plate at a density of 2 × 105 cells∙mL−1 and cultured to 70%-80% confluence. The cells were rinsed with fresh medium and stimulated with 1 μg∙mL−1 lipopolysaccharides (LPS). After incubation for 24 h, the supernatant was collected as macrophage-conditioned medium (CM) and used in subsequent experiments.
Cardiac fibroblasts (CFs) were separated from the ventricles of rats aged 2-3 days according to the kit manufacturer’s protocol (88279; Thermo Fisher Scientific, USA) [
34]. After isolation, the CFs were grown in DMEM supplemented with 10% FBS. We established a macrophage-CM-induced myocardial fibrosis cell model. Specifically, macrophage-CM collected from LPS-stimulated BMDMs was used to treat CFs. In this study, CM/4 represents one-fourth of the original concentration diluted in DMEM, and CM/8 represents one-eighth of the original concentration diluted in DMEM.
2.10. Transfection and metabolomic analysis
A gene-silencing small interfering RNA (siRNA) sequence targeting Nlrp3 (si-Nlrp3) and a control siRNA sequence (si-NC) were engineered by GenePharma (China). The strands were subsequently sequenced as follows: si-Nlrp3, 5′-CCAACUGGUCAAGGAGCAUTT-3′, and si-NC, 5′-UUCUCCGAACGUGUCACGUTT-3′. At a cellular density of 5 × 105 cells∙mL−1, BMDMs were plated into six-well plates and transfected with 4 μL Lipofectamine 8000 (C0533; Beyotime Biotechnology, China) and 5 μL siRNA for 6 h. After this procedure, the growth medium was replaced with DMEM supplemented with 2% FBS without antibiotics for 18 h before the cells received a 1 μg∙mL−1 dose of LPS. The efficiency of gene suppression by siRNA intervention was established through Western blotting assays.
In the targeted metabolomic workflow, BMDMs were lysed in 200 μL of a 1:1 methanol:acetonitrile mixture, which included an internal calibration standard. The cells were vigorously shaken for 5 min, cooled at -20 °C for 30 min, and centrifuged at 12 000 r∙min−1 for 10 min at 4 °C. The clear supernatant fluid was then carefully separated and subjected to eicosanoid extraction. After the analytes were concentrated, they were reintroduced to 100 μL of a methanol-water solution at equal volumes for subsequent LC-MS/MS analysis. The eicosanoid compounds were profiled using QTRAP 6500 LC-MS/MS system under specified conditions, including a specific solvent gradient and temperature-controlled column environment. The MS/MS apparatus, fitted with an electrospray ionization (ESI) Turbo Ion Spray interface operating under negative ion mode, was constructed using the Analyst 1.6.3 software suite. The source conditions for the ESI, such as temperature and ion spray voltage, were meticulously set. Eicosanoids were quantitatively analyzed via a scheduled multiple reaction monitoring method, and the resulting data were processed using the Analyst 1.6.3 and Multiquant 3.0.3 software programs.
2.11. Western blot analysis
Proteins were extracted from myocardial tissue, BMDMs, and CFs according to the manufacturer’s instructions [
29], [
30]. The protein concentrations were determined using a bicinchoninic acid (BCA) protein assay, and the proteins were separated by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. First, the membranes were incubated with primary antibodies (Table S2 in Appendix A) at 4 °C, incubated with the corresponding secondary antibodies at room temperature for 1 h, and then incubated with enhanced chemiluminescence (ECL) reagent at room temperature for 1 min. Western blotting bands were analyzed using Image Lab and quantified using Image Lab software.
2.12. Data analysis
The data are expressed as the means ± standard deviations (SDs), and P < 0.05 indicated statistical significance. Analysis of the differences between two or more groups was performed using either an unpaired two-tailed t test or one-way analysis of variance (ANOVA) followed by a Bonferroni-corrected post hoc test. All analyses were performed with GraphPad Prism 7 software.
3. Results
3.1. Time-course evaluation of cardiac function after LAD ligation
Temporal analysis of cardiac functionality post-LAD artery ligation is illustrated.
Fig. 1(a) depicts the procedural flowchart of the study.
Fig. 1(b) presents exemplary cardiac images and M-mode echocardiograms across various MI intervals. The echocardiographic data (Fig. S1 in Appendix A) revealed a temporal decrease in cardiac performance within the experimental cohorts compared to the homeostasis (Hom)-operated controls, as evidenced by markedly reduced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) (Fig. S2 in Appendix A). Histological examinations via H&E and Masson’s trichrome staining indicated organized cardiomyocytes in the Hom group and disorganized cardiomyocytes in the experimental groups (
Fig. 1(c)). On the third day post-MI, there was an increase in inflammatory infiltration and cell cytolysis, culminating in collagen deposition and subsequent interstitial fibrosis. Under physiological conditions, biomarkers such as cTn-I, CK, CK-MB, and LDH are present within the cardiomyocyte cytoplasm, and elevated serum levels of these markers serve as clinical indicators of HF. The study findings demonstrated a significant increase in the levels of these biomarkers in the experimental group relative to the Hom group (
Fig. 1(d)).
3.2. The NLRP3 inflammasome was dynamically activated in MI model mice
Dynamic activation of NLRP3 in MI model mice has been documented, with mounting evidence underscoring the essential role of the inflammasome in MI [
22], [
35], [
36], [
37]. However, the specific timeline for NLRP3 inflammasome activation throughout the process of MI has yet to be clarified. Our research evaluated the expression levels of proteins associated with the cardiac NLRP3 inflammasome and the levels of the inflammatory cytokines IL-18 and IL-1β at different time points following MI.
Fig. 2(a) shows a marked increase in the serum levels of IL-18 and IL-1β in the model group mice compared to those in the mice of the sham group. Through immunofluorescence, we observed that NLRP3 inflammasome activation post-MI was directly correlated with the duration of MI (
Fig. 2(b)). Furthermore, Western blot analyses revealed a temporal increase in the protein expression of NLRP3, ASC, cleaved caspase-1, cleaved IL-18, and gasdermin D N-terminal fragment (GSDMD-NT) post-MI, peaking on the third day and then gradually decreasing until day 14 (
Fig. 2(c)). These observations corroborate the temporal activation pattern of the NLRP3 inflammasome following MI.
3.3. Nlrp3 knockout inhibits MI-induced myocardial injury
Reflecting the dynamic nature of inflammasome activation in MI, an MI model was successfully established, including mice with or without expression of the
Nlrp3 gene, as depicted in
Fig. 3(a). LAD artery ligation led to an increase in left ventricular internal dimension diastole (LVID;d) and left ventricular internal dimension systole (LVID;s) and a decrease in LVEF and LVFS, respectively. In line with earlier observations, H&E and Masson’s trichrome staining corroborated the myocardial damage induced by MI (
Fig. 3(b)). Crucially, knockout of the
Nlrp3 gene curtailed the LAD-triggered activation of NLRP3 (
Fig. 3(c)) and subsequent IL-1β and IL-18 production (Fig. S3 in Appendix A), thereby mitigating cardiac dysfunction and myocardial injury induced by LAD. The role of the inflammasome
in vivo was further corroborated by intraperitoneal administration of the NLRP3 inhibitor MCC950 (10 mg∙kg
−1) (
Figs. 3(d)-(f); Fig. S4 in Appendix A), indicating that NLRP3 inflammasome activation is paramount in LAD-induced MI, while its inhibition offers cardioprotection.
3.4. Activation of the NLRP3 inflammasome predominantly occurs in macrophages on the third day after MI
Activation of the NLRP3 inflammasome in macrophages was predominantly observed on the third day post-MI. Following the observation of temporally dynamic NLRP3 inflammasome activation in MI model mice, we investigated whether activation varied across different cell types. Using database-supported single-cell sequencing,
Fig. 4(a) illustrates the alterations in NLRP3 inflammasome activation across various cell types and timepoints post-MI. The
t-SNE plot combines cardiovascular cell data from days 1, 3, and 7, identifying six cell types: fibroblasts, mural cells, endothelial cells, macrophages, lymphoid cells, and cycling cells. Notably, the proportion of macrophages increased significantly on day 3 post-MI. Immunofluorescence staining, as shown in
Fig. 4(b), revealed a marked increase in the number of ASC-immunopositive cells in the myocardia of MI model mice compared with that in the sham group. Labeling of different cell types revealed the most substantial increase in ASC fluorescence intensity within macrophages on day 3 post-MI, underscoring the crucial role of macrophages in the innate immune response and tissue repair following myocardial injury.
3.5. Myocardial fibrosis was induced by LPS-stimulated macrophage medium in vitro
To explore the impact of macrophage-associated inflammasome activation on fibroblast conversion, an
in vitro setup employing LPS-treated BMDMs was developed as previously described [
30], [
31]. In this study, BMDMs were exposed to LPS to mimic the molecular patterns of infection by microbial pathogens
in vitro (
Fig. 5(a)). The control group exhibited minimal NLRP3 protein expression and infrequent NLRP3 inflammasome complex formation. Conversely, post-LPS exposure, there was a marked increase in the expression of NLRP3 inflammasome-related proteins, including ASC, cleaved caspase-1, IL-1β, and IL-18 (Fig. S5 in Appendix A). Immunofluorescence analysis of NLRP3 expression in BMDMs treated with or without LPS corroborated the Western blot findings (
Figs. 5(b) and
(c)). Notably, LPS-triggered inflammasome activation without inducing pyroptosis in BMDMs, potentially due to the nature of extracellular stimulation by LPS [
32]. After verifying the LPS-induced activation of NLRP3 in macrophages, we further investigated the role of the macrophage-associated inflammasome in myocardial fibrosis
in vitro. A novel myocardial fibrosis model was generated by treating CFs extracted from rat ventricles with macrophage-CM. CM derived from BMDMs stimulated with LPS (1 μg∙mL
−1) was mixed with the CFs for 8 h. This CM was then diluted with DMEM to one-quarter (CM/4) and one-eighth (CM/8) of its original concentration. The transition of fibroblasts to myofibroblasts, marked by collagen production and the presence of α-smooth muscle actin (α-SMA) expression, serves as a primary indicator of myocardial fibrosis. Collagen I and α-SMA expression in CFs treated with CM/4 or CM/8 was significantly higher than that in LPS-treated or control cells (
Fig. 5(d)). Moreover, immunofluorescence staining notably confirmed that CM/4 induced myocardial fibrosis (
Fig. 5(e)). These findings confirmed the successful
in vitro induction of a cell model of myocardial fibrosis by CM.
3.6. Nlrp3 silencing in macrophages effectively inhibited myocardial fibrosis in vitro
To further elucidate the role of NLRP3 in macrophage-associated inflammasome activity, BMDMs were treated with si-
Nlrp3. Fig. S6 in Appendix A illustrates the effective knockdown of
Nlrp3 expression in BMDMs using si-
Nlrp3. Western blot results indicated that LPS stimulation increased the expression of inflammasome activation-associated proteins. However, subsequent LPS challenge post-si-
Nlrp3 treatment did not trigger inflammasome activation (
Fig. 6(a)). Immunofluorescence analysis of NLRP3 activation was consistent with the Western blot findings (
Fig. 6(b)), suggesting that si-
Nlrp3 transfection significantly dampened inflammasome activation in macrophages. Further examination of the impact of macrophage-associated inflammasome activation on myocardial fibrosis involving CFs stimulated with macrophage-CM following si-
Nlrp3 treatment of BMDMs. Dual immunofluorescence shows that CM/4 administration after si-
Nlrp3 treatment markedly reduced α-SMA and vimentin expression in CFs (
Fig. 6(c)). Western blot results of collagen I/III mirrored the immunofluorescence results (
Fig. 6(d)), indicating a pronounced suppressive effect of si-
Nlrp3 on myocardial fibrosis in macrophages.
3.7. Activation of the 15-HETE/small mother against decapentaplegic (Smad) pathway is the main mechanism of myofibroblast transformation induced by the activation of macrophage-associated inflammasomes
Oxidized lipid metabolomics analysis of the macrophage culture supernatant-induced cardiac fibrosis model revealed distinct expression differences among the control, model, and
Nlrp3-silenced groups, with orthogonal partial least squares-discriminant analysis (OPLS-DA) indicating high reliability (
R2X = 0.54,
R2Y = 0.93,
Q2 = 0.84, ANOVA of the cross-validated residuals (CV-ANOVA)
P = 4.43 × 10
−8) (
Fig. 7(a)). Elevated 15-HETE levels in the macrophage culture supernatant underscored its potential involvement in NLRP3 inflammasome-induced cardiac damage. Consistent with these findings, 15-HETE was found to amplify α-SMA and vimentin expression (
Fig. 7(b)) and upregulate phosphorylated (p)-Smad2/3, collagen I, and collagen III expression in fibroblasts (
Fig. 7(c)), further validating the role of this pathway in fibrosis. Finally, we investigated whether 15-HETE directly interacts with transforming growth factor-beta-activated kinase 1(TAK1). We performed molecular docking analysis of 15-HETE with the TAK1 protein, in which it exhibited suitable steric complementarity with the binding site of TAK1. Hydrogen bonds formed between TAK1 and 15-HETE (ARG-392 and TYR-587). These data suggest that si-
Tak1 has a significant inhibitory effect on collagen I/III expression in fibroblasts (
Fig. 7(d)).
3.8. 15-H ETE levels in patients with MI and ACS
To investigate the altered distribution of 15-HETE in human subjects, five MI patients who underwent surgical resection of ventricular aneurysms were recruited. The presence of cardiac lesions was confirmed by H&E staining (
Fig. 8(a)). The distribution of 15-HETE in paraffinized myocardial tissue was determined by MSI, and the levels of 15-HETE in infarct and peri-infarct heart tissues were significantly greater than those in normal tissues (
Fig. 8(b)). In addition, we recruited 231 patients with ACS (including ST-segment elevation MI and unstable angina), whose pathological characteristics were consistent with those of LAD ligation model mice, and measured their serum 15-HETE levels. Compared with those in healthy controls, the serum 15-HETE levels were significantly greater in individuals with ACS, and the difference was statistically significant (
Fig. 8(c)). In conclusion, our mouse MI study and preliminary human subject study highlight the potential role of 15-HETE in myocardial fibrosis and ventricular function.
4. Discussion
In this study, we investigated the temporal dynamics of NLRP3 inflammasome activation post-MI in mice and revealed that inhibiting NLRP3 activation confers protection against MI. We observed early activation of NLRP3 in macrophages following MI, a process implicated in the transformation of myocardial fibroblasts into myofibroblasts. This transformation contributes to increased susceptibility to myocardial fibrosis and cardiac function decline. Our results represent novel insights into the interaction between macrophage inflammasome activation and myocardial fibrosis post-MI, highlighting the significance of inflammasome-mediated fibroblast-to-myofibroblast conversion, a mechanism previously understudied in this context.
The intricate pathology of MI involves various inflammatory cellular actors within the injured heart tissue, influencing the repair process and progression to clinical HF [
38], [
39], [
40]. The dynamic interplay among different cell types and the extracellular matrix dictates cardiac function [
41], [
42], [
43], [
44]. Our study utilized database-based single-cell sequencing at different timepoints post-MI to investigate NLRP3 inflammasome activation and revealed an increase in the proportion of macrophages by the third day post-MI. We also employed immunofluorescence to label expression of key proteins across cell types and detected inflammasome activation predominantly in macrophages at this critical juncture. To elucidate the link between macrophage-associated inflammasome activation and myocardial fibrosis, we established an
in vitro myocardial fibrosis model using macrophage-CM. Our findings indicated that LPS-induced macrophage-associated inflammasome activation leads to fibrosis in fibroblasts in response to treatment with macrophage-CM. Notably, targeting the macrophage NLRP3 inflammasome significantly downregulated α-SMA and collagen I expression in fibroblasts, suggesting that inflammasome-targeting interventions could ameliorate myocardial fibrosis by modulating the macrophage-associated inflammasome activation pathway.
Some studies have shown that 15-HETE can induce pulmonary fibrosis, but whether it can cause myocardial fibrosis remains unclear [
45], [
46], [
47]. Our study also explored the role of 15-HETE, a metabolite implicated in inflammation [
47], [
48], [
49] whose levels were elevated in macrophage supernatants post-LPS stimulation and in heart tissues and blood samples from patients with ACS. The 15-HETE-induced upregulation of α-SMA, vimentin, p-Smad2/3, and collagen types I and III expression in fibroblasts underscores the potential mechanism by which NLRP3 activation in macrophages may drive myofibroblast transformation via the 15-HETE-mediated TAK1 pathway, thereby exacerbating myocardial fibrosis and impairing cardiac function.
The NLRP3 inflammasome is a macromolecular structure assembled by the chemotaxis of infiltrating inflammatory cells that induces not only self-cleavage and the activation of caspase-1 but also the conversion of pro-IL-1β and pro-IL-18 into IL-1β and IL-18 [
12], [
21], [
28]. IL-1β and IL-18 are key factors in cardiac inflammation and play important roles in patient prognosis after MI [
12], [
50], [
51]. Studies have shown that IL-1β is involved in myocardial fibrosis, cardiac remodeling, and HF by remodeling the extracellular matrix and regulating the function of fibroblasts [
52], [
53]. Increased levels of IL-18 have been detected in the plasma of MI patients and model animals, and intervention with an IL-18-neutralizing antibody can reduce the infarct size in mice with MI [
54], [
55]. Consistent with previous reports, our results suggest that the dynamic changes in the expression of the inflammatory cytokines IL-1β and IL-18 are consistent with the changes in NLRP3 expression. The dynamic expression of these inflammatory cytokines plays an important role in the activation of the NLRP3 inflammasome in macrophages.
This investigation illuminated the temporal and spatial dynamics of inflammasome activation following MI, revealing a previously underrecognized connection between macrophage inflammasome activation and myocardial fibrosis pathophysiology. Notably, early activation of NLRP3 in macrophages post-MI may promote the conversion of myocardial fibroblasts to myofibroblasts via the 15-HETE-mediated TAK1 pathway. This process potentially heightens vulnerability to myocardial fibrosis, thereby exacerbating cardiac function degradation. Our research revealed that the role of macrophage NLRP3 in the development of myocardial fibrosis is important for the precise prevention and treatment of MI, which provides a valuable perspective for developing therapeutic approaches for HF treatment post-MI.
However, the study has some limitations. These factors include the intricacies of the relationship between 15-HETE and the NLRP3 inflammasome and the impact of targeted macrophage-associated inflammasome interventions on cardiac function in MI model mice. Despite these challenges, this research provides a foundational understanding of the role of the inflammasome in myocardial injury and fibrosis, setting the stage for future explorations aimed at mitigating HF progression post-MI.
5. Conclusions
In conclusion, this study revealed temporal and spatial dynamic alterations in inflammasome activation after MI. Furthermore, we found early activation of the NLRP3 inflammasome in macrophages after MI, which could result in the transformation of myocardial fibroblasts into myofibroblasts through the 15-HETE-mediated Smad pathway, thereby exacerbating susceptibility to myocardial fibrosis and leading to deterioration of cardiac function. These data demonstrate that activation of the macrophage inflammasome contributes to myocardial fibrosis after MI, which provides insights for the development of treatment strategies for patients with HF after MI.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (82222075, 82374420, 82305025, and 82230126).
The authors express their gratitude to Professor Yang Chen for providing the homozygous Nlrp3-knockout mice.
Authors’ contribution
Yong Wang and Xu Chen conceived and designed the project and managed the study. Zhiyong Du performed the metabolomics and data analysis. Dongqing Guo revised the manuscript. Jincheng Guo performed the bioinformatics analyses. Qianbin Sun and Tiantian Liu contributed to the collection of heart and blood and partial animal experiments. Kun Hua made the clinical diagnoses and recruited the subjects. Xu Chen, Chun Li, Yong Wang, and Wei Wang wrote the manuscript. All the authors have read and approved the final manuscript.
Compliance with ethics guidelines
Xu Chen, Zhiyong Du, Dongqing Guo, Jincheng Guo, Qianbin Sun, Tiantian Liu, Kun Hua, Chun Li, Yong Wang, and Wei Wang declare that they have no conflict of interest or financial conflicts to disclose.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.eng.2024.05.015.
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