Neutrophil methylmalonic acid promotes microthrombus formation and adverse cardiac remodeling post-myocardial infarction through activating IL-6 signaling pathway-mediated NETosis

Abstract

Background

Neutrophils contribute critically to adverse cardiac remodeling following acute myocardial infarction (AMI), yet the precise regulatory mechanisms remain unclear. Our previous findings identified methylmalonic acid (MMA) as a novel cardiovascular prognostic biomarker. Thus, we aimed to investigate whether neutrophil-derived MMA mediates neutrophil extracellular trap (NET) formation and subsequent adverse cardiac remodeling post-MI, and to elucidate potential underlying mechanisms.

Methods

Serum and neutrophil MMA levels were measured in humans and mice with AMI. Neutrophil-specific Mmut knockout mice (S100a8^Cre Mmut^flox/flox) were treated with GSK484 (PAD4 inhibitor) or DNase I (NET-degrading agent) to evaluate the role of neutrophil-derived MMA in cardiac NET formation and adverse remodeling after MI. High-throughput RNA sequencing was performed on isolated neutrophils to identify molecular mechanisms.

Results

Compared with patients with angina, patients with AMI displayed significantly increased MMA levels in serum and neutrophils, particularly pronounced in neutrophils. Elevated NET markers were observed in thrombus tissue from patients with AMI with higher neutrophil MMA. Similarly, Mmut knockout mice exhibited increased NET formation, greater microthrombus burden, and worsened cardiac dysfunction 4 weeks after MI compared with S100a8Cre controls. NETosis-targeted interventions (GSK484 or DNase I) substantially reduced microthrombus formation and adverse cardiac remodeling, especially in Mmut knockout mice. Integrated transcriptomic and multifactorial analyses revealed that activation of the neutrophil IL-6/JAK1/STAT3 signaling pathway plays a key role in MMA-induced NETosis, which was largely compromised by the treatment with an IL-6 neutralizing antibody. Moreover, colchicine, an FDA-approved anti-inflammatory agent, significantly inhibited neutrophilic IL-6 expression, NETosis, and microthrombus formation, thereby attenuating post-MI cardiac remodeling against the hazards of neutrophil MMA elevation.

Conclusions

Neutrophil-derived MMA promotes NETosis and microthrombus formation through IL-6 activation, contributing to maladaptive cardiac remodeling post-MI. These findings identify neutrophil MMA as a novel immunometabolic trigger driving NET-mediated adverse cardiac remodeling and suggest colchicine as a promising therapeutic strategy to prevent heart failure post-MI, particularly in patients with elevated neutrophil MMA contents.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12916-026-04659-5.

Background

Although reperfusion therapies have substantially reduced acute infarct size for patients with acute myocardial infarction (AMI), adverse ventricular remodeling remains a leading complication of morbidity and mortality worldwide [1–3]. Beta receptor blockers and RAS inhibitors, targeting the excessive activation of the neuroendocrine system, have been proven to improve cardiac remodeling after MI and reduce the risk of adverse cardiovascular events [4]. However, the residual risks such as heart failure post-infarction remain significant. There is still an urgent need to identify novel mechanistic drivers of adverse remodeling post-MI. Immune dysregulation is gradually regarded as an important driving factor for cardiac remodeling after MI [5, 6]. Neutrophils represent the earliest responders recruited to the infarcted myocardium, where they release proteases, reactive oxygen species, and neutrophil extracellular traps (NETs) [7]. Accumulative evidence suggests that NETosis is implicated in the pathogenesis of chronic vascular remodeling. However, the molecular mechanisms driving NET formation in the context of heart remodeling post-MI remain incompletely defined [8–10].

Metabolic alteration may be an important mechanism for the complex phenotypes of immune cells, while the understanding remains in its infancy. Methylmalonic acid (MMA), a mitochondrial metabolic intermediate, was first identified in hereditary metabolic disease methylmalonic acidemia, mainly due to the mutation of methylmalonyl-CoA mutase (Mmut), a vitamin B₁₂-dependent mitochondrial enzyme [11, 12]. We and others recently found that MMA was significantly associated with poor prognosis across a spectrum of cardiometabolic diseases such as coronary artery disease, heart failure, and diabetes, even after adjusting for conventional cardiovascular risk factors, suggesting an unheeded impact of MMA [13–15]. A recent proteomics study reported that differential proteins in the serum of patients with inborn methylmalonic acidemia are enriched in neutrophil-related signals [12]. Nevertheless, whether MMA accumulation specifically mediates NETs and subsequent adverse remodeling in heart tissues post-MI remains unknown.

The present study elucidated that a neutrophil-intrinsic MMA metabolism defect promoted the formation of microthrombus in heart tissues of MI mice and caused a deteriorated cardiac function via activating IL-6/JAK1/STAT3 signaling mediated NETosis. We further assessed the translational relevance of targeting the IL-6 signaling pathway, by IL-6 neutralizing antibody or colchicine, to reduce microvascular thrombosis and prevent chronic cardiac impairment post-MI.

Methods

Human samples

This research was approved by the Ethics Committee of Harbin Medical University (YJSKY2023-503) and adhered to the principles outlined in the Helsinki Declaration regarding the use of human tissues or subjects. All samples were obtained after informed consent was provided. AMI was diagnosed according to the Fourth Universal Definition of Myocardial Infarction. While patients with angina were included, matched by age and sex as controls, patients with angina were enrolled based on ischemic symptoms and cardiologist adjudication, and did not meet diagnostic criteria for AMI/NSTEMI. The detailed clinical characteristics of patients with AMI and angina are presented in Supplementary Table 3. Blood samples were collected before percutaneous coronary intervention (PCI), and serum and neutrophils were subsequently isolated. Thrombus tissues were obtained from patients with ST-segment elevation myocardial infarction (STEMI) who successfully underwent PCI within 12 h of chest symptom onset. Immediately after the lesion was navigated through with a guidewire, the thrombus tissue at the occlusion site was aspirated, fixed in 4% paraformaldehyde (PFA), and embedded for subsequent analysis.

Generation of neutrophil-specific Mmut knockout mice

All animal experiments were approved by the Animal Care and Use Committee of Renmin Hospital of Harbin Medical University and performed in compliance with the Guidelines for Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and permitted by the Animal Care and Use Committee of the Harbin Medical University Renmin Hospital (number: YJSDW2023-245). To achieve specific knockout of the MMUT gene in neutrophils, this study employed a Cre-Flox system. Conditional gene knockout mice for MMUT were provided by Saier Biotechnology Co., Ltd. (Anhui, China), with a C57BL/6 J background. Neutrophil-specific knockout mice used S100a8^Cre mice, which were obtained by inserting the human S100a8^Cre gene promoter-regulated CRE recombinase and EGFP fluorescent protein gene expression elements into the mouse genome. When S100a8^Cre mice were crossed with MMUT mice carrying LOXP sites, CRE recombinase specifically recombined during differentiation to delete the MMUT gene sequence between LOXP sites in neutrophils and their granulocyte–monocyte progenitors (GMP), achieving specific knockout of the MMUT gene in offspring. All mice were maintained in a specific pathogen-free barrier system and were provided with standard laboratory feed ad libitum.

MI model

Adult male mice aged 8–12 weeks old (according to previous studies in the cardiovascular field, interference factors such as estrogen secretion may mask protective effects, so only adult male mice are used for experiments) were randomly divided into groups. After intraperitoneal injection of tribromoethanol (200 mg/kg), ventilation was performed using a standard rodent ventilator. Our selection of tribromoethanol is based on its applicability in specific experimental models, such as its excellent performance in ultrasonic measurements after anesthetizing mice or in establishing myocardial infarction (MI) models. Compared to isoflurane or ketamine combinations, tribromoethanol anesthesia takes effect more rapidly, more effectively avoids issues such as oversedation or metabolic interference, and ensures the accuracy and consistency of experimental data [16]. The chest cavity was opened through an incision between the third and fourth ribs, and the left anterior descending branch (LAD) was permanently ligated using a 7–0 polypropylene suture. Rapid blanching of the left ventricular anterior wall confirmed LAD occlusion [17, 18]. The sham-operated group underwent similar procedures but without ligation. Euthanasia was performed in a manner approved by the ethics committee. Before cervical dislocation, deep surgical anesthesia was induced via intraperitoneal injection of tribromoethanol (2.5% w/v solution in 0.9% saline, 200 mg/kg body weight) [19].

Echocardiography analysis

The heart structure and function were evaluated by using a Visual Sonics Vevo 3100LT Imaging System equipped with a 30-MHz MX400 transducer probe. The animals were anesthetized by intraperitoneal injection of tribromoethanol (200 mg/kg) to maintain a heart rate of around 500 bpm. The chest was shaved and standard long-axis B-mode and M-mode measurements were recorded. These included ejection fraction (EF), shortening fraction (FS), left ventricular end-diastolic diameter (LVIDd), and left ventricular end-systolic diameter (LVIDs) [19].

Platelet isolation and platelet suspension preparation

Blood was collected from the orbital sinus of mice after whisker trimming. It was mixed with ACD (85 mM sodium citrate, 71.38 mM citric acid, and 27.78 mM glucose) solution at a ratio of 9:1 (vol/vol). A centrifuge was used to prepare platelet-rich plasma (PRP) and platelet-poor plasma (PPP). Briefly, the anticoagulated whole blood was centrifuged at 300 g for 10 min to separate the PRP. The PRP was centrifuged at 900 g for 10 min, the supernatant was PPP, and the precipitate was platelets. The precipitate was collected and resuspended in Tyrode’s buffer (137 mM NaCl, 2.65 mM NaHCO3, 2 mM KCl, 0.34 mM Na2HPO4, 1 mM MgCl2, 5.5 mM glucose, and 15 mM HEPES; pH 7.4) to obtain a platelet concentration of 3 × 10⁸ platelets/mL [20].

Platelet aggregation studies

At 37 °C, platelet aggregation of mice stimulated with thrombin (0.05 U/mL) was detected in the 700-type Lumi-Aggregometer (Chrono-log Corporation, Havertown, PA, USA). The measurement amplitude can be presented by the aggregation track and calculated as the aggregation percentage, which is used to reflect the degree of platelet aggregation [20, 21].

Clot retraction assay

For clot retraction, platelet treatment was performed as previously described. Briefly, 0.3 mL of washed platelets were mixed with 0.1 mL of PPP and suspended in Tyrode’s buffer (3 × 10⁸ platelets/mL). At 37 °C, clot contraction was induced by stimulation with thrombin (1.0 U/mL) and monitored by taking photographs at designated time points using a digital camera. The area of the clot was quantified using ImageJ software. The results were expressed as the percentage of contraction (% = area/area × 100%) [21].

Liquid chromatography-mass spectrometry (LC–MS)

Serum samples are treated with a cold 20% acetonitrile/methanol solution containing the internal standard D3-MMA. After centrifugation, the supernatant is freeze-dried to precipitate, redissolved in 25 μL of a 50% water/methanol mixture, and centrifuged again to yield a clear supernatant. Cardiac tissues were collected from the left ventricular infarct zone of the model group or the corresponding normal zone of the sham group in S100a8^Cre or S100a8^Cre-Mmut^fl/fl mice. Tissues were flash-frozen in liquid nitrogen and stored at − 80 °C. For processing, 20 mg of tissue was weighed and homogenized in 1 mL of pre-chilled methanol/water (80% methanol, 0.1% formic acid) at 4 °C using a Precellys 24 homogenizer (three cycles of 30 s each with intermittent cooling). The homogenate was centrifuged (12,000 × g, 4 °C, 15 min), filtered through a 0.22-μm membrane, lyophilized, and reconstituted in 50 μL of 50% methanol/water containing 10 μM D3-MMA as an internal standard [22]. Bone marrow-derived neutrophils and monocytes were isolated (2 × 10⁶ cells/well) and stimulated with 50 nM phorbol myristate acetate (PMA) for 2 h. Supernatants were collected, centrifuged (300 × g, 4 °C, 10 min) to remove debris, filtered through a 0.22-μm membrane, and stored at − 80 °C. For analysis, 100 μL of supernatant was mixed with 400 μL of cold acetonitrile containing D3-MMA, centrifuged (12,000 × g, 4 °C, 15 min), lyophilized, and reconstituted in 25 μL of 50% methanol/water [23]. Bone marrow-derived neutrophils and monocytes (2 × 10⁶ cells/well) were washed with PBS and lysed in 200 μL of 80% methanol/water containing 0.1% formic acid. Lysates were centrifuged (12,000 × g, 4 °C, 10 min), and the supernatants were lyophilized and reconstituted in 20 μL of 50% methanol/water containing D3-MMA. Supernatants are analyzed via LC–MS/MS using the UltiMate 3000 UHPLC system and TSQ Quantis mass spectrometer from Thermo Fisher Scientific. Separation is performed on an ACQUITY HSS T3 column from Waters. The method uses negative mode ESI-SRM to track MMA and D3-MMA mass transitions. Standard curves and QC samples are included per batch for precision and accuracy. Inter-batch variability is assessed through the coefficient of variation (CV), with CV values all below 10%, indicating good inter-batch consistency. Data analysis is performed using Trace Finder software (Thermo Fisher Scientific).

Animals and experimental protocols

Experimental mice were divided into the Mmut knockout group and the S100a8^Cre control group. The following treatments were carried out when evaluating the improvement effect of the NETs inhibitor: The mice were divided into the following six groups (n = 6): S100a8^Cre + vehicle, S100a8^Cre-Mmut^fl/fl + vehicle, S100a8^Cre + DNase I, S100a8^Cre-Mmut^fl/fl DNase I, S100a8^Cre + GSK484, and S100a8^Cre-Mmut^fl/fl + GSK484. All mice were given vehicle, GSK484 (4 mg/kg) [24], or DNase I (100 U/mL) [25] daily for 10 consecutive days. The left anterior descending artery was then ligated to construct a myocardial infarction model, and evaluations of echocardiography, histology, and immunological markers were conducted on days 3, 7, and 14. To investigate the role of IL-6, mice were divided into four groups (n = 6): S100a8^Cre + IgG, S100a8^Cre + anti-IL-6, S100a8^Cre-Mmut^fl/fl + IgG, and S100a8^Cre-Mmut^fl/fl + anti-IL-6. IL-6 neutralizing antibody (2 mg/kg) or vehicle (IgG) was administered intraperitoneally on days − 1, 0, 1, 3, 7, and 10. All mice underwent left anterior descending artery ligation to establish a myocardial infarction model. Cardiac function was assessed by echocardiography at 3 and 14 days post-MI, and tissues were harvested for analysis. When evaluating the therapeutic effect of colchicine, the following treatments were carried out: The mice were divided into the following 4 groups (n = 6): S100a8^Cre + vehicle, S100a8^Cre + colchicine, S100a8^Cre-Mmut^fl/fl + vehicle, and S100a8^Cre-Mmut^fl/fl + colchicine. Colchicine was administered orally by intragastric gavage at a dose of 0.1 mg/kg once daily for 10 consecutive days before MI surgery [25]. After the final pre-treatment dose, MI was induced by permanent ligation of the LAD coronary artery as previously described. Mice were monitored postoperatively, and endpoint assessments were performed 14 days after MI.

Isolation of primary neutrophils

Human neutrophils were isolated from the blood by using the Human Neutrophil Isolation Kit (TBD) in accordance with the manufacturer’s protocol. Human neutrophil purity (> 80%) was confirmed using CD66b⁺ and CD16⁺ staining. Mouse bone marrow neutrophils were isolated by applying the Solarbio Neutrophil Isolation Kit (Solarbio, P8550). Purity was validated using Ly6G and CD11b⁺ staining (> 80%). Neutrophils were resuspended in RPMI 1640 for further use.

Quantification of neutrophils producing and releasing NETs

After obtaining neutrophils, they were stimulated with complete RPMI 1640 medium (supplemented with 10% FBS and 1% penicillin/streptomycin) containing 50 nM PMA for 4 h to induce NETs production [25]. Briefly, cells were fixed with 4% PFA, permeabilized with 0.5% Triton-X100, blocked with staining blocking solution (P0262, Beyotime), and then stained with citH3 (ab5103, Abcam) and ly6g (ab25377, Abcam) at 4 °C overnight. After washing with PBS, the secondary antibodies were incubated at room temperature for 1 h. Finally, the cell nuclei were stained with DAPI (C1005, Beyotime) for 5 min, and the staining was observed using a laser scanning confocal microscope (Zeiss). For microthrombus quantification, CD31-positive vascular regions were first segmented to define the intravascular compartment. Within these CD31-defined microvessels, vWF-positive areas were identified to represent microthrombus regions. The images underwent analysis with ImageJ software, and NETs were quantified as the percentage of positive events (neutrophils undergoing NETosis) out of total neutrophils in an average of 3 fields [26].

Histological analysis

For the analysis of fibrosis after myocardial infarction, 4-μm-thick paraffin sections of the heart were stained with Masson trichrome staining (Solarbio, G1340) and Sirius Red staining (Solarbio, G1473). The area of myocardial fibrosis was measured using ImageJ software. To observe immune cell infiltration, H&E staining (Solarbio, G1120) was performed on 4-μm Sects. (3 sections/heart) with image acquisition of 3 randomly selected fields per section across distinct myocardial zones [27].

Immunofluorescence analysis

The 4-μm paraffin sections were dewaxed with xylene, hydrated with gradient ethanol, subjected to antigen retrieval using the citrate-sodium-EDTA antigen retrieval solution (P0086, Beyotime), and blocked with the staining blocking solution (P0262, Beyotime), followed by incubation with the primary antibody at 4 °C overnight. Then, they were incubated with the fluorescently labeled secondary antibody for 1 h, and the nuclei were counterstained with DAPI (Beyotime, C1005). The staining of cultured neutrophils was performed by the previously reported method. Briefly, neutrophils were rinsed with PBS, fixed with 4% PFA, permeabilized with 0.1% Triton X-100, blocked with the staining blocking solution (P0262, Beyotime), and incubated with the primary antibody at 4 °C overnight. Then, they were incubated with the fluorescently labeled secondary antibody for 1 h, and the nuclei were counterstained with DAPI (Beyotime, C1005). The above tissue sections and cells were imaged using a laser scanning confocal microscope (Zeiss). ImageJ software was used to process the calculation of intensities and positive area for quantification. For the statistics of immunofluorescence images, there were 6 independent replicates in each group, and 5 random fields were selected for each replicate for statistics, and then the values were averaged [19].

In vivo vein thrombosis model

The mice were anesthetized using intraperitoneal injection of tribromoethanol (200 mg/kg). Thereafter, the mesenteric vein was exposed. FeCl₃ (1 μL of 10% FeCl₃ soaked in 1 mm² Whatman paper) was topically applied for 4 min to the mesenteric vein. Prior to the FeCl₃ application, platelets were labeled through intravenous injection of Rhodamine 6G (0.5 mg/mL; 100 μL) via the mice’s tail vein. Intravital microscopy was employed to observe thrombosis in the mesenteric veins of mice with FeCl₃-induced injury. Fluorescent images were acquired sequentially (1 image/30 s) and recorded for 30 min. Thrombus size and kinetics (i.e., time to occlusion, duration of occlusion) were analyzed using ImageJ software [21].

Thrombus formation under flow conditions ex vivo

The experimental preparation for thrombus formation under ex vivo flow conditions was as described previously. Briefly, a microfluidic whole blood perfusion experiment was performed on the BioFlux 200 flow system (Fluxion, CA) under arterial shear conditions (1000 s⁻¹) to assess thrombus formation using the fibrin-collagen mechanism. The BioFlux plate was coated with fibrinogen (40 µg/mL) overnight and blocked with 5% BSA. Anticoagulated blood samples were collected from mice and perfused onto the fibrin-collagen-coated BioFlux plate (Fluxion, CA) at a shear stress of 40 dyne/cm² for 3 min. Three brightfield images were captured using a laser scanning confocal microscope (Zeiss) and analyzed in a blinded manner using ImageJ software [28].

Flow cytometry analysis

Flow cytometry was used to quantify peripheral blood cells. Blood was collected from the orbital sinus of mice and anticoagulated with ACD (85 mM sodium citrate, 71.38 mM citric acid, and 27.78 mM glucose) solution (9:1 vol/vol). The samples were subjected to red blood cell lysis, centrifugation, washing, and resuspension in FACS buffer. For quantification of myeloid cells in the bone marrow by flow cytometry, bone marrow samples were obtained by flushing the bone marrow cavity with a cold 25-gauge 200-needle and PBS, passed through a 70-μm filter, and centrifuged at 500 g for 10 min at 4 °C to obtain bone marrow pellets. After red blood cell lysis, centrifugation, and washing, the cells were resuspended in FACS buffer. When analyzing mouse cardiac cells by flow cytometry, cardiac tissues were excised, minced for mechanical disruption, and digested with type I collagenase. A single-cell suspension was obtained by filtering through a 70-μm filter, rinsed with FACS buffer, and centrifuged at 500 g for 5 min to collect cell pellets. The fluorescent antibodies used in this study: Cells were stained with the Zombie AquaTM Fixable Viability Kit to exclude dead cells. Subsequent staining was performed using the following fluorochrome-conjugated antibodies: CD45-FITC, CD11b-APC, Ly6G-PE, and Ly6C-PerCP. These antibodies were diluted in FACS buffer for flow cytometric analysis. Data were acquired using the LSRFortessa flow cytometer (BD Biosciences) and processed using FlowJo software (version 10.6.2). The CD45^hiCD11b^hiLy6C^hi subset was defined as monocytes, and the CD45^hiCD11b^hiLy6G^hi subset was defined as neutrophils. All experimental samples were coded and analyzed in a blinded manner by investigators [29].

Western blotting

Cellular and tissue proteins were isolated under standardized protocols. Cells were lysed in RIPA buffer (Solarbio) containing phosphatase and protease inhibitors with protein concentration determined by BCA assay (ThermoFisher). Myocardial tissues from the left ventricular free wall (LVFW) were snap-frozen in liquid nitrogen, mechanically pulverized, and homogenized in RIPA buffer for whole lysates. For subcellular fractionation, tissues were first suspended in a hypotonic buffer to isolate cytoplasmic proteins, followed by nuclear extraction with NP-40 buffer. Equal amounts of protein (30 μg/lane; within linear detection range) were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. After blocking with 5% non-fat milk, membranes were incubated overnight at 4 °C with primary antibodies against JAK1, phospho-JAK1, STAT3, phospho-STAT3, IL-6, and citH3, as well as loading controls: β-actin (total), GAPDH (cytoplasm), and β-actin (nucleus). HRP-conjugated secondary antibodies (Jackson ImmunoResearch) were used for 1 h at room temperature. Signals were visualized with ECL (ThermoFisher) and quantified using ImageJ (v1.53e) [30].

Quantitative real-time PCR

Total RNA was isolated from myocardial tissues from the left ventricular free wall (20–30 mg per sample) using TRIzol reagent (Invitrogen). Tissues were snap-frozen in liquid nitrogen immediately after collection, mechanically pulverized, and lysed in 1 mL TRIzol. RNA was reverse transcribed into cDNA using the ReverTra Ace qPCR RT kit (FSQ-101, TOYOBO) according to the manufacturer’s instructions. The obtained cDNA was mixed with SYBR RT-PCR Master Mix (Q711-02, Vozyme) and specific primers for amplification [31]. The relative expression level of mRNA was normalized to β-actin (mouse) [32, 33]. Specific primers for mouse MMUT, IL-6, Col1a1l, Col3al, TIMP1, and MMP9 were employed. Primer sequences are presented in Supplementary Table 2.

ELISA assay

For ELISA, human and mouse serum were separated by centrifugation. The concentrations of NE, MPO, Cit-H3, MPO-DNA complex, and IL-6 in serum were measured using ELISA kits in accordance with the manufacturers’ instructions. To assess cytokines in cardiac tissue, excised hearts were homogenized in ice-cold PBS containing protease inhibitors and centrifuged at 12,000 × g for 15 min at 4 °C; the supernatants were collected and total protein quantified by BCA assay. Levels of IL-6, TNF-α, IL-1β, and NLRP3 in the tissue lysates were then determined by ELISA and expressed per milligram of total protein [34]. Details of ELISA kits are provided in Supplementary Table S5.

RNA-seq

After a 2-h treatment with PMA, neutrophils were collected, and total RNA was extracted using TRIzol reagent. After evaluating the library quality, qualified samples were selected for library construction and sequencing. OE Biotechnology Co., Ltd. (Shanghai, China) was responsible for transcriptome sequencing and analysis. Several R packages were employed to analyze the data and visualize the outcomes. The DESeq2 package was utilized to calculate the differentially expressed genes between groups. Genes with Log2 (FC) > 1 and a P value < 0.05 were regarded as upregulated genes, while genes with Log2 (FC) < − 1 and a P value < 0.05 were considered as downregulated genes. The volcano plot of differentially expressed genes was generated through the ggplot2 package. The combination of GO enrichment analysis and KEGG enrichment analysis was adopted to search for the key pathways enriched by differentially expressed genes [35].

Morphological by electron microscopy

Blood neutrophils were harvested from S100a8^Cre and S100a8^Cre-Mmut^fl/fl mice via density gradient centrifugation. Purified neutrophils were cultured in RPMI-1640 medium (Gibco) supplemented with 50 nM PMA (Sigma-Aldrich) and 10% fetal bovine serum (HyClone) for 2 h under standard conditions (37 °C, 5% CO₂). Neutrophils were fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences) buffered with 0.1 M sodium cacodylate (pH 7.2) at 4 °C for 12 h. Cells were pelleted by centrifugation (300 × g, 10 min) and adhered to poly-lysine-coated coverslips. Dehydration was performed through a graded ethanol series (30%, 50%, 70%, 90%, 100%), followed by critical-point drying with liquid CO₂. Samples were sputter-coated with 8-nm gold–palladium (Quorum Q150T) and imaged using a Hitachi Regulus 8220 SEM (Hitachi High-Tech).

Statistical analysis

All quantitative data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism software (version 10.1). Normality of data distribution was assessed using the Shapiro–Wilk test. For comparisons between two groups, either a two-tailed unpaired Student’s t-test or a Mann–Whitney U test was applied, as appropriate. For analyses involving more than two groups, one-way or two-way ANOVA followed by post hoc testing was used. Correlation analyses were conducted using Pearson’s or Spearman’s method depending on data distribution, and results are reported as correlation coefficients (r) with corresponding P values. Survival analyses were evaluated using the log-rank test. A P value < 0.05 was considered statistically significant.

Results

Neutrophil MMA accumulation post-infarction correlated with cardiac dysfunction and thrombosis biomarkers in human and mice

Serum MMA levels were higher in patients with AMI compared to age- and sex-matched angina controls as quantified by LC–MS (Fig. 1A; Table S3). Notably, intracellular MMA accumulation was especially significant in neutrophils from patients with AMI, compared to that in monocytes, platelets, and serum (Fig. 1B–E). We next assessed the expression of mitochondrial protein methylmalonyl-CoA mutase (Mmut), the key metabolic enzyme of methylmalate in coronary artery thrombi from patients with AMI. Confocal microscopy revealed a preferential colocalization of Mmut with neutrophil marker CD66b, indicating dysregulation of MMA metabolism within neutrophils, particularly under post-MI conditions (Fig. 1F). Moreover, neutrophil MMA levels showed significantly inverse correlations with cardiac markers TnI and NT-proBNP, and 1-year left ventricular ejection fraction change (ΔEF) and had a positive association with the thrombosis marker D-dimer (Fig. 1G–J). These clinical data suggested that neutrophil MMA might be involved in cardiac dysfunction and thrombotic burden post-MI.

Fig. 1.

Elevated neutrophil MMA levels in patients with AMI correlate with myocardial injury, adverse remodeling, and thrombosis. A Serum MMA concentrations in patients with AMI (n = 208) and angina controls (n = 208), as measured by LC–MS. Neutrophil (B), monocyte (C), and platelet (D) MMA concentrations determined by LC–MS in patients with AMI (n = 30) and angina controls (n = 30). E Bar plots display the fold change of MMA concentrations across different peripheral blood cell populations between patients with AMI and angina controls. F Representative gross image of thrombus tissue from patients with AMI (scale bar = 5 mm), H&E staining (scale bar = 500 μm and 100 μm), and immunofluorescence colocalization staining for CD66b (red), Mmut (green), and DAPI (blue) (scale bar = 20 μm). Correlation analysis of the relationships between the level of MMA and the levels of TnI (G), NT-proBNP (H), ΔEF (J), and D-dimer (I). Statistical significance was determined by the Mann–Whitney test (A), two-tailed unpaired Student’s t-test (B–D), Pearson’s correlation analysis (I), and Spearman’s correlation analysis (G, H, J). Data are presented as mean ± SD. **P < 0.01, ****P < 0.0001

To further investigate whether extracellular MMA contributes to MMA accumulation in neutrophils, we isolated peripheral neutrophils from healthy donors and incubated them with elevated MMA concentrations equivalent to those in patients with AMI (0.5 μmol/L, 1 μmol/L, and 2 μmol/L) for 6 h. Notably, no significant alterations in neutrophil viability or intracellular MMA levels were observed (Supplementary Fig. 1A, B), suggesting an endogenous source of MMA accumulation in neutrophils. To validate these findings in animals, we established an MI model in C57BL/6 mice. LC–MS analysis revealed significantly elevated MMA concentrations in both serum and bone marrow-derived neutrophils from MI mice compared with sham controls (Supplementary Fig. 1C, D). In contrast, MMA levels in monocytes remained comparable (Supplementary Fig. 1E). These results confirm neutrophil-intrinsic MMA accumulation, rather than a passive consequence of elevated circulating MMA levels post-MI.

Neutrophil-specific Mmut deletion promoted thrombosis and exacerbated cardiac dysfunction post-MI

To delineate neutrophil-specific MMA metabolism, neutrophil-targeted Mmut knockout mice (Mmut^flox/floxS100a8^Cre, Mmut CKO) were generated and validated by PCR-based genotyping (Supplemental Fig. 2A, B). Effective neutrophil-specific deletion of Mmut protein was further confirmed (Supplemental Fig. 2C, D). LC–MS analysis revealed significantly elevated intracellular MMA concentrations specifically in neutrophils from Mmut CKO mice compared with S100a8^Cre controls (Supplemental Fig. 2E), whereas MMA levels remained unchanged in monocytes (Supplemental Fig. 2F). Subsequently, both Mmut CKO and S100a8^Cre mice were subjected to the MI model induced by left anterior descending coronary artery ligation. Mmut CKO mice exhibited significantly reduced survival compared with the S100a8^Cre controls during the follow-up of 14 days post-MI (Fig. 2A, B). Additionally, Mmut CKO mice showed more deteriorated cardiac remodeling, as evidenced by the reduced LVEF and aggravated LV dilation from day 3 to day 28 post-MI (Fig. 2C–G), extensive myocardial fibrosis at day 28 on Masson’s and Sirius Red staining (Fig. 2H, J, K), and upregulation of fibrotic markers (Col1a1, Col3a1, TIMP-1, and MMP-9; Fig. 2M). To determine the association between MMA and thrombotic burden post-MI, we investigated that microthrombi areas were increased in peri-infarct zones of Mmut CKO compared with the S100a8^Cre mice at day 3 post-MI (Fig. 2I, L). Furthermore, Mmut CKO mice exhibited accelerated and prolonged thrombosis in mesenteric veins induced by FeCl₃ at day 3 post-MI (Supplementary Fig. 3A, C, D), and increased thrombus formation in microfluidic whole-blood perfusion assays (Supplementary Fig. 3B, E). Moreover, platelets isolated from Mmut CKO mice exhibited enhanced aggregation (Supplementary Fig. 3F, H) and accelerated clot retraction (Supplementary Fig. 3G, I). These findings indicated that neutrophil-specific MMA accumulation may contribute to post-MI cardiac dysfunction through augmented thrombotic responses.

Fig. 2.

Neutrophil-specific Mmut deletion exacerbates post-MI cardiac dysfunction, fibrosis, and thrombus formation in mice. A Schematic overview of the experimental design in the mouse model. B Survival rate of S100a8^Cre and S100a8^Cre-Mmut^fl/fl mice undergoing MI surgery for 14 days (n = 58). Representative M-mode images (C) and statistical analysis of EF (D), FS (E), LVEDV (F), and LVESV (G) from S100a8^Cre and S100a8^Cre-Mmut^fl/fl mice subjected to sham or MI surgery for 3, 7, 14, and 28 days (n = 6). H, J Representative image of Masson’s trichrome staining and quantitative analysis of fibrosis area (scale bar = 1 mm, n = 6). I, L Representative image of microthrombus staining in mouse heart slices and quantitative analysis, triple immunofluorescence colocalization analysis demonstrating clustered infiltration of CD31 (red color), vWF (green color), and DAPI (blue color) (scale bar = 20 μm, n = 6). K Quantitative analysis of Sirius Red staining (n = 6). M RT-PCR detection of cardiac fibrosis markers collagen I, collagen III, TIMP1, and MMP9 (n = 6). Statistical analyses were conducted using a two-tailed unpaired Student’s t-test (J–L), one-way ANOVA with post hoc analysis (D–G, M), and mouse survival analysis was performed using a log-rank test (B). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant

Neutrophil-intrinsic MMA accumulation enhanced NET formation following MI

Neutrophil extracellular traps (NETs) have been reported as critical mediators of thrombosis [36–38]. We investigated whether MMA accumulation promotes microthrombosis by increasing NET formation. Serum MMA levels in patients with AMI correlated positively with circulating NET biomarkers, including citH3, MPO, and NE (Fig. 3A–C). Additionally, neutrophils isolated from patients with AMI exhibited more release of NET upon stimulation with PMA (50 nM, 4 h) compared to neutrophils from angina controls (Fig. 3D, E). In mice subjected to MI, serum levels of NET-related biomarkers (citH3, MPO, NE, and MPO-DNA complex) were markedly elevated in Mmut CKO mice compared with S100a8^Cre mice (Fig. 4B–D). Consistently, heart-infiltrated citH3 was also significantly increased in the Mmut CKO mice, as determined by western blotting and flow cytometry (Fig. 4F–I). Immunofluorescence staining further identified a higher proportion of infiltrating Ly6G + citH3 + double-positive neutrophils within the infarcted myocardium of Mmut CKO mice, indicative of enhanced NETosis in vivo (Fig. 4J, K). Notably, no differences were observed in the neutrophil counts within the infarcted myocardial tissues or bone marrow between Mmut CKO and S100a8^Cre mice (Fig. 4L–N, Supplementary Fig. 4A–C), suggesting that increased NETosis driven by neutrophil-intrinsic MMA accumulation occurred independently of neutrophil biogenesis and recruitment. Next, we found that Mmut-deficient neutrophils markedly increased NET formation compared with controls in vitro (Fig. 4O, P). Ultrastructural analysis by confocal fluorescence microscopy and transmission electron microscopy (TEM) revealed exacerbated NETosis in Mmut-deficient neutrophils, characterized by enhanced cytoplasmic vesiculation and prominent “beads-on-a-string” morphology (Supplementary Fig. 4D, E). Collectively, these results suggest that neutrophil-intrinsic MMA accumulation promotes NETosis following MI, independent of neutrophil recruitment.

Fig. 3.

Association between plasma MMA levels and NETs markers in patients with AMI. Correlation analysis of the relationships between the level of MMA and the levels of citH3 (A), MPO (B), and NE (C) in patients with AMI (n = 30). Representative immunofluorescence images (D) and quantitative analysis (E) of NET-positive neutrophils in control patients versus patients with AMI (n = 6). Triple immunofluorescence analysis demonstrates colocalization of CD66b (red), citH3 (green), and DAPI (blue) (scale bar = 20 μm, n = 6). Statistical analyses were conducted using two-tailed unpaired Student’s t-test (E), Spearman’s correlation analysis (A, C), and Pearson’s correlation analysis (B). Data are presented as mean ± SD. ****P < 0.0001

Fig. 4.

Neutrophil-intrinsic MMA accumulation enhances NETosis following myocardial infarction independent of neutrophil recruitment. A Schematic overview of the experimental design in the mouse model. ELISA analysis of NETs markers NE (B), MPO (C), citH3 (D), and MPO-DNA complex (E) in serum from S100a8^Cre and S100a8^Cre-Mmut^fl/fl mice 3 days after MI (n = 6). F, G Representative western blot and quantification of citH3 expression in left ventricular tissue from S100a8^Cre and S100a8^Cre-Mmut^fl/fl mice 3 days post-MI (n = 3). H, I Representative flow cytometric images and quantitative analysis of NETs in left ventricular tissue from S100a8^Cre and S100a8^Cre-Mmut^fl/fl mice 3 days after MI (n = 6). J, K Representative immunofluorescence images of NETs staining in the cardiac peri-infarct zone from S100a8^Cre and S100a8^Cre-Mmut^fl/fl mice 3 days post-MI. Triple immunofluorescence analysis shows colocalization of Ly6G (red), cit-H3 (green), and DAPI (blue) (scale bar = 20 μm; n = 6). L–N Representative flow cytometric images and quantitative analysis of heart neutrophils from S100a8^Cre and S100a8^Cre-Mmut^fl/fl mice subjected to sham or MI surgery for 1 and 3 days (n = 6). O, P Representative immunofluorescence images of bone marrow-derived neutrophils stimulated with PMA from S100a8^Cre and S100a8^Cre-Mmut^fl/fl mice 3 days post-MI. Triple immunofluorescence analysis shows colocalization of Ly6G (red), cit-H3 (green), and DAPI (blue) (scale bar = 20 μm; n = 6). Statistical analyses were conducted using one-way ANOVA with post hoc analysis (B–E, H–K, M, N). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant

Targeted NET inhibition mitigated MMA-driven cardiac dysfunction and thrombosis post-infarction

To investigate the therapeutic benefits of NETosis inhibitors on MMA accumulation, mice received GSK484 (PAD4 inhibitor) or DNase I (NET-degrading agent) starting 10 days pre-MI induction (Fig. 5A). Administration of GSK484 and DNase I substantially decreased circulating NET markers in both S100a8^Cre and Mmut CKO mice (Supplementary Fig. 5Q–S). The elevated cardiac NET formation observed in Mmut CKO mice post-MI was effectively attenuated by treatment with GSK484 and DNase I (Fig. 5B, F). Echocardiographic measurements at days 3, 7, and 14 post-MI showed that the NET inhibitors significantly improved cardiac dysfunction caused by neutrophil-intrinsic MMA accumulation (Fig. 5C–J; Supplementary Fig. 5A–N). Masson’s trichrome staining and Sirius Red staining revealed a pronounced reduction in cardiac fibrosis in Mmut CKO mice by day 14 post-MI with NET inhibition (Fig. 5E; Supplementary Fig. 5 M, N). Immunofluorescence analyses further confirmed that NET inhibitors reduced microthrombus formation within the myocardium of Mmut CKO mice (Fig. 5K, N). Similarly, the increased NET release observed in bone marrow-derived neutrophils from Mmut CKO mice was also attenuated by NETosis inhibitors (Supplementary Fig. 5 J–L). In addition, NETosis inhibition attenuated the prothrombotic effects of neutrophil-intrinsic MMA, including platelet aggregation, clot retraction, and platelet adhesion (Fig. 5L–P; Supplementary Fig. 5O, P). Collectively, NET inhibition may mitigate MMA-induced cardiac dysfunction and thrombosis following MI.

Fig. 5.

NET inhibition attenuates MMA-induced cardiac dysfunction, remodeling, and thrombosis post-MI. A Schematic overview of the experimental protocol. B, F Representative immunofluorescence images and quantitative analysis of NETs in the peri-infarct zone at day 3 post-MI in each group (scale bar = 20 μm, n = 6). Representative M-mode echocardiography images at day 3 (C) and day 14 (D) after MI in the six groups. G–J Quantitative analysis of echocardiographic parameters, including ejection fraction (EF), fractional shortening (FS), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV) at day 14 post-MI (n = 6). E Representative Masson’s trichrome staining of cardiac sections (scale bar = 1 mm, n = 6). K, N Representative immunofluorescence images and quantification of microthrombus formation in the six groups. Triple immunofluorescence analysis demonstrates colocalization of CD31 (red), vWF (green), and DAPI (blue) (scale bar = 10 μm, n = 6). Representative images of clot retraction (L) and platelet aggregation (M), with corresponding quantitative analysis (O, P) (n = 6). Statistical significance was determined by two-way ANOVA with post hoc analysis. * indicates within-group comparisons and # indicates between-group comparisons. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001

MMA elevation induced NET formation via the IL-6 signaling pathway in neutrophils

To elucidate mechanisms underlying MMA-mediated NET formation, RNA sequencing was performed on bone marrow-derived neutrophils isolated from MI-operated S100a8Cre and neutrophil-specific Mmut CKO mice. Differential expression analysis identified 2492 upregulated and 1504 downregulated genes in Mmut-deficient neutrophils compared to controls. The increased mRNA expression of IL-6 was one of the most prominent features (adjusted P < 0.05, log2 FC > 1; Fig. 6A). GSEA and KEGG enrichment analyses of neutrophil DEGs consistently indicated significant upregulation of inflammatory signaling, highlighting activation of the IL-6/JAK1/STAT3 axis in Mmut CKO neutrophils (Fig. 6B, C). To directly explore IL-6’s role in MMA-driven cardiac pathology, IL-6-neutralizing antibody (2 mg/kg i.p.) was administered to conditional Mmut-deficient mice (Supplementary Fig. 6A). This intervention significantly reduced MMA-induced inflammatory cytokine production, including IL-6, TNF-α, and IL-1β, in cardiac tissues of Mmut-CKO mice (Supplementary Fig. 6B–D). Correspondingly, administration of IL-6 antibody significantly decreased NETosis and microthrombus burden in Mmut CKO mice (Fig. 6D, G; Supplementary Fig. 6E, F). Furthermore, blockade of IL-6 substantially mitigated the exacerbation of cardiac dysfunction from day 3 through day 14 post-MI in Mmut-deficient mice (Supplementary Fig. 6G–K). Collectively, these findings suggested that neutrophil-intrinsic MMA accumulation exacerbates NETosis and subsequent cardiac remodeling at least partly by amplifying IL-6 signals.

Fig. 6.

IL-6/JAK1/STAT3 signaling mediates MMA-driven NET formation and subsequent thrombotic outcomes. A Volcano plot showing differentially expressed genes (DEGs) in neutrophils isolated from S100a8^Cre and S100a8^Cre-Mmut^fl/fl mice (n = 4; adjusted P < 0.05, log₂ FC > 1). Red and blue dots indicate upregulated and downregulated genes, respectively (e.g., IL6, JAK1). B GSEA showing activation of the IL-6/JAK1/STAT3 pathway in S100a8^Cre and S100a8^Cre-Mmut^fl/fl neutrophils. C KEGG pathway enrichment analysis of DEGs (top 30 pathways). D, G Representative images and quantitative analysis of microthrombus immunofluorescence staining in cardiac tissue from the four groups of mice, with triple immunofluorescence for CD31 (red), vWF (green), and DAPI (blue) (scale bar = 20 μm, n = 3). E, I–L Mouse neutrophils were pretreated with neutralizing anti-IL-6 antibody (6 h), then stimulated with PMA (50 nM, 1 h). Representative immunoblots and quantification of IL-6 pathway markers including IL-6, p-JAK1, JAK1, p-STAT3, STAT3, and citH3 (n = 3). M–S Neutrophils were pretreated with the JAK1 inhibitor ruxolitinib (5 μM) or the STAT3 inhibitor cryptotanshinone (10 μM) for 6 h, followed by PMA (50 nM, 1 h) stimulation. Representative immunoblots and quantification of IL-6 pathway markers (n = 3). F, H Representative images and quantification of nuclear p-STAT3 localization in neutrophils from S100a8^Cre and S100a8^Cre-Mmut^fl/fl mice (scale bar = 10 μm, n = 6). Immunofluorescence images show p-STAT3 (red), F-actin (green), and DAPI (blue). Statistical analysis was performed using two-tailed unpaired Student’s t-test (G), one-way ANOVA with post hoc analysis (H), and two-way ANOVA with post hoc analysis (I–L, N–S). * indicates within-group comparisons and # indicates between-group comparisons. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant

Next, we confirmed the critical role of IL-6/JAK1/STAT3 pathway in MMA-induced NET formation. MMA-overexposed neutrophils exhibited markedly elevated IL-6 expression, increased phosphorylation of JAK1 and STAT3, and enhanced NET formation compared with S100a8^Cre controls following PMA stimulation for 2 h (Fig. 6E, I, J). Consistent with previous findings, neutralization of IL-6 significantly suppressed JAK1 and STAT3 phosphorylation and effectively reduced citH3 levels, confirming IL-6 as an essential mediator upstream in this signaling cascade pathway (Fig. 6I, J). Furthermore, neutrophils were treated with the JAK1 inhibitor ruxolitinib or the STAT3 inhibitor cryptotanshinone. Ruxolitinib suppressed the overactivation of JAK1 and STAT3 induced by intracellular MMA accumulation in vitro, whereas cryptotanshinone selectively inhibited STAT3 phosphorylation (Fig. 6M–O). Importantly, both inhibitors markedly attenuated the increased NETs biomarker citH3 expression in Mmut-deficient neutrophil (Fig. 6Q). Immunofluorescence analyses revealed the upregulation of nuclear translocation of p-STAT3 in Mmut-deficient neutrophils compared with wildtype counterparts (Fig. 6F, H). Notably, this MMA-induced nuclear translocation could also be abolished by the inhibition of JAK1 or STAT3 (Fig. 6R, S). Collectively, these findings suggested that neutrophil-specific MMA dysmetabolism drove IL-6 pathway activation, leading to nuclear STAT3 translocation and subsequent NETosis.

Colchicine attenuates MMA-induced cardiac remodeling and thrombosis by suppressing IL-6-mediated NETosis

Previous studies have established colchicine as an effective and non-specific anti-inflammatory agent, including the inhibitory effects on IL-6 signaling, with proven safety and low cost in clinical practice [39–41]. To directly assess the protective potential of IL-6 suppression in neutrophil MMA-related heart damage, colchicine (0.1 mg/kg, orally once daily) was administered to mice for 10 consecutive days before MI surgery (Fig. 7A). This low-dose regimen of colchicine treatment was well tolerated and did not affect body weight in either S100a8^Cre or Mmut CKO mice (Fig. 7C). Colchicine administration resulted in a significant reduction in circulating IL-6 levels in both WT and Mmut-deficient groups, with a more pronounced effect observed in Mmut-CKO mice (Fig. 7D). The intervention also substantially attenuated MMA-triggered cardiac inflammation (Supplementary Fig. 7A–C) and reduced NET formation within infarcted heart tissue (Supplementary Fig. 7D, E). Furthermore, colchicine treatment significantly preserved cardiac function compromised by neutrophil MMA accumulation (Fig. 7B, E–H). Next, the extent of cardiac fibrosis was evaluated at day 14 post-MI. Colchicine treatment effectively attenuated collagen deposition and fibrotic remodeling, particularly in Mmut CKO mice (Fig. 7I, J, M). In addition, colchicine diminished microthrombus burden within the infarcted myocardium of Mmut-deficient mice (Fig. 7K, L). Collectively, these data support colchicine as a feasible translational strategy to counteract MMA-exacerbated cardiac harms via inhibiting IL-6-mediated NETosis.

Fig. 7.

Colchicine inhibits IL-6-mediated NETosis and cardiac remodeling post-infarction in mice with neutrophil MMA elevation. A Schematic illustration of the experimental design. B Representative B-mode and M-mode echocardiographic images and quantitative analysis of cardiac function, including FS (E), EF (F), LVEDV (G), and LVESV (H) at day 14 post-MI (n = 6). I, J Representative image of Masson’s trichrome staining and quantification of fibrosis area (scale bar = 1 mm, n = 6). C Body weight of mice in the four groups (n = 6). D ELISA analysis of IL-6 in serum (n = 6). K, L Representative images and quantification of microthrombus staining in the four groups, with triple immunofluorescence showing colocalization of CD31 (red), vWF (green), and DAPI (blue) (scale bar = 10 μm, n = 6). M RT-PCR analysis of cardiac fibrosis markers, including collagen I, collagen III, TIMP1, and MMP9 (n = 3). Statistical significance was determined by two-way ANOVA with post hoc analysis. * indicates within-group comparisons and # indicates between-group comparisons. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant

Discussion

In this study, we observed that the elevated neutrophil MMA contents were in patients with AMI and were associated with worse cardiac function indicators. Moreover, the expression of NET biomarkers in thrombotic tissue from patients with AMI was positively correlated with neutrophil MMA level. Neutrophil-specific MMA metabolism defect promoted the formation of microthrombus in heart tissues of MI mice and caused a deteriorated cardiac function via activating IL-6/JAK1/STAT3 signaling mediated NETosis. Inhibiting IL-6 post-MI markedly suppressed NETosis and alleviated adverse cardiac remodeling in Mmut CKO mice, suggesting a promising therapeutic target for patients with AMI. Moreover, colchicine, an FDA-approved anti-inflammatory agent, significantly inhibited neutrophilic IL-6 expression, NETosis, and microthrombus formation, thereby attenuating post-MI cardiac remodeling in both S100a8^Cre and S100a8^CreMmut^fl/fl mice. A schematic illustration of the proposed neutrophil MMA–IL-6–NET axis underlying post-MI microthrombosis and adverse cardiac remodeling is shown in Fig. 8. Collectively, neutrophil MMA represents a novel immunometabolic trigger driving NET-mediated adverse cardiac remodeling and suggests colchicine as a promising therapeutic strategy to prevent heart failure post-MI, particularly in patients with elevated MMA content in neutrophils. These results provide new insights that the therapeutic potential of targeting neutrophil metabolic dysregulation and the subsequent inflammatory cascade may represent a promising approach for improving clinical outcomes in patients following MI.

Fig. 8.

Mechanism diagram. Methylmalonic acid promotes the release of NETs via the IL-6 signaling pathway, resulting in microthrombosis formation and exacerbating adverse cardiac remodeling following MI

The role of neutrophils during the acute phase of MI has been widely delineated [42, 43], while their contribution to long-term cardiac remodeling post-MI remains largely unknown. Mitochondrial metabolism has emerged as a critical determinant in the functional regulation of immunocytes, including neutrophils [44]. Our previous epidemiologic studies found that mitochondrial-derived metabolite MMA predicts cardiovascular mortality independent of conventional risk factors [45]. In this study, we found a significant elevation of MMA in neutrophils isolated from patients with AMI. Although circulating serum MMA concentrations were also increased in patients with AMI compared to controls, neutrophil-intrinsic MMA metabolism undergoes profound dysregulation following AMI, with intracellular accumulation exceeding that in serum or other immunocytes. Crucially, the exposure to pathophysiological concentrations of MMA (equivalent to serum levels in patients with AMI) failed to induce significant intracellular MMA elevation in neutrophils derived from individuals with no AMI. These data collectively attribute elevated neutrophil MMA levels post-AMI to neutrophil-autonomous metabolic alteration rather than extrinsic influx. Therefore, we employed a neutrophil-specific MMA accumulation model to confirm the neutrophil-specific consequences of MMA accumulation. Collectively, these findings delineate a potential role for neutrophil-intrinsic mitochondrial metabolic dysregulation, specifically involving MMA accumulation, in contributing to long-term cardiac dysfunction following MI.

NETs are involved in the progression of several cardiovascular pathologies, including vascular injury, atherosclerosis, and heart failure [46–49]. However, the specific role of NETs was always controversial in cardiovascular diseases. Some studies reported that NETs amplified the degree of inflammation and worsened outcomes in patients with STEMI [50]. Conversely, NET-associated anti-inflammatory cytokines (IL-10, TGF-β) were associated with the recovery of heart function post-revascularization [51, 52]. NETs may play heterogeneous roles under different pathological backgrounds, and an in-depth investigation into their precise regulatory mechanisms is needed. In the present study, we found that NETs induced by neutrophil-intrinsic MMA accumulation work as a critical trigger to aggravate adverse cardiac remodeling post-MI. Mice with neutrophil-specific MMA accumulation exhibited increased NETs and microthrombosis at infarct border zones, while NET inhibition significantly mitigated MMA-induced microthrombosis and cardiac dysfunction. These findings substantially advance the understanding of neutrophil-driven metabolic inflammation post-MI.

Next, we identified the IL-6/JAK1/STAT3 signaling as the key mechanism for MMA inducing NETosis. Our RNA sequencing analysis of bone marrow-derived neutrophils from MI-operated S100a8^Cre-Mmut^fl/fl mice identified the significant activation of the IL-6 pathway. Prior studies have demonstrated the pivotal role of IL-6 signaling in cardiovascular injury [53]. Ziltivekimab, a newly developed IL-6 ligand inhibitor specifically for the treatment of atherosclerotic cardiovascular disease [54, 55], has been reported to reduce biomarkers of inflammation and thrombosis. Our data indicate that neutrophil-derived MMA is associated with IL-6 pathway activation, NETosis, and thrombus burden post-MI in mouse models, thus extending previous understanding of metabolic-inflammatory crosstalk in cardiovascular disease.

Recent work has further underscored the importance of myeloid-derived IL-6 in promoting oxidative stress and vascular dysfunction. Conditional IL-6 overexpression in myeloid cells was shown to augment vascular ROS production and impair endothelial function [56], emphasizing the feed-forward nature of IL-6-driven redox signaling. Moreover, myeloid coagulation signaling, particularly through the MAPK pathway, has been identified as a key contributor to post-MI inflammation and fibrotic remodeling [57, 58]. These findings support the broader view that neutrophil-derived IL-6 acts in concert with other myeloid cytokines and coagulation pathways to amplify oxidative and thrombo-inflammatory injury within the infarcted heart. Given the substantial cost of ziltivekimab treatment and undetermined mortality benefits in its clinical application, we conducted further evaluation on the value of low-dose colchicine, which has been demonstrated to reduce cardiovascular events in both primary and secondary cardiovascular prevention settings [59, 60]. Notably, the precise molecular mechanisms underlying these benefits remain incompletely understood. Previous research primarily attributed colchicine’s effects to the inhibition of microtubule polymerization, suppression of NLRP3 inflammasome formation, and the resulting reduction in IL-1β secretion [61]. These processes are fundamentally linked to neutrophilic inflammation and pathological remodeling. Here, we found that colchicine treatment attenuated NETosis through modulation of IL-6/JAK1/STAT3 signaling, further highlighting its capability to counteract MMA-induced cardiac harm. NETs are known to promote platelet aggregation and thrombotic processes through thrombin generation and activation of proinflammatory transcriptional programs [62–64]. However, therapeutic interventions directly targeting thrombogenesis, such as antiplatelet agents including aspirin, ticagrelor, clopidogrel, and tirofiban, potentially incur hemorrhagic complications. Aspirin, as the cornerstone of antiplatelet therapy, has been reported to have insignificant benefits in primary prevention, mainly due to the potential risk of bleeding [65, 66]. Our findings showed that colchicine administration provided cardioprotection and NET suppression, especially in mice with neutrophil MMA metabolic defect, expanding the novel mechanistic relevance of neutrophil metabolic modulation in colchicine’s therapeutic benefit.

Our study has several limitations. First, while the use of Mmut CKO mice allowed precise modeling of neutrophil-specific MMA accumulation, this approach does not recapitulate common clinical comorbidities prevalent in acute myocardial infarction (AMI) patients, such as diabetes and renal dysfunction. These comorbidities could significantly modulate NETosis through alternative pathways, including HMGB1/TLR4 signaling, thus potentially influencing our observations. Second, although we specifically investigated the IL-6/JAK1/STAT3 signaling axis, additional compensatory or parallel mechanisms—such as MMA-induced reactive oxygen species (ROS) production, histone-mediated inflammation, or platelet-neutrophil interactions—remain unexplored in this study. Although these limitations currently remain unresolved, future studies will actively address these knowledge gaps to provide a more comprehensive understanding of neutrophil-driven MMA metabolism post-MI.

Conclusions

Collectively, our findings reveal the novel concept that neutrophil-intrinsic MMA accumulation exacerbates post-MI cardiac injury through IL-6-mediated NET formation and subsequent microvascular thrombosis. Neutrophil MMA represents a novel immunometabolic trigger driving NET-mediated adverse cardiac remodeling. The FDA-approved anti-inflammatory agent colchicine may be a promising therapeutic strategy to prevent heart failure post-MI, particularly in patients with elevated MMA content in neutrophils. These findings provide new insights that the therapeutic potential of neutrophil metabolic dysregulation and the subsequent inflammatory cascade represent a promising and readily translatable approach for improving clinical outcomes in patients following MI.

Abbreviations

AMI

Acute myocardial infarction

CV

Coefficient of variation

DEGs

Differentially expressed genes

DM

Diabetes mellitus

EF

Ejection fraction

FS

Fractional shortening

GMP

Granulocyte–monocyte progenitors

LAD

Left anterior descending branch

LC-MS

Liquid chromatography-mass spectrometry

LVIDd

Left ventricular end-diastolic diameter

LVIDs

Left ventricular end-systolic diameter

LVFW

Left ventricular free wall

MMA

Methylmalonic acid

MI

Myocardial infarction

Mmut

Methylmalonyl-CoA mutase

NETs

Neutrophil extracellular traps

PCI

Percutaneous coronary intervention

PFA

Paraformaldehyde

PMA

Phorbol 12-myristate 13-acetate

PPP

Platelet-poor plasma

PRP

Platelet-rich plasma

ROS

Reactive oxygen species

SD

Standard deviation

TEM

Transmission electron microscopy

Authors’ contributions

SF and BY conceived and supervised the overall project. YL, JW, and PW performed the experiments and interpreted the data. YL and QS analyzed the data and drafted the manuscript. HC provided guidance on data analysis and graphing. ZW and MW assisted in conducting animal experiments. RL and XL participated in vitro experiments. WW and XW provided clinical materials. ZZ and LX performed the bioinformatic analysis. YL and JW wrote the manuscript. JG and GM edited the manuscript. All authors edited and approved the final version. SF, SW, ZL, and XG supervised and took responsibility for the integrity and accuracy of the study. All authors read and approved the final manuscript.

Funding

Dr. Fang was supported by the National Natural Science Foundation of China (82170262 and 82370265). Dr. Yu was supported by the National Natural Science Foundation of China (62135002). Dr. Wang was funded by the National Natural Science Foundation of China (82200396). Dr. Li was funded by the National Natural Science Foundation of China (82500407). Dr. Cai was funded by the Heilongjiang Provincial Natural Science Foundation of China (QC2025H010). Dr. Liu was funded by the Open Research Project of the Key Laboratory of Myocardial Ischemia, Ministry of Education (No. KF202009).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All studies involving human participants were approved by the Ethics Committee of Harbin Medical University (YJSKY2023-503) and conducted in accordance with the principles of the Helsinki Declaration. Written informed consent was obtained from all subjects prior to sample collection. Animal experiments were approved by the Animal Care and Use Committee of Renmin Hospital of Harbin Medical University (YJSDW2023-245) and performed in compliance with the US NIH Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 1996).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.