Data processing

We downloaded one heart failure (HF) RNA-seq dataset (GSE76701) and one HF single-cell RNA-seq dataset (GSE161740) from the Gene Expression Omnibus (GEO) database. After standardization, the samples without grouping information were excluded, and ultimately, 5 samples were obtained from the GSE161740 dataset, and 4 paired tissue samples were obtained from the GSE76701 dataset.

Single-cell quality control and dimension reduction clustering

We selected cells with more than 300 expressed genes, fewer than 5500 expressed genes in total. Among these genes, with fewer than 10% of the candidates were mitochondrial genes, and less than 3% were red blood cell genes. A total of 15,749 cells were retained for analysis. Then, 3000 hypervariable genes were selected for analysis, and the number of principal components (PCs) was set to 15 for cell clustering. These clusters were displayed in the form of a t-distributed stochastic neighbor embedding (t-SNE) diagram. First, we identified the 10 markers that were the most significantly differently expressed (Supplementary file 1) in each cluster using the “FindAllmarkers” function. After identifying a group of macrophages, we isolated the macrophages and ultimately obtained 11 macrophage subclusters. The identified of the ten markers most closely associated with a macrophage subtypes were confirmed (Supplementary file 2).

Human samples

The Clinical Trials Ethics Committee of the Huazhong University of Science and Technology approved a clinical trial. All the samples were recruited from the Union Hospital affiliated with Tongji Medical College of Huazhong University of Science and Technology. Fibrotic left ventricular myocardium (FLVM) samples were obtained from end-stage HF patients at the time of heart transplantation. Control normal left ventricular myocardium (NLVM) samples were obtained from the left ventricular myocardium of nonfailing unused donor hearts. Written informed consent was obtained from all patients.

Animals

Male wild-type C57BL/6J mice and 8-week-old nude mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). For the study, all mice were maintained under specific-pathogen-free (SPF) conditions at the Animal Care and Use Committee of Tongji Medical College. Euthanasia of the animals was performed in a carbon dioxide chamber followed by cervical dislocation.

MI induction and study design

Male C57BL/6J wild-type (WT) littermate mice (8 weeks) were used to establish models of MI, which was induced as described previously (Gao et al. 2010). Briefly, mice were exposed to a 5% isoflurane inhaler at an oxygen flow rate of 1 L/min. The mice were then continuously monitored throughout the procedure, and complete anesthesia was confirmed by the lack of pedal reflexes. After the left chest was incised, the pectoralis major and pectoralis minor were directly incised. The fourth intercostal space was gently opened with a clamp, and the heart was gently squeezed. The left anterior descending coronary artery was ligated with 6–0 silk thread. Immediately after the ligation, the heart was placed back into the proper position, the chest was closed, and air was expelled. NaW was administered at a concentration of 100 mg kg−1 from days 11 to 38 after MI. To determine cardiac function, echocardiograms were performed on days 1 and 38 after MI induction using a Vevo 2100 high-resolution microimaging system. The scarred area was evaluated using Masson’s trichrome method and calculated as the ratio of fibrotic tissue area to the total left ventricular tissue area.

Immunofluorescence staining

Briefly, after being dried at room temperature for 15 min, frozen sections of aortic valves were fixed in 4% paraformaldehyde (PFA) for 20 min and then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS) for another 20 min. Next, the tissues were incubated with primary antibody, followed by incubation with fluorescently conjugated secondary antibody (Abcam) and counterstaining with 4′,6‐diamidino‐2‐phenylindole (DAPI).

Cell isolation and stimulation

Macrophages were isolated from the infarcted LV tissues on day 38 post-MI, and cardiac fibroblasts were isolated from control (noninfarcted) LV tissues. Macrophages and cardiac fibroblasts were isolated as previously described (Jung et al. 2017).

Reagents and antibodies

CHIR99021 (S1263), SB15286 (S2729), NaW (10213-10-2), GPI (S0031), NH4Cl (A9434), 2-NBDG (72987), and LysoSensor Green DND-189 (L7535) were purchased from Thermo Fisher Scientific. The following primary antibodies were purchased from Cell Signaling Technology: anti-GSK3β (#12456, 1:1000), anti-p-GSK-3β (Ser9) (#5558, 1:1000), anti-Glycogen Synthase (#3886, 1:1000), anti-p-Glycogen Synthase (Ser641) (#47043, 1:1000), anti-iNOS (#13120, 1:1000), anti-arginase1 (#93668, 1:1000), anti-phospho-STAT1-Tyr701 (#9167, 1:1000), anti-STAT1 (#9176, 1:1000), and anti-β-actin (#3700, 1:1000). The following primary antibodies were purchased from BioLegend: anti-human CD68 (333810, 1:100), anti-human CD206 (321104, 1:100), anti-mouse F4/80 (123108, 1:100), and anti-mouse CD206 (141701, 1:100). The following primary antibodies were purchased from Abcam: anti-Ugp2 (ab154817, 1:1000), anti-Pygl (ab190243, 1:1000), anti-TFEB (ab264421, 1:1000), and anti-Col I (ab270993). The primary TMEM175 (19925-1-AP) antibody was purchased from Proteintech. The primary anti-α-SMA (af1032) antibody was purchased from Affinity. The primary anti-Col III (A0817) antibody was purchased from ABclonal.

Preparation of mouse macrophages

Bone marrow cells isolated from C57BL/6J mice were cultured for 5 days in complete RPMI-1640 medium containing 20 ng mL−1 mouse recombinant macrophage colony-stimulating factor (M-CSF; 315-02, PeproTech). On day 6, bone marrow-derived macrophages (BMDMs) were stimulated with 10 ng mL−1 IL-4 (214-14, PeproTech) or 100 ng mL−1 LPS plus 20 ng mL−1 IFN-γ (315-05, PeproTech) for 24 h to generate anti-inflammatory (M2) or inflammatory (M1) macrophages. Mouse peritoneal macrophages were harvested via peritoneal lavage. Briefly, cold PBS was injected into the peritoneal cavity and extracted after gentle agitation. The peritoneal cell suspension was centrifuged at 1300 rpm, and the cell pellet was mixed with 2 mL of red blood cell lysis buffer for 5 min at room temperature. After washing, the cells were cultured in six-well plates for 3 h. Cells that adhered to the plates were peritoneal macrophages and collected.

Real-time PCR

Total RNA extraction was prepared with TRIzol reagent (15596026, Invitrogen), and cDNA was generated with a ReverTra Ace qPCR RT Kit (FSQ-101, Toyobo). Real-time PCR was performed for all genes with primers on a Bio–Rad CFX Connect instrument, and data were captured using Bio–Rad CFX Manager 2.0 software. The expression of mRNA for genes of interest was normalized to the level of Actb (Mus) expression. The entire procedure was repeated with at least three biologically independent samples. The primer sequences are shown in Supplementary Table 1.

Glycogen level assay

The level of glycogen was measured with a glycogen assay kit (KA0861, Abnova) according to the manufacturer’s instructions.

Western blot analysis and ELISAs

Cell lysates and prestained molecular weight markers were separated via SDS‒PAGE, and then, the proteins were transferred to nitrocellulose membranes. The membranes were blocked in Tris-buffered saline with 0.5% Tween 20 (TBST) with 5% bull serum albumin (BSA) and probed with specific antibodies overnight at 4 °C. The membranes were washed three times and incubated with horseradish peroxidase-conjugated secondary antibodies. Immunoreactivity was visualized via enhanced chemiluminescence (ECL) according to a kit manufacturer’s protocol (ECL Kit, 34577, Pierce). Mouse IL-10 (431425, BioLegend), TGFβ (433007, BioLegend), TNF-α (430904, BioLegend) and IL-6 (431307, BioLegend) levels in supernatants were quantified with ELISA kits according to the manufacturer’s protocol.

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis

LC–MS/MS analysis was performed on a Q-Exactive mass spectrometer (Thermo Fisher) equipped with a heated electrospray ionization (HESI) probe with the relevant parameters set as follows: heater temperature, 120 °C; sheath gas, 30; auxiliary gas, 10; sweep gas, 3; spray voltage, 2.5 kV in negative mode. A full scan range from 80 to 350 (mz−1) was used. The resolution was set to 70,000. The data were captured using Xcalibur™ software, version 3.0 (Thermo Fisher), and quantified by integrating the area underneath the curve of each compound using the Xcalibur Qual browser (Thermo Fisher). Accurate mass ion and subsequent isotopic ion data for each metabolite were extracted using a extracted ion chromatogram (EIC) window of 10 ppm.

Intracellular Ca 2+measurement

Cells were cultured in 24-well plates at a density of 5 × 104 cells/well in RPMI 1640 medium overnight. Briefly, macrophages were stained with 5 μM Fluo-4 AM, and 200 nM ionomycin was used to promote calcium release, as previously described (Wei et al. 2023). After complete cell lysis, the supernatant was centrifuged at 14,000×g for 5 min and quantified using a calcium colorimetric kit (S1063S, Beyotime) according to the manufacturer’s protocol.

RNA sequencing

Two groups of macrophage samples (a Ctrl group and an NaW group, 3 samples each group) were sent to Berry Genomics (Beijing, China) for RNA-seq and bioinformatics analyses.

Lysosomal pH value assay

LysoSensor Green is commonly used to qualitatively measure the pH of acidic organelles. The green fluorescence emitted by the probe becomes more intense in increasingly acidic environments and less intense in increasingly alkaline environments. The cells (3 × 106 cells per mL) were loaded with 1 μM LysoSensor Green in prewarmed 1640 medium for 30 min at 37 °C, washed twice with PBS and immediately analyzed via fluorescence microscopy. Quantification of lysosomal pH was performed using the ratiometric lysosomal pH dye LysoSensor Yellow/Blue. A pH calibration curve was generated according to the manufacturer's protocol. Cells were trypsinized and labeled with 10 μM LysoSensor Yellow/Blue for 5 min at 37 °C in 1640 medium and washed with PBS. The labeled cells were treated with 10 μM monensin and 10 μM nigericin for 10 min in 25 mM MES calibration buffer (pH 4.5–7.5) containing 5 mM NaCl, 115 mM KCl and 1.2 mM MgSO4. Quantitative comparisons were performed in a 96-well plate, and the fluorescence was measured with a microplate reader (Synergy H1, BioTek) at Ex-360/Em-440 and Ex-360/Em-550.

Reactive oxygen species (ROS) level measurements

ROS levels were measured using CellROX Green flow cytometry assay kits (C10444, Invitrogen). Cells were trypsinized and then loaded with 500 nM CellROX Green for 30 min at 37 °C in the dark. The cells were washed with PBS, scraped, maintained in PBS and immediately analyzed using flow cytometry at 488-nm excitation to induce CellROX Green fluorescence.

Gene silencing experiments

Short interfering RNAs (siRNAs) targeting mouse Pygl, Tmem175, and Tfeb and negative control siRNAs (NC) were purchased from RiboBio (Guangzhou, China). siRNA (50 nM) was transfected into macrophages using Lipofectamine™ RNAiMAX Transfection Reagent according to the manufacturer’s instructions. The siRNA sequences are shown in Supplementary Table 2.

Intracellular Ca 2+measurement

Cells were cultured in 24-well plates at a density of 5 × 104 cells/well in RPMI 1640 medium overnight. Before Ca2+ measurements were taken, cells were washed with PBS 3 times and incubated for 60 min in Hanks’ balanced salt solution containing 5 μM Fluo-4 AM in the dark at room temperature. The cells were then washed with Hanks’ balanced salt solution three times and incubated at room temperature for another 10 min. Then, 200 nM ionomycin was added extracellularly and incubated with the cells for 30 s, and cytosolic calcium release was recorded via low speed by flow cytometry. For an intracellular calcium concentration assay, the cells were washed with PBS and then treated with 100 μL of sample lysate. After cell lysis was completed, the samples were centrifuged for 5 min at 14,000×g, and the supernatants were quantified with a calcium colorimetric assay kit (S1063S, Beyotime) according to the manufacturer’s protocol.

Wound healing assay

In brief, the wound healing technique involved creating a thin linear scratch “wound” (creating a gap) in a confluent monolayer of cells. Periodically, images of the cells were taken, and the reduction in the gap width was measured as described previously (Grada et al. 2017).

Statistical analysis

All experiments were performed at least three times. The results are expressed as the mean ± SEM and were analyzed by two-tailed unpaired Student’s t test or one-way ANOVA. In all tests, p values of less than 0.05 were considered statistically significant. The analysis was conducted using GraphPad Prism 8.0 software.

Data availability

The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information files or from a corresponding author on reasonable request. The raw data used to generate the figures and supplementary figures are presented in a Source Data file.