Methods

Mitochondrial isolation

Mitochondria were isolated from skeletal muscle tissues of SD rats using the Tissue Mitochondria Isolation Kit (Cat. No. C3606, Beyotime). Fresh muscle tissues were rinsed with ice-cold PBS to remove contaminants and minced into small pieces. The minced tissues were digested with pre-cooled trypsin and incubated on ice for 20 min. After brief centrifugation to collect tissue pellets, samples were homogenized on ice in pre-chilled Mitochondrial Isolation Reagent B (800 μL per 100 mg tissue) with 20 gentle strokes using a Dounce homogenizer. The homogenate was centrifuged at 1000 g for 5 min at 4 °C, and the resulting supernatant was further centrifuged at 3500 g for 10 min at 4 °C. The final sediment was collected as purified mitochondria and resuspended in mitochondrial respiratory substrate solution containing 10 mM glutamate, 5 mM malate, and 10 mM ADP.

MQgel@Mito preparation

MQgel was prepared by dissolving MQ peptide in double-distilled water (ddH2O) to achieve final concentrations of 20, 30, and 40 mg mL−1, followed by the addition of an appropriate volume of saturated NaCl solution and immediate shaking at room temperature. For fabrication of the MQgel@Mito composite system, the isolated mitochondrial sediment was first resuspended in a moderate volume of respiratory substrate solution and then thoroughly mixed with pre-formed MQgel.

Isolation and cultivation of BMDMs

BMDMs were isolated from 6-week-old female C57BL/6J mice. Briefly, femurs and tibiae were aseptically dissected, and bone marrow was flushed with ice-cold PBS using a sterile syringe. The resulting cell suspension was filtered through a 70-μm cell strainer to remove bone fragments and tissue debris. Cells were pelleted by centrifugation at 800_g_ for 8 min at 4 °C and resuspended in erythrocyte lysis buffer (Cat. No. C3702, Beyotime). Following 2-min lysis, the reaction was terminated with ice-cold PBS. Cells were then centrifuged at 450 g for 8 min at 4 °C and resuspended in RPMI-1640 medium (Cat. No. C11875500BT, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, inactivated at 56 °C for 30 min; Cat. No. 164210, Procell), 1% penicillin-streptomycin solution (Cat. No. P1400, Solarbio), and 20 ng mL−1 recombinant macrophage colony-stimulating factor (M-CSF, Cat. No. HY-P7085, MCE). Cells were seeded in culture dishes and incubated at 37 °C in a humidified atmosphere containing 5% CO2. On day 3, half of the culture medium was replaced with an equal volume of fresh complete medium containing M-CSF. After 6 days of differentiation, adherent mature macrophages were harvested for subsequent experiments.

Internalization of mitochondria by BMDMs

To investigate the effect of different concentrations of MQ peptide on mitochondrial uptake by macrophages, BMDMs were isolated and cultured in 6-well plates at a density of 1 × 106 cells per well until day 6. The cells were divided into three groups and treated with MQ peptide at concentrations of 0, 10, and 25 μM, respectively. After 24 h of treatment, MTR (Cat. No. C1032, Beyotime)-labeled isolated mitochondria were co-incubated with BMDMs at a dose of 1 × 106 mitochondrial particles per well. At 2, 4, 8, and 12 h post-incubation, BMDMs were harvested and washed three times with PBS. The MFI (FL4-A) of BMDMs was quantified by flow cytometry using a flow cytometer (C6 Plus, BD Biosciences, San Jose, CA, USA) to evaluate the number of internalized mitochondria. To visually characterize the cell uptake of mitochondria, the BMDMs were stained by MTG (Cat. No. C1048, Beyotime) to label the endogenous mitochondria and then incubated with MTR-labeled exogenous mitochondria (1 × 106 particles per confocal dish) for 2 and 12 h. The fluorescent images were acquired using a laser scanning confocal microscope (TCS SP5II, Leica, Germany).

To investigate the effect and underlying mechanism of MQ on mitochondrial uptake by macrophages, BMDMs were divided into three groups: mitochondria (Mito), 25 μM MQ with mitochondria (MQ@Mito), and MQ@mito+si-AMPKα1. MTR-labeled mitochondria (3 × 105 particles per well) were added for co-incubation with BMDMs. Other procedures were performed as described above.

To investigate the effect and underlying mechanism of MQ on mitochondrial uptake by M1-polarized macrophages, BMDMs were induced into M1-like macrophages by stimulation with 200 ng mL−1 LPS (Cat. No. L8880, Solarbio) for 48 h and then divided into three groups: Mito, MQ@Mito, and MQ@Mito+si-AMPKα1. Other procedures were performed as described above.

Western blot

Total proteins were extracted from purified mitochondria or BMDMs using RIPA lysis buffer (Cat. No. P0013B, Beyotime) supplemented with protease and phosphatase inhibitors (Cat. No. P1045, Beyotime). Proteins were separated by SDS-PAGE and electrotransferred onto 0.22-μm-thick PVDF membranes. Membranes were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature, and then incubated overnight at 4 °C with primary antibodies. All Western blot primary antibodies involved in the entire study are listed as follows: COXIV (Cat. No. ab202554, Abcam), β-tubulin (Cat. No. LF203S, Epizyme), AMPK (Cat. No. 2532, CST), p-AMPK (Cat. No. 2535, CST), β-actin (Cat. No. 20536-1-AP, Proteintech). After six TBST washes, membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse IgG (H + L) secondary antibodies (Cat. No. AS014, Abclonal; Cat. No. 7076, CST) for 1 h at room temperature. Following six TBST washes, protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Cat. No. P10100, NCM Biotech). Band intensities were densitometrically analyzed using ImageJ software with β-actin or β-tubulin as internal references.

Flow cytometric analysis of macrophage polarization

BMDMs were polarized into M1 macrophages via stimulation with 200 ng mL−1 LPS for 48 h. To evaluate the effects of MQ@Mito on macrophage polarization, MQ peptide, isolated mitochondria, or MQ@Mito were administered to BMDMs at three different time points: 24 h prior to LPS exposure, simultaneously with LPS stimulation, or 24 h after LPS treatment. Subsequently, cells were harvested and co-stained with PE anti-mouse F4/80 antibody (Cat. No. 123110, BioLegend) and FITC anti-mouse CD86 antibody (Cat. No. 105005, BioLegend) at room temperature for 40 min in the dark. After three washes with PBS, BMDMs were resuspended in 4% paraformaldehyde (PFA, Cat. No. P1110, Solarbio) fixative and analyzed by flow cytometry. The proportion of M1 macrophages was quantified as the percentage of F4/80+CD86+ BMDMs using appropriate gating strategies with FlowJo software v10.8.1.

For M2 polarization induction, BMDMs were treated with MQ peptide, isolated mitochondria, or MQ@Mito for 48 h, with 40 ng/mL IL-4 treatment serving as the positive control. After treatment, BMDMs were harvested and stained with F4/80 antibody. Subsequently, the cells were fixed and permeabilized with 0.1% Triton X-100, followed by incubation with APC anti-mouse CD206 antibody (Cat. No. 141708, BioLegend) for 40 min in the dark. All other experimental procedures were consistent with those used for M1 polarization. The proportion of M2 macrophages was quantified as the percentage of F4/80+CD206+ BMDMs.

Metabolic profiling analysis

For targeted energy metabolomics analysis, BMDMs subjected to different treatments (control, LPS stimulation, LPS + MQgel@Mito intervention) were collected. Cells were washed twice with ice-cold PBS to remove residual medium and debris, and cellular metabolism was rapidly quenched by pre-chilled extraction solvent before cell lysis to preserve endogenous metabolites. Targeted energy metabolomics profiling was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) by Metware Biotechnology Co., Ltd. (Wuhan, China). Three independent biological replicates were included for each group, with each sample containing **>**1 × 106 BMDMs to ensure sufficient metabolite abundance and detection reliability. Raw data were processed for peak identification, metabolite annotation, and quantitative normalization. Metabolic profiling, multivariate statistical analysis, and data visualization were performed using online analytical platforms: Metware Cloud Platform (https://cloud.metware.cn/) and CNSknowall (https://www.cnsknowall.com/).

Animal models

All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Peking Union Medical College and approved by Institutional Animal Care and Use Committee (IACUC), Fuwai Hospital, Chinese Academy of Medical Sciences (IACUC Issue No.: 0109-7-1359-ZX(X)-30).

Male Sprague Dawley rats (Vital River Laboratory, Beijing, China) weighing 200 ± 20 g at 6 weeks of age were used to establish MI models. Anesthesia was induced via intramuscular injection of a mixed anesthetic agent containing Zoletil 50, xylazine hydrochloride injection, and normal saline at a volume ratio of 1:1:8, at a dose of 0.11 mL per 100 g body weight. Subsequently, endotracheal intubation was performed with a 16-G trocar, and mechanical ventilation was maintained with a ventilator. Thoracotomies were performed at the fourth intercostal space to expose the heart. Rats were randomly assigned to five groups: Sham, Saline, MQgel, Mito, and MQgel@Mito groups. The LAD coronary artery was ligated with a 6-0 nylon suture. The pallor of the cardiac apex indicated the successful establishment of MI model. Following 30 min of ischemia, 100 μL saline, MQgel (30 mg mL−1), Mito (mitochondrial suspension), or MQgel@Mito was injected into the infarct border zone in corresponding groups. The sham group underwent identical surgical procedures without LAD ligation or injection. Then, the thoracic cavity was carefully closed with 3-0 nylon sutures, followed by skin closure using the same suture material. Postoperatively, rats were placed in a temperature-controlled incubator for rewarming, and respiratory rhythm was monitored until full recovery from anesthesia. All rats were then housed in standard breeding cages. Intramuscular penicillin injection was administered for three consecutive days for postoperative anti-infection prophylaxis.

Echocardiography

Rats were anesthetized with the aforementioned combined anesthetic agent, and standard transthoracic echocardiography was performed using an ultrasound imaging system on day 7 and 28 post-treatment. Cardiac functional parameters including LVEF, LVFS, LVIDD, LVIDS, LVEDV, and LVESV were acquired and analyzed.

Immunofluorescence analysis

Rats were euthanized, and cardiac tissues were harvested, fixed in 4% PFA, paraffin-embedded, and sliced into 4-μm-thick sections. After baking at 60 °C for 1 h, dewaxing, rehydration, and antigen retrieval using citrate antigen retrieval powder (Cat No. E673002, Sangon Biotech), sections were permeabilized with 0.1% Triton X-100 (Cat. No. P0096, Beyotime) and blocked with goat serum. Subsequently, the sections were incubated overnight at 4 °C in the dark with primary antibodies. All immunofluorescent primary antibodies involved in the entire study are listed as follows: CD68 (Cat. No. ab283654, Abcam), CD86 (Cat. No. ab220188, Abcam), cTnT (Cat. No. ab209813, Abcam). Following three washes with PBS, sections were incubated for 2 h at room temperature in the dark with HRP-conjugated goat anti-rabbit or anti-mouse IgG (H + L) secondary antibodies (Cat. No. A11008, Invitrogen; Cat. No. ab175473, Abcam). After another three PBS washes, sections were mounted with DAPI-containing antifade mounting medium (Cat. No. ab104139, Abcam). Immunofluorescent images were acquired using a laser scanning confocal microscope.

Bulk RNA-seq

Cardiac tissues from rats in the Saline group and the MQgel@Mito group were collected for bulk RNA-seq. Library construction and transcriptome sequencing were performed by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Briefly, total RNA was extracted and purified from tissue samples, followed by quality control to assess RNA integrity and purity. Qualified RNA samples were used for cDNA library preparation, and high-throughput sequencing was conducted on an Illumina platform. Raw sequencing reads were filtered, aligned to the rat reference genome, and quantified for gene expression levels. Transcriptomic data analysis, including differential expression analysis, functional enrichment analysis, and data visualization, was performed using Majorbio Cloud Platform (https://www.majorbio.com/tools); and Chiplot online visualization tool (https://www.chiplot.online/).

Statistical analysis

All statistical analyses were performed using GraphPad Prism 9.5 software. Data are presented as mean ± SD. An unpaired Student's t-test was used to compare differences between two independent groups. One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test was utilized to compare each experimental group versus the control group, with statistical significance evaluated for each individual comparison. One-way ANOVA with Tukey's multiple comparisons test was employed for multiple comparisons among all groups. Statistical significance was defined as follows: ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001.