Cell lines and animal models
Rat cardiomyocytes (H9c2), human umbilical vein endothelial cells (HUVECs), and human mesenchymal stem cells (hMSCs) were obtained from the American Type Culture Collection (ATCC). All cell lines were cultured and maintained following ATCC’s standard protocols. Male Sprague-Dawley (SD) rats (6–8 weeks old, weighing 200–250 g) and male C57BL/6 mice (6–8 weeks old, weighing 18–22 g) were purchased from the Southern Medical University Experimental Animal Center. Animals were housed in a specific pathogen-free (SPF) environment under controlled conditions of 22 ± 2 °C, 50–60% relative humidity, and a 12 h light/dark cycle, with free access to food and water.
Pathological models of cells and animals and treatment
An I/R model was developed in H9c2 cardiomyocytes (ATCC, CRL-1446). The culture medium was removed, and cells were washed twice with glucose-free DMEM (Gibco, C11995500BT). Glucose-free, serum-free DMEM was then added, and the cells were incubated in a hypoxic chamber at 37 °C under 5% CO₂ and 95% N₂ for 6 h to induce hypoxia. After completing the hypoxia and glucose deprivation phase, the medium was replaced with high-glucose DMEM supplemented with 10% FBS. Exo-WT and Exo-I-S were added at a dosage of 3,000 particles per H9c2 cell (with an exosome protein concentration of 20 µg/mL), along with PBS as a control. The cells were subsequently cultured under standard conditions at 37 °C with 5% CO₂ for 12 h.
For the in vivo experiment was approved by Nan-fang hospital, southern medical university (IACUC-LAC-20240822-005), animal care and treatment were conducted following institutional procedures and national laws and regulations, male SD rats were anesthetized with isoflurane, and a thoracotomy was performed at the fourth intercostal space to expose the heart. A 6 − 0 polypropylene suture was placed 2–3 mm below the origin of the left anterior descending artery, between the left atrial appendage and the arterial cone, to induce myocardial ischemia/reperfusion injury. After 1 h of ischemia, the suture was removed to allow reperfusion. Intramyocardial injections of Exo-WT and Exo-I-S solutions (dose: 1 × 10¹¹ particles/kg body weight; exosome protein concentration: 20 µg/mL) or an equivalent volume of PBS were administered into the infarct area (identified by pale discoloration after ligation).
Construction of MSCIns cells
A dual-gene expression plasmid, pIRES-SIRT3/GPI + Insulin, was constructed using molecular cloning techniques. Human SIRT3 and insulin genes were amplified by PCR, with a GPI-anchoring sequence added to the 3’ end of the insulin gene. The amplified products were then cloned into MCS A and MCS B sites of the pIRES bicistronic expression vector. After successful sequencing verification, plasmid DNA was extracted and transfected into MSCs using Lipofectamine™ 2000 (ThermoFisher, 11668019) to generate SIRT2/GPI + Insulin expressed MSC (MSCIns). Transfected cells were harvested 12 and 24 h post-transfection for immunofluorescence and Western blot validation.
Preparation of Exo-I-S and Exo-WT
Both transfected and non-transfected MSCs (ATCC, PCS-500-012) were initially cultured for 48 h. The medium was then replaced with serum-free DMEM for an additional 24 h. The culture supernatant was collected and sequentially centrifuged for exosome isolation: 300 × g for 10 min, 2,000 × g for 10 min, and 100,000 × g for 70 min using an Optima MAX-XP ultracentrifuge (Beckman, USA). The supernatant was discarded, and the pellet was washed with PBS, followed by a final ultracentrifugation step at 100,000 × g for 70 min. Purified exosomes were stored at − 80 °C until analysis.
Characterization of Exo-I-S and Exo-WT
Exosome particle size analysis
Nanoparticle tracking analysis (NTA) was performed using the ZetaView PMX 110 instrument (Particle Metrix) and its accompanying software (ZetaView 8.02.28). Exosomes were diluted appropriately in particle-free PBS before being loaded into the sample chamber. The size distribution and concentration of exosomes were determined at a wavelength of 405 nm. Particle size data were quantified and recorded.
Protein quantification
Protein concentration was determined using a BCA Protein Assay Kit (Beyotime, P0012), following the manufacturer’s protocol. Samples and standard proteins (BSA) were added to a 96-well plate, mixed with BCA working reagent, and incubated at 37 °C for 30 min. Absorbance was measured at 562 nm in microplate reader, and protein concentration was calculated based on a standard curve (0–2,000 µg/mL).
Transmission electron microscopy
Exosome samples were dropped onto carbon-coated copper grids, air-dried at room temperature, and negatively stained with 2% phosphotungstic acid for 5 min. Excess stain was removed with filter paper, and the grids were examined under a transmission electron microscope (TEM) (JEM-1230, JEOL) to assess the morphology and size of the exosomes.
Western blot
Total protein was extracted from cells and exosomes and separated on 10–15% SDS-polyacrylamide gels, based on the molecular weight of the target proteins. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, IPVH00010) and blocked with 5% skim milk for 1 h. Membranes were incubated overnight at 4 °C with primary antibodies against: SIRT3 (1:1500, ABclonal, A5419), Insulin (1:2000, HUABIO, EM80714), CD63 (1:1500, Abcam, ab217345), CD9 (1:1800, Abcam, ab2215), ALIX (1:1500, Abcam, ab186429), Calnexin (1:1500, Abcam, ab22595), GAPDH (1:20000, Abcam, ab181602), AKT (1:1000, Abcam, ab179463), p-AKT (1:1000, Abcam, ab38449), PI3K (1:1000, Abcam, ab86714), p-PI3K (1:800, Abcam, ab182651), and Glut4 (1:1500, ABclonal, A25174). Secondary antibodies such as goat anti-mouse IgG (1:3000, Abcam, ab205719) and goat anti-rabbit IgG (1:3000, Abcam, ab205718) were applied. Protein bands were visualized using enhanced chemiluminescence (ECL) reagents (Beyotime, P0018S) and imaged using a ChemiDoc MP system (Bio-Rad).
Exosome uptake assay
The uptake of exosomes by H9c2 cardiomyocytes and HUVEC endothelial cells was monitored using PKH26-labeled (200 µg/mL, Sigma, MINI26) control Exo (Exo-WT) and Exo-I-S. Labeled exosomes were added to the H9c2 culture medium, and cells were incubated for 3, 6, 12, 24, and 36 h. After incubation, cells were washed with DPBS and fixed with 4% paraformaldehyde (PFA). Cell nuclei were stained with 4’,6-Diamidino-2-phenylindole (DAPI, Invitrogen) for visualization. Fluorescence imaging was performed using a confocal laser scanning microscope (LSM 880, Zeiss). Images of three independent samples were captured for each group, and ImageJ was applied to quantify the relative fluorescence intensity using the automatic threshold method to select the fluorescent areas.
GPI-Insulin molecular modeling and Docking prediction
The three-dimensional structure of the GPI-Insulin fusion protein was predicted using AlphaFold 3.0. By inputting the amino acid sequence, AlphaFold 3.0 generated a 3D structural model and provided the predicted Template Modeling Score (pTM). The predicted structure was then imported into AutoDock Vina for molecular docking analysis with the insulin receptor (IR). The structure of the insulin receptor was obtained from the Protein Data Bank (PDB). Prior to docking, preprocessing was performed using AutoDock Tools, which included removing water molecules, adding polar hydrogen atoms, calculating Gasteiger charges, and saving the files in PDBQT format. During the docking process, a grid box was set to cover the receptor’s active site, and the Lamarckian genetic algorithm was employed to complete the docking. The binding mode and binding energy of the GPI-Insulin fusion protein with the insulin receptor were subsequently determined.
Biocompatibility evaluation
Tube formation assay
HUVECs (ATCC, CRL-1730) were seeded into 96-well plates pre-coated with 10 mg/mL Matrigel matrix (Corning, 354234) to simulate an in vitro angiogenesis environment. After seeding at an appropriate density, culture medium containing exosomes (e.g., Exo-I-S, Exo-WT, or control) was added. After 6–12 h of incubation, the formation of tubular structures was observed and imaged using an inverted microscope (Leica, DMi8). Tube length and the number of branch points were analyzed using ImageJ software to evaluate the effects of different treatments on angiogenesis.
Live/Dead cell assay
H9c2 cells at appropriate confluency were treated with culture medium containing Exo-I-S, Exo-WT, or PBS for 24 h. After removing the medium and washing the cells with PBS, a dye working solution was prepared according to the manufacturer’s instructions: Calcein-AM (2 µM, Invitrogen, C3099) and PI (5 µg/mL, Invitrogen, P1304MP). Cells were incubated at 37 °C for 15–30 min, and fluorescence images were captured using a fluorescence microscope.
Organ toxicity assessment
Sterile Exo-I-S, Exo-WT, or PBS solutions were prepared and administered to C57BL/6 mice via tail vein injection at a volume of 10 mL/kg body weight. The control group received an equivalent volume of PBS. Seven days after administration, mice were sacrificed under excessive anesthesia, and major organs were rapidly harvested and fixed in 4% paraformaldehyde. Hematoxylin and eosin (H&E) staining was performed to assess histopathological changes.
Immunofluorescence stain
Cells were permeabilized with 0.1% Triton X-100 at room temperature for 10 min, followed by blocking with 1% BSA/PBST buffer for 1 h. The cells were incubated overnight at 4 °C with the primary antibody Glut4 (1:200, ABclonal, A25174), SIRT3 (1:200, ABclonal, A5419), Insulin (1:200, Abcam, ab46707). After washing with PBS, cells were incubated with Anti-rabbit Alexa Fluor 488-conjugated secondary antibody (1:1000, Cell Signaling, 4412) Anti-mouse Alexa Fluor 594-conjugated secondary antibody (1:1000, Cell Signaling, 8890) and in the dark for 2 h. Nuclei were stained with DAPI (1 µg/mL, 1:1000, Sigma-Aldrich, D9542).
Mitochondrial morphology assessment
Mitochondrial Electron microscopy
Cells were washed with serum-free medium and fixed with 4% glutaraldehyde (Sigma-Aldrich, 814393) and 4% paraformaldehyde (Sigma- Aldrich, 818715). Samples were dehydrated in a graded ethanol series and embedded in LX-812 resin (Ladd Research Industries Inc.). Ultrathin sections were stained with uranyl acetate for 30 min and lead citrate for 10 min, and observed using a FEI Tecnai G12 Spirit BioTwin transmission electron microscope (FEI Company, Hillsboro) at an acceleration voltage of 120 kV.
JC-1 staining
To evaluate mitochondrial membrane potential under different Exo treatments, JC-1 staining was performed using the MitoProbe JC-1 Assay Kit (Invitrogen, M34152). Cells were incubated with JC-1 (10 µL, 200 µM) for 30 min, and fluorescence was detected at 488 nm and 633 nm using a confocal laser scanning microscope (Zeiss LSM 880).
Mito-Tracker analysis
A 1 mM stock solution of Mito-Tracker Red was prepared in anhydrous DMSO and stored at − 20 °C in the dark. After removing the culture medium, pre-warmed Mito-Tracker Red CMXRos staining solution (Beyotime, C1999S) was added to the cells, which were incubated at 37 °C for 30 min. Following incubation, the staining solution was replaced with fresh medium, and fluorescence was observed using a confocal laser scanning microscope (Zeiss LSM 880).
Mitochondrial oxidative stress detection
ROS measurement
Reactive oxygen species (ROS) were measured using the DCFH-DA probe (ab113851). The DCFH-DA solution was diluted 1:1000 with serum-free medium and stored protected from light. Cells were washed twice, then incubated with 100 µL of the diluted DCFH-DA solution at 37 °C for 30 min. After incubation, cells from the oxidative stress model were washed three times with serum-free medium to remove excess probe not taken up by cells, followed by addition of fresh medium and further incubation for 30 min. Cells were then collected by centrifugation, resuspended in the diluted DCFH-DA solution, and analyzed by flow cytometry. A total of 10,000 cells per sample were measured for fluorescence intensity, and the average fluorescence intensity was calculated. Results were expressed as the ratio of fluorescence intensity between experimental and normal groups.
SOD1 and SOD2 measurement
SOD1 and SOD2 levels were measured using ELISA kits (CBIBO BIO, CB11409-Ra and CB10619-Ra). Cells were washed three times with PBS and centrifuged (3000 rpm, 5 min, 4 °C). After adding phosphate-buffered saline and extraction buffer (1:1000), cells were ultrasonicated (3 s bursts, 10 s intervals, 30 cycles) and centrifuged again (3000 rpm, 20 min, 4 °C). Supernatants were collected and kept on ice. Standards (50 µL) and samples (40 µL dilution buffer with 10 µL sample, 5-fold diluted) were added to wells. Except for blanks, 100 µL enzyme conjugate was added; plates were sealed and incubated at 37 °C for 60 min. Wells were washed 5 times with 20-fold diluted wash buffer, then incubated with 50 µL each of substrate A and B for 15 min at 37 °C in the dark. The reaction was stopped with 50 µL stop solution, turning color from blue to yellow. Absorbance was read at 450 nm within 15 min, and results were expressed as experimental-to-normal group ratios.
Apoptosis detection
Cell apoptosis was analyzed using Annexin V-FITC/PI dual staining. Cells were collected by trypsinization, washed twice with PBS, and resuspended in 1× Annexin V binding buffer (BD Biosciences, 556547). Annexin V-FITC (5 µL) and PI dye (5 µL) were added, and the samples were incubated in the dark for 15 min at room temperature. Fluorescence signals were detected using a flow cytometer (BD FACSCanto II).
Cell metabolic activity analysis
Cellular oxygen consumption rate (OCR) was measured using the Seahorse XFe24 extracellular flux analyzer (Agilent, Seahorse XFe24) and the Mito Stress Test Kit (Agilent, 103015-100). H9c2 cells were seeded onto culture plates (Agilent, 100882-004) and subjected to I/R treatment. On the day of measurement, cells were washed with XFe24 medium and incubated in a CO₂-free incubator at 37 °C for 2 h to equilibrate. OCR was measured before and after the sequential addition of glucose (10 mmol/L), oligomycin (1 µmol/L), FCCP (4 µmol/L), and rotenone/antimycin A (0.5 µmol/L). Basal respiration, ATP production, and maximal respiration were calculated.
Mitochondrial enzymatic detection
For the activities of glycolysis enzymes, We used the 6-phosphofructokinase (PFK-1) activity kit (Solarbio, BC0530), lactate dehydrogenase (LDH) activity kit (Solarbio, BC0685), lactate content kit (Solarbio, BC2230), pyruvate dehydrogenase (PDH) activity kit (Abcam, ab287837), and ATP content kit (Solarbio, BC0300). and RT-qPCR was conducted to assess the enzymes in the tricarboxylic acid (TCA) cycle, specifically citrate synthase (CS) and isocitrate dehydrogenase (IDH), as well as components of the oxidative phosphorylation complex, namely NADH dehydrogenase 1-β3 (NDUFB3) and succinate dehydrogenase complex subunit B (SDHB).The reaction mix included 5 µL of SsoFast Eva Green Supermix, 0.5 µL of each upstream and downstream primer (10 µM), 2 µL of cDNA template, and 2 µL of ultrapure water, totaling 10 µL. We used β-tubulin as the reference gene, and the relative expression levels of the target genes were calculated using the 2-ΔΔCT method. Primers sourced from Shanghai Shenggong were as follows: (See Table 1).
Serum detection of CK-MB, CTnT, and LDH
Blood samples were collected from the inner canthus vein 12 h after myocardial infarction modeling, and serum was separated by centrifugation at 3000 rpm for 10 min. The levels of CK-MB, CTnT, and LDH were measured using enzyme-linked immunosorbent assay (ELISA) kits. The following specific kits were used: CK-MB (Abcam, ab285275), CTnT (Abcam, ab246529), and LDH (Sigma-Aldrich, MAK066). Following the instructions of the kits, serum samples were added to the enzyme-linked plate, incubated, and then the enzyme conjugate and substrate were added for color development. After terminating the reaction, the absorbance was measured at 450 nm. The concentrations of each biomarker were calculated based on the standard curve to assess the extent of myocardial injury.
Electrocardiogram
Prior to the experiment and 12 h after myocardial infarction modeling, rats were anesthetized (with intraperitoneal injection of pentobarbital sodium at a dose of 40 mg/kg) and fixed on the experimental platform. Their limbs were exposed, and electrocardiogram electrodes were connected (one for each forelimb and hindlimb). Care was taken to ensure that the electrodes had good contact with the skin. An animal-specific electrocardiogram device (ADInstruments, ML136) was used to record.
Echocardiography
Using the Vevo high-resolution small animal ultrasound imaging system and a high-frequency transducer, cardiac ultrasound examinations were conducted through the sternum on the 14th day post-surgery. SD rats were anesthetized while maintaining spontaneous respiration, and two-dimensional images of the left ventricle were obtained in the short-axis view at the level of the left ventricular papillary muscles. Key measurements included heart rate, left ventricular systolic wall thickness (Awsth; Awdth), diastolic wall thickness (Pwsth; Pwdth), left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVESd), left ventricular end-diastolic external diameter (EXLVDd), and left ventricular short-axis fractional shortening (FS%). The end-diastolic volume (EDV) and end-systolic volume (ESV) were then calculated using built-in correction formulas.
FS was calculated using the formula: FS = [(LVEDd – LVESd) / LVEDd] × 100%, and ejection fraction (EF) was determined using the formula: EF = (EDV – ESV) × 100% / EDV.
Histological analysis
H&E staining
Heart tissue Sect. (5 μm thick) were prepared from paraffin-embedded blocks. Sections were deparaffinized, stained with hematoxylin (Beyotime, C0105S), differentiated in acidic water, and counterstained with eosin. After dehydration with ethanol and clearing with xylene, the slides were mounted and analyzed under a microscope.
WGA staining
Fixed tissue samples were incubated in PBS containing WGA fluorescent dye at room temperature for 2 h, followed by washing three times with PBS. Nuclei were counterstained with DAPI for 30 min, and the slides were washed again before imaging.
Sirius red staining
Fixed tissue samples in 4% paraformaldehyde were dehydrated, embedded in paraffin, and sectioned. After deparaffinization and rehydration, the sections were stained with Sirius Red solution (Sigma-Aldrich, 365548) for 1 h. Following staining, the sections were dehydrated with absolute ethanol, cleared with xylene, and mounted with a coverslip for microscopic analysis.
Tissue Immunofluorescence
Tissue sections were baked at 70 °C for 30 min using an in situ hybridization instrument. The processed sections were quickly immersed in xylene twice, for 5 min each, for deparaffinization. Gradient rehydration was performed by immersing the sections for 5 min each in 100%, 90%, 80%, and 70% ethanol, followed by distilled water. Endogenous peroxidase activity was blocked, and antigen retrieval was performed as required. Sections were blocked with 1% BSA at room temperature for 2 h, then incubated with a primary antibody at 37 °C for 2 h or overnight at 4 °C. The following primary antibodies were applied: SOD2(1:500, abcam, ab13534), cTnl (1:500, abcam, ab47002), CD31(1:500, Abcam, ab9498), and α-SMA (1:500, CST, 19245T), Glut4 (1:1500, ABclonal, A25174).After washing with PBS, the sections were incubated with a fluorescently labeled secondary antibody at room temperature in the dark for 1 h. At last, the cells were stained with DAPI for 5 min. After another wash with PBS, an appropriate amount of a fluorescence quenching agent was added to cover the slides for 90 min. Following another PBS wash, an anti-quenching mounting medium (G1221-5ML, Servicebio) was applied to mount the sections for subsequent observation and analysis.
Quantification of Immunofluorescence
For the fluorescent staining of SOD2 and ROS, we selected tissue surrounding the infarct area for image acquisition. For the fluorescent staining of CTnI, αSMA, and CD31, images were acquired directly from the infarct region. The collected images were analyzed for fluorescence intensity using ImageJ. After splitting the fluorescence channels corresponding to the target proteins, an automatic threshold segmentation was applied to select the appropriate highlighted signal regions. The average fluorescence intensity of these regions was then calculated, and the data were exported.
Co-localization analysis of Glut4 and WGA was performed based on the overlap of the red and green channels. ImageJ was used for co-localization analysis following these steps: splitting the fluorescence channels for Glut4 and WGA, aligning them, subtracting background, and obtaining the grayscale values of the co-localized regions.The extent of vascular growth in tissues was assessed by comparing immunofluorescence staining of CD31 and αSMA in tissues from each experimental group. Fluorescence channels were separated in ImageJ, and the fluorescence threshold was adjusted to an appropriate range. The CD31 and αSMA channel images were then stacked separately. Using the segmented line tool, traces along CD31-positive vessels were recorded, and the positive area ratios were calculated by comparing them with those of the normal group.
Statistical analysis
All data are presented as mean ± standard deviation (SD). Statistical analysis was performed using SPSS software (version 27.0) and GraphPad Prism software (version 9.5). For comparisons among multiple groups, one-way analysis of variance (ANOVA) was employed, provided that the data met the assumptions of normality and homogeneity of variance. A significance level of p < 0.05 was set. For data that did not satisfy these assumptions, non-parametric tests such as the Kruskal-Wallis test were used for analysis. Additionally, for comparisons between two independent groups, a two-sample t-test was conducted, assuming normal distribution and equal variances.