Cell strains and animal fashions
Rat cardiomyocytes (H9c2), human umbilical vein endothelial cells (HUVECs), and human mesenchymal stem cells (hMSCs) had been obtained from the American Sort Tradition Assortment (ATCC). All cell strains had been cultured and maintained following ATCC’s normal protocols. Male Sprague-Dawley (SD) rats (6–8 weeks outdated, weighing 200–250 g) and male C57BL/6 mice (6–8 weeks outdated, weighing 18–22 g) had been bought from the Southern Medical College Experimental Animal Middle. Animals had been housed in a particular pathogen-free (SPF) atmosphere underneath managed circumstances of twenty-two ± 2 °C, 50–60% relative humidity, and a 12 h gentle/darkish cycle, with free entry to meals and water.
Pathological fashions of cells and animals and therapy
An I/R mannequin was developed in H9c2 cardiomyocytes (ATCC, CRL-1446). The tradition medium was eliminated, and cells had been washed twice with glucose-free DMEM (Gibco, C11995500BT). Glucose-free, serum-free DMEM was then added, and the cells had been incubated in a hypoxic chamber at 37 °C underneath 5% CO₂ and 95% N₂ for six h to induce hypoxia. After finishing the hypoxia and glucose deprivation section, the medium was changed with high-glucose DMEM supplemented with 10% FBS. Exo-WT and Exo-I-S had been added at a dosage of three,000 particles per H9c2 cell (with an exosome protein focus of 20 µg/mL), together with PBS as a management. The cells had been subsequently cultured underneath normal circumstances at 37 °C with 5% CO₂ for 12 h.
For the in vivo experiment was permitted by Nan-fang hospital, southern medical college (IACUC-LAC-20240822-005), animal care and therapy had been performed following institutional procedures and nationwide legal guidelines and laws, male SD rats had been anesthetized with isoflurane, and a thoracotomy was carried out on the fourth intercostal house to reveal the guts. A 6 − 0 polypropylene suture was positioned 2–3 mm under the origin of the left anterior descending artery, between the left atrial appendage and the arterial cone, to induce myocardial ischemia/reperfusion damage. After 1 h of ischemia, the suture was eliminated to permit reperfusion. Intramyocardial injections of Exo-WT and Exo-I-S options (dose: 1 × 10¹¹ particles/kg physique weight; exosome protein focus: 20 µg/mL) or an equal quantity of PBS had been administered into the infarct space (recognized by pale discoloration after ligation).
Development of MSCIns cells
A dual-gene expression plasmid, pIRES-SIRT3/GPI + Insulin, was constructed utilizing molecular cloning strategies. Human SIRT3 and insulin genes had been amplified by PCR, with a GPI-anchoring sequence added to the three’ finish of the insulin gene. The amplified merchandise had been then cloned into MCS A and MCS B websites of the pIRES bicistronic expression vector. After profitable sequencing verification, plasmid DNA was extracted and transfected into MSCs utilizing Lipofectamine™ 2000 (ThermoFisher, 11668019) to generate SIRT2/GPI + Insulin expressed MSC (MSCIns). Transfected cells had been harvested 12 and 24 h post-transfection for immunofluorescence and Western blot validation.
Preparation of Exo-I-S and Exo-WT
Each transfected and non-transfected MSCs (ATCC, PCS-500-012) had been initially cultured for 48 h. The medium was then changed with serum-free DMEM for a further 24 h. The tradition 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 utilizing an Optima MAX-XP ultracentrifuge (Beckman, USA). The supernatant was discarded, and the pellet was washed with PBS, adopted by a last ultracentrifugation step at 100,000 × g for 70 min. Purified exosomes had been saved at − 80 °C till evaluation.
Characterization of Exo-I-S and Exo-WT
Exosome particle dimension evaluation
Nanoparticle monitoring evaluation (NTA) was carried out utilizing the ZetaView PMX 110 instrument (Particle Metrix) and its accompanying software program (ZetaView 8.02.28). Exosomes had been diluted appropriately in particle-free PBS earlier than being loaded into the pattern chamber. The dimensions distribution and focus of exosomes had been decided at a wavelength of 405 nm. Particle dimension knowledge had been quantified and recorded.
Protein quantification
Protein focus was decided utilizing a BCA Protein Assay Equipment (Beyotime, P0012), following the producer’s protocol. Samples and normal proteins (BSA) had been added to a 96-well plate, combined with BCA working reagent, and incubated at 37 °C for 30 min. Absorbance was measured at 562 nm in microplate reader, and protein focus was calculated based mostly on a normal curve (0–2,000 µg/mL).
Transmission electron microscopy
Exosome samples had been dropped onto carbon-coated copper grids, air-dried at room temperature, and negatively stained with 2% phosphotungstic acid for five min. Extra stain was eliminated with filter paper, and the grids had been examined underneath a transmission electron microscope (TEM) (JEM-1230, JEOL) to evaluate the morphology and dimension of the exosomes.
Western blot
Whole protein was extracted from cells and exosomes and separated on 10–15% SDS-polyacrylamide gels, based mostly on the molecular weight of the goal proteins. Proteins had been transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore, IPVH00010) and blocked with 5% skim milk for 1 h. Membranes had been incubated in a single day at 4 °C with major antibodies towards: 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 comparable to goat anti-mouse IgG (1:3000, Abcam, ab205719) and goat anti-rabbit IgG (1:3000, Abcam, ab205718) had been utilized. Protein bands had been visualized utilizing enhanced chemiluminescence (ECL) reagents (Beyotime, P0018S) and imaged utilizing a ChemiDoc MP system (Bio-Rad).
Exosome uptake assay
The uptake of exosomes by H9c2 cardiomyocytes and HUVEC endothelial cells was monitored utilizing PKH26-labeled (200 µg/mL, Sigma, MINI26) management Exo (Exo-WT) and Exo-I-S. Labeled exosomes had been added to the H9c2 tradition medium, and cells had been incubated for 3, 6, 12, 24, and 36 h. After incubation, cells had been washed with DPBS and stuck with 4% paraformaldehyde (PFA). Cell nuclei had been stained with 4’,6-Diamidino-2-phenylindole (DAPI, Invitrogen) for visualization. Fluorescence imaging was carried out utilizing a confocal laser scanning microscope (LSM 880, Zeiss). Photographs of three unbiased samples had been captured for every group, and ImageJ was utilized to quantify the relative fluorescence depth utilizing the automated threshold technique to pick out the fluorescent areas.
GPI-Insulin molecular modeling and Docking prediction
The three-dimensional construction of the GPI-Insulin fusion protein was predicted utilizing AlphaFold 3.0. By inputting the amino acid sequence, AlphaFold 3.0 generated a 3D structural mannequin and offered the expected Template Modeling Rating (pTM). The expected construction was then imported into AutoDock Vina for molecular docking evaluation with the insulin receptor (IR). The construction of the insulin receptor was obtained from the Protein Knowledge Financial institution (PDB). Previous to docking, preprocessing was carried out utilizing AutoDock Instruments, which included eradicating water molecules, including polar hydrogen atoms, calculating Gasteiger costs, and saving the recordsdata in PDBQT format. Through the docking course of, a grid field was set to cowl the receptor’s lively web site, and the Lamarckian genetic algorithm was employed to finish the docking. The binding mode and binding vitality of the GPI-Insulin fusion protein with the insulin receptor had been subsequently decided.
Biocompatibility analysis
Tube formation assay
HUVECs (ATCC, CRL-1730) had been seeded into 96-well plates pre-coated with 10 mg/mL Matrigel matrix (Corning, 354234) to simulate an in vitro angiogenesis atmosphere. After seeding at an applicable density, tradition medium containing exosomes (e.g., Exo-I-S, Exo-WT, or management) was added. After 6–12 h of incubation, the formation of tubular buildings was noticed and imaged utilizing an inverted microscope (Leica, DMi8). Tube size and the variety of department factors had been analyzed utilizing ImageJ software program to judge the consequences of various remedies on angiogenesis.
Stay/Useless cell assay
H9c2 cells at applicable confluency had been handled with tradition medium containing Exo-I-S, Exo-WT, or PBS for twenty-four h. After eradicating the medium and washing the cells with PBS, a dye working answer was ready in keeping with the producer’s directions: Calcein-AM (2 µM, Invitrogen, C3099) and PI (5 µg/mL, Invitrogen, P1304MP). Cells had been incubated at 37 °C for 15–30 min, and fluorescence photos had been captured utilizing a fluorescence microscope.
Organ toxicity evaluation
Sterile Exo-I-S, Exo-WT, or PBS options had been ready and administered to C57BL/6 mice through tail vein injection at a quantity of 10 mL/kg physique weight. The management group obtained an equal quantity of PBS. Seven days after administration, mice had been sacrificed underneath extreme anesthesia, and main organs had been quickly harvested and stuck in 4% paraformaldehyde. Hematoxylin and eosin (H&E) staining was carried out to evaluate histopathological modifications.
Immunofluorescence stain
Cells had been permeabilized with 0.1% Triton X-100 at room temperature for 10 min, adopted by blocking with 1% BSA/PBST buffer for 1 h. The cells had been incubated in a single day at 4 °C with the first antibody Glut4 (1:200, ABclonal, A25174), SIRT3 (1:200, ABclonal, A5419), Insulin (1:200, Abcam, ab46707). After washing with PBS, cells had been 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 at nighttime for two h. Nuclei had been stained with DAPI (1 µg/mL, 1:1000, Sigma-Aldrich, D9542).
Mitochondrial morphology evaluation
Mitochondrial Electron microscopy
Cells had been washed with serum-free medium and stuck with 4% glutaraldehyde (Sigma-Aldrich, 814393) and 4% paraformaldehyde (Sigma- Aldrich, 818715). Samples had been dehydrated in a graded ethanol sequence and embedded in LX-812 resin (Ladd Analysis Industries Inc.). Ultrathin sections had been stained with uranyl acetate for 30 min and lead citrate for 10 min, and noticed utilizing a FEI Tecnai G12 Spirit BioTwin transmission electron microscope (FEI Firm, Hillsboro) at an acceleration voltage of 120 kV.
JC-1 staining
To guage mitochondrial membrane potential underneath totally different Exo remedies, JC-1 staining was carried out utilizing the MitoProbe JC-1 Assay Equipment (Invitrogen, M34152). Cells had been incubated with JC-1 (10 µL, 200 µM) for 30 min, and fluorescence was detected at 488 nm and 633 nm utilizing a confocal laser scanning microscope (Zeiss LSM 880).
Mito-Tracker evaluation
A 1 mM inventory answer of Mito-Tracker Crimson was ready in anhydrous DMSO and saved at − 20 °C at nighttime. After eradicating the tradition medium, pre-warmed Mito-Tracker Crimson CMXRos staining answer (Beyotime, C1999S) was added to the cells, which had been incubated at 37 °C for 30 min. Following incubation, the staining answer was changed with contemporary medium, and fluorescence was noticed utilizing a confocal laser scanning microscope (Zeiss LSM 880).
Mitochondrial oxidative stress detection
ROS measurement
Reactive oxygen species (ROS) had been measured utilizing the DCFH-DA probe (ab113851). The DCFH-DA answer was diluted 1:1000 with serum-free medium and saved protected against gentle. Cells had been washed twice, then incubated with 100 µL of the diluted DCFH-DA answer at 37 °C for 30 min. After incubation, cells from the oxidative stress mannequin had been washed 3 times with serum-free medium to take away extra probe not taken up by cells, adopted by addition of contemporary medium and additional incubation for 30 min. Cells had been then collected by centrifugation, resuspended within the diluted DCFH-DA answer, and analyzed by circulate cytometry. A complete of 10,000 cells per pattern had been measured for fluorescence depth, and the typical fluorescence depth was calculated. Outcomes had been expressed because the ratio of fluorescence depth between experimental and regular teams.
SOD1 and SOD2 measurement
SOD1 and SOD2 ranges had been measured utilizing ELISA kits (CBIBO BIO, CB11409-Ra and CB10619-Ra). Cells had been washed 3 times with PBS and centrifuged (3000 rpm, 5 min, 4 °C). After including phosphate-buffered saline and extraction buffer (1:1000), cells had been ultrasonicated (3 s bursts, 10 s intervals, 30 cycles) and centrifuged once more (3000 rpm, 20 min, 4 °C). Supernatants had been collected and saved on ice. Requirements (50 µL) and samples (40 µL dilution buffer with 10 µL pattern, 5-fold diluted) had been added to wells. Apart from blanks, 100 µL enzyme conjugate was added; plates had been sealed and incubated at 37 °C for 60 min. Wells had been washed 5 occasions with 20-fold diluted wash buffer, then incubated with 50 µL every of substrate A and B for 15 min at 37 °C at nighttime. The response was stopped with 50 µL cease answer, turning colour from blue to yellow. Absorbance was learn at 450 nm inside 15 min, and outcomes had been expressed as experimental-to-normal group ratios.
Apoptosis detection
Cell apoptosis was analyzed utilizing Annexin V-FITC/PI twin staining. Cells had been 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) had been added, and the samples had been incubated at nighttime for 15 min at room temperature. Fluorescence alerts had been detected utilizing a circulate cytometer (BD FACSCanto II).
Cell metabolic exercise evaluation
Mobile oxygen consumption charge (OCR) was measured utilizing the Seahorse XFe24 extracellular flux analyzer (Agilent, Seahorse XFe24) and the Mito Stress Take a look at Equipment (Agilent, 103015-100). H9c2 cells had been seeded onto tradition plates (Agilent, 100882-004) and subjected to I/R therapy. On the day of measurement, cells had been washed with XFe24 medium and incubated in a CO₂-free incubator at 37 °C for two h to equilibrate. OCR was measured earlier than 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 manufacturing, and maximal respiration had been calculated.
Mitochondrial enzymatic detection
For the actions of glycolysis enzymes, We used the 6-phosphofructokinase (PFK-1) exercise package (Solarbio, BC0530), lactate dehydrogenase (LDH) exercise package (Solarbio, BC0685), lactate content material package (Solarbio, BC2230), pyruvate dehydrogenase (PDH) exercise package (Abcam, ab287837), and ATP content material package (Solarbio, BC0300). and RT-qPCR was performed to evaluate the enzymes within the tricarboxylic acid (TCA) cycle, particularly citrate synthase (CS) and isocitrate dehydrogenase (IDH), in addition to elements of the oxidative phosphorylation complicated, specifically NADH dehydrogenase 1-β3 (NDUFB3) and succinate dehydrogenase complicated subunit B (SDHB).The response combine included 5 µL of SsoFast Eva Inexperienced Supermix, 0.5 µL of every upstream and downstream primer (10 µM), 2 µL of cDNA template, and a pair of µL of ultrapure water, totaling 10 µL. We used β-tubulin because the reference gene, and the relative expression ranges of the goal genes had been calculated utilizing the 2-ΔΔCT technique. Primers sourced from Shanghai Shenggong had been as follows: (See Desk 1).
Serum detection of CK-MB, CTnT, and LDH
Blood samples had been collected from the internal canthus vein 12 h after myocardial infarction modeling, and serum was separated by centrifugation at 3000 rpm for 10 min. The degrees of CK-MB, CTnT, and LDH had been measured utilizing enzyme-linked immunosorbent assay (ELISA) kits. The next particular kits had been used: CK-MB (Abcam, ab285275), CTnT (Abcam, ab246529), and LDH (Sigma-Aldrich, MAK066). Following the directions of the kits, serum samples had been added to the enzyme-linked plate, incubated, after which the enzyme conjugate and substrate had been added for colour growth. After terminating the response, the absorbance was measured at 450 nm. The concentrations of every biomarker had been calculated based mostly on the usual curve to evaluate the extent of myocardial damage.
Electrocardiogram
Previous to the experiment and 12 h after myocardial infarction modeling, rats had been anesthetized (with intraperitoneal injection of pentobarbital sodium at a dose of 40 mg/kg) and stuck on the experimental platform. Their limbs had been uncovered, and electrocardiogram electrodes had been related (one for every forelimb and hindlimb). Care was taken to make sure that the electrodes had good contact with the pores and skin. An animal-specific electrocardiogram system (ADInstruments, ML136) was used to report.
Echocardiography
Utilizing the Vevo high-resolution small animal ultrasound imaging system and a high-frequency transducer, cardiac ultrasound examinations had been performed by means of the sternum on the 14th day post-surgery. SD rats had been anesthetized whereas sustaining spontaneous respiration, and two-dimensional photos of the left ventricle had been obtained within the short-axis view on the stage of the left ventricular papillary muscular tissues. Key measurements included coronary heart charge, 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 exterior diameter (EXLVDd), and left ventricular short-axis fractional shortening (FS%). The top-diastolic quantity (EDV) and end-systolic quantity (ESV) had been then calculated utilizing built-in correction formulation.
FS was calculated utilizing the components: FS = [(LVEDd – LVESd) / LVEDd] × 100%, and ejection fraction (EF) was decided utilizing the components: EF = (EDV – ESV) × 100% / EDV.
Histological evaluation
H&E staining
Coronary heart tissue Sect. (5 μm thick) had been ready from paraffin-embedded blocks. Sections had been deparaffinized, stained with hematoxylin (Beyotime, C0105S), differentiated in acidic water, and counterstained with eosin. After dehydration with ethanol and clearing with xylene, the slides had been mounted and analyzed underneath a microscope.
WGA staining
Mounted tissue samples had been incubated in PBS containing WGA fluorescent dye at room temperature for two h, adopted by washing 3 times with PBS. Nuclei had been counterstained with DAPI for 30 min, and the slides had been washed once more earlier than imaging.
Sirius crimson staining
Mounted tissue samples in 4% paraformaldehyde had been dehydrated, embedded in paraffin, and sectioned. After deparaffinization and rehydration, the sections had been stained with Sirius Crimson answer (Sigma-Aldrich, 365548) for 1 h. Following staining, the sections had been dehydrated with absolute ethanol, cleared with xylene, and mounted with a coverslip for microscopic evaluation.
Tissue Immunofluorescence
Tissue sections had been baked at 70 °C for 30 min utilizing an in situ hybridization instrument. The processed sections had been rapidly immersed in xylene twice, for five min every, for deparaffinization. Gradient rehydration was carried out by immersing the sections for five min every in 100%, 90%, 80%, and 70% ethanol, adopted by distilled water. Endogenous peroxidase exercise was blocked, and antigen retrieval was carried out as required. Sections had been blocked with 1% BSA at room temperature for two h, then incubated with a major antibody at 37 °C for two h or in a single day at 4 °C. The next major antibodies had been utilized: 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 had been incubated with a fluorescently labeled secondary antibody at room temperature at nighttime for 1 h. Eventually, the cells had been stained with DAPI for five min. After one other wash with PBS, an applicable quantity of a fluorescence quenching agent was added to cowl the slides for 90 min. Following one other PBS wash, an anti-quenching mounting medium (G1221-5ML, Servicebio) was utilized to mount the sections for subsequent statement and evaluation.
Quantification of Immunofluorescence
For the fluorescent staining of SOD2 and ROS, we chosen tissue surrounding the infarct space for picture acquisition. For the fluorescent staining of CTnI, αSMA, and CD31, photos had been acquired instantly from the infarct area. The collected photos had been analyzed for fluorescence depth utilizing ImageJ. After splitting the fluorescence channels comparable to the goal proteins, an automated threshold segmentation was utilized to pick out the suitable highlighted sign areas. The typical fluorescence depth of those areas was then calculated, and the info had been exported.
Co-localization evaluation of Glut4 and WGA was carried out based mostly on the overlap of the crimson and inexperienced channels. ImageJ was used for co-localization evaluation following these steps: splitting the fluorescence channels for Glut4 and WGA, aligning them, subtracting background, and acquiring the grayscale values of the co-localized areas.The extent of vascular development in tissues was assessed by evaluating immunofluorescence staining of CD31 and αSMA in tissues from every experimental group. Fluorescence channels had been separated in ImageJ, and the fluorescence threshold was adjusted to an applicable vary. The CD31 and αSMA channel photos had been then stacked individually. Utilizing the segmented line software, traces alongside CD31-positive vessels had been recorded, and the constructive space ratios had been calculated by evaluating them with these of the traditional group.
Statistical evaluation
All knowledge are offered as imply ± normal deviation (SD). Statistical evaluation was carried out utilizing SPSS software program (model 27.0) and GraphPad Prism software program (model 9.5). For comparisons amongst a number of teams, one-way evaluation of variance (ANOVA) was employed, offered that the info met the assumptions of normality and homogeneity of variance. A significance stage of p < 0.05 was set. For knowledge that didn’t fulfill these assumptions, non-parametric exams such because the Kruskal-Wallis take a look at had been used for evaluation. Moreover, for comparisons between two unbiased teams, a two-sample t-test was performed, assuming regular distribution and equal variances.