Engineered exosomes improve myocardial cell membrane integrity and heart function in dystrophic cardiomyopathy

FIGURE 1. DmdΔ4 mice develop progressive cardiac dysfunction. (A) Sanger sequencing analysis of DmdΔ4 mice containing a 4‐bp deletion in dystrophin gene. (B) Detection of dystrophin protein loss in heart, diaphragm and gastrocnemius of DmdΔ4 mice via immunoblotting. Vinculin was used as a loading control and a molecular weight standard was marked on the left. (C) Representative flow gate strategy and result of single‐cell suspension from cardiac tissue digestion. CD11b+/F4/80+ double positive cells represent macrophages in the DmdΔ4 mice's heart. Scale bars: 50 µm. (D) Pooled data from (C) reveal a higher number of CD11b+F4/80+ cells in DmdΔ4 mice compared with age‐matched wild‐type (WT) mice (n = 4). (E and F) Single‐cell transcriptome sequencing of cardiac CD45+ immune cells. Uniform manifold approximation and projection (UMAP) analysis cell clusters (E). The percentage of cells for each cell population in DmdΔ4 mice and age‐matched WT mice (F). (G) Representative Masson staining images from the DmdΔ4 mice and age‐matched WT mice at 4, 8, 16, 24 and 40 weeks old. Scale bars: 500 µm. (H) Pooled data from (G) revealed a higher fibrosis in DmdΔ4 mice compared with age‐matched WT mice (n =…

FIGURE 2. Preparation and analysis of engineered exosomes (exosomes‐cardiac homing peptide [Exo‐CHP]). (A) Illustration depicting the preparation process of Exo‐CHP. (B) Representative images of cell membranes labelled with DSPE‐CHP‐RED. RED indicates DSPE‐CHP‐5‐TAMRA. Scale bars: 100 µm. (C) Representative images of cells labelled with DSPE‐CHP‐RED (n = 3). Scale bars: 20 µm. (D) Transmission electron microscopy (TEM) images of Exo and Exo‐CHP. Scale bars: 100 nm. (E) Representative immunoblots showing the expression of exosome markers CD9 and CD63 in Exo and Exo‐CHP (n = 3). (F) Nano‐flow cytometry analysis of the size distribution of Exo and Exo‐CHP. (G‒I) miRNA sequencing analysis Exo and Exo‐CHP. (G) Venn diagram showing shared and differential miRNAs between Exo and Exo‐CHP. (H) Gene Ontology (GO) pathway enrichment analysis of common miRNA target genes. (I) Heatmap showing highly expressed common miRNA molecules in Exo and Exo‐CHP.

FIGURE 3. Exosomes‐cardiac homing peptide (Exo‐CHP) targets DmdΔ4 heart in vivo and inhibits cardiomyocyte injury in vitro. (A) Illustration depicting Exo‐CHP targeting the mouse heart via tail vein injection. (B) H9C2 cells were pretreated with H2O2, and then treated with Exo‐CHP (50 µg/mL) and Exo‐Src (50 µg/mL) for 24 h (n = 3). Representative images of Dio‐labelled Exo‐CHP and Exo‐Src uptake by H9C2. Scale bars: 20 µm. (C) Quantitative analysis from (B) reveals a higher fluorescence signal in Exo‐CHP‐treated H9C2 cells compared to the Src‐Exo group (n = 3). (D) Representative in vivo imaging system (IVIS) images of in vivo imaging DmdΔ4 mice and isolated hearts, and the biological distribution results of Exo‐CHP in mice (n = 3). (E) Quantitative analysis from (D) reveals a higher fluorescence count in Exo‐CHP‐treated mice compared with Src‐Exo group (n = 3). (F) H9C2 were pretreated with H2O2, and then treated with Exo‐CHP (50 µg/mL) and phosphate‐buffered solution (PBS) for 24 h. Representative images showing reactive oxygen species (ROS) fluorescence imaging images. Scale bars: 100 µm. (G) Quantitative analysis of fluorescence intensity in the PBS‐treated group and Exo‐CHP‐treated…

FIGURE 4. Exosomes‐cardiac homing peptide (Exo‐CHP) inhibits myocardial pathological changes and improves heart function in DmdΔ4 cardiomyopathy mice. (A) Illustration depicting the experimental design. (B) Representative Masson's trichrome‐stained micrographs from wild‐type (WT), vehicle and Exo‐CHP treatment groups. Scale bars: 20 µm. (C) Quantitative analysis of data from (B) showing reduced interstitial fibrosis in Exo‐CHP‐treated DmdΔ4 hearts (n = 6). (D) Transmission electron microscopy (TEM) examination of the submicroscopic structure of the heart in WT, vehicle and Exo‐CHP treatment groups. (E‒J) Echocardiographic measurements of left ventricular ejection fraction (LVEF) (E), left ventricular fractional shortening (LVFS) (F), diastolic diameter of left ventricle (Diam.(d)) (G), diastolic volume of left ventricle (Vol.(d)) (H), left ventricular posterior wall thickness at systole (LVPW.(s)) (I) and left ventricular posterior wall thickness at end‐diastole (LVPW.(d)) (J) in Exo‐CHP‐treated DmdΔ4 mice after 30 days of treatment (n = 6). (K and L) Assessment of serum creatine kinase (CK) (K) and CK‐MB (L) levels in DmdΔ4 mice after treatment with Exo‐CHP (n = 6). * p < .05; ** p < .01.

FIGURE 5. Intravenous injection of exosomes‐cardiac homing peptide (Exo‐CHP) reduces DmdΔ4 mice cardiac immune cell infiltration and inflammation in DmdΔ4 mice's heart. (A) Illustration depicting the experimental design. (B) Representative haematoxylin and eosin (H&E) staining images of DmdΔ4 mice heart from wild‐type (WT), vehicle and Exo‐CHP treatment groups (n = 4). Scale bars: 20 µm. (C) Representative immunofluorescence (IF) staining images of heart macrophages from WT, vehicle and Exo‐CHP treatment groups. CD68+ cells indicate macrophages. Scale bars: 20 µm. (D) Quantitative analysis of data from (C) showing fewer CD68+ cells in Exo‐CHP‐treated DmdΔ4 hearts (n = 4). (E) Representative immunohistochemistry (IHC) staining images of heart T cells from WT, vehicle and Exo‐CHP treatment groups. CD3+ cells indicate T cells. Scale bars: 20 µm. (F) Quantitative analysis of data from (E) showing fewer CD3+ cells in Exo‐CHP‐treated DmdΔ4 hearts (n = 4). (G) Representative immunoblot images from WT, Exo‐CHP and vehicle treatment groups. (H) Quantitative analysis of data from (G) reveals reduced expression of phosphorylated NF‐κB (p‐NF‐κB) in Exo‐CHP treatment group (n = 3). Relative protein…

FIGURE 6. Exosomes‐cardiac homing peptide (Exo‐CHP) inhibits DAPC degradation, calpain activity, and intracellular calcium influx in DmdΔ4 cardiomyopathy mice. (A and B) Western blot analysis to examine the expression of α‐sarcoglycan (A) and its quantitative analysis (B) in the hearts (n = 3). (C and D) Western blot analysis to examine the expression of β‐dystroglycan (C) and its quantitative analysis (D) in the hearts (n = 3). Relative protein levels were normalised to GAPDH and shown as fold change versus control. (E and F) Immunofluorescence (IF) staining was used to detect the co‐localisation of DAPC complex α‐sarcoglycan or β‐dystroglycan with Exo‐CHP in the hearts of DmdΔ4 mice, respectively (n = 3). The white arrow represents co‐localisation. (G) Evaluation of calpain activity in hearts of DmdΔ4 treated with Exo‐CHP (n = 4). (H) Quantitative analysis of calcium content in the hearts of DmdΔ4 mice treated with Exo‐CHP (n = 5). (I) Measurement of lactate dehydrogenase (LDH) activity in the heats of DmdΔ4 mice treated with Exo‐CHP (n = 5). (J) Calcium content kit detected the calcium content of primary cardiomyocytes derived from DMD mice under different treatment conditions. (K)…

FIGURE 7. Safety evaluation of exosomes‐cardiac homing peptide (Exo‐CHP) in vivo. After Exo‐CHP treatment for 24 h, samples of heart, liver, spleen, lung and kidney were collected for haematoxylin and eosin (H&E) staining (n = 6). Representative H&E staining images (A). Detection of the expression level of alanine aminotransferase (ALT), Urea, aspartate aminotransferase (AST), creat, creatine kinase (CK) and lactate dehydrogenase (LDH) in the serum of mice (B). Flow cytometry was used to detect the numbers of T cells, B cells and monocytes in the peripheral blood mononuclear cell (PBMC) of mice (C). Long‐term safety evaluation of Exo‐CHP in vivo. After Exo‐CHP treatment for 30 days, samples of heart, liver, spleen, lung and kidney were collected for H&E staining. Representative H&E staining images (D). Flow cytometry was used to detect the numbers of T cells, B cells and monocytes in the peripheral blood PBMC of mice (E) (n = 6).