Hypoxia-preconditioned mesenchymal stem cell-derived exosomes attenuate myocardial ischemia/reperfusion injury by miR-29b-3p via PINK1/Parkin-mediated mitophagy

Figure 1 Characterization of exosome and hypoxia-preconditioned mesenchymal stem cell-derived exosomes. A: Schematic diagram of the isolation process for exosomes derived from mesenchymal stem cells; B: Size distribution chart of exosome (Exo) and hypoxia-preconditioned mesenchymal stem cell-derived Exo; C: Transmission electron micrographs of Exo and hypoxia-preconditioned mesenchymal stem cell-derived Exo. Scale bar: 200 nm; D: Western blot analysis of Alix, Tsg101, and CD9 expression in Exo and HPC-Exo. BM-MSC: Bone marrow mesenchymal stem cell; Exo: Normoxic exosomes; HPC-Exo: Hypoxia-preconditioned mesenchymal stem cells-derived exosomes; MSCs: Mesenchymal stem cells.

Figure 2 Exosome therapy can improve myocardial ischemia/reperfusion injury in mice. A: Schematic diagram of intramyocardial injection of exosome (Exo) and hypoxia-preconditioned mesenchymal stem cell-derived Exo (HPC-Exo) in the ischemia/reperfusion (I/R) mouse model; B: Evans blue and triphenyltetrazolium chloride double staining of hearts 28 days after different treatments. Blue represents non-ischemic areas. Red represents ischemic areas. White represents infarcted areas (n = 6); C and D: Echocardiographic measurements of left ventricular end-diastolic diameter, left ventricular end-systolic diameter, ejection fraction, and fractional shortening in mice subjected to sham surgery, I/R, Exo treatment, and HPC-Exo treatment from 2 days to 4 weeks post-myocardial I/R injury (n = 8); E and F: Representative fluorescent micrographs of cardiomyocyte apoptosis (E) and quantitative analysis of apoptosis rates (F); G: Schematic diagram of co-incubation of Exo and HPC-Exo with cardiomyocytes (n = 3); H: Dihydroethidium staining of myocardial cells; I and J: Flow cytometric analysis of dihydroethidium-stained myocardial cells (I) and quantitative analysis of oxidative stress in myocardial cells (J); K and L: Representative flow cytometry plots showing quantitative analysis of the apoptosis rate (K) and the effects of different treatments on cell apoptosis (L). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Con: Control; DAPI: 4’,6-diamidino-2-phenylindole; DHE: Dihydroethidium; Exo: Normoxic exosomes; HPC-Exo: Hypoxia-preconditioned mesenchymal stem cells-derived exosomes; H/R: Hypoxia/reperfusion; I/R: Ischemia/reperfusion; LVEDd: Left ventricular end-diastolic diameter; LVESd: Left ventricular end-systolic diameter; MFI: Mean fluorescent intensity; PI: Propidium iodide; TUNEL: Transferase dUTP nick end labeling.

Figure 3 Profiling and identification of upregulated microRNAs in exosome and hypoxia-preconditioned mesenchymal stem cell-derived exosome. A: Schematic diagram of next-generation sequencing; B and C: Twenty-two microRNAs (miRNAs) that were differently expressed between exosome (Exo) and hypoxia-preconditioned mesenchymal stem cell-derived Exo as displayed in the volcano plot (C) and hierarchical clustering heat map (B); D: Verification for the miRNA expressions in Exo and hypoxia-preconditioned mesenchymal stem cell-derived Exo by quantitative PCR analysis; E and F: Representative flow cytometry plots show the effects of highly expressed miRNAs identified (Figure 3D) on cell apoptosis (E) and quantitative analysis of apoptosis rate (F); G and H: Flow cytometric analysis of dihydroethidium-stained myocardial cells (G) and quantitative analysis of oxidative stress in myocardial cells (H); I: Dihydroethidium staining of myocardial cells. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Con: Control; DAPI: 4’,6-diamidino-2-phenylindole; DHE: Dihydroethidium; Exo: Normoxic exosomes; HPC-Exo: Hypoxia-preconditioned mesenchymal stem cells-derived exosomes; MFI: Mean fluorescent intensity; miR: MicroRNA; miRNAs: MicroRNAs; NC: Negative control; NGS: Next-generation sequencing; NS: Not significant; PI: Propidium iodide; q-PCR: Quantitative PCR; H/R: Hypoxia/reperfusion.

Figure 4 miR-29b-3p alleviates mitochondrial damage. A: Schematic protocol for transfection of cardiomyocytes with miR-NC and miR-29b-3p; B: Gene Ontology enrichment analysis of differentially expressed target genes between the model and miR-29b-3p treatment groups (P < 0.05); C: Transmission electron microscopy images showing mitochondrial damage; D: Double immunofluorescence staining of mitochondria and LC3B. Scale bar: 10 μm; E: MitoSOX Red staining of myocardial cells. Scale bar: 10 μm; F and G: Study of myocardial cells labeled with MitoSOX Red using flow cytometry; H: Mitochondrial membrane potential staining. Red, JC-1 aggregates. Green, JC-1 monomers; I and J: Representative flow cytometry plots showing the effects of different treatments on mitochondrial membrane potential (I) and quantitative analysis of JC-1 monomers (J); K and L: Western blot assay revealed the protein expression of PINK1, Parkin, and Tim23 and quantitative analysis. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Con: Control; H/R: Hypoxia/reperfusion; DAPI: 4’,6-diamidino-2-phenylindole; JC-1: 5,5‘,6,6’-Tetrachloro-1,1‘,3,3’-tetraethylbenzimidazolylcarbocyanine iodide; MFI: Mean fluorescent intensity; miR: MicroRNA; NC: Negative control; NS: Not significant.

Figure 5 miR-29b-3p alleviates myocardial ischemia/reperfusion injury by inducing mitophagy. A: Mdivi-1 inhibited mitochondrial autophagy; B: Flow cytometric analysis of MitoSOX Red-stained myocardial cells; C and D: Representative flow cytometry plots showing the effects of different treatments on cell apoptosis (C) and quantitative analysis of apoptosis rate (D); E and F: Western blot assay showed the effect of PINK1 siRNA on the protein expression of PINK1, Parkin, Tim23, Tom20, and LC3 and quantitative analysis; G and H: Representative flow cytometry plots showing the effects of different treatments on cell apoptosis (G) and quantitative analysis of apoptosis rate (H); I and J: Mitochondrial oxidative stress detected by flow cytometry (I) and its quantitative analysis (J); K: Schematic diagram of animal model establishment; L: Evans blue and triphenyltetrazolium chloride double staining of hearts 28 days after different treatments. Blue represent non-ischemic areas. Red represents ischemic areas. White represents infarcted areas; M: Echocardiographic measurements of left ventricular end-diastolic diameter, left ventricular end-systolic diameter, ejection fraction, and fractional shortening in mice subjected to sham surgery, ischemia/reperfusion (I/R), I/R + microRNA (miR)-negative control, I/R + miR-29b-3p, I/R + AAV9-sh-negative control, I/R + AAV9-sh-PINK1, and I/R + AAV9-sh-PINK1 + miR-29b-3p from 2 days to 4 weeks post-myocardial I/R injury (n = 10). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Con: Control; DHE: Dihydroethidium; H/R: Hypoxia/reperfusion; I/R: Ischemia/reperfusion; LVEDd: Left ventricular end-diastolic diameter; LVESd: Left ventricular end-systolic diameter; MFI: Mean fluorescent intensity; miR: MicroRNA; NC: Negative control; NS: Not significant; PI: Propidium iodide.