Cardiac progenitor cell-derived exosomes prevent cardiomyocytes apoptosis through exosomal miR-21 by targeting PDCD4

Abstract

Cardiac progenitor cells derived from adult heart have emerged as one of the most promising stem cell types for cardiac protection and repair. Exosomes are known to mediate cell-cell communication by transporting cell-derived proteins and nucleic acids, including various microRNAs (miRNAs). Here we investigated the cardiac progenitor cell (CPC)-derived exosomal miRNAs on protecting myocardium under oxidative stress. Sca1(+)CPCs-derived exosomes were purified from conditional medium, and identified by nanoparticle trafficking analysis (NTA), transmission electron microscopy and western blotting using CD63, CD9 and Alix as markers. Exosomes production was measured by NTA, the result showed that oxidative stress-induced CPCs secrete more exosomes compared with normal condition. Although six apoptosis-related miRNAs could be detected in two different treatment-derived exosomes, only miR-21 was significantly upregulated in oxidative stress-induced exosomes compared with normal exosomes. The same oxidative stress could cause low miR-21 and high cleaved caspase-3 expression in H9C2 cardiac cells. But the cleaved caspase-3 was significantly decreased when miR-21 was overexpressed by transfecting miR-21 mimic. Furthermore, miR-21 mimic or inhibitor transfection and luciferase activity assay confirmed that programmed cell death 4 (PDCD4) was a target gene of miR-21, and miR-21/PDCD4 axis has an important role in anti-apoptotic effect of H9C2 cell. Western blotting and Annexin V/PI results demonstrated that exosomes pre-treated H9C2 exhibited increased miR-21 whereas decreased PDCD4, and had more resistant potential to the apoptosis induced by the oxidative stress, compared with non-treated cells. These findings revealed that CPC-derived exosomal miR-21 had an inhibiting role in the apoptosis pathway through downregulating PDCD4. Restored miR-21/PDCD4 pathway using CPC-derived exosomes could protect myocardial cells against oxidative stress-related apoptosis. Therefore, exosomes could be used as a new therapeutic vehicle for ischemic cardiac disease.

Figure 1

Characteristics of cardiac progenitor cells and exosomes. (a) The phase morphology of isolated CPCs growing on gelatin-coated dish; scale bar, 100 μm. (b) Flow Cytometry analyzed purified Sca-1⁺CPCs from the first preparation. Typical purity of isolation is >95% after magnetic beads sorting. (c) Nanoparticle trafficking analyzed the diameters and concentration of exosomes; 1 is a representative screen shot of the NTA videos, the bright white dot indicates one moving particle, (2) NTA estimated the size of the EVs between 90 and 300 nm, and the mode of these particles is 85 nm, and predict the proper concentration is around 1.04 × 10⁸ particles per ml, the dilution is 1 : 80, 3 is a heat map pattern of 2, and 4 is a detail statistical report. (d) Electron micrograph analyzed CPC-derived exosomes. The image showed small vesicles of approximately 100 nm in diameter. Scale bar, 100 nm. (e) Western blotting characterized CPC-exosomes. CPC-exosomes preparation was separated by SDS-polyacrylamide gel electrophoresis, and electroblotted to the poly vinylidene fluoride (PVDF) membrane, and probed with exosomes marker CD63, CD9, Alix

Figure 2

H₂O₂ affects the cells' apoptosis and exosomes' production. (a) Western blotting analyzed procaspase-3 and cleaved caspase-3 in H9C2 cells after treatment of different H₂O₂ concentrations (N=3, *P<0.05, **P<0.01 versus 0 μM). (b) NTA counted the production of exosomes derived from normal cultured CPCs (Nor-exo) and exosomes from 100 μM H₂O₂ treated CPCs (H₂O₂-exo), H₂O₂-CPC produce more exosomes ((3.36±0.66) × 10⁹ versus (1.31±0.29) × 10⁹, N=5; *P<0.05 versus Nor-CPC)

Figure 3

H₂O₂ changes the levels of miRNAs in CPC-exosomes. (a) Main reported miRNAs participated in cardiomyocytes apoptosis, H₂O₂ induced apoptosis and miRNAs contained in the EVs after myocardial infarction, some miRNAs are multifunctional in both fields, such as miR-21, miR-214 and miR-34a. (b) Quantitative PCR analysis of main reported miRNAs in Nor-exo and H₂O_2-exo, six miRNAs (miR-21, 24, 214, 132, 195, 210) are founded in H₂O₂-exo, and only miR-21 was significantly upregulated (N=6, **P<0.01 versus Nor-exo). (c) RT-qPCR analyzed and miR-21 in H9C2 cells after different concentrations of H₂O₂ treated (N=3, *P<0.05, **P<0.01 versus 0 μM). (d) 100 μM H₂O₂-treated H9C2 cells transfected with miR-21 mimics, inhibitors and both negative controls, and western blotting analyzed procaspase-3 and cleaved caspase-3 proteins levels in H9C2 cells (N=5, **P<0.01, ***P<0.001 versus blank group)

Figure 4

PDCD4 is a target gene of miR-21. (a) Western blotting analyzed PDCD4 in H9C2 cells after different concentrations of H₂O₂ treated (N=4, **P<0.01, ***P<0.001 versus 0 μM). (b) One hundred micromolar H₂O₂-treated H9C2 cells transfected with miR-21 mimics, inhibitors and both negative controls, western blotting analyzed PDCD4 protein levels in H9C2 cells (N=3, *P<0.05, **P<0.01, ***P<0.001 versus blank group). (c) Schematic representation of 3′-UTR of PDCD4 mRNA reporter with and without the miR-21 seed-binding site (red). (d) Luciferase activity assay of HEK293T cells transfected with luciferase constructs containing WT-3′UTR and Mut-3′UTR of PDCD4 (N=3, ***P<0.001 versus blank group). (e) Western blot analyzed the protein levels of caspase-3 and PDCD4 after H9C2 transfected with siRNA-PDCD4 (N=3, **P<0.01 versus H₂O₂ group, ^#P<0.05, ^##P<0.01 versus siRNA-PDCD4 group). (f–h) Representative dot plots of cell apoptosis were showed after Annexin V/PI dual staining. The proportion of dead cells (Annexin V−/PI+), live cells (Annexin V−/PI−), early apoptotic cells (Annexin V+/PI−) and late apoptotic/necrotic cells (Annexin V+/PI+) was respectively measured for comparison. (j)The percentage of apoptotic cells was represent for both early and late apoptotic cells (N=3, **P<0.01 versus siRNA-PDCD4 group)

Figure 5

CPC-exosomes inhibit the apoptotic function caused by H₂O₂ in cardiomyocytes. (a) RT-qPCR analyzed miR-21 in H9C2 cells under 100 μM H₂O₂ after CPCs co-culture, Nor-exo and H₂O₂-exo pre-protection (N=4, *P<0.05, ***P<0.001 versus control group). (b) Western blotting analyzed procaspase-3, cleaved caspase-3 and PDCD4 in H9C2 cells under 100 μM H₂O₂ after CPCs co-culture, Nor-exo and H₂O₂-exo pre-protection (N=4, **P<0.01, ***P<0.001 versus control groups). (c–g) Representative dot plots of cell apoptosis were showed after Annexin V/PI dual staining. (h) The percentage of apoptotic cells was represent for both early and late apoptotic cells (N=3, *P<0.05, **P<0.01 versus H₂O₂ groups)

Figure 6

Schematic of our working hypothesis. In some oxidative stress-related cardiac injury, CPCs secreted a considerable amount of exosomes, which can be taken up by cardiomyocytes, and miR-21 contained in CPC-exosomes had an inhibiting role in the apoptosis pathway through downregulating PDCD4