Exosomes Derived from miR-214-Enriched Bone Marrow-Derived Mesenchymal Stem Cells Regulate Oxidative Damage in Cardiac Stem Cells by Targeting CaMKII

Figure 6

Change in CaMKII expression during BMSC-exo-induced antiapoptotic effect in CSCs under oxidative stress. Cultured CSCs were transfected with CaMKII3 overexpression cDNA or siRCaMKII3 for 48 h. Then, the cells were treated with BMSC-exos under different conditions for 24 h and/or cultured with 100 μM H₂O₂ for 2 h. (a) RT-qPCR were carried out to detect CaMKII mRNA levels. Compared with other treatments, CaMKII3 transfection significantly upregulated CaMKII expression, and SiRCaMKII3 transfection significantly downregulated CaMKII mRNA levels in CSCs. (b-c) Western blotting revealed that compared with H₂O₂ treatment, CaMKII3 transfection upregulated CaMKII protein levels, while SiRCaMKII3 transfection downregulated CaMKII protein levels in CSCs. Additionally, compared with Hypoxic-exos, Hypoxic-exos + CaMKII3 upregulated CaMKII protein levels. (d) Representative dot plots of cell apoptosis after Annexin V/PI dual staining are shown. The upper left quadrant (% gated) shows necrotic cells (Annexin V−/PI+); the upper right quadrant (% gated) shows late apoptotic cells (Annexin V+/PI+); the left lower quadrant (% gated) shows live cells (Annexin V−/PI−); and the right lower quadrant (% gated) shows early apoptotic cells (Annexin V+/PI-). These cells were measured for comparison. (e) The percentage of apoptotic cells represents both early and late apoptotic cells. Compared with the H₂O₂ group, the SiRCaMKII3-transfected group displayed a decreased percentage of apoptotic cells. In addition, the Hypoxic-exo-induced protective effect on CSC apoptosis under oxidative stress was suppressed by CaMKII3 overexpression. (f) Intracellular ROS level was determined by FCM. The P2 percentage indicates the proportion of cells with increased ROS production, with fluorescence levels above background DCF fluorescence levels. (g) Compared with that in H₂O₂-treated CSCs, fluorescence intensity of intracellular ROS was decreased in SiRCaMKII3-treated CSCs. In addition, the Hypoxic-exo-induced protective effect on CSCs against oxidative stress injury was suppressed by CaMKII3 overexpression. ; compared with the H₂O₂ group. compared with Hypoxic-exos group.

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