FIG 5.

Mfn1 and miR-140 are able to regulate mitochondrial fission in cardiomyocytes. (A and B) Analysis of mitochondrial fission and apoptosis from cardiomyocytes infected with adenoviruses harboring the Mfn1 or β-galactosidase (β-gal) construct and then exposed to H₂O₂ (A) or doxorubicin (B) treatment. (A) Mitochondrial fission was analyzed 6 h after treatment. Apoptosis was analyzed 12 h after treatment by a TUNEL assay. (B) Mitochondrial fission was analyzed 5 h after treatment. Apoptosis was analyzed 15 h after treatment by a TUNEL assay. *, P < 0.05. (C) Effect of knockdown miR-140 on anoxia-induced apoptosis and mitochondrial fission. Cardiomyocytes were transfected with anti-miR-140 or the negative control (anti-miR-NC) and then exposed to anoxia. Data represent three separate experiments. *, P < 0.05. (D) Mitochondrial morphology in cardiomyocytes transfected with anti-miR of miR-140 or the negative control (anti-miR-NC) and then treated with H₂O₂. (E) Counting of cells with mitochondrial fission and apoptosis. Cardiomyocytes were treated as described for panel D. *, P < 0.05. (F) Analysis of Mfn1 expression by immunofluorescence. Cardiomyocytes were transfected with anti-miR of miR-140 or its negative control (NC) and then exposed to 100 μmol/liter H₂O₂. Immunofluorescence and TUNEL assays were performed after H₂O₂ treatment. Red, Mfn1 expression; green, TUNEL-positive cell; blue, DAPI staining. Bar, 10 mm. (G) Effect of miR-140 on mitochondria morphology and cell death in cardiomyocytes. Cardiomyocytes were infected with adenoviral miR-140 or the control (miR-con) at the indicated titers, and the percentage of cells with mitochondrial fission or undergoing cell death was determined. *, P < 0.05. (H) Counting of cardiomyocytes with mitochondrial fission and apoptotic cardiomyocytes infected with adenoviral miR-140 or the control (miR-con) and then treated with 20 μM H₂O₂. Data represent three separate experiments. *, P < 0.05 compared with the control. Data are expressed as the means ± standard errors of the means of three independent experiments.