Figure 3: miR-421 regulates mitochondrial fragmentation and apoptosis.

(a) miR-421 antagomir inhibits the elevation of miR-421 levels on H₂O₂. Cardiomyocytes were transfected with anta-421 or anta-NC, then exposed to H₂O₂. Cells were harvested for qRT–PCR analysis of miR-421 levels. Data are shown as mean±s.e.m. of four independent experiments. Analysis was performed with one-way analysis of variance (ANOVA) followed by Tukey–Kramer post hoc analysis. *P<0.05 versus H₂O₂ alone. (b,c) Knockdown of miR-421 prevents mitochondrial fragmentation and apoptosis induced by H₂O₂. Cardiomyocytes were treated as described in a, the cells were stained with MitoTracker Red (b), Bar=20 μm. The cells with fragmented mitochondria were counted (b). Apoptosis was analysed by TdT-mediated dUTP nick end labelling (TUNEL) assay (c). Data are shown as mean±s.e.m. of three independent experiments. Analysis was performed with one-way ANOVA followed by Tukey–Kramer post hoc analysis. *P<0.05 versus H₂O₂ alone. (d) miR-421 is upregulated in response to I/R injury. Mice were subjected to I/R as described in Methods. MiR-421 levels were detected by qRT–PCR. Data are shown as mean±s.e.m. of five independent experiments. *P<0.05 versus sham in Student’s t-test. (e,f) Knockdown of miR-421 attenuates mitochondrial fragmentation on I/R. Adult male C57BL/6 mice (8 weeks old) were delivered in 3 consecutive days, intravenous injections of miR-421 antagomir (anta-421) or antagomir control (anta-NC) at doses of 30 mg kg⁻¹ body weight. Three days after injection, the mice were subjected to I/R. The fragmented mitochondria were shown in e and counting of the fragmented mitochondria was shown in f. Data are shown as mean±s.e.m. of six independent experiments. Analysis was performed with one-way ANOVA followed by Tukey–Kramer post hoc analysis. *P<0.05 versus I/R alone. (g) Knockdown of miR-421 attenuates apoptosis on I/R. Counting of the apoptotic cell was shown. TUNEL-positive cardiomyocyte nuclei (apoptotic cells) were green. Nuclei stained by DAPI showed blue. Cardiomyocytes were labelled with α-actinin. Data are shown as mean±s.e.m. of six independent experiments. Analysis was performed with one-way ANOVA followed by Tukey–Kramer post hoc analysis. *P<0.05 versus I/R alone. DAPI, 4,6-diamidino-2-phenylindole.