Figure 3. miR-361 provokes mitochondrial fission program.

A. Knockdown of miR-361 prevents mitochondrial fission induced by A/R. Cardiomyocytes were transfected with miR-361 antagomir (anta-361) or the antagomir negative control (anta-NC), then exposed to A/R. The cells were stained with MitoTracker Green (left panel), Bar = 20 µm. The cells with fragmented mitochondria were counted (right panel). *p<0.05 versus A/R alone. B. Knockdown of miR-361 prevents apoptosis induced by A/R. Cardiomyocytes were treated as described for (A). Apoptosis was analyzed by TUNEL assay. *p<0.05 versus A/R alone. C. Knockdown of miR-361 attenuates mitochondrial fission upon I/R. Adult male C57BL/6 mice (8 weeks old) were delivered in three consecutive days, intravenous injections of miR-361 antagomir (anta-361) or antagomir control (anta-NC) at doses of 35 mg/kg body weight. 3 days after injection the mice were exposed to 45 min of ischemia and 3 hours of reperfusion. Counting of the fragmented mitochondria was shown in upper panel. Counting of the apoptotic cell was shown in low panel and right panel. TUNEL-positive myocyte nuclei (apoptotic cells) are green. Nuclei stained by DAPI show blue. Cardiomyocytes were labeled with α-actinin. n = 6, *p<0.05 versus I/R alone. D and E. miR-361 transgenic mice exhibited increased mitochondrial fission, apoptosis and myocardial infarction sizes in response to ischemia/reperfusion (I/R). Wild type C57BL/6 mice and miR-361 transgenic mice (8 weeks old) were exposed to 45 min of ischemia and 3 hours of reperfusion. Mitochondrial fission, apoptosis (D) and myocardial infarction sizes (E) were analyzed. n = 8, *p<0.05 versus WT+I/R. F. The knockdown of miR-484 attenuates the inhibitory effect of miR-361 knockdown on mitochondria fission and apoptosis. Cardiomyocytes were transfeted with the miR-361 antagomir, miR-484 antagomir or antagomir negative control, and then treated with A/R. Mitochondria fission and apoptosis were analyzed. *p<0.05.