MicroRNA-21 knockout blocked the regulatory effects of isoflurane preconditioning on endothelial nitric-oxide synthase and mitochondrial nicotinamide adenine dinucleotide in ischemic myocardium. (A) MicroRNA-21 knockout blocked isoflurane-induced increases in the ratio of endothelial nitric-oxide synthase dimers/monomers in myocardium 5 min after ischemia (mean ± SD, n = 5 hearts/group). (B) Representative Western blot bands showing the expression of endothelial nitric-oxide synthase dimers (230 kDa) and monomers (130 kDa) and glyceraldehyde-3-phosphate dehydrogenase (37 kDa) as a loading control in myocardium 5 min after ischemia (n = 3 hearts/blot). (C) MicroRNA-21 knockout blocked isoflurane-induced decreases in mitochondrial nicotinamide adenine dinucleotide levels 5 min after ischemia (n = 9 hearts in control, microRNA-21 knockout, and isoflurane groups and 8 hearts in microRNA-21 knockout+isoflurane group). All hearts were stabilized for 30 min in a Langendorff apparatus and subjected to 5 min of global ischemia. Control = C57BL/6 mouse hearts underlying 5 min of ischemia; microRNA-21 knockout, microRNA-21 knockout hearts subjected to 5 min of ischemia; isoflurane = C57BL/6 mouse hearts treated with isoflurane and subsequently underlying 5 min of ischemia; microRNA-21 knockout+isoflurane = microRNA-21 knockout hearts treated with isoflurane and subsequently subjected to 5 min of ischemia. *P < 0.05 versus control groups; †P < 0.05 versus microRNA-21 knockout groups; #P < 0.05 versus isoflurane groups. (D) Endothelial nitric-oxide synthase knockout blocked isoflurane-induced decreases in mitochondrial nicotinamide adenine dinucleotide levels 5 min after ischemia (n = 10 hearts in control, 9 hearts in endothelial nitric-oxide synthase knockout and isoflurane groups, and 8 hearts in endothelial nitric-oxide synthase knockout+isoflurane group). Control = C57BL/6 mouse hearts underlying 5 min of ischemia; endothelial nitric-oxide synthase knockout, endothelial nitric-oxide synthase knockout hearts subjected to 5 min of ischemia; isoflurane = C57BL/6 mouse hearts treated with isoflurane and subsequently subjected to 5 min of ischemia; endothelial nitric-oxide synthase knockout+isoflurane = endothelial nitric-oxide synthase knockout hearts treated with isoflurane and subsequently subjected to 5 min of ischemia. *P < 0.05 versus control groups; †P < 0.05 versus endothelial nitric-oxide synthase knockout groups; #P < 0.05 versus isoflurane groups.