ps at 30 min, 3 hours, and 24 hours of reperfusion. Plasma concentrations of pitavastatin were significantly higher in the Pitavastatin-NP group than in the pitavastatin group 30 min after reperfusion. There were no differences in pitavastatin concentrations in the lung between the Pitavastatin-NP and pitavastatin groups. Effects of Pitavastatin-NP on MI size Intravenous treatment with Pitavastatin-NP containing pitavastatin 1 mg/kg at the time of reperfusion significantly reduced MI size 24 hours after reperfusion. FITC-NP was used as a control and showed no effects on MI size. As previously reported by other groups using rosuvastatin or fluvastatin, intravenous treatment with pitavastatin at 1 and 10 mg/kg at the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19741728 time of reperfusion did not reduce MI size. Treatment with Pitavastatin-NP or pitavastatin alone did not affect AAR in the hearts, plasma biochemical data except CPK, or hemodynamic parameters. Decreased plasma CPK levels in the Pitavastatin-NP group are consistent with therapeutic effects of Pitavastatin-NP on MI size. 9 / 23 Nanomedicine for Myocardial Reperfusion Injury Fig 3. We performed mitochondria swelling assays to examine the effects of pitavastatin-NP on mPTP opening and found that pitavastatin-NP did not affect mitochondria swelling, while the pretreatment with cyclosporine A reduced the mitochondria swelling. Pitavastatin-NP induced phosphorylation of Akt 3 hours after IR in a PI3K-dependent manner, but not 15 minutes and 30 minutes after IR Data are expressed as the mean SEM. doi:10.1371/journal.pone.0132451.t003 11 / 23 Nanomedicine for Myocardial Reperfusion Injury Fig 4. Effects of Pitavastatin-NP on mitochondrial permeability transition pore openning., Effects of WM on therapeutic effects of Pitavastatin-NP on MI size. Data are expressed as the meanSEM. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests., Effects of Pitavastatin-NP at the time of reperfusion on cytosolic cytochrome C in IR myocardium 30 minutes after reperfusion. N = 6 per group. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests., Effects of Pitavastatin-NP at the time of reperfusion on mitochondrial cytochrome C in IR myocardium 30 minutes after reperfusion. Data are meanSEM. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests., Effects of Pitavastatin-NP at the time of reperfusion on mitochondrial swelling in IR myocardium 10 minutes after reperfusion. N = 5 per group. Data are compared using one-way ANOVA followed by Bonferroni’s multiple comparison tests. doi:10.1371/journal.pone.0132451.g004 when mPTP opening plays a role in cytochrome C leakage and myocardial necrosis. Immunohistochemistry revealed that the phosphorylated Akt localized mainly in cardiomyocytes within the AAR. Treatment with Pitavastatin-NP also induced GSK3 12 / 23 Nanomedicine for Myocardial Reperfusion Injury Nanomedicine for Myocardial Reperfusion Injury Fig 6. Effects of Pitavastatin-NP on cell death after IR., Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with Cyclosporine A every 12 hours starting 36 hours before MedChemExpress NVP-BKM120 ischemia on MI size. N = 7 per group. Data are compared using one-way ANOVA followed by Bonferroni’s PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19741130 multiple comparison tests., Effects of Pitavastatin-NP at the time of reperfusion after pretreatment with CsA every 12 hours starting 36 hours before ischemia on cytosolic cytochrome C in IR myocardium 30 minutes af