Background-Whether alterations in mitochondrial morphology affect the susceptibility of the heart to ischemia/ reperfusion injury is unknown. We hypothesized that modulating mitochondrial morphology protects the heart against ischemia/reperfusion injury. Methods and Results-In response to ischemia, mitochondria in HL-1 cells (a cardiac-derived cell line) undergo fragmentation, a process that is dependent on the mitochondrial fission protein dynamin-related protein 1 (Drp1). Transfection of HL-1 cells with the mitochondrial fusion proteins mitofusin 1 or 2 or with Drp1 K38A , a dominantnegative mutant form of Drp1, increased the percentage of cells containing elongated mitochondria (65Ϯ4%, 69Ϯ5%, and 63Ϯ6%, respectively, versus 46Ϯ6% in control: nϭ80 cells per group; PϽ0.05), decreased mitochondrial permeability transition pore sensitivity (by 2.4Ϯ0.5-, 2.3Ϯ0.7-, and 2.4Ϯ0.3-fold, respectively; nϭ80 cells per group; PϽ0.05), and reduced cell death after simulated ischemia/reperfusion injury (11.6Ϯ3.9%, 16.2Ϯ3.9%, and 12.1Ϯ2.9%, respectively, versus 41.8Ϯ4.1% in control: nϭ320 cells per group; PϽ0.05). Treatment of HL-1 cells with mitochondrial division inhibitor-1, a pharmacological inhibitor of Drp1, replicated these beneficial effects. Interestingly, elongated interfibrillar mitochondria were identified in the adult rodent heart with confocal and electron microscopy, and in vivo treatment with mitochondrial division inhibitor-1 increased the percentage of elongated mitochondria from 3.6Ϯ0.5% to 14.5Ϯ2.8% (Pϭ0.023). Finally, treatment of adult murine cardiomyocytes with mitochondrial division inhibitor-1 reduced cell death and inhibited mitochondrial permeability transition pore opening after simulated ischemia/reperfusion injury, and in vivo treatment with mitochondrial division inhibitor-1 reduced myocardial infarct size in mice subject to coronary artery occlusion and reperfusion (21.0Ϯ2.2% with mitochondrial division inhibitor-1 versus 48.0Ϯ4.5% in control; nϭ6 animals per group; PϽ0.05). Conclusion-Inhibiting mitochondrial fission protects the heart against ischemia/reperfusion injury, suggesting a novel pharmacological strategy for cardioprotection. Key Words: cardiomyocytes Ⅲ hypoxia Ⅲ ischemia Ⅲ myocardial infarction Ⅲ reperfusion I nnovative treatment strategies for protecting the heart from ischemia/reperfusion injury (IRI) are needed to improve clinical outcomes in patients with coronary heart disease. Previous studies suggest that mitochondria are highly dynamic and that changes in mitochondrial shape can affect a variety of biological processes such as apoptosis, respiration, mitosis, and development. 1,2 Mitochondria change their morphology by undergoing either fusion or fission, resulting in either elongated, tubular, interconnected mitochondrial networks or fragmented, discontinuous mitochondria, respectively. 1,2 These 2 opposing processes are regulated by the mitochondrial fusion proteins mitofusin (Mfn) 1, Mfn2, and optic atrophy protein 1 and the mitochondrial fission proteins dynamin-related...

Smith CC, Dixon RA, Wynne AM, Theodorou L, Ong SG, Subrayan S, Davidson SM, Hausenloy DJ, Yellon DM. Leptininduced cardioprotection involves JAK/STAT signaling that may be linked to the mitochondrial permeability transition pore. Am J Physiol Heart Circ Physiol 299: H1265-H1270, 2010. First published July 23, 2010; doi:10.1152/ajpheart.00092.2010.-Leptin-induced protection against myocardial ischemia-reperfusion (I/R) injury involves the activation of the reperfusion injury salvage kinase pathway, incorporating phosphatidylinositol 3-kinase-Akt/protein kinase B and p44/42 MAPK, and the inhibition of the mitochondrial permeability transition pore (MPTP). Recently published data indicate that the JAK/STAT signaling pathway, which mediates the metabolic actions of leptin, also plays a pivotal role in cardioprotection. Consequently, in the present study we considered the possibility that JAK/STAT signaling linked to the MPTP may be involved in modulating the cardioprotective actions of leptin. Employing rat in vitro models (Langendorffperfused hearts and cardiomyocytes) of I/R injury, we investigated the actions of leptin (10 nM), administered at reperfusion, in the presence or absence of the JAK2 inhibitor, AG-490 (5 M). Leptin reduced infarct size significantly (control, 60.05 Ϯ 7.41% vs. leptin treated, 29.9 Ϯ 3.24%, P Ͻ 0.05), protection being abolished by AG-490. Time course studies revealed that leptin caused a 171% (P Ͻ 0.001) increase in STAT3/tyrosine-705 phosphorylation at 2.5 min reperfusion; however, increases were not seen at 5, 10, 15, or 30 min reperfusion. Contrasting with STAT3, Akt/serine-473 phosphorylation was not significantly increased until 15 min into the reperfusion phase (140%, P Ͻ 0.05). AG-490 blocked the leptin-induced rise in STAT3 phosphorylation seen at 2.5 min reperfusion but did not influence Akt/serine-473 phosphorylation at 15 min. Leptin reduced the MPTP opening (P Ͻ 0.001), which was blocked by AG-490. This is the first study to yield evidence that JAK/STAT signaling linked to the MPTP plays a role in leptin-induced cardioprotection. Under the experimental conditions employed, STAT3 phosphorylation appears to have occurred earlier during reperfusion than that of Akt. Further research into the interactions between these two signaling pathways in the setting of I/R injury is, however, required. myocardium; ischemia-reperfusion injury; Janus-activated kinase/signal transducer and activator of transcription signaling

Background The restoration of blood and oxygen to ischaemic myocardium under threat of infarction is of paramount importance, but reperfusion paradoxically exacerbates injury (IR injury). Exosomes are extracellular, lipid bilayer vesicles that range from 30 to 100nm in diameter. Exosomes released from in vitro cultured stem cells have been shown to be cardioprotective. Exosomes are also present in the blood of healthy individuals, but their properties are unknown. We hypothesized that plasma exosomes have cardioprotective properties. Aim To characterize the circulating exosomes of healthy rats, and to determine whether they protect against IR injury in HL-1 cardiac cells, in a Langendorff isolated, perfused rat heart model, and in an in vivo rat model of IR injury. Methods and Results Exosomes were isolated from rat plasma using the standard ultracentrifugation protocol. Their identity as exosomes was confirmed by their typical diameter of 87±2 nm measured by nanoparticle tracking analysis, their typical-shape morphology by electron microscopy, and by their expression of exosome marker proteins CD63 and HSP70. An average concentration of 2.5±1.1x10