We used proteomics to detect regional differences in protein expression levels from mitochondrial fractions of control, ischemia-reperfusion (IR), and ischemic preconditioned (IPC) rabbit hearts. Using 2-DE, we identified 25 mitochondrial proteins that were differentially expressed in the IR heart compared with the control and IPC hearts. For three of the spots, the expression patterns were confirmed by Western blotting analysis. These proteins included 3-hydroxybutyrate dehydrogenase, prohibitin, 2-oxoglutarate dehydrogenase, adenosine triphosphate synthases, the reduced form of nicotinamide adenine dinucleotide (NADH) oxidoreductase, translation elongation factor, actin alpha, malate dehydrogenase, NADH dehydrogenase, pyruvate dehydrogenase and the voltage-dependent anion channel. Interestingly, most of these proteins are associated with the mitochondrial respiratory chain and energy metabolism. The successful use of multiple techniques, including 2-DE, MALDI-TOF-MS and Western blotting analysis demonstrates that proteomic analysis provides appropriate means for identifying cardiac markers for detection of ischemia-induced cardiac injury.

. Nitric oxidecGMP-protein kinase G signaling pathway induces anoxic preconditioning through activation of ATP-sensitive K ϩ channels in rat hearts. Am J Physiol Heart Circ Physiol 290: H1808 -H1817, 2006. First published December 9, 2005 doi:10.1152/ajpheart.00772.2005.-Nitric oxide (NO) plays an important role in anoxic preconditioning to protect the heart against ischemia-reperfusion injuries. The present work was performed to study better the NO-cGMP-protein kinase G (PKG) signaling pathway in the activation of both sarcolemmal and mitochondrial ATP-sensitive K ϩ (KATP) channels during anoxic preconditioning (APC) and final influence on reducing anoxia-reperfusion (A/R)-induced cardiac damage in rat hearts. The upstream regulating elements controlling NO-cGMP-PKG signal-induced KATP channel opening that leads to cardioprotection were investigated. The involvement of both inducible and endothelial NO synthases (iNOS and eNOS) in the progression of this signaling pathway was followed. Final cellular outcomes of ischemia-induced injury after different preconditioning in the form of lactate dehydrogenase release, DNA strand breaks, and malondialdehyde formation as indexes of cell injury and lipid peroxidation, respectively, were investigated. The lactate dehydrogenase and malondialdehyde values decreased in the groups that underwent preconditioning periods with specific mitochondrial KATP channels opener diazoxide (100 M), nonspecific mitochondrial KATP channels opener pinacidil (50 M), S-nitroso-Nacetylpenicillamine (SNAP, 300 M), or ␤-phenyl-1,N 2 -etheno-8-bromoguanosine-3Ј,5Ј-cyclicmonophosphorothioate, Sp-isomer (10 M) before the A/R period. Preconditioning with SNAP significantly reduced the DNA damage. The effect was blocked by glibenclamide (50 M), 5-hydroxydecanoate (100 M), N G -nitro-L-arginine methyl ester (200 M), and ␤-phenyl-1,N 2 -etheno-8-bromoguanosine-3Ј,5Ј-cyclic monophosphorothioate, Rp-isomer (1 M). The results suggest iNOS, rather than eNOS, as the major contributing NO synthase during APC treatment. Moreover, the PKG shows priority over NO as the upstream regulator of NO-cGMP-PKG signal-induced KATP channel opening that leads to cardioprotection during APC treatment.guanosine 3Ј,5Ј-cyclic monophosphate; adenosine 5Ј-triphosphate; oxidative damage ISCHEMIC PRECONDITIONING, in which short-term occlusion and reperfusion of a coronary artery are followed by long-term occlusion, can reduce subsequent ischemia-induced injury to the heart (42). Nitric oxide (NO), protein kinase G (PKG), and ATP-sensitive K ϩ (K ATP ) channels (both the sarcolemmal and mitochondrial subtypes) can mimic the effects of ischemic preconditioning in the heart, and mitochondrial K ATP channels appear to be the end effectors (19,20,25). The activation of these channels may improve the recovery of regional contractility of myocardium by shortening the duration of action potentials and by attenuating membrane depolarization, both of which would decrease myocardial contractility and reduce energy expenditure during ischem...

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In this study, we investigated the effects of melatonin on adriamycin-induced cardiotoxicity both in vivo in rats and in vitro, and on the antitumor activities of adriamycin on MDA-231 and NCI breast cancer cells. Rats that received a single intraperitoneal injection of 25 mg/kg adriamycin showed a mortality rate of 86%, which was reduced to 20% by melatonin treatment (10 mg/kg, SC for 6 days). Melatonin attenuated adriamycin-induced body-weight loss, hemodynamic dysfunction, and the morphologic and biochemical alterations caused by adriamycin. Melatonin also reduced adriamycin-induced nuclear DNA fragmentation, as assessed by the comet assay. In addition, the antitumor activity of adriamycin could be maintained using lower doses of this drug in combination with melatonin. Melatonin treatment in the concentration range of 0.1-2.5 mM inhibited the growth of human breast cancer cells. In terms of oncolytic activity, the combination of adriamycin and melatonin improved the antitumor activity of adriamycin, as indicated by an increase in the number of long-term survivors as well as decreases in body-weight losses resulting from adriamycin treatment. These results indicate that melatonin not only protects against adriamycin-induced cardiotoxicity but also enhances its antitumor activity. This combination of melatonin and adriamycin represents a potentially useful regimen for the treatment of human neoplasms because it allows the use of lower doses of adriamycin, thereby avoiding the toxic side effects associated with this drug.

J. Dynamic changes in nitric oxide and mitochondrial oxidative stress with site-dependent differential tissue response during anoxic preconditioning in rat heart. Am J Physiol Heart Circ Physiol 293: H1457-H1465, 2007. First published June 1, 2007; doi:10.1152/ajpheart.01282.2006.-In this study, dynamic changes in nitric oxide (NO) and mitochondrial superoxide (O 2 •Ϫ ) were examined during anoxic preconditioning (AP) in rat heart model. AP and anoxia-reoxygenation (A/R) were performed on isolated hearts and single cardiomyocytes. The cellular insult in the form of infarct size and DNA damage were localized and correlated with NO synthases (endothelial and inducible) expression levels. The results showed that endocardium was the most affected region in AP groups, whereas the larger area of infarct was confined to mid-and epicardium in the A/R group. Interestingly, a high-level expression of immunofluorescent NO synthases was restricted to viable areas in the AP. In contrast to the gradual increase in O 2•Ϫ level that occurred in the AP group, a sudden massive increase in its level was demonstrated at the onset of reoxygenation in the A/R group. The observed increase in NO production during reoxygenation in the AP group was attenuated by inducible NO synthase inhibitor. The study revealed, on a real-time basis, the role played by preconditioning for modulating NO and O 2•Ϫ levels on behalf of cell survival. The results afford a better understanding of cardiac-adapting mechanism during AP and the role of inducible NO synthase in this important phenomenon. mitochondrial superoxide; endocardium; inducible nitric oxide synthase HEART FAILURE DUE TO ischemic-reperfusion injury is one of the most serious problems in heart disease. Ischemic preconditioning, in which short-term ischemia and reperfusion are followed by long-term ischemia, can protect the heart against ischemia and reperfusion-induced cardiac injuries (25). Although preconditioning by a nitric oxide (NO) donor can mimic the anoxic preconditioning (AP) phenomenon to protect the heart from anoxia-reoxygenation (A/R) injuries, the evidence that a change in NO concentration contributes to either pro-or antiapoptotic effects remains equivocal (13). NO stimulates soluble guanylate cyclase, leading to an activation of both the sarcolemmal and mitochondrial ATP-sensitive K ϩ (K ATP ) channels, thus reducing ischemia and reperfusion-induced heart damage (10, 18). However, this is a double-edged sword, since NO interacts with the reactive oxygen species (ROS) superoxide (O 2•Ϫ ) to form peroxynitrite (ONOO Ϫ ), which is a reactive nitrogen species (RNS) that causes oxidative DNA damage (4,16, 27). An early state of reoxygenation produces a massive increase in ROS levels, and the resulting apoptosis and/or necrosis impair cardiac function through arrhythmia or heart failure (26).Recent studies have demonstrated that differences in O 2•Ϫ dismutase distribution, action potential duration, and myosinactin dynamics lead to regional differences in ischemia and reperfus...