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Cardiac tissue undergoes changes during ischemia-reperfusion (I-R) that compromise its normal function. Cell death is one of the consequences of such damage, as well as diminution in nitric oxide (NO) content. This signaling molecule regulates the function of the cardiovascular system through dependent and independent effects of cyclic guanosine monophosphate (cGMP). The independent cGMP pathway involves post-translational modification of proteins by S-nitrosylation. Studies in vitro have shown that NO inhibits the activity of caspases and calpains through S-nitrosylation of a cysteine located in their catalytic site, so we propose to elucidate if the regulatory mechanisms of NO are related with changes in S-nitrosylation of cell death proteins in the ischemic-reperfused myocardium. We used two compounds that increase the levels of NO by different mechanisms: Prolame, an amino-estrogenic compound with antiplatelet and anticoagulant effects that induces the increase of NO levels in vivo by activating the endothelial nitric oxide synthase (eNOS) and that has not been tested as a potential inhibitor of apoptosis. On the other hand, S-Nitroso- N -acetylpenicillamine (SNAP), a synthetic NO donor that has been shown to decrease cell death after inducing hypoxia-reoxygenation in cell cultures. Main experimental groups were Control, I-R, I-R+Prolame and I-R+SNAP. Additional groups were used to evaluate the NO action pathways. Contractile function represented as heart rate and ventricular pressure was evaluated in a Langendorff system. Infarct size was measured with 2,3,5-triphenyltetrazolium chloride stain. NO content was determined indirectly by measuring nitrite levels with the Griess reaction and cGMP content was measured by Enzyme-Linked ImmunoSorbent Assay. DNA integrity was evaluated by DNA laddering visualized on an agarose gel and by Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling assay. Activities of caspase-3, caspase-8, caspase-9 and calpain-1 were evaluated spectrophotometrically and the content of caspase-3 and calpain-1 by western blot. S-nitrosylation of caspase-3 and calpain-1 was evaluated by labeling S-nitrosylated cysteines. Our results show that both Prolame and SNAP increased NO content and improved functional recovery in post-ischemic hearts. cGMP-dependent and S-nitrosylation pathways were activated in both groups, but the cGMP-independent pathway was preferentially activated by SNAP, which induced higher levels of NO than Prolame. Although SNAP effectively diminished the activity of all the proteases, a correlative link between the activity of these proteases and S-nitrosylation was not fully established., Competing Interests: The authors declare that they have no competing interests.

Cytidine-5'-diphosphocholine (CDP-choline) participates as an intermediary in the synthesis of phosphatidylcholine, an essential component of cellular membranes. Citicoline treatment has shown beneficial effects in cerebral ischemia, but its potential to diminish reperfusion damage in liver has not been explored. In this work, we evaluated the hepatoprotective effect of citicoline and its possible association with inflammatory/oxidative stress and mitochondrial function because they are the main cellular features of reperfusion damage. Ischemia/reperfusion (I/R) in rat livers was performed with the Pringle's maneuver, clamping the 3 elements of the pedicle (hepatic artery, portal vein, and biliary tract) for 30 minutes and then removing the clamp to allow hepatic reperfusion for 60 minutes. The I/R + citicoline group received the compound before I/R. Liver injury was evaluated by measuring aspartate aminotransferase and alanine aminotransferase as well as lactic acid levels in serum; proinflammatory cytokines, proresolving lipid mediators, and nuclear factor kappa B content were determined as indicators of the inflammatory response. Antioxidant effects were evaluated by measuring markers of oxidative stress and antioxidant molecules. Oxygen consumption and the activities of the respiratory chain were used to monitor mitochondrial function. CDP-choline reduced aspartate aminotransferase (AST), alanine aminotransferase (ALT), as well as lactic acid levels in blood samples from reperfused rats. Diminution in tumor necrosis factor alpha (TNF-α) and increase in the proresolving lipid mediator resolvin D1 were also observed in the I/R+citicoline group, in comparison with the I/R group. Oxidative/nitroxidative stress in hepatic mitochondria concurred with deregulation of oxidative phosphorylation, which was associated with the loss of complex III and complex IV activities. In conclusion, CDP-choline attenuates liver damage caused by ischemia and reperfusion by reducing oxidative stress and maintaining mitochondrial function. Liver Transplantation XX XX-XX 2018 AASLD., (© 2018 The Authors. Liver Transplantation published by Wiley Periodicals, Inc. on behalf of American Association for the Study of Liver Diseases.)

Ceramide accumulation in mitochondria has been associated with reperfusion damage, but the underlying mechanisms are not clearly elucidated. In this work we investigate the role of sphingomyelinases in mitochondrial ceramide accumulation, its effect on reactive oxygen species production, as well as on mitochondrial function by using the sphingomyelinase inhibitor, tricyclodecan-9-yl-xanthogenate (D609). Correlation between neutral sphingomyelinase (nSMase) activity and changes in ceramide content were performed in whole tissue and in isolated mitochondria from reperfused hearts. Overall results demonstrated that D609 treatment attenuates cardiac dysfuncion, mitochondrial injury and oxidative stress. Ceramide was accumulated in mitochondria, but not in the microsomal fraction of the ischemic-reperfused (I/R) group. In close association, the activity of nSMase increased, whereas glutathione (GSH) levels diminished in mitochondria after reperfusion. On the other hand, reduction of ceramide levels in mitochondria from I/R+D609 hearts correlated with diminished nSMase activity, coupling of oxidative phosphorylation and with mitochondrial integrity maintenance. These results suggest that mitochondrial nSMase activity contributes to compartmentation and further accumulation of ceramide in mitochondria, deregulating their function during reperfusion., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A high proportion of primary percutaneous coronary interventions performed in the setting of acute myocardial infarction, concur with inadequate myocardial perfusion at the microvascular level. This phenomenon, known as "no-reflow" contributes to reperfusion injury, poor prognosis and to unfavorable clinical outcome. In this study, we evaluated the hypothesis that the synthetic 17β-aminoestrogen Prolame, may confer cardioprotection and prevent against no-reflow. In an open-chest model of 30-min ischemia and 90-min reperfusion, male Wistar rats were randomly assigned to different groups: Control, Prolame, Prolame followed by the nitric oxide synthase inhibitor (L-NAME), and 17β-estradiol. Areas of risk, infarct size and no-reflow were determined by planimetry with triphenyltetrazolium chloride and thioflavin-S stains. Structural damage of the vasculature was measured as capillary compression in clarified tissue after intra-atrial injection of Microfil. Hemodynamic function was obtained at the end of stabilization, ischemia and reperfusion; nitric oxide (NO·) content was determined indirectly using the Griess reaction. Activation of the eNOS signaling cascade was determined by western blot. Prolame reduced the infarcted area, decreased the zones of no-reflow and capillary compression by activating the PI3K/Akt/eNOS signaling pathway in correlation with NO· increase. Prolame also activated endothelial cells augmenting NO· production, which was inhibited by ICI182780 (a selective estrogen receptor down-regulator), supporting the notion that the cardioprotective effect of Prolame involves the preservation of endothelium through the activation of estrogen receptor downstream signaling. Our results provide evidence that Prolame has potential therapeutic application in patients with AMI, as it prevents from both vascular and cardiac tissue damage.