Protection by Nitric Oxide Donors of Isolated Rat Hearts Is Associated with Activation of Redox Metabolism and Ferritin Accumulation.

Preconditioning (PC) procedures (ischemic or pharmacological) are powerful procedures used for attaining protection against prolonged ischemia and reperfusion (I/R) injury, in a variety of organs, including the heart. The detailed molecular mechanisms underlying the protection by PC are however, complex and only partially understood. Recently, an 'iron-based mechanism' (IBM), that includes de novo ferritin synthesis and accumulation, was proposed to explain the specific steps in cardioprotection generated by IPC. The current study investigated whether nitric oxide (NO), generated by exogenous NO-donors, could play a role in the observed IBM of cardioprotection by IPC. Therefore, three distinct NO-donors were investigated at different concentrations (1-10 µM): sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP). Isolated rat hearts were retrogradely perfused using the Langendorff configuration and subjected to prolonged ischemia and reperfusion with or without pretreatment by NO-donors. Hemodynamic parameters, infarct sizes and proteins of the methionine-centered redox cycle (MCRC) were analyzed, as well as cytosolic aconitase (CA) activity and ferritin protein levels. All NO-donors had significant effects on proteins involved in the MCRC system. Nonetheless, pretreatment with 10 µM SNAP was found to evoke the strongest effects on Msr activity, thioredoxin and thioredoxin reductase protein levels. These effects were accompanied with a significant reduction in infarct size, increased CA activity, and ferritin accumulation. Conversely, pretreatment with 2 µM SIN-1 increased infarct size and was associated with slightly lower ferritin protein levels. In conclusion, the abovementioned findings indicate that NO, depending on its bio-active redox form, can regulate iron metabolism and plays a role in the IBM of cardioprotection against reperfusion injury.

Time window of nitroxide effect on myocardial ischemic-reperfusion injury potentiated by iron.

Transition metals such as iron and copper potentiate the postischemic reperfusion (I/R) injury induced by oxygen-derived radical and nonradical toxic species (ROS). Various natural and synthetic antioxidants have been previously tested to ameliorate such injury, yet the limitations of the common antioxidants are well known. An alternative strategy for combating oxidative damage is presented wherein cell-permeable, nitroxide stable radicals, which act as SOD-mimics and oxidize reduced metals thus prompting the Fenton-like chemistry, are investigated for utility in ameliorating I/R injury. Our study concentrates on the early effect of nitroxide on the myocardial I/R injury. Isolated rat hearts in the Langendorff configuration were equilibrated with Krebs-Henseleit buffer and then subjected to 18 min of normothermic global ischemia followed by 20 min reperfusion. Iron administered as Fe(III)-citrate (10 microM) did not affect the cardiac function under normoxia but did potentiate I/R injury and decreased the recovery during reperfusion. The iron-induced damage was manifested by further deterioration of the cardiac hemodynamic function and the energy status as reflected by decreased tissue level of phosphorylated nucleotides. Nitroxide at 200 microM protected against the iron-potentiated I/R injury by improving the recovery of the hemodynamic function and the cardiac energy status. Exogenously added iron requires bioreduction to form deleterious Fe(II) bound to critical cellular sites. The nitroxide, which enters the cell and oxidizes the reduced metal instantaneously, provided protection even when administered 2 or 3.5, but not 5 min, after the onset of reperfusion. Thus, its narrow therapeutic time window provides insight into the schedule of the I/R injurious process.