Alterations of iron metabolism during hearth ischemia/reperfusion injury. Cytoprotective effects of simvastatin

Ischemic heart disease, the main cause of mortality and morbidity in industrialized countries, is a metabolic phenomenon due to an inadequate oxygenation of heart tissue caused by the closing or narrowing of the coronary arteries. However, the ischemic condition and the subsequent tissue reperfusion, lead to several functional and metabolic changes that globally define the so-called “ischemia/reperfusion injury”. This injury leads to metabolic and functional alterations, in particular due to the production of the Oxygen Reactive Species (ROS) that are able to promote cell damage. Because iron is involved in the ROS production by the Haber-Weiss-Fenton reaction, the aim of this study was to elucidate the molecular mechanisms underlying the iron metabolism during the cardiac ischemia/reperfusion. To this aim it has been analyzed the activity and the expression of the main proteins involved in iron homeostasis, such as the Iron Regulatory Proteins, Transferrin Receptor 1 (TfR1), and ferritin in an in vivo model of cardiac ischemia/reperfusion. The results show that in rats hearts subjected to the ischemic/reperfusion injury, the activity of IRP1 was altered without changing its cellular content. The evaluation of the TfR1 levels showed an evident decrease of the expression of this protein during ischemia followed by a marked increase after the reperfusion phase, while regarding the ferritin expression it was observed a considerable decrease of the cytosolic levels of this protein only after the reperfusion phase. Moreover, using rat cardyomyoblasts (H9c2 cell line) in an in vitro model of hypoxia and reoxygenation, it was evaluated the cellular levels of the “Labile Iron Pool” (LIP), showing a “free iron” increase after the reoxygenation phase, in accordance with the observed changes of the TfR1 and ferritin expression. In addition, it was observed an increased ROS production after the hypoxia/reoxygenation damage and, using the iron chelator SIH (Salicylaldehyde Isonicotinoyl Hydrazone), it was showed that a significant part of these ROS depend by the higher levels of the LIP, strongly suggesting that iron is involved in the development of the cardiac damage induced by ischemia/reperfusion conditions. Other aim of this study has been to evaluate the cytoprotective role of the cholesterol-lowering drug Simvastatin, during the ischemic/reperfusion injury, because of its anti-inflammatory and antioxidant effects (“pleiotropic effects”). Simvastatin, at concentration of 0,01µM, reduced the reactive nitrogen species levels and ROS productions in rat cardyomyoblasts (H9c2 cell line) subjected to hypoxia/reoxygenation conditions and also was able to reduce the cellular levels of the “Labile Iron Pool”, justifying the reduced production of the ROS and the resulting increased cell viability, observed after the drug treatment. Moreover, Simvastatin increased the ferritin levels, in particular during hypoxia conditions, thus explaining the LIP reduction after treatment with this drug. In conclusion, these results not only clarify the crucial role that iron plays in the progression of ischemic damage, but also show that proteins regulating the homeostasis of this metal, such as ferritin, may be a target of the Simvastatin, which could be used for the prevention of oxidative damage induced by cardiac ischemic conditions. Should this be the case, a new horizon as an antioxiodant opens for Simvastatin.