Cardiac protection by preconditioning is generated via an iron-signal created by proteasomal degradation of iron proteins.

Ischemia associated injury of the myocardium is caused by oxidative damage during reperfusion. Myocardial protection by ischemic preconditioning (IPC) was shown to be mediated by a transient 'iron-signal' that leads to the accumulation of apoferritin and sequestration of reactive iron released during the ischemia. Here we identified the source of this 'iron signal' and evaluated its role in the mechanisms of cardiac protection by hypoxic preconditioning. Rat hearts were retrogradely perfused and the effect of proteasomal and lysosomal protease inhibitors on ferritin levels were measured. The iron-signal was abolished, ferritin levels were not increased and cardiac protection was diminished by inhibition of the proteasome prior to IPC. Similarly, double amounts of ferritin and better recovery after ex vivo ischemia-and-reperfusion (I/R) were found in hearts from in vivo hypoxia pre-conditioned animals. IPC followed by normoxic perfusion for 30 min ('delay') prior to I/R caused a reduced ferritin accumulation at the end of the ischemia phase and reduced protection. Full restoration of the IPC-mediated cardiac protection was achieved by employing lysosomal inhibitors during the 'delay'. In conclusion, proteasomal protein degradation of iron-proteins causes the generation of the 'iron-signal' by IPC, ensuing de-novo apoferritin synthesis and thus, sequestering reactive iron. Lysosomal proteases are involved in subsequent ferritin breakdown as revealed by the use of specific pathway inhibitors during the 'delay'. We suggest that proteasomal iron-protein degradation is a stress response causing an expeditious cytosolic iron release thus, altering iron homeostasis to protect the myocardium during I/R, while lysosomal ferritin degradation is part of housekeeping iron homeostasis.

Aging is an organ-specific process: changes in homeostasis of iron and redox proteins in the rat.

Organ-specific changes of iron- and redox-related proteins occur with age in the rat. Ferritin, the major iron storage and detoxifying protein, as well as the proteins of the methionine-centered redox cycle (MCRC) were examined in old and young animals, and showed organ-dependent changes. In spleens and livers of aged rats, ferritin (protein) levels were greater than in young ones, and their iron saturation increased, rendering higher ferritin-bound iron (FtBI). Iron saturation of the ferritin molecule in the tongues and sternohyoids of old rats was lower but ferritin level was higher than in young rats, resulting in increased FtBI with age. Ferritin level in the esophagus of older rats was lower than in young rats but its molecular iron content higher thus the total FtBI remained the same. In the larynx, both ferritin and its iron content were the same in young and old animals. MCRC proteins were measured in livers and spleens only. With aging, methionine sulfoxide reductase A and B (MsrA and MsrB) levels in livers and spleens decreased. Thioredoxin1 (Trx) and Trx-reductase1 were elevated in old spleens, but reduced in livers. Aged spleens showed reduced Msr isozyme activity; but in the liver, its activity increased. mRNA changes with age were monitored and found to be organ specific. These organ-specific changes could reflect the different challenges and the selective pathways of each organ and its resultant capacity to cope with aging.

Heart protection by ischemic preconditioning: a novel pathway initiated by iron and mediated by ferritin.

Ischemic preconditioning is a well-known procedure transiently protecting the heart against injury associated with prolonged ischemia, through mechanism/s only partly understood. The aim of this study was to test whether preconditioning-induced protection of the heart involves an iron-based mechanism, including the generation of an iron signal followed by accumulation of ferritin. In isolated rat hearts perfused in the Langendorff configuration, we measured heart contractility, ferritin levels, ferritin-iron content, and mRNA levels of ferritin subunits. Ischemic preconditioning caused rapid accumulation of ferritin, reaching 359% of the baseline value (set at 100%). This was accompanied by a parallel decline in ferritin-bound iron: from 2191+/-548 down to 760+/-34 Fe atoms/ferritin molecule, p<0.05. Ferritin levels remained high during the subsequent period of prolonged ischemia, and returned to nearly the baseline value during the reperfusion phase. Selective iron chelators (acetyl hydroxamate or Zn-desferrioxamine) abrogated the functional protection and suppressed ferritin accumulation, thus demonstrating the essentiality of an iron signal in the preconditioning-induced protective mechanism. Moreover, introduction of an iron-containing ternary complex, known to import iron into cells, caused a three-fold accumulation of ferritin and simulated the preconditioning-induced functional protection against prolonged myocardial ischemia. The ischemic preconditioning-and-ischemia-induced increase in ferritin levels correlated well with the accumulation of ferritin L-subunit mRNA: 5.44+/-0.47 vs 1.23+/-0.15 (units) in the baseline, p<0.05, suggesting that transcriptional control of ferritin L-subunit synthesis had been activated. Ischemic preconditioning initiates de novo synthesis of ferritin in the heart; the extra ferritin is proposed to serve a 'sink' for redox-active iron, thus protecting the heart from iron-mediated oxidative damage associated with ischemia-reperfusion injury. The present results substantiate a novel iron-based mechanism of ischemic preconditioning and could pave the way for the development of new modalities of heart protection.

Iron-chelator complexes as iron sources for early developing human erythroid precursors.

Developing erythroid cells are dependent on transferrin (Tf) to acquire iron in amounts sufficient for hemoglobin production. Previously, we showed that although these cells cannot grow in culture in the absence of Tf, ferritin (Ft) can substitute Tf to some extent and support the development of hemoglobin-containing cells. In the current study, we investigated the ability of various iron sources to replace Tf in cultures of normal human erythroid precursors. The results showed that whereas Ft and hemin supported erythroid cell proliferation and hemoglobinization in Tf-free cultures to some extent, ferric amonium citrate and iron complexed with several chelators had little or no effect. Although salicylaldehyde-isonicotinoyl-hydrazone, which is a tridentate lipid-soluble chelator, complexed with iron increased both cytosolic and mitochondrial labile iron pools, it failed to support heme synthesis and did not decrease the surface Tf receptors, suggesting that its iron is not recognized by the cells. Moreover, this iron-chelator complex did not support erythroid precursor proliferation and hemoglobinization. Thus, although under normal conditions, Tf is the major route of iron uptake, Ft and hemin, but not iron-chelator complexes, may serve as alternative iron sources under Tf-poor conditions.

Ischemic preconditioning of the rat retina: protective role of ferritin.

Ischemic preconditioning (IPC) of the retina, accomplished by ischemia of short duration, is highly effective in preventing subsequent severe injury caused by iron-dependent free radical burst after prolonged ischemia. To investigate the mechanistic basis for IPC rescue, we examined changes in the levels of the retinal redox-active and labile iron pool, ferritin, and ferritin-bound iron. Prolonged ischemia severely impaired retinal function, with total loss of the full-field electroretinographic response. IPC provided marked protection against such injury. Histological examination revealed that ischemia-associated structural damage and loss of cells in the outer and inner nuclear layers were largely prevented by IPC. Ferritin levels decreased after prolonged ischemia but remained close to normal when the ischemic episode was preceded by IPC. The protective effect of IPC on retinal function and ferritin was blocked by a zinc-desferrioxamine complex known to interfere with iron signaling. The results suggest a mechanism whereby IPC activates an iron signaling pathway leading to a marked increase in ferritin levels, which mediates resistance to prolonged ischemia.

Macrophages function as a ferritin iron source for cultured human erythroid precursors.

Iron is essential for the survival as well as the proliferation and maturation of developing erythroid precursors (EP) into hemoglobin-containing red blood cells. The transferrin-transferrin receptor pathway is the main route for erythroid iron uptake. Using a two-phase culture system, we have previously shown that placental ferritin as well as macrophages derived from peripheral blood monocytes could partially replace transferrin and support EP growth in a transferrin-free medium. We now demonstrate that in the absence of transferrin, ferritin synthesized and secreted by macrophages can serve as an iron source for EP. Macrophages trigger an increase in both the cytosolic and the mitochondrial labile iron pools, in heme and in hemoglobin synthesis, along with a decrease in surface transferrin receptors. Inhibiting macrophage exocytosis, binding extracellular ferritin with specific antibodies, inhibiting EP receptor-mediated endocytosis or acidification of EP lysosomes, all resulted in a decreased EP growth when co-cultured with macrophages under transferrin-free conditions. The results suggest that iron taken up by macrophages is incorporated mainly into their ferritin, which is subsequently secreted by exocytosis. Nearby EP are able to take up this ferritin probably through clathrin-dependent, receptor-mediated endocytosis into endosomes, which following acidification and proteolysis release the iron from the ferritin, making it available for regulatory and synthetic purposes. Thus, macrophages support EP development under transferrin-free conditions by delivering essential iron in the form of metabolizable ferritin.

Action of chelators in iron-loaded cardiac cells: Accessibility to intracellular labile iron and functional consequences.

Labile iron in hemosiderotic plasma and tissue are sources of iron toxicity. We compared the iron chelators deferoxamine, deferiprone, and deferasirox as scavengers of labile iron in plasma and cardiomyocytes at therapeutic concentrations. This comprised chelation of labile plasma iron (LPI) in samples from thalassemia patients; extraction of total cellular iron; accessing labile iron accumulated in organelles and preventing formation of reactive-oxidant species; and restoring impaired cardiac contractility. Neonatal rat cardiomyocytes were used for monitoring chelator extraction of LCI (labile cell iron) as 59Fe; assessing in situ cell iron chelation by epifluorescence microscope imaging using novel fluorescent sensors for iron and reactive oxygen species (ROS) selectively targeted to organelles, and monitoring contractility by time-lapse microscopy. At plasma concentrations attained therapeutically, all 3 chelators eliminated LPI but the orally active chelators rapidly gained access to the LCI pools of cardiomyocytes, bound labile iron, attenuated ROS formation, extracted accumulated iron, and restored contractility impaired by iron overload. The effect of deferoxamine at therapeutically relevant concentrations was primarily by elimination of LPI. The rapid accessibility of the oral chelators deferasirox and deferiprone to intracellular labile iron compartments renders them potentially efficacious for protection from and possibly reversal of cardiac damage induced by iron overload.

Objectives and mechanism of iron chelation therapy.

Prevention of cardiac mortality is the most important beneficial effect of iron chelation therapy. Unfortunately, compliance with the rigorous requirements of daily subcutaneous deferoxamine (DFO) infusions is still a serious limiting factor in treatment success. The development of orally effective iron chelators such as deferiprone and ICL670 is intended to improve compliance. Although total iron excretion with deferiprone is somewhat less than with DFO, deferiprone may have a better cardioprotective effect than DFO due to deferiprone's ability to penetrate cell membranes. Recent clinical studies indicate that oral ICL670 treatment is well tolerated and is as effective as parenteral DFO used at the standard dose of 40 mg/kg of body weight/day. Thus, for the patient with transfusional iron overload in whom results of DFO treatment are unsatisfactory, several orally effective agents are now available to avoid serious organ damage. Finally, combined chelation treatment is emerging as a reasonable alternative to chelator monotherapy. Combining a weak chelator that has a better ability to penetrate cells with a stronger chelator that penetrates cells poorly but has a more efficient urinary excretion may result in improved therapeutic effect through iron shuttling between the two compounds. The efficacy of combined chelation treatment is additive and offers an increased likelihood of success in patients previously failing DFO or deferiprone monotherapy.

Iron overload and chelation.

Iron is one of the most common elements in nature. As a transition metal it is very efficient in electron transport and redox reactions. The proteins and enzymes in which iron is an essential component play a key role in respiration, energy production, detoxification of harmful oxygen species and cell replication. Despite the abundance of iron in nature, the solubility of its stable ferric form is extremely low. Hence, living organisms were compelled to develop efficient mechanisms for iron transport and storage.

Iron chelation therapy.

Although iron chelation therapy with deferoxamine (DFO) has changed life expectancy in thalassemic patients, compliance with the rigorous requirements of long-term subcutaneous DFO infusions is unsatisfactory. This problem underlines the current efforts for developing alternative, orally effective chelators to improve compliance and treatment results. For the patient with transfusional iron overload in whom results of DFO treatment are unsatisfactory, several orally effective agents are now available. The most important of the new generation of oral chelators are deferiprone and ICL670. Total iron excretion with deferiprone is less than with DFO, but deferiprone has a better ability to penetrate cell membranes and may have a better cardioprotective effect than DFO. Current studies of the clinical efficacy and tolerability of ICL670 indicate that at a single oral dose of 20 mg/kg daily, it may be as effective as parenteral DFO used at the standard dose of 40 mg/kg daily. Combined chelation treatment, employing a weak chelator that penetrates cells better, and a stronger chelator with efficient urinary excretion, may result in improved therapeutic effect through iron shuttling between the two compounds. The efficacy of combined chelation treatment is additive and offers an increased likelihood of success in patients previously failing DFO or deferiprone monotherapy.

Effects of combined chelation treatment with pyridoxal isonicotinoyl hydrazone analogs and deferoxamine in hypertransfused rats and in iron-loaded rat heart cells.

Although iron chelation therapy with deferoxamine (DFO) results in improved life expectancy of patients with thalassemia, compliance with parenteral DFO treatment is unsatisfactory, underlining the need for alternative drugs and innovative ways of drug administration. We examined the chelating potential of pyridoxal isonicotinoyl hydrazone (PIH) analogs, alone or in combination with DFO, using hypertransfused rats with labeled hepatocellular iron stores and cultured iron-loaded rat heart cells. Our in vivo studies using 2 representative PIH analogs, 108-o and 109-o, have shown that PIH analogs given orally are 2.6 to 2.8 times more effective in mobilizing hepatocellular iron in rats, on a weight-per-weight basis, than parenteral DFO administered intraperitoneally. The combined effect of DFO and 108-o on hepatocellular iron excretion was additive, and response at a dose range of 25 to 200 mg/kg was linear. In vitro studies in heart cells showed that DFO was more effective in heart cell iron mobilization than all PIH analogs studied. Response to joint chelation with DFO and PIH analogs was similar to an increase in the equivalent molar dose of DFO alone, rather than the sum of the separate effects of the PIH analog and DFO. This finding was most likely the result of iron transfer from PIH analogs to DFO, a conclusion supported directly by iron-shuttle experiments using fluorescent DFO. These findings provide a rationale for the combined, simultaneous use of iron-chelating drugs and may have useful, practical implications for designing novel strategies of iron chelation therapy.

Roles of ferritin and iron in ischemic preconditioning of the heart.

Iron and copper play major roles in biological systems, catalyzing free radical production and consequently causing damage. The relatively high levels of these metals, which are mobilized into the coronary flow following prolonged ischemia, have been incriminated as key players in reperfusion injury to the heart. In the present communication we investigated other roles of iron - providing protection to the ischemic heart via preconditioning (PC). PC was accomplished by subjecting isolated rat hearts to three episodes of 2 min ischemia separated by 3 min of reperfusion. Prolonged ischemia followed the PC phase. PC hearts (group I) were compared to hearts subjected to normal perfusion (group II, no ischemia) and to ischemia without PC (group III). Group I showed a marked improvement in the recovery of hemodynamic function vs. group III. Biochemical parameters further substantiated the PC protection provided to group I against prolonged ischemia. Correspondingly, group I presented markedly lower re-distribution and mobilization of iron and copper into the coronary flow, following prolonged ischemia, as evinced from the decrease in total levels, and in the 'free' fraction of iron and copper. During the PC phase no loss of cardiac function was observed. A small wave of re-distribution and mobilization of iron (typically less than 4-8% of the value of 35 min ischemia) was recorded. The cellular content of ferritin (Ft) measured in the heart was significantly higher in group I than in group III (0.90 and 0.54 microg/mg, respectively). Also, iron-saturation of Ft was significantly lower for PC hearts, compared to both groups II and III (0.22 vs. 0.32 and 0.31 microg/mg, for 35 min ischemia, respectively). These findings are in accord with the proposal that intracellular re-distribution and mobilization of small levels of iron, during PC, cause rapid accumulation of ferritin - the major iron-storage protein. It is proposed that iron play a dual role: (i) It serves as a signaling pathway for the accumulation of Ft following the PC phase. This iron is not involved in cardiac injury, but rather prepares the heart against future high levels of 'free' iron, thus reducing the degree of myocardial damage after prolonged ischemia. (ii) High levels of iron (and copper) are mobilized following prolonged ischemia and cause tissue damage.

Acquisition, storage and release of iron by cultured human hepatoma cells.

BACKGROUND/AIMS: The recovery from iron overload is hampered by the limited number of pathways and therapeutic agents available for the augmentation of iron secretion/excretion. The present study was aimed to investigate the process of iron storage and release by cultured human hepatoma cells, the role of transferrin receptors and ferritin in this process as well as the effect of iron chelators. METHODS: We followed the acquisition, storage and release of iron by cultured cells HepG2 and Hep3B by biochemical means and electron microscopy. RESULTS: The uptake of iron from diferric transferrin (Trf) was extremely low, while iron as ferric-ammonium-citrate (FAC) was taken up readily, especially by Hep3B cells. Up to 80% of the iron taken up by hepatoma cells was released to the medium. The rate of spontaneous iron release depended on the extent of iron loading. ApoTrf and deferoxamine facilitated release after 1- and 7-day iron-exposure. Up to a third of the radio-iron released from the cells was associated with ferritin. The release of ferritin-iron was not enhanced by either deferoxamine or Trf. CONCLUSIONS: Ferritin-iron release appeared to be an important mechanism of iron discarding in cultured human hepatoma cells, independent of the activity of chelating agents.

The labile iron pool of hepatocytes in chronic and acute iron overload and chelator-induced iron deprivation.

BACKGROUND: The cytosolic labile iron pool (LIP) is a transitory, catalytically active compartment that has been implicated in cell iron homeostasis and in metal-induced cytotoxicity. AIMS: We attempted to define LIP levels in living hepatocytes derived from chronic overloaded rats and from normal hepatocytes either acutely loaded with iron or depleted by chelation. METHODS: LIP levels were measured in living rat hepatocytes derived from normal and iron-fed rats. RESULTS: Steady-state LIP levels in untreated hepatocytes ( approximately 0.2 microM) were raised by 1.8-fold following iron loading and were reduced by 0.66-fold by short-term chelation treatment. Changes in LIP were accompanied by the corresponding changes in iron-responsive protein (IRP) activity and ferritin levels, that, in rat hepatocytes isolated from chronically loaded animals, raised by approximately 19-fold. CONCLUSIONS: Whereas ferritin levels provide an index of long-term or cumulative iron loading, LIP measurements provide an "instantaneous" parameter of iron availability within hepatocytes. The latter was associated with the cell chelatable pool in cells derived from normal and iron-loaded animals, both of which showed similar accessibility to iron chelators.