The MCK mouse heart model of Friedreich's ataxia: Alterations in iron-regulated proteins and cardiac hypertrophy are limited by iron chelation

Abstract

There is no effective treatment for the cardiomyopathy of the most common autosomal recessive ataxia, Friedreich's ataxia (FA). The identification of potentially toxic mitochondrial (MIT) iron (Fe) deposits in FA suggests that Fe plays a role in its pathogenesis. This study used the muscle creatine kinase conditional frataxin (Fxn) knockout (mutant) mouse model that reproduces the classical traits associated with cardiomyopathy in FA. We examined the mechanisms responsible for the increased cardiac MIT Fe loading in mutants. Moreover, we explored the effect of Fe chelation on the pathogenesis of the cardiomyopathy. Our investigation showed that increased MIT Fe in the myocardium of mutants was due to marked transferrin Fe uptake, which was the result of enhanced transferrin receptor 1 expression. In contrast to the mitochondrion, cytosolic ferritin expression and the proportion of cytosolic Fe were decreased in mutant mice, indicating cytosolic Fe deprivation and markedly increased MIT Fe targeting. These studies demonstrated that loss of Fxn alters cardiac Fe metabolism due to pronounced changes in Fe trafficking away from the cytosol to the mitochondrion. Further work showed that combining the MIT-permeable ligand pyridoxal isonicotinoyl hydrazone with the hydrophilic chelator desferrioxamine prevented cardiac Fe loading and limited cardiac hypertrophy in mutants but did not lead to overt cardiac Fe depletion or toxicity. Fe chelation did not prevent decreased succinate dehydrogenase expression in the mutants or loss of cardiac function. In summary, we show that loss of Fxn markedly alters cellular Fe trafficking and that Fe chelation limits myocardial hypertrophy in the mutant.

Friedreich's ataxia (FA) is the most common autosomal recessive neuro- and cardiodegenerative disorder, resulting from insufficient expression of the mitochondrial (MIT) protein frataxin (Fxn) (1). Many studies infer a role for Fxn in MIT iron (Fe) metabolism, particularly iron–sulfur cluster (ISC) biosynthesis (2, 3). Deletion of the yeast Fxn homolog 1 gene or loss of Fxn in FA patients or Fxn knockout (mutant) mice promotes MIT Fe accumulation, hypersensitivity to oxidants, and the loss of MIT DNA and ISC-containing enzymes (2–5). Increased cardiac Fe deposition and perturbations in heme biosynthesis and ATP production suggest that FA pathogenesis is linked to MIT Fe overload (6). Furthermore, the antioxidant idebenone improves heart and neurological function in FA patients, suggesting the presence of oxidant stress (7).

Iron loading can lead to toxicity in Fe overload disease due to the ability of Fe to redox cycle, leading to cytotoxic radicals (6). The potential of Fe loading to become toxic may be more pronounced in the highly redox-active MIT environment (3). There is no effective treatment for FA, although evidence of MIT Fe loading within cardiomyocytes from FA patients supports the use of chelation as a therapeutic strategy (6). The Fe chelator desferrioxamine (DFO) has some ability to rescue FA fibroblasts from oxidant stress (8). However, the inability of DFO to permeate the mitochondrion means it is ineffective for FA treatment (6, 9).

To circumvent the poor permeability of DFO (6), the lipophilic, membrane-permeable chelator pyridoxal isonicotinoyl hydrazone (PIH), which mobilizes MIT Fe (9), was developed (10). Studies in vitro, in vivo, and in a clinical trial have shown the marked chelation efficacy and high tolerability of PIH (6, 9–11).

Cardiomyopathy is a frequent cause of death in FA. To test the potential of Fe chelation therapy for FA treatment, we have used muscle creatine kinase (MCK) conditional Fxn knockout mice lacking Fxn in cardiomyocytes (5). This model exhibits classical phenotypic traits of the cardiomyopathy in FA, including marked cardiac MIT Fe accumulation and deficiency in ISC enzymes (5).

In this study, we show that Fxn deficiency leads to abnormal Fe metabolism in vivo in MCK mutant mice, and that treatment with PIH and DFO prevents cardiac Fe loading and limits myocardial hypertrophy.

Results

Altered Cardiac Fe Metabolism in the Mutant.

Little is known regarding the function of Fxn in vivo and the role of Fe loading in FA pathogenesis. To this end, we examined the Fe metabolism of MCK mutants and the potential of Fe chelation to prevent cardiomyopathy. This model was chosen as it closely reflects the alterations in the heart of FA patients, including MIT Fe loading (5). The Fe accumulation is observed only after 7 weeks of age, with no detectable deposits being found earlier (5).

In initial studies, cardiac Fe uptake was assessed by using the physiological serum Fe binding protein ⁵⁹Fe–transferrin (Tf) to radiolabel 9-week-old wild-type (WT) and mutant mice via tail vein (i.v.) injection. Incorporation of ⁵⁹Fe into the whole heart was measured after 24 h. A significant (P < 0.001) increase in the level of cardiac ⁵⁹Fe was observed in mutant mice compared to WT controls (Fig. 1A). To assess the intracellular distribution of ⁵⁹Fe, hearts were fractionated to yield cytosolic and stromal MIT membrane (SMM) fractions. It was evident in mutant compared to WT mice that there was a marked decrease in the proportion of cytosolic ⁵⁹Fe, although there was a significant (P < 0.001) increase of ⁵⁹Fe in the SMM (Fig. 1B). This result directly confirmed that deletion of Fxn leads to MIT Fe loading in the mutant (5) and results in a relative cytosolic Fe deficiency.

Fig. 1.

Marked alterations in ⁵⁹Fe uptake and distribution and expression of molecules that play key roles in Fe metabolism occur in mutant relative to WT mice. (A) Iron uptake from ⁵⁹Fe-Tf into the heart is increased in the mutant. Mutant and WT mice (9 weeks old) were injected i.v. with ⁵⁹Fe-Tf and killed 24 h later. Hearts were washed and assessed for ⁵⁹Fe activity. (B) Mutant hearts show a cytosolic Fe deficiency relative to the stromal-mitochondrial membrane (SMM). Hearts from (A) were processed as described in the Materials and Methods and the cytosol and SMM separated and assessed for ⁵⁹Fe activity. (C and D) Examination of (C) mRNA and (D) protein expression of Fxn and molecules involved in Fe metabolism in hearts from 4- and 9-week-old WT and mutant mice. mRNA and protein expression were assessed as per Materials and Methods. Densitometry is expressed relative to the respective WT at each age and GAPDH. Results in A and B are mean ± SD (3 experiments). The RT-PCR and Western panels in C and D are typical experiments, whereas the densitometry is mean ± SD (3–5 experiments). ***, P < 0.001.

Considering the alterations in ⁵⁹Fe uptake and distribution, we examined the changes that occur at the mRNA and protein levels of molecules involved in cardiac Fe metabolism at 4 and 9 weeks of age. These ages were chosen because at 4 weeks no overt phenotype was present, whereas at 9 weeks a severe cardiomyopathy was found (5). There was a significant (P < 0.001) decrease in Fxn expression at the mRNA (Fig. 1C) and protein levels (Fig. 1D) in 4- and 9-week-old mutants. However, Fxn expression was not totally ablated, due to other cells within the total heart homogenate apart from cardiomyocytes (e.g., fibroblasts), which are not targeted by the conditional knockout strategy (5).

Transferrin receptor 1 (TfR1) was significantly (P < 0.001) up-regulated in 4- and 9-week-old mutants relative to WT mice at the mRNA (Fig. 1C) and protein (Fig. 1D) levels. The extent of TfR1 protein up-regulation in mutants was significantly (P < 0.001) more pronounced at 9 than 4 weeks of age (Fig. 1D).

The Fe-storage protein ferritin is composed of heavy (H) and light (L) chains (12). Comparing WT and mutant mice at 4 and 9 weeks of age, H- and L-ferritin mRNA expression was not markedly or significantly altered (Fig. 1C). In contrast, Western analysis showed a significant (P < 0.001) decrease in H- and L-ferritin protein expression in mutants compared to WT mice at 9 weeks of age (Fig. 1D). The molecular alterations in TfR1 and ferritin expression suggest the cytosol was in an Fe-deficient state. This hypothesis was supported by the significantly (P < 0.001) decreased expression of the Fe export molecule ferroportin1 in mutant compared to WT mice at the mRNA and protein levels at 9 weeks of age (Fig. 1 C and D). Because ferroportin1 protein expression is down-regulated by decreased cellular Fe levels (13), this further supports the idea that cardiomyocytes are experiencing a cytosolic Fe deficiency.

MIT ferritin is thought to act as an Fe-storage protein within this organelle (14). Similar to cytosolic H- and L-ferritin expression, there was no alteration in MIT ferritin mRNA between mutant and WT mice at both ages (Fig. 1C), although a decrease in protein expression was detected in mutant mice, particularly at 9 weeks of age (Fig. 1D). It is notable that to detect this mRNA, 40 cycles of PCR were necessary in both genotypes, suggesting it was very poorly expressed. This observation suggested MIT ferritin was not playing a role in storing Fe in the mutant, as a marked increase in its expression would be expected to accommodate the Fe loading.

The observations above indicate pronounced alterations in cardiac Fe homeostasis caused by deletion of Fxn. Given that iron regulatory protein (IRP)-1 and -2 are key regulators of TfR1, ferritin and ferroportin1 expression (12), we examined IRP–RNA binding activity in mutant and WT mice.

IRP2- RNA-Binding Activity is Increased in Mutant Heart.

Iron metabolism is regulated, in part, by IRP1 and IRP2, which interact with iron-response elements (IREs) in the 3′ untranslated region (UTR) of TfR1 mRNA or 5′ UTR of H- and L-ferritin or ferroportin1 mRNA, in response to cytosolic Fe (12). IRP binding to the 3′ UTR IRE stabilizes TfR1 mRNA, increasing its expression, whereas binding to the 5′ IREs of ferritin or ferroportin1 mRNA inhibits translation (12, 15, 16).

Gel-retardation analysis indicated significantly (P < 0.01) increased IRP2–RNA binding activity in mutant relative to WT mice only at 9 weeks of age, while there was little alteration in IRP1-RNA binding (Fig. 2). It has been demonstrated that 2-mercaptoethanol (β-ME) converts IRP1 to its RNA-binding state (15, 16). However, its addition to lysates did not significantly increase IRP1-binding activity (Fig. 2). This indicated that IRP1 was largely in its active RNA-binding form in WT and mutant mice at 4 and 9 weeks of age.

Fig. 2.

IRP2–RNA-binding activity in the heart is increased in mutant mice relative to the WT at 9, but not 4, weeks of age, whereas IRP1-RNA binding does not alter. (A and B) IRP–RNA-binding activity was assayed by gel-retardation analysis. At 4 and 9 weeks of age WT and mutant mice were killed, and the heart was washed and homogenized in Munro buffer. Equal amounts of heart extract were assayed in the absence and presence of 2-mercaptoethanol (β-ME). Results in (A) are from a typical experiment, whereas the densitometry in (B) is mean ± SD of 3 experiments. **, P < 0.01; ***, P < 0.001.

Previous studies indicated that IRP2 is crucial in controlling Fe-regulated gene expression in vivo at physiological oxygen tension (15, 16). This is because in contrast to IRP1, only IRP2 registers intracellular Fe levels and modulates its RNA-binding activity at physiological oxygen levels (15, 16). Indeed, our results are consistent with the theory of Rouault and colleagues (15, 16) that IRP2 primarily regulates mammalian Fe homeostasis in vivo despite its lower expression compared to IRP1.

The increased IRP2 activity in 9-week mutants confirms a cytosolic Fe deficiency, which could mediate the marked up-regulation of TfR1 and down-regulation of ferritin and ferroportin1 protein (Fig. 1D). As there was no alteration in IRP1 or IRP2 RNA-binding activity at 4 weeks of age (Fig. 2), other factors (e.g., at the transcriptional level) could be responsible for the less pronounced alteration in TfR1 and ferritin expression at this time.

The elevated IRP2–RNA-binding activity in mutant relative to WT mice (Fig. 2) and increased Fe uptake into the heart (Fig. 1A) and SMM (Fig. 1B) suggest that Fxn deficiency leads to a major alteration in Fe trafficking. Specifically, lack of Fxn leads to a cytosolic Fe deficiency relative to MIT Fe loading.

Marked Alterations in Cytosolic ⁵⁹Fe Distribution Within Ferritin in Mutant Heart.

To directly assess the altered distribution of cardiac Fe within subcellular compartments, 9-week-old WT and mutant mice were radio-labeled via injection of ⁵⁹Fe–Tf and then killed 2–96 h later. The cytosol was extracted from whole heart and separated by using 3%–12% native gradient PAGE; ⁵⁹Fe distribution was assessed by autoradiography (Fig. 3A) (11).

Fig. 3.

Intracellular ⁵⁹Fe distribution in cytosolic ferritin is markedly different in mutant mice relative to WT animals. (A) Native PAGE ⁵⁹Fe autoradiography identifies two ⁵⁹Fe-containing bands in the cytosol of WT mice (bands A and B) and one (band C) in the mutant. Anti-ferritin antibody (anti-L or anti-H) supershifts bands A and B in WT and band C in mutant mice. WT or mutants were injected i.v. with ⁵⁹Fe-Tf and killed after 2–96 h. The hearts were washed and homogenized at 4°C, and cytosol was prepared for native PAGE-⁵⁹Fe autoradiography by centrifugation at 16,500 g for 45 min at 4°C. Anti-H- or anti-L-ferritin antibody was added to cytosolic lysates and incubated for 1 h at room temperature before native PAGE ⁵⁹Fe autoradiography was performed. (B) Densitometry of the bands in the WT and mutant mice shown in panel A. The density of bands A and B in the WT were added and are compared to band C in the mutant. (C) Anion-exchange chromatography of the cytosolic fraction prepared in A demonstrates two major forms of ⁵⁹Fe in the WT (A and B) relative to 1 major peak (C) in the mutant. Proteins were eluted by a linear gradient of 0–1 M NaCl and ⁵⁹Fe measured by a γ-counter. Results are typical experiments from three performed.

A clear difference in the cytosolic ⁵⁹Fe distribution was found in mutants compared to their WT counterparts at all time points (Fig. 3A). In the cytosol of WT mice, a major ⁵⁹Fe-containing band (band A) and a smaller more diffuse band (band B) were observed (Fig. 3A). Band A comigrated with horse spleen ferritin (data not shown). This ⁵⁹Fe distribution is consistent with previous studies assessing ⁵⁹Fe distribution in primary cultures of WT cardiomyocytes, in which ⁵⁹Fe-labeled ferritin was present as two bands (11). In contrast, the majority of ⁵⁹Fe in mutants appeared as a more faint and smeared band (band C; Fig. 3A). We showed that bands A, B, and C correspond to ⁵⁹Fe–ferritin, as they could be supershifted with anti-H- or L-ferritin antibodies (Fig. 3A). Control antibodies to other proteins (e.g., cyclin D1) had no effect on supershifting these bands (data not shown).

Densitometric analysis showed that ⁵⁹Fe–ferritin in the mutant, expressed as a percentage of the WT, increased from 60% after 2 h incubation with ⁵⁹Fe–Tf to 74% after 4 h (Fig. 3B). After 24–96 h incubation, cytosolic ⁵⁹Fe–ferritin in the mutant increased to the same level as that of the WT (Fig. 3B). This result appears to conflict with the data in Fig. 1B. However, we note that the results in Fig. 3B represent ⁵⁹Fe–ferritin, which is only a subcomponent of the total Fe shown in Fig. 1B.

To characterize the ⁵⁹Fe–ferritin distribution, we performed anion exchange chromatography using cytosol from ⁵⁹Fe-labeled heart (Fig. 3C). Interestingly, the WT lysate showed 2 major ⁵⁹Fe-containing peaks in fractions 14–16 and 18–20 (Fig. 3C) that appeared to correspond to bands B and A observed after native PAGE ⁵⁹Fe autoradiography (Fig. 3A). In contrast, the mutant lysate had only one major peak in fractions 14–17 (Fig. 3B), which appeared to correspond to band C in Fig. 3A.

Collectively, these data indicate differences exist in the molecular distribution of ⁵⁹Fe in ferritin of mutant compared to WT mice.

Iron Chelation.

Considering the potential role of MIT Fe accumulation in the mutant cardiomyopathy and the marked alterations in Fe metabolism, we initated experiments to assess the effect of chelation on body weight loss and cardiac hypertrophy. In these studies, PIH (150 mg/kg) (9) was combined with the more hydrophilic DFO (150 mg/kg) (6). This combination is known to increase Fe mobilization due to the ability of lipid-soluble PIH to mobilize cell Fe and donate it to the largely extracellular ligand DFO (17).

Chelator treatment was initiated at 4.5 weeks of age, before visible signs of the disease were evident, and continued 5 days/week until animals reached 8.5 weeks of age, when the phenotype was pronounced (5). Initially, male and female mouse data were separated due to the difference in Fe metabolism between the sexes (18). However, the results obtained for both sexes were similar and thus were combined.

Chelation Reduces Heart Fe in the Mutant.

Heart Fe levels were significantly (P < 0.001) greater in mutants (462 ± 19 μg/g; n = 27) than in WT mice (328 ± 9 μg/g; n = 34) when treated with vehicle control alone (Fig. 4A).

Fig. 4.

Combination of the MIT-permeable chelator, PIH, with the hydrophilic ligand, DFO, prevents cardiomyocyte Fe loading in the mutant and limits cardiac hypertrophy. However, it does not rescue weight loss or decreased SDHA expression in the mutant relative to WT. (A and B) PIH/DFO prevents cardiac Fe accumulation in mutants. WT and mutant mice (4.5 weeks old) were injected i.p. with 150 mg/kg PIH and DFO 5 days a week for 4 weeks. After the animals were killed, the heart was washed and Fe was measured by (A) inductively coupled plasma atomic emission spectrometry for total Fe, or (B) histological quantitation of Prussian blue staining using Zeiss KS-400 software (Mag. 100x). The arrow denotes blue Fe deposits in cardiomyocytes. The plot illustrates the area of blue Fe deposits per section (30 sections per group; mean ± SEM). (C) Mutant mice lose body weight irrespective of treatment with vehicle or PIH/DFO. WT and mutants were treated as in (A), and their weight was measured. (D and E) PIH/DFO significantly limits cardiac hypertrophy in the mutant as measured by (D) heart weight or (E) heart to body weight ratio. Animals were treated as in (A). (F) PIH/DFO does not prevent loss of SDHA expression in mutants but up-regulates TfR1 in WT mice. Mice were treated as in (A), and the hearts were used for Western analysis. Results in A, C, D, and E are mean ± SEM (n = 21–35 mice). Histology in (B) and results in (F) are typical experiments from four performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Treatment of mutants with PIH/DFO resulted in a significant (P < 0.001) decrease in cardiac Fe (282 ± 13 μg/g; n = 21) compared to mutants treated with the vehicle at 8.5-weeks of age (Fig. 4A). This was confirmed by Prussian blue staining, which was significantly (P < 0.001) decreased in mutants following treatment with chelators to a level comparable to that of the vehicle-treated WT (Fig. 4B). These results showed that chelators effectively decreased cardiac Fe in the mutants to the level in WT mice treated with vehicle alone, but did not lead to overt cardiac Fe depletion (Fig. 4 A and B). In mutant and WT mice, chelation reduced Fe levels in the liver, spleen, and kidney by 21%–28%, 12%–26%, and 6%–10%, respectively, of that found for the WT vehicle control. However, this reduction was only significant (P < 0.05) for the liver. Treatment with chelators had no effect on histology of the heart (Fig. 4B) or any other major organs (data not shown), indicating that it was well tolerated. Assessment of hematological indices between chelator- and vehicle-treated mice showed that the 4-week treatment did not lead to decreased erythrocyte count, hemoglobin concentration, or hematocrit (supporting information (SI) Table S1).

Growth After Chelation.

The first evidence of weight loss and retarded animal growth in vehicle-treated mutants was at 6 weeks of age, as similarly reported (5). The body weight of vehicle-treated mutants continued to decline until the study endpoint, when they were 8.5 weeks of age (Fig. 4C).

Treatment of WT mice with 150 mg/kg PIH/DFO had no significant negative impact on body weight as determined through comparison to WT mice treated with vehicle alone (Fig. 4C). These data indicated that the chelators were well tolerated. However, treatment of mutant mice with PIH/DFO did not significantly prevent the decline in body weight observed in mutants treated with vehicle from 6–8.5 weeks of age (Fig. 4C). Phenotypically, chelator-treated mutants were indistinguishable from those treated with vehicle; both groups experienced weight loss (Fig. 4C) and adopted a hunched stance at 8.5 weeks of age (data not shown).

Chelation Limits Heart Hypertrophy.

Heart weight (Fig. 4D) and the heart to body weight ratio (Fig. 4E) are markers of hypertrophy (5) and were measured to assess the effects of Fe chelation. The increase in heart weight and heart to body weight ratio in the mutants compared to the WT mice were clearly evident and significant (P < 0.001) when mice were treated with the vehicle.

In the chelator-treated mice, there was a significant (P < 0.001) difference between the heart weight (Fig. 4D) and heart to body weight ratio (Fig. 4E) in mutant and control animals. However, assessing chelator-treated mutants, there was a significant decrease in both heart weight (P < 0.01; Fig. 4D) and heart to body weight ratio (P < 0.05; Fig. 4E) compared to mutant mice treated with vehicle. Hence, the chelator-treatment limited the cardiomyopathy but did not totally rescue the phenotype. Indeed, echocardiography showed no difference in ventricular function between mutants treated with chelators or vehicle at 8.5 weeks of age (data not shown).

The chelator combination was well tolerated by the mice and did not have any negative effect on the heart, there being no difference in heart weight (Fig. 4D) or heart to body weight ratios (Fig. 4E) in WT animals treated with the vehicle alone or PIH/DFO.

The effect of chelators on rescuing the metabolic defect in FA was also examined by assessing myocardial expression of succinate dehydrogenase (SDHA). This ISC enzyme is critical for MIT energy metabolism and is markedly decreased in the mutant compared to WT (5, 19). When we examined 8.5-week-old mice treated with vehicle or PIH/DFO, it was clear the chelators did not prevent the decrease in SDHA expression (Fig. 4F). This again confirmed that chelator treatment limited cardiac hypertrophy, but could not rescue the metabolic defect. Further, in WT animals, chelator treatment was effective as it increased (P < 0.001) TfR1 by 225% relative to WT mice treated with vehicle (Fig. 4F). In mutants, TfR1 expression was far greater than in the WT. However, chelator treatment in the mutant did not further increase TfR1 relative to the vehicle, probably because its expression was already very pronounced relative to that of WT mice (Fig. 4F).

Discussion

At present, there is no effective treatment for the severe debilitating cardiac effects observed in FA. Considering that Fe accumulation is toxic (6), the discovery that MIT Fe accumulation occurs in Fxn knockout mice (5) and in FA patients (6) provides a potential target for therapeutic intervention (6).

We show that Fe accumulation in MCK mutants is mediated by increased Fe uptake from Tf due to TfR1 up-regulation. The decreased Fxn expression leads to Fe targeted to the mitochondrion and a relative cytosolic Fe deficiency, low ferritin expression, and decreased expression of the Fe exporter ferroportin1. Hence, Fxn deficiency leads to decreased ISC synthesis, which results in a compensatory increase in Fe transport to the mitochondrion. This response may be an attempt to rescue the decreased ISC synthesis that is vital for energy generation etc. A model summarizing the altered Fe trafficking observed is presented in Fig. 5. These alterations in Fe metabolism were important to delineate, in order to understand the mechanisms responsible for the MIT Fe loading and, in part, the cardiac pathology observed.

Fig. 5.

Schematic of the alterations in Fe trafficking in mutant compared to WT mice. Ablation of Fxn decreases ISC synthesis that is vital for energy generation. There is increased TfR1 and decreased ferritin and ferroportin1 in the mutant relative to the WT mice. These changes are likely mediated in older mutants by increased IRP2–RNA binding.

Similar changes in the expression of molecules involved in the transport and storage of Fe have been observed in cells hyperexpressing MIT ferritin (20, 21). In this case, there was down-regulation of Fxn expression, up-regulation of TfR1, down-regulation of ferritin, cytosolic Fe deficiency, and MIT Fe overload. Furthermore, ISC synthesis and heme biogenesis were markedly reduced. This may be due to MIT ferritin sequestering Fe, making it unavailable for these essential MIT processes (20, 21). Hence, the defect caused by forced MIT ferritin expression resembles some consequences of Fxn deficiency.

Previous data in combination with the current study indicate that a major control of Fe uptake is MIT Fe utilization for functions such as ISC synthesis, which is depressed in the absence of Fxn (22). The mechanism of communication between the mitochondrion and cytosol that alters Fe trafficking to “satisfy” the MIT demand remains unknown. However, the lack of ISC synthesis and its decreased release into the cytosol could be involved. It is relevant that mutations in ABCB7, which is involved in MIT ISC export, result in MIT Fe accumulation leading to sideroblastic anemia with ataxia (23). Further, it can be speculated that alterations in endosomal/organelle or Fe-chaperone trafficking may be involved in directing Fe targeting between the cytosol and mitochondrion. Direct endosomal and MIT contact to effect MIT Fe targeting may occur in erythroid cells (24, 25). In addition, it is well known that organelle and transporter trafficking is involved in copper transport (26). Thus, further studies are required to define the alterations in Fe trafficking in the mutants.

Considering the changes in MCK mutant Fe metabolism and the MIT Fe accumulation that could be toxic, we combined PIH and DFO to treat mice. We showed that Fe chelation prevents cardiac Fe accumulation in mutants and limits cardiac hypertrophy. However, this therapy did not totally rescue the defect due to loss of Fxn, with mutants still suffering decreased cardiac function. Deterioration of mutants despite Fe chelation therapy indicates chelator-insensitive or Fe-independent events occur that contribute to death. Clearly, chelation cannot replace Fxn in ISC synthesis, but it may prevent the oxidative damage due to MIT Fe accumulation. Hence, this could explain the partial rescue observed.

It is significant that chelation prevented Fe accumulation in mutants but did not lead to overt cardiac Fe depletion or toxicity. These observations have implications for FA patients. Indeed, a valid criticism against chelation therapy for FA is that it could result in whole-body Fe depletion (6). This would be the case if chelation therapy was implemented using high-dose DFO to treat gross Fe overload (6). Clearly, this approach is not appropriate for patients with FA, in which Fe loading is not as marked as in untreated β-thalassemia (6). It is well known that DFO is a hydrophilic drug that does not penetrate MIT membranes to bind intra-MIT Fe deposits (9). The goal of an effective chelation regime for FA patients is to prevent MIT Fe accumulation using chelators that permeate the mitochondrion without causing marked body Fe depletion and hematological toxicity (6).

In this study, both PIH and DFO were used to limit Fe accumulation, as the phenotype is very severe in mutant mice, with death occurring at 10 weeks of age (5). It is likely that such intense chelation therapy would not be required in human FA, as the progression is far slower (27). Hence, only minimal chelation therapy with PIH alone via the oral route may be enough to prevent MIT Fe accumulation and its pathological effects.

Recently, a small clinical trial suggested that the chelator deferiprone may have favorable effects on preventing the neurological symptoms of FA (28). However, the role of Fe chelation in producing this outcome was difficult to ascertain, as deferiprone was given at the same time as idebenone, which is known to ameliorate the neurological effects of FA (29). Therefore, further studies with chelators alone in FA patients are essential.

In summary, there was a marked change in cardiac Fe trafficking in MCK mutants, and our studies show the mechanisms responsible for MIT Fe loading. Further, chelation therapy limits cardiac hypertrophy in the mutants, suggesting it may be beneficial for FA patients.

Materials and Methods

Animals.

Mutants homozygous for deletion of Frda exon 4 were used and genotyped as in ref. 5. All animal work was approved by the University of Sydney Animal Ethics Committee.

Chelator Studies.

PIH was synthesized (10), and DFO was from Novartis. Chelators were dissolved in 20% propylene glycol/0.9% saline. Treatment commenced in 4.5-week-old mice, which were injected intraperitoneally with vehicle or vehicle containing DFO and PIH (both at 150 mg/kg) for 5 days/week. Injections continued until mice were 8.5 weeks old. Histochemistry, tissue Fe stores, hematological indices, and organ weights were then assessed by standard methods (30). Image analysis was done using Zeiss KS-400 software to quantitate Prussian blue-stained sections.

RT-PCR, Western Analysis, and IRP–RNA Binding Activity.

RNA was isolated and RT-PCR performed (31) using the primers in Table S2. Western analysis was achieved (31) using antibodies against Fxn (US Biological); TfR1 (Invitrogen); ferroportin1 (D. Haile, University Texas Health Science Center); SDHA (Santa Cruz Biotechnology); and H-, L-, and MIT-ferritin (S. Levi, San Raffaele Institute). IRP–RNA-binding activity was performed via gel-retardation analysis (20).

Labeling of Transferrin with ⁵⁹Fe and ⁵⁹Fe Uptake In Vivo and its Intracellular Distribution.

Established methods (32) were used to label apo-Tf (Sigma) with ⁵⁹Fe (Perkin-Elmer). Standard procedures were used to measure organ ⁵⁹Fe uptake after i.v. injection of ⁵⁹Fe–Tf (0.6 mg) (32). After 2–96 h, mice were killed and heart and organs removed and placed on ice (32). The heart was washed, homogenized at 4°C and centrifuged at 800 g/20 min/4°C. The supernatant was centrifuged at 16,500 g for 45 min at 4°C to separate cytosolic and SMM fractions. Cytosolic ⁵⁹Fe distribution was determined by using native PAGE ⁵⁹Fe autoradiography (11, 24). Native supershifts using the latter technique were performed by using anti-H- and L-ferritin. Anion-exchange chromatography of cytosolic samples was performed by using a Mono Q 5/50 GL column (Amersham Biosciences) via a BioLogic Chromatography System (Bio-Rad) (33). ⁵⁹Fe was monitored using a Wallac WIZARD 1480 γ-counter.

Statistical Analysis.

Data were compared by using Student's t test. Data were considered statistically significant when P < 0.05. Results were expressed as mean ± SD or mean ± SEM.