The MCK mouse heart model of Friedreich’s ataxia:Alterations in iron-regulated proteins and cardiachypertrophy are limited by iron chelationMegan Whitnall*, Yohan Suryo Rahmanto*, Robert Sutak*, Xiangcong Xu*, Erika M. Becker*, Marc R. Mikhael†,Prem Ponka†‡, and Des R. Richardson*‡

*Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia; †Lady Davis Institute, Jewish GeneralHospital and Department of Physiology, McGill University, Montreal, QC, Canada H3T 1E2

Communicated by Pamela J. Bjorkman, California Institute of Technology, Pasadena, CA, May 1, 2008 (received for review April 10, 2008)

There is no effective treatment for the cardiomyopathy of the mostcommon autosomal recessive ataxia, Friedreich’s ataxia (FA). Theidentification of potentially toxic mitochondrial (MIT) iron (Fe)deposits in FA suggests that Fe plays a role in its pathogenesis. Thisstudy used the muscle creatine kinase conditional frataxin (Fxn)knockout (mutant) mouse model that reproduces the classical traitsassociated with cardiomyopathy in FA. We examined the mecha-nisms responsible for the increased cardiac MIT Fe loading inmutants. Moreover, we explored the effect of Fe chelation on thepathogenesis of the cardiomyopathy. Our investigation showedthat increased MIT Fe in the myocardium of mutants was due tomarked transferrin Fe uptake, which was the result of enhancedtransferrin receptor 1 expression. In contrast to the mitochondrion,cytosolic ferritin expression and the proportion of cytosolic Fewere decreased in mutant mice, indicating cytosolic Fe deprivationand markedly increased MIT Fe targeting. These studies demon-strated that loss of Fxn alters cardiac Fe metabolism due topronounced changes in Fe trafficking away from the cytosol to themitochondrion. Further work showed that combining the MIT-permeable ligand pyridoxal isonicotinoyl hydrazone with the hy-drophilic chelator desferrioxamine prevented cardiac Fe loadingand limited cardiac hypertrophy in mutants but did not lead toovert cardiac Fe depletion or toxicity. Fe chelation did not preventdecreased succinate dehydrogenase expression in the mutants orloss of cardiac function. In summary, we show that loss of Fxnmarkedly alters cellular Fe trafficking and that Fe chelation limitsmyocardial hypertrophy in the mutant.

ferritin � iron chelators � mitochondria � transferrin receptor � frataxin

Friedreich’s ataxia (FA) is the most common autosomalrecessive neuro- and cardiodegenerative disorder, resulting

from insufficient expression of the mitochondrial (MIT) proteinfrataxin (Fxn) (1). Many studies infer a role for Fxn in MIT iron(Fe) metabolism, particularly iron–sulfur cluster (ISC) biosyn-thesis (2, 3). Deletion of the yeast Fxn homolog 1 gene or loss ofFxn in FA patients or Fxn knockout (mutant) mice promotesMIT Fe accumulation, hypersensitivity to oxidants, and the lossof MIT DNA and ISC-containing enzymes (2–5). Increasedcardiac Fe deposition and perturbations in heme biosynthesisand ATP production suggest that FA pathogenesis is linked toMIT Fe overload (6). Furthermore, the antioxidant idebenoneimproves 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 tothe ability of Fe to redox cycle, leading to cytotoxic radicals (6).The potential of Fe loading to become toxic may be morepronounced in the highly redox-active MIT environment (3).There is no effective treatment for FA, although evidence ofMIT Fe loading within cardiomyocytes from FA patients sup-ports the use of chelation as a therapeutic strategy (6). The Fechelator desferrioxamine (DFO) has some ability to rescue FAfibroblasts from oxidant stress (8). However, the inability of

DFO to permeate the mitochondrion means it is ineffective forFA treatment (6, 9).

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

Cardiomyopathy is a frequent cause of death in FA. To test thepotential of Fe chelation therapy for FA treatment, we have usedmuscle creatine kinase (MCK) conditional Fxn knockout micelacking Fxn in cardiomyocytes (5). This model exhibits classicalphenotypic traits of the cardiomyopathy in FA, including markedcardiac MIT Fe accumulation and deficiency in ISC enzymes (5).

In this study, we show that Fxn deficiency leads to abnormalFe metabolism in vivo in MCK mutant mice, and that treatmentwith PIH and DFO prevents cardiac Fe loading and limitsmyocardial hypertrophy.

ResultsAltered Cardiac Fe Metabolism in the Mutant. Little is knownregarding the function of Fxn in vivo and the role of Fe loadingin FA pathogenesis. To this end, we examined the Fe metabolismof MCK mutants and the potential of Fe chelation to preventcardiomyopathy. This model was chosen as it closely reflects thealterations 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 thephysiological serum Fe binding protein 59Fe–transferrin (Tf) toradiolabel 9-week-old wild-type (WT) and mutant mice via tailvein (i.v.) injection. Incorporation of 59Fe into the whole heartwas measured after 24 h. A significant (P � 0.001) increase in thelevel of cardiac 59Fe was observed in mutant mice compared toWT controls (Fig. 1A). To assess the intracellular distribution of59Fe, hearts were fractionated to yield cytosolic and stromal MITmembrane (SMM) fractions. It was evident in mutant comparedto WT mice that there was a marked decrease in the proportionof cytosolic 59Fe, although there was a significant (P � 0.001)increase of 59Fe in the SMM (Fig. 1B). This result directlyconfirmed that deletion of Fxn leads to MIT Fe loading in themutant (5) and results in a relative cytosolic Fe deficiency.

Considering the alterations in 59Fe uptake and distribution, we

Author contributions: M.W., Y.S.R., R.S., X.X., E.M.B., P.P., and D.R.R. designed research;M.W., Y.S.R., R.S., X.X., E.M.B., and M.R.M. performed research; M.R.M. contributed newreagents/analytic tools; M.W., Y.S.R., R.S., P.P., and D.R.R. analyzed data; and M.W., Y.S.R.,P.P., and D.R.R. wrote the paper.

The authors declare no conflict of interest.

‡To whom correspondence may be addressed. Email: prem.ponka@mcgill.ca ord.richardson@med.usyd.edu.au.

This article contains supporting information online at www.pnas.org/cgi/content/full/0804261105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

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examined the changes that occur at the mRNA and protein levelsof molecules involved in cardiac Fe metabolism at 4 and 9 weeksof age. These ages were chosen because at 4 weeks no overtphenotype was present, whereas at 9 weeks a severe cardiomy-opathy was found (5). There was a significant (P � 0.001)decrease in Fxn expression at the mRNA (Fig. 1C) and proteinlevels (Fig. 1D) in 4- and 9-week-old mutants. However, Fxnexpression was not totally ablated, due to other cells within thetotal heart homogenate apart from cardiomyocytes (e.g., fibro-blasts), which are not targeted by the conditional knockoutstrategy (5).

Transferrin receptor 1 (TfR1) was significantly (P � 0.001)up-regulated in 4- and 9-week-old mutants relative to WT miceat the mRNA (Fig. 1C) and protein (Fig. 1D) levels. The extentof 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) andlight (L) chains (12). Comparing WT and mutant mice at 4 and9 weeks of age, H- and L-ferritin mRNA expression was notmarkedly or significantly altered (Fig. 1C). In contrast, Westernanalysis showed a significant (P � 0.001) decrease in H- andL-ferritin protein expression in mutants compared to WT miceat 9 weeks of age (Fig. 1D). The molecular alterations in TfR1and ferritin expression suggest the cytosol was in an Fe-deficientstate. This hypothesis was supported by the significantly (P �0.001) decreased expression of the Fe export molecule ferro-portin1 in mutant compared to WT mice at the mRNA andprotein levels at 9 weeks of age (Fig. 1 C and D). Becauseferroportin1 protein expression is down-regulated by decreasedcellular Fe levels (13), this further supports the idea thatcardiomyocytes are experiencing a cytosolic Fe deficiency.

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

The observations above indicate pronounced alterations incardiac Fe homeostasis caused by deletion of Fxn. Given thatiron regulatory protein (IRP)-1 and -2 are key regulators ofTfR1, ferritin and ferroportin1 expression (12), we examinedIRP–RNA binding activity in mutant and WT mice.

IRP2- RNA-Binding Activity is Increased in Mutant Heart. Iron me-tabolism is regulated, in part, by IRP1 and IRP2, which interactwith iron-response elements (IREs) in the 3� untranslated region(UTR) of TfR1 mRNA or 5� UTR of H- and L-ferritin orferroportin1 mRNA, in response to cytosolic Fe (12). IRP bindingto the 3� UTR IRE stabilizes TfR1 mRNA, increasing itsexpression, whereas binding to the 5� IREs of ferritin or ferro-portin1 mRNA inhibits translation (12, 15, 16).

Gel-retardation analysis indicated significantly (P � 0.01)increased IRP2–RNA binding activity in mutant relative to WTmice only at 9 weeks of age, while there was little alteration inIRP1-RNA binding (Fig. 2). It has been demonstrated that2-mercaptoethanol (�-ME) converts IRP1 to its RNA-bindingstate (15, 16). However, its addition to lysates did not signifi-cantly increase IRP1-binding activity (Fig. 2). This indicated thatIRP1 was largely in its active RNA-binding form in WT andmutant mice at 4 and 9 weeks of age.

Previous studies indicated that IRP2 is crucial in controllingFe-regulated gene expression in vivo at physiological oxygentension (15, 16). This is because in contrast to IRP1, only IRP2registers intracellular Fe levels and modulates its RNA-bindingactivity at physiological oxygen levels (15, 16). Indeed, ourresults are consistent with the theory of Rouault and colleagues(15, 16) that IRP2 primarily regulates mammalian Fe homeosta-sis in vivo despite its lower expression compared to IRP1.

The increased IRP2 activity in 9-week mutants confirms acytosolic Fe deficiency, which could mediate the marked up-regulation of TfR1 and down-regulation of ferritin and ferro-portin1 protein (Fig. 1D). As there was no alteration in IRP1 orIRP2 RNA-binding activity at 4 weeks of age (Fig. 2), otherfactors (e.g., at the transcriptional level) could be responsible forthe less pronounced alteration in TfR1 and ferritin expression atthis time.

The elevated IRP2–RNA-binding activity in mutant relative toWT mice (Fig. 2) and increased Fe uptake into the heart (Fig.1A) and SMM (Fig. 1B) suggest that Fxn deficiency leads to a

Fig. 1. Marked alterations in 59Fe uptake and distribution and expression ofmolecules that play key roles in Fe metabolism occur in mutant relative to WTmice. (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 killed24 h later. Hearts were washed and assessed for 59Fe activity. (B) Mutant heartsshow a cytosolic Fe deficiency relative to the stromal-mitochondrial mem-brane (SMM). Hearts from (A) were processed as described in the Materialsand Methods and the cytosol and SMM separated and assessed for 59Feactivity. (C and D) Examination of (C) mRNA and (D) protein expression of Fxnand molecules involved in Fe metabolism in hearts from 4- and 9-week-old WTand mutant mice. mRNA and protein expression were assessed as per Mate-rials and Methods. Densitometry is expressed relative to the respective WT ateach age and GAPDH. Results in A and B are mean � SD (3 experiments). TheRT-PCR and Western panels in C and D are typical experiments, whereas thedensitometry is mean � SD (3–5 experiments). ***, P � 0.001.

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major alteration in Fe trafficking. Specifically, lack of Fxn leadsto a cytosolic Fe deficiency relative to MIT Fe loading.

Marked Alterations in Cytosolic 59Fe Distribution Within Ferritin inMutant Heart. To directly assess the altered distribution of cardiacFe within subcellular compartments, 9-week-old WT and mutantmice were radio-labeled via injection of 59Fe–Tf and then killed2–96 h later. The cytosol was extracted from whole heart andseparated by using 3%–12% native gradient PAGE; 59Fedistribution was assessed by autoradiography (Fig. 3A) (11).

A clear difference in the cytosolic 59Fe distribution was foundin mutants compared to their WT counterparts at all time points(Fig. 3A). In the cytosol of WT mice, a major 59Fe-containingband (band A) and a smaller more diffuse band (band B) wereobserved (Fig. 3A). Band A comigrated with horse spleen ferritin(data not shown). This 59Fe distribution is consistent withprevious studies assessing 59Fe distribution in primary cultures ofWT cardiomyocytes, in which 59Fe-labeled ferritin was present astwo bands (11). In contrast, the majority of 59Fe in mutantsappeared 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 antibod-ies (Fig. 3A). Control antibodies to other proteins (e.g., cyclinD1) had no effect on supershifting these bands (data not shown).

Densitometric analysis showed that 59Fe–ferritin in the mu-tant, 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 mutantincreased to the same level as that of the WT (Fig. 3B). Thisresult appears to conflict with the data in Fig. 1B. However, wenote that the results in Fig. 3B represent 59Fe–ferritin, which isonly 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-labeledheart (Fig. 3C). Interestingly, the WT lysate showed 2 major59Fe-containing peaks in fractions 14–16 and 18–20 (Fig. 3C)that appeared to correspond to bands B and A observed afternative PAGE 59Fe autoradiography (Fig. 3A). In contrast, themutant 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 mo-lecular distribution of 59Fe in ferritin of mutant compared to WTmice.

Iron Chelation. Considering the potential role of MIT Fe accu-mulation in the mutant cardiomyopathy and the marked alter-ations in Fe metabolism, we initated experiments to assess theeffect of chelation on body weight loss and cardiac hypertrophy.In these studies, PIH (150 mg/kg) (9) was combined with themore hydrophilic DFO (150 mg/kg) (6). This combination isknown to increase Fe mobilization due to the ability of lipid-soluble PIH to mobilize cell Fe and donate it to the largelyextracellular ligand DFO (17).

Chelator treatment was initiated at 4.5 weeks of age, beforevisible signs of the disease were evident, and continued 5days/week until animals reached 8.5 weeks of age, when thephenotype was pronounced (5). Initially, male and female mousedata were separated due to the difference in Fe metabolismbetween the sexes (18). However, the results obtained for bothsexes were similar and thus were combined.

Chelation Reduces Heart Fe in the Mutant. Heart Fe levels weresignificantly (P � 0.001) greater in mutants (462 � 19 �g/g; n �

Fig. 2. IRP2–RNA-binding activity in the heart is increased in mutant micerelative to the WT at 9, but not 4, weeks of age, whereas IRP1-RNA bindingdoes 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 amountsof heart extract were assayed in the absence and presence of 2-mercapto-ethanol (�-ME). Results in (A) are from a typical experiment, whereas thedensitometry in (B) is mean � SD of 3 experiments. **, P � 0.01; ***, P � 0.001.

Fig. 3. Intracellular 59Fe distribution in cytosolic ferritin is markedly differentin mutant mice relative to WT animals. (A) Native PAGE 59Fe autoradiographyidentifies two 59Fe-containing bands in the cytosol of WT mice (bands A andB) 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 mutantswere injected i.v. with 59Fe-Tf and killed after 2–96 h. The hearts were washedand homogenized at 4°C, and cytosol was prepared for native PAGE-59Feautoradiography by centrifugation at 16,500 g for 45 min at 4°C. Anti-H- oranti-L-ferritin antibody was added to cytosolic lysates and incubated for 1 h atroom 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 bandC in the mutant. (C) Anion-exchange chromatography of the cytosolic fractionprepared 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 lineargradient of 0–1 M NaCl and 59Fe measured by a �-counter. Results are typicalexperiments from three performed.

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27) than in WT mice (328 � 9 �g/g; n � 34) when treated withvehicle control alone (Fig. 4A).

Treatment of mutants with PIH/DFO resulted in a significant(P � 0.001) decrease in cardiac Fe (282 � 13 �g/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, whichwas significantly (P � 0.001) decreased in mutants followingtreatment with chelators to a level comparable to that of thevehicle-treated WT (Fig. 4B). These results showed that chela-tors effectively decreased cardiac Fe in the mutants to the levelin WT mice treated with vehicle alone, but did not lead to overtcardiac 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 foundfor the WT vehicle control. However, this reduction was onlysignificant (P � 0.05) for the liver. Treatment with chelators hadno effect on histology of the heart (Fig. 4B) or any other majororgans (data not shown), indicating that it was well tolerated.Assessment of hematological indices between chelator- andvehicle-treated mice showed that the 4-week treatment did notlead to decreased erythrocyte count, hemoglobin concentration,or hematocrit (supporting information (SI) Table S1).

Growth After Chelation. The first evidence of weight loss andretarded animal growth in vehicle-treated mutants was at 6weeks of age, as similarly reported (5). The body weight ofvehicle-treated mutants continued to decline until the studyendpoint, when they were 8.5 weeks of age (Fig. 4C).

Treatment of WT mice with 150 mg/kg PIH/DFO had nosignificant negative impact on body weight as determinedthrough 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 notsignificantly prevent the decline in body weight observed inmutants treated with vehicle from 6–8.5 weeks of age (Fig. 4C).Phenotypically, chelator-treated mutants were indistinguishablefrom those treated with vehicle; both groups experienced weightloss (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 theheart to body weight ratio (Fig. 4E) are markers of hypertrophy(5) and were measured to assess the effects of Fe chelation. Theincrease in heart weight and heart to body weight ratio in themutants compared to the WT mice were clearly evident andsignificant (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 heartto body weight ratio (Fig. 4E) in mutant and control animals.However, assessing chelator-treated mutants, there was a sig-nificant decrease in both heart weight (P � 0.01; Fig. 4D) andheart to body weight ratio (P � 0.05; Fig. 4E) compared tomutant mice treated with vehicle. Hence, the chelator-treatmentlimited the cardiomyopathy but did not totally rescue the phe-notype. Indeed, echocardiography showed no difference inventricular function between mutants treated with chelators orvehicle at 8.5 weeks of age (data not shown).

The chelator combination was well tolerated by the mice anddid not have any negative effect on the heart, there being nodifference in heart weight (Fig. 4D) or heart to body weight ratios(Fig. 4E) in WT animals treated with the vehicle alone orPIH/DFO.

The effect of chelators on rescuing the metabolic defect in FAwas also examined by assessing myocardial expression of succi-nate dehydrogenase (SDHA). This ISC enzyme is critical forMIT energy metabolism and is markedly decreased in themutant compared to WT (5, 19). When we examined 8.5-week-old mice treated with vehicle or PIH/DFO, it was clear thechelators did not prevent the decrease in SDHA expression (Fig.4F). This again confirmed that chelator treatment limited car-diac hypertrophy, but could not rescue the metabolic defect.Further, in WT animals, chelator treatment was effective as itincreased (P � 0.001) TfR1 by 225% relative to WT mice treatedwith vehicle (Fig. 4F). In mutants, TfR1 expression was fargreater than in the WT. However, chelator treatment in themutant did not further increase TfR1 relative to the vehicle,probably because its expression was already very pronouncedrelative to that of WT mice (Fig. 4F).

Fig. 4. Combination of the MIT-permeable chelator, PIH, with the hydro-philic ligand, DFO, prevents cardiomyocyte Fe loading in the mutant and limitscardiac hypertrophy. However, it does not rescue weight loss or decreasedSDHA expression in the mutant relative to WT. (A and B) PIH/DFO preventscardiac Fe accumulation in mutants. WT and mutant mice (4.5 weeks old) wereinjected i.p. with 150 mg/kg PIH and DFO 5 days a week for 4 weeks. After theanimals 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 plotillustrates the area of blue Fe deposits per section (30 sections per group;mean � SEM). (C) Mutant mice lose body weight irrespective of treatmentwith vehicle or PIH/DFO. WT and mutants were treated as in (A), and theirweight was measured. (D and E) PIH/DFO significantly limits cardiac hypertro-phy in the mutant as measured by (D) heart weight or (E) heart to body weightratio. Animals were treated as in (A). (F) PIH/DFO does not prevent loss of SDHAexpression in mutants but up-regulates TfR1 in WT mice. Mice were treated asin (A), and the hearts were used for Western analysis. Results in A, C, D, and Eare mean � SEM (n � 21–35 mice). Histology in (B) and results in (F) are typicalexperiments from four performed. *, P � 0.05; **, P � 0.01; ***, P � 0.001.

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DiscussionAt present, there is no effective treatment for the severedebilitating cardiac effects observed in FA. Considering that Feaccumulation is toxic (6), the discovery that MIT Fe accumu-lation 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 mediatedby increased Fe uptake from Tf due to TfR1 up-regulation. Thedecreased Fxn expression leads to Fe targeted to the mitochon-drion and a relative cytosolic Fe deficiency, low ferritin expres-sion, and decreased expression of the Fe exporter ferroportin1.Hence, Fxn deficiency leads to decreased ISC synthesis, whichresults in a compensatory increase in Fe transport to themitochondrion. This response may be an attempt to rescue thedecreased ISC synthesis that is vital for energy generation etc. Amodel summarizing the altered Fe trafficking observed is pre-sented in Fig. 5. These alterations in Fe metabolism wereimportant to delineate, in order to understand the mechanismsresponsible for the MIT Fe loading and, in part, the cardiacpathology observed.

Similar changes in the expression of molecules involved in thetransport and storage of Fe have been observed in cells hyper-expressing 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 Feoverload. Furthermore, ISC synthesis and heme biogenesis weremarkedly reduced. This may be due to MIT ferritin sequesteringFe, making it unavailable for these essential MIT processes (20,21). Hence, the defect caused by forced MIT ferritin expressionresembles some consequences of Fxn deficiency.

Previous data in combination with the current study indicatethat a major control of Fe uptake is MIT Fe utilization forfunctions such as ISC synthesis, which is depressed in the absenceof Fxn (22). The mechanism of communication between themitochondrion and cytosol that alters Fe trafficking to ‘‘satisfy’’the MIT demand remains unknown. However, the lack of ISCsynthesis and its decreased release into the cytosol could beinvolved. It is relevant that mutations in ABCB7, which isinvolved in MIT ISC export, result in MIT Fe accumulationleading to sideroblastic anemia with ataxia (23). Further, it canbe speculated that alterations in endosomal/organelle or Fe-chaperone trafficking may be involved in directing Fe targetingbetween the cytosol and mitochondrion. Direct endosomal and

MIT contact to effect MIT Fe targeting may occur in erythroidcells (24, 25). In addition, it is well known that organelle andtransporter trafficking is involved in copper transport (26). Thus,further studies are required to define the alterations in Fetrafficking in the mutants.

Considering the changes in MCK mutant Fe metabolism andthe MIT Fe accumulation that could be toxic, we combined PIHand DFO to treat mice. We showed that Fe chelation preventscardiac Fe accumulation in mutants and limits cardiac hyper-trophy. However, this therapy did not totally rescue the defectdue to loss of Fxn, with mutants still suffering decreased cardiacfunction. Deterioration of mutants despite Fe chelation therapyindicates chelator-insensitive or Fe-independent events occurthat contribute to death. Clearly, chelation cannot replace Fxnin ISC synthesis, but it may prevent the oxidative damage due toMIT Fe accumulation. Hence, this could explain the partialrescue observed.

It is significant that chelation prevented Fe accumulation inmutants but did not lead to overt cardiac Fe depletion or toxicity.These observations have implications for FA patients. Indeed, avalid criticism against chelation therapy for FA is that it couldresult in whole-body Fe depletion (6). This would be the case ifchelation therapy was implemented using high-dose DFO totreat gross Fe overload (6). Clearly, this approach is not appro-priate for patients with FA, in which Fe loading is not as markedas in untreated �-thalassemia (6). It is well known that DFO isa hydrophilic drug that does not penetrate MIT membranes tobind intra-MIT Fe deposits (9). The goal of an effective chelationregime for FA patients is to prevent MIT Fe accumulation usingchelators that permeate the mitochondrion without causingmarked body Fe depletion and hematological toxicity (6).

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

Recently, a small clinical trial suggested that the chelatordeferiprone may have favorable effects on preventing the neu-rological symptoms of FA (28). However, the role of Fe chelationin producing this outcome was difficult to ascertain, as de-

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 byincreased IRP2–RNA binding.

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feriprone was given at the same time as idebenone, which isknown to ameliorate the neurological effects of FA (29). There-fore, further studies with chelators alone in FA patients areessential.

In summary, there was a marked change in cardiac Fetrafficking in MCK mutants, and our studies show the mecha-nisms responsible for MIT Fe loading. Further, chelation therapylimits cardiac hypertrophy in the mutants, suggesting it may bebeneficial for FA patients.

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

Chelator Studies. PIH was synthesized (10), and DFO was from Novartis.Chelators were dissolved in 20% propylene glycol/0.9% saline. Treatmentcommenced in 4.5-week-old mice, which were injected intraperitoneally withvehicle 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 Festores, hematological indices, and organ weights were then assessed bystandard methods (30). Image analysis was done using Zeiss KS-400 softwareto quantitate Prussian blue-stained sections.

RT-PCR, Western Analysis, and IRP–RNA Binding Activity. RNA was isolated andRT-PCR performed (31) using the primers in Table S2. Western analysis wasachieved (31) using antibodies against Fxn (US Biological); TfR1 (Invitrogen);

ferroportin1 (D. Haile, University Texas Health Science Center); SDHA (Santa CruzBiotechnology); 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 IntracellularDistribution. Established methods (32) were used to label apo-Tf (Sigma) with59Fe (Perkin-Elmer). Standard procedures were used to measure organ 59Feuptake after i.v. injection of 59Fe–Tf (0.6 mg) (32). After 2–96 h, mice werekilled and heart and organs removed and placed on ice (32). The heart waswashed, homogenized at 4°C and centrifuged at 800 g/20 min/4°C. Thesupernatant was centrifuged at 16,500 g for 45 min at 4°C to separate cytosolicand SMM fractions. Cytosolic 59Fe distribution was determined by using nativePAGE 59Fe autoradiography (11, 24). Native supershifts using the latter tech-nique were performed by using anti-H- and L-ferritin. Anion-exchange chro-matography of cytosolic samples was performed by using a Mono Q 5/50 GLcolumn (Amersham Biosciences) via a BioLogic Chromatography System (Bio-Rad) (33). 59Fe was monitored using a Wallac WIZARD 1480 �-counter.

Statistical Analysis. Data were compared by using Student’s t test. Data wereconsidered statistically significant when P � 0.05. Results were expressed asmean � SD or mean � SEM.

ACKNOWLEDGMENTS. We thank H. Puccio and M. Koenig (Institute de Ge-netique et de Biologie Moleculaire et Cellulaire, Strasborg, France) for theMCK mice. This research was supported by the National Health and MedicalResearch Council (Australia), Muscular Dystrophy Association (USA), Fried-reich’s Ataxia Research Alliance (USA), Friedreich’s Ataxia Research Associa-tion (Australia), and the Canadian Institutes of Health Research.

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