Characterization of mitochondrial ferritin-deficient mice.

Ferritins are highly conserved multi-subunit proteins that detoxify and reversibly store intracellular iron(1). Mice lacking the cytosolic ferritin heavy chain (H) exhibit embryonic lethality; mice with a conditional ferritin H deletion exhibit severe tissue damage(2,3). Mitochondrial ferritin (Ftmt) is structurally and functionally homologous to ferritin H chain(4,5). Elevated Ftmt expression in erythroblasts from sideroblastic anemia patients suggests a role for Ftmt in these anemias, a heterogeneous group of inherited and acquired disorders characterized by mitochondrial iron deposition(6). While cytosolic ferritin H and light (L) chains are ubiquitous in mammals, Ftmt mRNA is found largely in the testes(7,8). The lack of an iron responsive element in the Ftmt mRNA, while present in ferritin H and L mRNAs, suggests that Ftmt may function independently of iron metabolism. Studies to date have focused on protective effects of Ftmt overexpression against iron overload and oxidative stress in tissue culture, yeast and flies(9–15). To investigate the in vivo role of Ftmt, we deleted the Ftmt gene in mice and examined baseline hematology, iron metabolism and male fertility phenotypes. In addition, we subjected them to vitamin B6 (pyridoxine) deprivation to induce sideroblast/siderocyte formation. In all cases, we observed no significant defects in mice lacking mitochondrial ferritin. To construct Ftmt-deficient mice, we replaced the entire open reading frame (ORF) of the intronless Ftmt gene with a neomycin-resistance cassette (Fig. 1A). Southern blotting using probes flanking the region encompassed by the targeting construct indicated successful recombination at the Ftmt locus in embryonic stem (ES) cells (data not shown). We confirmed Ftmt deletion using PCR and primers specific to the flanking regions and the neomycin cassette (Fig. 1B). To measure Ftmt expression, we used reverse transcriptase-polymerase chain reaction (RT-PCR) and primers specific to β-actin and Ftmt; control reactions without reverse transcription were performed to ensure that PCR products did not represent amplification of genomic DNA. As Ftmt is most abundantly expressed in the testis, we measured expression in RNA isolated from testes of wild-type and homozygous littermates; no expression was detected in homozygous mutant animals (Fig. 1C). We could not detect Ftmt expression in bone marrow from either genotype, even after pyridoxine deprivation (data not shown; see below). Figure 1 Construction and analysis of Ftmt-deficient mice Ftmt−/− animals on the C57BL/6J genetic background at backcross generation greater than N7 were obtained at expected Mendelian frequencies from heterozygous pairings and no gross anatomic or histological abnormalities, including iron staining, were present in the mutant offspring (data not shown). Given the testis-specific expression, we assessed male fertility by pairing mutant and wild type males with two wild type females for 14 days and counted the number of pregnancies and total number of offspring obtained(16); based on this assessment, there was no difference in male fertility between mutants and controls (data not shown). To determine the effect of Ftmt deletion on baseline murine iron metabolism and hematology, we measured serum, spleen and liver iron concentrations, liver hepcidin expression and red blood cell parameters. There were no significant differences between wild-type and homozygous animals (Fig. 1D, 2A–G). To assess the role of genetic background, we bred the Ftmt deletion onto 129/SvEvTac for five generations (N5); we identified no significant differences between wild-type and homozygous animals on this background as well(data not shown). Given that baseline parameters of iron metabolism differ between these strains, this result suggests that alterations in serum or liver iron levels do not impact the steady state phenotype of Ftmt-deficient mice. Figure 2 Analysis of Ftmt-deficient mice on a pyridoxine-deficient diet We next placed a cohort of wild-type and homozygous mice on a pyridoxine-deficient diet for four months beginning at one month of age to induce formation of sideroblast or siderocyte (iron-granule positive nucleated or enucleated erythrocytes) (Fig. 2A–2G). Similar to observations made in C57BL/6J x C3H (B6C3F1) mice maintained on a pyridoxine-deficient diet(17), we noted a decreased MCV and hematocrit, along with decreased MCH, reticulocyte counts and CHR. The lack of decrease in HGB levels in our mice, while noted in the study in B6C3F1 mice, may have been due to strain-specific differences. While a small but statistically significant increase in red cell distribution width and an increase in number of siderocytes were noted in the homozygous animals early in the course of the experiment, these differences did not persist. Notably, the number of siderocytes was greater in homozygous than wild-type animals after three months on a pyridoxine-deficient diet; the decrease in siderocyte number thereafter in the homozygous animals may reflect an unknown mechanism of chronic adaptation to the pyridoxine-deficient diet (Fig. 2H). Siderocytes had similar appearance by light microscopy in wild-type and homozygous mutant animals (Fig. 3A–B), as did reticulocyte mitochondria by electron microscopy (Fig. 3C–D). As is true of most mouse sideroblastic anemia models, bone marrow ringed sideroblasts were not present in wild type or mutant animals (data not shown). Overall, there were no pronounced differences in red cell parameters between wild-type and homozygous mice maintained on the pyridoxine-deficient diet. Figure 3 Microscopic evaluation of Ftmt-deficient erythrocytes Our results do not contradict the relatively small body of Ftmt literature. Most, if not all, functional studies to date have relied on overexpression to demonstrate that Ftmt can alter the cellular effects of challenges such as oxidative stress under iron-loaded conditions. We have shown that Ftmt is not essential for viability or male fertility and that Ftmt deletion does not alter several parameters of iron metabolism under baseline conditions, nor does it affect the hematological response to a pyridoxine-deficient diet. Intriguingly, even in the absence of Ftmt, iron-rich deposits can be formed in erythroid cells, suggesting that Ftmt is not essential for siderocyte formation in mice. Furthermore, there may be redundant systems in mice to compensate for the lack of Ftmt function, as is the case with endothelial nitric synthase- or myoglobin-deficient mice in which adaptive mechanisms thoroughly compensate for the respective gene defects(18). One can also speculate that a failure to induce Ftmt expression on a pyridoxine-deficient diet might account for the fact that bone marrow ringed sideroblasts are not typically seen in mice. This demonstrates some of the limitations of using the mouse as a model for sideroblastic anemia—a failure to induce sideroblast formation in mice prevents us from effectively determining if Ftmt plays a role in detoxification of erythroid mitochondrial iron.