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. 2012 Mar 13;109(11):4152-7.
doi: 10.1073/pnas.1119338109. Epub 2012 Feb 28.

Disruption of ATP-binding cassette B8 in mice leads to cardiomyopathy through a decrease in mitochondrial iron export

Yoshihiko Ichikawa  1 , Marina Bayeva, Mohsen Ghanefar, Vishnu Potini, Lin Sun, R Kannan Mutharasan, Rongxue Wu, Arineh Khechaduri, Tejaswitha Jairaj Naik, Hossein Ardehali
Affiliations

Disruption of ATP-binding cassette B8 in mice leads to cardiomyopathy through a decrease in mitochondrial iron export

Yoshihiko Ichikawa et al. Proc Natl Acad Sci U S A. .

Abstract

Mitochondrial iron levels are tightly regulated, as iron is essential for the synthesis of Fe/S clusters and heme in the mitochondria, but high levels can cause oxidative stress. The ATP-binding cassette (ABC) transporter ABCB8 is a mitochondrial inner membrane protein with an unknown function. Here, we show that ABCB8 is involved in mitochondrial iron export and is essential for baseline cardiac function. Induced genetic deletion of ABCB8 in mouse hearts resulted in mitochondrial iron accumulation and cardiomyopathy, as assessed by echocardiography and invasive hemodynamics. Mice with ABCB8 deletion in the heart also displayed mitochondrial damage, and higher levels of reactive oxygen species and cell death. Down-regulation of ABCB8 in vitro resulted in decreased iron export from isolated mitochondria, whereas its overexpression had the opposite effect. Furthermore, ABCB8 is needed for the maturation of the cytosolic Fe/S proteins, as its deletion in vitro and in vivo led to decreased activity of cytosolic, but not mitochondrial, iron-sulfur-containing enzymes. These results indicate that ABCB8 is essential for normal cardiac function, maintenance of mitochondrial iron homeostasis and maturation of cytosolic Fe/S proteins. In summary, this report provides characterization of a protein involved in mitochondrial iron export.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
ABCB8 KO in the heart. (A) Southern blot analysis of WT (lane 1) and targeted ES cells (lane 2). Homologous recombination results in a detection of a smaller band after genomic DNA is digested with Sca I restriction enzyme, as shown in A. (B) PCR results of the 5′ arm of ABCB8 heterozygote and homozygote floxed mice. Presence of LoxP sequence results in a larger fragment. (C) Tamoxifen-mediated genetic ablation of ABCB8 in the heart. DNA samples from two untreated (labeled 1 and 2) and two tamoxifen-treated (labeled 3 and 4) mice are shown. (D) ABCB8 mRNA levels are significantly lower in MCM × ABCB8f/f mice 2 wk after treatment with per-oral (PO) tamoxifen compared with WT ABCB8+/+ mice. (E) Levels of ABCB8 protein after treatment of MCM × ABCB8f/f mice with per-oral tamoxifen for 2 wk are significantly lower than in control mice. (F) Summary of densitometry analysis of Western blot (n = 3). Data are presented as mean ± SEM (*P < 0.05).
Fig. 2.
Fig. 2.
Cardiac function is compromised in ABCB8 KO mice. (A) M-mode echocardiography images of control and ABCB8 KO mice. Two sets of control animals are used in these studies: MCM × ABCB8+/+ mice treated with tamoxifen chow (white bars) and MCM × ABCB8f/+ mice treated with regular chow (gray bars). (B–E) Control and ABCB8 KO mice were analyzed by echocardiography 4 wk after tamoxifen treatment. Both percentage of FS (measure of ventricular contractility) and CO/BW (the amount of blood pumped by the heart) are significantly reduced, whereas LVDd (measure of chamber dilation) and HW/BW (measure of cardiac size) are not different in ABCB8 KO mice compared with control (n = 6). (F–I) Echocardiographic analysis of control and ABCB8 KO mice at a delayed phase (8 wk after tamoxifen treatment; n = 6). (J) Invasive hemodynamic measurements 8 wk after tamoxifen treatment. (K) dP/dtmax (contractility) and dp/dtmin (relaxation) in WT and ABCB8Δ/Δ mice 8 wk after tamoxifen treatment. (L) Heart rate during hemodynamic measurements in WT and KO mice (n = 6). Data are presented as mean ± SEM (*P < 0.05).
Fig. 3.
Fig. 3.
ABCB8 KO hearts display structural abnormalities. (A) Histological analysis of hearts from control (MCM × ABCB8+/+ treated with tamoxifen) and ABCB8Δ/Δ mice 4 and 8 wk after tamoxifen treatment. Upper: H&E eosin staining. Lower: Masson trichrome staining. Blue staining represents cardiac fibrosis. (B) Representative EM images of hearts from ABCB8+/+ and ABCB8Δ/Δ mice. Arrows indicate mitochondria. The graph below shows quantification of mitochondrial number and size (n = 5). (C) Representative EM images of hearts from ABCB8+/+ and ABCB8Δ/Δ mice. Hearts from ABCB8+/+ mice show well aligned mitochondria with clearly distinguishable cristae. Mitochondria from ABCB8Δ/Δ mouse hearts show accumulation of electron-dense material (asterisks), disruption of cristae, and deformed morphology. (D) LV myocardial sections from ABCB8+/+ and ABCB8Δ/Δ mice subjected to TUNEL and DAPI (nuclei) staining after 4 wk reveal higher cell death in ABCB8Δ/Δ mice. (E) Quantitative analysis of five fields as shown in D (n = 6). (F) Mitochondrial nonheme iron levels are higher in ABCB8 KO in the heart (n = 5). (G) The levels of mitochondrial heme are not altered in the hearts of ABCB8Δ/Δ mice (n = 3). Data are presented as mean ± SEM (*P < 0.05).
Fig. 4.
Fig. 4.
ABCB8 down-regulation and overexpression in NRCMs result in an increase and a decrease in mitochondrial iron levels, respectively. (A) Western blot of cytosolic and mitochondrial fractions from NRCMs. (B) Western blot of NRCMs treated with control and ABCB8 siRNA. Actin is used as an internal control (n = 3). (C) Densitometric analysis of the Western blot in B (n = 3). (D) Mitochondrial nonheme iron levels are increased in NRCMs treated with ABCB8 siRNA compared with control (n = 4). (E) Radiotracer (55Fe) in the mitochondrial fraction is in a higher amount in cells treated with ABCB8 siRNA compared with control siRNA (n = 3). (F) Confocal images of NRCMs transduced at a multiplicity of infection (MOI) of 0, 1, 5, and 10 of ABCB8 adenovirus with a GFP coding sequence. (G) Western blot of extracts from NRCMs transduced with increasing amounts of the ABCB8 or GFP adenovirus. (H) Mitochondrial nonheme iron levels are lower in NRCMs transduced at an MOI of 5 of ABCB8 adenovirus compared with GFP-transduced cells (n = 4). (I) Mitochondrial 55Fe levels in NRCMs treated with ABCB8 adenovirus are lower than in GFP (n = 4). Data are presented as mean ± SEM (*P < 0.05).
Fig. 5.
Fig. 5.
ABCB8 modulation alters mitochondrial iron export. (A) Western blot of HEK293 cells treated with control and ABCB8 siRNA. (B) Flow cytometry analysis of rhodamine 123-stained mitochondria revealed no difference in mitochondrial membrane potential between ABCB8 and control siRNA treated HEK293 cells (n = 3). Unstained mitochondria were used as a blank. (C) Export of 55Fe was reduced in isolated mitochondria from ABCB8 siRNA-treated cells compared with control siRNA-treated cells. Radioactivity in the soluble fraction was measured and normalized to mitochondrial protein and total cellular radioactivity (n = 3). (D) Export of 55Fe was increased in isolated mitochondria from ABCB8 adenovirus-transduced cells compared with GFP (n = 4). (E) ABCB8 siRNA treatment did not alter 32P export, suggesting the effect on 55Fe export is specific to ABCB8 modulation (n = 3). Data are presented as mean ± SEM (*P < 0.05).
Fig. 6.
Fig. 6.
ABCB8 knockdown results in the decreased activity of cytosolic Fe/S proteins. (A) XO activity (n = 8), (B) cytosolic aconitase activity (n = 6), and (C) GPAT levels (n = 3) in WT and ABCB8Δ/Δ hearts. (D) XO activity (n = 4) and (E) GPAT levels (n = 3) in NRCMs treated with ABCB8 siRNA. Data are presented as mean ± SEM (*P < 0.05).
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