ATP-binding cassette B10 regulates early steps of heme synthesis

Abstract

Rationale: Heme plays a critical role in gas exchange, mitochondrial energy production, and antioxidant defense in cardiovascular system. The mitochondrial transporter ATP-binding cassette (ABC) B10 has been suggested to export heme out of the mitochondria and is required for normal hemoglobinization of erythropoietic cells and protection against ischemia-reperfusion injury in the heart; however, its primary function has not been established.

Objective: The aim of this study was to identify the function of ABCB10 in heme synthesis in cardiac cells.

Methods and results: Knockdown of ABCB10 in cardiac myoblasts significantly reduced heme levels and the activities of heme-containing proteins, whereas supplementation with δ-aminolevulinic acid reversed these defects. Overexpression of mitochondrial δ-aminolevulinic acid synthase 2, the rate-limiting enzyme upstream of δ-aminolevulinic acid export, failed to restore heme levels in cells with ABCB10 downregulation. ABCB10 and heme levels were increased by hypoxia, and reversal of ABCB10 upregulation caused oxidative stress and cell death. Furthermore, ABCB10 knockdown in neonatal rat cardiomyocytes resulted in a significant delay of calcium removal from the cytoplasm, suggesting a relaxation defect. Finally, ABCB10 expression and heme levels were altered in failing human hearts and mice with ischemic cardiomyopathy.

Conclusions: ABCB10 plays a critical role in heme synthesis pathway by facilitating δ-aminolevulinic acid production or export from the mitochondria. In contrast to previous reports, we show that ABCB10 is not a heme exporter and instead is required for the early mitochondrial steps of heme biosynthesis.

Keywords: ATP-binding cassette transporters; cardiomyopathies; heme; mitochondria; δ-aminolevulinic acid.

Figure 1. ABCB10 regulates heme levels in cardiac myoblasts

A,B, Cellular (A) and mitochondrial (B) heme content with ABCB10 overexpression in H9c2 cells (n=6). C,D, Cellular (C) and mitochondrial (D) heme levels with ABCB10 knockdown in H9c2 cells (n=6). Ad-GFP, adenovirus encoding GFP, Ad-ABCB10, adenovirus encoding ABCB10 protein. Data are presented as mean ± SEM. * p<0.05 vs. control.

Figure 2. Heme-containing enzyme activities are reduced by ABCB10 knockdown

A-C, Catalase (A), peroxidase (B) and mitochondrial complex IV (C) activities in H9c2 with ABCB10 knockdown (n=3-6). D-F, Heme-containing enzyme activities with ABCB10 overexpression in H9c2 cells (n=3-6). Data are presented as mean ± SEM. * p<0.05 vs. control.

Figure 3. ABCB10 does not regulate heme synthesis

A, Protein levels of ALAS 1, the rate-limiting enzyme in heme synthesis. B, Densitometry analysis of ALAS1 Western blots (n=6). C,D, Total porphyrin levels with ABCB10 knockdown alone (C) or with ABCB10 knockdown and ALA supplementation (D) (n=6). E, Incorporation of ⁵⁵Fe into protoporphyrin IX (PPIX) with ABCB10 knockdown in H9c2 cells. Cells are treated with ALA to promote porphyrin synthesis, and incubated with ⁵⁵Fe, followed by quantification of ⁵⁵Fe saturation of PPIX in the organic fraction containing porphyrins (n=6). Data are presented as mean ± SEM. * p<0.05 vs. control.

Figure 4. ABCB10 promotes mitochondrial ALA export

A,B, Cellular (A) and mitochondrial (B) heme levels in ABCB10 knockdown cells incubated with 1.2mM ALA for 6 hours (n=6-18). ALA treatment reversed the decrease in heme levels associated with ABCB10 knockdown (as shown in Fig. 1A and B). C-E, Activities of heme-containing enzymes in cells with ABCB10 knockdown following supplementation with ALA (n=4-6). ALA supplementation restored the activities of heme-containing enzymes to control levels in ABCB10 knockdown cells. F, Cellular heme levels with ABCB10 knockdown and supplementation with 500μM methyl-succinate (m-succinate) or vehicle (n=6). G, Cellular heme levels with overexpression of zALAS2 and knockdown of ABCB10 (n=6). Data are presented as mean ± SEM. * p<0.05 vs. control, # p<0.05 vs. zALAS2 overexpressing, control shRNA transfected cells.

Figure 5. Hypoxic upregulation of ABCB10 is required for cell survival

A, Western blot analysis showing that ABCB10 is upregulated by hypoxia in H9c2 cells, and this increase is prevented by treatment of hypoxic cells with ABCB10 siRNA (n=3). B, Heme levels in H9c2 subjected to 48 hours of hypoxia (1% O₂) or normoxia, with or without suppression of hypoxic ABCB10 upregulation by siRNA (n=6). C, MitoSox staining (red) of H9c2 cells with and without reversal of ABCB10 upregulation by siRNA subjected to 48 hours of hypoxia followed by 30 minutes of reoxygenation. Nuclei are counterstained with Hoescht (blue). Representative images are shown on the left, and quantification is presented on the right (n=6, 4 fields per sample). D,E, Cell death as assessed by propidium iodine (PI) and Annexin V staining and quantified by flow cytometry in H9c2 cells with and without ABCB10 siRNA treatment subjected to hypoxia-reoxygenation injury as in C (n=9) (D), or with additional incubation with 200μM hydrogen peroxide for 12 hours in hypoxia prior to reoxygenation (n=6) (E). Data are presented as mean ± SEM. * p<0.05 vs. control.

Figure 6. ABCB10 is upregulated in the ischemic heart and modulates Ca²⁺ transients

A, ABCB10 protein levels in sham-operated mice and mice subjected to MI injury one week and one month following the surgery. Densitometric analysis is presented below the Western blot (n=3-4). B,C, Total cellular (B) and mitochondrial (C) heme levels in mouse hearts 1 month following MI or sham operation (n=3). D, ABCB10 protein levels in explanted human hearts with ischemic cardiomyopathy or in noncardiomyopathic control human hearts. Densitometric analysis is shown on the right. E, Heme levels in myopathic and non-myopathic human hearts (n=10). F-H, Analysis of Ca²⁺ transients in NRCM with ABCB10 and control siRNA: representative images (F), F/F0 as a measure of transient magnitude (n=10-11 cells from 3 coverslips) (G), and rise and decay times (n=15 cells from 4 coverslips) (H). NM, nonmyopathic; ICM, ischemic cardiomyopathy. Data are presented as mean ± SEM. * p<0.05 vs. control.

Figure 7

ABCB10 and heme synthesis pathway