Essential role for mitochondrial thioredoxin reductase in hematopoiesis, heart development, and heart function

Abstract

Oxygen radicals regulate many physiological processes, such as signaling, proliferation, and apoptosis, and thus play a pivotal role in pathophysiology and disease development. There are at least two thioredoxin reductase/thioredoxin/peroxiredoxin systems participating in the cellular defense against oxygen radicals. At present, relatively little is known about the contribution of individual enzymes to the redox metabolism in different cell types. To begin to address this question, we generated and characterized mice lacking functional mitochondrial thioredoxin reductase (TrxR2). Ubiquitous Cre-mediated inactivation of TrxR2 is associated with embryonic death at embryonic day 13. TrxR2(TrxR2(-/-)minus;/TrxR2(-/-)minus;) embryos are smaller and severely anemic and show increased apoptosis in the liver. The size of hematopoietic colonies cultured ex vivo is dramatically reduced. TrxR2-deficient embryonic fibroblasts are highly sensitive to endogenous oxygen radicals when glutathione synthesis is inhibited. Besides the defect in hematopoiesis, the ventricular heart wall of TrxR2(TrxR2(-/-)minus;/TrxR2(-/-)minus;) embryos is thinned and proliferation of cardiomyocytes is decreased. Cardiac tissue-restricted ablation of TrxR2 results in fatal dilated cardiomyopathy, a condition reminiscent of that in Keshan disease and Friedreich's ataxia. We conclude that TrxR2 plays a pivotal role in both hematopoiesis and heart function.

FIG. 1.

Gene targeting and expression pattern of TrxR2 in mice. (A) Strategy for conditional disruption of the TrxR2 gene. The 3′ region of the TrxR2 gene including exons 12 to 18 is shown in the upper line. The SeCys codon UGA is marked with an asterisk. For conditional gene targeting, exons 15 to 18 were flanked with loxP sites (open triangles). Homologous recombination, subsequent Flp-mediated removal of the frt (black triangles)-flanked neomycin phosphotransferase gene (neo), and Cre-mediated deletion of the C-terminally located redox-center are outlined below. Underneath, the expected DNA fragments from characteristic restriction digests (E, EcoRI; K, KpnI) detected with the corresponding 5′ (pMC67), 3′ (pMC68) and internal (int.) probes (striped rectangles) are schematically depicted. In the targeting vector the thymidine kinase gene (TK) is placed downstream from the 3′ arm for negative selection. (B) Homologous recombination in ES cell clones was confirmed by Southern blotting of EcoRI-digested DNA hybridized with 3′ (lanes 1 and 2) and 5′ (lane 3) external probes and after removal of the neo gene (lane 2). (C) Germ line transmission of the modified TrxR2 allele was verified by PCR genotyping of tail DNA (lanes 1 and 2). The floxed TrxR2 allele gives rise to a 40-bp-longer product than the WT allele (lane 1; lane 2, 100-bp ladder). Southern blotting of KpnI-digested DNA, isolated from E12.5 embryos of TrxR2^+/− intercrosses, was hybridized with the internal probe (lanes 3 to 5) revealing heterozygous (lane 3), WT (lane 4), and KO (lane 5) embryos. (D) Semiquantitative RT-PCR analyses of mRNA obtained from E12.5 embryos of TrxR2^+/− intercrosses. (E) Additional gene targeting strategy to study the TrxR2 expression pattern with β-galactosidase as a reporter gene. TrxR2^{+/−[lacZk.i.]} mice were generated and identified by Southern blotting as described in panels B and C. (F to I) Immunohistochemistry of TrxR2^+/+ (F and H) and TrxR2^{+/−[lacZ k.i.]} (G and I) embryos stained with an anti-lacZ antibody (brown color; arrows) revealed strong expression in the atrium (A) and ventricle (V) of the heart (F and G) and lower expression in the liver (H and I). Cryosections were counterstained with hematoxylin. Scale bars (F to I), 100 μm.

FIG. 2.

Anemic appearance of TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} embryos. (A) TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} embryos (right) are anemic and smaller than WT littermates (left) but do not display any gross developmental abnormalities. (B to E) The blood vessels of freshly prepared TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} embryos (E13.5) (C and E) were less supplied with blood than TrxR2^+/+ littermates (B and D) as best seen in the yolk sac (B and C) and developing brain (D and E). Placenta formation was not affected (B and E), as also confirmed by histological examination (data not shown).

FIG. 3.

Histological examination of TrxR2-deficient embryos. (A and B) H&E staining of transversal sections through the heart region of TrxR2^+/+ (A) and TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} (B) embryos at E13.0. All basic structures of the heart are formed, but the ventricular chamber walls are thinned and the trabeculae are less prominent. The endocardial cushions and valves are not affected. BrdU incorporation (C and D) and nuclear PCNA staining (E and F) revealed reduced proliferation of myocardial cells in TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} (D and F) versus TrxR2^+/+ (C and E) embryos. Hematoxylin and eosin (G and H) and ISEL staining (I and K) of liver sections revealed pleiomorphic, spongiform-like structures (H) and increased apoptosis (K) in TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} embryos. ISEL-(brown) and antibody (blue)-double staining of TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} liver sections revealed that part of the apoptotic cells (brown) are of hematopoietic origin (CD45-positive, L), whereas the majority of nonhematopoietic keratin 8 (m)-, and keratin 18-positive cells (N) were negative for ISEL staining. Scale bars: A and B, 100 μm; C, D, I, and K, 25 μm; E to H, 20 μm.

FIG.4.

Hematopoietic colony formation (A to E), fibroblast proliferation (F) and removal of ROS (G to L) are severely affected in TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} cells. (A to E) CFU assays were performed with isolated liver cells from E12.5 embryos. The liver cells were cultivated for 1 week. Colonies were classified as macrophages (group 1); burst-forming units (group 2); mixed colonies composed of macrophages, granulocytes, and burst-forming units (group 3); granulocytes and macrophages (group 4); and pure granulocyte colonies (group 5). (A) Differentiation into the different colony types was not altered in TrxR2^+/− and TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} cells. (B to E) The size of individual TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} colonies (C and E) was, however, dramatically reduced compared to WT controls (B and D). (F) Decreased proliferation of MEFs of TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} compared to TrxR2^+/+ mice. (H) When GSH synthesis was inhibited in MEFs by 50 μM BSO, cell death was rapidly induced in TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} but not in TrxR2^+/+ cells. (G) The main cellular defense systems against ROS that may compensate for the loss of the TrxR2 are schematically depicted. (I to L) Triple staining with Hoechst 33342 (blue nuclear staining), MitoTracker Green FM (green mitochondrial staining), and RedoxSensor Red CC-1 (red) after 48 h of BSO treatment revealed predominant lysosomal localization of the RedoxSensor Red CC-1 in WT cells (I) and mitochondrial localization in TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} cells (K). (L) The distribution of the dyes in the different cellular compartments is quantified. (M) NAC treatment (5 mM) rescued TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} MEFs from BSO-induced apoptosis. A strongly increasing number of detaching and dying cells was apparent only in KO cells (days 3 and 5) without NAC treatment. Photographs were taken at days 1, 3, and 5 after BSO and NAC treatment. (H) Quantification of the results.

FIG. 5.

Dilated congestive cardiomyopathy in heart-specific TrxR2 KO mice. (A) Southern blot analysis revealed a decrease of the floxed allele (arrow) relative to the KO band in heart (lane H) compared to brain tissue (lane B). (B and C) Myocardial thinning and dilatation in cardiac tissue-specific TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} mice (C). The ventricles and lung vessels were dilated and congested with blood cells. (D and E) Higher magnification of ventricular myocard (boxed in panels B and C) revealed nuclei of various sizes, massive vacuolization and perturbed directional arrangement of TrxR2^{TrxR2−/−minus;/TrxR2−/−minus;} cardiomyocytes. (F and G) Phase-contrast imaging demonstrated strong reduction of cross-striation in KO cardiomyocytes (G) compared to control tissue (arrow in panel F). (H to L) Transmission electron microscopy revealed severe mitochondrial malformation and swelling with destruction or loss of cristae in KO myocardial (I and L) versus WT (H and K) cells at low (H and I) and high (K and L) magnifications. Scale bars: B and C, 250 μm; D to G, 10 μm.