Impairment or ablation of antioxidant enzymes leading to the accumulation of toxic levels of ROS might initiate or promote a variety of human diseases, e.g., neurodegeneration, cancer development, arthritis, and atherosclerosis. Many studies have addressed the role of GSH and GSH peroxidases as central regulators in the maintenance of the cellular redox balance. To assess the importance of the thioredoxin/thioredoxin reductase system in this process, we generated and analyzed mice with a nonfunctional TrxR2, a key enzyme of the mitochondrial redox system. At present, very little is known about the physiological role of TrxR2. TrxR2 belongs to the family of selenoproteins that are characterized by one or several catalytically indispensable SeCys residues (16
). Since cotranslational SeCys incorporation at the UGA codon is very inefficient (35
), overexpression studies are strongly limited in vitro and in vivo. For this reason, gene inactivation methods in mice offer a promising strategy.
Anticipating that loss of TrxR2 might be associated with embryonic death, we used the cre/loxP technology for the generation of TrxR2
KO mice. Cre-mediated excision of the last four exons leads to the removal of the final 100 amino acids including the C terminally located redox-center consisting of Cys-SeCys-Gly. Mutational and biochemical studies have previously shown that SeCys is essential for the enzymatic function of TrxRs (9
). Also, this approach deemed necessary because the 5′ region of TrxR2
overlaps with the first exon of the catechol-o-methyltransferase
gene, and there is also alternative first exon usage in the TrxR2
We found that during development TrxR2
is mainly expressed in the embryonic heart and liver, reflecting the adult situation (22
). This expression profile associates TrxR2 function with organs characterized by high metabolic activity and further corroborates a crucial role for TrxR2 in the control of harmful intracellular ROS.
Ubiquitous deletion of the TrxR2 gene leads to embryonic lethality at E13. The phenotypic characteristics of TrxR2TrxR2−/−minus;/TrxR2−/−minus; embryos reflected the expression pattern of TrxR2 in normal development. TrxR2-null embryos were highly anemic and smaller compared to WT littermates. Histological examination of the heart revealed that the ventricular walls and the trabeculae were thinner in KO mice than in WT and TrxR2+/− mice. The number of ISEL-positive cells was increased in the liver but not in the heart. In CFU assays established from fetal liver at E13, the size of all types of hematopoietic colonies was dramatically reduced, whereas hematopoietic differentiation was not affected by TrxR2 deficiency.
Outgrowth of TrxR2TrxR2−/−minus;/TrxR2−/−minus;
MEFs in tissue culture was also impaired but not to the same extent as observed for hematopoietic cells. TrxR2TrxR2−/−minus;/TrxR2−/−minus;
fibroblasts could be propagated in vitro; they were, however, much more sensitive to oxidative stress imposed to the cells by GSH depletion than TrxR2+/+
cells. These data indicate that under standard cell culture conditions hematopoietic cells are more dependent on TrxR2 than fibroblasts. Several mechanisms, either alone or in combination, may account for the proposed hierarchy in TrxR2 dependence. Cells may differ in their ability to cope with oxygen radicals due to different degrees of pathway redundancy and/or overall antioxidant capacity. Alternatively, oxygen radical production of different cell types may vary greatly due to differences in their cellular metabolism. Finally, different cell types may differ widely in their intrinsic susceptibility versus resistance to oxygen radical-induced apoptosis due to different expression patterns of pro- and antiapoptotic proteins. The hematopoietic system is well known for its selective sensitivity to ionizing irradiation. Our finding that TrxR2
KO mice die of anemia at day 13 of embryonic development may thus emphasize the particular susceptibility of hematopoietic cells to oxygen radical-induced apoptosis. This interpretation is in line with the fact that deletion of Trx2
in the chicken B-cell line DT40 leads to oxygen-radical induced apoptosis mediated by cytochrome c
release and caspase-3 activation (37
The TrxR2 inactivation phenotype is less severe than the one observed in the KO mice of Trx2
, the main substrate of TrxR2. Trx2−/−
mice die at E10.5 due to massive apoptosis (26
). This suggests that other enzymes, such as, for example, TrxR1 or the GSH-dependent redox system, can partially substitute for the TrxR2
Besides the defect in hematopoiesis, TrxR2
KO mice exhibited morphological changes in the heart. To discriminate whether TrxR2 plays an intrinsic role in heart development and function or whether the defect in heart morphology is a consequence of the defect in hematopoiesis, we bypassed the hematopoietic phenotype and established cardiac tissue-restricted TrxR2
-deficient mice. These mice died shortly after birth from dilated cardiomyopathy and congestive heart failure. High-magnification and ultrastructural examination revealed severe distortion in the morphology of cardiomyocytes, i.e., pycnotic nuclei, partial loss of cross-striation, swelling of the mitochondria, and destruction of mitochondrial cristae. In contrast to the severe changes in morphology, no signs of apoptosis were detected in the myocardium by ISEL staining. Difficulties in detecting apoptotic cells in morphologically severely perturbed heart tissue have also been noted by others. Narula et al. found that cardiomyocytes of patients with idiopathic dilated or ischemic cardiomyopathy exhibit severe mitochondrial swelling and accumulation of cytochrome c
in the cytosol in the absence of apoptotic changes (24
). Likewise, the apoptotic index as defined by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay was found to be very low in models of chronic heart failure (12
). We suggest that cardiac tissue-restricted TrxR2
KO mice die of congestive heart failure due to mitochondrial dysfunction of cardiomyocytes before DNA degradation and cell death of cardiomyocytes can be detected to a significant extent.
The fact that mice with cardiac tissue-specific deletion of TrxR2 die shortly after birth has two implications. First, it indicates that the defect in heart morphology observed in TrxR2TrxR2−/−minus;/TrxR2−/−minus; mice at E13 is not only a consequence of the hematopoietic defect. It demonstrates for the first time that loss of one member of the Trx/TrxR system is indispensable for normal heart development and function. Second, lethality in complete TrxR2TrxR2−/−minus;/TrxR2−/−minus; embryos is delayed from E13 until birth in mice with a cardiac tissue-specific deletion of TrxR2. This suggests that the heart-specific defect observed in TrxR2TrxR2−/−minus;/TrxR2−/−minus; mice at E13 is not limiting on its own for survival during embryonic development. However, TrxR2 deficiency in cardiomyocytes becomes fatal when an entirely self-sustaining circulatory system has to be maintained by the heart. It is conceivable that in mice with ubiquitous deletion of TrxR2, anemia and the heart-specific defect enhance each other and, thus, may lead to an earlier lethal compound phenotype compared to the respective tissue specific deletions. To clarify this issue, it would be necessary to generate a hematopoiesis-specific KO of TrxR2.
Using transgenic approaches, Yamamoto et al. demonstrated that Trx1
is involved in cardiac myocyte growth of mice (39
). Heart-specific overexpression of a dominant-negative form of Trx1 led to increased oxidative stress in cardiomyocytes associated with cardiac hypertrophy, whereas overexpression of WT Trx1
reduced the extent of ROS-induced hypertrophy in response to pressure overload. Since the dominant-negative Trx1 may impair Trx2 activity as well, a putative contribution of Trx2 in protection of cardiomyocytes in this model seems likely. The perinatal lethality in our heart-specific TrxR2
KO model precludes at present a detailed study of heart function parameters. Crossing the conditional TrxR2
KO mice with the inducible heart-specific MERCreMER mice might generate a valuable tool for studying the impact of TrxR2
deficiency on adult heart physiology (32
A first indication that selenium is essential for heart function came from patients suffering from Keshan disease, a selenium deficiency disease endemic in China. Severely selenium-depleted humans show dilated congestive cardiomyopathy resembling the histopathologic phenotype of Friedreich's cardiomyopathy (28
) and the phenotype observed in the TrxR2TrxR2−/−minus;/TrxR2−/−minus;
mice. It is plausible that TrxR2 is the most likely candidate whose function is impaired in Keshan disease due to selenium deficiency. Whether functional impairment of other selenoproteins also contributes to the pathophysiology of this disease remains an open question. It is also noteworthy that TrxR2
is localized to chromosome 22q11
, a region deleted in the human haploinsufficiency velo-cardio-facial/DiGeorge Syndrome (VCS/DGS) (13
). Hallmarks of VCS/DGS have not been noticed in hemizygous/homozygous or in cardiac tissue-restricted TrxR2
KO mice (data not shown). Further studies will address the question whether loss of one TrxR2
allele may contribute to the compound VCS/DGS phenotype.