Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 ...

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$Heart failure with preserved ejection fraction - European Heart Journal$

1128 A59T mutant was mitochondrial in Txnrd2 2/2 MEFs as shown by co-staining of Prx III and the TAPe-tagged mutant (Figure 3B). The G375R mutant, however, was barely detectable immunocytochemically in accordance with the data obtained by immunoblotting (compare Figure 3A), rendering unequivocal assignment to a distinct cellular compartment difficult. In Txnrd2 2/2 cells, this mutant also appeared to be localized solely or predominantly in mitochondria (Figure 3B). Next, we studied the function of both Txnrd2 mutants D. Sibbing et al. in Txnrd2 2/2 MEFs. To this end, we used an assay that is based on the fact that Txnrd2 is indispensible for cell survival of fibroblasts when the cells are depleted of GSH by treatment with the g-glutamyl-cysteine-synthetase inhibitor BSO. 18 As illustrated in Figure 3C, reconstitution of wt Txnrd2 in Txnrd2 2/2 cells fully rescued cell death induced by GSH depletion. In contrast, cells expressing the mutants A59T and G375R died at very low BSO concentrations similarly to mock-transduced Txnrd2 2/2 cells Downloaded from http://eurheartj.oxfordjournals.org/ by guest on June 7, 2013
TXNRD2 and dilated cardiomyopathy 1129 (Figure 3C). We conclude from this experiment that both mutations abolish the function of Txnrd2. Both mutants impair the function of endogenous wild-type Txnrd2 Since Txnrd2 is enzymatically active as a homodimer and since all three patients were heterozygous for the mutations, it was important to see whether the mutant allele also impacts on the function of the wt enzyme allele by a dominant-negative mechanism. To address this question, the lentiviral vectors encoding wt as well as the A59T and G375R Txnrd2 mutants were transduced into Txnrd2 +/+ cells. Subcellular localization and cell survival in the presence of BSO were monitored in the same manner as described above for lentivirally transduced Txnrd2 2/2 cells. Although wt Txnrd2 and the A59T mutant were expressed at high level in Txnrd2 +/+ cells, the mutant G375R was barely detectable by western blotting (Figure 4A) as well as immunofluorescent staining (Figure 4B). Like in Txnrd2 2/2 cells, wt Txnrd2 and the A59T mutant localized to mitochondria in Txnrd2 +/+ cells, whereas the low expression of mutant G375R in Txnrd2 +/+ cells precluded any conclusion regarding its subcellular localization (Figure 4B). Although mock-transduced and wt Txnrd2-transduced cells were resistant to GSH depletion over a wide range of BSO concentrations, cell survival of A59T- and G375R-transduced Txnrd2 +/+ cells was significantly impaired in the presence of increasing BSO concentrations (Figure 4C). The fact that both mutants had a similar impact on cell survival despite different levels of expression suggests that mutant G375R is more toxic and that a non-toxic threshold of expression is selected for upon retroviral transduction. Notably, the introduction of the mutant alleles into Txnrd2 +/2 cells would have been closer to the in vivo situation in the patients. But, as it is impossible to adjust the expression level to that of the endogenous Txnrd2 alleles in the patients in vivo (which has been unknown anyway), it was of lesser importance whether one or two copies of the wt allele have resided in the cells. Apparently, it is more difficult to detect a dominant-negative effect in cells with two wt Txnrd2 copies left than in cells with one copy. Yet, the dominant-negative effect was already visible in an unequivocal manner in cells harbouring two wt alleles precluding the necessity of further breeding and of establishing cell lines heterozygous for Txnrd2. Ultrastructural analysis of cells expressing the Txnrd2 mutants A59T and G375R We then performed ultrastructural analyses to address the questions (i) whether morphological differences in mitochondria can be observed in knockout when compared with wt fibroblasts, similarly to those observed in heart-specific Txnrd2 2/2 cardiomyocytes ex vivo, 18 and (ii) whether the add-back of wt Txnrd2 or of the different mutants would affect mitochondrial morphology (Figure 5). We found that the size of mitochondria varied to a greater extent in knockout (Figure 5B) when compared with wt mitochondria (Figure 5A). Ultrastructural changes, ranging from swelling and loss of cristae to deterioration of matrix and mitochondrial membranes, were sometimes detectable in knockout mitochondria (Figure 5B). The add-back of wt Txnrd2 and of the different mutants, however, did not induce major changes in the overall morphology of mitochondria (Figure 5A and B). From this, we conclude that the functional parameters described in Figures 3 and 4 are more sensitive and more readily quantifiable than ultrastructural parameters. Discussion For the first time, we describe rare mutations in human TXNRD2 in patients with DCM. Both identified mutations result in amino acid substitutions in the highly conserved and functionally essential FAD-binding domain of the enzyme. A major influence of mutations located in FAD-binding domains on enzyme activity of 30 – 32 oxidoreductases has been repeatedly demonstrated. Figure 3 Expression of wild-type (wt) Txnrd2 and the novel mutations (A59T and G375R) in Txnrd2 2/2 cells. Immunoblotting using a Txnrd2-specific antibody (A) and confocal microscopy of immunocytochemical staining of transduced cells with an anti-Prx III-specific antibody (Alexa488) for mitochondria, an anti-FLAG-specific antibody (Alexa568) for reconstituted wt Txnrd2 and the A59T and G375R mutants and a nuclear counterstain with DAPI (B) revealed similar expression of wt Txnrd2 and A59T in both wild-type and knockout cells, whereas the G375R mutant was only weakly expressed inTxnrd2 2/2 cells. (B) Wild-type Txnrd2 and the A59T mutant predominantly localized to mitochondria [co-localization (yellow) of Prx III (green) and TAPe-tagged protein (red)] in knockout cells as shown in the false colour merge. The G375R mutant was only weakly expressed [compare (A)], but appeared also to localize to mitochondria. Shown are optical sections acquired by confocal microscopy of transduced cells stained with a nuclear DAPI counterstaining (blue), an anti-Prx III-specific antibody (Alexa488 shown in green) and an anti-FLAG-specific antibody for TAPe-tagged Txnrd2 (Alexa568 shown in red). Co-localization is indicated by a yellow colour arising from Prx III (green) and TAPe-tagged Txnrd2 (red) staining of knockout and wild-type cells as shown in the false colour merge. Mock ¼ empty virus-transduced cells. Scale bars, 10 mm. (C) The novel mutations did not restore the function of Txnrd2 in Txnrd2 2/2 fibroblasts under oxidative stress. Both transduced mutants A59T and G375R failed to rescue cell death of Txnrd2 2/2 cells under glutathione depletion induced by increasing L-buthionine sulfoximine concentrations. Txnrd2 2/2 cells transduced with the mutant forms were even more sensitive to glutathione depletion than mock-transduced Txnrd2 2/2 cells. Cell numbers were determined by trypan blue exclusion 48 h after treatment with the indicated L-buthionine sulfoximine concentrations. Shown are results of one out of three independent experiments with similar results. Cell numbers are presented as relative to the number of mock-transfected Txnrd2 +/+ cells in the absence of L-buthionine sulfoximine. Bars represent means and error bars standard deviations derived from cell counting of three different wells for each condition. P-values ,0.001 were considered as highly significant and are marked with two asterisks. Values between mock-transfected Txnrd2 +/+ cells (black bars) and Txnrd2 2/2 cells (empty bars) treated with different concentrations of L-buthionine sulfoximine were always highly significant (not shown for a simpler illustration). Downloaded from http://eurheartj.oxfordjournals.org/ by guest on June 7, 2013
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