Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 ...

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