Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 ...

More documents

Recommendations

Info

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

1130 D. Sibbing et al. Figure 4 Expression of wild-type (wt) Txnrd2 and the novel mutations (A59T and G375R) in Txnrd2 +/+ 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 wild-type (wt) Txnrd2 and the A59T and G375R mutants and a nuclear counterstain with DAPI (B) revealed comparable expression of wt Txnrd2 and A59T in wild-type cells, whereas the G375R mutant was barely detectable in Txnrd2 +/+ cells. (B) Wild-type Txnrd2 and the A59T mutant predominantly localized to mitochondria in Txnrd2 +/+ cells as shown in the false colour merge. The low expression of mutant G375R in Txnrd2 +/+ cells, however, precluded any conclusion regarding its subcellular localization (for details regarding staining and illustration, see Figure 3 and the Methods section). Mock ¼ empty virustransduced cells. Scale bars, 10 mm. (C) Cell survival of Txnrd2 +/+ cells transduced with A59T or G375R was impaired in response to increasing L-buthionine sulfoximine concentrations, indicating that both forms harbour a dominant-negative function. 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 Bars represent means and error bars standard deviations derived from cell counting of three different wells for each condition. P-values ,0.05 were considered as significant and are marked with an asterisk, and P-values ,0.001 were considered as highly significant and are marked with two asterisks (for experimental details and statistical analysis, see the legend of Figure 3). Downloaded from http://eurheartj.oxfordjournals.org/ by guest on June 7, 2013
TXNRD2 and dilated cardiomyopathy 1131 Figure 5 Ultrastructural analysis of Txnrd2 +/+ and Txnrd2 2/2 cells expressing wt Txnrd2 and the different Txnrd2 mutants. (A) Empty virustransduced (mock), wt Txnrd2-, and A59T- and G375R-transduced Txnrd2 +/+ mouse embryonic fibroblasts did not reveal major differences in mitochondrial structures. (B) Although the submitochondrial structures (cristae) were less distinct in Txnrd2 2/2 mouse embryonic fibroblasts compared with wild-type cells, the add-back of the different Txnrd2 variants did not grossly change the overall mitochondrial structure. The scale bar represents 500 nm. Functional analysis of the two identified mutants reconstructed in murine fibroblasts revealed that both forms do not rescue Txnrd2 2/2 cells from cell death induced by GSH depletion and that they exerted a dominant-negative effect when expressed in Txnrd2 +/+ cells. Regarding a possible causative role of the mutations for DCM, two questions had to be answered: first, are the mutations rare polymorphisms that are functionally silent or do they abolish or impair the function of Txnrd2? And second, as the mutations were heterozygous in all three patients, do they exert a dominant- negative function on the wt enzyme? Both questions raise fundamental issues regarding the enzymatic function of Txnrd2 that is shared among different cell types and tissues and should therefore be independent of the cellular model system. Apparently, the first question could only be addressed in Txnrd2 2/2 cells, thus precluding the use of cardiomyocytes. We reasoned that if the mutant alleles could rescue the Txnrd2 2/2 phenotype in a manner similar to the wt allele, the mutations would be functionally silent. Conversely, a functional significance of the mutations could be disclosed if the mutant alleles were not able to rescue Downloaded from http://eurheartj.oxfordjournals.org/ by guest on June 7, 2013
Page 1 and 2: European Heart Journal (2011) 32, 1
Page 3 and 4: TXNRD2 and dilated cardiomyopathy 1
Page 9: TXNRD2 and dilated cardiomyopathy 1
Page 13: TXNRD2 and dilated cardiomyopathy 1

Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 ...

Create successful ePaper yourself

Delete template?

Save as template?