Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 ...

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

1132 the phenotype of Txnrd2 2/2 cells as the wt Txnrd2 gene does. The phenotype of Txnrd2 2/2 cells is defined as high sensitivity to GSH depletion, a phenotype that is highly sensitive and well quantifiable. The necessity to assess the function of Txnrd2 quantitatively in a homogeneous population of cells precluded the use of primary cells. Given these experimental constraints, we considered the question whether conclusions drawn from fibroblasts might be irrelevant or relevant for cardiomyocytes. At least two points suggest that a phenotype observed in fibroblasts will also be relevant for cardiomyocytes. First, cardiomyocytes are extremely dependent on energy production by mitochondria, probably more so than fibroblasts. Findings made in fibroblasts are therefore likely to impact also on cardiomyocytes. Secondly, fibroblasts express Txnrd1 as well as Txnrd2, whereas cardiomyocytes only show low expression of Txnrd1 33 and depend on Txnrd2 for reduction of thioredoxin. 18 If there is some redundancy between the thioredoxin-1 and thioredoxin-2 system, which is suggested by the phenotype of mouse knockouts in both systems, cardiomyocytes should be functionally affected by mutations at least as severely as fibroblasts. We did not reconstruct or functionally test any of the five nonsynonymous variants identified in both patients and controls to rule out a major pathological effect by them. Structural considerations combined with evolutionary studies on 46 aligned FADbinding oxidoreductases (Figure 2A and B; see Supplementary material online, Figure S1) including 13 Txnrd2 genes revealed that A59 and G375 are located in two helices contributing to the formation of the FAD-binding pocket. The mutations A59T and G375R are predicted not to be tolerated (SIFT; see Supplementary material online, Table S3) and to disrupt the formation of the FAD-binding pocket. G375 is conserved in all FAD-binding oxidoreductases across evolution that we have studied. In all but two oxidoreductase genes, A59 is conserved and only two Drosophila thioredoxin reductases harbour valine as another hydrophobic amino acid at this position. Both mutations A59T and G375R stand out in that they are unique across all in silico analyses performed which discriminates them from the other five variants and supports their causality (see Supplementary material online, Table S3). None of the five non-synonymous variants observed in patients and controls are conserved in evolution. Four of them are located at the surface of the molecule and are thus irrelevant for NADPH/FAD binding and enzymatic activity. The fifth I370T is located in the same helix as G375R, but the side chain points away from FAD. Most importantly, T370 is present at this position in mouse Txnrd2 used for modelling human TXNRD2, and sequence alignments have furthermore provided conclusive evidence that threonine at this position is the evolutionary ancient allele. In summary, two mechanisms are likely to account for the observed phenotype in the human DCM patients: first, increased oxidative stress due to reduced expression of fully functional TXNRD2. Even though heterozygosity of Txnrd2 (Txnrd2 +/2 )or of mitochondrial thioredoxin (Txn2 +/2 ) has no gross effect on mouse development or maturation, 18,34 gene dosage may contribute to the capacity to cope with oxidative stress under challenge. Secondly, increased oxidative stress due to a dominant-negative effect of the toxic mutants impairing the function of the wt protein that is plausible because thioredoxin reductases act as homodimers. 27 All three patients were heterozygous carriers of the two TXNRD2 mutations. Although having severely impaired LV function when diagnosed, they died at rather advanced age. This suggests that heterozygous carriage of the mutations has resulted in a moderate phenotype and accumulation of cardiac tissue damage during life that has long been compensated. Similar to previous observations for other already established DCM causing mutations in different genes, 4 – 7 TXNRD2 mutations described here explain only a small fraction of overall disease burden. As TXNRD2 is a Sec-containing enzyme, the presence of selenium is essential for proper enzyme function. In this context, TXNRD2 in addition to GPX1 may represent the link between selenium deficiency and Keshan’s disease, an endemic cardiomyopathy prevalent in China. 18,35,36 Limitations The following limitations of our study must be acknowledged: due to the fact that mostly rare mutations in more than 20 different disease genes have been described in inherited cases of DCM, we are unable to fully exclude the presence of already known mutations in our study cohort. In addition, due to their advanced age in none of the three patients with novel amino acid residue-altering TXNRD2 mutations, parents were available for study. Thus, the question of an inherited vs. a de novo mutation and cosegregation within the family cannot be resolved in either of them. The fact that we observed only three TXNRD2 mutation carrying patients precludes to reliably describing a TXNRD2 mutant DCM subphenotype. The answer to this question will have to await the identification of further TXNRD2 mutation carrying patients in other cohorts and the present study may provide the rationale for further investigations. Experimental limitations of our study include the fact that cardiomyocytes would have been the optimal model system. Yet, there are experimental constraints regarding the use of cardiomyocytes and therefore we had to choose the MEFs as the experimental system. Conclusions Our data underline the essential role of TXNRD2 for mitochondrial redox homeostasis in cardiomyocytes and normal heart function 18 and point to a novel pathophysiological mechanism for heart failure: failure of the cellular mechanisms that counterbalance the production of ROS. For the first time, we describe mutations in DCM patients in a gene involved in the regulation of cellular redox state. TXNRD2 mutations may explain a fraction of human DCM disease burden and further studies are needed to corroborate the present results. Supplementary material Supplementary material is available at European Heart Journal online. Acknowledgements D. Sibbing et al. We thank Dr Antonio Miranda-Vizuete (Universidad Pablo de Olavide, Sevilla, Spain) for providing the plasmid encoding the Downloaded from http://eurheartj.oxfordjournals.org/ by guest on June 7, 2013
TXNRD2 and dilated cardiomyopathy 1133 murine Txnrd2 cDNA and Luise Jennen for excellent technical assistance. We are most grateful to Stefan Rahlfs for his suggestion to include glutathione reductases in the definition of the FADbinding domain in TXNRD2. Funding This work has been supported by a grant from the Technische Universität München (KKF 69-04) to N.B. and A.P. as well as by grants from the German Research Foundation (DFG) Priority Programme 1087 ‘Selenoproteins’ to G.W.B. and M.C. The KORA research platform was initiated and financed by the Helmholtz Center Munich, German Research Center for Environmental Health, which is funded by the German Federal Ministry of Education and Research and by the State of Bavaria. The work of KORA is supported by the German Federal Ministry of Education and Research (BMBF) in the context of the German National Genome Research Network (NGFN-2 and NGFN-plus). Our research was also supported within the Munich Center of Health Sciences (MC Health) as part of LMUinnovativ. Additional funding was obtained by A.P. from the German National Genome Research Network NGFN 01GR0803 and the BMBF German Federal Ministry of Research BMBF 01EZ0874, T.M. German National Genome Research Network NGFN and S.K. from German National Genome Research Network NGFN BMBF 01GS0838; Leducq Foundation 07-CVD 03, LMU Excellence Initiative. The sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or approval of the manuscript. Conflict of interest: none declared. References 1. Towbin JA, Bowles NE. The failing heart. Nature 2002;415:227–233. 2. Grunig E, Tasman JA, Kucherer H, Franz W, Kubler W, Katus HA. Frequency and phenotypes of familial dilated cardiomyopathy. J Am Coll Cardiol 1998;31:186–194. 3. 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