Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 ...

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

1122 Conclusion 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. ----------------------------------------------------------------------------------------------------------------------------------------------------------- Keywords Dilated cardiomyopathy † Genetics † TXNRD2 Introduction Dilated cardiomyopathy (DCM) is a frequent cause of congestive heart failure and is the most common diagnosis in patients undergoing heart transplantation. According to systematic studies, famil- 1 – 3 ial transmission is observed in about 20–30% of DCM cases. Rare mutations in more than 20 different disease genes, most of them encoding for structural proteins of cardiomyocytes, have been identified. 4 – 7 Mutations in known disease genes, though, explain ,20% of inherited cases. 8 Thioredoxin reductases are essential components of the thioredoxin system 9 and are therefore crucial for the control of cellular redox balance. They are selenocysteine (Sec)-containing, homodimeric flavoenzymes that maintain thioredoxins, small proteins that catalyse redox reactions, in their reduced state using the reducing power of NADPH. 10 Three mammalian thioredoxin reductases exist; a cytosolic (TXNRD1), a mitochondrial (TXNRD2), and a testis-specific thioredoxin reductase (TXNRD3). The Sec-residue, encoded by a UGA codon, is the penultimate amino acid of the Cterminal catalytic centre of all thioredoxin reductases and is essential for enzyme activity. The premature termination of protein synthesis at the UGA codon is prevented by a stem-loop-like structure, the selenocysteine insertion sequence (SECIS) element located in the 3 ′ UTR. 11,12 Cardiac energy requirement is met to a large extent by oxidative phosphorylation in mitochondria that are highly abundant in cardiac myocytes. 13 The mitochondrial thioredoxin reductase (TXNRD2), along with mitochondrial thioredoxin and peroxiredoxins III and V, is of paramount importance for mitochondrial scavenging of reactive oxygen species (ROS). 9,10 It is well established that excessive ROS causes oxidative stress and cell death. 14 On the other hand, compelling evidence has established a role for ROS as modulators of intracellular signalling cascades. 15 Beyond providing protection against ROS, thioredoxins are known to inhibit or activate apoptotic signalling molecules like apoptosis signal-regulating kinase 1 and Ras or transcription factors like NF-kB. 16 We showed that glutathione (GSH) peroxidase 4, along with GSH, senses and translates oxidative stress into a distinct cell death signalling cascade involving the activation of 12/ 15-lipoxygenase and apoptosis-inducing factor. 17 Recently, we generated and characterized transgenic mice deficient for Txnrd2. 18 Ubiquitous inactivation resulted in embryonic death of anaemic embryos exhibiting marked thinning of the ventricular heart walls. Analysis of fibroblasts isolated from Txnrd2 2/2 embryos revealed a critical role for Txnrd2 in the removal of toxic ROS species. Heart-specific inactivation of Txnrd2 resulted in a phenotype reminiscent of human DCM with dilatation of heart chambers and thinning of ventricular walls and death shortly after birth. 18 This study set out to search for and characterize rare TXNRD2 mutations associated with DCM in humans. Methods DCM patients From January 1996 to July 2004, we collected blood from 227 consecutive patients with DCM at three participating centres in Germany: Deutsches Herzzentrum München, 1. Medizinische Klinik, Klinikum rechts der Isar, München, and Zentrum für Innere Medizin, Klinikum Garmisch-Partenkirchen. Cardiac catheterization and echocardiography was performed in all patients. Patients’ charts were used as the main source for clinical information. Diagnosis of DCM was based on the ‘Guidelines for the study of familial dilated cardiomyopathies’. 19 Patients were included if they had an ejection fraction of the left ventricle (LV) of ,45% and an LV end-diastolic diameter of .117% of the predicted value corrected for age and body surface area according to the equation of Henry et al. 20 Patients with coronary heart disease (.50% stenosis of at least one coronary artery or a major branch), a history of severe systemic arterial hypertension (arterial blood pressure .160/100 mmHg documented at repeated measurements), myocarditis (suspected or confirmed), persistent high-rate supraventricular arrhythmias, systemic disease, pericardial disease, congenital heart disease, or cor pulmonale were excluded. All patients had given written informed consent for participation in the study. The investigation conforms to the principles outlined in the Declaration of Helsinki and was approved by the institutional Ethics Committee. General population control sample D. Sibbing et al. Between 1999 and 2001, we conducted an epidemiological survey of the general population living in or near the city of Augsburg, Germany (KORA S4). 21 This was the fourth in a series of populationbased surveys originating from our participation in the World Health Organisation (WHO) Multinational MONItoring of trends and determinants in CArdiovascular disease (MONICA) project. The study population consisted of residents of German nationality born between 1 July 1925 and 30 June 1975 and identified through the registration office. A sample of 6640 subjects was drawn with 10 strata of equal size according to gender and age. Following a pilot study of 100 individuals, 4261 individuals (66.8%) agreed to participate in the survey, which were ethnic Germans with very few exceptions (.99.5%). During 2002 and 2003, we reinvestigated a subsurvey of 880 persons specifically for cardiovascular diseases. Seven hundred and two individuals from that subsurvey were selected as population-based controls. In nineteen (2.7%) individuals, an ejection fraction of the LV of ,45% was determined by echocardiography or symptoms/signs of heart failure were observed. The remaining 683 individuals were used as a DCM and congestive heart failure-free control sample. Blood samples were drawn after informed consent had been obtained. DNA extraction and DNA sequencing Peripheral venous whole blood samples were collected using EDTAvials (Sarstedt, Numbrecht, Germany). DNA was extracted from Downloaded from http://eurheartj.oxfordjournals.org/ by guest on June 7, 2013
TXNRD2 and dilated cardiomyopathy 1123 these samples using the commercially available QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). A randomly selected subset of 96 patients of the entire DCM study population was subjected to capillary Sanger sequencing. Primers, flanking the 17 coding exons plus the 3 ′ SECIS (exon 18) element of TXNRD2, were designed using the ExonPrimer software based on Primer3 (http://primer3.sourceforge.net/) according to the human genome build hg17. Amplifications were conducted following standard polymerase chain reaction (PCR) protocol procedures. PCR products were purified with both Exonuclease I and Shrimp alkaline phosphatase (Fermentas, St Leon-Rot, Germany) to digest the remaining primers and to reduce the remaining dNTPs. Subsequent cycle sequencing was performed including the respective forward and reverse primers and BigDye-Terminator 3.1 (Qiagen). Thereafter, products were cleaned by DyeEx (Qiagen). Both forward and reverse strands were sequenced for all 18 TXNRD2 exons in the population of 96 patients. Sequencing was performed with an ABI 3730 Automated Sequencer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s recommendations. Results of sequencing were analysed with Mutation Surveyor v3.00 (Softgenetics, State College, PA, USA). Matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry genotyping All synonymous and non-synonymous exonic sequence variants identified by direct sequencing in the population of 96 DCM patients were subsequently genotyped in all DCM patients (n ¼ 227) and in the KORA controls (n ¼ 683) using matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry (MALDI-TOF, Autoflex HT, Sequenom, San Diego, CA, USA) in a 384-well format using the standard protocols supplied by the manufacturer as described previously. 22 Genotype-analyses were performed with the Sequenom MassARRAY Typer Analyzer software v3.4.0.6 (Sequenom). Cloning of full-length wild-type Txnrd2, generation of both Txnrd2 mutants, and cloning into the lentiviral expression system Vector pGEM-Teasy-Txnrd2 was a kind gift from Dr Antonio Miranda- Vizuete (Universidad Pablo de Olavide, Sevilla, Spain). The vector contained most parts of the 5 ′ region of murine Txnrd2 cDNA including the mitochondrial leader sequence; however, it lacked the 3 ′ UTR including the SECIS element. The missing sequence was amplified by PCR from mouse liver cDNA 18 with the primers Oligo-SECIS-BglII-for (5 ′ -TCTCAGAGATCTGAGAAGATGTGGATGGAAC-3 ′ ) and Oligo- SECIS-rev (5 ′ -GTTTGAACCCCTGGCATTTCTAGAGCACT-3 ′ ). The resulting 160 bp PCR product was purified, cloned into pDrive via PaeI and BglII (Qiagen) and sequenced. The 3 ′ region of Txnrd2 was cloned via PaeI and BglII in pGEM-T-Easy-Txnrd2 (4702 bp), yielding pGEM-T-Easy-Txnrd2 [in the following referred to as wild-type (wt)]. Site-directed PCR mutagenesis using pGEM-T-Easy-Txnrd2 as a template was performed to generate the Txnrd2-A59T and Txnrd2-G375R mutant forms. Codons were chosen according to the highest murine codon usage of the respective amino acid. The Ala codon (GCC) at position 175 of Txnrd2 gene was replaced with Thr (ACC) using the primer pair Oligo-Txnrd2-A59T-for (5 ′ -AGCGGGAATCGATTATAA AGAT-3 ′ )/Oligo-Txnrd2-A59T-rev (5 ′ -GCCAGAAGCTTTCTTGCCT TGATAGC-3 ′ ); the Gly codon (GGG) at position 375 was mutated to Arg (AGG) with the primer pair Oligo-Txnrd2-G375R-for 5 ′ -GCTATCAAGGCAAGAAAGCTTCTGGC-3 ′ )/Oligo-Txnrd2-G375Rrev (5 ′ -GCCAGAAGCTTTCTTGCCTTGATAGC-3 ′ ). Mutations were verified by sequencing. All three forms were additionally tagged by a novel N-terminal TAPe (tandem affinity purification tag enhanced) 23 for detection of the tagged proteins with a FLAG-specific antibody in immunocytochemistry. The lentiviral expression vector 442L1 17 was used for efficient gene transfer and expression of wt Txnrd2 and the two mutant variants in mouse embryonic fibroblasts (MEFs). XhoI and EcoRI were used for digestion of 442L1 (7870 bp) and pcDNA3-NTAPe-Txnrd2 (1992 bp). Two shorter fragments carrying the mitochondrial NTAPe version of Txnrd2 were transferred into the backbone of 442L1, yielding the different NTAPe mitochondrial Txnrd2 forms. In any of these vectors, the VENUS nuclear membrane anchor protein in the bicistronic expression cassette was replaced by the puromycin acetyltransferase gene from the plasmid 442L1-NTAPe-Txnrd1 using BsrGI and SnaBI. This allowed stable expression of the various forms under puromycin selection. Isolation, maintenance of mouse embryonic fibroblasts, and stable expression of the different Txnrd2 forms in mouse embryonic fibroblasts Mouse embryonic fibroblasts were isolated from E12.5 Txnrd2 2/2 and Txnrd2 +/+ embryos and cultured in DMEM supplemented with 10% FCS, 1% glutamine, 50 U/mL penicillin G, and 50 mg/mL streptomycin as described. For stable expression of wt Txnrd2, Txnrd2-A59T, and Txnrd2-G375R in wt and knockout cells in a bicistronic manner, a thirdgeneration lentiviral expression system was utilized as described. 17 Transduced cells were selected with puromycin (1 mg/mL) for at least 3 weeks prior to the start of the experiment. Immunoblotting and production of an antibody directed against murine Txnrd2 Cell lysis, protein determination, SDS–PAGE, and protein transfer were performed as described. 17 A peptide sequence next to the Cterminus of Txnrd2 (VKLHISKRSGLEPTVTG) lacking the three Cterminal amino acids Cys, Sec, and Gly was used to raise monoclonal antibodies against Txnrd2 in rats. The peptide was obtained from Peptide Specialty Laboratories (Heidelberg, Germany) and was coupled to ovalbumin (OVA) or bovine serum albumin (BSA) at the C-terminus (peptide-OVA/KLH). Immunocytochemistry and confocal microscopy Mouse embryonic fibroblasts were seeded onto cover slips in six-well cell culture dishes at ≏45 000 cells per well, cultured for 24 h in standard DMEM and then fixed for 15 min in 2% PFA solution. Cells were washed twice with PBS and then permeabilized by treatment with 0.15% Nonidet P-40 (NP-40) in PBS for 3 min. Unspecific antibody binding was minimized by washing three times in PBS+ [PBS containing 1% (w/v) BSA, 0.15% (w/v) glycine]. After incubation of cells over night with the primary antibody (FLAG; Sigma-Aldrich, Deisenhofen, Germany; F1804 and Prx III; LabFrontier; LP-PA0030) at 48C in a humidified chamber, cells were washed with PBS, treated twice with PBS/NP-40 (0.15%), and transferred to PBS+. Cells were then incubated with fluorophore-labelled secondary antibodies (anti-Mouse- Alexa Fluor 568; A-10037 and anti-Rabbit-Alexa Fluor 488 A-21441; Invitrogen, Karlsruhe, Germany) in the dark for 45 min. Cells were then washed twice in PBS/NP-40 (0.15%) and twice in PBS. Finally, DAPI solution (1:10 000 in PBS) was briefly added, and cells were washed twice in PBS and mounted in mounting medium (Dako, Glostrup, Denmark). Cover slips were sealed with nail polish to prevent drying and they were kept overnight at 48C in the dark. Downloaded from http://eurheartj.oxfordjournals.org/ by guest on June 7, 2013
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