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• W2003575797 abstract "Prolylhydroxylase domain proteins (PHD) are cellular oxygen-sensing molecules that regulate the stability of the α-subunit of the transcription factor hypoxia inducible factor (HIF)-1. HIF-1 affects cardiac development as well as adaptation of the heart toward increased pressure overload or myocardial infarction. We have disrupted PHD2 in cardiomyocytes (cPhd −/−) using Phd2flox/flox mice in combination with MLCvCre mice, which resulted in HIF-1α stabilization and activation of HIF target genes in the heart. Although cPhd2−/− mice showed no gross abnormalities in cardiac filament structure or function, we observed a significant increased cardiac capillary area in those mice. cPhd2 −/− mice did not respond differently to increased mechanical load by transverse aortic constriction compared with their wild-type (wt) littermates. After ligation of the left anterior descending artery, however, the area at risk and area of necrosis were significantly smaller in the cPhd2−/− mice compared with Phd2 wt mice in line with the described pivotal role of HIF-1α for tissue protection in case of myocardial infarction. This correlated with a decreased number of apoptotic cells in the infarcted myocardium in the cPhd2−/− mice and significantly improved cardiac function 3 weeks after myocardial infarction. Prolylhydroxylase domain proteins (PHD) are cellular oxygen-sensing molecules that regulate the stability of the α-subunit of the transcription factor hypoxia inducible factor (HIF)-1. HIF-1 affects cardiac development as well as adaptation of the heart toward increased pressure overload or myocardial infarction. We have disrupted PHD2 in cardiomyocytes (cPhd −/−) using Phd2flox/flox mice in combination with MLCvCre mice, which resulted in HIF-1α stabilization and activation of HIF target genes in the heart. Although cPhd2−/− mice showed no gross abnormalities in cardiac filament structure or function, we observed a significant increased cardiac capillary area in those mice. cPhd2 −/− mice did not respond differently to increased mechanical load by transverse aortic constriction compared with their wild-type (wt) littermates. After ligation of the left anterior descending artery, however, the area at risk and area of necrosis were significantly smaller in the cPhd2−/− mice compared with Phd2 wt mice in line with the described pivotal role of HIF-1α for tissue protection in case of myocardial infarction. This correlated with a decreased number of apoptotic cells in the infarcted myocardium in the cPhd2−/− mice and significantly improved cardiac function 3 weeks after myocardial infarction. When oxygen availability is impaired, the resulting hypoxia activates homeostatic mechanisms at the systemic and cellular level (1Schofield C.J. Ratcliffe P.J. Biochem. Biophys. Res. Commun. 2005; 30: 617-626Crossref Scopus (284) Google Scholar). Hypoxia-inducible factors (HIFs) 2The abbreviations used are: HIFhypoxia-inducible factorAARarea at riskAngangiopoietinAONarea of necrosisBnip3BCL2/adenovirus E1B 19-kDa protein-interacting protein 3BNPbrain natriuretic peptidecPhd2−/−cardiac-specific PHD2 knock outEembryonic dayFSfractional shorteningGlut-1glucose transporter-1Hox-1hemoxygenaseiNOSinducible nitric-oxide synthaseLADleft anterior descending arteryPDK1pyruvate dehydrogenase kinase 1pfk1phosphofruktokinase 1PGKphosphoglycerate kinasePHDprolylhydroxylase domainTACtransverse aortic constrictionTTC2,3,5-triphenyltetrazolium chloridewtwild type. are essential players in these responses because they regulate the transcription of a large number of genes that affect a myriad of cellular processes, including angiogenesis, metabolism, cell survival, and oxygen delivery (2Wenger R.H. FASEB J. 2002; 16: 1151-1162Crossref PubMed Scopus (1019) Google Scholar). HIF is a heterodimeric protein comprising the oxygen-sensitive α-subunit HIF-1α or the more cell type-specifically expressed HIF-2α or HIF-3α and the oxygen-insensitive β-subunit (3Wang G.L. Semenza G.L. J. Biol. Chem. 1995; 270: 1230-1237Abstract Full Text Full Text PDF PubMed Scopus (1737) Google Scholar). In the presence of oxygen, HIFα becomes hydroxylated at two critical proline residues by prolylhydroxylase domain (PHD) enzymes (4Ivan M. Kondo K. Yang H. Kim W. Valiando J. Ohh M. Salic A. Asara J.M. Lane W.S. Kaelin Jr., W.G. Science. 2001; 292: 464-468Crossref PubMed Scopus (3917) Google Scholar, 5Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. von Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4489) Google Scholar). The PHD protein family responsible for HIFα regulation consists of three members called prolylhydroxylase domain (PHD)1, PHD2, and PHD3 (6Epstein A.C. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2749) Google Scholar, 7Bruick R.K. McKnight S.L. Science. 2001; 294: 1337-1340Crossref PubMed Scopus (2130) Google Scholar). Following prolyl-4-hydroxylation of the critical prolyl residues under normoxic conditions, the ubiquitin ligase von Hippel-Lindau tumor suppressor protein recognizes HIF-1α subunits and targets them for rapid ubiquitination and proteasomal degradation (8Hon W.C. Wilson M.I. Harlos K. Claridge T.D. Schofield C.J. Pugh C.W. Maxwell P.H. Ratcliffe P.J. Stuart D.I. Jones E.Y. Nature. 2002; 417: 975-978Crossref PubMed Scopus (571) Google Scholar, 9Min J.H. Yang H. Ivan M. Gertler F. Kaelin Jr., W.G. Pavletich N.P. Science. 2002; 296: 1886-1889Crossref PubMed Scopus (590) Google Scholar, 10Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. Nature. 1999; 399: 271-275Crossref PubMed Scopus (4179) Google Scholar). hypoxia-inducible factor area at risk angiopoietin area of necrosis BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 brain natriuretic peptide cardiac-specific PHD2 knock out embryonic day fractional shortening glucose transporter-1 hemoxygenase inducible nitric-oxide synthase left anterior descending artery pyruvate dehydrogenase kinase 1 phosphofruktokinase 1 phosphoglycerate kinase prolylhydroxylase domain transverse aortic constriction 2,3,5-triphenyltetrazolium chloride wild type. Based on the ubiquitous expression pattern and its dominant effect in normoxia, it had to be assumed that PHD2 is the most critical HIF-1α-regulating PHD isoform in most tissues (11Berra E. Benizri E. Ginouvès A. Volmat V. Roux D. Pouysségur J. EMBO J. 2003; 22: 4082-4090Crossref PubMed Scopus (1101) Google Scholar, 12Lieb M.E. Menzies K. Moschella M.C. Ni R. Taubman M.B. Biochem. Cell Biol. 2002; 80: 421-426Crossref PubMed Scopus (159) Google Scholar, 13Appelhoff R.J. Tian Y.M. Raval R.R. Turley H. Harris A.L. Pugh C.W. Ratcliffe P.J. Gleadle J.M. J. Biol. Chem. 2004; 279: 38458-38465Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar). This notion, learned from in vitro studies, was confirmed by the up to now available genetically modified Phd2 mouse models (14Katschinski D.M. Acta Physiol. 2009; 195: 407-414Crossref PubMed Scopus (35) Google Scholar). Phd2 knock-out embryos die between embryonic day (E) 12.5 and E14.5 (15Takeda K. Ho V.C. Takeda H. Duan L.J. Nagy A. Fong G.H. Mol. Cell. Biol. 2006; 26: 8336-8346Crossref PubMed Scopus (323) Google Scholar). This time point coincides with the increased levels of PHD2 in wild-type (wt) mice starting from E9.0. A major role of PHD2 in regulating the HIF system is further underscored by mouse models with a somatic Phd2−/− knock out, which enable to analyze the in vivo function of PHD2 in the adult mice. Two independent inducible Phd2−/− mouse models were developed by Takeda et al. (16Takeda K. Cowan A. Fong G.H. Circulation. 2007; 116: 774-781Crossref PubMed Scopus (188) Google Scholar) and Minamishima et al. (17Minamishima Y.A. Moslehi J. Bardeesy N. Cullen D. Bronson R.T. Kaelin Jr., W.G. Blood. 2008; 111: 3236-3244Crossref PubMed Scopus (233) Google Scholar). The phenotype of these mice most obviously resembles the consequences of HIFα overexpression with increased angiogenesis, erythropoiesis, and extramedullar hematopoiesis (17Minamishima Y.A. Moslehi J. Bardeesy N. Cullen D. Bronson R.T. Kaelin Jr., W.G. Blood. 2008; 111: 3236-3244Crossref PubMed Scopus (233) Google Scholar, 18Takeda K. Aguila H.L. Parikh N.S. Li X. Lamothe K. Duan L.J. Takeda H. Lee F.S. Fong G.H. Blood. 2008; 111: 3229-3235Crossref PubMed Scopus (215) Google Scholar). Most interestingly, these mice also develop a cardiac phenotype with symptoms of dilated cardiomyopathy. In the heart, HIF-1α and thereby also the PHDs are known to influence key components of heart development, morphogenesis, and function (19Krishnan J. Ahuja P. Bodenmann S. Knapik D. Perriard E. Krek W. Perriard J.C. Circ. Res. 2008; 103: 1139-1146Crossref PubMed Scopus (92) Google Scholar, 20Krishnan J. Suter M. Windak R. Krebs T. Felley A. Montessuit C. Tokarska- Schlattner M. Aasum E. Bogdanova A. Perriard E. Perriard J.C. Larsen T. Pedrazzini T. Krek W. Cell Metab. 2009; 9: 512-524Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). Long term activation of HIF-1α in the heart seems to activate detrimental pathways resulting in the development of heart failure (21Bekeredjian R. Walton C.B. MacCannell K.A. Ecker J. Kruse F. Outten J.T. Sutcliffe D. Gerard R.D. Bruick R.K. Shohet R.V. PLoS One. 2010; 5: e11693Crossref PubMed Scopus (97) Google Scholar). Thus, it is tempting to speculate that loss of PHD2 in the heart is responsible for the dilated cardiomyopathy as observed in the inducible Phd2−/− mice. However, because these mice also develop an increased hematocrit, secondary blood hyperviscosity-dependent cardiac changes cannot be ruled out. Therefore we have generated cardiomyocyte-specific PHD2 knock-out mice (cPhd2−/−) by crossing MLCvCre mice with Phd2flox/flox mice. While this work was ongoing Moslehi et al. have recently reported the generation of cardiac-specific PHD2 knock-out mice using αMHCCre expressing mice as deleter mice in combination with Phd2flox/flox mice (22Moslehi J. Minamishima Y.A. Shi J. Neuberg D. Charytan D.M. Padera R.F. Signoretti S. Liao R. Kaelin Jr., W.G. Circulation. 2010; 122: 1004-1016Crossref PubMed Scopus (122) Google Scholar). In line with our observation, Moslehi et al. observed no striking differences in morphology or function of resting young cPhd2−/− mice. Moslehi et al. additionally analyzed older mice and demonstrated that with increasing age cardiomyocyte-specific PHD2 knock-out mice developed changes, which phenocopy ischemic cardiomyopathy. At a young age these animals responded to increased afterload with fortified heart failure despite the successful development of cardiac hypertrophy. In contrast to the results of Moslehi et al., we did not observe significant changes in the development of hypertrophy and heart failure of young PHD2-deficient mice toward increased afterload in our animal model. Moreover, we observed cardioprotective effects in the PHD2-lacking mice in a model of myocardial ischemia. Challenging the mice with permanent ligation of the left anterior descending artery (LAD) resulted in cardiac tissue protection and decreased myocardial infarct size in the cPhd2−/− mice compared with Phd2 wt littermates. Taken together, our results show for the first time that lack of PHD2 in cardiomyocytes protects the heart from an acute ischemic insult. This phenomenon is at least in part most likely due to an increased capillary area in the cPhd2−/− mice. The generation and detailed characterization of Phd2flox/flox mice will be reported elsewhere. 3B. Wielockx, K. Anastassiadis, A. F. Stewart, and G. Breier, unpublished observations. In brief, LoxP sites were cloned into the Phd2 gene via Red/ET recombination (23Zhang Y. Muyrers J.P. Testa G. Stewart A.F. Nat. Biotechnol. 2000; 18: 1314-1317Crossref PubMed Scopus (215) Google Scholar) flanking exons 2 and 3. To characterize the Phd2flox/flox mice, full inactivation of the Phd2 gene was achieved via an intercross with PGKCre mice. Consistent with earlier reports, no viable PHD2−/− mice were obtained (15Takeda K. Ho V.C. Takeda H. Duan L.J. Nagy A. Fong G.H. Mol. Cell. Biol. 2006; 26: 8336-8346Crossref PubMed Scopus (323) Google Scholar, 17Minamishima Y.A. Moslehi J. Bardeesy N. Cullen D. Bronson R.T. Kaelin Jr., W.G. Blood. 2008; 111: 3236-3244Crossref PubMed Scopus (233) Google Scholar). Cre-mediated excision of exons 2 and 3 results in a frameshift mutation from exon 1 to 4 leading to an early translational stop and subsequent Phd2 knock out. Exons 2 and 3 encode for almost the entire catalytic domain of Phd2. The successful deletion of Phd2 in the floxed mice was verified by Western blotting on protein extracts of embryonic hearts (E14.5) from PGKCre × Phd2flox/flox mice using a homemade Phd2 antibody against a C-terminal peptide (supplemental Fig. 1). No PHD2 protein could be detected in Phd2−/− hearts. Moreover, the fact that HIF-1α protein is stabilized in these samples verifies the loss of functionality of Phd2. To analyze, if a shorter PHD2 protein encoded by exon 1 is formed, we performed Western blotting using an antibody, which detects the N-terminal part of PHD2 (supplemental Fig. 2). There is indeed a shorter Phd2 protein detectable in the Phd2+/− and Phd2−/− mice, which in its apparent molecular mass matches the predicted number of 31.5 kDa. The putatively exon 1-encoded protein, however, was expressed compared with the wt PHD2 protein in wt or Phd2+/− embryos only at very low levels. Whether this shorter protein would have a dominant negative character and therefore would bind to its interaction partner (e.g. HIFα) and block its normal function has not been studied. However, it was suggested that in human PHD2 both Arg371 and Pro317 (in mice Arg348 and Pro294 located in exons 3 and 2, respectively) contribute to the HIF-1α or HIF-2α binding (24Percy M.J. Furlow P.W. Beer P.A. Lappin T.R. McMullin M.F. Lee F.S. Blood. 2007; 110: 2193-2196Crossref PubMed Scopus (124) Google Scholar). In our mice these sites are not present anymore, and a dominant negative interaction with HIF-1α is therefore unlikely. All animals in this study were backcrossed to C57BL/6 mice at least five times. Phd2flox/flox mice were crossed with MLCvCre+/− mice (25Minamisawa S. Gu Y. Ross Jr., J. Chien K.R. Chen J. J. Biol. Chem. 1999; 274: 10066-10070Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) to generate Phd2flox/flox × MLCvCre+/− mice within two generations. Phd2flox/flox × MLCvCre+/− mice were then crossed with Phd2flox/flox mice to obtain cPhd2−/− (Phd2flox/flox × MLCvCre+/−) mice and littermate control wild-type mice (Phd2flox/flox). Mice were genotyped by PCR using the following primers: Cre forward, 5′-CGTACTGACGGTGGGAGAAT-3′ and Cre reverse, 5′-CGGCAAAACAGGTAGTTA-3′; PHD2 forward, 5′-CTCACTGACCTACGCCGTGT-3′ and PHD2 reverse, 5′-CGCATCTTCCATCTCCATTT-3′. The deletion of floxed exons in cDNAs isolated from ventricles or atria by PCRs was shown with the following primers: PHD2 forward, 5′-TACAGGATAAACGGCCGAAC-3′ and PHD2 reverse, 5′-GGCAACTGAGAGGCTGTAGG-3′. The forward primer binds in exon 1, the reverse primer binds in exon 5. Animal experimentation was performed with cPhd2−/− mice and littermate Phd2 wt control mice. All protocols regarding animal experimentation were conducted according to the German animal protection laws and approved by the responsible governmental authority (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit in Oldenburg; animal experimentation numbers 33.942502-04-10/0024 and 33.9-42502-04-10/0069). Pressure overload was induced by transverse aortic constriction (TAC) and performed essentially as described previously by our group with blunted 27-gauge needles as placeholders in 10-week-old female mice (26Silter M. Kögler H. Zieseniss A. Wilting J. Schafer K. Toischer K. Rokita A.G. Breves G. Maier L.S. Katschinski D.M. Eur. J. Physiol. 2010; 459: 569-577Crossref PubMed Scopus (17) Google Scholar). LAD ligations were performed by an investigator who was blinded regarding the genotypes of the mice. The measurement of infarct size was performed by an additional investigator, who was likewise blinded. LAD ligation was performed on female mice having a minimum weight of 21 g (8–12 weeks of age). Mice were anesthetized (2% isoflurane). The trachea was surgically exposed, and tracheal intubation was performed. A blunt intubation cannula (intubation cannula, stainless steel with Y-adapter, 1.2-mm outer diameter, 30-mm length; Hugo Sachs Elektronik, Harvard Apparatus GmbH) was inserted into the trachea. Correct tube placement was confirmed by direct visualization of the cannula within the previously exposed trachea. The tracheal tube was connected to a mechanical ventilator (MiniVent; Hugo Sachs Elektronik, Harvard Apparatus GmbH), and the animals were ventilated by using a pressure-controlled ventilation mode (stroke volume 150 μl, 150 strokes/min fractional inspired O2 = 0.3). After exposing the heart, the pericardium was removed, and a 9-0 polyamide suture with a U-shaped needle was passed under the left anterior descending artery. The suture was tied to occlude the artery. The chest was closed and the mouse recovered. Echocardiography and measurement of posterior wall thickness, septum thickness, left ventricular end systolic diameter, left ventricular end diastolic diameter, and fractional shortening (FS) were performed as described by Silter et al. (26Silter M. Kögler H. Zieseniss A. Wilting J. Schafer K. Toischer K. Rokita A.G. Breves G. Maier L.S. Katschinski D.M. Eur. J. Physiol. 2010; 459: 569-577Crossref PubMed Scopus (17) Google Scholar). Six hours after LAD ligation the mice were given heparin (250 units) and anesthetized, and the hearts were excised. Myocardial infarct size was determined by using Evans blue/2,3,5-triphenyltetrazolium chloride (TTC) staining as described by Bohl et al. (27Bohl S. Medway D.J. Schulz-Menger J. Schneider J.E. Neubauer S. Lygate C.A. Am. J. Physiol. Heart Circ. Physiol. 2009; 297: H2054-H2058Crossref PubMed Scopus (70) Google Scholar). Briefly, the ascending aorta was cannulated with a 20-gauge tubing adapter, and 1% Evans blue was perfused into the aorta and coronary arteries to delineate the “area at risk” (AAR). The Evans blue dye was distributed uniformly to those areas of the myocardium, which were well perfused; hence, the area of the myocardium that was not stained with Evans blue was defined as the AAR. The left ventricle was separated from the rest of the heart and sectioned into three transverse slices. Sections were incubated in 2% TTC for 20 min at 37 °C to identify viable tissue. Infarct quantification was performed on digital photographs (SMZ 100; Nikon, Tokyo, Japan) using ImageJ (National Institutes of Health). The area of necrosis (AON) and AAR were determined as the average percent area per slice from the middle section and the lowest section. Also, myocardial samples were collected for cyrosectioning and assessment of apoptosis. Heart tissue was rapidly homogenized in a buffer containing 4 m urea, 140 mm Tris (pH 6.8), 1% SDS, 2% Nonidet P-40, and protease inhibitors (Roche Applied Science). Protein concentrations were quantified (DC Protein Assay; Bio-Rad). For immunoblot analysis protein samples were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Biosciences) by semidry blotting (PeqLab). Primary antibodies used were: anti-PHD2 (NB100-2219; Novus), anti-HIF-1α (NB100-479; Novus), and anti-vinculin (hVin-1, V9264; Sigma). For detection of immunocomplexes, horseradish peroxidase-conjugated secondary goat anti-rabbit or goat anti-mouse antibodies (Santa Cruz Biotechnology) were used, and membranes were incubated with 100 mm Tris-HCl (pH 8.5), 2.65 mm H2O2, 0.45 mm luminol, and 0.625 mm coumaric acid for 1 min. Chemiluminescence signals were detected with the LAS3000 camera system (Fuji Film Europe, Düsseldorf, Germany). After RNA extraction reverse transcription (RT) was performed with 2 μg of RNA and a first strand cDNA synthesis kit (Fermentas, St. Leon-Rot). mRNA levels were quantified by using 1 μl of the cDNA reaction and a SYBR Green qPCR reaction kit (Clontech) in combination with a MX3000P light cycler (Stratagene). The initial template concentration of each sample was calculated by comparison with serial dilutions of a calibrated standard. Primers were as follows: glucose transporter-1 (Glut-1) forward, 5′-TGGCCTTGCTGGAACGGCTG and Glut-1 reverse, 5′-TCCTTGGGCTGCAGGGAGCA-3′; mS12 forward, 5′-GAAGCTGCCAAGGCCTTAGA-3 and mS12 reverse, 5′-AACTGCAACCAACCACCTTC-3′; PHD2 forward, 5′-TTGCTGACATTGAACCCAAA-3′ and PHD2 reverse, 5′-GGCAACTGAGAGGCTGTAGG-3′; PHD1 forward, 5′-GCTAGGCTGAGGGAGGAAGT-3′ and PHD1 reverse, 5′-TCTACCCAGGCAATCTGGTC-3′; PHD3 forward, 5′-GGCCGCTGTATCACCTGTAT-3 and PHD3 reverse, 5′-TTCTGCCCTTTCTTCAGCAT-3′; brain natriuretic peptide (BNP) forward, 5′-AAGTCCTAGCCAGTCTCCAGA-3′ and BNP reverse, 5′-GAGCTGTCTCTGGGCCATTTC-3′; phosphofruktokinase 1 (Pfkl) forward, 5′-ACGAGGCCATCCAGCTCCGT-3′ and Pfkl reverse 5′-TGGGGCTTGGGCAGTGTCCT-3′; pyruvate dehydrogenase kinase 1 (PDK-1) forward, 5′-GTTCACGTCACGCTGGGCGA-3′ and PDK-1 reverse, 5′-CCAGGCGTCCCATGTGCGTT-3′; phosphoglycerate kinase (PGK) forward, 5′-ACGTCTGCCGCGCTGTTCTC-3′ and PGK reverse, 5′-ACCATGGAGCTATGGGCTCGGT-3′; VEGF forward, 5′-CGAACGTACTGCCGATTGAGA-3 and VEGF reverse, 5′-TGGTGAGGTTTGATCCGCATGATCTG-3′; angiopoietin (Ang)-1 forward, 5′-CGCTCTCATGCTAACAGGAGGTTGG-3′ and Ang-1 reverse, 5′-GCATTCTCTGGACCCAAGTGGCG-3′; Ang-2 forward, 5′-CACCCAACTCCAAGAGCTCGG-3′ and Ang-2 reverse, 5′-CACGTAGCGGTGCTGACCGG-3′; BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (Bnip3) forward, 5′-GTCCAGTGTCGCCTGGCCTC-3′ and Bnip3 reverse, 5′-TGGGAGCGAGGTGGGCTGTC-3′; iNOS, inducible nitric-oxide synthase; (iNOS) forward, 5′-GCTGCCTTCCTGCTGTCGCA-3′ and iNOS reverse, 5′-GGAGCCGCTGCTGCCAGAAA-3; hemoxygenase (Hox)-1 forward, 5′-TGGTGCAAGATACTGCCCCTGC-3′ and Hox-1 reverse, 5′-TGGGGGACAGCAGTCGTGGT-3′, apelin forward, 5′-TGCAGTTTGTGGAGTGCCACTG-3′ and apelin reverse, 5′-GCACCGGGAGGCACT-3′; adrenomedullin forward, 5′-TGGCCCCCTACAAGCCAGCAAT-3′ and adrenomedullin reverse, 5′-GCCAACGGGATACTCGCCCG-3′. The sets of primers for adenosine receptor A2b and CD73 were according to previously published sequences (28Eckle T. Köhler D. Lehmann R. El Kasmi K. Eltzschig H.K. Circulation. 2008; 118: 166-175Crossref PubMed Scopus (335) Google Scholar). Plasma BNP levels were measured using a commercially available enzyme immunoassay kit (catalog number EK-011-23; Phoenix Pharmaceuticals) according to the manufacturer's instructions. For Trichrome staining, heart tissue was fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and embedded in paraffin before sectioning. Freshly isolated hearts were treated as described before (29Zieseniss A. Schroeder U. Buchmeier S. Schoenenberger C.A. van den Heuvel J. Jockusch B.M. Illenberger S. Cell Tissue Res. 2007; 327: 583-594Crossref PubMed Scopus (16) Google Scholar). Briefly, hearts were sectioned (5–10 μm) in a cryostat, mounted on glass slides, and dried for at least 30 min. After being washed with PBS, the sections were briefly incubated with 1% Triton X-100 and then fixed with 4% formaldehyde and permeabilized with 0.2% Triton X-100. Nonspecific binding of antibodies was blocked by incubation with 1% bovine serum albumin for 1 h. The sections were then incubated with the polyclonal anti-CD31 antibodies (DIA 310, diluted 1:20; Dianova) and anti-vinculin antibodies (hVin-1, diluted 1:100; Sigma) at room temperature for 1 h, washed three times in PBS, and then incubated with anti-mouse TR (diluted 1:200; Santa Cruz Biotechnology) and anti-rat FITC (1:200; Santa Cruz Biotechnology). Samples were counterstained for DNA (Hoechst). Finally, samples were washed extensively in PBS, mounted in Mowiol, and examined by fluorescence microscopy (Axio Observer D1; Carl Zeiss, Göttingen, Germany). The capillary area was determined by analyzing CD31-positive pixels using ImageJ. In addition, the number of capillaries were counted per view of field. Paraffin-embedded sections were immunostained as described by others (22Moslehi J. Minamishima Y.A. Shi J. Neuberg D. Charytan D.M. Padera R.F. Signoretti S. Liao R. Kaelin Jr., W.G. Circulation. 2010; 122: 1004-1016Crossref PubMed Scopus (122) Google Scholar). The anti-HIF-1α primary antibody (Novus NB100-123) was used at a 1:1000 dilution. Standard procedures were applied for fixation (1.5% glutaraldehye and 1.5% paraformaldehyde in phosphate buffer) of hearts of cPhd2−/− and Phd2 wt mice. The hearts were then washed in PBS and fixed in 2% osmium tetroxide. The samples were dehydrated in a graded series of alcohol and embedded in Araldite. Ultrathin sections were stained with 2% methanolic uranyl acetate. Samples were then examined in a transmission electron microscope equipped with photodocumentation. Apoptotic cells were detected with a TUNEL assay according to the manufacturer's protocol (In Situ Cell Death Detection kit, catalog number 11 684 795 001; Roche Applied Science). Three hearts of each genotype were analyzed, and five random high power fields from each heart sample were quantified. Data were analyzed by two-tailed Student's t test or (in case of repeated echocardiography analysis after TAC treatment) as paired t test and presented as mean ± S.E. A p value < 0.05 was considered statistically significant. For generating cPhd2−/− mice, we crossed Phd2flox/flox mice with mice that express the Cre recombinase under the control of the MLCv promoter (25Minamisawa S. Gu Y. Ross Jr., J. Chien K.R. Chen J. J. Biol. Chem. 1999; 274: 10066-10070Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The MLCv-driven Cre permits recombination of the floxed Phd2 exons 2 and 3 in ventricular cardiomyocytes (Fig. 1A). Isolation and PCR analysis of cDNAs from atria and ventricles of cPhd2−/− and littermate Phd2 wt control mice verified successful recombination in the ventricles but not in the atria of the cPhd2−/− mice (Fig. 1B). The additional bands seen in the PCR analysis of Phd2 wt mice most likely are the result of described PHD2 splice variants (30Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). Quantification of PHD2 RNA levels in the left ventricles and atria of cPhd2−/− mice compared with Phd2 wt mice revealed a roughly 5-fold reduction in the knock-out ventricles but not the atria, confirming the MLCvCre-mediated knock out in the ventricular cardiomyocytes (Fig. 1C). Considering that the RNA was isolated from the whole left ventricles, which besides the cardiomyocytes also contain especially cardiac fibroblasts but also endothelial cells, smooth muscle cells etc., this reduction in PHD2 expression is in accordance with a successful recombination in the Cre-expressing cardiomyocytes. In line, cardiac PHD2 expression was also reduced at the protein level in left ventricles but not the atria of cPhd2−/− mice as determined by immunoblot analysis (Fig. 1D). As a consequence of the PHD2 knock out, HIF-1α protein levels were slightly increased (Fig. 1D). Immunohistochemical analysis confirmed the accumulation of HIF-1α protein in cell nuclei of the cardiomyocytes (Fig. 2). Detection of HIF-2α by immunohistochemistry was hampered by varying qualities of the batches of the commercially available polyclonal anti-HIF-2α antibodies. At least in vitro it was described that in contrast to PHD3, PHD2 seems to have higher activity toward HIF-1α compared with HIF-2α (30Hirsilä M. Koivunen P. Günzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (656) Google Scholar). Therefore the increased levels of HIF-1α in the cardiomyocytes are in line with an effective PHD2 knock out in our mouse model. A moderate activation of the HIF-signaling pathway in the hearts of cPhd2−/− mice was likewise detected by analyzing the expression of the HIF target genes PHD3 (Fig. 1C), Glut-1, Pfk1, and PDK1 (Fig. 3A). Whereas increased levels of Glut-1, Pfk1, and PDK1 reflect the impact of HIF on glucose transport and glucose metabolism, respectively (31Wenger R.H. Stiehl D.P. Camenisch G. Sci. STKE. 2005; 2005: re12PubMed Google Scholar), the elevated levels of PHD3 are indicative of the described negative HIF/PHD feedback loop, which involves the HIF-dependent transcription of PHD3 (32Stiehl D.P. Wirthner R. Köditz J. Spielmann P. Camenisch G. Wenger R.H. J. Biol. Chem. 2006; 281: 23482-23491Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). In contrast, PHD1 RNA levels, which are not inducible by hypoxia (13Appelhoff R.J. Tian Y.M. Raval R.R. Turley H. Harris A.L. Pugh C.W. Ratcliffe P.J. Gleadle J.M. J. Biol. Chem. 2004; 279: 38458-38465Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar), were not affected in the hearts of cPhd2−/− mice (Fig. 1C). HIF target genes involved in angiogenesis (VEGF, Ang-1, Ang-2), vasotonus regulation (adrenomedullin, apelin, iNOS, and Hox-1), purinergic signaling (CD73, and adenosine receptor A2b), and apoptosis (Bnip3) were found to be unchanged (Fig. 3B). BNP is produced in the ventricles and is regarded as surrogate marker for heart failure (33Palazzuoli A. Gallotta M. Quatrini I. Nuti R. Vasc. Health Risk Manag. 2010; 6: 411-418Crossref PubMed Google Scholar). Analyzing BNP RNA levels in the left ventricles o" @default.
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• W2003575797 cites W1555471169 @default.
• W2003575797 cites W1749022239 @default.
• W2003575797 cites W1963922085 @default.
• W2003575797 cites W1982291271 @default.
• W2003575797 cites W2005729255 @default.
• W2003575797 cites W2005868568 @default.
• W2003575797 cites W2010356452 @default.
• W2003575797 cites W2014740708 @default.
• W2003575797 cites W2016160962 @default.
• W2003575797 cites W2017575850 @default.
• W2003575797 cites W2019521269 @default.
• W2003575797 cites W2021741218 @default.
• W2003575797 cites W2024206845 @default.
• W2003575797 cites W2030334534 @default.
• W2003575797 cites W2031385405 @default.
• W2003575797 cites W2032268426 @default.
• W2003575797 cites W2039959602 @default.
• W2003575797 cites W2040031153 @default.
• W2003575797 cites W2041492515 @default.
• W2003575797 cites W2042216712 @default.
• W2003575797 cites W2046429224 @default.
• W2003575797 cites W2050140657 @default.
• W2003575797 cites W2052201224 @default.
• W2003575797 cites W2052553321 @default.
• W2003575797 cites W2056714071 @default.
• W2003575797 cites W2056951282 @default.
• W2003575797 cites W2058021996 @default.
• W2003575797 cites W2064266243 @default.
• W2003575797 cites W2070683625 @default.
• W2003575797 cites W2072526492 @default.
• W2003575797 cites W2072727898 @default.
• W2003575797 cites W2083988122 @default.
• W2003575797 cites W2098209017 @default.
• W2003575797 cites W2113259370 @default.
• W2003575797 cites W2115095194 @default.
• W2003575797 cites W2116571550 @default.
• W2003575797 cites W2130456602 @default.
• W2003575797 cites W2144625075 @default.
• W2003575797 cites W2150332104 @default.
• W2003575797 cites W2167786713 @default.
• W2003575797 cites W2596045242 @default.
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