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Protection of Cardiomyocytes from Ischemic/Hypoxic Cell Death via Drbp1 and pMe2GlyDH in Cardio-specific ARC Transgenic Mice

2008; Elsevier BV; Volume: 283; Issue: 45 Linguagem: Inglês

10.1074/jbc.m804209200

ISSN

1083-351X

Autores

Jong-Ok Pyo, Jihoon Nah, Hyojin Kim, Jae‐Woong Chang, Young-Wha Song, D Yang, Dong‐Gyu Jo, Hyung‐Ryong Kim, Han‐Jung Chae, Soo‐Wan Chae, Seung‐Yong Hwang, Seung-Jun Kim, Hyo Joon Kim, Chunghee Cho, Changgyu Oh, Woo Jin Park, Yong‐Keun Jung,

Tópico(s)

Cardiac Ischemia and Reperfusion

Resumo

The ischemic death of cardiomyocytes is associated in heart disease and heart failure. However, the molecular mechanism underlying ischemic cell death is not well defined. To examine the function of apoptosis repressor with a caspase recruitment domain (ARC) in the ischemic/hypoxic damage of cardiomyocytes, we generated cardio-specific ARC transgenic mice using a mouse α-myosin heavy chain promoter. Compared with the control, the hearts of ARC transgenic mice showed a 3-fold overexpression of ARC. Langendoff preparation showed that the hearts isolated from ARC transgenic mice exhibited improved recovery of contractile performance during reperfusion. The cardiomyocytes cultured from neonatal ARC transgenic mice were significantly resistant to hypoxic cell death. Furthermore, the ARC C-terminal calcium-binding domain was as potent to protect cardiomyocytes from hypoxic cell death as ARC. Genome-wide RNA expression profiling uncovered a list of genes whose expression was changed (>2-fold) in ARC transgenic mice. Among them, expressional regulation of developmentally regulated RNA-binding protein 1 (Drbp1) or the dimethylglycine dehydrogenase precursor (pMe2GlyDH) affected hypoxic death of cardiomyocytes. These results suggest that ARC may protect cardiomyocytes from hypoxic cell death by regulating its downstream, Drbp1 and pMe2GlyDH, shedding new insights into the protection of heart from hypoxic damages. The ischemic death of cardiomyocytes is associated in heart disease and heart failure. However, the molecular mechanism underlying ischemic cell death is not well defined. To examine the function of apoptosis repressor with a caspase recruitment domain (ARC) in the ischemic/hypoxic damage of cardiomyocytes, we generated cardio-specific ARC transgenic mice using a mouse α-myosin heavy chain promoter. Compared with the control, the hearts of ARC transgenic mice showed a 3-fold overexpression of ARC. Langendoff preparation showed that the hearts isolated from ARC transgenic mice exhibited improved recovery of contractile performance during reperfusion. The cardiomyocytes cultured from neonatal ARC transgenic mice were significantly resistant to hypoxic cell death. Furthermore, the ARC C-terminal calcium-binding domain was as potent to protect cardiomyocytes from hypoxic cell death as ARC. Genome-wide RNA expression profiling uncovered a list of genes whose expression was changed (>2-fold) in ARC transgenic mice. Among them, expressional regulation of developmentally regulated RNA-binding protein 1 (Drbp1) or the dimethylglycine dehydrogenase precursor (pMe2GlyDH) affected hypoxic death of cardiomyocytes. These results suggest that ARC may protect cardiomyocytes from hypoxic cell death by regulating its downstream, Drbp1 and pMe2GlyDH, shedding new insights into the protection of heart from hypoxic damages. Programmed cell death, or apoptosis, is an evolutionarily conserved process that plays a critical role in embryonic development and adult tissue homeostasis. In humans and mice, dysregulated apoptosis has been implicated in the pathogenesis of cancer and in autoimmune, cardiovascular, and neurodegenerative diseases (1Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6205) Google Scholar). Recently, apoptosis of cardiomyocytes has been recognized as a cellular mechanism of ischemic injury in the heart. Furthermore, a large body of research has focused on identifying the signaling molecules that might protect the myocardium from ischemic damage (2Bishopric N.H. Andreka P. Slepak T. Webster K.A. Curr. Opin. Pharmacol. 2001; 1: 141-150Crossref PubMed Scopus (231) Google Scholar). For example, signaling molecules enhance apoptosis of cardiomyocytes, such as p38, c-Jun N-terminal kinase, tumor necrosis factor-α, p53, β-adrenergic receptors, and nitric oxide (2Bishopric N.H. Andreka P. Slepak T. Webster K.A. Curr. Opin. Pharmacol. 2001; 1: 141-150Crossref PubMed Scopus (231) Google Scholar, 3Dawn B. Bolli R. Ann. N. Y. Acad. Sci. 2002; 962: 18-41Crossref PubMed Scopus (166) Google Scholar, 4Aoki H. Kang P.M. Hampe J. Yoshimura K. Noma T. Matsuzaki M. Izumo S. J. Biol. Chem. 2002; 277: 10244-10250Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). In contrast, other signaling pathways have been demonstrated to protect the heart from apoptosis, such as cardiotrophin-1 through the gp130 receptor, p38β, insulin-like growth factor-1, Akt/protein kinase B, protein kinase C, and extracellular signal-regulated kinase 1/2 (2Bishopric N.H. Andreka P. Slepak T. Webster K.A. Curr. 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Thus, an increased understanding of the signaling pathways that are regulated during ischemia/reperfusion is important for the development of effective therapies (10Hamacher-Brady A. Brady N.R. Logue S.E. Sayen M.R. Jinno M. Kirshenbaum L.A. Gottlieb R.A. Gustafsson A.B. Cell Death Differ. 2007; 14: 146-157Crossref PubMed Scopus (517) Google Scholar). ARC 3The abbreviations used are: ARC, apoptosis repressor with a caspase recruitment domain; CARD, caspase recruitment domain;H&E, hematoxylin and eosin; LVDp, left ventricular development pressure; LVEDp, left ventricular end-diastolic pressure; MHC, myosin heavy chain; Tg, transgenic; shRNA, short hairpin RNA; WT, wild type; RT, reverse transcriptase; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling. (apoptosis repressor with CARD) is a caspase recruitment domain (CARD) protein that is expressed almost exclusively in long-lived tissues, such as heart, skeletal muscles, and brain. ARC selectively interacts with the initiator caspases-2 and -8, and significantly attenuates death receptor-induced apoptosis dependent on the activation of these caspases (11Koseki T. Inohara N. Chen S. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5156-5160Crossref PubMed Scopus (311) Google Scholar). In the H9c2 cell line, ectopic expression of ARC suppressed apoptosis, the protection being mediated, in total or in part, through the blockade of hypoxia-induced cytochrome c released from the mitochondria (12Ekhterae D. Lin Z. Lundberg M.S. Crow M.T. Brosius 3rd, F.C. Nunez G. Circ. Res. 1999; 85: e70-e77Crossref PubMed Google Scholar). Therefore, specific interference with both receptor and mitochondria death pathways, as well as its high cardiac expression, makes ARC a unique and central cardiac death repressor. This supposition is supported by the observation that viral gene transfer or TAT-mediated transduction of ARC reduces infarct size after the ischemia/reperfusion injury of isolated rat hearts and blocks the development of post-ischemic cardiomyopathy (13Chatterjee S. Bish L.T. Jayasankar V. Stewart A.S. Woo Y.J. Crow M.T. Gardner T.J. Sweeney H.L. J. Thorac. Cardiovasc. Surg. 2003; 125: 1461-1469Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 14Gustafsson A.B. Sayen M.R. Williams S.D. Crow M.T. Gottlieb R.A. Circulation. 2002; 106: 735-739Crossref PubMed Scopus (116) Google Scholar). Recently, ARC knock-out mice also exhibited enhanced sensitivity to hypoxic injury in the heart (15Donath S. Li P. Willenbockel C. Al-Saadi N. Gross V. Willnow T. Bader M. Martin U. Bauersachs J. Wollert K.C. Dietz R. von Harsdorf R. Circulation. 2006; 113: 1203-1212Crossref PubMed Scopus (101) Google Scholar). However, the detailed function of ARC as a suppressor of ischemic damage in the heart needs to be clarified. In this study, by generating cardio-specific ARC transgenic (Tg) mice and global gene expression analysis, we found that ARC protects cardiomyocytes from ischemic/hypoxic damages via its C-terminal Pro/Glu-rich region and expressional regulation of Drbp1 and pMe2GlyDH. Plasmid Construction and shRNA Analysis—The mouse ARC cDNA was subcloned into an expression vector under the control of a cardio-specific mouse α-myosin heavy chain (αMHC) promoter (16Gulick J. Subramaniam A. Neumann J. Robbins J. J. Biol. Chem. 1991; 266: 9180-9185Abstract Full Text PDF PubMed Google Scholar). ARC, NARC-(1–98), and CARC-(99–208) were subcloned into pcDNA3.1 (Invitrogen) and pEGFP (Clontech) (17Jo D.G. Lee J.Y. Hong Y.M. Song S. Mook-Jung I. Koh J.Y. Jung Y.K. J. Neurochem. 2004; 88: 604-611Crossref PubMed Scopus (51) Google Scholar). Drbp1 cDNA was kindly provided by Dr. H. Endo (Jichi Medical School, Japan) and Alas2 cDNA was subcloned into pcDNA3.1. For vector expressing shRNAs against pMe2GlyDH, oligonucleotides (number 1 forward, 5′-GATCC CCGGG ATAAA CTTGA AGAAG ATTCA AGAGA TCTTC TTCAA GTTTA TCCCT TTTTG GAAA-3′ and reverse, 5′-AGCTT TTCCA AAAAG GGATA AACTT GAAGA AGATC TCTTG AATCT TCTTC AAGTT TATCC CGGG-3′; number 2 forward, 5′-GATCC CCCTG AAAGG GTGGA CGAAT TTTCA AGAGA AATTC GTCCA CCCTT TCAGT TTTTG GAAA-3′ and reverse, 5′-AGCTT TTCCA AAAAC TGAAA GGGTG GACGA ATTTC TCTTG AAAAT TCGTC CACCC TTTCA GGGG-3′) containing the target sequence (Dharmacon) were synthesized, annealed, and cloned into BglII and HindIII sites of pSuper (OligoEngine). Generation of ARC Tg Mice—The mouse ARC expression construct was injected into embryos and positive F0 mice were identified by PCR analysis using a synthetic oligonucleotide (forward, 5′-CCACA TTCTT CAGGA TTCTC-3′) corresponding to the MHC promoter and a oligonucleotide (reverse, 5′-CTTCT GGCGT CCAGT GG-3′) of ARC cDNA (Macrogen Inc., Korea). Three independent founder lines were identified and mated to FVB wild type (WT) mice to generate a pure FVB genetic background WT for the F2 generation, and then F3 Tg offspring were then backcrossed to Balb/c 3T3 mice under a 12:12 h light:dark cycle with access to food and water ad libitum. The animal protocols were approved by the Gwangju Institute of Science and Technology and Seoul National University Standing Committees on Animals. Genomic DNA PCR and Reverse Transcription-PCR (RT-PCR)—Genomic DNA PCR from the tails of Tg mice and RT-PCR from the whole tissues of Tg mice were carried out as described previously (18Tian R. Miao W. Spindler M. Javadpour M.M. McKinney R. Bowman J.C. Buttrick P.M. Ingwall J.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 96: 13536-13541Crossref Scopus (25) Google Scholar, 19Zhang Z. He Y. Tuteja D. Xu D. Timofeyev V. Zhang Q. Glatter K.A. Xu Y. Shin H.S. Low R. Chiamvimonvat N. Circulation. 2005; 112: 1936-1944Crossref PubMed Scopus (111) Google Scholar). Gene-specific oligonucleotides were synthesized: Alas2 forward, 5′-GTATT GGACG CTGCC CCATC C-3′ and reverse, 5′-CTTCA GGGTC TCCTC TATGG C-3′; Drbp1 forward, 5′-GTGAT CAGCA AGCAC ACATC C-3′ and reverse 5′-CGCAC GTAGC CCAAG CCTTT A-3′; pMe2GlyDH forward, 5′-GACAG AGCAG AGACT GTGAT-3′ and reverse, 5′-CATCT CCTGG GTTAT ACAGT C-3′; ARC forward, 5′-ATGGG CAACG TGCAG GAG-3′ and reverse, 5′-CTTCT GGCGT CCAGT GG-3′; and β-actin forward, 5′-GAGCT GCCTG ACGGC CAGG-3′ and reverse, 5′-CATCT GCTGG AAGGT GGAC-3′; atrial natriuretic factor forward, 5′-CCATA TTGGA GCAAA TCCTG-3′ and reverse, 5′-CGGCA TCTTC TCCTC CAGG-3′; β-MHC forward, 5′-TGCAA AGGCT CCAGG TCTGA GGGC-3′ and reverse, 5′-GCCAA CACCA ACCTG TCCAA GTTC-3′; MMP2 forward, 5′-GATAC CCTCA AGAAG ATGCA GAAGT-3′ and reverse, 5′-ATCTT GGCTT CCGCA TGGT-3′ (20Abmayr S. Crawford R.W. Chamberlain J.S. Hum. Mol. Genet. 2004; 13: 213-221Crossref PubMed Scopus (39) Google Scholar). Western Blot Analysis—Samples were homogenized in lysis buffer (120 mm NaCl, 1 mm EGTA, 1 mm EDTA, 1 mm MgCl2, 1 mm Na3VO3, 10 mm Na4P2O7, 10 mm sodium fluoride, 1% Triton, 10% glycerol, and 50 mm Tris-HCl, pH 8.0) and the amounts of protein were determined using the method of Bradford (Bio-Rad). Total 30–50 μg of cell extracts in a buffer (60 mm Tris-HCl, pH 6.8, 1% SDS, 10% glycerol, and 0.5% β-mercaptoethanol) were subjected to SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes using the Semi-Dry Transfer system (Bio-Rad). The membranes were blocked with TBST buffer (20 mm Tris-Cl, pH 7.5, 150 mm NaCl, and 0.2% Tween 20) containing 3% bovine serum albumin. Proteins were then visualized using Enhanced Chemiluminescence. Anti-ARC antibody was purified by ARC affinity chromatography. Mouse anti-FADD and anti-poly(ADP-ribose)polymerase antibody were purchased from Pharmingen. Histological Analysis and Immunohistochemistry—After perfusion, the hearts were fixed with 4% formaldehyde, embedded, and thin-sectioned, followed by deparaffinization and rehydration. The paraffin sections were blocked in 5% rabbit serum and then incubated overnight at 4 °C with anti-ARC antibody. For the preparation of frozen sections, hearts were isolated after perfusion and immediately frozen in liquid nitrogen-cooled OCT embedding medium (Tissue-Tek). The frozen sections were cut to a 5-μm thickness and mounted on silane-coated slides. Langendoff Preparation of Isolated Mouse Hearts—The mice were anesthetized with pentobarbital (50 mg/kg, intraperitoneal injection) and their hearts were excised and perfused with oxygenated buffer (21Yet S.F. Tian R. Layne M.D. Wang Z.Y. Maemura K. Solovyeva M. Ith B. Melo L.G. Zhang L. Ingwall J.S. Dzau V.J. Lee M.E. Perrella M.A. Circ. Res. 2001; 89: 168-173Crossref PubMed Scopus (378) Google Scholar, 22Serviddio G. Di Venosa N. Federici A. D'Agostino D. Rollo T. Prigigallo F. Altomare E. Fiore T. Vendemiale G. FASEB J. 2005; 19: 354-361Crossref PubMed Scopus (100) Google Scholar). The hearts were retrogradely perfused on a Langendoff apparatus with Krebs-Henseleit solution (118 mm NaCl, 4.7 mm KCl, 1.2 mm MgSO4, 1.2 mm KH2PO4, 25 mm NaHCO3, 11 mm glucose, 1 mm CaCl2, and 10 mm HEPES) with a gas mixture of 95% O2 and 5% CO2 at 37 °C. Through a left atrial incision, a latex balloon connected to a pressure transducer was inserted into the left ventricular (LV) cavity for the measurement of the LV isovolumic pressure. The LVEDp was identified as the lowest value from the LV pressure curve. The LVDp and heart rate were continuously monitored using a polygraph and a computer analysis system (PolyView, GRASS Co.). Induction of Myocardial Infarction—After 1 h of ischemia and 24 h of reperfusion, the left anterior descending coronary artery was reoccluded, and 1 ml of 1.0% Evans blue was injected into the apex of each heart to stain nonischemic tissue. The hearts were then excised, washed with phosphate-buffered saline, embedded in agarose, and cut into five transverse slices for 15 min of incubation at room temperature with 1.5% 2,3,5-triphenyltetrazolium chloride to measure viable myocardium (red staining). Slices were photographed (each side) under a microscope and the left ventricular area, the area at risk, and the infarct area were determined by digital planimetry (14Gustafsson A.B. Sayen M.R. Williams S.D. Crow M.T. Gottlieb R.A. Circulation. 2002; 106: 735-739Crossref PubMed Scopus (116) Google Scholar). TUNEL Assay—Accumulated internucleosomal DNA fragments (apoptosis) were detected using an in situ apoptosis detection kit (Roche Applied Science). Percentages of positively stained cells were determined by counting the numbers of labeled cells and total cells in cross-sections (15Donath S. Li P. Willenbockel C. Al-Saadi N. Gross V. Willnow T. Bader M. Martin U. Bauersachs J. Wollert K.C. Dietz R. von Harsdorf R. Circulation. 2006; 113: 1203-1212Crossref PubMed Scopus (101) Google Scholar). Isolation of Neonatal Cardiomyocytes and DNA Transfection—ARC homozygous Tg mice for this study were produced by mating heterozygous ARC Tg mice in the Balb/c 3T3 background to heterozygous ARC Tg mice and used for primary culture. A procedure for culturing ventricular cardiomyocytes from neonatal mice was modified (23Nakamura T.Y. Goda K. Okamoto T. Nakamura T. Goshima K. Circ. Res. 1993; 73: 758-770Crossref PubMed Scopus (48) Google Scholar). For transfection, cardiomyocytes isolated from neonatal ARC Tg mice were allowed to stabilize for 3 days and then transfected with plasmids using PolyFectamine reagent according to the manufacturer's instruction (Qiagen). In Vitro Hypoxia of Cultured Cardiomyocytes and Cell Death Assays—Cardiomyocytes were cultured in fetal bovine serum/minimal essential medium. Hypoxia was simulated by incubating cells in a hypoxic buffer (125 mm NaCl, 8 mm KCl, 1.2 mm KH2PO4, 1.25 mm MgSO4, 1.2 mm CaCl2, 6.25 mm NaHCO3, 20 mm 2-deoxyglucose, 5 mm sodium lactate, and 20 mm HEPES, pH 6.6) and by placing the cells under hypoxic pouches (GasPak™ EZ, BD Biosciences) at 37 °C (10Hamacher-Brady A. Brady N.R. Logue S.E. Sayen M.R. Jinno M. Kirshenbaum L.A. Gottlieb R.A. Gustafsson A.B. Cell Death Differ. 2007; 14: 146-157Crossref PubMed Scopus (517) Google Scholar). Cell death assays were performed using the Live/Dead cell viability kit (Molecular Probes). Microarray Using GenePix 4000B Gene Expression Profiling— Total RNA samples were prepared from the hearts of 3- and 30-week-old mice by using an RNeasy Mini Kit (Qiagen). RNA samples (30 μg) were labeled with cyanine (Cy3) or cyanine (Cy5)-conjugated dCTP (Amersham) by a reverse transcription reaction using SuperScript II (Invitrogen). The labeled cDNA mixture was resuspended and mixed in 10 μl of hybridization solution (GenoCheck, Korea) and placed on an OpArray mouse genome 35K (OPMMV4, Operon Biotechnologies, GmbH). The hybridized slides were washed in a buffer (2× SSC, 0.1% SDS for 2 min, 1× SSC for 3 min, and then 0.2× SSC for 2 min) at room temperature. Microarray Data Analysis—Microarray data analysis was carried out as described previously (24Irizarry R.A. Warren D. Spencer F. Kim I.F. Biswal S. Frank B.C. Gabrielson E. Garcia J.G. Geoghegan J. Germino G. Griffin C. Hilmer S.C. Hoffman E. Jedlicka A.E. Kawasaki E. Martinez-Murillo F. Morsberger L. Lee H. Petersen D. Quackenbush J. Scott A. Wilson M. Yang Y. Ye S.Q. Yu W. Nat. Meth. 2005; 2: 329-330Crossref PubMed Scopus (37) Google Scholar). Statistical Analyses—All statistical analyses were performed using a two-tailed Student's t test or one-way analysis of variance followed by SigmaStat software. Generation of Tg Mice Overexpressing ARC in the Heart—To examine the molecular function of ARC in the hypoxic injury of heart, we generated cardio-specific Tg mice overexpressing mouse ARC using the promoter of mouse MHC gene (Fig. 1). ARC Tg mice were born normal and grew to adulthood without any abnormalities in their health and appearance. Compared with WT, an analysis of tissue extracts using Western blotting (Fig. 1A) and polymerase chain reaction (Fig. 1B) demonstrated an increased level of ARC protein and RNA with about 3-fold in the whole heart tissues of ARC Tg mice, but not in the kidney as well as in liver and brain. 4J.-O. Pyo, J. Nah, H.-J. Kim, J.-W. Chang, Y.-W. Song, D.-K. Yang, D.-G. Jo, H.-R. Kim, H.-J. Chae, S.-W. Chae, S.-Y. Hwang, S.-J. Kim, H.-J. Kim, C. Cho, C.-G. Oh, W. J. Park, and Y.-K. Jung, unpublished observations. Similar expression levels of ARC were observed in the other two lines of ARC Tg mice we generated. 4J.-O. Pyo, J. Nah, H.-J. Kim, J.-W. Chang, Y.-W. Song, D.-K. Yang, D.-G. Jo, H.-R. Kim, H.-J. Chae, S.-W. Chae, S.-Y. Hwang, S.-J. Kim, H.-J. Kim, C. Cho, C.-G. Oh, W. J. Park, and Y.-K. Jung, unpublished observations. Immunostaining analysis also revealed the increased expression of ARC in the left ventricle of the heart of ARC Tg mice (Fig. 1C). In ARC Tg mice, strong immunoreactivity against ARC was restricted to ∼40% of individual cardiomyocytes, which is consistent with the previous report demonstrating the expression pattern of heme oxygenase-1 under the control of MHC promoter (21Yet S.F. Tian R. Layne M.D. Wang Z.Y. Maemura K. Solovyeva M. Ith B. Melo L.G. Zhang L. Ingwall J.S. Dzau V.J. Lee M.E. Perrella M.A. Circ. Res. 2001; 89: 168-173Crossref PubMed Scopus (378) Google Scholar). Although histological examination with hematoxylin and eosin (H & E) staining revealed no severe cardiac morphological defects, cardiac remodeling was evident in the hearts of ARC Tg mice with the relative increase in cell numbers (Fig. 1D). Comparison of heart sizes revealed that under resting conditions, the hearts of ARC Tg mice were apparently larger than those of age-matched WT mice (Fig. 1F). There was a 20% difference in the ratios of heart/body weights between 8-week-old ARC Tg and WT mice, whereas no detectible difference was observed with respect to kidney weight (Fig. 1E). In addition, examination of the expression of hypertrophic markers, such as atrial natriuretic factor and β-MHC, and MMP2, the sign of fibrosis, with RT-PCR analysis revealed no obvious increase in the hearts of ARC Tg mice (Fig. 1G). These findings suggest that heart enlargement observed in ARC Tg mice may result from the principal cause of the increased cell numbers. Hemodynamic Function of the Isolated Hearts of ARC Tg Mice—Under normal living conditions, ARC Tg mice displayed no apparent failure of the heart after a 12-month follow-up. To investigate the cardiac function of ARC, the hemodynamic parameters were recorded for 50 min of reperfusion after global ischemia. The baseline heart rates were similar between two groups, WT and ARC Tg mice.4 Compared with WT mice, however, the left ventricular development pressure (LVDp) and the left ventricular end-diastolic pressure (LVEDp) were significantly improved in the hearts of ARC Tg mice (Fig. 2, A and B). These findings indicate that ARC functions to suppress ischemia/reperfusion-induced deterioration of the Langendoff preparation exhibiting cardiac dysfunction and overt heart failure. We then assessed the myocardial infarct after occluding left anterior descending coronary artery followed by reperfusion for 24 h. Total left ventricular area, the area at risk, and the infarct area were measured. Large infarcts were observed in the hearts of WT mice (Fig. 2C). Infarct size (infarct/risk area) was significantly reduced in Tg mice compared with WT mice (about 40%).4 This was accompanied by a significant decrease in apoptosis as measured with proteolytic cleavage of poly-(ADP-ribose)polymerase, a hallmark of apoptosis (Fig. 2D). Similarly, an evaluation of death rates using the TUNEL assay revealed reduced cell death (5.2-fold decrease) in the hearts of ARC Tg mice compared with WT mice during ischemic damages (Fig. 2E). Suppression of Hypoxia-induced Cell Death by ARC in Cardiomyocytes—We then addressed sensitivity of the primary cardiomyocytes isolated from ARC Tg mice to hypoxia-induced cell death. Although the incubation of control cardiomyocytes in the hypoxic conditions induced 42% of cell death at 36 h, the hypoxic death rate in the same condition decreased to 20% in the cardiomyocytes prepared from ARC Tg mice (Fig. 3, A and B). These results show that the primary cardiomyocytes expressing ARC derived from ARC Tg mice are resistant to hypoxic cell death in vitro. Previously, we found that ARC may be a calcium-binding protein (25Jo D.G. Jun J.I. Chang J.W. Hong Y.M. Song S. Cho D.H. Shim S.M. Lee H.J. Cho C. Kim D.H. Jung Y.K. Mol. Cell. Biol. 2004; 22: 9763-9770Crossref Scopus (45) Google Scholar). Thus, we addressed whether ARC affects calcium overload-induced cell death of the primary cardiomyocytes. Incubation with thapsigargin, an inhibitor of Ca2+-ATPase in the endoplasmic reticulum, induced 67% of cell death in WT cardiomyocytes but 30% in the cardiomyocytes cultured from the neonatal ARC Tg mice, indicating that ARC suppresses calcium overload-induced death of cardiomyocytes (Fig. 3, C and D). We then analyzed the domain(s) of ARC responsible for the anti-hypoxic cell death activity using its deletion mutants (Fig. 4). The primary cardiomyocytes transiently transfected with ARC or ARC C terminus rich in proline and glutamate (Pro/Glu-rich domain) (CARC, amino acid residues, 99–208) were resistant to hypoxic cell death to a degree comparable with that of Bcl-2, an anti-apoptotic protein, whereas the ARC N terminus (CARD domain) (NARC, amino acid residues, 1–98) failed to show such inhibitory effects on hypoxic cell death (Fig. 4, B and C). These results indicate that ARC exhibits anti-hypoxic cell death activity in cardiomyocytes through its C-terminal Pro/Glu-rich region, a calcium-binding region. Altered Profiles of Gene Expression in the Heart of ARC Tg Mice—Given the vast number of effectors that might potentially influence the survival versus the apoptotic decision of myocardial cells after ischemia-reperfusion injury, we performed a large-scale genomic screening for altered gene expression. Specifically, hearts harvested from 3- and 30-week-old control and ARC Tg mice were analyzed for gene expression profiling using the GenePix 4000B array. This array contains all of the genes in the murine Unigene data base that have been functionally characterized (∼40,000). Heart samples were cross-compared between WT and ARC Tg mice, resulting in 170 genes being significantly detected in one or more groups after internal normalization. Of these genes, 39 genes exhibited significantly altered expression between the hearts of ARC Tg and WT mice (Table 1).TABLE 1Relative expression ratios of the selected groups of genes assessed by RNA microarray analysis Cardiac RNA was collected from two wild type and two ARC Tg mice (Tg 1, 3-week-old; Tg 2, 30-week-old) and subjected to expression profiling using the GenePix 4000B array. –Fold change filters include the requirement that the genes be present in at least 200% of the controls for up-regulated genes and in less than 50% of the controls for down-regulated genes. Absolute normalized expression data are shown for each sample in the right-hand columns along with the GenBank accession number.Relative gene expression (ratio)Tg 1Tg 2GenBank™Enzyme activityN-Acylsphingosine amidohydrolase (acid ceramidase)-like (Asahl)0.2121074210.178740707NM_025972Carbonic anhydrase 32.8052915792.709155955NM_007606Methionine aminopeptidase-like 12.217968691.83970689NM_025633Dimethylglycine dehydrogenase precursor (pMe2GlyDH)0.0488536250.052353731NM_028772Adipsin2.9540597361.977203728NM_013459Aminolevulinic acid synthase 2, erythroid (Alas2)2.1608855482.036045855NM_0096536-Phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (Pfkfb3), transcript variant 10.4122293470.450426963NM_133232UnknownMus musculus RIKEN cDNA 4930550L24 gene0.2521369450.466819994XM_621012Bromodomain and WD repeat domain containing 30.04851310.056798788XM_356350Bardet-Biedl syndrome 4 homolog (human)0.3319058260.359647179NM_175325Protein bindingNeurofibromatosis 22.4333806221.551598644NM_010898PREDICTED: DMRT-like family B with proline-rich C-terminal0.3658157770.433494475XM_205469WW domain binding protein 10.3024903450.373305013NM_016757Fras1-related extracellular matrix 22.0645051251.519267093NM_172862Protocadherin β4 (Pcdhb4)2.0049287951.566438906NM_053129Vitamin D receptor interacting protein (Vdrip)0.2309638550.145157177NM_026119FYVE, RhoGEF, and PH domain containing 10.1803714620.239611358NM_008001Ligand of numb-protein X 1 (Lnx1)0.3896376990.367762517NM_010727Synuclein, α (Snca)2.019657981.90034372NM_009221Signal transducerPeriod homolog 3 (Drosophila) (Per3)2.5865910921.821002595NM_011067Regulator of G protein signaling 7 (Rgs7)2.19001241.738080612NM_011880OtherRIKEN cDNA 1600012F09 gene2.6171330212.601587207NM_025904Ribosomal protein S6 kinase polypeptide 32.3888338731.727797544NM_148945UDP-GlcNAc:βGal β-1,3-N-acetylglucosaminyltransferase 12.1974760711.886600148NM_175383Signal recognition particle 542.0702499641.75692098NM_028527PREDICTED: M. musculus similar to surface sperm protein P26h0.4143724940.475688281CK137444RIKEN cDNA 1810008A14 gene0.4127437420.499033244NM_025457Sad1 and UNC84 domain containing 10.3955202990.300412406NM_177576Hypothetical protein 4931417A200.3564622990.458295539NM_145380Par-3 (partitioning defective 3) homolog (Caenorhabditis elegans)0.3271919140.354896792NM_031235Similar to transmembrane protein induced by tumor necrosis factor α0.2375746870.283793964AK_132283RIKEN cDNA 2310056P07 gene0.2347900790.175357973NM_027342RIKEN cDNA 4930451I11 gene0.2336468470.2583342NM_183131RIKEN cDNA 4930563C04 gene, mRNA0.1069732510.160551176NM_029231Transcription activityHairy and enhancer of split 7 (Drosophila)0.3310727360.428150795NM_033041Ankyrin repeat and SOCS box-containing protein 120.3277400740.367856146NM_080858Nucleic acid bindingDevelopmentally regulated RNA binding protein 1 (Drbp1)4.8432070454.596722765NM_153405G patch domain containing 3, mRNA0.2307905270.329046637NM_172876Receptor activityLeucine-rich repeat-containing G protein-coupled receptor 7 (Lgr7)2.4056362052.106995516NM_212452 Open table in a new tab The raw data shown in Table 1 represent the 2n relationship of expression between 3- and 30-week-old ARC Tg mice hearts compared with those of WT mice. The expressions of 23 genes, including the dimethylglycine dehydrogenase precursor (pMe2GlyDH), bromodomain, WD repeat domain containing 3, RIKEN cDNA 4930563C04, and FYVE RhoGEF were significantly down-regulated in the hearts of ARC Tg mice. On the contrary, 16 genes, including the developmentally regulated RNA-binding protein 1 (Drbp1), Alas2, and carbonic anhydrase 3, exhibite

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