Introduction 2001 2003 2003 2007 ATP  2003 2005 2004 Gillichthys mirabilis 2001 2005 2003 In this study, we have identified CCH-induced gene expression changes in the zebrafish heart by looking at over half of the zebrafish genome. We have compared several of these novel changes described in other species and tissues. We have here identified the heart-specific molecular adaptations to CCH. Future functional experiments are warranted to determine whether some of the findings can be used to better adapt mammalian hearts to CCH. Material and methods Animal handling Danio rerio Haplochromis piceatus Hypoxia treatment . 2  2  2 2  Perfusion of cichlid hearts In order to minimize blood clotting, a perfusion protocol was developed in which the whole blood volume of clinically dead animals was initially replaced with isotonic buffer and with a fixative solution secondarily. Heart dissection The fish were killed with an overdose of anesthetic (MS-222; Tricaine Methanesulfonate from Argent Chemical Laboratories, USA). Hearts were dissected from the fish immediately after the anesthetic worked. For RNA preparation the hearts were immediately shock-freezed in liquid nitrogen. For histology and microscopy, the hearts were left intact and fixed immediately in Karnovsky fixative (4% paraformaldehyde (PFA) and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2) for 4 h at 4°C. After three washes in 0.1 M phosphate buffer, pH 7.2, they were transferred to 70% ethanol. Histology of adult fish hearts, statistical analysis and scanning electron microscopy 2006 2005 t P Immunohistochemistry and statistical analysis 2  http://rsb.info.nih.gov/nih-image/ t P RNA preparation and biological sampling After dissection hearts were homogenized in a Dounce homogenizer using 1 ml Trizol solution (GibcoBrl, Life technologies). The whole heart was used and for each biological sample hearts were pooled from five different animals. After Trizol extraction, total RNA was further purified using RNAeasy columns (Qiagen). RNA samples were analyzed for quality control by Lab-on-a-chip analysis (Agilent) and on agarose gels. For the array experiment five arrays were done for normoxic and 5 arrays for the hypoxic condition. Biological samples (BS) came from two independent experiments and one technical replicate (TR) was included (2BS + 2BS + TR for normoxia and 2BS + 2BS + TR for hypoxia). As mentioned above for each BS, hearts from five different animals were pooled. Microarray analysis ® P ≤  http://www.ncbi.nlm.nih.gov/geo 1 Table 1 Danio rerio Haplochromis piceatus Number of cardiomyocyte nuclei per section  2 2 Normoxia Hypoxia Normoxia Hypoxia Mean 9.81 13.67 14.7 24 SD 0.30 0.39 0.50 0.68 P −17 −12   D. rerio 2 H. piceatus 2 t P Gene ontology analysis http://www.genetools.microarray.ntnu.no/egon/ P < Real-time quantitative RT-PCR http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi Results 2 2 2  1 Hypoxia treatment 2  2 Material and methods Fig. 1 n n 2  Phenotypic changes in the heart of adult teleosts under chronic constant hypoxia (CCH) Danio rerio 2 Haplochromis piceatus 2 2 1 3 Fig. 2 a A B E C D F dark  (dark blue light  (pink A C E F B D D a v vo  ca b A–F G H A–F Azan blue G H A B C G D E F H  Fig. 3 a c d b e f bigger images smaller images scale v vo  Gene expression changes in the heart of adult zebrafish under chronic constant hypoxia (CCH) In this study, we used microarrays for the transcriptional profiling of up and downregulated genes in response to hypoxia in the zebrafish heart. We identified 376 genes that were differentially expressed under hypoxic conditions, out of which 116 genes showed a decrease in gene expression (30.9%) in comparison to 260 genes which showed increased expression levels (69.1%). All 376 differentially expressed genes, including the ones with oligo sequences which could not be annotated so far (referred therein either as transcribed locus or zebrafish clone) are shown in the complete file (Supplemental Table 1). Functional groups are color coded and if possible, gene functions are briefly summarized and OMIM links given. Functional groups of differentially expressed genes in the heart 2 Table 2 Functional groups of differentially expressed genes UniGene GeneBank Fold Gene name Upregulated genes Angiogenesis Dr.11575 NM_173244 2.3 T-cell acute lymphocytic leukemia 1; TAL1 Dr.845 BG729013 2.8 Fibrinogen alpha/alpha-E chain  Dr.4907 BC045868 4.2 Fibrinogen, gamma polypeptide Apoptosis Dr.15862 AF493987 2.1 BCL2adenovirus E1b 19 interacting protein3 DrAffx.1.39 AF302789 2.3 Death receptor Dr.20106 AI722277 2.8 Apoptosis inhibitor 5 Dr.4039 BQ480688 21.8 BAX inhibitor 1 Cell adhesion Dr.6007 NM_131820 2.9 Cadherin 1 Dr.25140 BQ262802 3.3 Tumor-associated calcium signal transducer  Dr.4409 BC049036 4.4 CD9 antigen Dr.25140 BQ262802 7.7 Tumor-associated calcium signal transducer glycoprotein Development Dr.11575 NM_1732 2.3 T-cell acute lymphocyte leukemia 1 (tal 1) Dr.23348 BE201653 2.6 Bone morphogenetic protein 3b; (bmp 3) Dr25405 BC013923 2.8 SOX2 SRY-box 2 Dr.6382 AW165053 2.9 Hedgehog-interacting protein Dr.10879 U97669 3.0 Drosophila Dr.15055 BC050172 3.6 Chemokine receptor 4a Dr.6787 BI533426 5.5 Noelin Dr.16720 BI980847 6.3 notch 2 Disease related Dr.6349 AW116668 2.4 Eparin cofactor II Dr.21064 BC046075 4.5 4hydroxyphenylpyruvate dioxygenase HPD Dr.12584 NM_131211 5.4 Gata binding protein 3 (GATA3) Dr.3530 AI497545 79.3 Prion protein (prp) gene Growth regulation Dr.8145 NM_13143 2.2 Insulin like growth factor 2 (IGF-2) Dr.7609 BI475857 2.4 Prolactin receptor Dr.8285 NM_13136 2.4 Mad homolog 2  Dr.8947 CD594735 2.5 Spint 2 Dr.822 BM184127 2.5 Spint 2 Dr.3563 CD014488 2.8 Tetraspan membrane protein IL-TMP Dr.8587 NM_17328 2.9 Insulin-like growth factor binding protein 1  Dr.2596 BM342901 3.2 Cyclin I Dr.8587 AL910822 3.4 Insulin-like growth factor binding protein 1 Dr.26458 BC053206 5.6 m-ras Heart related Dr.15088 BM181749 4.3 Lectin galactoside-binding soluble 1; (galectin10-like 3) Dr.4867 AI496840 5.5 Haptoglobin Dr.3585 AY049731 6.6 Angiotensinogen  Dr.2452 BQ284848 4.3 Complement component C9  Dr.18453 BC044525 4.8 Uridine phosphorylase Dr.3025 BG738204 2.7 Alpha-2 macro-globulin; A2MG Inflammation Dr.12491 BI672168 2.1 Complement C4–2 Dr.4047 NM_131627 2.3 Small inducible cytokine A (scyba) Dr.5053 NM_131723 2.3 Kruppel-like factor 4  Dr25207 X06465 2.5 Complement component 8, gamma polypeptide Dr.6845 K02765 2.9 C3 complement component 3 Dr.5741 BU710482 3.2 Complement component b fb Dr.7722 BI878414 3.5 Complement C3-H1 Dr.22244 AW019781 3.6 Complement C1s Dr.22133 AW076768 3.7 c1rs-A and clrs-B Dr.5528 AI497212 4.2 Complement component C9 Dr.2452 BQ284848 4.3 Complement component C9  Dr.1730 AI721528 4.8 cfI-B complement control protein factor I-B  Dr.2452 BM778002 5.8 Complement component C9 Dr.20291 BM036389 6.5 Complement C3-S Dr.190 NM_131338 7.9 Complement component factor B  Dr.1192 AB071601 2. Lipocalin-type prostaglandin D synthase-like protein Metabolism Dr.9492 BI882244 2.0 Sulfide dehydrogenase like Dr.15574 BM571467 2.1 Hypoxanthine _hosphor-ribosyltransferase 1 Dr.3332 AI943053 2.2 Angiopoietin 5  Dr.16130 CD014898 2.3 Alcohol dehydrogenase 8 b Dr.3959 BI43001 2.5 5′-nucleotidase Dr.22205 AW019477 2.6 Oxidoreductase Dr.1699 AI667249 2.7 Pyruvate kinase Dr.5504 BI879550 3.2 Cystathionine-beta-synthase Dr.1202 AJ245491 3.9 Apolipoprotein A-I Dr.4111 BC053267 4.2 Fructose-1,6-bisphosphatase 1 Dr.18834 AW019321 4.2 Urate oxidase Dr.19224 BC050167 4.3 Aldolase b Dr.4938 NM_131645 4.4 Fatty acid desaturase 2 Dr.12654 BC046901 14.8 ELOVL family member 6, Dr.5488 AI545593 17.3 Apolipoprotein A-IV  Muscle related Dr.3585 AY049731 6.6 Angiotensinogen Dr.2452 BQ284848 4.3 Complement component C9 Proteolysis Dr.20934 AF541952 2.6 Trypsin precursor Dr.3025 BG738204 2.7 Alpha-2-macroglobulin  Dr.22139 AW018965 3.0 Alpha-1-antitrypsin Dr.25331 AI658072 4.1 Alpha-2-macroglobulin-2 Dr.12602 NM_139180 4.3 Lysozyme Dr.1605 BM185388 4.4 Protease inhibitor 1 Dr.17459 CD586837 4.8 Inter-alpha-trypsin inhibitor heavy chain H3  Dr.3073 AI585030 5.0 Serine protease inhibitor alpha 1 Dr.26371 AI667676 5.4 Prostasin Dr.3025 BM530427 5.6 Alpha-2-macroglobulin-1 Dr.3025 BM316867 6.5 Alpha-2-macroglobulin-2 Dr.2960 X67055 3.5 ITIH3 pre-alpha (globulin) inhibitor, H3 polypeptide Dr.25379 BI326783 6.7 Alpha-2-macroglobulin Dr.4797 AI959534 7.8 26–29 kD-Proteinase protein ROS protection Dr.20068 NM_131075 2.1 Metallothionein (mt) Dr.5399 AI957765 2.3 Biliverdin I Beta Reductase Dr.14058 CD015351 3.5 S Dr.25160 BC049475 5.9 Metallothionein 2 Dr.3613 BC048037 6.0 Cerulopasmin Dr.4905.1 BC045464 6.5 Uncoupling protein 4 Signal transduction Dr.9852 AW826425 2.1 CAM kinase 1 Dr.8591 BM186508 2.9 Rho guanine nucleotide exchange factor 10 Dr.6236 AW115973 3.1 Rho guanine nucleotide exchange factor 5 Dr.1267 BC051157 3.4 Phospholipase C delta Dr.22129 BC016668 3.9 RRAGC Rag C (Ras-related GTP binding C) Dr.7255 AW116479 4.4 Protein phosphatase 1, Dr.4453 BC044421 5.8 Phosphoprotein phosphatase Translation Dr.13234 BM036471 2.0 Ribonuclease P  Dr.382 CB363830 2.1 Nucleolin Dr.6949 AW078116 2.1 RNA 3′-terminal phosphate cyclase-like protein (HSPC338) Dr.13563 BI890729 2.3 Methionyl aminopeptidase 2 Dr.26328 AL723696 2.3 Eukaryotic translation initiation factor 4A, Dr.17693 BQ078285 3.7 40 S ribosomal protein S6 Dr.20270 BI674050 5.9 Ribosomal protein L12  Dr.25224 CD015330 20.4 Ribosomal protein L12  Dr.12439 BM533848 17.5 Heterogeneous nuclear ribonucleoprotein K  Dr.12439 BM533848 24.2 Heterogeneous nuclear ribonucleoprotein K Dr.14821 BM071714 33.8 Heterogeneous nuclear ribonucleoprotein K  Dr.12502 BQ284686 40.7 Heterogeneous nuclear ribonucleoprotein K  Dr.12439.  BM534432 40.9 Heterogeneous nuclear ribonucleoprotein K Dr.12439 BQ616930 45.4 Heterogeneous nuclear ribonucleoprotein K Transport Dr.1084 BQ109772 3.0 Clathrin coat assembly protein AP19 Dr.5562 X04506 3.0 APOB apolipoprotein B (including Ag(x) antigen) Dr.13231 BM778646 4.2 Solute carrier family 22 Dr.30444 AY329629 4.3 Embryonic globin beta e2 Dr.24250 AF489105 2.0 Uroporphyrinogen III synthase Dr.10343 NM_131687 4.7 Na+K+ transporting, alpha 1a.2 polypeptide Dr.7634 AW115757 11.3 Hemopexin Downregulated genes Angiogenesis Dr.26411 BQ783571 −8.9 Fast muscle troponin I Dr.15501 BM316040 −2.1 Similar to CYR6 HUMAN CYR61 protein precursor, Insulin-like growth factor-binding protein 10 Cell adhesion Dr.251 BQ285646 −2.3 Cadherin 11 Disease related Dr.22774 AW280206 −5.7 ras-like GTP-binding protein RAB27A Dr.1816 AL720262 −4.4 Ataxin 2-binding protein Dr.9893 BM036473 −2.3 Fibrillarin Dr.16726 BI429372 −2.0 netrin G1 Growth regulation Dr.12986 CA787334 −5.3 v-fos Dr.12986 BI881979 −5.0 v-fos Dr.12986 BM957279 −4.5 v-fos Dr.1221 AW510198 −4.3 Pmx-1b (PHOX-1) Dr.12986 BI881979 −4.2 v-fos Dr.12410 NM_131826 −2.4 Sprouty homolog 4  Dr.6431 BC049326 −2.3 Suppressors of cytokine signaling 3 Dr.6511 NM_130922 −2.2 B-cell translocation gene 2 Dr.5365 AI601685 −2.2 Dual specificity phosphatase 5 Dr.12062 BC047814 −2.1 Epidermal growth factor receptor kinase substrate EPS8 Dr.17286 BM777144 −2.0 Hormone-regulated proliferation-associated 20 kDa protein Dr.9448 BM156058 −2.0 TGF-beta-inducible early growth response protein 2 Heart related Dr.20010 BQ826502 −7.0 ATPase, Ca++ transporting, cardiac muscle (ATP2A1) Dr.1448 AL717344 −3.5 Fast skeletal myosin light chain 1a Dr.20990 AY033829 AY081167 −2.4–2.1 Titin Metabolism Dr.24950 BC053305 −4.1 Creatine kinase CKM3 Dr.9528 BC045993 −3.5 Pyruvate dehydrogenase kinase Dr.146 AI477401 −2.9 O Dr.21501 AI667180 −2.4 Short-chain acyl-CoA dehydrogenase  Dr.19643 AL918850 −2.4 FabG beta-ketoacyl -reductase Dr.15059 BM530407 −2.2 Elongation of very long chain fatty acids (Cig30) Dr.21040 BC045479 −2.1 Glucose-6-phosphatase, transport protein 1 Dr.988 AW154697 −2.1 Dodecenoyl-coenzyme A delta isomerase  Dr.11971 BG727588 −2.0 O Dr.4777 AW420997 −2.0 Succinate-CoA ligase Dr.11252 BC047826 −2.0 Creatine kinase, mitochondrial 1  Muscle related Dr.21800 AI883923 −5.0 Myosin binding protein C Dr.5066 AF524840 −3.4 Alpha-actinin 3 Dr.24260 NM_131619 −3.0 Myosin, light polypeptide 3 Dr.2914 BC045520 −2.5 Myosin light polypeptide 2; mylz2 Dr.20990 AY033829 AY081167 −2.4–2.1 Titin Dr.1435 AI353817 −2.0 Caveolin 3 Dr.18657 BQ479700 −2.1 Carbonic anhydrase II Dr.26411 BQ783571 −8.9 Troponin I Proteolysis Dr.3581 BM101561 −8.3 Chymotrypsinogen B1  Dr.3581 BM101561 −7.5 Chymotrypsinogen B1 Signal transduction Dr.22841 AI641080 −2.4 Serum deprivation response protein (SDPR) Translation Dr.7939 AW281840 −2.7 Mitochondrial elongation factor G1 Dr.1286 BM036808 −2.2 Mitochondrial ribosomal protein L48 Dr.18218 AL909921 −2.1 Mitochondrial 28 S ribosomal protein S12  Transport Dr.676 BC050956 −4.4 ADT2, ADP,ATP carrier protein Dr.25199 CD014403 −2.1 Calcium-binding mitochondrial carrier protein Aralar2 (Citrin)  Dr.2784 AI942949 −2.0 Solute carrier family 25  Dr.11127 BG306498 −3.7 Synaptotagmin I Dr.11127 AW826278 −3.2 Synaptotagmin I  Dr.13273 BI885460 −2.3 GTP-binding protein rab15  Dr.22748 AW280026 −2.7 trpn1 Dr.11302 BG306530 −2.1 ATPase (Ca++ transporting plasma membrane 2) Proteinbiosynthesis (Translation) 2 Metabolism 2 Protection against reactive oxygen species (ROS) Apoptosis 1997 1998 Growth regulation Discussion 2 Inflammation Heart-related function 2 Discussion Muscle-related function 2 Discussion Development Transport (cellular and vascular) Angiogenesis 2004 Expression changes of known hypoxia responsive genes 1999 2005 1995 2005a 2000 Evaluation of microarray results by quantitative real-time RT-PCR 4 2005 2003 2005 Fig. 4 Verification of gene expression changes by quantitative real-time PCR. 10. selected genes, which were found to be differentially expressed on the microarrays, were further analyzed by quantitative real-time RT-PCR. Relative expression is given based on normalization to β-actin. A standard curve for β-actin was included in each experiment and data represents three independent experiments each done in triplicates. The primers used are given in Supplemental Table 2  Assessment of microarray results for the IGF/PI3K/Akt pathway by comparing phospho-Akt levels in cardiac myocytes of normoxic versus hypoxic zebrafish hearts 5 5 5 P Fig. 5 a b c d e  Discussion In the aquatic environment, oxygen concentrations can often vary, and being able to adapt to changes in oxygen levels can be advantageous for the survival of aquatic animals. This might be in part the reason why some teleosts have developed the ability to withstand extreme hypoxic conditions. In this study, we have focused on the long-term response to hypoxia in the fish heart. The hypothesis is that the zebrafish heart, in contrast to most mammalian hearts, which are characterized by relative intolerance to injury or the lack of oxygen, are able to adapt to extreme hypoxic conditions. 2000a 2007 2001 2002 2002 2005 1997 2003 1993 2000 2 2004 2006 2006 1996 2002 2005 2002 2 S 2001 2006 2 2005b 1996 1996 2006 2002 c 2005 2006 2007 2005 2005 2 2003 Electronic supplementary material Below is the link to the electronic supplementary material.
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