On-line supplement
Sex-Specific Pathways in Early Cardiac
Response to Pressure Overload in Mice
Henning Witt, PhD 1,2,4-*; Carola Schubert, PhD1,2-*; Juliane Jaekel, VetD1,2; Daniela Fliegner, PhD1,2; Adam Penkalla, MD2; Klaus Tiemann, MD3; Jens Stypmann, MD3; Stefan Roepcke, PhD4; Sebastian Brokat, MD1,2; Shokoufeh Mahmoodzadeh, PhD1,2; Eva Brozova, PhD1,2; Mercy M. Davidson, PhD6, Patricia Ruiz Noppinger, PhD1,2,4; Christian Grohé, MD3 and Vera Regitz-Zagrosek, MD1,2,5
1 Berlin Institute of Gender in Medicine (GiM), Charité – Universitaetsmedizin Berlin, Luisenstraße 65, 10117 Berlin; 2Center for Cardiovascular Research (CCR), Charité – Universitaetsmedizin Berlin, Hessische Str. 3-4, 10115 Berlin; 3Medizinische Klinik und Polyklinik II, Universitaetsklinikum Bonn, Sigmund-Freud-Str. 25, 53105 Bonn; 4Max-Planck-Institute for Molecular Genetics Berlin, Ihnestr. 63-73, 14195 Berlin; 5German Heart Institute Berlin (DHZB), Augustenburger Platz 1, 13353 Berlin, 6 College of Physicians and Surgeons, Columbia University, New York
* Authors contributed equally
Prof. Dr. med Vera Regitz-Zagrosek,
Center for Gender in Medicine, Center for Cardiovascular Research
Hessische Str. 3-4, 10115 Berlin, Germany
Tel.: +49 (0)30 450 525 172, Fax: +49 (0)30 450 525 972
Email:
Figure e1 (electronic supplement): 2nd best male network. Ingenuity Pathways Analysis was used to create networks. For the gene set with a relative upregulation in males the network with the 2nd highest score (17) is shown. Different combinations of the directions of deregulation in the comparison of TAC and sham for males and females could lead to a sex-specific expression pattern as indicated by the color code. Linking nodes not contained in the two sets of genes are colored grey. In our experiment the central node of this network TGFß1 was not detected with microarray technology (absent calls for all probes). As reported by Briest et al TGFß2 and TGFß3 expression resemble TGFß1 in function 42, TGFß2 displayed stronger induction in male mice whereas as similar degree of induction in the TAC animals was detected for TGFß3.
Table e1
Primer sequences of real-time PCR
Table e2
Exact designation and chromosomal localization of genes in Figure 3A
Table e3
All genes regulated differently by TAC
Table e4
Confirmation of selected genes. Relative up- and downregulation of sex-specific genes in Microarray- and real-time PCR analyses. Ratios correspond to quotients of TAC-f/sham-f divided by TAC-m/sham-m.
Table e5
Genes relatively upregulated in females within the gene ontology categories of mitochondrion, oxidoreductases or electron carrier
Table e6
Genes with sex differences in their expression pattern were GO-annotated in the categories "Biological Process" and "Cellular Localization" and sorted in KEGG pathways. Significance of the enrichment of genes in categories and pathways was calculated with Fisher`s exact test.
Table e7
Exact designation of genes in networks
Figure e1
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Table e1.
Gene Symbol / Gene Title / Forward Primer Sequence / Reverse Primer Sequence / Size / Probe Set IDAcadl / acyl-Coenzyme A dehydrogenase, long chain / 5´- aag tga ttc ctc acc aca cag a - 3´ / 5´- cag ctt ttt ccc aga cct ctc - 3´ / 69 bp / 1448987_at
Acta2 / actin, alpha 2, smooth muscle, aorta / 5´- cca acc ggg aga aaa tga c - 3´ / 5´- cag ttg tac gtc cag agg cat a - 3´ / 108 bp / 1416454_s_at
Actg1 / actin, gamma, cytoplasmic 1 / 5´- gag agc acg ctg tag atg aga a - 3´ / 5´- aat acc ctg cac aga tca ctc a - 3´ / 77 bp / 1415779_s_at
Calm3 / calmodulin 3 / 5´- gga gct gca gga cat gat taa tg - 3´ / 5´- tgg tca aga act ctg gga agt ca - 3´ / 74 bp / 1426710_at
Cebpb / CCAAT/enhancer binding protein (C/EBP), beta / 5´- aag ctg agc gac gag tac aag a - 3´ / 5´- gtc agc tcc agc acc ttg tg - 3´ / 116 bp / 1418901_at
Cks2 / CDC28 protein kinase regulatory subunit 2 / 5´- caa cag agc cta gga tgg gtt cat t - 3´ / 5´- tgg agg agg tcg tct aaa gag aag a - 3´ / 78 bp / 1417457_at
Col1a2 / procollagen, type I, alpha 2 / 5´- tag tgg tcc tca agg cat cc - 3´ / 5´- acc atg tag gcc agggc aag ac - 3´ / 124 bp / 1423110_at
Col3a1 / collagen, type III, alpha 1 / 5´- ctc acc ctt ctt cat ccc act ctt a - 3´ / 5´- aca tgg ttc tgg ctt cca gac at - 3´ / 117 bp / 1427883_a_at
Col5a2 / procollagen, type V, alpha 2 / 5´- cag ctg aag atg caa tca aag ttt - 3´ / 5´- aag acc gta cca cac agg ctt att - 3´ / 137 bp / 1450625_at
Corin / corin / 5´- gaa ctc aaa agg aag cgt caa t - 3´ / 5´- gga tgc gtt gtc ctg tat tca c - 3´ / 141 bp / 1419017_at
Cox5A / cytochrome c oxidase subunit Va / 5´- tta aat gaa ttg gga atc tcc ac - 3´ / 5´- tgc ctt agg aag ccc atc g - 3´ / 81 bp / 1415933_a_at
Ctnnbl1 / catenin, beta like 1 / 5´- atg cca aca agc tgt act gta gtg aa - 3´ / 5´- ggg ttg tgt ctt tta aat acg gat aac t -3´ / 136 bp / 1448582_at
Cxcl12 / chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1) / 5´- tcc tct tgc tgt cca gct ct - 3´ / 5´- cag gct gac tgg ttt acc g - 3´ / 129 bp / 1448823_at
Epas1 / endothelial PAS domain protein 1 / 5´- ggt taa gga acc cag gtg ct - 3´ / 5´- ggg att tct cct tcc tca gc - 3´ / 104 bp / 1449888_at
Fkbp5 / FK506 binding protein 5 / 5´- gag gga tac cca aac cca aac g - 3´ / 5´- gga atg tcg tgg tct tct cct t - 3´ / 128 bp / 1416125_at
Flt1 / fms-related tyrosine kinase 1 (vascular endothelial growth factor) / 5´- ggc cat cat ctg aat gtg gt - 3´ / 5´- tgc agt att cca cga tca cc - 3´ / 82 bp / 1419300_at
Gapdh / glyceraldehyde-3-phosphate dehydrogenase / 5´- atg gtg aag gtc ggt gtg a - 3´ / 5´- aat ctc cac ttt gcc act gc – 3´ / 84 bp
Ghr / growth hormone receptor / 5´- ggc tac acc agc tac tct tgg - 3´ / 5´- gtg aat cga ggc ttt cca ga - 3´ / 93 bp / 1451871_a_at
Gyk / glycerol kinase / 5´- cct cct gac aac cgt agc at - 3´ / 5´- cag cta tag cca cgg aac ct - 3´ / 80 bp / 1422703_at
Ide / Insulin degrading enzyme / 5´- aaa cgg gaa tac cgt gga cta ga - 3´ / 5´- cag gaa tat ttg gag ggt ctg aca - 3´ / 133 bp / 1435140_at
Igf2r / insulin-like growth factor 2 receptor / 5´- tca gta tgc aaa gtt cct gtg g - 3´ / 5´- ttg aat att gga ggg cct gt - 3´ / 71 bp / 1424111_at
Il1b / interleukin 1, beta / 5´- agt tga cgg acc cca aaa g - 3´ / 5´- gaa gct gga tgc tct cat ca - 3´ / 77 bp / 1449399_a_at
Itga7 / integrin alpha 7 / 5´- aga agg tgg agc cta gca ca - 3´ / 5´- gct gaa cac cac aca ctt gg - 3´ / 104 bp / 1418393_a_at
Klf16 / Kruppel-like factor 16 / 5´- cgc cca aag cct att aca ag - 3´ / 5´- cag aac ggg cga act tct t - 3´ / 114 bp / 1416350_at
Mef2c / myocyte enhancer factor 2C / 5´- caa atc tcc tcc ccc tat gaa tct a - 3´ / 5´- ctg agc cga ctg gga gtt att tat c - 3´ / 130 bp / NM 025282
Meox2 / mesenchyme homeobox 2 / 5´- acg agg agg gca tgt ttg - 3´ / 5´- gtg cca gtt gct ttg cag - 3´ / 113 bp / 1424233_at
Mknk1 / MAP kinase interacting serine/threonine kinase 1 / 5´- tgg ctg gaa gac acc agt c - 3´ / 5´- tca ctg ctg ccc atc tcc - 3´ / 75 bp / 1417630_at
Mmp2 / matrix metallopeptidase 2 / 5´- ggt tta ttt ggc gga cag tga ca - 3´ / 5´- act cat tcc ctg cga aga aca ca - 3´ / 130 bp / 1416136_at
Myl1 / myosin, light polypeptide 1 / 5´- a cc aat gca gag gtc aag aag gtt - 3´ / 5´- cat ggg cag aaa ctg ttc aaa ctc - 3´ / 90 bp / 1452651_a_at
Myl9 / myosin, light polypeptide 9, regulatory / 5´- aag gcc aag acc acc aag aag a - 3´ / 5´- tcg tgc aag tcc tcc tta tca a - 3´ / 90 bp / 1452670_at
Pdgfra / platelet-derived growth factor receptor, alpha polypeptide / 5´- aag acc tgg gca aga gga ac - 3´ / 5´- gaa cct gtc tcg atg gca ct - 3´ / 67 bp / 1421917_at
Pdk4 / pyruvate dehydrogenase kinase, isoenzyme 4 / 5´- cgc tta gtg aac act cct tcg - 3´ / 5´- ctt ctg ggc tct tct cat gg - 3´ / 94 bp / 1417273_at
Plcb1 / phospholipase C, beta 1 (phosphoinositide-specific) / 5´- gcc aaa aag gac agc aag aa - 3´ / 5´- ggt ctt gct caa tgg ctg at - 3´ / 73 bp / 1425782_at
Ppargc1a / peroxisome proliferative activated receptor, gamma, coactivator 1, alpha / 5´- gaa agg gcc aaa cag aga ga - 3´ / 5´- gta aat cac acg gcg ctc tt - 3´ / 63 bp / 1456395_at
Pten / phosphatase and tensin homolog (mutated in multiple advanced cancers 1) / 5´- cca caa aca gaa caa gat gct c - 3´ / 5´- cca ttt tcc act ttt tct gag g - 3´ / 105 bp / 1422553_at
Pxn / paxillin / 5´- ggc gtc act gtc aga ttt ca - 3´ / 5´- act ccc caa cat gct gtc c - 3´ / 109 bp / 1424027_at
Rarb / retinoic acid receptor, beta / 5´- caccggcatactgctcaa - 3´ / 5´- caaacgaagcagggcttg - 3´ / 113 bp / 1454906_at
Slc25a20 / solute carrier family 25 (carnitine/acylcarnitine translocase), member 20 / 5´- gagccgaaacccatcagtc - 3´ / 5´- tgtgtctgcagtcggacct - 3´ / 113 bp / 1423109_s_at
Smad2 / MAD homolog 2 (Drosophila) / 5´- tgaaccaaagtatggacacagg - 3´ / 5´- ccaagctgtgattaacaggaga - 3´ / 75 bp / 1420634_a_at
Smad4 / MAD homolog 4 (Drosophila) / 5´- actgtggatggctatgtggat - 3´ / 5´- cctttgcctatgtgcaacct - 3´ / 113 bp / 1422487_at
Tgfb2 / transforming growth factor, beta 2 / 5´- tactgcaggagaaggcaagc - 3´ / 5´- tagaaagtgggcgggatg - 3´ / 139 bp / 1423250_a_at
Thrb / thyroid hormone receptor beta / 5´- ggagtgggagctcatcaaga - 3´ / 5´- tgggtgcttgtccaatgtc - 3´ / 113 bp / 1422202_at
Timp2 / tissue inhibitor of metalloproteinase 2 / 5´- cct ctg tga ctt cat tgt gc - 3´ / 5´- tga tgc tct tct ctg tga cc - 3´ / 175 bp / 1420924_at
Timp3 / tissue inhibitor of metalloproteinase 3 / 5´- cttgggcttgtcgtgctc - 3´ / 5´- cttctttcccaccactttgg - 3´ / 138 bp / 1449335_at
Timp4 / tissue inhibitor of metalloproteinase 4 / 5`- gag gga gag cct gaa tca tca cta -3` / 5`- tac cca tag agc ttc cgt tcc a -3` / 138 bp / 1423405_at
Txn / thioredoxin / 5´- tgaagctgatcgagagcaag - 3´ / 5´- agaagtccaccacgacaagc - 3´ / 78 bp / 1416119_at
Table e2.
1427262_at / inactive X specific transcripts / Xist / X D / 69,44 / 5,52
1451090_a_at / eukaryotic translation initiation factor 2, subunit 3, structural gene X-linked / Eif2s3x / X A3.1; X C-D|X 32.0 cM / 1,87 / 5,52
1423042_at / fibroblast growth factor inducible 14 / LOC100045923 / X A1.1|X 42.0 cM / 1,56 / 5,52
1423043_s_at / DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked / Ddx3x / X A1.1|X 42.0 cM / 1,66 / 5,52
1421346_a_at / solute carrier family 6 (neurotransmitter transporter, taurine), member 6 / Slc6a6 / 6 D1|6 38.2 cM / 1,48 / 5,52
1434888_a_at / matrin 3 / Matr3 / 18 C|18 15.0 cM / 1,45 / 5,52
1417210_at / eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked / Eif2s3y / Y A1 / 0,01 / 5,52
1426438_at / DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, Y-linked / Ddx3y / Y A1|Y 2.07 cM / 0,01 / 5,52
1424903_at / jumonji, AT rich interactive domain 1D (Rbp2 like) / Jarid1d / Y A1|Y 2.03 cM / 0,25 / 5,52
1426598_at / ubiquitously transcribed tetratricopeptide repeat gene, Y chromosome / Uty / Y E|Y 2.06 cM /// Y A1 / 0,31 / 5,52
1435943_at / Dipeptidase 1 (renal) / Dpep1 / 8 E1|8 67.0 cM / 0,61 / 5,52
1448775_at / interferon activated gene 203 / Ifi203 / 1 H3|1 95.31 cM / 0,56 / 7,22
1419714_at / CD274 antigen / Cd274 / 19 C2 / 0,29 / 7,22
1451564_at / poly (ADP-ribose) polymerase family, member 14 / Parp14 / 16 B3 / 0,51 / 7,22
1431591_s_at / ISG15 ubiquitin-like modifier / Isg15 / 17 C; 4 E2 / 0,43 / 7,22
1452349_x_at / interferon activated gene 205 /// myeloid cell nuclear differentiation antigen / Ifi205 / 1 H3|1 95.3 cM / 0,52 / 7,22
1418533_s_at / frizzled homolog 2 (Drosophila) / Fzd2 / 11 E1 / 0,62 / 7,22
1449025_at / interferon-induced protein with tetratricopeptide repeats 3 / Ifit3 / 19 C3 / 0,30 / 7,22
1421998_at / torsin family 3, member A / Tor3a / 1 H1 / 0,65 / 7,22
1426276_at / interferon induced with helicase C domain 1 / Ifih1 / 2 C3 / 0,52 / 7,22
Table 3.
(TAC/sham) / Ratio / FDR [%] / Chromosome
--- / --- / 1438954_x_at / 0.68 / 1.42 / 0.48 / 0.001 / ---
--- / --- / 1434618_at / 0.92 / 1.67 / 0.55 / 0.002 / ---
--- / --- / 1450941_at / 1.34 / 0.77 / 1.74 / 0.008 / ---
--- / --- / 1442025_a_at / 1.60 / 0.45 / 3.60 / 0.016 / ---
--- / --- / 1452292_at / 1.24 / 0.97 / 1.28 / 0.018 / 11
--- / --- / 1442026_at / 1.62 / 0.60 / 2.71 / 0.019 / ---
--- / --- / 1452654_at / 1.03 / 1.87 / 0.55 / 0.020 / ---
--- / --- / 1438758_at / 0.86 / 0.70 / 1.22 / 0.021 / ---
--- / --- / 1420942_s_at / 0.28 / 0.39 / 0.71 / 0.034 / ---
Oas1a / 2'-5' oligoadenylate synthetase 1A / 1424775_at / 1.47 / 0.62 / 2.38 / 0.020 / 5
Oasl1 / 2'-5' oligoadenylate synthetase-like 1 / 1424339_at / 1.15 / 0.59 / 1.94 / 0.028 / 5
Oasl2 / 2'-5' oligoadenylate synthetase-like 2 / 1453196_a_at / 0.81 / 0.35 / 2.35 / 0.002 / 5
Bdh / 3-hydroxybutyrate dehydrogenase (heart, mitochondrial) / 1426959_at / 3.18 / 1.31 / 2.43 / 0.002 / 16
Ywhag / 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma polypeptide / 1450012_x_at / 1.30 / 0.95 / 1.38 / 0.026 / 5
Pfkfb1 / 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 / 1427213_at / 0.36 / 0.24 / 1.51 / 0.037 / X
Abhd4 / abhydrolase domain containing 4 / 1439259_x_at / 1.48 / 1.00 / 1.47 / 0.027 / 14
D930038M13Rik / ABI gene family, member 3 (NESH) binding protein / 1427053_at / 0.83 / 1.89 / 0.44 / 0.027 / 16
Acacb / acetyl-Coenzyme A carboxylase beta / 1427052_at / 0.71 / 0.62 / 1.15 / 0.024 / 5
Acadl / acetyl-Coenzyme A dehydrogenase, long-chain / 1448987_at / 0.92 / 0.72 / 1.29 / 0.048 / 1
Acp1 / acid phosphatase 1, soluble / 1450721_at / 1.19 / 0.97 / 1.22 / 0.047 / 12