HIF-P4H-2 deficiency protects against skeletal muscle ischemia-reperfusion injury

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J Mol Med 2015 HIF-P4H-2 deficiency protects against skeletal muscle ischemia-reperfusion injury Sara Karsikas; Mikko Myllymäki; Minna Heikkilä; Raija Sormunen; Kari I Kivirikko; Johanna Myllyharju; Raisa Serpi; Peppi Koivunen Supplementary methods 1 Quantitative Real Time RT-PCR (qpcr) The skeletal muscles (M. gastrocnemius and M. soleus) were dissected out immediately after sacrifice of the mice, snap-frozen in liquid nitrogen and stored at -70 C. The muscle tissue was homogenized in a TissueLyser LT (QIAGEN) device and the RNA was isolated using the TriPure isolation reagent (Roche Applied Science) and purified using an EZNA total RNA kit (OMEGA Bio-Tek). Reverse transcription was performed with an iscript cdna synthesis kit (Bio-Rad) and qpcr was performed in a CFX96 Real-Time System (Bio-Rad). The sequences of the primers are presented in Supplementary Table 1. Transmission electron microscopy (TEM) Skeletal muscle samples were fixed in 1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer, postfixed in 1% osmium tetroxide, dehydrated in acetone, and embedded in Epon LX 112 (Ladd Research Industries). Thin sections were cut with a Leica Ultracut UCT ultramicrotome, stained in uranyl acetate and lead citrate, and examined in a Tecnai Spirit transmission electron microscope (Fei Europe). The images were captured using a Quemesa CCD (Olympus Soft Imaging Solutions). Histological analyses Hematoxylin-eosin staining was used to study the morphometry of the muscle. 5-8 images per sample were taken using a magnification of 20x. The central nuclei in muscle sections were counted at a magnification of 40x. Anti-fast myosin skeletal heavy chain staining (ab91506; Abcam) was used to study the ratio of fast to slow myosin fibers, the proportion of fast myocytes was determined in a blinded manner from 5 pictures/sample. The proportion of glycogen-rich myocytes was determined using Periodic acid-schiff (PAS) staining. In muscle 8-10 pictures/sample and in liver 5 pictures/sample were analyzed in a blinded manner. 1

The livers were analyzed after 12 h fasting. Apoptosis in muscle was analyzed with In Situ Cell Death Detection kit, Fluorescein (Roche). Number of neutrophils was evaluated using anti-neutrophil antibody (ab2557; Abcam). Muscle capillaries were visualized using an anti-cd31 antibody (Pecam-1; sc-1506; Santa Cruz), and capillary density and mean capillary lumen area (referred to as capillary size) were measured from five randomly chosen CD31 stained sections/mouse (NIS-Elements BR software, Nikon). Masson-Trichrome staining was used to calculate the percentage of injured myocytes after hind limb ischemia-reperfusion by scoring at least 300 myocytes in 5-10 randomly chosen fields/mouse as injured/noninjured. Glycogen amount The amount of glycogen in skeletal muscles was also analyzed using a chemical method (Glycogen Assay Kit, #700480, Cayman Chemical) according to manufacturer s instructions. 2

Suppl. fig. 1 Absolute ADP and ATP levels of the Hif-p4h-2 gt/gt (gt/gt) and wild-type (wt) gastrocnemius muscles in baseline, n = 5 for both genotypes. 3

Suppl. fig. 2 Number of neutrophils per unit area in gastrocnemius muscles 48 hours after ischemiareperfusion, based on quantification of anti-neutrophil staining. n = 8 for wild-type mice (wt), n = 7 for hypomorphic mice (gt/gt). 4

Suppl. fig. 3 Liver glycogen content 48h post I/R using. Scoring of glycogen content is shown, p = 1.0, n = 4 for both genotypes. Representative figures of both wilt-type (wt) and hypomorphic (gt/gt) livers, PAS staining. Bar, 100 µm. 5

Supplementary table 1. Sequences of the qpcr primers. Gene Forward primer Reverse primer Hif-p4h-2 GGCAACTACGTCATCAATG ACCTTAACATCCCAGTTCTGA Hif-p4h-2 CTGGGCAACTACAGGATAAAC GCGTCCCAGTCTTTATTTAGATA Hif-p4h-3 AGGCCCAACGTACCCCACCT GTGTGTTGGCTGGGAGCCCA β-actin AGAGGGAAATCGTGCGTGAC CAATAGTGATGACCTGGCCGT Cox1 CTGAGCGGGAATAGTGGGTA TGGGGCTCCGATTATTAGTG Pfkl Quantitect primer assays (Qiagen) Pdk1 Quantitect primer assays (Qiagen) Pdk4 Quantitect primer assays (Qiagen) Gbe1 ACTGCTTTGATGGCTTCCGT AACCTTGAACCCATTCCGTGG Gyg GCTGGTCACTTACTCAGTATTCC AGGGTTGATAGACAAAGACTCCA Gsk3b AAGCGATTTAAGAACCGAGAGC AGAAATACCGCAGTCGGACTAT Pygl GTGTCCCAAGAGGGTGTATTACC GGCATTTTGTAAGCCAAGGTTGA Epm2a CGCGTTGACACGTTCTGGTA CATGGTGAGGTCCATTTCCTTC Pgm2 CATCCCGACCCCAATCTCAC ATGTTTCGATCCCCGTCACC Calm1 CAACGAAGTGGATGCTGATG TCCGTCGCCATCAATATCTG Calm2 TCTAAGCCCGTCTGCACATCT TCCACAGTCCACGCAGAGTTA Phka1 CCTGGGTCCGAGATAATGTGT CGGTCAGCATTTTTGCGATATG Phkb GCTAGCATACAGGCGCATTG TATCAGCCTGCCGCATGTAG Ppp2ca GTTCAAGAGGTTCGATGTCCAG AGCTACAAGCAGTGTAACTGTTT PGC-1a AGCCGTGACCACTGACAACGAG GCTGCATGGTTCTGAGTGCTAAG enos GACCCTCACCGCTACAACAT GCTCATTTTCCAGGTGCTTC inos CTCACTGGGACAGCACAGAA TGGTCAAACTCTTGGGGTTC Ang1 TCATCATCCAGGAGTTGGAGAAGCA TGTGTCCATGAGCTCCAGTTGTTGC Ang2 CATCTGCAAGTGTTCCCAGA CGTCGTGTTCTGGAAGAATGA Tie2 ATGTGGAAGTCGAGAGGCGAT CGAATAGCCATCCACTATTGT Vegfa CGACGTCAGAGAGCAACATCA TCATTCTCTCTATGTGCTGGCT Vegfb CCAGCCCCGCTCTGT GAGTGGGATGGATGATGTCA Vegfc TTGCTGCAACAGCGAGGGGC GACATGCACCGGCAGGAAGTGT Vegf-R1 GAGGAGGATGAGGGTGTCTATAGGT GTGATCAGCTCCAGGTTTGACTT Vegf-R2 GCCCTGCTGTGGTCTCACTAC CAAAGCATTGCCCATTCGAT Apelin CATAAGGGCCCCATGCCT GCACCGGGAGGGCACT APJ GGTTACAACTACTATGGGGCTGA AGCTGAGCGTCTCTTTTCGC Adm GCTGATGGTCACGGTAACCC TTCCAGACTACAGGCTGAAGG Ldha GGATGAGCTTGCCCTTGTTGA GACCAGCTTGGAGTTCGCAGTTA Ldhb TGCGTCCGTTGCAGATGAT TTTCGGAGTCTGGAGGAACAA Fbp1 CTTGCCATGGATTGTGGTGT TTGATGGCAGGGTCAAAGTC Fbp2 TGGCCTCCATCGGAACTATATT CAGGGTTGCACTACCATACAG Pepck1 CGATGACATTGCCTGGATGAAG TCTTCACTGAGGTGCCAGGAG Ca9 GCCTTGCAGTTGCATCTACAC GGGAAACGGTGACCATTGACT PC GATGACCTCACAGCCAAGCA GGGTACCTCTGTGTCCAAAGGA Nhe1 TCATCCACCTCGGATCTTCCC TCCTGAGAACAGGTAGCAGTC Mpc1 ACCTCGAAACTGGCTTTTGTT TCGTAGTTGATAAGTCGTCCTCC Mpc2 AATTGAGGCCGCTTTACAACC CACACACCAATCCCCATTTCA Mct4 TGGAATGGGGCTGACATTTTT ATTGGCTGTGGATGGGTCTTA LonP1 ATGACCGTCCCGGATGTGT CCTCCACGATCTTGATAAAGCG Nadufa2 CTGGGGTTGCGTGAGATTC CCGTTGCACGATGAAATCCC Cox4i1 ATTGGCAAGAGAGCCATTTCTAC TGGGGAAAGCATAGTCTTCACT Cox4i2 CTGCCCGGAGTCTGGTAATG CGTAGCAGTCAACGTAGGGG G6Pc AAAAAGCCAACGTATGGATTCCG CAGCAAGGTAGATCCGGGA Mct1 GGTGGGCAGTGTTAGTCGG GATAGGACCTCCAGCATACATGA 6

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