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• W1979581862 abstract "The expression of ankyrin repeat domain protein 1 (Ankrd1), a transcriptional cofactor and sarcomeric component, is strongly elevated by wounding and tissue injury. We developed a conditional Ankrd1fl/fl mouse, performed global deletion with Sox2-cre, and assessed the role of this protein in cutaneous wound healing. Although global deletion of Ankrd1 did not affect mouse viability or development, Ankrd1−/− mice had at least two significant wound-healing phenotypes: extensive necrosis of ischemic skin flaps, which was reversed by adenoviral expression of ANKRD1, and delayed excisional wound closure, which was characterized by decreased contraction and reduced granulation tissue thickness. Skin fibroblasts isolated from Ankrd1−/− mice did not spread or migrate on collagen- or fibronectin-coated surfaces as efficiently as fibroblasts isolated from Ankrd1fl/fl mice. More important, Ankrd1−/− fibroblasts failed to contract three-dimensional floating collagen gels. Reconstitution of ANKRD1 by adenoviral infection stimulated both collagen gel contraction and actin fiber organization. These in vitro data were consistent with in vivo wound closure studies, and suggest that ANKRD1 is important for the proper interaction of fibroblasts with a compliant collagenous matrix both in vitro and in vivo. The expression of ankyrin repeat domain protein 1 (Ankrd1), a transcriptional cofactor and sarcomeric component, is strongly elevated by wounding and tissue injury. We developed a conditional Ankrd1fl/fl mouse, performed global deletion with Sox2-cre, and assessed the role of this protein in cutaneous wound healing. Although global deletion of Ankrd1 did not affect mouse viability or development, Ankrd1−/− mice had at least two significant wound-healing phenotypes: extensive necrosis of ischemic skin flaps, which was reversed by adenoviral expression of ANKRD1, and delayed excisional wound closure, which was characterized by decreased contraction and reduced granulation tissue thickness. Skin fibroblasts isolated from Ankrd1−/− mice did not spread or migrate on collagen- or fibronectin-coated surfaces as efficiently as fibroblasts isolated from Ankrd1fl/fl mice. More important, Ankrd1−/− fibroblasts failed to contract three-dimensional floating collagen gels. Reconstitution of ANKRD1 by adenoviral infection stimulated both collagen gel contraction and actin fiber organization. These in vitro data were consistent with in vivo wound closure studies, and suggest that ANKRD1 is important for the proper interaction of fibroblasts with a compliant collagenous matrix both in vitro and in vivo. As part of a gene expression profiling program to identify genes altered after dermal wounding in mice, we reported that ankyrin repeat domain protein 1 (Ankrd1; alias cardiac ankyrin repeat protein) mRNA and protein levels were highly induced and remained elevated during the healing process.1Shi Y. Reitmaier B. Regenbogen J. Slowey R.M. Opalenik S.R. Wolf E. Goppelt A. Davidson J.M. CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.Am J Pathol. 2005; 166: 303-312Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Cells of the epidermis and dermis that showed induced expression included vascular endothelial cells, cells of the hair follicle bulb, the panniculus carnosus, keratinocytes, monocytes, and fibroblasts, indicating increased ANKRD1 may be a generalized stress response in many cell types of the skin. Overexpression of ANKRD1, a member of the muscle ankyrin repeat protein (MARP) family, which includes Ankrd2 and Ankrd23,2Miller M.K. Bang M.L. Witt C.C. Labeit D. Trombitas C. Watanabe K. Granzier H. McElhinny A.S. Gregorio C.C. Labeit S. The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules.J Mol Biol. 2003; 333: 951-964Crossref PubMed Scopus (274) Google Scholar also improved many aspects of wound healing in several animal models.1Shi Y. Reitmaier B. Regenbogen J. Slowey R.M. Opalenik S.R. Wolf E. Goppelt A. Davidson J.M. CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.Am J Pathol. 2005; 166: 303-312Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar The dynamics of MARP gene expression during both normal muscle development and function and those seen under pathological stress conditions in muscle and many other tissues3Kojic S. Radojkovic D. Faulkner G. Muscle ankyrin repeat proteins: their role in striated muscle function in health and disease.Crit Rev Clin Lab Sci. 2011; 48: 269-294Crossref PubMed Scopus (56) Google Scholar, 4Mikhailov A.T. Torrado M. The enigmatic role of the ankyrin repeat domain 1 gene in heart development and disease.Int J Dev Biol. 2008; 52: 811-821Crossref PubMed Scopus (59) Google Scholar, 5Samaras S.E. Shi Y. Davidson J.M. CARP: fishing for novel mechanisms of neovascularization.J Investig Dermatol Symp Proc. 2006; 11: 124-131Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar suggested that disruptions in normal MARP function could have dire consequences to the organism. However, a recent report observed that deletions of the MARP family members, both individually and in combination, produced a subtle phenotype.6Barash I.A. Bang M.L. Mathew L. Greaser M.L. Chen J. Lieber R.L. Structural and regulatory roles of muscle ankyrin repeat protein family in skeletal muscle.Am J Physiol Cell Physiol. 2007; 293: C218-C227Crossref PubMed Scopus (75) Google Scholar On the basis of the altered skeletal muscle response to eccentric contraction, the authors suggested that the MARPs are not essential for development or basal function of skeletal muscle, but that they play a role in mechanical behavior and stability of muscle after exercise. It was also concluded that the three MARP proteins are structurally and functionally redundant, because it required deletion of all three genes to see a significant effect. A recent study with the same strains further concluded that the MARPs were not involved in the response to cardiac pressure overload.7Bang M.L. Gu Y. Dalton N.D. Peterson K.L. Chien K.R. Chen J. The muscle ankyrin repeat proteins CARP, Ankrd2, and DARP are not essential for normal cardiac development and function at basal conditions and in response to pressure overload.PLoS One. 2014; 9: e93638Crossref PubMed Scopus (44) Google Scholar We developed a conditional Ankrd1fl/fl mouse with the ultimate goal of determining the role of ANKRD1 in various cell types. As an initial approach, we generated a global deletion for the purpose of studying ANKRD1 function in all cell types involved in wound healing. We now report that deletion of Ankrd1 results in a viable mouse with significant wound-healing phenotypes that are not compensated by intact Ankrd2 or Ankrd23. Results of cell culture studies suggest that part of this phenotype results from dysfunctional cell-matrix interaction in fibroblasts. We designed a targeting vector to delete the mouse Ankrd1 gene basal promoter and protein-coding exons 1 and 2 (1.87 kb) when targeted mice were crossed with cre-recombinase–expressing mice (Figure 1A). The targeting vector was constructed using 15 kb of Ankrd1 sequence that was retrieved from strain 129S7/SvEvBrd-Hptrb-m2 mouse bacterial artificial chromosome (BAC) library (bMQ-382E2; BACPAC Resource Center, Children's Hospital Oakland Research Institute, Oakland, CA).8Adams D.J. Quail M.A. Cox T. van der Weyden L. Gorick B.D. Su Q. Chan W.I. Davies R. Bonfield J.K. Law F. Humphray S. Plumb B. Liu P. Rogers J. Bradley A. A genome-wide, end-sequenced 129Sv BAC library resource for targeting vector construction.Genomics. 2005; 86: 753-758Crossref PubMed Scopus (95) Google Scholar Two cre-recombinase recognition sequences (loxP) were introduced into the 15-kb BAC sequence: the first loxP was located approximately 7 kb upstream of the 5′ end, and the second loxP was inserted into intron 2 approximately 6 kb upstream from the 3′ end (Figure 1A). A neomycin gene cassette flanked by flp-recombinase recognition (frt) sites was inserted 5′ of the second loxP site. Herpes simplex virus–thymidine kinase was also included as a negative selection marker. Internal EcoRV and SmaI sites were introduced into this 15-kb BAC DNA for confirmation of the targeted allele (Figure 1B). Embryonic stem cells (129S6) were electroporated with the linearized targeting vector in the Vanderbilt Transgenic/Embryonic Stem Cell Core (Nashville, TN), and isolated colonies were screened by Southern blot analysis. Positive clones were injected into C57Bl/6J blastocysts. Resulting male pups that were ≥70% chimeric were tested for germ-line transmission by breeding to C57Bl/6J females. The neomycin cassette was removed by breeding hemizygous (Ankrd1wt/fl) mice to actin promoter-driven, eflp-recombinase C57Bl/6J mice. Hemizygous (Ankrd1wt/fl) mice with the neomycin cassette deleted were bred to homozygosity [Ankrd1fl/fl; flanked by loxP (FLOX)], followed by breeding to Sox2 promoter-driven cre-recombinase C57Bl/6J mice. Because the Sox2 promoter is expressed in the oocyte,9Hayashi S. Tenzen T. McMahon A.P. Maternal inheritance of Cre activity in a Sox2Cre deleter strain.Genesis. 2003; 37: 51-53Crossref PubMed Scopus (84) Google Scholar all offspring acquired a recombined allele, even when the Sox2 cre-recombinase transgene did not segregate to the pup. Mice with deletion of one Ankrd1 allele [Ankrd1wt/−; wild type (WT/−)] were crossbred to produce homozygous [Ankrd1−/−; knockout (KO)] mice. Mice lacking the Sox2-cre recombinase and eflp-recombinase transgenes were selectively used for breeding to eliminate the transgenes from the KO background. Initial phenotyping studies used mixed-breed C57Bl/6J/129S6 FLOX, WT/−, and KO mice. Mice were further backcrossed to C57Bl/6J to generate a pure inbred strain. The mice used for the excisional wound studies had been backcrossed for eight generations. Adult FLOX, WT/−, and KO mice were weighed and euthanized. The spleen, three lobes of the liver, one kidney, brain, cardiac ventricles, and the gastrocnemius muscle (skeletal muscle) were dissected and weighed. Organ/body weight ratios were calculated. Differences among the three genotypes were tested for significance using Graphpad Prism version 5.03 (GraphPad Software, Inc., La Jolla, CA) and two-way analysis of variance, with significance set at P < 0.05. Samples of tissue from all major organ systems of young (5 to 6 months old) and aged (>1 year) adult male and female Ankrd1-null mice and FLOX age-matched controls were collected, fixed overnight in formalin, processed routinely, divided into sections (5 μm thick), and stained with hematoxylin and eosin. The specimens underwent thorough gross and microscopic evaluation of all organ systems by a veterinary pathologist in the Division of Comparative Pathology, Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine (Nashville, TN). No abnormalities were found. Studies were performed in the Association for Assessment and Accreditation of Laboratory Animal Care International–approved facilities of Vanderbilt University, under approval of the Institutional Animal Care and Use Committee at Vanderbilt University School of Medicine. FLOX (control) and KO male mice (aged 15 to 20 weeks; n = 8, each genotype) in a mixed background and in eighth-generation C57Bl/6J background received two 6-mm, full-thickness excisional wounds on the dorsum. Wounds were photographed with a Canon PC1234 camera (Canon USA, Lake Success, NY). Wound areas were determined using ImageJ software version 1.48 (NIH, Bethesda, MD) and expressed as percentage of initial wound size. At completion of the experiment, mice were euthanized, and the complete wounds, including 2 mm of the wound margin, were harvested and prepared for histological analysis. Large excisional wounds were used to follow wound closure rates in 15 C57BL/6 and 10 KO age-matched female mice weighing 19 to 22 g. Under isoflurane anesthesia, the dorsal surface was shaved with electric clippers, cleaned successively with betadine and 70% isopropanol (3×), and then incised with a scalpel to produce a 15 × 15-mm, full-thickness wound. Each site was covered with a Tegaderm (3M, St. Paul, MN) dressing to prevent dehydration and infection, and each mouse was fitted with a fabric sleeve (1.6-cm Surgitube; Western Medical Ltd, Tenafly, NJ) to prevent the wound site from being disturbed. Animals were maintained in cages with a 37°C warming pad covering 50% of the cage bottom during a recovery period of 4 to 6 hours, after which time cages were returned to the animal facility. Each subject received 1 mL i.p. of warm 0.9% sodium chloride for 3 days to avoid dehydration. The dressing was removed at day 7. Wound dimensions were measured with an electronic digital caliper (15-cm; Harbor Freight Tools, Camarillo, CA) on alternate days, beginning at day 7 until healing was complete. An ischemic skin flap was generated on the dorsum by making 1.5 × 2.0-cm skin flaps with a rostral pedicle and underlying the full flap with a 0.13-mm thick silicone sheet (Invotec International, Jacksonville, FL) that extended s.c. 0.25 cm beyond the flap boundaries.10Tepper O.M. Galiano R.D. Capla J.M. Kalka C. Gagne P.J. Jacobowitz G.R. Levine J.P. Gurtner G.C. Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures.Circulation. 2002; 106: 2781-2786Crossref PubMed Scopus (1296) Google Scholar The silicone sheet was sutured to the internal fascia, and the flap margins were sutured to close the incisions. This paradigm effectively prevented subdermal blood vessel infiltration into the flap from anywhere other than the pedicle, generating a graded, proximodistal ischemia. Mice were euthanized at indicated time points, and the flaps were removed, cut either transversely or longitudinally, and prepared for histological analysis. Fibrin clots containing virus were generated by mixing 10 mg/mL fibrinogen (Sigma-Aldrich, St. Louis, MO), 100 U/mL thrombin (Sigma-Aldrich), and adenovirus expressing either luciferase plus green fluorescent protein (GFP; adLuc-GFP; control) or ANKRD1 plus GFP (adAnkrd1-GFP)1Shi Y. Reitmaier B. Regenbogen J. Slowey R.M. Opalenik S.R. Wolf E. Goppelt A. Davidson J.M. CARP, a cardiac ankyrin repeat protein, is up-regulated during wound healing and induces angiogenesis in experimental granulation tissue.Am J Pathol. 2005; 166: 303-312Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar so that the final concentrations in the vehicle were 5 mg/mL fibrinogen, 20 U/mL thrombin, and 2 × 1010 plaque-forming units of adenovirus per flap. Control flaps were treated with an equivalent dose of adLuc-GFP in fibrin vehicle. Sterile saline was added to adjust the volume of the solutions as needed. Virus (300 μL in fibrin vehicle) was injected between the skin and the silicone sheet of each closed flap. Mice were euthanized by CO2 asphyxiation at the times indicated later. The wounds plus surrounding skin were excised and fixed in neutral-buffered formalin overnight at 4°C, embedded in paraffin, divided into sections, stained with hematoxylin and eosin or Masson's trichrome green stain, and imaged (Olympus BX50 microscope, DP71 camera, and Software CellSens Standard 1.6; Olympus Corporation, Center Valley, PA). Digital images of each excisional wound were used to determine the distance between the edges of the panniculus carnosus as a measure of original wound gap using ImageJ software. Digital images of the wounds were also used to measure the granulation tissue thickness and cross-sectional area and the extent of ischemic flap necrosis with a 4× objective. Mouse dermal fibroblasts were isolated from FLOX and KO neonatal skin, as previously described.11Normand J. Karasek M.A. A method for the isolation and serial propagation of keratinocytes, endothelial cells, and fibroblasts from a single punch biopsy of human skin.In Vitro Cell Dev Biol Anim. 1995; 31: 447-455Crossref PubMed Scopus (120) Google Scholar Early-passage populations of the isolated skin fibroblasts were immortalized with a simian virus (SV)40 large T-antigen plasmid.12Chang L.S. Pan S. Pater M.M. Di Mayorca G. Differential requirement for SV40 early genes in immortalization and transformation of primary rat and human embryonic cells.Virology. 1985; 146: 246-261Crossref PubMed Scopus (35) Google Scholar Briefly, 100-mm dishes of primary cells at 80% to 90% confluence were transfected with 8 μg of plasmid using Lipofectamine (Life Technologies, Grand Island, NY) overnight at 37°C in 5% CO2, after which the transfection reagent was removed and growth medium was added.12Chang L.S. Pan S. Pater M.M. Di Mayorca G. Differential requirement for SV40 early genes in immortalization and transformation of primary rat and human embryonic cells.Virology. 1985; 146: 246-261Crossref PubMed Scopus (35) Google Scholar Both primary and immortalized cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin-streptomycin (P/S), 1% antibiotic-antimycotic (AA), and 2 mmol/L l-glutamine. Of a 12-well tissue culture plate, 4 wells were coated with extracellular matrix (ECM) molecules by incubation in a tissue culture hood at room temperature. In each plate, two wells were coated with collagen (rat tail type I; 100 μg/mL; BD Biosciences, Bedford, MA) and two wells were coated with fibronectin (10 μg/mL; Santa Cruz Biotechnology, Santa Cruz, CA), both in phosphate-buffered saline (PBS). ECM solutions were removed after 2 hours, and the wells were washed with sterile PBS and incubated with serum-free DMEM at 37°C in 5% CO2. FLOX and KO fibroblasts at 80% confluence were trypsinized from flasks and counted and diluted to a concentration of 6 × 105 cells/mL, a concentration that permitted cells to reach confluence 24 hours after plating. ECM-coated cell culture plates were secured onto a magnetic array that aligned magnets in the center of each of four coated wells. The media were removed from the four coated wells, 500 μL of fresh growth medium was added, and sterile, magnetically adherent stencils13Ashby W.J. Wikswo J.P. Zijlstra A. Magnetically attachable stencils and the non-destructive analysis of the contribution made by the underlying matrix to cell migration.Biomaterials. 2012; 33: 8189-8203Crossref PubMed Scopus (16) Google Scholar (MAtS) (Supplemental Figure S1) were placed in the center of each well. Cell suspensions (500 μL each) were equally distributed around the MAtS, one cell type per ECM, and the plates were incubated for 24 hours to allow cells to attach and reach confluence. Plates were removed from the magnetic array, and the medium was carefully aspirated, to remove floating cells, and replaced with fresh growth medium. The MAtS were carefully removed to leave a cell-free, undisturbed, four-arm, ECM-coated area in the center of each well. The cell-free wells were filled with warm PBS to help prevent drying of open plates, and the plate was placed in a humidified stage incubator (Bioscience Tools, San Diego, CA) infused with 5% CO2, attached to a Zeiss Axiovert 200M microscope (Carl Zeiss Microscopy LLC, Thornwood, NY) with an automated stage driven by the Ludl Mac 2000 driver module (Ludl Electronic Products Ltd., Hawthorne, NY). Images were acquired every 10 minutes for 5 hours with a Tucsen 3.3 MP cooled charge-coupled device digital microscope camera (OnFocus Laboratories, Lilburn, GA) using a 10× objective. ImagePro Plus 3D with StagePro software version 6.0 (Media Cybernetics Inc., Bethesda, MD) for time-lapse image acquisition was used to acquire and process images. Data analysis to determine percentage of total pixels that were not covered (percentage open area) was done using TScratch software version 1.0 for Windows without Matlab Compiler Runtime (Swiss Federal Institute of Technology in Zürich, Zürich, Switzerland).14Geback T. Schulz M.M. Koumoutsakos P. Detmar M. TScratch: a novel and simple software tool for automated analysis of monolayer wound healing assays.Biotechniques. 2009; 46: 265-274PubMed Google Scholar Three-dimensional collagen lattices were prepared as previously described.15Ngo P. Ramalingam P. Phillips J.A. Furuta G.T. Collagen gel contraction assay.Methods Mol Biol. 2006; 341: 103-109PubMed Google Scholar Briefly, type I rat tail collagen (BD Biosciences, San Jose, CA) was diluted with 20 mmol/L acetic acid to 3 mg/mL. Primary or immortalized skin fibroblasts isolated from neonatal FLOX and KO mice were cultured in DMEM (10% FBS, 1% AA, and 1% P/S), trypsinized, and counted. Collagen (200 μL) neutralized to pH 7.0 with 1N sodium hydroxide was loaded into each well of a 24-well plate. Cells (1.5 × 105) were suspended in 400 μL of DMEM (10% FBS, 1% AA, and 1% P/S) and added to the wells containing 200 μL of collagen solution to yield a final concentration of 1 mg/mL. The gels were then incubated at 5% CO2 and 37°C for 30 minutes to allow the collagen to polymerize. After collagen polymerization, 500 μL of DMEM (10% FBS, 1% AA, and 1% P/S) was added to each well. By using a sterile spatula, each gel was mechanically released from the wall and bottom of the wells. Collagen lattices were imaged (GelLogic200 Imaging System, Molecular Imaging System version 7.1; Carestream Health, Inc., Woodbridge, CT) every 24 hours, and lattice area was analyzed using the Molecular Imaging software (Carestream Health, Inc.). Reduction in lattice area due to contraction was determined at daily intervals up to 7 days. FLOX and KO immortalized skin fibroblasts were infected with either adAnkrd1-GFP or adLuc-GFP as a control, at a multiplicity of infection of 100. Cells were harvested 48 hours after infection, and used for collagen gel contraction assays or protein isolation. Whole cell extracts were prepared in radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich, St. Louis, MO) containing a protease inhibitor cocktail (Complete Mini Protease Inhibitor Tablets; Roche, Mannheim, Germany), followed by sonication at 4°C using a Branson 250 Sonifier (Emerson Industrial Automation, Danbury, CT) with a water bath cup horn attachment. Protein concentration in cleared lysates was determined by BCA protein assay kit (ThermoScientific, Rockford, IL), and lysates were stored at −80°C. Aliquots containing 30 μg protein from cell extracts were separated by 10% polyacrylamide SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore, Billerica, MA) using the NuPage (Life Technologies, Carlsbad, CA) blotting apparatus, following the manufacturer's protocol. After blocking with a solution of 10 mmol/L Tris-HCl, pH 8, 150 mmol/L NaCl, 0.05% Tween 20, and 5% milk powder, the membrane was incubated with anti-ANKRD1 antibody (1:2000) and anti-cyclophilin (1:20,000; BML-SA296; Enzo, Farmingdale, NY) at 4°C overnight, followed by incubation with anti-rabbit IgG (C2609; Santa Cruz Biotechnology) at room temperature for 30 minutes. The membrane was washed and incubated with Western Lightning Plus Enhanced Chemiluminescent Reagent (Perkin Elmer, Waltham, MA), and protein bands were visualized and quantified using a Kodak Image Station 4000 MM Pro with Kodak MI software version 5.3.3.17476 (Standard Editon; Carestream Health, Inc.). For the virus reconstitution studies, protein was isolated from cells 48 hours after infection. RNA was isolated using the Illustra RNAspin Mini Isolation Kit (GE Healthcare, Piscataway, NJ), according to manufacturer's instructions. RNA concentration was determined spectrophotometrically (Nanodrop; Thermo Scientific, Wilmington, DE), and samples were stored at −80°C. TaqMan real-time quantitative PCR for Ankrd1, Ankrd2, and Ankrd23 (see probe and primer sequences below) and cyclophilin (Applied Biosystems, Foster City, CA), as the housekeeping control, was performed on 50 ng reverse-transcribed RNA with the following primers and probes: Ankrd1, 5′-AGACTCCTTCAGCCAACATGATG-3′ (forward), 5′-CTCTCCATCTCTGAAATCCTCAGG-3′ (reverse), and 5′-CCCCTGCCTCCCCATTGCCATTCT-3′ (probe); Ankrd2, 5′-GCAGTGGAGGGGAAAATGAAAG-3′ (forward), 5′-CTGTCCGACGGAACTCATCAC-3′ (reverse), and 5′-TCCGCTGAACCTCCQTCCGCCA-3′ (probe); and Ankrd23, 5′-ACTGCCTAGAGCACCTTATCG-3′ (forward), 5′-GGAAGCCACATTCTTCACACC-3′ (reverse), and 5′-GCCCACATCAACGCACAGGATAAG-3′ (probe). Collagen lattices were fixed in 4% paraformaldehyde in phosphate buffer after 3 days of culture. Gels were stained following the manufacturer's instructions. Briefly, fixed collagen gels were treated with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 5 minutes and washed twice with PBS. To reduce nonspecific staining, 1% bovine serum albumin in PBS was added to the gels for 30 minutes before staining with rhodamine phalloidin (Invitrogen, Carlsbad, CA) and SYTOX green nucleic acid staining (Invitrogen). SYTOX green nucleic acid stain (1 μmol/L) and 12.5 μL of rhodamine phalloidin were diluted in 500 μL PBS per well and incubated with the gels for 30 minutes at room temperature. Gels were washed twice with PBS, followed by confocal analysis under ×20 magnification using the Perkin Elmer Opera QEHS Automated Confocal Microscopy System (PerkinElmer, Waltham, MA) at the Vanderbilt Institute for Integrative Biosystems Research and Education Core Facility. Confocal images were analyzed using Columbus Software version 3.2 (PerkinElmer). To analyze the effects of Ankrd1 deletion on wound healing, we placed loxP sites 600 bp upstream of the Ankrd1 proximal promoter and in intron 2, thereby excising the transcription start site and exon 1 and 2 coding sequences (Figure 1A). This design ensured that Ankrd1 could, in future studies, be selectively deleted in any tissue that expressed cre-recombinase. To achieve global deletion of the protein, Sox2 promoter-driven, cre-recombinase–expressing, C57Bl/6J mice were bred to FLOX mice. Mixed background FLOX, WT/−, and KO adult mice were evaluated. All litters had a normal numbers of pups, and genotyping confirmed that each genotype was born at the expected allelic distribution with no obvious effects on development, growth, or reproduction as a result of either the insertion of the loxP sites or the deletion of Ankrd1, suggesting that Ankrd1 is expendable under normal physiological conditions in mixed background mice. However, backcrossing for eight generations into the pure inbred C57Bl/6J strain resulted in a growth-diminishing effect of Ankrd1 deletion that was observed in the neonate and carried through to the adult (Supplemental Figure S2). We confirmed the Ankrd1 deletion by mRNA analysis in skin. Because Ankrd1 expression is low in normal skin, but sharply and strongly induced after wounding, we generated full-thickness, dorsal excisional wounds in FLOX, WT/−, and KO adult mice. Tissue was harvested 18 hours after injury for mRNA and protein analysis. WT/− wound tissue had >50% reduction of Ankrd1 mRNA, and transcripts were essentially undetectable in KO tissue (Figure 2A). Similarly, the ANKRD1 protein content of FLOX wound tissue was strongly amplified over that in intact (0 hours) skin, significantly diminished in WT/− wound tissue, and undetectable in wounds of KO mice (Figure 2B). Wound contraction in all three genotypes was similar at 18 hours, although there was a mild impairment in wound contraction in mice with deletion of either one or both Ankrd1 alleles (Supplemental Figure S3). These data confirmed the complete deletion of Ankrd1 mRNA and protein expression in skin of KO mice and showed that there was no compensatory overexpression from the WT allele in the WT/− mouse. Because we saw no dramatic effects of Ankrd1 deletion on development or viability, effects on organ development were studied. Organ weights, expressed as a percentage of body weight, of the spleen, liver, kidney, brain, and skeletal muscle from FLOX, WT/−, and KO adult male and female mice showed no differences (Supplemental Figure S4). However, the weight of the cardiac ventricles relative to body weight was moderately increased in female mice with a single Ankrd1 allele, and relative ventricular weight in both sexes increased significantly with the deletion of both alleles (Supplemental Figure S4). To evaluate histopathological changes that might occur with aging after Ankrd1 deletion, young (5 to 6 months old) and aged (>1 year) adult male and female, FLOX and KO mice were examined in the Vanderbilt Comparative Pathology Core. Gross visual examination by a veterinary pathologist found no abnormalities in any tissues. Microscopic examination of hematoxylin and eosin–stained sections from all major organ systems also revealed no abnormalities. The three members of the MARP family are functionally redundant, at least in skeletal muscle.6Barash I.A. Bang M.L. Mathew L. Greaser M.L. Chen J. Lieber R.L. Structural and regulatory roles of muscle ankyrin repeat protein family in skeletal muscle.Am J Physiol Cell Physiol. 2007; 293: C218-C227Crossref PubMed Scopus (75) Google Scholar To investigate possible compensation for Ankrd1 deletion by one or both of the other MARPs, skin, heart, and skeletal muscle samples were collected from FLOX and KO mice for quantification of Ankrd2 and Ankrd23 mRNA. There was no difference in Ankrd23 mRNA levels in any of the three tissues. Ankrd2 mRNA, the primary MARP in skeletal muscle, decreased in both skeletal muscle tissue and skin from KO mice (Supplemental Figure S5). This response was consistent with evidence for an involvement of ANKRD1 in MyoD-dependent transcription of Ankrd2.16Belgrano A. Rakicevic L. Mittempergher L. Campanaro S. Martinelli V.C. Mouly V. Valle G. Kojic S. Fa" @default.
• W1979581862 created "2016-06-24" @default.
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• W1979581862 date "2015-01-01" @default.
• W1979581862 modified "2023-09-24" @default.
• W1979581862 title "Global Deletion of Ankrd1 Results in a Wound-Healing Phenotype Associated with Dermal Fibroblast Dysfunction" @default.
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