title: Decreased Desmin expression in the developing diaphragm of the nitrofen-induced congenital diaphragmatic hernia rat model.
abstract: OBJECTIVE
  Congenital diaphragmatic hernia (CDH) is presumed to originate from defects in the primordial diaphragmatic mesenchyme, mainly comprising of muscle connective tissue (MCT). Thus, normal diaphragmatic morphogenesis depends on the structural integrity of the underlying MCT. Developmental mutations that inhibit normal formation of diaphragmatic MCT have been shown to result in CDH. Desmin (DES) is a major filament protein in the MCT, which is essential for the tensile strength of the developing diaphragm muscle. DES-/-knockout mice exhibit significant reductions in stiffness and elasticity of the developing diaphragmatic muscle tissue. Furthermore, sequence changes in the DES gene have recently been identified in human cases of CDH, suggesting that alterations in DES expression may lead to diaphragmatic defects. This study was designed to investigate the hypothesis that diaphragmatic DES expression is decreased in fetal rats with nitrofen-induced CDH.
  
  
  METHODS
  Time-mated Sprague-Dawley rats were exposed to either nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on selected time-points D13, D15 and D18, and dissected diaphragms (n = 72) were divided into control and nitrofen-exposed specimens (n = 12 per time-point and experimental group, respectively). Laser-capture microdissection was used to obtain diaphragmatic tissue elements. Diaphragmatic gene expression of DES was analyzed by quantitative real-time polymerase chain reaction. Immunofluorescence double staining for DES was combined with the mesenchymal marker GATA4 to evaluate protein expression and localization in developing fetal diaphragms.
  
  
  RESULTS
  Relative mRNA expression levels of DES were significantly decreased in pleuroperitoneal folds on D13 (1.49 ± 1.79 vs. 3.47 ± 2.32; p < 0.05), developing diaphragms on D15 (1.49 ± 1.41 vs. 3.94 ± 3.06; p < 0.05) and fully muscularized diaphragms on D18 (2.45 ± 1.47 vs. 5.12 ± 3.37; p < 0.05) of nitrofen-exposed fetuses compared to controls. Confocal laser scanning microscopy demonstrated markedly diminished immunofluorescence of DES mainly in diaphragmatic MCT, which was associated with a reduction of proliferating mesenchymal cells in nitrofen-exposed fetuses on D13, D15 and D18 compared to controls.
  
  
  CONCLUSIONS
  Decreased expression of DES in the fetal diaphragm may disturb the basic integrity of myofibrils and the cytoskeletal network during myogenesis, causing malformed MCT and leading to diaphragmatic defects in the nitrofen-induced CDH model.
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Takahashi
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: Florian
authors.initials: F
authors.lastname: Friedmacher
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
authors.forename: Julia
authors.initials: J
authors.lastname: Zimmer
authors.affiliation: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland. prem.puri@ncrc.ie.|Conway Institute of Biomolecular and Biomedical Research, School of Medicine and Medical Science, University College Dublin, Dublin, Ireland. prem.puri@ncrc.ie.
authors.forename: Prem
authors.initials: P
authors.lastname: Puri
mesh_terms: D000818:Animals; D003893:Desmin / Q000378:metabolism*; D003964:Diaphragm / Q000196:embryology*; D004195:Disease Models, Animal; D005260:Female; D047109:Fetal Development; D005455:Fluorescent Antibody Technique; D065630:Hernias, Diaphragmatic, Congenital / Q000196:embryology* / Q000378:metabolism*; D010647:Phenyl Ethers; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D003893:Desmin; D010647:Phenyl Ethers; C007350:nitrofen
keywords: Congenital diaphragmatic hernia; Desmin; Diaphragm; Muscle connective tissue; Nitrofen