title: Impact of resuscitation strategies on the acetylation status of cardiac histones in a swine model of hemorrhage.
abstract: BACKGROUND
  Chromatin remodeling through histone acetylation is a key control mechanism in gene transcription. We have shown previously that fluid resuscitation in rodents is coupled with highly structured post-translational modifications of cardiac histones. The current experiment was performed to validate this concept in a clinically relevant large animal model of hemorrhage and resuscitation, and to correlate the changes in histone acetylation with altered expression of immediate-early response genes.
  
  
  METHODS
  Yorkshire swine (n=49, 7/group, weight=40-58kg) were subjected to combined uncontrolled and controlled hemorrhage (40% of estimated blood volume) and randomly assigned to the following resuscitation groups: (1) 0.9% saline (NS), (2) racemic lactated Ringer's (dl-LR), (3) l-isomer lactated Ringer's (l-LR), (4) Ketone Ringer's (KR), (5) 6% hetastarch in saline (Hespan). KR contained an equimolar substitution of lactate with beta-hydroxybutyrate. No hemorrhage (NH) and no resuscitation (NR) groups were included as controls. Cardiac protein was used in Western blotting to analyze total protein acetylation and histone acetylation specifically. Lysine residue-specific acetylation of histone subunits H3 and H4 was further evaluated. In addition, Chromatin Immunoprecipitation (ChIP) technique was used to separate the DNA bound to acetylated histones (H3 and H4 subunits), followed by measurement of genes that are altered by hemorrhage/resuscitation, including immediate-early response genes (c-fos and c-myc), and heat shock protein (HSP) 70.
  
  
  RESULTS
  The type of fluid used for resuscitation influenced the patterns of cardiac histone acetylation. Resuscitation with dl-LR and KR induced hyperacetylation on H3K9. KR resuscitation was also associated with increased acetylation on H3K14 and H4K5, and hypoacetylation on H3K18. The expression of genes was also fluid specific, with the largest number of changes following KR resuscitation (increased c-fos and c-myc, HSP 70 linked with H3; and increased c-myc linked with H4). Among the histone subunits studied, altered H3 acetylations were associated with the majority of changes in immediate-early gene expression.
  
  
  CONCLUSIONS
  Acetylation status of cardiac histones, affected by hemorrhage, is further modulated by resuscitation producing a fluid-specific code that is preserved in different species. Resuscitation with KR causes histone acetylation at the largest number of lysine sites (predominately H3 subunit), and has the most pronounced impact on the transcriptional regulation of selected (immediate-early response) genes.
authors.affiliation: Trauma Research and Readiness Institute for Surgery, Uniformed Services University of the Health Sciences, Bethesda, MD, United States. hbalam@partners.org
authors.forename: Hasan B
authors.initials: HB
authors.lastname: Alam
authors.forename: Christian
authors.initials: C
authors.lastname: Shults
authors.forename: Naresh
authors.initials: N
authors.lastname: Ahuja
authors.forename: Eduardo C
authors.initials: EC
authors.lastname: Ayuste
authors.forename: Huazhen
authors.initials: H
authors.lastname: Chen
authors.forename: Elena
authors.initials: E
authors.lastname: Koustova
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Sailhamer
authors.forename: Yongqing
authors.initials: Y
authors.lastname: Li
authors.forename: Baoling
authors.initials: B
authors.lastname: Liu
authors.forename: Marc
authors.initials: M
authors.lastname: de Moya
authors.forename: George C
authors.initials: GC
authors.lastname: Velmahos
mesh_terms: D000107:Acetylation; D000818:Animals; D015153:Blotting, Western; D015816:Cell Adhesion Molecules, Neuronal / Q000235:genetics / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D058966:Contactins; D004195:Disease Models, Animal; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D012151:Resuscitation / Q000379:methods*; D012771:Shock, Hemorrhagic / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D013552:Swine; D014158:Transcription, Genetic; D029961:Zebrafish Proteins