title: Differentially regulated functional gene clusters identified during ischemia and reperfusion in isolated cardiac myocytes using coverslip hypoxia.
abstract: BACKGROUND
  Coverslip hypoxia (CSH) is a recently described method for producing rapid and severe ischemia derived from the metabolic activity of synchronously contracting isolated neonatal rat ventricular myocytes (NRVMs). While the effect of acute ischemia produced by CSH is documented, the contribution of reperfusion to cell viability has not been fully studied.
  
  
  METHODS
  We therefore used fluorescence microscopy and expression profiling by microarray to determine the morphological and genetic effects in NRVMs of both the ischemic and reperfusion events of CSH.
  
  
  RESULTS
  Fluorescence microscopy studies in coverslipped NRVMs showed cell death at 1 h as previously reported. Matched samples coverslipped for up to 2 h and then reperfused 18 h showed myocyte recovery prior to but not beyond 1 h upon post-staining, suggesting a limited window of recovery. Expression profiling of more than 30,000 genes using total RNA collected from NRVMs subjected to varying periods of ischemia and reperfusion revealed 103 genes regulated at least 2-fold at p<10(-7). These genes fall into discrete functional groups including apoptosis, metabolism, and hypoxia/acidosis. The regulation of a subset of genes from these groups was confirmed by RT-PCR. Interestingly, the hypoxia/acidosis gene BNip3 (a Bcl-2 family member implicated in hypoxia/acidosis-associated cell death) was upregulated early during ischemia and persisted throughout reperfusion. In addition, other hypoxia/acidosis genes such as heme oxygenase 1, pyruvate dehydrogenase kinase 1, prolyl hydroxylases, and hypoxia-inducible protein 2 were upregulated.
  
  
  CONCLUSIONS
  These data suggest that the ischemic and reperfusion events created by CSH induce gene regulation within distinct functional groups related to in vivo ischemia.
authors.affiliation: Inflammation Research, Amgen Inc., Seattle, WA 98119, USA. kelly.pitts@gilead.com
authors.forename: Kelly R
authors.initials: KR
authors.lastname: Pitts
authors.forename: Jonathan M J
authors.initials: JM
authors.lastname: Derry
authors.forename: Keith
authors.initials: K
authors.lastname: Kerkof
authors.forename: William A
authors.initials: WA
authors.lastname: Lawrence
authors.forename: Christopher F
authors.initials: CF
authors.lastname: Toombs
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D016923:Cell Death; D015687:Cell Hypoxia / Q000502:physiology; D002470:Cell Survival; D002478:Cells, Cultured; D016000:Cluster Analysis; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006352:Heart Ventricles / Q000166:cytology; D008856:Microscopy, Fluorescence; D017202:Myocardial Ischemia; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism*; D040081:Protein Array Analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury