title: Assessing experimental models in myocardial injury: lack of activation of the proteases TACE and calpain in brief ischaemia and reperfusion.
abstract: BACKGROUND
  Calpain inhibitors are reportedly cardioprotective. Furthermore, oxidative stress may acutely activate the sheddase tumour necrosis factor (TNF)-alpha-cleaving enzyme (TACE). The aim of this study was to examine whether myocardial reperfusion leads to activation of the proteases mu- and m-calpain, and to evaluate which cardiac cells act as a source of TNF-alpha.
  
  
  METHODS
  Isolated hearts (guinea pig) were subjected to global ischaemia (15 min) and reperfused. Calpain activity was determined by zymography. Calpastatin (inhibitor) and troponin I (substrate) were quantified by western blotting. Immunohistology of hearts and a human mast cell line (HMC-1) was used to localise expression of TNF-alpha and TACE. Shedding of TNF-alpha was assessed in Mono Mach, Jurkat-T, HMC-1 and peripheral blood leucocytes with and without oxidative stress.
  
  
  RESULTS
  Neither of the ubiquitous calpains (mu- and m-calpain) was significantly activated by brief ischaemia/reperfusion, nor were calpastatin and troponin degraded more than in extracts of control hearts. Cardiac TNF-alpha immunoreactivity was localised to mast cells. None of the tested cell lines shed TNF-alpha in response to non-toxic amounts of oxidants. However, HMC-1 cells showed poor expression of proTNF-alpha, while TACE was abundant.
  
  
  CONCLUSIONS
  Although the severity of ischaemia in the current model may have been insufficient, activation of calpain by ischaemia/reperfusion cannot be demonstrated simply in the Langendorff-mode perfused isolated heart. Mast cells are the prime source of myocardial TNF-alpha. A suitable whole-cell model remains to be found to demonstrate acute oxidative activation of TACE.
authors.affiliation: Department of Physiology, University of Munich, Munich, Germany.
authors.forename: Bernhard F
authors.initials: BF
authors.lastname: Becker
authors.forename: Stefanie
authors.initials: S
authors.lastname: Gilles
authors.forename: Vera
authors.initials: V
authors.lastname: Gonscherowski
authors.forename: Dusica
authors.initials: D
authors.lastname: Gabrijelcic-Geiger
authors.forename: Christian P
authors.initials: CP
authors.lastname: Sommerhoff
authors.forename: Ulrich
authors.initials: U
authors.lastname: Welsch
authors.forename: Stefan
authors.initials: S
authors.lastname: Zahler