title: Tetramethylphenylenediamine protects the isolated heart against ischaemia-induced apoptosis and reperfusion-induced necrosis.
abstract: OBJECTIVE
  Cytochrome c when released from mitochondria into cytosol triggers assembly of the apoptosome resulting in caspase activation. Recent evidence suggests that reduced cytochrome c is unable to activate the caspase cascade. In this study, we investigated whether a chemical reductant of cytochrome c, N,N,N',N'-tetramethylphenylene-1,4-diamine (TMPD), which we have previously shown to block cytochrome c-induced caspase activation, could prevent ischaemia-induced apoptosis in the rat perfused heart.
  
  
  METHODS
  The Langendorff-perfused rat hearts were pretreated with TMPD and subjected to stop-flow ischaemia or ischaemia/reperfusion. The activation of caspases (measured as DEVD-p-nitroanilide-cleaving activity), nuclear apoptosis of cardiomyocytes (measured by dUTP nick end labelling assay), mitochondrial and cytosolic levels of cytochrome c (measured spectrophotometrically and by elisa), and reperfusion-induced necrosis (measured as the activity of creatine kinase released into perfusate) were assessed.
  
  
  RESULTS
  We found that perfusion of the hearts with TMPD strongly inhibited ischaemia- or ischaemia/reperfusion-induced activation of caspases and partially prevented nuclear apoptosis in cardiomyocytes. TMPD did not prevent ischaemia- or ischaemia/reperfusion-induced release of cytochrome c from mitochondria into cytosol. TMPD also inhibited ischaemia/reperfusion-induced necrosis.
  
  
  CONCLUSIONS
  These results suggest that TMPD or related molecules might be used to protect the heart against damage induced by ischaemia/reperfusion. The mechanism of this protective effect of TMPD probably involves electron reduction of cytochrome c (without decreasing its release) which then inhibits the activation of caspases.
authors.affiliation: Institute for Biomedical Research, Lithuanian University of Health Sciences, Kaunas, Lithuania.
authors.forename: Jurgita
authors.initials: J
authors.lastname: Barauskaite
authors.forename: Regina
authors.initials: R
authors.lastname: Grybauskiene
authors.forename: Ramune
authors.initials: R
authors.lastname: Morkuniene
authors.forename: Vilmante
authors.initials: V
authors.lastname: Borutaite
authors.forename: Guy C
authors.initials: GC
authors.lastname: Brown
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002316:Cardiotonic Agents / Q000494:pharmacology*; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D008297:Male; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D009336:Necrosis / Q000517:prevention & control; D051381:Rats; D017208:Rats, Wistar; D013772:Tetramethylphenylenediamine / Q000494:pharmacology*
chemical_list: D002316:Cardiotonic Agents; D045304:Cytochromes c; D020169:Caspases; D013772:Tetramethylphenylenediamine