title: BNIP3 promotes calcium and calpain-dependent cell death.
abstract: OBJECTIVE
  Loss of cardiac muscle by programmed cell death contributes to the progression of ischemic heart disease. Hypoxia, metabolite waste buildup and energy depletion are components of ischemia which may initiate caspase dependent and independent cell death pathways. Previous work from our laboratory has shown that combined hypoxia with acidosis, a hallmark of ischemia promotes cardiac myocyte injury with increasing severity as the pH declines. Hypoxia-acidosis was demonstrated to activate the pro-apoptotic Bcl-2 protein BNIP3 which initiated opening of the mitochondrial permeability transition pore and cell death in the absence of caspase activation. Because calpains are known to contribute to ischemic myocardial damage in some models, we hypothesized that they are intermediates in the BNIP3-mediated death caused by hypoxia-acidosis.
  
  
  METHODS
  Neonatal rat cardiac myocytes were subjected to hypoxia with and without acidosis and the contribution of calpains to hypoxia-acidosis cell death determined.
  
  
  RESULTS
  Here we report that the death pathway activated by hypoxia-acidosis is driven by a combination of calcium-activated calpains and pro-death factors (DNases) secreted by the mitochondria. Cytochrome c accumulated in the cytoplasm during hypoxia-acidosis but caspase activity was repressed through a calpain-dependent process that prevents the cleavage of procaspase 3. Calpain inhibitors provide vigorous protection against hypoxia-acidosis-induced programmed death. Knockdown of BNIP3 with siRNA prevented calpain activation confirming a central role of BNIP3 in this pathway.
  
  
  CONCLUSIONS
  The results implicate BNIP3 and calpain as dependent components of cardiac myocyte death caused by hypoxia-acidosis.
authors.affiliation: Department of Molecular and Cellular Pharmacology, Vascular Biology Institute, Miller School of Medicine, University of Miami, Miami, FL 33136, United States.
authors.forename: Regina M
authors.initials: RM
authors.lastname: Graham
authors.affiliation: Department of Molecular and Cellular Pharmacology, Vascular Biology Institute, Miller School of Medicine, University of Miami, Miami, FL 33136, United States.
authors.forename: John W
authors.initials: JW
authors.lastname: Thompson
authors.affiliation: Department of Molecular and Cellular Pharmacology, Vascular Biology Institute, Miller School of Medicine, University of Miami, Miami, FL 33136, United States. Electronic address: kwebster@med.miami.edu.
authors.forename: Keith A
authors.initials: KA
authors.lastname: Webster
mesh_terms: D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D017202:Myocardial Ischemia / Q000378:metabolism* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction
chemical_list: C505650:BNIP3 protein, rat; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D002154:Calpain; C505410:Casp3 protein, rat; D053148:Caspase 3; D002118:Calcium
keywords: Acidosis; Apoptosis; BNIP3; Calcium; Calpain; Cardiac myocytes; Caspases; Heart; Hypoxia; Ischemia; Mitochondrial permeability transition pore