title: Mitochondrial m-calpain opens the mitochondrial permeability transition pore in ischemia-reperfusion.
abstract: OBJECTIVE
  Opening of the mitochondrial permeability transition pore (mPTP) is involved in ischemia-reperfusion injury. Isoforms of Ca(2+)-activated cysteine proteases, calpains, are implicated in the development of myocardial infarction in ischemia-reperfusion. Growing evidence has revealed the presence of calpains in the mitochondria. We aimed to characterize mitochondrial calpains in the rat heart and to investigate the roles of calpains in mPTP opening after ischemia-reperfusion.
  
  
  RESULTS
  Western blotting analysis showed the expression of μ-calpain, m-calpain and calpain 10 in mitochondria isolated from male Sprague-Dawley rats, but casein zymography detected only m-calpain activity. Subcellular fractionation of mitochondria demonstrated the distribution of m-calpain to the matrix fraction. Addition of >500μM of Ca(2+) to isolated mitochondria induced mitochondrial swelling, reflecting mPTP opening, and calpain activation. Ca(2+)-induced mitochondrial swelling was inhibited partially by the calpain inhibitor calpeptin. These results support a partial contribution of calpain in the opening of the mPTP. The addition of Ca(2+) to the mitochondria induced inactivation of complex I of the electron transport chain, and cleavage of the ND6 complex I subunit, which were inhibited by calpeptin. Mitochondria isolated from rat hearts that underwent 30min of coronary occlusion followed by 30min of reperfusion showed activation of mitochondrial calpains, ND6 cleavage, complex I inactivation, and mPTP opening, which were inhibited by pretreatment with calpain inhibitor 1.
  
  
  CONCLUSIONS
  We demonstrated for the first time the presence of mitochondrial matrix m-calpain, and its contribution to complex I inactivation and mPTP opening after postischemic reperfusion in the rat heart.
authors.affiliation: Department of Forensic Medicine, Graduate School of Medicine, the University of Tokyo, Japan. Electronic address: kaori@koto.kpu-m.ac.jp.
authors.forename: Kaori
authors.initials: K
authors.lastname: Shintani-Ishida
authors.affiliation: Department of Forensic Medicine, Graduate School of Medicine, the University of Tokyo, Japan.
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Yoshida
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D004151:Dipeptides / Q000494:pharmacology; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D008929:Mitochondria, Heart / Q000201:enzymology*; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism*; D000083162:Mitochondrial Permeability Transition Pore; D015428:Myocardial Reperfusion Injury / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D055815:Young Adult
chemical_list: D004151:Dipeptides; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; C071482:calpeptin; D002154:Calpain; C449000:m-calpain; D002118:Calcium
keywords: Calcium; Calpain; Ischemia–reperfusion injury; Mitochondria; Mitochondrial permeability transition pore