title: Ischemic preconditioning attenuates calpain-mediated degradation of structural proteins through a protein kinase A-dependent mechanism.
abstract: OBJECTIVE
  It has been shown that sarcolemmal rupture can occur during reenergization in cardiomyocytes in which previous ischemia has induced sarcolemmal fragility by calpain-dependent hydrolysis of structural proteins. We tested the hypothesis that attenuated calpain activation contributes to the protection against reperfusion-induced cell death afforded by ischemic preconditioning (IPC), and investigated the involvement of protein kinase A (PKA) in this effect.
  
  
  METHODS
  Calpain activity and degradation of different structural proteins were studied along with the extent of necrosis in isolated rat hearts submitted to 60 min of ischemia and 30 min of reperfusion with or without previous IPC (two cycles of 5 min ischemia-5 min reperfusion), and the ability of different treatments to mimic or blunt the effects of IPC were analyzed.
  
  
  RESULTS
  IPC accelerated ATP depletion and rigor onset during ischemia but reduced LDH release during reperfusion by 69% (P<0.001). At the end off reperfusion, calpain activity was reduced by 66% (P<0.001) in IPC, and calpain-dependent degradation of sarcolemmal proteins was attenuated. Addition of the calpain inhibitor MDL-28170 mimicked the effects of IPC on protein degradation and reduced LDH release by 48% (P<0.001). The effects of IPC on calpain, alpha-fodrin, and LDH release were blunted by the application of the PKA inhibitor H89 or alprenolol during IPC, while transient stimulation of PKA with CPT-cAMP or isoproterenol before ischemia attenuated calpain activation, alpha-fodrin degradation, and markedly reduced LDH release (P<0.001). In hearts exposed to Na(+)-free perfusion, IPC attenuated calpain activation by 67% (P<0.001) and reduced by 56% (P<0.001) LDH release associated to massive edema occurring during Na(+) readmission without modifying its magnitude.
  
  
  CONCLUSIONS
  These results are consistent with PKA-dependent attenuation of calpain-mediated degradation of structural proteins being an end-effector mechanism of the protection afforded by IPC.
authors.affiliation: Servicio de Cardiologia, Hospital Universitari Vall d'Hebron, Passeig Vall d'Hebron 119-129, Barcelona 08035, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Inserte
authors.forename: David
authors.initials: D
authors.lastname: Garcia-Dorado
authors.forename: Marisol
authors.initials: M
authors.lastname: Ruiz-Meana
authors.forename: Luis
authors.initials: L
authors.lastname: Agulló
authors.forename: Pilar
authors.initials: P
authors.lastname: Pina
authors.forename: Jordi
authors.initials: J
authors.lastname: Soler-Soler
mesh_terms: D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000526:Alprenolol / Q000494:pharmacology; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004151:Dipeptides / Q000494:pharmacology; D004789:Enzyme Activation; D019157:Ischemic Preconditioning, Myocardial; D007546:Isoquinolines / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D009270:Naloxone / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D009336:Necrosis; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley; D012964:Sodium / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D013049:Spectrin
chemical_list: D000319:Adrenergic beta-Antagonists; D000526:Alprenolol; D002154:Calpain; D002352:Carrier Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D004151:Dipeptides; D007546:Isoquinolines; D007770:L-Lactate Dehydrogenase; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009270:Naloxone; D009292:Narcotic Antagonists; D012964:Sodium; D013449:Sulfonamides; D013049:Spectrin; C109742:SLMAP protein, human; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C058076:calpain inhibitor III