title: The significance of the washout period in preconditioning.
abstract: OBJECTIVE
  Exposure of the heart to 5 min global ischaemia (I) followed by 5 min reperfusion (R) (ischaemic preconditioning, IPC) or transient Beta 2-adrenergic receptor (B2-AR) stimulation with formoterol (B2PC), followed by 5 min washout before index ischaemia, elicits cardioprotection against subsequent sustained ischaemia. As the washout period during preconditioning is essential for subsequent cardioprotection, the aim of this study was to investigate the involvement of protein kinase A (PKA), reactive oxygen species (ROS), extracellular signal-regulated kinase (ERK), PKB/Akt, p38 MAPK and c-jun N-terminal kinase (JNK) during this period.
  
  
  METHODS
  Isolated perfused rat hearts were exposed to IPC (1x5min I / 5min R) or B2PC (1x5min Formoterol / 5min R) followed by 35 min regional ischaemia and reperfusion. Inhibitors for PKA (Rp-8CPT-cAMP)(16μM), ROS (NAC)(300μM), PKB (A-6730)(2.5μM), ERKp44/p42 (PD98,059)(10μM), p38MAPK (SB239063)(1μM) or JNK (SP600125)(10μM) were administered for 5 minutes before 5 minutes global ischaemia / 5 min reperfusion (IPC) or for 5 minutes before and during administration of formoterol (B2PC) prior to regional ischaemia, reperfusion and infarct size (IS) determination. Hearts exposed to B2PC or IPC were freeze-clamped during the washout period for Western blots analysis of PKB, ERKp44/p42, p38MAPK and JNK.
  
  
  RESULTS
  The PKA blocker abolished both B2PC and IPC, while NAC significantly increased IS of IPC but not of B2PC. Western blot analysis showed that ERKp44/p42 and PKB activation during washout after B2PC compared to IPC was significantly increased. IPC compared to B2PC showed significant p38MAPK and JNKp54/p46 activation. PKB and ERK inhibition or p38MAPK and JNK inhibition during the washout period of B2PC and IPC respectively, significantly increased IS.
  
  
  CONCLUSIONS
  PKA activation before regional ischaemia is a prerequisite for cardioprotection in both B2PC and IPC. However, ROS was crucial only in IPC. Kinase activation during the washout phase of IPC and B2PC, albeit different, affords the same cardioprotective response.
authors.affiliation: Biomedical Research and Innovation Platform, South African Medical Research Council, Parow Valley, Cape Town, South Africa.|Faculty of Medicine and Health Sciences, Division of Medical Physiology, University of Stellenbosch, Tygerberg, Cape Town, South Africa.
authors.forename: Ruduwaan
authors.identifier: http://orcid.org/0000-0001-9396-4025
authors.initials: R
authors.lastname: Salie
authors.affiliation: Faculty of Medicine and Health Sciences, Division of Medical Physiology, University of Stellenbosch, Tygerberg, Cape Town, South Africa.
authors.forename: Amanda
authors.initials: A
authors.lastname: Lochner
authors.affiliation: Faculty of Medicine and Health Sciences, Division of Medical Physiology, University of Stellenbosch, Tygerberg, Cape Town, South Africa.
authors.forename: Dirk J
authors.initials: DJ
authors.lastname: Loubser
mesh_terms: D058666:Adrenergic beta-2 Receptor Agonists / Q000494:pharmacology*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004789:Enzyme Activation; D000068759:Formoterol Fumarate / Q000494:pharmacology*; D019157:Ischemic Preconditioning, Myocardial / Q000379:methods*; D000067268:Isolated Heart Preparation; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009203:Myocardial Infarction / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control*; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology / Q000517:prevention & control*; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors
chemical_list: D058666:Adrenergic beta-2 Receptor Agonists; D000975:Antioxidants; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D051057:Proto-Oncogene Proteins c-akt; D017868:Cyclic AMP-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D000068759:Formoterol Fumarate
keywords: Cardiac function; Infarct size; Myocardial ischemia/reperfusion injury; Preconditioning