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University of South Alabama Mobile, Alabama USA

University of South Alabama. Ischemic preconditioning: and its elusive mechanism James Downey, PhD Professor of Physiology. University of South Alabama Mobile, Alabama USA. In 1972 Eugene Braunwald noted

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University of South Alabama Mobile, Alabama USA

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  1. University of South Alabama Ischemic preconditioning: and its elusive mechanism James Downey, PhDProfessor of Physiology University of South AlabamaMobile, Alabama USA

  2. In 1972 Eugene Braunwald noted that morbidity and mortality following acute myocardial infarction was in direct proportion to the amount of myocardium infarcted. • He proposed that interventions be found to make the ischemic heart resistant to infarction. • 35 years later all we have is reperfusion therapy

  3. Why is that?

  4. All were ineffective in clinical trials All werecontroversial in preclinical studies • During the 80’s and 90’s many drugs were tested based on the popular theories of what killed ischemic myocardium • Free radical scavengers • Beta blockers • Calcium antagonists • Anti-inflammatory agents (anti-complement, anti-leukocyte, or low-dose adenosine) Was infarct size reduction even possible?

  5. (ischemia)

  6. Rosetta stone Created in 196 BC, It was discovered in 1799 at Rosetta. Using it hieroglyph writing was finally deciphered in 1822 by Thomas Young and Jean-François Champollion. It took 23 years to figure it out!

  7. Shows that infarct size can be modified Preconditioned ischemic preconditioning 40 min ischemia 50 40 30 % Ischemic Zone Infarcted 20 10 0 Control Murry et al, Circulation 1986;74:1124-1136

  8. Like the Rosetta stone it has taken 23 years to decipher preconditioning’s mechanism. How was that done?

  9. And developed them! 1957 Triumph Thunderbird Start with some rough ideas…..

  10. In 1991 we discovered that preconditioning is triggered by adenosine receptors and that PKC was required for the protection. That revealed that a signal transduction pathway was responsible for the protection. Here is the pathway as we understand it today.

  11. PLC/PLD Trigger pathway is activated before ischemia

  12. During the preconditioning ischemia three receptors are occupied

  13. PLC/PLD A1/ A3 adenosine receptors activate PKC directly through PLC

  14. BK and opioid activate PKC through a complex pathway involving the mitochondria

  15. Nitric oxide is produced PKG is activated PKG opens a mitochondrial potassium channel

  16. mitochondria swelling reveals K+ channel Opening. 1- 30 mM 300nM 100nM [cGMP] 0.20 ATP = 200 mM Purified PKG 0.19 cGMP + ATP Volume 0.18 0 520 λ 0.17 1 2 3 0 Time (min) PMT Isolated Mitochondria Garlid et al. Circ Res 97:329-36. 2005

  17. PKG ? PKC not sarc. KATP PKG mKATP KATP Garlid et al. Circ Res 97:329-36. 2005

  18. Even a weird idea sometimes works out ok. 1950 British Sunbeam S7

  19. We proposed PKC is activated by mitochondrial ROS

  20. Adult Rabbit Ventricular Myocytes We can observe ROS production Control ROS detected by Reduced MitoTracker, a ROS-sensitive fluorochrome Diazoxide

  21. KATP channel opener Potassium entry KATP channels Mitochondria BKN + 5-HD Valinomycin25nM BKN + Myxothiazol DIAZ 50μM BKN500nM Bradykinin Causes ROS Generation Adult Rabbit Cardiomyocytes 200 150 ROS Production (% of Control) 100 50 0 Control Oldenburg et al. Cardiovasc Res. 55:544-52, 2002

  22. Electron transport blocker myxothiazol reduces preconditioning’s protection in isolated hearts Yue et al. Am J Physiol Heart Circ Physiol 281: H590-H595, 2001;

  23. ROS scavenger N-(2-mercaptopropionyl)-glycine Group 4 Group 5 Group 6 Ischemia Reperfusion Group 1 IPC Group 2 Group 3 150 -20 -15 -10 -5 0 5 30 Time (min) MPG infusion Myocardial MPG Conc. Ischemia Hypoxic buffer

  24. Oxygen is required for redox signaling * Reperf. with hypox. Buffer Redox signaling occurs during reperfusion 100 80 Only MPG during early reperfusion blocks protection 60 Infarction (% of risk zone) # 40 # 20 * * * # # # 0 Control IPC MPG in Reperf. phase MPG in ischemia phase MPG in index ischemia Dost et al. Basic Res Cardiol 103:378–384 (2008)

  25. ROS signaling is also required for IPC’s protection at reperfusion. Giving antioxidants to AMI patients could increases infarction in those with inadvertent preconditioning. 60 50 300 uM MPG 40 Infarction (% of risk zone) 30 20 * 10 0 Control IPC IPC + MPG Liu et al. Basic Res Cardiol. 103:54, 2008

  26. * * IPC +Chel PMA PMA+MPG MPG We can determine the order of events in ROS signaling 60 50 50 pM PMA 40 Infarction (% of risk zone) 30 ROS must be upstream of PKC 20 * 10 0 Control IPC Liu et al. Basic Res Cardiol. 103:54, 2008

  27. The free radical mechanism explains why receptor population during ischemia without brief reperfusion does not protect Receptors mKATP ROS protection O2 during reperfusion fuels radical production protection Receptors populatedmKATP open but… no ROS, no protection Dost et al. Basic Res Cardiol 103:378–384 (2008)

  28. A Bovine Serum Albumen motorcycle???

  29. PKC leads to A2B receptor activity

  30. Adenosine A2B receptors are important at reperfusion in the IPC heart Occlusion PC Reperfusion 60 25’ocl - 15’rep Adenosine receptor 50 A1 involved A2B involved 40 Infarction (% of risk zone) 30 Not A2A 20 10 0 IPC +SPT IPC+MRS IPC + CSC Control IPC+DPCPX IPC 20 nM 1 uM 20 nM 100 nM Solenkova et al Am J Physiol 290:H441-H449, 2006

  31. Activation of PKC at reperfusion protects like preconditioning PKC Adenosine Ischemia * PMA PMA PMA0.05nM + + Chel MRS1754 PKC must be upstream of A2B 60 PKCActivator 40 % of ischemic zone Infarcted Really due to PKC 20 0 Control Philipp et al. Cardiovasc Res 70:308-14, 2006

  32. Protection from an A2B agonist is not PKC-dependent - again PKC is upstream of the A2B 60 Ischemia Chel NECA 40 Infarction (% of risk zone) * * 20 PKC must be upstream of A2B 0 IPC Control NECA 2ug/kg NECA + Chel +Chel Philipp et al. Cardiovasc Res 70:308-14, 2006

  33. A2B receptors activate survival kinases PI3 kinase and ERK

  34. Adenosine receptor blockade at reperfusion blocks PI3 kinase activation 1.4 30 min ischemia BL 10’ 1.2 10’ BL control 1.0 P-AKT IPC 0.8 (PI3-K Reporter) Density AU IPC+SPT 0.6 0.4 0.2 0.0 A similar response is seen for ERK control IPC+SPT IPC 10’ reperfusion Isolated Rabbit Heart Solenkova et al Am J Physiol 290:H441-H449, 2006

  35. PKC increases the affinity of the A2B receptors so endogenous adenosine can occupy them.

  36. 293 kidney cells expressing A2bAR PKC increases the sensitivity of the low-affinity A2B Akt ERK 0 0.3 1.0 3.0 0.3 1.0 3.0 NECA (uM) PMA(PKC on) Chelerythrine (PKC off) The A2B will not be populated unless PKC has been activated Kuno et al. J Mol Cell Cardiol. 43:262-71, 2007

  37. So what is the difference between a preconditioned and a naïve heart? 1966 BMW R27 (250cc)

  38. The A2b Adenosine receptor in its high affinity (phosphorylated) state. That way its own adenosine can protect at reperfusion.

  39. Survival kinases protect by inhibiting permeability transition pores

  40. 1967 Triumph T90 (350cc) UK domestic model Yes, good science but is it useful?

  41. How can we activate the mediator pathway at reperfusion? Preconditioning has not been clinically useful since acute myocardial infarction patients only present after ischemia has begun. ischemia precondition reperfuse See patient

  42. Can we protect with Adenosine at Reperfusion?

  43. Adenosine Reperfusion Ischemia 0' 30' 90' 210' hypotension -11 mmHg -17 mmHg -23 mmHg Intravenous Adenosine at Reperfusion in situ Does Not Protect the Rabbit Heart 70 60 50 40 % infarction of the risk zone 30 20 Any higher dose killed the rabbit! 10 0 Control Adenosine Adenosine Adenosine (0.15 mg/kg/min) (0.3 mg/kg/min) (0.4 mg/kg/min)

  44. BAY 60-6583, a non-purine, highly A2B-selective adenosine receptor agonist No A3 reactivity detected up to 10 uM 600 A2a receptor 500 A2b receptor A1 receptor 400 RLUx100 300 200 100 0 -13 -12 -11 -10 -9 -8 -7 -6 -5 EC50 10 nM BAY A2b (log M) CHO cells expressing human receptors

  45. Like preconditioning BAY 60-6583 treatment activates survival kinases at reperfusion 2.5 Akt ‡ ‡ ERK1 † ERK2 2.0 biopsy 1.5 Phosphorylation (X Baseline) 1.0 0.5 0.0 10 min rep BAY 60 + MRS1754 BAY 60

  46. There are many drugs that stimulate the mediator pathway but most cause unwanted hemodynamic effects. • Bradykinin • AMP579 and NECA (A1,A2A, A2B) • Angiotensin • Acetylcholine A2B receptors are found only on a few blood vessels so A2B-selective agonists have minimal hemodynamic effect

  47. A highly selective A2B agonist protects when given to open-chest rabbits at reperfusion with no hemodynamic effect in situ rabbit hearts 60 100μg/kg 40 A2B agonist Infarct size (% of risk zone) 20 * * 0 Postcondition BAY60-6583 ~250nM Control Kuno et al. J Mol Cell Cardiol 43:262-271.

  48. What are some other ways to activate the pathway?

  49. 8-(4-chlorophenylthio)-cGMP Cell permeantPKG activator A direct PKG activator given at reperfusion protects ischemia CPT 60 min

  50. Chelerythrine CPT 5HD MPG

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