1. Remote Ischemic Conditioning: An Update on Mechanisms Remote Ischemic Conditioning: An Update on Mechanisms Karin Przyklenk PhD Director, Cardiovascular Research Institute Professor, Departments of Physiology & Emergency Medicine Wayne State University School of Medicine Detroit MI ICT Focus Group Meeting: 17 th November, 2013

2. classic cardioprotective paradigms: ischemic preconditioning, postconditioning protective stimuli (brief antecedent ischemia; stuttered reflow) applied to the heart remote ischemic conditioning (RIC): protective stimulus applied at a remote site first evidence: remote preconditioning (Przyklenk et al, 1993) Myocardial ‘Conditioning’ coronary occlusionreperfusion remote ischemia

3. Przyklenk et al, Circulation 1993;87:893-99. Remote Ischemic Conditioning: First Evidence 1 h LAD Occl 4.5 h Reflow Control Cx Occl 1 h LAD Occl 4.5 h Reflow Circumflex (Cx) PC infarct size (% of risk region) Significant reduction of infarct size with ‘intra-cardiac’ remote ischemic preconditioning

4. Developing the Concept... intra-cardiac RIC inter-organ RIC

5. ‘Inter-organ’ RIC Dickson et al, Am J Physiol 1999;277:H2451-57. 40’ ischemia 1 h Reflow Donor: Control 40’ ischemia Donor: PC infarct size 40’ ischemia Acceptor: Control 40’ ischemia Acceptor: PC Effluent **p<0.01 vs Donor-Control model: isolated buffer- perfused rabbit PC stimulus: transfer of coronary effluent endpoint: infarct size

6. Kharbanda et al, Circulation 1997;106:2881-83. model: anesthetized pig PC stimulus: skeletal muscle ischemia endpoint: infarct size 40’ LAD Occl 2 h Reflow Control 40’ LAD Occl Hindlimb ischemia infarct size (% of risk region) ‘Inter-Organ’ RIC

7. intra-cardiac RIC inter-organ RIC Phase II clinical trials Phase III clinical trials Developing the Concept...

8. characterize (physiology) understand (mechanisms) apply Why is this important?

9. ~25 published Phase II clinical trials cardiac surgery; elective PCI; primary PCI in patients with STEMI stimulus: multiple (3-4) 5 min episodes of limb ischemia primary endpoint: infarct size or its surrogate outcomes have been mixed...... possibly a consequence of gaps in our understanding of the mechanisms of RPC Ovize, Thibault & Przyklenk, Circulation Research 2013;113:439-50.

10. Mechanisms For pre-, postconditioning: signaling receptor stimulation trigger effector CARDIOPROTECTION adenosine; bradykinin, opioids G-protein coupled receptors RISK, SAFE pathways (ERK, PI3 kinase/Akt, JAK, STAT3) mitochondria (mPTP)

11. Mechanisms For pre-, postconditioning: signaling receptor stimulation trigger effector CARDIOPROTECTION

12. Mechanisms For remote conditioning: signaling receptor stimulation trigger effector CARDIOPROTECTION COMMUNICATION For pre-, postconditioning: signaling receptor stimulation trigger effector CARDIOPROTECTION

13. Communication signaling receptor stimulation trigger effector CARDIOPROTECTION COMMUNICATION In 1993: the infarct-sparing effect of remote conditioning ‘... may be mediated by factor(s) activated, produced, or transported throughout the heart during brief ischemia-reperfusion.’ In 2013...

14. Communication: circulating humoral factor Candidates: adenosine, bradykinin, opiods by HPLC: ‘small (<15 kDa) hydrophobic molecule’ from proteomic screens: Apo-A1 Hilbert et al, PLoS 2013;8:e77211 Hepponstall et al, PLoS 2012;7:e48284 ‘educated guesses’: SDF1-α/CXCR4; microRNAs Davidson et al, Basic Res Cardiol 2013;108:377 Duan et al, Cardiology 2012;122:36-43

15. Davidson et al, Basic Res Cardiol 2013;108:377 SDF1-α/CXCR4 Signaling in vivo rat model plasma SDF1-α concentration increased following RIC CXCR4 inhibitor (AMD3100) attenuated the infarct-sparing effect of RIC

16. Candidates: adenosine, bradykinin, opiods by HPLC: ‘small (<15 kDa) hydrophobic molecule’ from proteomic screens: Apo-A1 ‘educated guesses’: SDF1-α/CXCR4; microRNAs Source: myocytes, endothelial cells? Transported to heart via: blood/perfusate exosomes, microparticles/microvesicles Communication: circulating humoral factor

17. : neuronal pathways Source: activation of sensory neurons stimulation of afferent neuronal pathways CNS Transported to heart via: activation of efferent neuronal pathways ‘Remote Preconditioning Reflex’ Mastitskaya et al, Cardiovasc Res 2012;95:487-94. Gourine & Gourine, Physiology 2013: in press. Communication: neuronal pathways

18. Source: activation of sensory neurons stimulation of afferent neuronal pathways CNS Transported to heart via: activation of efferent neuronal pathways Model-dependent? In some models: integrated (neurohumoral communication)? redundant? Communication: neuronal pathways

19. Summary signaling receptor stimulation trigger effector CARDIOPROTECTION COMMUNICATION In 1993: the infarct-sparing effect of remote conditioning ‘... may be mediated by factor(s) activated, produced, or transported throughout the heart during brief ischemia-reperfusion.’ In 2013... observations, associations... no integrated, unifying hypothesis

20. Collaborators Peter Whittaker, PhD Joe Wider Vishnu Undyala Eric Dickson, MD Michelle Maynard Craig Smith, MD Chad Darling, MD Dale Greiner, PhD

21. Signaling myocardial biopsies obtained from patients during coronary artery bypass surgery ‘unique signaling signature of RIPC’: increased STAT5 phosphorylation in patients that received RIPC vs Controls Heusch et al, Circ Res 2012;110:111-15.