1. Update on Remote Ischemic Conditioning: Basic and Translational Perspectives Update on Remote Ischemic Conditioning: Basic and Translational Perspectives Karin Przyklenk PhD Director, Cardiovascular Research Institute Professor, Departments of Physiology & Emergency Medicine Wayne State University School of Medicine Detroit MI ICT Webinar: 3 rd November, 2015 Disclosure: Scientific Advisor for LifeCuff, Inc.

2. preconditioning postconditioning remote conditioning initiate the up-regulation of endogenous protective mechanisms that render the heart resistant to ischemia-reperfusion injury; reduce infarct size Control‘Conditioned’ Ischemic ‘Conditioning’

3. classic cardioprotective paradigms: ischemic preconditioning, postconditioning protective stimuli (brief antecedent ischemia; stuttered reflow) applied to the heart Control Postconditioning Preconditioning sustained ischemia infarct size sustained ischemia Ischemic ‘Conditioning’

4. 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) Remote preconditioning sustained ischemia remote infarct size Ischemic ‘Conditioning’

5. 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

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

7. ‘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

8. 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

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

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

11. unprecedented preclinical agreement: pre- post- and remote conditioning reduce infarct size postconditioning, remote conditioning: poised for clinical translation... Ischemic ‘Conditioning’

12. unprecedented preclinical agreement: pre- post- and remote conditioning reduce infarct size however, results of Phase II trials have been mixed i.e., remote conditioning: outcomes have ranged from positive to neutral to deleterious Ovize, Thibault & Przyklenk, Circulation Research 2013;113:439-50. Ischemic ‘Conditioning’

13. unprecedented preclinical agreement: pre- post- and remote conditioning reduce infarct size however: results of Phase II trials have been mixed remote conditioning: outcomes have ranged from positive to neutral to deleterious recent meta-analyses: outcomes have been mixed results of recent Phase III trials have been negative remote conditioning: ERICCA, RIPHeart Ischemic ‘Conditioning’

14. unprecedented preclinical agreement: pre- post- and remote conditioning reduce infarct size however: recent Phase III trials have been negative ERICCA Trial: Hausenloy DJ et al. N Engl J Med 2015;373:1408-17.

15. unprecedented preclinical agreement: pre- post- and remote conditioning reduce infarct size however: progress toward clinical translation ‘somewhere between frustrating and disappointing’ (Shevchuck & Laskey, Circulation Cardiovasc Interv 2013;6:484-492)... possibly a consequence of gaps in our understanding of the characteristics and mechanisms of RIC Ischemic ‘Conditioning’

16. Characteristics site of remote ischemia? number and duration of remote ischemic episodes? timing of stimulus relative to onset of sustained ischemia? threshold required to evoke myocardial protection? ‘dose response’ and possibility of ‘hyperconditioning’? (Whittaker & Przyklenk, Dose Response 2014;12:650-63) Remote preconditioning sustained ischemia remote infarct size

17. Characteristics “As for every drug and therapy, the principle of Paracelsus, dosis sola venenum facit (the dose alone makes the poison), applies to remote ischemic preconditioning. Unfortunately, we do not know the right dose of ischemia to deliver.” Zaugg & Lucchinetti, N Engl J Med 2015;373;1470-72. Remote preconditioning sustained ischemia remote infarct size

18. 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, STAT) mitochondria (mPTP)

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

20. 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 2015...

21. Pickard et al., Basic Res Cardiol 2015; 110:453. Communication: neuronal and/or humoral

22. Pickard et al., Basic Res Cardiol 2015; 110:453. Communication: neuronal activation of sensory neurons stimulation of afferent pathways CNS transported to heart via activation of efferent neuronal pathways activation of sensory neurons stimulation of afferent pathways CNS transported to heart via activation of efferent neuronal pathways

23. Pickard et al., Basic Res Cardiol 2015; 110:453. Communication: humoral as-yet unidentified ‘circulating protective factor’ multiple candidates... as-yet unidentified ‘circulating protective factor’ multiple candidates...

24. Candidates include: 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 targeted hypotheses: SDF (stromal cell derived factor)1-α/ CXCR4; one or more miRNAs; exosomes Davidson et al, Basic Res Cardiol 2013;108:377 Duan et al, Cardiology 2012;122:36-43 Slagsvold et al, Circ Res 2014;114:851-9 Li et al, Basic Res Cardiol 2014;109:423 Giricz et al, JMCC 2014;68:75-8 Communication: humoral

25. isolated buffer-perfused rat hearts exosomes detected in effluent from preconditioned hearts transfer of effluent from preconditioned hearts to naïve acceptors: cardioprotective depletion of exosomes: loss of ‘transferred’ protection Giricz et al, JMCC 2014;68:75-8.

26. 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 2015... observations, associations no integrated, unifying hypothesis in all likelihood: model-dependent

27. Signaling signaling receptor stimulation trigger effector CARDIOPROTECTION COMMUNICATION role of STAT5

28. 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.

29. Signaling With propofol anesthesia: RIPC failed to attenuate cTnI release no evidence of STAT5 phosphorylation Kottenberg et al et al, J Thorac Cardiovasc Surg 2014;147:376-82.

30. Signaling With propofol anesthesia: RIPC failed to attenuate cTnI release no evidence of STAT5 phosphorylation Propofol anesthesia was used in >90% of patients in ERICCA, 100% of patients in RIPHeart Kottenberg et al et al, J Thorac Cardiovasc Surg 2014;147:376-82.

31. characterize (physiology) understand (mechanisms) apply Ischemic ‘Conditioning’ From frustration and disappointment...... to successful translation

32. Collaborators Peter Whittaker, PhD Michelle Maynard Eric W. Dickson, MD Chad E. Darling, MD Craig Smith, MD Dale Greiner, PhD Thomas Sanderson, PhD Rita Kumar, PhD Yi Dong, MBBS PhD Christian Reynolds, PhD Joe Wider Andrew Kulek Vishnu Undyala, MS