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Role of 15-F2T-Isoprostane in the pathogenesis of myocardial ischemia-reperfusion injury : a novel therapeutic… Xia, Zhengyuan 2004

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R O L E OF 15-F -ISOPROSTANE IN T H E PATHOGENESIS OF MYOCARDIAL ISCHEMIA-REPERFUSION INJURY: A NOVEL THERAPEUTIC APPROACH TO CARDIOPROTECTION WITH PROPOFOL 2T  By ZHENGYUAN XIA M . B., Xian Ning Medical College, 1984 M . Sc., Hubei Medical University, 1991  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Department of Pharmacology & Therapeutics In Close Association with the Department of Anesthesiology We accept this thesis as confirming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A April 2004 © Zhengyuan Xia, 2004  11  ABSTRACT Myocardial ischemia-reperfusion injury (IRI) is a major pathophysiologic factor contributing to post-operative cardiac dysfunction in patients undergoing coronary artery bypass surgery utilizing cardiopulmonary bypass. Reactive oxygen species (ROS)mediated lipid peroxidation plays a critical role in mediating myocardial IRI. This thesis reports the results of four studies designed to investigate the role of 15-F2.-isoprostane, a reliable measure of lipid peroxidation that has bioactivity, in the pathogenesis of myocardial IRI and to explore the therapeutic potential of propofol. In the first study we demonstrated for the first time that significant in vivo lipid peroxidation occurs early during myocardial ischemia and continues during reperfusion rather than primarily only during reperfusion in patients undergoing cardiac surgery utilizing cardiopulmonary bypass. The plasma decay patterns of 15-F -isoprostane 2t  during reperfusion parallel post-operative cardiac functional recovery. In an in vitro study, a unique therapeutic regimen of propofol was developed to best utilize its antioxidant properties in order to attenuate ROS generation during myocardial ischemia and early reperfusion in isolated rat hearts. Propofol provides better cardiac protection when applied at clinically achievable high concentration before ischemia and during global myocardial ischemia and continued during early reperfusion, followed by a relatively lower concentration during the later phase of reperfusion. O f particular relevance is our identification, using the isolated perfused rat hearts, that 15F2 -isoprostane is produced in situ during global myocardial ischemia. This finding t  provides evidence to support the use of antioxidant interventions during ischemia which would target the coronary endothelium and/or the cardiomyocytes.  Ill  A third study investigated whether or not aging could be a factor that adversely j  affects the cardiac protective effect of propofol on myocardial IRI. The results showed that propofol equally preserved myocardial endogenous antioxidant capacity in the young and middle-aged rat hearts and, more significantly, enhanced post-ischemic myocardial functional recovery in the middle-aged rat hearts relative to that in the young rat hearts. This finding provides evidence to support the notion that drug(s) with antioxidant properties (such as propofol) could be more effective in attenuating myocardial IRI in populations suffering from insufficient or decreased endogenous antioxidant capacity, such as the elderly. In our study, we identified a strong inverse correlation between myocardial 15-F isoprostane levels and post-ischemic cardiac function in the isolated 2r  rat heart, suggesting 15-F2 -isoprostane itself could be a factor mediating myocardial IRI. t  In the last study, we further explored whether 15-F -isoprostane can directly 2t  mediate myocardial IRI and i f 15-F -isoprostane antagonism could be a potential adjunct 2t  therapy. We found that 15-F2 -isoprostane exacerbated myocardial IRI as evidenced by an t  increased myocardial infarct size, cellular damage and reduced post-ischemic myocardial function. 15-F2 -isoprostane antagonism abolished its deleterious effects. We also found t  evidence that 15-F2 -isoprostane may mediate myocardial IRI, at least in part, by t  increasing ET-1 production during later reperfusion. It is hoped that the studies described in the thesis have enhanced knowledge concerning the role of 15-F2 -isoprostane in the pathogenesis of myocardial IRI, from its t  mechanism(s) of action to its clinical relevance. It is hoped that our findings can aid in the development of novel and effective therapeutic interventions against myocardial ischemia-reperfusion injury.  TABLE OF CONTENTS Abstract T A B L E OF CONTENTS List of Tables List of Figures Acknowledgements Dedication  ii iv viii ix xii xiii  1. Chapter 1 Literature Review  1 1  1.1. Ischemic Heart Disease — A Leading Cause Of Death In North America 1.1.1. Advantages And Limitations O f Current Therapeutic Interventions 1.1.2. Sustained Significant Morbidity And Mortality  1 1 .2  1.2. Myocardial Ischemia-Reperfusion Injury (IRI) 1.2.1. Myocardial Ischemia 1.2.2. Myocardial Reperfusion: The Role Of Reactive Oxygen Species (ROS) In Mediating Myocardial IRI 1.2.3. ROS-Mediated Lipid Peroxidation: Available Measures And Their Limitations  3 3  7  1.3. F -Isoprostanes — A Reliable Index Of Endogenous Lipid Peroxidation 1.3.1. Mechanism O f F2-Isoprostane Formation 1.3.2. 15-F2t-Isoprostane — A Marker And Mediator O f Oxidant Injury 1.3.3. Bioactivity Of 15-F -Isoprostane (15-F -IsoP) 1.3.4. Methods Of Measurement Of 15-F2 -Isoprostane 1.3.5. 15-F -Isoprostane Metabolism 1.3.6. Evidence For A Unique 15-F -Isoprostane Receptor 1.3.7. 15-F -Isoprostane In Myocardial IRI  8 8 10 12 14 16 17 18  1.4. Antioxidant Interventions Against Myocardial IRI 1.4.1. Antioxidant Vitamins C And E 1.4.2. Allopurinol 1.4.3. N-Acetylcysteine 1.4.4. Propofol  19 19 20 21 21  1.5. Protective Effects And Limitations Of Ischemic Preconditioning On Myocardial IRI  22  1.6. Anesthesia And Myocardial IRI 1.6.1. Inhalational Anesthesia 1.6.2. Intravenous Anesthesia  23 24 25  2  2t  2t  t  2t  2t  2t  5  V  1.7. Propofol: An Anesthetic And Antioxidant 1.7.1. Structure 1.7.2. Basic Pharmacokinetics 1.7.3. Effects O f Cardiopulmonary Bypass On Propofol Pharmacokinetics 1.7.4. Propofol Antioxidant Activity 1.7.5. Propofol Effects On IRI In Isolated Hearts 1.7.6. Propofol Anesthesia In Cardiopulmonary Bypass Surgery  26 28 29 30 32 33  2. CHAPTER 2  36  The Relationship Between Plasma 15-F2-Isoprostane Concentration And Early Postoperative Cardiac Function Following Cardiac Surgery  36  2.1.  Preface  36  2.2.  Introduction  36  2.3.  Materials And Methods  38  t  2.3.1. Patient Data And Surgical Technique 2.3.2. Data And Samples Collection 2.3.3. 15-F2t-Isop Enzyme Immunoassay 2.3.4. Data Analysis  38 39 40 41  2.4. 2.4.1. 2.4.2. 2.4.3. 2.4.4.  Results Patient Profile And Perioperative Data Evaluation O f The Assay Plasma Free 15-F2 -Isoprostane Concentration 15-F2 -Isoprostane And Postoperative Cardiac Index  2.5.  Discussion  52  2.6.  Conclusion  57  3.  t  t  CHAPTER 3  41 41 42 47 52  59  Dose-Dependent Protection Of Propofol In Myocardial IRI In Rats: Effects On 15-F -Isop formation  59  3.1.  Preface  .59  3.2.  Introduction  59  3.3.  Materials And Methods  60  3.3.1. 3.3.2. 3.3.3. 3.3.4. 3.3.5.  Heart Preparation Experimental Protocol Heart Tissue Antioxidant Capacity Determination 15-F2 -Isop Assays Statistical Analysis  60 61 63 64 64  2t  t  vi  3.4. 3.4.1. 3.4.2. 3.4.3. 3.4.4. 3.4.5. 3.4.6. 3.4.7.  Results 65 15-F2 -Isop Generation During Ischemia-Reperfusion 65 Tissue Antioxidant Capacity 65 Contracture Development During Ischemia 70 Functional Response To Ischemia -Reperfusion 70 Coronary Perfusion Pressure 72 Lipid Peroxidation And Post-ischemic Myocardial Function 75 Pre-Ischemic Myocardial Depression And Post-ischemic Myocardial Function..78  3.5.  Discussion  78  3.6.  Conclusion  82  t  4. C H A P T E R 4  84  Propofol Effects On Ischemic Tolerance Of Middle-Aged Rat Hearts: Effects Of 15-F2t-Isop Formation And Tissue Antioxidant Capacity  84  4.1.  Preface  84  4.2.  Introduction  84  4.3.  Materials And Methods  86  4.3.1. 4.3.2. 4.3.3. 4.3.4. 4.3.5.  Heart Perfusion Experimental Protocol 15-F Isop Assays Heart Tissue Antioxidant Capacity Determination Data Analysis  86 87 88 89 90  4.4. 4.4.1. 4.4.2. 4.4.3.  2r  Results 15-F2 -Isop Generation During Ischemia-Reperfusion Tissue Antioxidant Capacity Effects Of Propofol On Contracture Development During Ischemia And Reperfusion 4.4.4. Coronary Perfusion Pressure 4.4.5. Left Ventricular Mechanics 4.4.6. Correlation Analysis t  90 90 91 94 97 97 98  4.5.  Discussion  103  4.6.  Conclusion  109  5. CHAPTER 5  110  Effects of 15-F -isoP on myocardial IRI in isolated rat hearts: potential mechanism of 15-F2 -isoP action 2t  t  110  5.1.  Preface  110  5.2.  Introduction  110  5.3.  Materials and methods  113  5.3.1. 5.3.2. 5.3.3. 5.3.4. 5.3.5. 5.3.6. 5.3.7.  Heart preparation Experimental Protocol Measurement of Endothelin-1 Measurement of C K - M B 15-F -IsoP Assays Myocardial Infarct Size Measurement Statistical Analysis  113 114 115 117 117 118 118  5.4. 5.4.1. 5.4.2. 5.4.3. 5.4.4. 5.4.5. 5.4.6. 5.4.7.  Results Endothelin-1 Release and its Relation with 15-F -IsoP 15-F -IsoP Generation During Ischemia-reperfusion. C K - M B Release During Ischemia-reperfusion Contracture Development during Ischemia Functional Response to Ischemia-reperfusion Coronary Perfusion Pressure Myocardial Infarct Size  5.5.  Discussion  131  5.6  Conclusion  135  2t  2t  2t  ,  118 118 119 122 126 130 130  6. CHAPTER 6  136  General summary and conclusions  136  6.1.  Summary  136  6.2.  Conclusions  140  6.3.  Future directions for research  141  References  144 .  Appendix I:  192  Propofol inhibition of TNF-alpha- induced vascular endothelial cell apoptosis: effects on Bcl-2 and Bax protein expression  192  LIST OF TABLES Table 2.1.  Patients demographic and perioperative data  44  Table 2.2. Cardioplegic perfusion data for cardiac surgery patients who do not need inotrope or those who need > 2 inotropes support post-operatively 45 Table 2.3.  Plasma free 15-F risoprostane assay precision analysis  46  Table 3.1. Changes of left ventricular developed pressure (LVDP), L V systolic pressure (LVSP) and coronary perfusion pressure (CPP) (mm hg) during myocardial ischemia and reperfusion  73  Table 4.1. Variations of coronary perfusion pressure (CPP) (mm Hg) of the ischemic-reperfused rat hearts  99  2  IX  LIST OF FIGURES Figure 1.1.  15-F2 -isoprostane formation pathway t  Figure 1.2. Molecular structures of 2,6-diisopropylphenol (propofol) and alphatocopherol (vitamin E)  9  27  Figure 2.1. Changes of plasma free 15-F2 -Isoprostane during ischemia-reperfusion in patients undergoing coronary artery bypass graft surgery utilizing cardiopulmonary bypass 48 t  Figure 2.2. Changes of plasma free 15-F2 -Isoprostane during ischemia-reperfusion in patients who do not need inotrope or those who need > 2 inotropes support post-operatively 49 t  Figure 2.3. Plasma free 15-F2 -Isoprostane in the non-inotrope group decays exponentially after global myocardial ischemia-reperfusion  50  Figure 2.4. Correlation between post-operative cardiac index (CI) and changes of plasma free 15-F t-Isoprostane during early reperfusion  51  Figure 3.1.  67  t  2  15-F2 -isoprostane release during ischemia and reperfusion t  Figure 3.2. Formation of thiobarbituric acid reactive substances (TBARS), a measure of tissue antioxidant capacity, in heart tissues (represented as absorbance at 532 nm) in the presence of 1 m M t-butylhydroperoxide  68  Figure 3.3-A. Effect of Propofol on left ventricular end-diastolic pressure (LVEDP), reflecting myocardial contracture (ventricular stiffness), during myocardial ischemia 69 Figure 3.3-B. Effect of Propofol on left ventricular end-diastolic pressure (LVEDP), during postischemic reperfusion  69  Figure 3.4. Relationship between 15-F -IsoP generation upon reperfusion and coronary perfusion pressure (CPP) at 90 minutes of reperfusion  74  Figure 3.5. Correlation between the recovery of left ventricular developed pressure (LVDP) after 90 min of reperfusion and the formation of heart tissue thiobarbituric acid reactive substances (TBARS), a measure of tissue antioxidant capacity  76  2t  Figure 3.6. Correlation between the changes of left ventricular developed pressure (LVDP) after reperfusion (from reperfusion 60 to 90 min) and the formation of heart tissue thiobarbituric acid reactive substances (TBARS) in the presence of 1 m M t-butylhydroperide 77 Figure 4.1-A. Coronary effluent 15-F2.-isoprostane release during reperfusion of the ischemic-reperfused young and middle-age rat hearts  92  Figure 4.1-B. Coronary effluent 15-F2 -isoprostane release during ischemia of the ischemic-reperfused young and middle-age rat hearts  92  Figure 4.2. Thiobarbituric acid reactive substances (TBARS) formation (as reflected by absorbance at 532 nm) as a function of t-BHP concentration for heart tissues at reperfusion 90 min  93  t  Figure 4.3. Left ventricular end diastolic pressure (LVEDP), reflecting myocardial contracture during ischemia of the ischemic-reperfused young and middle-age rat hearts 95 Figure 4.4. Left ventricular end diastolic pressure (LVEDP), reflecting myocardial contracture during reperfusion following 40 min of global ischemia of the ischemic-reperfused young and middle-age rat hearts 96 Figure 4.5.  Post-ischemic Left ventricular developed pressure (LVDP)  100  Figure 4.6. Correlation between coronary effluent 15-F -isoprostane release during ischemia and heart tissue antioxidant capacity. Heart tissue was sampled after 40 min of ischemia and 90 min of reperfusion  101  Figure 4.7. Correlation between left ventricular developed pressure (LVDP) at 90 min of reperfusion (Re-90) and 15-F -isoprostane release during the first 30 min of ischemia  102  Figure 5.1-A. Coronary Effluent Endothelin-1 (ET-1) concentrations during myocardial ischemia-reperfusion  120  Figure 5.1-B. Relationship between 15-F2 -isoprostane (15-F2 -isoP) and ET-1 concentration during the first 30 min of ischemia in the untreated control group  120  Figure. 5.2. Effect of SQ 29548 (SQ) on 15-F -isoprostane (15-F -isoP) release during myocardial ischemia-reperfusion  121  Figure 5.3. Coronary effluent C K - M B concentration during myocardial ischemia-reperfusion  123  2t  2t  t  t  2t  2t  Figure 5.4-A. Development of left ventricular end-diastolic pressure (LVEDP), reflecting myocardial contracture, during ischemia Figure 5.4-B. Ischemic contracture onset time during myocardial ischemia Figure 5.5. Variations of left ventricular end-diastolic pressure (LVEDP), reflecting myocardial stiffness, during post-ischemic reperfusion Figure 5.6. Recovery of left ventricular developed pressure (LVDP), reflecting effective myocardial contractility, during reperfusion  124 124  125 128  Figure 5.7. Myocardial infarct size after 60 min of reperfusion following 40 min of ischemia  129  Figure A - l . Tumor necrosis factor (TNF) -alpha-induced apoptotic cell death in cultured human umbilical vein endothelial cells (HUVECs) measured by T U N E L staining  201  Figure A-2. The expression of anti-apoptotic Bcl-2 protein evaluated by immunoperoxidase technique  202  Figure A-3. The expression of pro-apoptotic Bax protein evaluated by immunoperoxidase technique  203  Figure A - 4-A. Changes of Bcl-2/Bax ratio of cultured H U V E C s after TNF-alpha stimulation and propofol treatment  204  Figure A - 4-B. Inverse correlation between endothelial cell apoptotic index (Al) and the ratio of bcl-2 over Bax proteins expression  204  Figure A-5. Nitric oxide (NO) concentration in the culture medium  205  Figure A-6. Representative electron microscopy of endothelial cells (HUVECs) apoptotic conformational changes  206  Figure A-7. H2O2 and TNF-alpha synergistically induced apoptotic cell death in cultured H U V E C s  208  Figure A-8-A. Effects of H 0 2 on TNF-mediated changes in Bcl-2 expression  210  Figure A-8-B. Effects of H2O2 on TNF-mediated changes in Bax (B) expression  210  2  Xll  ACKNOWLEDGEMENTS  I would like to express my gratitude to the many individuals who assisted me during the course of this study. In particular, I would like to acknowledge the Centre for Anesthesia & Analgesia, Department of Pharmacology & Therapeutics for the use of office and laboratory facilities and for the support of my study through Dr. Jean Templeton Hugill funding.  I would like to express my sincere gratitude to my supervisors Dr. David Ansley and Dr. David Godin for teaching me an incredible amount about both science and life. I am grateful for their never-ending support and encouragement throughout the course of my studies. Their friendship has made my years at U.B.C. most memorable. I would like to thank Dr. Ernie Puil and Dr. Karim Qayumi for giving me the opportunity to join their laboratory group meetings and for their teaching and support. I would also like to thank Dr. Michael Walker and Dr. Thomas Chang for allowing me to use their laboratory facilities and for their teaching.  Xlll  DEDICATION  To my wife Lihui and daughter Weiyi who continually provide inspiration.  1  CHAPTER 1 LITERATURE REVIEW  1.1. Ischemic heart disease — A Leading Cause Of Death In North America  Cardiac disease remains the leading cause of death for men and women in North America. Ischemic heart disease accounts for more than one in five cardiac-related deaths, annually. Consequently, much effort has been made with the aim of optimising 1  conditions to effectively re-establish the blood perfusion (reperfusion) to the ischemic myocardium in order to salvage tissue at risk of irreversible damage.  1.1.1. Advantage And Limitations Of Current Therapeutic Interventions  The advent of treatments such as angioplasty, thrombolysis and coronary artery bypass grafting have improved the overall survival of individuals receiving treatment within the first few hours of a myocardial infarction. However, the mortality rate is increased within the first 24 hours following the onset of reperfusion compared to the situation if no reperfusion therapy is given. This may be attributable to the extension of 2  subsequent myocardial damage and the "no-flow" phenomenon during reperfusion.  3 ;4 ; 5  Intravenous thrombolytic therapy is the standard approach for managing patients with a cute m yocardial infarction, b ased upon its widespread availability and ability to reduce patient mortality as demonstrated in randomised trials. Despite its proven efficacy,  thrombolytic therapy has limitations. Many patients are ineligible for treatment with thrombolytics. O f those given thrombolytic therapy, 10 to 15 percent have persistent occlusion or reocclusion of the infarct-related artery.  Recent studies have shown that angioplasty provides a short-term clinical advantage over thrombolysis which may not be sustained.  6  Therefore, for patients with  acute ST-segment elevation acute myocardial infarction (AMI) and contraindications to thrombolytics, coronary artery bypass grafting surgery is likely a better choice for therapeutic management.  7  In fact, the rate of coronary artery bypass grafting (CABG)  surgery has more than quadrupled in the last 20 years in Canada.  8  1.1.2. Sustained Significant Morbidity A n d Mortality  Despite advances in modern surgical and anesthetic techniques, the risks of morbidity and mortality associated with C A B G surgery are significant since physicians encounter, with increasing frequency, patients who are elderly, suffering from increased disease severity, and concomitant medical illnesses.  9-11  Myocardial ischemia-reperfusion injury (IRI) is a major pathophysiologic factor in the high mortality rate from diseases of the cardiovascular system. '  12 13  Myocardial IRI  contributes to early postoperative myocardial ischemia and dysfunction, two factors associated with increased morbidity, and prolonged intensive care unit (ICU) stay that have not been significantly reduced by current forms of therapy. " 14  17  3  Myocardial IRI is a complex phenomenon, the mechanisms of which are incompletely understood. It is, however, well accepted that lipid peroxidation mediated by reactive oxygen species (ROS) plays a critical role in myocardial IRI.  1 8 - 2 7  1.2. Myocardial Ischemia-Reperfusion Injury (IRI)  1.2.1. Myocardial Ischemia  Myocardial ischemia and infarction are typically caused by a substantial reduction or complete interruption of regional blood supply following occlusion of its coronary artery. During C A B G surgery utilizing cardiopulmonary bypass (CPB), the whole heart is subjected to ischemia (global myocardial ischemia) during the period of aortic crossclamping. This deprives the heart of oxygen and metabolic nutrients essential for the maintenance of myocardial tissue integrity.  As with most living tissues, heart muscle harnesses and utilizes the energy in the form of adenosine triphosphate (ATP). Energy transfer within the cardiac myocyte is mediated through a series of interconnected cycles. The consumption of ATP:  (1)  provides the energy for the cross-bridge cycle of contractile elements; (2) decreases the proton gradient across the inner mitochondrial membrane (which drives ATP synthesis); (3) increases the oxidation of N A D H ; (4) increases flux through the Krebs cycle (the major source of reducing equivalents); (5) increases acetyl-CoA use; and finally (6) increases substrate consumption.  4 The major source of ATP in heart muscle is via phosphorylation of A D P coupled to respiratory chain activity. The heart's content of ATP is normally about 20 umol/g dry weight. At an oxygen consumption rate of about 80 umol/min/g dry weight phosphorus/oxygen ratio of approximately 2 . 5 ,  29  2 8  and a  heart muscle replenishes about 33 umol  of ATP/g dry weight each minute. Therefore, ATP must be re-synthesized as quickly as it is broken down. Without continuous replenishment, the intracellular stores of ATP would be expected to be exhausted within <1 minute.  The deprivation of oxygen during myocardial ischemia interrupts mitochondrial oxidative phosphorylation, thereby compromising the supply of ATP. When cellular ATP is depleted during ischemia, the energy deficiency leads to the disruption of normal ion gradients (e.g. involving sodium, calcium, potassium and the hydrogen ion), resulting in intracellular edema and m orphological alterations. Disruption of the plasma membrane integrity causes further changes in ionic gradients by a massive influx of sodium and • efflux of potassium. The loss of the ion concentration gradients and the resultant cellular depolarization  activate  voltage-gated  uncontrolled calcium influx.  3 0  calcium channels,  resulting  in a  massive  The subsequent cytosolic calcium overload leads to the  activation of intracellular Ca -dependent proteases and phospholipases, resulting in 2+  extensive cardiomyocyte cellular damage, and ultimately irreversible necrosis.  31  Re-establishment of blood flow (i.e. reperfusion) to the ischemic myocardium is necessary in order to salvage dying cardiomyocytes.  5 1.2.2. Myocardial Reperfusion: The Role Of Reactive Oxygen Species (ROS) In Mediating Myocardial IRI  Reperfusion of the ischemic myocardium initiates two disparate types of cellular processes in the previously ischemic tissue as a result of oxygen re-introduction. Upon reperfusion, cells adjust to the reintroduction of molecular oxygen, and cellular repair begins. Paradoxically, however, in the meantime, a second mechanism triggers the immediate over-production of reactive oxygen species (ROS), which causes further cell injury, referred to as "ischemia-reperfusion injury" (IRI).  Reactive oxygen species are molecules or fragments of molecules containing unpaired electrons in their outermost orbits. Unpaired electrons tend to acquire an electron to form a pair; therefore, most ROS are highly chemically reactive and, as a result, short-lived. Important ROS in biological systems include superoxide anion (0 "), 2  hydrogen peroxide ( H 0 ) , hydroxyl radical (OH), and peroxynitrite (ONOO"), 2  2  product of nitric oxide (NO) combining with 0 " .  3 2  the  3 3 - 3 8  2  The release of high levels of ROS associated with reperfusion can trigger lipid peroxidation of the unsaturated phospholipid components of cellular membranes, which is a crucial event determining the onset of irreversible cellular necrosis in the ischemicreperfused tissue. ' 39  40  Lipid peroxidation and the subsequent activation of phopholipases  can initiate the formation and release of inflammatory mediators such as tumor necrosis factor-alpha, thromboxanes,  leukotrienes and platelet activating factor, which can  6 adversely affect hemodynamic homeostasis and promote vascular endothelial cell and cardiomyocyte apoptotic cell death during reperfusion.  Previous  studies  have  shown  that  during  cardiac  surgery  cardiopulmonary bypass, ROS are generated during myocardial reperfusion, are important contributors to tissue injury.  4 2  4 1  utilizing and these  Moreover, a number of recent studies,  including our own, have shown that profound systemic oxidative stress and lipid peroxidation occur before as well as during myocardial ischemia shortly after the onset of CPB in humans. ' 8  subsequently  4 3 ;  4 4  This is likely due to the activation of leukocytes which  release substantial amounts of cytotoxic ROS. ' 4 5  4 6  Superoxide and NO,  when present in equimolar concentrations, can combine to form ONOO", a highly reactive and injurious free radical.  4 7  Myocardial antioxidant enzymes, including glutathione reductase, superoxide dismutase (SOD), and catalase, are activated in proportion to the degree of myocardial IRI.  4 8  Host antioxidants can become depleted during CPB, presumably as a result of  consumption by ROS.  4 9 ;  5 0  When ROS production exceeds host defense scavenging  capacity, cellular injury results.  4 1  '  5 1  A n inverse correlation has been identified between  preoperative total plasma antioxidant capacity and lipid peroxidation, the latter being an index of myocardial cellular injury . Furthermore, post-CPB coronary endothelial cell A  dysfunction appears to be partially mediated by ROS.  This is of significance. A recent  study has shown that apoptosis of endothelial cells precedes myocyte cell apoptosis in ischemia-reperfusion injury.  This suggests that reperfusion induces the release of  7 soluble pro-apoptotic mediators (including ROS) from endothelial cells that promote myocyte apoptotic cell death.  1.2.3. ROS-Mediated  Lipid  Peroxidation:  Available  Measures  And  Their  Limitations  The d etection a nd m easurement o f 1 ipid p eroxidation h as b een m ost f requently used to support the involvement of the ROS reactions in pathophysiologic processes. Furthermore, these indices have been the basis for the development and use of antioxidant interventions to prevent oxidative injury.  Part of the difficulty in determining the role of oxidant stress in human disease has been the lack of a sensitive and specific indicator of oxidative damage.  54  The  thiobarbituric acid (TBA) assay that measures an aldehydic breakdown product of lipid hydroperoxides, namely malondialdehyde (MDA), has been used most frequently. However, T B A reacts with a variety of other biological compounds, such as prostaglandins, thromboxanes, carbohydrates, and sialic acids. Consequently, T B A and other commonly used assays, such as diene conjugation, are neither sufficiently specific nor sensitive enough to monitor small changes in free radical status and lipid peroxidation in vivo. Therefore, it has been recognized that one of the greatest needs in the field of oxygen-derived free radical research is the availability of a reliable noninvasive method to assess oxidative stress status in vivo in humans.  8 In 1990, Morrow and colleagues reported that a series of prostaglandin (PG) F255  like compounds are produced by the free radical-catalyzed peroxidation of arachidonic acid independent of the cyclooxygenase enzyme. These compounds are termed F2isoprostanes,  5 6  and these provide a specific and unique measure of in vivo lipid  peroxidation (see below).  1.3. F2-Is0pr0stan.es — A Reliable Index Of Endogenous Lipid Peroxidation  1.3.1. Mechanism Of F -Isoprostane Formation 2  A n interesting aspect related to the formation of F -isoprostanes is that they are 2  esterified phospholipids formed in situ and they are subsequently released in the. free form by phospholipases.  56-58  Since only small amounts of arachidonic acid are present in  the unesterified state and the vast majority of arachidonate is esterified to phospholipids 58  , the level of F2-isoprostane formation could therefore serve as an ideal indicator of in vivo lipid peroxidation.  Indeed, recently, a substantial body of evidence has been obtained indicating that the measurement of F -isoprostanes, in urine or plasma, provides a reliable non-invasive 2  approach to assessing lipid peroxidation status in vivo and represents a major advance in our ability to assess oxidative stress in humans.  56;59  X Free radical attack  Arachidonovl radicals ^  Peroxidation  H2-isoprostanes endoperoxides  12-series  5-series  8-series  15-series  15-F t-isoprostane 2  Figure 1.1. The isoprostane pathway. Free radical (i.e. reactive oxygen species) attack on arachidonic acid results in the formation of arachidonoyl radicals, which, following peroxidation, form 4 prostaglandin-F£2-like compounds that can then be fully reduced to form 4 prostaglandin F2 regioisomers [those of thel 5-series, 8-series, 12a  series and 5-series], or rearrange to form  prostaglandin E2 and D2 regioisomers. Each  regioisomer comprises 8 diastereoisomers and so 64 different F2-isoprostanes can be generated.  10 Depending on which of the labile hydrogen atoms of arachidonic acid is first abstracted by reactive oxygen species (ROS), three initial arachidonoyl radicals can be formed following ROS attack. These radicals form four  prostaglandin-H2-like  compounds that can then be fully reduced to form 4 prostaglandin F  2 a  regioisomers (i.e.  15-series, 5 series, 8-series and 12-series F2-isoprostanes, Figure 1.1), or rearranged to 60  form prostaglandin E and D regioisomers. Because each F2-isoprostane regioisomer 2  2  comprises 8 diastereoisomers, 64 different F -isoprostanes can be generated. 2  Most studies have focused on 15-F2.-isoprostane (15-F2 -IsoP), which is one of the t  most abundant F2-isoprostanes produced in vivo, and one of the few isoprostanes commercially available.  1.3.2.15-F Isoprostane — A Marker And Mediator Of Oxidant Injury 2r  15-F2 -isoprostane (previously 8-epi-PGF2 ), t  a  6 1  has been the most extensively  studied isomer of the F2-isoprostane family. Much of the interest in this compound derives from the fact that it is not only one of the most abundant F2-isoprostanes that is produced in vivo  6 2  but it also possesses potent bioactivity.  6 3  Measurement of 15-F2t-IsoP  has been shown to represent a sensitive and reliable marker of oxidative stress because: (1) it can be specifically and accurately measured in biological samples; (2) the level is increased in response to pro-oxidants, and (3) levels can be suppressed by dietary supplementation with antioxidants.  6 4  11  1.3.2.1.15-F2 -Isoprostane As An Index Of Oxidant Stress t  Reactive oxygen species-mediated non-enzymatic oxidation o f arachidonic acid accounts for almost all of the formation of 15-F2 -isoprostane in vivo, t  6 3  although minute  amounts of this compound may be produced as a by-product of the cyclooxygenase enzyme.  6 5  Studies have shown that the physiological formation of 15-F2 -isoprostane in t  vivo is not affected by cyclooxygenase inhibition in humans, even when high doses of cyclooxygenase inhibitors are administrated.  6 3 ;  6 6  This indicates that the relative  contribution of enzymatic generation of 15-F2 -isoprostane in vivo is inconsequential t  compared with the amounts formed via the ROS-mediated non-enzymatic pathway.  15-F2 -isoprostane is detectable in significant amounts in human plasma from t  normal volunteers at levels of 35 ± 6 pg/ml (n=12).  '  Plasma levels of 15-F2 t  isoprostane are known to increase during oxidant stress. It has been reported that there is a tendency for 15-F2 -isoprostane formation to increase with age in humans. t  68  This lends  some support to the hypothesis that the normal aging process is associated with enhanced oxidant damage to important biological molecules over time.  69  Measurements of 15-F2 -isoprostane should potentially allow for exploration of t  the role of ROS in the pathophysiology of a wide range of human diseases. In particular, it could provide a valuable tool to define the clinical pharmacology of antioxidant agents. Previous clinical trials examining the effect of antioxidants to prevent or ameliorate some  12 of the pathology of diseases in which ROS have been implicated were hampered by insufficient information regarding what doses and combinations of antioxidants are maximally effective. Studies have shown that the formation of 15-F -isoprostane 2t  increases significantly in animals deficient in vitamin E  6 3  '  7 0  and administration of  antioxidants decreases the formation of 15-F2 -isoprostane in animal models of oxidant t  stress, and in humans with increased oxidative activity. ' 7 1  7 2 ; 7 3  Of interest, vitamin E  dose-dependently reduced plasma levels of 15-F2 -isoprostane in normal volunteers.  5 6  t  This suggests that measurements of 15-F2 -isoprostane can be used to quantitatively t  define the effects of antioxidants to inhibit free radical processes in vivo in humans.  1.3.3. Bioactivity O f 15-F -Isoprostane 2t  The discovery that 15-F2 -isoprostane can exert biological effects t  6 3  '  7 1  reveals that  this compound may not simply be a marker of lipid peroxidation, but may also participate as a mediator of oxidant injury.  In the early 1990's, studies showed that renal ischemia-reperfusion injury results in increased urinary excretion of 15-F -isoprostane by 300% over baseline levels. 1574  2t  F2 -isoprostane, when administrated to rats via intrarenal arterial infusion at low t  nanomolar concentrations, reduces glomerular filtration rate and renal blood flow by 40 50%.  7 1 ; 7 4  However, systemic infusion of 15-F2 -isoprostane produced a fall in renal t  blood flow o f - 5 0 % without alterations in systemic blood pressure, suggesting a selective effect of 15-F2 -isoprostane on renal vasculature. The primary action of 15-F t-isoprostane t  2  13 in the glomerulus is constriction of the afferent renal arteriole, leading to a drop in glomerular capillary pressure.  74;  7 5  More recently, experimental studies have shown that 15-F2 -isoprostane can exert t  effects on the heart.  7 6  Concentration-dependent coronary vasoconstriction with parallel  decreases in left ventricular developed pressure (LVDP) were seen in the isolated perfused guinea pig heart at a dose range of 10" - 10" M 15-F -isoprostane. 8  5  7 6  2t  Interestingly, in the isolated perfused rat heart, 15-F -isoprostane did not affect coronary 2t  flow at concentrations up to 3 x 10" M . 6  7 7  However, 15-F -isoprostane exerted a 2t  vasoconstriction at concentrations as low as 10 n M when rat hearts were either subjected to a short period (30 min) of low flow ischemia and subsequent re-establishment of normal perfusion, or when the hearts were subjected to varying periods of oxidant stress. 77  Furthermore, 15-F -isoprostane-mediated vasoconstriction increases with the duration 2t  of oxidant stress in the isolated perfused rat heart. This supports the notion that 15-F 77  2t  isoprostane can function as a mediator that can exacerbate oxidant injury. Increasing evidence supports the possibility that 15-F -isoprostane could be a 2t  mediator o f o xidant i njury i n h umans. E levated 1 evels o f 1 5-F isoprostane h ave b een 2r  demonstrated in the pericardial fluid of patients suffering from congestive heart failure, the degree of elevation correlating with the functional severity of disease.  7 8  15-F 2t  isoprostane was found to accumulate in coronary arteries from patients with coronary artery disease and it may, therefore, be involved in the process of atherogenesis possibly in the pathology of ischemic heart syndromes.  7 9  and  14 1.3.4. Methods Of Measurement Of 15-F2-Isoprostane t  1.3.4.1. Gas chromatography (GC)-mass spectrometry (MS)  Gas chromatography-mass spectrometry (GC-MS) has been widely used for the analysis of the 15-F -isoprostane (15-F -IsoP). Compared to the classical procedure for 2t  2t  the isolation and quantitative determination of plasma prostanoids, which involves chromatography on a Cig and Si cartridge followed by thin-layer chromatography (TLC) prior to final determination, the procedure for GC-MS measurement of 15-F -IsoP has 2t  been improved.  The improvements include the following:  6 4  (1) recovery of  prostaglandin(PG)-F -like compounds during the Cig chromatography step is improved; 2  (2) the Si cartridge and the T L C steps are replaced with an aminopropyl cartridge, further improving the recovery of PGF2-like compounds; and (3)  gas chromatographic  separation of PGF2-like compounds is improved.  However, despite the improvements mentioned above, the overall recovery of PGF -like compounds from GC-MS procedures 2  6 4  is not complete (-75%). The assay is  still time consuming. The availability of the instrument and the capacity to analyse large amounts of clinical material has significantly limited the application of this method.  15 1.3.4.2. Radioimmunoassay And Enzyme Immunoassay  Immunoassay has always been an important tool for the measurement of low levels of endogenous prostaglandins or their exogenous analogues. Enzyme immunoassay (EIA) is more readily available. However, the accuracy of EIA for the analysis of 15-F2 t  IsoP has been questioned, due to the potential for cross-reactivity of the antibody with other isoprostane isomers.  Recently, an enzyme immunoassay and a radioimmunoassay (RIA) for measuring urinary concentrations of 15-F -IsoP have been developed by raising antibodies against 2t  this compound. The antisera had high titers (>l/300,000) and provided highly sensitive 80  assays (IC50, 8 and 24 pg/ml, for EIA and RIA, respectively), and cross-reactivity with other isoprostane isomers was negligible. The intra-assay precision (<10%) is acceptable. Measurements of urinary 15-F2 -IsoP by immunoassay were validated using different t  antisera and by comparison with GC-MS. The results obtained by EIA or R I A were highly correlated with those obtained from GC-MS.  It is important to note that the measurements of F2-isoprostanes by EIA or R I A and G C - M S are not equivalent. G C - M S estimates F2-isoprostane concentration from a peak encompassing a number of F2-isoprostanes i somers (15-F2 -IsoP being one of the t  major F2-isoprostanes isomers detected), while EIA or RIA methods measure a specific isoprostane (i.e., 15-F -IsoP in most studies). Therefore, direct comparison of F 2t  2  16 isoprostane levels derived from GC-MS and levels derived from EIA or R I A may be inappropriate.  The work described in this thesis utilizes the EIA method to measure 15-F -IsoP 2t  in studies designed to specifically address the role of 15-F2 -IsoP in the pathogenesis of t  myocardial IRI.  1.3.5.15-F -Isoprostane Metabolism 2t  In general, knowledge about the metabolic fate of 15-F2.-isoprostane is very limited. The t_/ of the clearance of 15-F2.-isoprostane from the circulation in the rat is 2  57  ~16 min.  It has been postulated that, analogous to the metabolism of other prostanoids,  the lung is the major site of metabolic clearance of F2-isoprostanes from the circulation. 5 6  This postulation is supported by the finding that the creation of a porta-caval shunt and  the ligation of the hepatic artery in rats, completely eliminating clearance of 15-F 2t  isoprostane by the liver, but only prolonged the tm of the clearance of 15-F2 -isoprostane t  from the circulation by an incremental amount of 5 minutes - i.e., from 16 to 21 min.  5 7  In rabbits, p lasma 1 5-F2 -isoprostane concentration was maximum at 1.5 m i n after the t  intravenous administration of the substance.  81  The level decreased rapidly thereafter. The  plasma distribution phase half-life (a-phase) of 15-F2.-isoprostane was found to be about 1 min and the terminal elimination phase half-life (P -phase) was 4 m i n .  81  17 Experimental studies have shown that 15-F2 -isoprostane is readily transported by t  a prostaglandin transporter (PGT), tissues, most notably the lung.  which is expressed at high levels in various rat  8 2  This indicates that PGT probably represents the  8 3  predominant route by which certain prostanoids, including F2-isoprostanes, are transported across plasma membranes.  The urinary concentration of 15-F -isoprostane reaches its height at 20 min 2t  following the intravenous administration of the substance in rabbits. The total excretion 81  of radioactivity in the urine was about 80% at 4 hours after the administration of tritiumlabelled 15-F -isoprostane. This suggests that, at least in the rabbit, urinary elimination 81  2t  is an important route for 15-F2 -isoprostane clearance. The major urinary metabolite of t  15-F -isoprostane in the rabbit is a-tetranor-15-keto-13,14-dihydro-15-F t-isoprostane 2t  2  and about 7% 15-F2 -isoprostane is found unconverted in the urine. t  major  urinary  metabolite  of  15-F2 -isoprostane t  is  8 1  In humans, the  2,3-dinor-5,6-dihydro-15-F2 T  isoprostane, which represented 29% of the radiolabeled 15-F -isoprostane.  84  2t  1.3.6. Evidence For A Unique 15-F -Isoprostane Receptor 2t  It has been previously determined  7 4  that the renal vasoconstricting actions of 15-  F -isoprostane a re c ompletely p revented o r r eversed b y S Q 2 9548, a t hromboxane A 2 2t  (TXA2)  receptor antagonist.  8 5  This suggests that 15-F -isoprostane may exert its 2t  vasoconstricting effect via activation of the T x A receptor. 2  18 Fukunaga and colleagues, using cultured rat aortic smooth muscle cells, found 75  specific binding sites for [ H]SQ 29548 and for [ I] BOP, a T x A agonist. They found 3  125  2  that both ligands were displaced from these binding sites by 15-F2 -isoprostane, but with t  significantly lesser potency than labelled SQ 29548 or I-BOP. In contrast, 15-F 2t  isoprostane stimulated inositol 1,4,5-trisphosphate production and D N A synthesis in these cells with significantly greater potency than any of the T x A agonists tested, effects 2  only partially inhibited by SQ 29548. In human T x A receptor cDNA-transfected cells, 2  competition by 15-F -isoprostane for specific [ H]SQ 29548 binding was negligible. 3  7 5  2t  These findings suggest the existence of distinct F -isoprostane binding sites, i.e., F 2  2  isoprostane receptors, although these receptors bear homology to the T x A receptor. The 2  existence of a unique receptor for F -isoprostane was further evidenced by radiolabeled 2  binding studies.  86  1.3.7.15-F -Isoprostane In Myocardial IRI 2t  Using GC-MS as a measure of 15-F -isoprostane, 2t  8 7  it was first documented  clinically in 1997 that urinary levels of 15-F -isoprostane increased during myocardial 2t  reperfusion in patients undergoing elective coronary artery bypass graft (CABG) surgery using cardiopulmonary bypass (n=5). However, due to the rapid clearance from the circulation of 15-F -isoprostane and its relatively delayed appearance in the urine,  8 1  2t  urinary levels of 15-F -isoprostane might not accurately reflect the acute changes of 2t  circulatory 15-F -isoprostane levels during myocardial IRI. Furthermore, the clinical 2t  19 relevance of the role of 15-F2 -isoprostane in myocardial IRI could not be addressed due t  to the limited number of patients studied.  Most recent studies  4 3  '  6 6  have shown that plasma levels of 15-F2 -isoprostane t  significantly increased within 3 min and continued for 50 min during CPB, or until 30 min after aorta de-clamping. Also, coronary endothelial and myocardial tissue 15-F 2t  isoprostane levels are increased during CPB in patients.  1 9  Furthermore, the decay  patterns of plasma 15-F -isoprostane are predictive the recovery of post-operative 2t  cardiac function.  4 3  These studies suggest that 15-F -isoprostane may play an important 2t  role in the pathogenesis of myocardial IRI.  1.4. Antioxidant Interventions Against Myocardial IRI  Theoretically, ROS scavengers, such as enzymatic scavengers, antioxidants, and iron chelators, may be useful therapeutic adjuncts to control or attenuate ROS-mediated myocardial IRI, as demonstrated by numerous experimental studies.  8 8 - 9 0  However,  enzymatic scavengers or antioxidants used experimentally are not all clinically applicable.  1.4.1. Antioxidant Vitamins C And E  Clinially, high-dose vitamin C (ascorbic acid) has been demonstrated to effectively scavenge ROS, decreasing cell membrane lipid peroxidation,  4 1 ; 9 1  indices of  20 myocardial injury, improving pulmonary endothelial function hemodynamics.  9 1  9 2  and post-operative  Controversy exists regarding whether endogenous vitamin E (alpha-  tocopherol) is depleted d uring CPB surgery.  5 0 ; 9 3 ; 9 4  Previous studies have shown that  vitamin E supplementation reduces plasma concentrations of hydrogen peroxide decreases cell membrane lipid peroxidation following CPB.  4 1  9 5  and  It has been demonstrated  that preoperative supplementation with a combination of vitamin E, vitamin C and allopurinol in patients undergoing C A B G  9 6  reduced cardiovascular dysfunction and  decreased the incidence of perioperative myocardial infarction.  Most recent clinical trials using a combination of vitamin E and C supplements in C A B G surgery revealed no detectable attenuation in peri-operative myocardial injury.  9 7  Of interest, long-term vitamin E supplementation failed to reduce lipid peroxidation in individuals at cardiovascular risk.  no  In summary, despite the general acceptance of ROS-mediated lipid peroxidation as an important contributor to myocardial IRI, clinically available antioxidant interventions have yielded no convincing clinical benefit.  1.4.2. Allopurinol  Allopurinol is an inhibitor of the enzyme xanthine oxidase, a pivotal generator of ROS during reperfusion injury.  9 9 ; 1 0 0  Studies have suggested that in the heart, xanthine  oxidase is a major source of free radical formation. decrease myocardial formation of cytotoxic R O S , ' 5 1  1 0 1  1 0 2  Allopurinol has been shown to to lower markers of myocardial  21 cellular injury, and to improve post-operative myocardial functional recovery following CPB.  9 6 ;  1 0 2  However, other studies have demonstrated no improvement in either 22  myocardial function or myocardial cellular injury with allopurinol,  casting doubt on its  therapeutic p otential, a lthough t heoretically i t s hould b e a good c hoice for a ntioxidant intervention. In some cases, differences in the nature of the allopurinol treatment regimen may explain these discrepancies. However, the lack of myocardial xanthine oxidase activity in some species, notably the human h e a r t ' ,03  104  , unlike the rat heart, may account  for some of the discrepancies regarding the effects of allopurinol on myocardial IRI. However, allopurinol has been shown to attenuate myocardial IRI of the rabbit heart that lacks xanthine oxidase  105  . This would suggest that mechanisms other than inhibition of  xanthine oxidase can contribute to the protective effects of allopurinol against myocardial IRI. 1.4.3. N-acetylcysteine  N-acetylcysteine has been used as a radical scavenger to prevent liver damage associated with acetaminophen overdose.  1 0 6 - 1 0 8  Recently, N-acetylcysteine has come into  use during cardiac surgery. High-dose N-acetylcysteine, before or during bypass surgery, appears to act as a free radical scavenger and reduce the neutrophil oxidative burst response.  1 0 9  However, the therapeutic potential of N-acetylcysteine in attenuating  myocardial IRI has yet to be proved.  1.4.4. Propofol  22 Propofol is an intravenous anesthetic that has antioxidant properties and is now commonly used for anesthesia to patients undergoing C A B G surgery. It may provide a promising antioxidant therapy for myocardial IRI in patients undergoing cardiac surgery utilizing CPB (see section 1.7 of this Chapter, as well as Chapters 3 and 4 for a detailed discussion).  1.5. Protective Effects And Limitations Of Ischemic Preconditioning On Myocardial IRI  It has been shown that repeated brief coronary occlusions increase myocardial resistance towards prolonged episodes of ischemia. This phenomenon, which renders the heart more tolerant to ischemia with subsequent limitation of infarct size, has been termed "ischemic preconditioning". It has been described in a variety of species, including humans.  110;111  "  115  Preconditioning may also protect the heart against  postischemic dysfunction and ventricular arrhythmias. Although the beneficial effects seem to be transient, they re-appear at 24 hours, representing a "second window of protection." Ischemic preconditioning has been considered one of the most powerful mechanisms of myocardial protection so far identified.  Ischemic preconditioning, however, has several disadvantages that may limit its clinical application. Firstly, brief periods of ischemia could induce numerous untoward changes in the myocardium, including some that may persist for days.  1 1 6  '  1 1 7  One of these  is referred to as the "stunned myocardium", which represents "prolonged postischemic  23 contractile dysfunction of myocardium salvaged by reperfusion." The mechanism of stunning is believed to involve generation of oxygen radicals as well as alterations in calcium homeostasis. Stunning has been observed in several clinical scenarios, including after percutaneous transluminal coronary angioplasty, unstable angina, stress-induced ischemia, after thrombolysis, and after cardiopulmonary bypass.  1 1 6 ; 1 1 7  It is likely that  classical ischemic preconditioning might not be sufficient to attenuate the stunning of surviving myocardium that renders the ventricle akinetic during early reperfusion.  118  Secondly, a recent study has shown that pathological conditions such as diabetes mellitus prevent ischemic preconditioning in patients with a first acute anterior wall myocardial infarction. ischemic  1 1 9  preconditioning  Similarly, oral sulfonylurea hypoglycemic agents prevent in human  myocardium,  and  may  cause  excess  cardiovascular mortality in these patients. Consequently, the application of ischemic preconditioning to diabetic patients, a high-risk population, is limited.  In addition, in patients with chronic coronary stenosis, their myocardium may have  already been in a naturally ischemic preconditioned status. It is, therefore,  questionable whether further ischemic preconditioning prior to cardiopulmonary bypass surgery in these patients would be beneficial or even detrimental.  1.6. Anesthesia A n d Myocardial IRI  The contribution of anesthesia to the course of myocardial IRI has not been adequately addressed. Controversies exist about the potential risks or benefits of  24 anesthetics on myocardial IRI in the clinincal setting. It is generally agreed that volatile anesthetics (such as isoflurane and sevoflurane) and intravenous anesthetics (e.g., propofol) reduce the myocardial damage caused by ischemia and reperfusion.  1 2 1  The  proposed mechanisms for protection by anesthetic agents may include an anesthetic preconditioning (APC) effect by volatile anesthetics propofol.  ,24  "  1 2 2 ; 1 2 3  and an antioxidant effect by  126  1.6.1. Inhalational Anesthesia  Laboratory studies have shown that volatile anesthetics such as sevoflurane and isoflurane can reduce post-ischemic myocardial infarct size.  1 2 7  Volatile anesthetics could  exert myocardial protective effects through an ischemic preconditioning-like mechanism (i.e. anesthetic preconditioning). It should be noted, however, that volatile anestheticmediated anesthetic preconditioning (APC) primarily requires the generation of ROS in advance (i.e., before ischemia) as a trigger of myocardial preconditioning.  1 2 7  1 2 8  Theoretically, the additional generation of ROS in a population with preexisting high degrees of oxidative stress, for example, chronic heart failure and diabetes, may stimulate increased mitochondrial permeability transition, releasing large amounts of ROS (involving radical-induced radical release).  1 2 9  This could overwhelm endogenous  antioxidant defenses, resulting in extensive lipid peroxidation and cellular destruction.  Indeed, experimental studies  I 3 0 ;  1 3 1  1 2 9  have shown that myocardial IRI can be  accentuated by the interaction of inhalational anesthetics and the xanthine oxidase system, a major source of free radical formation.  1 0 1  This is an important issue, given the  25 fact that during CPB surgery patients are already under oxidative stress resulting from dramatically increased ROS originating from the ischemic-reperfused heart and systemic circulation.  4 2  '  4 4  This systemic oxidative stress is likely to give rise to secondary  myocardial damage. In addition, isoflurane-induced preconditioning may be attenuated or prevented by diabetes or acute hyperglycemia, associated with increased post-operative  1 3 2  a pathological condition that is  cardiac dysfunction.  133  Of interest, one  experimental study has shown that N-acetylcysteine can restore isoflurane-induced preconditioning against myocardial infarction during hyperglycemia in rats (on a time limited basis, ie. less than 40 min of ischemia).  1 3 4  It is yet to be proven i f this could be  clinically applicable. Hence, the potential clinical utility of A P C may be limited and alternative approaches to A P C are required.  Sevoflurane, a new inhalational anesthetic  that is in clinical use, decreased superoxide during ischemia and reperfusion in the isolated guinea pig heart and attenuated post-ischemic myocardial dysfunction.  1 2 7  This is  promising. Further studies are needed to confirm the cardioprotective effects of sevoflurane in clinical settings. Taken together, these studies imply a preference for the use o f i ntravenous a nesthetic a gents i n s ettings where o xidative s tress m ay a lready b e elevated.  1.6.2. Intravenous Anesthesia  The intravenous anesthetic propofol may have potential benefit over isoflurane anesthesia in facilitating post-operative myocardial functional recovery.  1 2 5  However,  propofol, when administrated to achieve a plasma concentration between 2 to 4 ug/ml,  26 did not demonstrate greater effects than volatile anesthetic sevoflurane or desflurane in attenuating post-operative myocardial cellular damage using myocardial troponin I as a specific indicator.  1 3 5  '  1 3 6  The dosage and the timing of propofol application may need to  be adjusted in order to optimize cardiac protection.  1 2 4  1.7. Propofol: An Anesthetic and Antioxidant  Propofol is a short-acting intravenous anesthetic, which is highly lipid-soluble. It is widely used in both ambulatory and hospitalized patients and is increasingly used for cardiac anesthesia and for perioperative sedation.  1 3 7 - 1 3 9  Propofol permits both efficient  control of anesthetic depth as well as rapid and controllable recovery antioxidant activity  1 4 0 ; 1 4 1  139  . Propofol's  may have contributed to its protective effects in attenuating  myocardial IRI in animal models.  1 4 2 ; 1 4 3  1.7.1. Structure  Propofol, 2,6-diisopropylphenol (molecular weight 178.27), is chemically similar to phenol-based free radical scavengers such as the endogenous antioxidant vitamin E (alpha-tocophenol) (Figure 1.2).  1 4 1  '  1 4 4  A l l these compounds carry a hydroxyl substituent  on the phenyl ring, which is known to confer free radical scavenging properties  1 4 1  .  CH  3  CH  3  Vitamin E  Figure 1.2. Molecular structures of 2,6-diisopropylphenol (propofol) and alphatocopherol (vitamin E).  28  1.7.2. Basic Pharmacokinetics  Propofol anesthesia offers several advantages over other intravenous anesthetics, notably rapid onset and emergence, rapid return to spontaneous ventilation and reduction in time to tracheal extubation. ' 145  1 4 6  Administration of propofol, 2-2.5 mg/kg,  intravenously over 15 seconds or less, produces unconsciousness within about 30 seconds. Awakening is very rapid and complete, with minimal residual central nervous system effects - this is likely the most important advantage over other drugs used for anesthesia induction, especially for ambulatory patients.  After intravenous administration of propofol, distribution occurs with a half-life (ti/2a) of 2-8 minutes. The elimination half-life (ti/ (3) of propofol is approximately 30- 60 2  minutes. The drug is rapidly metabolized in the liver by conjugation to glucuronide and sulfate derivatives which are excreted in the urine.  14;  15;  1 4 7  Less than 1% of the  administered dose of drug is excreted unchanged in the urine.  The b lood c learance o f propofol i s greater than hepatic b lood flow, suggesting that tissue uptake and metabolism  l 4 8  '  1 4 9  contribute to the removal of this drug from the  blood. This property of propofol is useful in patients with impaired ability to metabolize other anesthetics.  29 Despite the rapid clearance of propofol by metabolism, there is no evidence of impaired elimination in patients with moderate cirrhosis  1 5 0  151  or renal dysfunction.  1 5 2  Patients older than 60 years of age exhibit a reduced plasma clearance of propofol. Therefore, there may be a modest cumulative effect in elderly patients receiving continuous intravenous infusions of propofol.  1.7.3. Effects Of Cardiopulmonary Bypass On Propofol Pharmacokinetics  Propofol is a weak organic acid that is bound extensively (97-98%) to plasma albumin, with a free fraction in the plasma of about 2-3%.  151  It is likely the unbound,  rather than the total plasma, concentration that is related to the anesthetic action of propofol. It has been reported that hemodilution during cardiopulmonary bypass (CPB) is associated with decreases in the concentration of plasma proteins, a decrease in total plasma propofol concentration disproportional to the decreases in the concentration of plasma proteins,  1 5 3  '  1 5 4  and an increase in the fraction of unbound propofol. ' 155  156  As a  result, the unbound concentration of propofol may remain relatively constant during CPB.  1 5 7  The decrease in total propofol concentration with the initiation of CPB is probably mainly a result of the hemodiluting effect of the priming solution in the extra-corporeal circuit. Binding of propofol to the extracorporeal circuit may be an additional factor in decreasing plasma concentration of propofol in patients undergoing C P B ,  155  since the  decrease of propofol concentration is up to 50% more than that predicted by hemodilution alone.  1 5 3 ; 1 5 4  30  During the later phase of CPB, total propofol concentration increases to prebypass values. This increase in propofol concentration may result from decreases in volume of distribution during hypothermia.  1 5 5 ; 1 5 8  The increase in the fraction of unbound propofol during C P B may be a consequence of heparin administration. The administration of heparin during CPB tends to increase the free fraction of propofol in plasma and these changes were reversed after protamine.  155  Heparin activates lipoprotein lipase, resulting in the hydrolysis of plasma  triglycerides into non-esterified fatty acids.  1 5 9 ; 1 6 0  Non-esterified fatty acids could then  competitively inhibit the binding of various drugs to the plasma proteins and may contribute to the increase in the unbond fraction of the drug  1 6 1  as noted above.  1.7.4. Propofol Antioxidant Activity  Tissue damage caused by the activity of reactive oxygen species (ROS) contributes to myocardial IRI.  1 6 2 ;  1 6 3 - 1 6 6  As a consequence of the action of ROS, lipid  peroxidation can occur. Under physiological conditions, the polyunsaturated fatty acids in the cell membrane are protected against lipid peroxidation by endogenous antioxidants including the lipid soluble chain-breaking antioxidants such as vitamin E (a-tocopherol). 167, 168  T  n  e  hydj-Qxyj (OH) group of a-tocopherol donates the hydrogen atom, a-  Tocopherol terminates the chain reaction of lipid peroxidation by scavenging lipid  31  peroxyl radicals (LOO') by a process of hydrogen donation. In this reaction, a-tocopherol itself becomes a phenoxyl radical which is much less reactive than LOO' (reaction 1).  a-tocopherol-OH + LOO' - » a-tocopherol-O' + L O O H  1 6 7  (reaction 1)  Propofol (2,6-diisopropopylphenol) also contains a phenolic OH-group (figure 1.2) and it has been reported to act as an antioxidant via the similar mechanism to atocopherol according to spin resonance spectroscopy,  1 4 1 ;  1 6 9  one of the most specific  methods for characterizing radical species . Propofol reacts with peroxynitrite (ONOO-), a reactive radical formed from the reaction of superoxide anion with nitric oxide, form a phenoxyl radical as demonstrated by electron spin resonance.  1 7 2  1 7 0 ;1 7 1  to  This might be a  very important aspect of protocol's antioxidant capacity, since peroxynitrite is more potent than superoxide anion or nitric oxide in mediating myocardial ischemiareperfusion injury.  4 7 ; 163  "  165;  173  "  175  Propofol has also been shown to directly and dose-dependently scavenge ROS generated either by stimulated human leucocytes or in cell-free systems,  1 7 6  as examined  using luminal chemiluminescence, a sensitive assay for monitoring free radicals and reactive oxygen metabolites produced by enzymes, cell or organ systems.  1 7 7 ; 1 7 8  A n in vitro study has demonstrated that propofol, when applied at a concentration of 8 u M , can completely suppress lipid peroxidation in isolated liver mitochondria from  32 rats.  1 6 9  Propofol's capability to inhibit mitochondrial lipid peroxidation may prove to be  an important mechanism of its protective effect against myocardial IRI. A recent study has shown that the capacity of mitochondria to produce both ROS and lipid peroxidation increases upon reperfusion in rat hearts subjected to ischemia and reperfusion.  1 7 9  1.7.5. Propofol Effects On IRI In Isolated Hearts  Propofol, at 25 and 50 uM, attenuated both mechanical dysfunction and metabolic damage induced by exogenous hydrogen peroxide in isolated rat hearts.  1 4 3  Hydrogen  peroxide significantly decreased left ventricular developed pressure (i.e., it produced mechanical dysfunction) and decreased tissue concentrations of A T P and creatine phosphate and also increased tissue lipid peroxidation. Propofol (25 u M and 50 uM) completely suppressed  hydrogen peroxide-induced tissue  lipid peroxidation and  significantly attenuated mechanical and metabolic alterations induced by hydrogen peroxide.  Propofol significantly improved functional and metabolic recovery in ischemicreperfused isolated rat hearts at 25 uM, 50 u M  1 4 3  or 100 u M  1 4 2  when the isolated hearts  were subjected to 15 or 25 min of global ischemia followed by 20 to 30 min of reperfusion. 11 is unknown whether propofol could exert cardioprotective effects i f the duration of global ischemia is prolonged.  33 However, when applied at 3 or 10 u M , propofol had no significant effect on myocardial function before or after global ischemia, nor did it suppress free radical formation in isolated rat hearts during reperfusion as measured w ith high performance liquid chromatography.  1 8 0  These concentrations of propofol (3 or 10 uM) are likely  lower than the concentration needed to attenuate coronary artery endothelial damage induced by inflammatory cytokines during myocardial ischemia and reperfusion. Recent studies have shown that apoptosis of coronary artery endothelial cells precedes and affects myocyte cell apoptosis in ischemia-reperfusion injury in the rat heart. Our recent 53  study h as d emonstrated that p ropofol s uppresses c ytokine-induced v ascular e ndothelial cell apoptosis at concentrations > 12. 5 u M  181  (see Appendix I).  1.7.6. Propofol Anesthesia In Cardiopulmonary Bypass Surgery  Propofol anesthesia has the advantage of rapid and complete awakening, resulting in early extubation after surgery and early discharge from the intensive care unit.  '  It  has been used widely since the early 1990's to produce total intravenous anesthesia in cardiac surgeries using CPB.  1 8 4 - 1 8 7  Hypotension is considered to be a major "adverse" effect of propofol anesthesia. This is a result of a decrease in peripheral vascular resistance.  1 8 8 - 1 9 0  However, propofol  did not significantly depress myocardial contractility as compared to the inhalational anesthetics isoflurane or enflurane during cardiac surgery.  34  Propofol significantly attenuated, but did not completely prevent, myocardial lipid peroxidation during C A B G surgery using CPB when it was infused continuously at 3-6 mg/kg/hr.  191  This dosage of propofol achieves plasma concentrations of up to about 1  5ug/ml (28 uM) based on a previous study in our laboratory.  ~) S  Interestingly, enhancing  red blood cell antioxidant capacity during CPB with propofol, an intravenous anesthetic with antioxidant properties, functional recovery.  140;  1 4 1  was related to improved post-operative cardiac  1 2 5  Recent studies have shown that 15-F2 -isoprostane is a vasoconstrictor in human t  saphenous veins and internal mammary arteries.  1 9 2  '  1 9 3  Taken together with the fact that  level of 15-F2 -isoprostane is increased during coronary reperfusion clinically, t  8 7  '  1 9 4  one  may postulate that 15-F2 -isoprostane produced at sites of free radical generation may t  play an important role in internal mammary artery and/or coronary spasm in situations of oxidant stress such as coronary bypass surgery, contributing to post-ischemic reperfusion injury. It can be hypothesized that 15-F -isoprostane may not only serve as a specific 2t  marker of oxidative myocardial damage, but also play a causative role in exacerbating myocardial IRI. One can further hypothesize that interventions aimed to reducing the formation, or antagonizing the action, of 15-F -isoprostane during myocardial ischemia2t  reperfusion should attenuate post-ischemic myocardial dysfunction.  The overall aims in this thesis were: 1) to investigate the role of 15-F2 t  isoprostane in myocardial ischemia-reperfusion injury during cardiac surgery, and, 2) to  35 develop a clinically applicable therapeutic regimen for propofol in order to effectively target ROS-mediated myocardial IRI. Furthermore, the potential mechanism(s) whereby 15-F2 -isoprostane may exacerbate t  explored.  post-ischemic myocardial dysfunction will be  36  CHAPTER 2 THE  RELATIONSHIP  BETWEEN  PLASMA  15-F -ISOPROSTANE 2T  CONCENTRATION AND EARLY POSTOPERATIVE CARDIAC FUNCTION FOLLOWING CARDIAC SURGERY  2.1. Preface  This investigation was supported by internal department development funding to Dr. D . M . Ansley from the Department of Anesthesia, University of British Columbia. A manuscript reporting the studies in this Chapter has been published in The Journal Thoracic and Cardiovascular Surgery 2003;126:1222-3  (Ref. ), co-authored with 43  D . M . Ansley and B.S. Dhaliwal. B.S. Dhaliwal contributed to plasma sample collection and clinical hemodynamic data analysis  2.2. Introduction  Warm heart surgery has changed the nature of cardiac surgery, and is commonly employed today.  195  Although the overall mortality rate has declined, significant morbidity  is still be associated with the procedure. The reported risk of early postoperative myocardial dysfunction is 6-25%.  1 9 6 ; 1 9 7  Low cardiac output syndrome is associated with  increased operative mortality, myocardial infarction rate, prolonged intensive c are unit and hospital lengths of stay.  196  This problem  reflects inadequate intraoperative  myocardial protection and/or incomplete revascularization.  37  Ischemia-reperfusion injury (IRI) is a major pathophysiologic factor.  This is a  complex phenomenon, whose mechanisms are incompletely understood. The generation of oxygen derived free radicals initiates a series o f events, which culminate in loss of cellular integrity and function.  Although well documented in animal studies using different methods, the role of oxidant stress in humans, particularly myocardial IRI, remains unclear. Part of the difficulty has been the lack of a sensitive and specific indicator of oxidative damage. The measurement of  F -Isoprostanes appears to be both a specific and sensitive index of 2  oxidative s tress status in vivo.  56;  isoP)  I 9 8 ;  1 9 9  O f special interest, 1 5-F -isoprostane (15-F 2t  2t  exerts biologic activity on the heart. Concentration dependent coronary  vasoconstriction, with parallel decreases in cardiac function have been documented in experimental models. ' 76  200  Evidence documenting the clinical relevance of oxidant stress in cardiac surgery is lacking. 15-F -isoP formation has been implicated in P T C A - associated complications 2t  such as coronary vasospasm, required.  201  but investigation of a role in myocardial stunning is  The relationship between 15-F -isoP formation and postoperative cardiac 2t  function, i f one exists, is unknown.  Our overall goal was to establish the role of 15-F -isoP in the pathogenesis of 2t  myocardial IRI, in an effort to develop a novel strategy to improve outcomes following  38 cardiac surgery. We hypothesized that the intraoperative level of plasma 15-F2 -isoP is a t  determinant of postoperative cardiac function. We tested this hypothesis in the setting of warm heart surgery.  2.3. Materials and methods 2.3.1. Patient data and surgical technique  Following institutional ethics board approval and written informed consent, 30 patients scheduled for C A B G (n=24) or combined valve replacement/CABG (n=6) procedures were enrolled in this study. Patients with a history of preoperative use of Vitamin C or E, acute or evolving myocardial infarction, preoperative hemodynamic instability, hepato-renal dysfunction, and age > 80 years, were excluded.  Cardiac catheterization was performed in all patients between 1 and 12 weeks preoperatively to determine the extent of coronary artery disease and left ventricular function. Left ventricular ejection fraction was measured by contrast ventriculography.  Patients continued their cardiac medications up to the time of surgery. Perioperative monitoring included five lead E C G for ST-segment analysis ( Marquette® Solar 8000) of leads 1,11,111,V , aVR, aVL,aVF. 5  diagnose perioperative myocardial ischemia. included  arterial,  central  venous  and/or  2 0 2  Standard criteria were utilized to Invasive hemodynamic monitoring  pulmonary  artery  determination of mean arterial, central pressures, and cardiac output.  catheterization  for  39 After median sternotomy and systemic heparinization, aortic and venous cannulae were inserted. P atients received epsilon aminocaproic acid (AMICAR®) 1 0 grams i n two divided doses prior to, and at the initiation of, cardiopulmonary bypass. cardiopulmonary bypass was established at a patient  temperature  (esophageal) with a non-pulsatile flow rate of 2 1/min/m  2  Full  of 34-37°C  and use of a membrane  oxygenator . T he h eart w as arrested b y antegrade i nfusion o f i ntermittent w arm h igh potassium blood cardioplegia (8:1 with Normosol R; potassium 20 meq/liter; mean, hematocrit approximately 80 g/L) via an aortic root catheter at 300 ml/min and continuous cross clamping of the aorta in all patients.  In 9 patients, additional  intermittent cold (10°C ) low blood potassium cardioplegia was subsequently applied. Intermittent w arm 1 ow b lood p otassium c ardioplegia w as applied i n 2 1 patients. L ow potassium cardioplegia was used throughout the procedure, unless electrical activity dictated the need to use the high potassium solution. Distal anastomoses were initially performed, then cardioplegia was delivered down the graft as each was completed. The last anastomosis in each patient was internal mammary to anterior descending coronary artery. Proximal anastomoses were performed during continuous aortic side clamping.  2.3.2. D a t a  a n d samples collection  A l l patient data were collected prospectively in our data base. Cardiac output was determined in triplicate, by the thermodilution technique prebypass, 30, 60, 120, 240 and 360 min following surgery. Cardiac index was derived from mean cardiac output.  40 Central venous blood sampling was conducted at baseline, 30 min global ischemia, and at 10, 30, and 120 min reperfusion. Blood was draw into Vacutainer® tubes containing E D T A , temporarily stored on ice (during operation room to laboratory transportation) and immediately centrifuged at 0°C to separate the plasma. The plasma was snap frozen with liquid nitrogen and stored at -70 °C until analysis.  Low cardiac output syndrome was defined by cardiac index < 2.2 L.min.m despite optimization of preload, afterload and heart rate.  1 9 6  2  The hemodynamic support  required to achieve a cardiac index greater than or equal to 2.2 1/min/m was recorded. 2  Inotropic support was defined as any use of dopamine > 4 (ig/kg/min, epinephrine > 0.04 (ig/kg/min, or milrinone 0.25-1.0 fag/kg/min, alone or in combination, for greater than 30 min duration during the first six postoperative hours.  2.3.3.15-F -IsoP enzyme immunoassay 2t  Unlablelled 15-F -IsoP (8-iso-PGF ) standard and the enzyme immunoassay 2t  2a  (EIA) kit were purchased from Cayman Chemicals (Ann Arbor, MI, USA). The EIA kit was tested for assay accuracy prior to direct assay for plasma free 15-F -IsoP 2t  concentration. The EIA procedures used to measure free plasma F -IsoP were according 2  to the methods provided by the manufacturer with a minor modification. In brief, plasma samples were removed from -70 °C storage and thawed on ice. For the assay, 50 (al standards and samples were first added in duplicate to the 96-well plate provided in the kit, followed by addition of isoprostane acetycholinesterase tracer and antibody. The prepared plates were then incubated for 18 hours at room temperature. On the next day,  41 the plates were washed 5 times with wash buffer, followed by addition of Ellman's reagent. After optimal development by using an orbital shaker, the plates were read at 405 nm, and the values of the unknowns were expressed as picograms per milliliter plasma. The plasma samples were re-analyzed in duplicate or triplicate after being diluted with phosphate-buffered saline (PBS) whenever the first direct assay values of free plasma 15-F2 -IsoP were above 250 pg/mL or inconsistent results occurred. t  2.3.4. Data analysis  Patient data were analysed as a whole (n=30) and by subgroups according to inotrope requirements for postoperative hemodynamic stabilization:  Group I (no  inotropes, n=7); Group II ( > 2 inotropes, n=6). A l l data are presented as mean ± S E M . Student t test was used to compare pre-anesthesia and during CPB patient data. Isoprostane data were c ompared b y A N O V A w ith B onferroni's c orrections ( GraphPad Prism; NCSS). One-way repeated measure A N O V A and Tukey's Multiple Comparison test was applied for within group comparison. Correlations were evaluated by the Pearson test. The differences were considered significant at P < 0.05.  2.4. Results  2.4.1. Patient profile and perioperative data  42 The patient demographic and perioperative data for thirty patients is presented in Table 2.1. Four patients were monitored with cental venous pressure only. Cardiac index data was complete in 26/30 patients. No perioperative myocardial ischemia was detected in any patient. Differences in maintenance cardioplegia techniques were not associated with differences in outcome variables.  Seven patients separated easily from CPB and remained stable postoperatively (group I). Six patients needed two or more inotropes for hemodynamic stabilization (group II). Patients in the two subgroups were demographically similar in terms of age (60.3 ± 2.3 vs 69.0±3.4 yr), gender (male/female, 5/2 vs 4/2), preoperative left ventricular ejection fraction (LVEF) (58.2±3.0 vs 55.7±6.3%), incidence of recent M I (one patient in group I vs three patients in group II) and dyslipidemia (five patients in group I vs four in group II).  There was no significant difference between group I and group II in duration of A C C , C P B , mean systemic oxygenation during surgery, hemoglobin content of cardioplegia, time between doses of cardioplegia, or systemic oxygen tension during CPB (Table 2.2).  2.4.2. Evaluation of the assay  To assess the linearity and accuracy of the assay, EIA buffer spiked with 15-F2tIsoP standard was assayed using serial dilutions. The intra-assay mean values (triplicate  43 assays) were 98%, 90%, 103%, 103% of the expected values of 7.8, 15.6, 62.5, 125 pg/mL, respectively. Values were 144% and 118% for the expected values of 3.9 and 250 pg/mL. The best assay linearity relationship exists between 7.8 to 125 pg/mL (R2 = 0.9998).  Three plasma samples of known concentrations of 15-F2 -IsoP (obtained after t  preliminary direct EIA assay in duplicate) were assayed six times on one sample plate, or assayed in 12 separate assays to assess intra-assay and inter-assay precision. The intraand inter-assay coefficient of variation was less than 9% or less than 14.7% when plasma free 15-F -IsoP was in the range of about 14 or 40 to 58 pg/mL, respectively (Table 2.3). 2t  Table 2.1. Demographic and perioperative data [mean ± SEM]  (N = 30) Age (yr)  67.7 ± 1.6  Sex (male/female)  22/8  Body surface area (m )  1.97 ± 0.04  N Y H A Class  II-IV  Pre-operative L V E F (%)  56.3 ± 2.2  Duration of A C C (min)  100.9+ 8.5  Duration of CPB (min)  132.1+ 10.6  Volume of cardioplegia (ml)  5874 ± 7 8 5  2  A C C = Aorta Cross-clamping CPB = Cardiopulmonary Bypass L V E F = left ventricular ejection fraction  Table 2.2. Perfusion data for the non-inotropes and > 2 inotropes group [mean ± SEM]  Non-inotrope  >2 inotropes  (N=7)  (N=6)  Ratio (x: 1)  7.6±0.4  8±0.0  Intra-op Hct  0.24±0.01  0.23±0.01  Plegia Hb (g/dL)  6.91+0.31  6.82±0.37  20.9±2.3  19.5±2.8  P02 of plegia (mmHg)  198.1±9.1  176.8+7.3  Duration of ACC(min)  112.7±31.3  108.5±16.1  Duration of CPB(min)  141.0±36.6  150.8±25.0  Plegia(blood:crystalloid)  Time interval for plegia (min)  A C C = Aorta Cross-clamping; CPB = Cardiopulmonary Bypass. P > 0.05 between groups for all parameters.  Table 2.3. Plasma free 15-F2t-Isoprostane assay precision  N Intra-assay Sample 1 Sample 2 Sample 3 Inter-assay Sample 1 Sample 2 Sample 3  Mean (pg/mL)  S D  C V ( % )  6 6 6  13.81 42.35 58.40  0.97 5.71 7.75  7.0 13.48 13.27  12 12 12  13.54 40.11 55.00  1.21 5.15 8.06  8.96 12.85 14.65  N: number of measures; SD: standard deviation; CV: coefficient of variation  47  2.4.3. Plasma free 15-F -isoprostane concentration 2t  The lowest detected plasma free 15-F2 -isoP concentration was 12 pg/mL at t  baseline in one patient. Eleven out of a total of 150 plasma samples were re-analyzed at different dilutions due to free 15-F2t-isoprostane values exceeding 250 pg/mL.  For the group as a whole (n = 30), plasma 15-F -isoP increased significantly 2t  during ischemia, and remained significantly elevated after 10 min of reperfusion (PO.001 vs baseline, Figure 2.1). 15-F2 -isoP did not differ from baseline at reperfusion t  30 min and onwards (P>0.05 vs baseline). There was no relation between plasma 15-F 2t  isoP and duration of A C C or CPB.  Figure 2.2 depicts the different patterns of plasma 15-F2 -isoP formation in group t  I and group II. Plasma 15-F2 -isoP in group I underwent exponential decay (11/2 = 71.4 t  min, Figure 2.3) after ischemia, and did not differ significantly from  baseline  at  reperfusion 10 min and onwards (P>0.05 vs baseline, Figure 2.2). In contrast, plasma 15F2 -isoP remained elevated during reperfusion in group II, with levels unchanged in 2 t  patients and increased in 4 patients between 10-30 min reperfusion. In 6/7 patients requiring no inotropes, the isoprostane concentration decreased primarily between 10-30 min reperfusion.  48  300-1 v c  Baseline Ischemia Rep-10 Rep-30 Rep-120 Sampling time  Figure 2.1. Changes of plasma free 15-F2t-lsoprostane during ischemiareperfusion. Values were mean ± S E M . N=30. Rep-10, Rep-30 and Rep-120 represent 10, 30, and 120 min of reperfusion. * P < 0.001 vs baseline. * P < 0.01 vs ischemia.  49  500400300200100- JL  i  Baseline Ischemia  Wi  Rep-10  I • Rep-30  Rep-120  Sampling time  Figure 2.2.  Changes of plasma free 15-F2 -Isoprostane during ischemia-reperfusion in t  non-inotrope and > 2 inotropes groups. Values were mean ± S E M . Light bar indicates non-inotrope group (N = 7), solid bar indicates > 2 inotropes group (N = 6). * P < 0.05, ** P < 0.001 vs baseline. P < 0.01 vs ischemia. Difference between groups was not #  significant (P > 0.05).  50  Figure 2.3.  Plasma free  15-F2 -Isoprostane t  in the non-inotrope  group  decays  exponentially after global myocardial ischemia-reperfusion. Values were the mean of seven (N = 7). Isoprostane decay half-life (tl/2) 71.4 minutes (Goodness of fit of the decay curve: R = 0.97. GraphPad Prism program). Average duration of ischemia =122 2  min.  51  Figure  free  2.4. Correlation between post-operative cardiac index (CI) and changes of plasma  15-F2 -Isoprostane during early reperfusion. CI was significantly negatively t  correlated with percentage increase of 8-F -isoprostane from reperfusion (rep) 10 to rep 2a  30 min (r = -0.73, 95%CI: -0.87 to -0.47, P < 0.0001). N = 26. CI data not available in 4 patients.  52  2.4.4.15-F2 -isoprostane and postoperative cardiac index t  A significant negative correlation was observed between postoperative cardiac index and percentage change in plasma 15-F -isoP concentration from 10 to 30 min 2t  reperfusion for n=26 patients (Figure 2.4, r = 0.8361; 95% CI: -0.9496 to -0.5258J? = 0.0004). Postoperative cardiac index did not correlate with baseline 15-F2 -isoP, nor the t  15-F -isoP concentration during ischemia (P > 0.1). 2t  2.5. Discussion  The aim of our study was to determine the relationship between plasma 15-F 2t  isoP generation and early postoperative cardiac depression following warm heart surgery. The principal findings include : 1) 15-F -isoP generation occurs despite application of 2t  warm blood cardioplegia; 2) the pattern of intraoperative 15-F -isoP generation varies 2t  between hemodynamically stable and unstable patients; 3) an inverse relationship exists between early postoperative c ardiac function and the percentage change i n 1 5-F -isoP 2t  during early reperfusion.  The use of warm heart techniques in C A B G surgery is associated with superior postoperative cardiac performance, reduced incidence of low cardiac output syndrome and increased tolerance to prolonged duration of aortic crossclamping, compared to  53 hypothermic protection and C P B .  203  '  2 0 4  This clinical practice is common and has  changed the outcome of cardiac surgery.  Low cardiac output syndrome is recognized as a high risk scenario complicating postoperative recovery of patients undergoing cardiac surgery. myocardial protection during warm heart surgery is important.  196  The approach to  The incidence of low  output syndrome increases beyond 8-10% when time between doses of cardioplegia exceeds 13 min, and/or hemoglobin content of cardioplegia is less than 8 g / d l . ' ' 97  204  The  mechanism(s) of cardiac dysfunction has yet to be elucidated. The systemic inflammatory response to cardiopulmonary bypass has been previously implicated. The direct cardiodepressant effects of pro-inflammatory cytokines, nitric oxide, or the vasoconstrictor substance endothelin-1 instability. ' ' 42  205  206  (ET-1)  are  associated  with  postoperative  Oxidant stress influences the release of these factors.  hemodynamic 207  In an effort to find a sensitive and specific in vivo marker of oxidant stress, recent attention has focused on the measurement of prostaglandin isomers known as isoprostanes .  5 6  '  1 9 8 ;  Isoprostanes are chemically stable end products of lipid  1 9 9  peroxidation formed in vivo, that circulate in plasma, exert biologic activity(coronary vasoconstriction, platelet activation and adhesion) and are excreted in the urine.  Using G C M S , Delanty and Reilly et al. reported an increase in urinary 8-epiprostaglandin F2 (15-F2 -isoP) during cardiac surgery or angioplasty. '  They used  194 208  a  t  sampling intervals different from our study.  They did not measure 15-F2 -isoP levels t  54 during the ischemic interval. Their results are limited because of the small patient sample size and the lack of documented clinical sequelae in response to 15-F2 -isoP formation. t  Iuliano et al provided first evidence of cardiac 15-F2 -isoP formation.  They  t  measured increased levels of F2-isoprostane in the coronary sinus blood of 12 patients during percutaneous transluminal coronary angioplasty. They suggested a potential role for isoprostane in PTCA- associated complications like vasospasm and myocardial stunning.  Clermont et al. recently described the systemic production of free radicals during hypothermic CPB in eleven patients with normal preoperative ejection fraction.  44  The  pattern and amount of free radical activity measured in peripheral and in coronary sinus blood during the ischemia and early reperfusion (25 min) was characterized by ESR spin trapping. The amount of oxidant stress was correlated to the duration of CPB and was associated with a decrease in plasma antioxidant status despite adequate systemic antioxidant reserve. They concluded that systemic oxidant stress during CPB participates in myocardial damage. The clinical effect of oxidant stress on cardiac function was not evaluated.  Our study characterizes isoprostane formation predominantly during global ischemia, subject to variation in exponential decay (metabolism/excretion ) during the course of surgery.  Our findings demonstrate that 15-F -IsoP formation is clinically 2t  significant, correlating inversely with early postoperative myocardial function when it  55 remains e levated f ollowing s urgery. W e o bserved a n a verage t ime o f 1 9 m in b etween doses of cardioplegia containing slightly less than 80g/L hemoglobin. This may explain the decline in postoperative cardiac index observed in 20% of our patients. Since these factors were similar between subgroups, our findings suggest that the recovery of postoperative cardiac function may be related, at least in part, to isoprostane formation and its elimination. The half-life of elimination of 15-F2 -IsoP is relevant, reflecting the t  duration of early postoperative myocardial depression previously characterized by Breisblatt et a l . We feel it is significant that cardiac depression occurred without any 16  evidence of perioperative myocardial infarction. Whether isoprostane acts directly or is involved in another mechanism of cardiac depression, is beyond the scope of this study but is the subject of current investigation.  GC-Mass spectrometry is considered the "gold standard" for 15-F2 -IsoP analysis, t  but is expensive and technically difficult.  209  The availability of the instrument and the  need to analyse large amounts of clinical material has limited its clinical application. Enzyme immunoassay (EIA) is more readily available, but the use of EIA to measure isoprostane is controversial. The accuracy of E I A has been questioned, due to the potential for cross-reactivity of antibodies in immunoassays, differential metabolism of different isomers, and differential loss during sample preparation.  The use of EIA for 15-F2 -IsoP has recently been validated by GC-Mass t  80  spectrometry and the results from both methods were significantly correlated.  The  measurement  via  of  free  15-F2 -IsoP t  has  been  performed  and  validated  56 210  radioimmunoassay of unextracted plasma.  We used the same rabbit 15-F2 -IsoP t  antibody to directly measure 15-F2 -IsoP in unextracted plasma. This approach to E I A t  yielded reliable and acceptable results. It is important to note however, we found that the EIA result is most reliable within a narrow range (7.8 to 250 pg/mL).  Plasma free F -IsoP is about one third of the total F2-Isoprostanes esterified to 2t  lipoproteins in humans.  71  The baseline free plasma 15-F2t-IsoP levels we measured are  comparable to the results measured in healthy adults by Mori and colleagues who used 209  capillary gas chromatography/electron capture negative ionization mass spectrometry (GC-ECNI-MS), and Iuliano and colleagues during P T C A .  201  Our findings are in  keeping with 15-F2 -IsoP being the most abundant isomer in plasma. Direct comparison t  of clinical results measured with EIA and G C M S is inappropriate, as they do not measure exactly the same compounds and the presence of co-migrating substances in GC/MS analysis may produce incorrect results. ' 211  212  Therefore, our data is meaningful in terms  of the patterns we identified.  We relied on central venous samples for our assay, w hich makes it difficult to localize the site of origin for 15-F2 -IsoP formation. The levels we measured are similar t  to those measured in coronary sinus blood by Iuliano et al,  suggesting cardiac  formation. In a separate pilot study mimicking conditions of C A B G in a non-working rat heart model, we found the ischemic myocardium to be a source of 15-F2 -IsoP t  213  formation.  While this does not exclude systemic formation of 15-F -IsoP, we suspect 2t  57 that myocardium is a major source, at least during reperfusion. Cardiac endothelium and coronary plaque are likely sites of origin.  78; 7 9  15-F2t-IsoP formation occurred in a manner unrelated to the duration of CPB. Alterations in its formation, metabolism and elimination during CPB, by as yet unknown mechanisms, may explain these effects. F2 isoprostanes are metabolized by the liver to a dinor metabolite, or cleared by the kidney. While we excluded patients with pre-existent hepato-renal dysfunction, we can not exclude that a prolonged low flow or cardiac output state could reduce the clearance of 15-F2rIsoprostane and lead to accumulation in plasma.  2.6. C o n c l u s i o n  In  summary,  we studied perioperative  15-F2 -IsoP formation in patients t  undergoing C A B G surgery. To the best of our knowledge, we provide first evidence that 15-F -IsoP formation and metabolism may be a factor in postoperative recovery of 2t  cardiac function. Although elevated primarily during ischemia, the pattern of 15-F2t-IsoP degradation d uring r eperfusion appears c linically r elevant.  Besides b eing a m arker o f  oxidant stress, 15-F2 -IsoP may be a prognostic indicator of postoperative cardiac t  dysfunction.  Based on our results, persistent intraoperative elevation of 15-F2 -IsoP t  during reperfusion is associated with increased need for hemodynamic stabilization following C A B G surgery in humans. This occurs independent of the timing and hemoglobin content of cardioplegic protection, two factors in low cardiac output syndrome. A role for 15-F2t-IsoP in the pathogenesis of myocardial IRI is suggested.  58 Postoperative myocardial ischemia was not evident. The mechanism of cardiac depression has yet to be determined.  Determination of the mechanism(s) will be  important in the development of new therapies that prevent or treat postoperative cardiac depression.  59  CHAPTER 3 DOSE-DEPENDENT PROTECTION OF PROPOFOL IN MYOCARDIAL IRI IN RATS: EFFECTS ON 15-F -ISOP FORMATION 2T  3.1. Preface  A manuscript reporting s tudies described in this Chapter has been published in 213  •  Canadian Journal of Physiology and Pharmacology 2003;81:14-21 (Ref.  ) and is co-  authored with D . V . Godin, T. K . Chang and D. M . Ansley. T. K . Chang provided technical assistance with the assay for 15-F2 -isoprostane. t  3.2. Introduction  Oxidant stress has been implicated in myocardial ischemia/reperfusion injury (IRI) and reactive oxygen species (ROS), such as the superoxide anion and hydroxyl radicals, whose formation increase during reperfusion, have been implicated. " 214  217  The  development of an effective antioxidant therapy for the treatment of myocardial IRI is of interest. However, study of the effects of "traditional" antioxidants vitamin E and C supplementation has yielded no beneficial effects in humans with major cardiovascular risk factors,  9 8  nor reduced myocardial injury after cardiac surgery.  9 7  60 Previous work from our laboratory has demonstrated that the intravenous anesthetic propofol enhances red cell and tissue antioxidant capacity both in vitro and in vivo. '  125 218  It is unknown, however, i f enhancing myocardial antioxidant capacity with  propofol protects against the oxidant stress associated with ischemia/reperfusion damage. A recent study reported that propofol failed to reduce ROS formation during reperfusion in the isolated reperfused rat heart.  1 8 0  The concentration of propofol used, however, may  not have been sufficient to enhance myocardial antioxidant capacity.  The purpose of the present study was to determine the effect of propofol on 15F2 -IsoP generation and functional recovery during myocardial ischemia-reperfusion in an t  isolated rat heart model and to explore a novel approach to antioxidant enhancement.  3.3. Materials and methods  3.3.1. Heart preparation  The study was approved by the Committee of Animal Care of the University of British Columbia. Animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. Male Sprague-Dawley rats (250~300g) were anesthetized with pentobarbital (70mg/kg intraperitoneally) and heparinized with sodium heparin (1000 IU/kg, intraperitoneally). After  median  thoracotomy, hearts were quickly excised and immersed in ice-cold Krebs-Henseleit (KH) solution to stop contractions.  Hearts were gently squeezed to remove residual  blood to prevent clot formation. Hearts were retrogradely perfused via the aorta in a non-  61 working "Langendorff' preparation at a constant flow rate of lOml/min using a peristaltic pump. The perfusion fluid (pH 7.4; temperature, 37°C) was K H solution that contained: 1 2 0 m M N a C l ; 2 0 m M N a H C O ; 4 . 6 3 m M K C l ; 1 . 2 m M M g C l ; 1 . 2 5 m M C a C l ; 1.17 3  2  2  m M K H PO4; 8 m M glucose. The perfusate was bubbled with a mixture of 95% 0 and 2  2  5% C 0 . The perfusate solution and the bath temperature were maintained at 37°C using 2  a thermostatically controlled water circulating system. Coronary perfusion pressure (CPP) was measured via a side arm of the perfusion cannula connected to a pressure transducer (Statham p23 ID, Gould Electronics, Cleveland). A latex water-filled balloon fixed to a pressure transducer was inserted through the mitral valve into the left ventricle for the determination of left ventricular (LV) developed pressure (LVDP), which was calculated by subtracting end-diastolic pressure (LVEDP) from L V peak systolic pressure (LVSP). Rates of maximum L V pressure development and relaxation (positive dp/dt and negative dp/dt, respectively) were calculated with a differentiator. L V E D P was adjusted to approximately 5 mmHg before the start of the experiment by adjusting the volume in the intraventricular balloon. Hearts were perfused within 30 to 40 seconds after excision. Exclusion criteria included heart preparation times longer than 60 seconds and/or L V S P lower than 70 mmHg after 10 min of equilibration.  3.3.2. Experimental Protocol  A l l hearts were initially equilibrated for 10 min (BS10), then they were randomly allocated one of the three experimental groups (n=6 each):  ischemia-reperfusion  untreated control group (control), low-concentration propofol group (lo-P) and highconcentration propofol group (hi-P). A sham perfused group (Sham, n=6) was included  62 to test i f coronary effluent 15-F -IsoP levels change over time during the period of 2t  observation for 15-F -IsoP. After BS10, propofol was applied for 10 min at 5 ixg/ml (lo2t  P) or 12 |o.g/ml (hi-P) before inducing global ischemia for 40 min by stopping perfusion flow. Control hearts were equilibrated for another 10 min before inducing global ischemia for 40 min. During ischemia, saline (control) or lo-P or hi-P in saline was perfused through the aorta at 60 ul/min using a mini-pump. K H was perfused during 90 min of reperfusion in the control group. Either lo-P or hi-P in K H was perfused for the first 15 min of reperfusion. To minimize the direct negative inotropic effects of propofol 219  on myocytes,  lo-P in K H was applied during reperfusion for 75 min in both groups.  The perfusion flow rate (10 ml/min) was based on the result of a pilot work which showed  that hearts sham-perfused  without  ischemia beat well  and  remained  hemodynamically stable for 150 min (the duration of the experiment) in our experimental set-up. It has been previously shown that up to 300 n M 15-F -IsoP had no effect on 2t  coronary flow in sham-perfused isolated rat hearts  7 7  and therefore we did not include this  experiment in our study. Hearts were electrically paced at a rate of 300 beats/min, prior to and following, but not during the ischemic period when hearts ceased to beat spontaneously.  Baseline effluent perfusate was sampled at BS10.  Effluent samples during  ischemia were collected during the first 30 min of ischemia (1-30). Also, effluent was sampled at 0.5 (Re-0.5), 5 (Re-5), 10 (Re-10) and 30 (Re-30) min of reperfusion in control and propofol-treated groups. Coronary effluent was sampled at the corresponding time points in the sham group. The effluent samples were stored at -70 °C until analysis  63 for free 15-F -IsoP. L V function was continuously monitored using a polygraph. At the 2t  end of the 90 min reperfusion period, hearts were immediately removed from the cannula, frozen with liquid nitrogen and stored at -70 °C. Hearts were assayed for tissue thiobarbituric acid-reactive substances (TBARS) following in vitro exposure to the peroxidizing agent t-butylhydroperoxide (f-BHP) within 48 hours of storage.  3.3.3. Heart tissue antioxidant capacity determination  Myocardial tissue antioxidant capacity was determined by exposure of tissue homogenates to the peroxidizing agent f-BHP. The oxidation of tissues by J-BHP results in the formation of numerous lipid byproducts, which form a chromogen when incubated with thiobarbituric acid (TBA) and are therefore collectively termed TBARS. Lower tissue antioxidant capacity will result in a greater amount of T B A R S formation in the presence of f-BHP. The level of TBARS in the sample is estimated from the absorbance at 532 nm. Heart tissue samples (300 mg) were thawed and homogenized on ice in 3 ml Tris-EDTA buffer using a Polytron homogenizer for 30 s at 25% power. The resulting homogenates were used for in vitro forced peroxidation using ?-BHP and subsequent determination of T B A R S , as previously described.  217  In brief, 400 p.L of tissue  homogenate was combined with 400 uL f-BHP (in 0.9% saline/2 m M sodium azide to produce final concentrations of f-BHP ranging from 0.5 to lOmM). These suspensions were incubated for 30 m i n at 37 °C, and then 400 \\L of cold 28% (w/v) TCA-0.1 M sodium arsenite was added. The mixture was centrifuged at 12,000g for 5 min at 4 °C, and 800 jaL of supernatant was removed and added to 400 uL of thiobarbituric acid  64 (0.5% in 25 m M NaOH). The samples were boiled for 15 min, and the absorbance at 532 nm was measured spectrophotometrically.  3.3.4.15-F2t-IsoP Assays  Enzyme-linked immunoassay (EIA) was used to measure free 15-F2 -IsoP levels t  according to the methods provided by the manufacturer (Cayman Chemical, Ann Arbor). EIA provides a sensitive measure for 15-F -IsoP with a limit of quantification as low as 2t  3.9 pg/ml. In brief, effluent samples were removed from -70 °C storage and thawed on ice. Fifty uL standards and samples were added in duplicate to the 96-well plate provided in the kit, followed by addition of 15-F2 -IsoP acetycholinesterase tracer and antibody. t  The prepared plates were then incubated overnight at room temperature. On the next day, the plates were washed 5 times with wash buffer, followed by addition of Ellman's reagent. After optimal development, the plates were read at 405 nm, and the values of the unknowns were expressed as picograms 15-F2 -IsoP per milliliter effluent. The samples t  were coded and the investigator responsible for 15-F2 -IsoP assays was blinded until the t  completion of the assay.  3.3.5. Statistical analysis  A l l data are presented as mean + S E M . 15-F -IsoP and hemodynamic data were 2t  compared by two-way A N O V A with Bonferroni's correction (GraphPad Prism). The  65 correlation between 15-F2 -IsoP concentrations and myocardial function was evaluated by t  the Pearson test. PO.05 was considered statistically significant.  3.4. Results 3.4.1.15-F2 -IsoP generation during ischemia-reperfusion t  As shown in Figure 3.1, baseline (BS10) 15-F2 -IsoP values did not differ among t  groups. 15-F2 -IsoP levels increased during ischemia (P < 0.01 vs BS10) and remained t  elevated at Re-0.5 (P < 0.05 vs BS10) in all the three experimental groups. Propofol reduced effluent 15-F -IsoP release during ischemia and the early phase of reperfusion. 2t  During ischemia, 15-F2 -IsoP levels were higher in the control than in the lo-P (P< 0.01 t  vs control) and hi-P groups (PO.05 vs control) groups. At Re-0.5, effluent 15-F -IsoP 2t  concentrations in the control group (22.5 ± 2.5 pg/ml) were also significantly higher than the corresponding values in the lo-P (14.6 ± 1.8 pg/ml, P < 0.05) and hi-P (12.0 ± 2.3 pg/ml, P < 0.05) groups. Levels of 15-F -IsoP decreased rapidly after reperfusion in the 2t  ischemic/reperfused groups and these were not statistically differ from baseline values at Re-5 (P > 0.05). 15-F -IsoP did not change over time during the observation period in the 2t  sham group.  3.4.2. Tissue antioxidant capacity  66 Heart tissue T B A R S formation following in vitro peroxide challenge (as reflected by absorbance at 532 nm) was significantly higher in the control group than in the propofol treatment groups (Figure 3.2) at I m M £-BHP, a concentration that provides a sensitive measure of rat tissue T B A R S .  217  The final £-BHP concentration of 1 m M was  considered critical as it is a compromise between concentrations sufficiently high to produce adequate levels of TBARS but sufficiently low to avoid non-specific bleaching of the color produced by the T B A reaction. Tissue antioxidant capacity was found to be lower in the control group. Tissue TBARS formation in hi-P was lower than that in lo-P (P < 0.05).  67  100-  a 0. o  m\m Control V7777A LO-P  75-  • Hi-P F=lSham  (0  50-  c  o =  LU  25-  La. BS10  Ische  la.  Re-0.5  [Qi. Re-5  Re-10  Re-30  Time during ischemia-reperfusion  Figure 3.1. 15-F -isoprostane (15-F -IsoP) release during ischemia and reperfusion. 2t  2t  BS10 indicates 10 minutes after equilibration; Ische indicates ischemia, samples were collected during the first 30 minutes of ischemia; Re-0.5, Re-5, Re-10 and RE-30 indicate 0.5, 5, 10 and minutes after reperfusion respectively. *P<0.05 or P O . O l vs control; +  P<0.05 or P O . O l vsBSlO.  68  Figure 3.2. Formation of thiobarbituric acid reactive substances (TBARS), a measure of tissue antioxidant capacity, in heart tissue (represented as absorbance at 532 run) in the presence of 1 m M t-butylhydroperoxide. Hearts were assayed for in vitro TBARS formation after 90 min of reperfusion following 40 min of ischemia. * P < 0.001 vs control; P < 0.05 vs Hi-P group. +  mmm Control ^^Lc-P • Hi-P  A 40-  30ef 20-  I  10-  I  15  IML. 20  25  30  35  Duration of ischemia (min)  40  B 100-, Control Lo-P Hi-P  E  B  *+ 25-  5  in.  i r i  i-  i n . 10 30 60 Duration of reperfusion (min)  i 90  Iri  Figure 3.3. Effect of Propofol on left ventricular end-diastolic pressure (LVEDP), reflecting myocardial contracture (ventricular stiffness), during ischemia (A) and reperfusion (B). Values were mean ± S E M , n=6 for all groups. *P<0.05, or P<0.01 control. P<0.05 Lo-P vs Hi-P. +  70  3.4.3. Contracture development during ischemia Increases in L V E D P were indicative of contracture (ventricular stiffness) of isolated hearts during ischemia ("ischemic contracture").  The L V E D P  increased  progressively during ischemia in the control group (Figure 3.3A). Propofol at the high (12 ia,g/ml) but not at the low (5 |ig/ml) concentration, reduced L V E D P (P < 0.05, hi-P vs control at 35 and 40 min ischemia). At 35 min of ischemia, the magnitude of L V E D P in the hi-P group was also less than in the lo-P group (7.3 ± 3.3 vs 26.5±7.1 mm Hg, P < 0.05). In both lo-P and hi-P groups, the latency to the onset of contracture was significantly increased (21.2 ± 2.1 and 24.5 ± 2.3 min in lo-P and hi-P, P < 0.05 vs 15.0 ± 1.1 min in control).  3.4.4. Functional response to ischemia -reperfusion  One of the main effects of propofol on functional recovery during reperfusion was a difference in L V E D P (an indicator of reperfusion-induced increase in "ventricular stiffness"). During reperfusion, L V E D P in the control group increased over time and peaked at 65.7 ± 10.4 mmHg at 90 min of reperfusion (Re-90) (Figure 3.3B). The low concentration of propofol attenuated the increase of L V E D P at 60 min of reperfusion (Re-60) and Re-90 (P < 0.05 vs control). The magnitude of L V E D P was less in the hi-P than in the lo-P group after Re-30 (P < 0.05, hi-P vs lo-P). In fact, the high concentration of propofol completely prevented the increase in ventricular stiffness during the experiment.  71 The L V D P in the untreated control group recovered to a maximum of 78.2 + 7.9 % of its baseline value at Re-30 (Table 3.1) and decreased progressively thereafter. At Re-90, L V D P in the control group was lower than its baseline value (P < 0.05, Table 3.1). The low concentration propofol prevented the progressive decrease of L V D P seen in the control group after Re-30. Application of hi-P for 10 min prior to ischemia and during the first 15 min of reperfusion was associated with a significant decrease in L V D P and L V S P prior to ischemia and at Re-10. However, L V D P rapidly recovered to baseline values after Re-30 in the hi-P group (Table 3.1), and was about 25% higher than that in the lo-P group at Re-30. The rapid recovery of L V D P observed in hi-P coincided with the reduction in propofol concentration from 12 jag/ml to 5 p.g/ml after 15 min of reperfusion. The L V D P , as a percentage of baseline values at Re-90, was significantly higher in the hi-P (P < 0.01) and lo-P groups (P < 0.05) than in the controls, despite the fact t hat p ropofol at 5 \xg/m\ m ay h ave i nhibited m yocardial c ontractility. P ropofol (5 p,g/mi) inhibition of myocardial contractility was evidenced by the significant decrease of L V D P from baseline to pre-ischemia in the lo-P group (Table 3.1).  Since one of the major goals of this study was to optimize the long-term protective effects of propofol, administration of propofol was not discontinued during reperfusion. This mimicked the clinical study of propofol used as the principal anesthetic and also possibly to increase myocardial antioxidant status during cardiopulmonary *  bypass surgery, and then continued at reduced dosage for post-operative sedation.  125  •  This  approach however, makes it inappropriate to compare post-ischemic L V D P values in the propofol treatment groups directly with that in the control group because of the potential confounding negative inotropic effect of propofol. The within-group percentage changes  72 of L V D P from Re-60 to Re-90 (the later phase of reperfusion in this study) best reflected the post-ischemic myocardial function preservation at different experimental conditions and served as a meaningful index for comparison between groups. The percentage decrease of L V D P from Re-60 to Re-90 in hi-P (-3.0±2.6%) was less than that in either control (-19.1±6.6%, P < 0.05) or in lo-P (-11.412.5%, P < 0.05). The value for lo-P did not differ statistically from that of the control group (P > 0.05).  After 90 min of reperfusion, L V relaxation (-dp/dt) in the control group was 44.2±4.7% of baseline, whereas L V development (+dp/dt) was 65.7±10.4% of its baseline (BS10) v alue. Recovery as a p ercentage o f t he b aseline (BS10) v alue f or t he rates of L V -dp/dt paralleled the responses observed with L V D P in the hi-P, lo-P and control groups. Hi-P, but not lo-P, also resulted i n significantly better recovery o f L V +dp/dt (P < 0.05) at Re-90 compared to the control group.  3.4.5. Coronary perfusion pressure  Coronary perfusion pressure (CPP) increased significantly after 60 min of reperfusion in the control and lo-P groups (P < 0.05 vs BS10, Table 3.1). The low concentration of propofol did not significantly reduce CPP during reperfusion (P > 0.05 vs control). However, the high concentration of propofol reduced CPP by 2 0% before ischemia (Table 3.1) and prevented the significant increase of CPP after reperfusion seen in all the other groups. The CPP value at Re-90 in the hi-P group was lower than that in the control (P < 0.05).  73  Table 3.1. Left Ventricular Developed Pressure (LVDP), L V Systolic Pressure (LVSP) and Coronary Perfusion Pressure (CPP) (mm Hg)  BS10  BS20  86.6 ±5.2  95.6 ±4.8 110.9 ±3.9  Re-10  Re-30  Re-60  Re-90  49.0 ±15.0*  67.3 ± 6.2  57.5 ±6.7  47.1 ±8.4*  57.3 ± 17.2  78.2 ±7.9  66.7 ± 7.7  54.3 ± 8.0  61.3 ± 1.9*  41.7 ±11.7*  56.5 ±12.0  69.0 ±7.7  60.3 ±4.0  77.1±2.0*  52.6 ± 14.7  73.9 ± 11.5  86.3 ± 8.5  75.8 ±4.3*  42.3 ±7.1**  32.5 ±6.1*  76.5 ±7.4  67.5 ±7.0  66.0 ±8.3  53.2 ±7.2*  41.5 ±7.4  97.6 ±6.4  85.9 ± 4.9*  83.8 ± 6.5*  85.2 ± 6.5  106.8 ±3.6  116.5 ±5.8*  112.8+ 5.0  LVDP Control %BS10 Lo-P  80.0 ±4.1  %BS10 Hi-P  78.2 ± 5.1  % BS10 LVSP Control  91.7 ± 5.3  100.5 ±4.8  Lo-P  84.8 ±4.0  65.7 ± 2.0**  59.2 ± 5.7**  77.0 ± 8.8*  92.8 ± 5.7*  87.5 ± 5.7*  Hi-P  83.0 ±5.1  46.8 ± 6.9**  38.7 ± 6.2**  82.8 ±7.1*  73.5 ± 7.2*  73.2 ± 9.2*  CPP Control  57.2 ± 6.6  58.3 ±6.9  60.0 ± 6.8  79.7 ± 11.2  95.7 ± 17.0*  103.8 ±14.7*  Lo-P  52.7 ± 1.6  48.5 ±2.6  50.5 ± 1.4  64.2 ±4.9  79.2 ± 11.2*  86.7 ± 10.6*  Hi-P  50.8 ±1.54  40.5 ± 2.5*  45.0 ±4.4  52.5 ±3.9  54.8 ±5.1  62.0 ± 6.9*  Values are mean ± S E M (n=6 for each group). BSlO^baseline, BS20=pre-ischemia; Re10, Re-30, Re-60 and Re-90 refer to 10, 30, 60 and 90 min after reperfusion. There is no significant difference in BS10 among groups for L V D P , L V S P and CPP. *P<0.05 or P O . 0 1 , vs control ; P<0.05 or PO.01 vs baseline. +  1  74  £  180  • Hi-P  |  160  OLo-P  ~  140  A Control  £  120  o  at  c 100 o '3 80  t  60  £ 40 TO 20 Q. Q0 O 10  100  15-F2t-lsoP ar reperfusion 0.5 min (pg/mL, log scale)  Figure 3.4. Relationship between 15-F2 -IsoP generation upon reperfusion and coronary t  perfusion pressure (CPP) at 90 minutes of reperfusion (n=18). CPP was positively correlated (r=0.74, 95%CI: 0.4180 to 0.8972, P=0.0004) with 15-F -IsoP generation 2t  upon reperfusion.  75  3.4.6. Lipid peroxidation and post-ischemic myocardial function  Figure 3.4 depicts a strong positive correlation between effluent 15-F2 -isoP levels t  at Re-0.5 and CPP at Re-90 (r = 0.74, P = 0.0004). A weak, but significant negative correlation was obtained between L V D P recovery at Re-90 as a percentage of baseline and effluent 15-F ,-isoP levels at Re-0.5 (r = -0.585, P = 0.011). In general, effluent 152  F2 -isoP levels at Re-0.5 correlated with tissue TBARS formation at Re-90 (n=18, r=0.66, t  P<0.003), but this association resulted primarily from a good correlation between 15-F2 t  isoP release and tissue T B A R S formation in the propofol treatment groups (n=12, r =0.74, P=0.005). On the contrary, two control hearts that released the least 15-F2 -isoP at t  Re-0.5 in the group ended up with highest tissue T B A R S formation (indicating lowest tissue antioxidant capacity preservation). Taking into consideration the immediate and rapid increase in CPP upon reperfusion in these two hearts, the lower release of 15-F r 2  isoP at Re-0.5 most likely resulted from the sequestration of 15-F -isoP in the 2t  inadequately (or non-) reperfused tissue.  A negative correlation was obtained between heart tissue T B A R S formation and L V D P recovery at Re-90 as a percentage of baseline (r= -0.71, Figure 3.5), and especially between tissue T B A R S formation and changes of L V D P from Re-60 to Re-90 (r = -0.72, P =0.0007, Figure 3.6), which best represented post-ischemic myocardial function preservation in this model.  76  120.0  A Control OLo-P • Hi-P  c  1 o o>  a> t=tu in ra  100.0  'oo  • 0  80.0  •  0  60.0  Q.-Q  ra  40.0  a.  a >  20.0  0.0 0.05  0.1  0.15  0.2  0.25  0.3  Tissue TBARS absorbance at 532 nm  Figure 3.5. Recovery of left ventricular developed pressure (LVDP) after 90 min of reperfusion inversely correlated with the formation of heart tissue thiobarbituric acid reactive substances (TBARS), a measure of tissue antioxidant capacity (r = -0.71, 95% CI: -0.8837 to -0.3629, P = 0.001). Heart tissue was assayed for in vitro T B A R S formation after 90 min of reperfusion following 40 min of ischemia, in the presence of I m M t-butylhydroperide.  77  • Hi-P  10.00  11 -10.00 n.  OLo-P  •ft  0.00  0.05  0.1  • Control 0.15  A  CJ^  0.25  0.3  o  Q »> > O _J ~ -20.00  *- °  O  A  o «>  w = -30.00  a) o  2>w 1 ^ O a  -40.00  v -50.00  a:  Heart tissue TBARS Absorbance at 532 nm  Figure 3.6. Changes of left ventricular developed pressure (LVDP) after reperfusion (from reperfusion 60 to 90 min) inversely correlated with the formation of heart tissue thiobarbituric acid reactive substances (TBARS) in the presence of 1 m M tbutylhydroperide (r = -0.72, 95% CI: -0.8898 to -0.3672, P = 0.0007). It indicated a positive correlation between post-ischemic preservation of myocardial function and tissue antioxidant capacity.  78  3.4.7.  Pre-ischemic myocardial depression and post-ischemic myocardial function  Administration of hi-P (12 u.g/mL) and lo-P (5 p.g/mL) for 10 min before ischemia decreased myocardial c ontraction as evidenced by the significantly lower pre-ischemic L V D P values in the propofol treatment groups than in the control (Table 3.1). Hi-P decreased L V D P (-46.8±7.2% reduction from baseline) more than lo-P (-22.912.0%, P < 0.05). This contrasts with a slight increase of pre-ischemic L V D P in the control group relative to its baseline value (P > 0.1).  In general, a weak negative correlation was  obtained between the percentage change of L V D P from baseline to pre-ischemia and the post-ischemic percentage recovery of L D V P at Re-90 (n=18, r=-0.52, P =0.03), the later phase of reperfusion. However, this relationship primarily existed in the lo-P and control groups (n=12, r = -0.61, P =0.04), but not in the two propofol treatment groups (r =0.13, P=0.6).  3.5. Discussion  To our knowledge, this is the first study to use 15-F2 -isoP as an index of the t  effects of propofol on lipid peroxidation in an isolated rat heart model of ischemiareperfusion. In this study, we found: 1) lipid peroxidation occurs during both global myocardial ischemia and reperfusion; 2) propofol (at 5 p.g/ml and 12 p.g/ml) significantly inhibits lipid peroxidation during myocardial IRI; 3) post-ischemic cardiac functional preservation positively correlated with heart tissue antioxidant capacity and inversely correlated with the extent of lipid peroxidation during reperfusion; 4) the protective effect  79 of propofol on post-ischemic preservation of cardiac function and heart tissue antioxidant capacity are concentration-dependent, being greater at 12 (ag/mL than at 5 jj.g/mL in this model.  Baseline 15-F2 -isoP values in this model were within the limits of quantification t  of the assay. The baseline generation of 15-F2 -isoP in our model likely represents release t  from normal myocardial tissue and not from ischemic insult during heart isolation, because of careful organ harvesting and reperfusion within 40 seconds after excision. 15F -isoP is detectable in the rat heart and plasma under control conditions. Since large 70  2t  quantities of isoprostanes can be generated ex vivo,  56  we wanted to ensure that the  measured 15-F2 -isoP was generated by the heart during ischemia, and not produced t  during collection or storage. We found that the values did not change when effluent was stored overnight at room temperature (data not shown). This is a similar finding to that of 990  Morrow and Robert,  who found that urinary 15-F2 -isoP levels did not increase when t  urine was incubated at 37 °C for 5 days. It is unlikely that 15-F -isoP would be generated 2t  in substantial amounts in samples containing very small amounts of lipid, ex vivo.  56  In our experimental model, a small volume of physiological saline was infused during ischemia in order to quantitatively assess the extent of lipid peroxidation during global ischemia and validate a novel antioxidant therapy regimen involving propofol. We found that this delayed the onset of ischemic contracture as compared to the observation of Ko et al  1 4  ' and of our own pilot work, where the onset of ischemic contracture was  about 5 min when saline infusion during ischemia was not incorporated (n = 3,' data not  80 shown). The precise mechanism for the delay of ischemic contrature onset by infusing saline during ischemia is uncertain. It might be attributable, at least in part, to the washout of 15-F -isoP during ischemia by the infused saline. 15-F -isoP is a potent 2t  2t  vasoconstrictor. Pretreatment with vasoconstrictors such as norepinephrine calcium channel agonist B A Y K 8644  2 2 2  2 2 1  or the  has been shown to accelerate and exacerbate  ischemic contracture in isolated rat hearts. In a pilot study we infused 15-F -isoP (100 2t  nM) during global myocardial ischemia.  This increased the magnitude of ischemic  contracture and shortened the time to peak contracture (data not shown). In addition, propofol, when administrated at a concentration of 5.3 ug/mL (30 uM) for 10 min immediately before global ischemia, did not slow the onset of ischemic contracture, nor did it reduce the magnitude of ischemic contracture.  1 4 2  Propofol at 5 ug/mL, when  applied both before and during ischemia, significantly slowed the onset of ischemic contracture in our study, and this was accompanied by a significant decrease in 15-F  2r  isoP release during ischemia as compared with the control group. These data implicate oxidative stress and the generation of 15-F -isoP as potential contributors to myocardial 2t  ischemic contracture.  We found that ROS-mediated lipid peroxidation (as indicated by release of 15-F 2t  isoP) occurred mainly during ischemia and in the early phase of reperfusion in this model (Figure 3.1). In our experimental design, we decreased the propofol concentration from 12 ug/mL (hi-P) to 5 ug/mL (lo-P) after 15 min of reperfusion in order to avoid or reduce the possible direct negative inotropic effects of propofol on myocytes.  219  The fact that  heart tissue T B A R S formation in the hi-P group was significantly lower than that in the lo-P group (Figure 3.2) after the completion of the experiment further indicated that  81 tissue antioxidant status was compromised mainly during ischemia and early reperfusion. This is because the major difference between these two groups is that propofol was applied at higher concentration during ischemia and the first 15 min of reperfusion. It validated the necessity for reducing propofol concentration during the late phase of reperfusion when antioxidant therapy is the major consideration to the use of high dose propofol. In fact, continuous application of hi-P (12 (ig/mL) beyond the early phase of reperfusion compromised or prevented myocardial function recovery, based on our pilot work. This may have potential clinical significance.  Forty minutes of ischemia induced a profound ischemic insult, as evidenced by the magnitude of the ischemic contracture (Figure 3.3A). Myocardial ischemia and the subsequent reperfusion were accompanied by significant myocardial lipid peroxidation as evidenced by the significant increase in coronary effluent 15-F -isoP concentration 2t  during ischemia and the early phase of reperfusion. It is noteworthy that coronary effluent 15-F2t-isoP levels at Re-0.5 were negatively correlated with myocardial functional recovery after reperfusion 90 min. Tissue TBARS formation negatively correlated with post-ischemic  myocardial function recovery  (Figure 3.5)  and cardiac  function  preservation after prolonged reperfusion (Figure 3.6). This provides indirect evidence that the d ecrease o f e ndogenous t issue a ntioxidant c apacity a nd t he s ubsequent i ncrease o f lipid peroxidation during myocardial ischemia and reperfusion are major mediators of myocardial IRI.  Applying hi-P during ischemia and the early phase of reperfusion provided more long-lasting myocardial protection than lo-P. This is evidenced by the significantly lower  82 percentage decrease of L V D P from Re-60 to Re-90 in the hi-P than in the lo-P group. Interestingly, in our study, hi-P (12 u.g/mL), but not lo-P (5 p.g/mL), completely prevented the increase of L V E D P during reperfusion (Figure 3.3B) which was apparently not completely achieved by Mathur et a l .  223  They applied propofol at 6.2 \xg/mL (35 uM)  plus H O E 642 (a sodium ion-hydrogen ion exchange inhibitor) for 15 min before global ischemia and throughout 60 min of reperfusion in isolated rat hearts and achieved greater post-ischemic myocardial functional recovery than using propofol or H O E 642 alone. Our findings indicate that oxidant stress might be a major and/or initial contributor to myocardial IRI, whereas other mechanisms such as ion imbalances may be secondary mediators of myocardial IRI. Our study indicated that hi-P enhanced heart tissue antioxidant capacity to a greater extent than lo-P (Figure 3.2).  It should be noted that antioxidant vitamins (vitamin E and C) are not able to prevent ROS production from other sources in whole blood despite their ability to reduce ROS release from polymorphonucleated cells. function in the experimental diabetic rat.  224  Vitamin E and C impaired vascular  In contrast, propofol effectively preserved  heart tissue antioxidant capacity and myocardial function. Our present study suggests that when applied in adequate amount, antioxidant therapy with propofol could potentially reduce myocardial I RI during c ardiac surgery in high-risk patients, such as those with diabetes.  3.2. Conclusion  83 Our results support the hypothesis that propofol facilitates myocardial functional recovery following ischemia and reperfusion primarily by preventing lipid peroxidation. This does not exclude the possible role of other actions of propofol, including "yyt  preservation o f h igh energy p hosphates  0  a nd f acilitation o f m etabolic r ecovery  Oft  as  intermediate mechanisms of cardio-protection. These, however, are not likely the major mechanism of protection, since the degree of pre-ischemic myocardial depression did not correlate with post-ischemic myocardial function recovery in the two propofol treatment groups.  In terms of clinical implications: clinically achievable high concentrations of  propofol (12 ug/mL),  125  administrated primarily during myocardial ischemia and the  early phase of reperfusion, can provide greater cardiac protection than when applied at a lower c one entration. Antioxidant therapy should be focused on the period o f ischemia and the earliest stages of reperfusion. The concern regarding cardiovascular depression with t he u se o f h igh-dose p ropofol w ould 1 ikely be m inimized i f i t w ere a dministered primarily during the period of cardiopulmonary bypass support.  84  CHAPTER 4 PROPOFOL EFFECTS ON ISCHEMIC TOLERANCE OF MIDDLE-AGED RAT HEARTS:  EFFECTS  OF  15-F -ISOP 2T  FORMATION  AND  TISSUE  ANTIOXIDANT CAPACITY  4.1. Preface  This investigation was supported, in part, by funding from the Centre for Anesthesia and Analgesia, Dept. of Pharmacology & Therapeutics, The University of British Columbia. A manuscript reporting studies described in this Chapter has been published in Cardiovascular Research 2003;59:113-21 (ref.  ) and is co-authored with  D. V . Godin and D. M . Ansley.  4.2. Introduction  Aging is known to be associated with biochemical and functional changes in the \  heart.  '  Animal studies have shown an age-related decrease in recovery of cardiac  function with post-ischemic reperfusion. '  230 231  Clinical studies have shown that age i s  one of the best predictors for operative mortality in patients undergoing cardiac surgery using cardiopulmonary bypass ( C P B ) . ' 232  2 3 3  However, the post-operative outcomes in  terms of long-term survival and freedom from angina were excellent in senescent as compared with middle-aged patients .  2 3 4  Thus, it is important to determine i f the  susceptibility of the myocardium to ischemia-reperfusion injury (IRI) varies with age, and to identify treatments that will effectively protect less tolerant myocardium.  85  Recent studies have shown that middle-aged rat hearts became more vulnerable to ischemic insult  2 3 5  and that rat coronary arteries became more sensitive to the  vasoconstrictor endothelin-1 before and after ischemia-reperfusion, particularly during the period of maturation from younth to adulthood.  236  Oxidative stress may occur during  myocardial ischemia-reperfusion and contribute to IRI secondary to lipid peroxidation of cell membranes. It has been reported that reactive oxygen species (ROS) and products of oxidation increase with age,  2 3 7 - 2 3 9  accompanied by a reduction in tissue antioxidant  capacity.  Based on these observations, we postulate that antioxidant intervention could increase ischemic tolerance and enhance postischemic myocardial functional recovery of the middle-aged rat hearts. Our previous work has demonstrated that the intravenous anesthetic propofol enhances red cell and tissue antioxidant capacity both in vitro and in vivo} '  25 218  We have recently found that enhancing myocardial antioxidant capacity with  propofol protects against the oxidant stress associated with ischemia-reperfusion damage 213  in young rat hearts.  We hypothesize that propofol may provide effective protection  against myocardial IRI of the more vulnerable middle-aged rat hearts  2 3 5 ; 2 3 6  and that this  protection is related to the reduction of lipid peroxiation during ischemia-reperfusion. The hypothesis was tested in an isolated rat heart model, using 15-F2 -isoprostane (15-F t  2t  isoP, previous name 8-epi-PGF2 ), a specific and reliable index of lipid peroxidation, ' 56  a  1 9 8  as a measure of oxidative injury.  86 4.3. Materials and methods  4.3.1. Heart perfusion  The study was approved by the Committee of Animal Care of the University of British Columbia. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 8 5-25, 1 996). Y oung ( Y , 1 0 w eeks, w eighing 2 50-300 g) o r m iddle-aged (M, 2 0 weeks, weighing 550-620 g) Male Sprague-Dawley rats were anesthetized with pentobarbital (70mg/kg, intraperitoneally) and heparinized with sodium heparin (1000 IU/kg, intraperitoneally). After thoracotomy, hearts were quickly excised and immersed in ice-cold Krebs-Henseleit (KH) solution to stop contractions.  Hearts were gently  squeezed to remove residual blood and thereby t o prevent clot formation. Hearts were retrogradely perfused via the aorta as non-working "Langendorff preparations at a constant flow rate using a peristaltic pump. The perfusion flow rate was 10 ml/min in young and 15 ml/min in middle-aged rat hearts. The choice of different perfusion flow rates was based on our pilot study results showing: 1) these flow rates yielded a comparable initial coronary perfusion pressure of about 50 mmHg in the hearts; 2) hearts beat well and remain hemodynamically stable for a duration of 150 min (the duration of our study) when sham-perfused without ischemia and reperfusion (n=3 each for young and middle-aged rat hearts). The flow rate for young rat hearts was the same as that used 223  by Mathur et al,  whereas the flow rate for the middle-aged rat hearts was comparable  to that achieved by Goodwin et a l .  236  The perfusion fluid (pH 7.4; temperature, 37°C)  was K H solution that contained: 120mM NaCl; 20mM N a H C 0 ; 4.63 m M KC1; 1.2mM 3  87 M g C l ; 1.25 m M CaCl ; 1.17 m M K H P 0 and 8 m M glucose. The perfusate was 2  2  bubbled with a mixture o f 95% 0  2  2  4  and 5% C 0 . The perfusate solution and the bath 2  temperature were maintained at 37°C using a thermostatically controlled water circulating system. During the experiment, the heart was in a chamber with circulating water in its jacket thermostatically controlled at 37°C. The chamber was properly sealed during the experiment, and the inside chamber environmental temperature  was  continuously monitored with a thermometer and maintained between 36.9 °C to 37.1 °C during ischemia. Coronary perfusion pressure (CPP) was measured via a side arm of the perfusion cannula connected to a pressure transducer (Statham p23 ID, Gould Electronics, Cleveland). A latex water-filled balloon fixed to a pressure transducer was inserted through the mitral valve into the left ventricle for the determination of left ventricular (LV) developed pressure (LVDP), which was calculated by subtracting enddiastolic pressure (LVEDP) from L V peak systolic pressure (LVSP). L V E D P was adjusted to approximately 5 mmHg before the start of the experiment by adjusting the volume in the intraventricular balloon. Hearts were perfused within 30 to 40 seconds after excision. Exclusion criteria included heart preparation times longer than 60 seconds and/or L V S P lower than 70 mmHg after 10 min equilibration.  4.3.2. Experimental Protocol  A l l hearts were initially equilibrated for 10 min (baseline, BS), and they were then randomly divided into one of four groups (n=6 each): ischemia-reperfusion untreated control groups of young (C-Y group) and middle-aged (C-M group) rat hearts, propofol treatment groups of young (P-Y group) and middle-aged (P-M group) rat hearts. Previous  88 studies in our laboratory have shown that the carrier vehicle for propofol was devoid of antioxidant activity, and therefore it was deemed unnecessary to include vehicle controls into an experimental protocol.  After the initial equilibration, propofol was applied for 10 min at 12 ug/ml (67 uM) in the P - Y and P - M groups prior to inducing global ischemia by stopping the perfusion flow for 40 min. Control (non-propofol-treated) hearts were equilibrated for another 1 0 m in p rior t o i nducing global i schemia f or 4 0 m in. During i schemia, s aline (controls) or propofol (P-Y and P-M) in saline (12 p.g/ml) was perfused through the aorta at 60 ul/min using a mini-pump. K H was perfused during the 90 min of reperfusion in the control groups. Propofol 12 p,g/ml in K H was perfused for the first 15 min of reperfusion, followed by propofol 5 p.g/ml in K H for 75 min in both propofol treatment groups. This reduction in propofol concentration during reperfusion was effected to avoid or reduce the possible direct negative effects of propofol on myocytes.  219  Hearts were  electrically paced at a rate of 300 beats/min, prior to and following, but not during the ischemic period. L V function was continuously monitored using a polygraph. At the end of the 90 min of reperfusion period, hearts were immediately removed from the cannula, precooled in liquid nitrogen and stored at -70 °C. Hearts were assayed for tissue thiobarbituric acid reactive substances (TBARS) formation within 48 hours of storage.  4.3.3.15-F2 -isoP Assays t  Effluent perfusate was sampled at baseline (BS), during the first 30 min of ischemia (1-30) and at 0.5 (Re-0.5), 5 (Re-5) and 30 (Re-30) min of reperfusion in control  89 and propofol-treated groups. Effluent perfusate samples at baseline and after reperfusion were collected over a 10-second period, while the samples during ischemia were collected over a period of 30 min. The effluent samples were stored at -70 °C until analysis for free 15-F2 -isoP. t  Enzyme-linked immunoassay (EIA) was used to measure free 15-F -isoP levels 2t  as mentioned before (Chapter 3.3.4).  4.3.4. Heart tissue antioxidant capacity determination Tissue antioxidant capacity was determined by exposure of tissue homogenates to the peroxidizing agent t-butylhydroperoxide (r-BHP). The oxidation of tissues by r-BHP results in the formation of numerous lipid byproducts, which are collectively termed T B A R S . The levels of T B A R S in the sample were estimated from the absorbance at 532 nm (18). Heart tissue samples (300 mg) were thawed and homogenized on ice in 3 ml Tris-EDTA buffer using a Polytron (PT-10, Brinkman Instruments, Canada) homogenizer for 30 s at 25% power. The resulting homogenates were used for in vitro forced peroxidation and subsequent determination of T B A R S as previously described.  240  In  brief, 400 uL of tissue homogenate were combined with 400 uL r-BHP (in 0.9% saline/2 m M sodium azide to produce r-BHP concentrations ranging from 0.5 to lOmM). These suspensions were incubated for 30 min at 37 °C, then 400 uL of cold 28% (w/v) TCA-0.1 M sodium arsenite was added. The mixture was centrifuged at 12,000g for 5 min at 4 °C, and 800 uL of supernatant was removed and added to 400 uL of thiobarbituric acid  90 (0.5% in 25 m M NaOH). The samples were boiled for 15 min, and the absorbance at 532 nm was measured spectrophotometrically.  4.3.5. Data Analysis  A l l data are presented as mean ± S E M . Effluent 15-F -isoP concentration and 2t  hemodynamic data were compared by two-way A N O V A with Bonferroni's correction (GraphPad Prism). One-way repeated measures A N O V A  and Tukey's Multiple  Comparison test were applied for within-group comparison. The correlation relationships were evaluated by the Pearson test. P < 0.05 (two-tailed) was considered significant.  4.4. Results  4.4.1.15-F2t-isoP generation during ischemia-reperfusion  Baseline 15-F -isoP values were low (5.0 + 0.5, 6.1 ± 1.0, 4.5 ± 0.4 and 4.0 ± 0.1 2t  pg/ml i n C - Y , P - Y , C - M a nd P - M groups, r espectively) and d id n ot d iffer among t he various experimental groups (P>0.1). Since perfusion flow rates were different between young and middle-aged rat hearts, 15-F -isoP production at baseline and during 2t  reperfusion was converted according to the flow rates and presented as the amount of 15F -isoP produced per minute (pg/min) in the effluent. 15-F -isoP production during 2t  2t  ischemia was, however, presented as pg/mL. As shown in Figure 4.1, 15-F -isoP levels increased significantly at Re-0.5 2t  (P<0.05 or PO.01 vs baseline) in all experimental groups.  It decreased to close to  91 baseline levels in P-Y and P - M groups (P>0.05 vs baseline) at Re-5 and in C - Y and C - M groups at Re-30 (Figure 4.1 A). During early reperfusion, 15-F2 -isoP levels fell more t  rapidly in the C - Y than in the C - M group. At Re-5 min, a significant decrease of 15-F2 t  isoP from Re-0.5 was seen in the C-Y, but not in the C - M group. Propofol significantly reduced 15-F2 -isoP production at 0.5 min of reperfusion in young (PO.05, P-Y vs C-Y) t  and at 5 min of reperfusion in the middle-aged rat hearts (PO.05, P - M vs C-M). 15-F2 t  isoP was produced in substantial amounts during ischemia in the C - Y and C - M groups (Figure 4.IB). Propofol significantly reduced 15-F2 -isoP production during ischemia in t  both young and middle-aged rat hearts.  4.4.2. Tissue antioxidant capacity  As shown in Figure 4.2, tissue T B A R S formation following in vitro peroxide challenge (as reflected by absorbance at 532 nm) was significantly higher in control groups than in propofol treatment groups at J-BHP concentrations higher than 0.5 m M (PO.01 or PO.001 P - Y vs C - Y or P - M vs C-M). At a f-BHP concentration of 2 m M , tissue T B A R S formation in C - M was found to be significantly higher than that in the C - Y group (P = 0.03, T B A R S absorbance = 0.519 + 0.019 in C - M vs 0.429 ± 0.028 in C-Y). This indicated lower tissue antioxidant preservation in the C - M group than in the C - Y group. Nevertheless, propofol treatment reduced T B A R S absorbance at 2 m M t-BHP to the same level in the P - M (0.214 ± 0.017) as in the P-Y (0.222 ± 0.014) group, suggesting that propofol could more effectively preserve tissue antioxidant capacity during ischemiareperfusion in middle-aged rat hearts than in young rat hearts.  92  ^ 3 C-Y group P-Y group C-M group mmm P-M group  X *#  X  Baseline Re-0.5 min Re-5 min Re-30 min Time points for effluent sampling  -  C-Y group P-Y group C-M group P-M group  T T  -  +  _  C-Y  Hi  P-Y C-M Groups  1 1  P-M  Figure 4.1. Coronary effluent  15-F -isoprostane 2t  release during ischemia (B) and  reperfusion (A). Effluent was sampled at baseline, 0.5 (Re-0.5), 5 (Re-5) and 30 (Re-30) min of reperfusion for a duration of 10 second. Effluent during ischemia was collected for the first 30 min. Values are mean ± S E M . *P < 0.05 or P < 0.01 vs baseline; P < +  0.05 P - Y vs C - Y or P - M vs C - M group; P<0.05 Re-5 or Re-30 vs Re-0.5 min; P > 0.05 #  P - M vs P-Y or C - M vs C-Y.  93  0.75n  C-Y group ^ P-Y group CZD C-M group muU P-M group EEEE9  CM CO  in  -M re  0.50-  0)  o c co  •S o w .Q  <  0.25-  0.00  0.5  10  t-BHP concentration ( mM)  Figure 4.2. Thiobarbituric acid reactive substances (TBARS) formation (as reflected by absorbance at 532 nm) as a function of t-BHP concentration for heart tissues at reperfususion 90 min. Heart tissue were sampled after 40 min of ischemia and 90 min of reperfusion. Values are mean ± S E M . P < 0.01 P-Y vs C - Y or P - M vs C - M group; P < +  0.05 C - M vs C-Y; P > 0.05 P - M vs P-Y.  #  94  4.4.3. Effects of propofol on contracture development during ischemia and reperfusion L V E D P increased progressively during ischemia in the two control groups (Figure 4.3). Maximum ischemic contracture (reflected as peak LVEDP) in the C - M group (38.3±3.8 mmHg) was not significantly different from that in the C - Y (37.8+3.0 mmHg) group (Table 1). There was no significant difference in the onset time of ischemic contracture and time to peak L V E D P between C - Y and C - M groups. L V E D P increased more rapidly in the C - M group after 25 min of ischemia. L V E D P at ischemia 30 min (and onwards) was significantly higher than that at ischemia 25 min (P = 0.03) in the C - M group (Figure 4.3). The gradual increase in L V E D P in the C - Y group only became significant after 40 min of ischemia. Propofol significantly reduced L V E D P in young rat hearts after 35 min of ischemia, and completely abolished ischemic contracture in middle-aged rat hearts during the 40 min period of ischemia (Figure 4.3).  Reperfusion was associated with a generalized elevation in L V E D P in both C - Y and C - M groups (Figure 4.4) when compared to the L V E D P of approximately 5 mmHg before ischemia. The L V E D P further increased over time after 30 min of reperfusion in the C - Y and C - M groups. The L V E D P at 5 min of reperfusion was 7.8 ± 1 . 2 mmHg in PY and 5.8 ± 0.5 mmHg in the P - M group and was significantly lower than the corresponding values in C - Y and C - M (Figure 4.4). L V E D P did not increase over time during the 90 min period of reperfusion in either the P-Y or the P - M group.  95  50-i  3 C-Y group P-Y group 1C-M group  40-  [ # rl J  I  30-  +  20-  I JL 10015  20  in. 25  - *  30  +  i  35  40  Duration of ischemia (min)  Figure 4.3. Left ventricular end diastolic pressure (LVEDP), reflecting myocardial contracture during ischemia. Values are mean ± S E M . *P < 0.05 vs C - Y group; P < 0.01 +  vs C - Y and C - M groups. P < 0.05 vs ischemia 25 min of the same group. Propofol (67 #  uM) completely abolished ischemic contracture (i.e., L V E D P = 0) in all six hearts in the P - M group.  96  100-1  C-Y group ^ P-Y group i i C-M group m\m P-M group  75-  I  50  JI  25-  *  5  I  s  E =  10  30  1  =  1  rd *  !• mi  1  IL 60  90  Duration of reperfusion (min)  Figure 4.4.  . Left ventricular end diastolic pressure (LVEDP), reflecting myocardial  contracture during reperfusion following 40 min of global ischemia. Values are mean ± S E M . *P < 0.05 or P < 0.01 vs C - Y or C - M groups. P > 0.05 C - Y vs C - M or P-Y vs P - M groups.  97  4.4.4. Coronary Perfusion Pressure  As shown in Table 4.1, initial CPP values were comparable in C-Y, P-Y, C - M and P - M groups. CPP values increased gradually after reperfusion in the C - Y and C - M groups and were significantly higher than baseline after 30 min of reperfusion in the C - M group and after 60 min of reperfusion in the C - Y group. Administration of propofol for 10 min before ischemia significantly reduced CPP in young but not in the middle-aged rat hearts. Propofol prevented the increase of CPP after reperfusion that was seen in the control groups. The CPP was significantly lower in P-Y and P - M than in C - Y and C - M , respectively, after 90 min of reperfusion.  4.4.5. Left ventricular mechanics Figure 4.5 depicts the pre- and post-ischemic values of L V D P , a measure of myocardial contractile function. Baseline L V D P values did not differ among the 4 experimental groups. Propofol administration for 10 min before ischemia reduced L V D P by 43.2% in P - Y and 36.5% in P - M groups. The pre-ischemia values of L V D P in P - Y and P - M were significantly lower than the corresponding values at baseline (PO.01) prior to inducing ischemia. The L V D P in the C - Y and C - M groups recovered to 82.0 ± 8.6 % and 87.2 ± 9.0% of the corresponding baseline values, respectively, at the 30 min (Re-30) reperfusion time-point and decreased progressively thereafter. At 90 min of reperfusion (Re-90), L V D P in the C - Y and C - M groups was significantly lower than their baseline values (PO.05). Propofol treatment resulted in better recovery of L V D P in middle-aged than in young rat hearts following reperfusion. L V D P in the P - M group was  98 significantly higher than that in the P - Y group after 60 min (Re-60) and 90 min of reperfusion. At Re-90, L V D P in the P - M was significantly higher than that in the C - M group (PO.05).  Since one of the major goals of this study was to optimize the long-term protective effects of propofol, administration of propofol was not discontinued during reperfusion. For a better comparison among groups, the percentage change of L V D P from Re-60 to Re-90 was calculated. The percentage decrease of L V D P from Re-60 to Re-90 in the C - M group (-27.811.8%) was significantly higher than that in the C - Y group (-17.9±3.9%) (PO.05), both being significantly higher than that in the corresponding propofol treatment groups. The percentage decrease of L V D P from Re-60 to Re-90 did not differ between P-Y (-3.6±2.7%) and P - M (-6.7±2.9%) groups.  4.4.6. Correlation Analysis  Figure 4.6 depicts the relationship between heart tissue antioxidant capacity (as reflected in peroxide-induced TBARS generation) at the end of 90 min of reperfusion and coronary  effluent  15-F2 -isoP production during ischemia. Tissue TBARS t  was  significantly positively correlated with effluent 1 5-F2 -isoP production during ischemia t  (R - 0.68, P = 0.0003), indicating an inverse correlation between tissue antioxidant capacity preservation and 15-F -isoP production during ischemia. Tissue TBARS levels 2t  did not correlate with 15-F2 -isoP generation during reperfusion. t  99  Table 4.1. Coronary perfusion pressure (CPP) (mm Hg)  BS  Pre-isch  Re-10  Re-30  Re-60  Re-90  C-Y group  53.0 ± 4.8  55.0 ± 5.2  P-Y group  50.8+ 1.5  40.8 ± 2.7  C-M group  54.2 ± 3.3  P-M group  53.5 ± 2.3  59.2 ± 5.5  79.7 ± 9.5  46.2 ± 4.9  53.0+ 4.2  56.5 ± 3.6  60.2 ± 2.2  71.3 ± 3.9*  84.2 ± 7.8**  48.0 ± 2.5  48.5 ± 3.0  58.8 ± 5.4  66.7+ 8.4  +  +  95.5 ± 15.1** +  53.0 ± 3.8  +  107.5 ± 13.2** 59.2 ± 4.7*  +  103.8+ 9.1** 71.7+ 7.9*  +  Values are mean ± S E M (n=6 for each group). BS = baseline, Pre-isch = pre-ischemia; Re-10, Re-30, Re-60 and Re-90 refer to 10, 30, 60 and 90 min after reperfusion. CPP did not differ among groups at baseline. *P<0.05, **P<0.01, vs BS; P<0.05 P-Y vs C-Y or +  P - M vs C - M group.  100  150-  ( = \ C-Y group P-Y group I I C-M group umW P-M group  ^ 100-  E, ST a >  I*  50-  BS  Pre-isch Re-10 Re-30  Re-60  Re-90  Time during ischemia-reperfusion  Figure 4.5.  Left ventricular developed pressure (LVDP). BS and Pre-isch indicate  baseline and pre-ischemia respectively. Re-10, Re-30, Re-60 and Re-90 indicate 10, 30, 60 and 90 min of reperfusion following 40 min of global myocardial ischemia. Values are mean ± S E M . * P < 0.05 or P < 0.01 vs BS; P < 0.05 or P < 0.01 P-Y vs C-Y group or +  P - M vs C - M group. P <0.05 P - M vs P-Y group. P > 0.05 C - M vs C - Y group. #  101  0)  o c  0.8  (0  .Q  0.6  k-  o  •Q E  0.4  CO CN CE CO  0.2  «  c  <  »  CQ fa  • C-Y group  y = 0.0046x+0.1505 R = 0.68  • P-Y group A C-M group A P-M group  •  'A  •  0  0) 3 (A  50  in  100  Effluent 15-F2t-isoP during ischemia (pg/mL)  Figure 4.6.  Correlation between coronary effluent 15-F2 -isoprostane release during t  ischemia and heart tissue antioxidant capacity. Heart tissue were sampled after 40 min of ischemia and 90 min of reperfusion. Tissue T B A R S formation in the presence of the peroxidizing a gent t -BHP (2 m M) w as s ignificantly p ositively correlated w ith e ffluent isoprostane generation during ischemia (R = 0.68, 95% CI: 0.3798 to 0.8498, P = 0.0003),  indicating an inverse  correlation between  preservation and isoprostane generation during ischemia.  tissue  antioxidant  capacity  102  100  y = -0.5O47x + 82.86 R = -0.65  80 A  60  •  A  P  • C-Y group • P-Y group A C-M group A P-M group  40 20  a. a >  0 0 25 50 75 100 Effluent 15-Rt-isoP during ischemia (pg/mL)  B  120 100  c. o  80  '55 .  t0)  A<6  AD  • C-Y group • P-Y group A C-M group A P-M group  60  3  Q.  y = -0.596x + 96.878 R = -0.65  40  I  £ ;  ra  20  Q. O  0  >  0 25 50 75 100 Effluent 15-F2t-isoP during ischemia (pg/mL)  Figure 4.7. Correlation between left ventricular developed pressure (LVDP) at 90 min of reperfusion (Re-90) and 15-F2t-isoprostane release during the first 30 min of ischemia. 15-F2 -isoP generation during ischemia was significantly negatively correlated with t  L V D P at Re-90 (R= -0.65, 95% CI: -0.8324 to -0.3277, P = 0.0007, A) and the percentage recovery of L V D P at Re-90 to baseline (R = -0.65, 95% CI: -0.8338 to 0.3318, P =0.0006, B).  103  At 90 min of reperfusion, L V D P , as well as its percentage recovery to baseline values, was highly negatively correlated with heart 15-F2 -isoP generation during t  ischemia (P < 0.001, Figure 4.7). A weak but significant correlation existed between 15F -isoP generation during ischemia and peak ischemic contracture (P = 0.04) and 2t  between 15-F2t-isoP production at reperfusion 5 min and coronary perfusion pressure at reperfusion 90 min (P = 0.04).  Changes of L V D P from Re-60 to Re-90 were inversely correlated with changes of CPP from Re-60 to Re-90 in ischemic-reperfused middle-aged rat hearts (R= -0.68, P=0.015, n=12). Such a relationship did not exist in the young rat hearts (P=0.8). This extends the finding of Goodwin et al  2 3 6  that middle-aged (5-month old) rat hearts became  more sensitive to vasoconstriction induced by endothlin-1, whose release was increased in isolated ischemic-reperfused rat hearts.  241  4.5. Discussion  Previous laboratory studies investigating the effect of propofol on myocardial IRI have focused primarily on young animal hearts. ' 142  143; 2 1 3 ; 2 1 8 ; 2 2 3 ; 2 2 6  To our knowledge,  this is the first study to compare the cardioprotective effects of propofol on isolated hearts from young and middle-aged rats and to use 15-F2 -isoP as an index of lipid peroxidation t  to explore the mechanism of the protection. This approach is important to understanding the effects of age, which could identify patient populations that could benefit most from antioxidant intervention during clinical settings of myocardial IRI.  104 The principal findings of this study include the following. 1) lipid peroxidation occurs during global ischemia and reperfusion in both young and middle-aged rat hearts; 2) myocardial tissue antioxidant capacity preservation following IRI is highly negatively correlated with the extent of lipid peroxidation that occurs during prolonged global ischemia rather than during reperfusion; 3) 15-F -isoP generation during ischemia is 2t  highly negatively correlated with post-ischemic myocardial functional recovery; 4) propofol  applied before and during ischemia as well as during early reperfusion  significantly reduced 15-F2 -isoP generation during ischemia and abolished ischemic t  contracture in middle-aged rat hearts.  In attempts to clarify the contribution of ROS to myocardial IRI, experiments using a wide array  of antioxidants  have  yielded conflicting  results. Certain  pathophysiological mechanisms may predominate depending on the conditions of the experiment, such as the duration of ischemia, the timing of the antioxidant intervention as well as the nature and dosage of the antioxidant used. If significant cellular necrosis has occurred during the preceding ischemic event,  antioxidant interventions during  reperfusion may have little effect. Cardiac mitochondria, critical to the energy status and function of the heart, are a source of ROS during ischemia and reperfusion 242  exhibit increased rates of ROS production with age. " 237  239  2 4 3 ; 2 4 4 ;  and  Based on previous studies from  125" 218  our laborotary,  '  we postulate that the cardioprotective effects of propofol on IRI are  mostly attributable to its antioxidant properties.  105 15-F2 -isoP is a chemically stable end product of lipid peroxidation, which is t  detectable in rat heart and plasma under control conditions. EIA provides a sensitive 70  measure for 15-F -isoP in biological specimens. Baseline 15-F2 -isoP values in our model 2t  t  of myocardial IRI were within the limits of detection of the assay. The baseline generation of 15-F2 -isoP in our model likely represents release from normal myocardial t  tissue rather than an ischemic insult during heart isolation, because of careful organ harvesting and reperfusion within 40 seconds after excision. Since large quantities of isoprostanes can be generated ex vivo, it was important to ensure that the measured 1556  F2 -isoP was generated by the heart during ischemia, and not produced during collection t  or storage. We found that the values did not change when effluent was stored overnight at 220  room temperature (data not shown). This is similar to a report by Morrow et al,  who  found that urinary F2-isoprostane levels did not increase when urine was incubated at 37 °C for 5 days. Forty minutes of ischemia induced a profound ischemic insult, as evidenced by the magnitude of the ischemic contracture in the two control groups. It is noteworthy that the magnitude of the ischemic contracture in the C - M group significantly increased every five minutes when the duration of ischemia exceeded 25 min, which was not the case in the C - Y group. This indicates that middle-aged rat hearts are more vulnerable to ischemic insult after prolonged ischemia. Interestingly, propofol abolished ischemic contracture in middle-aged but not in young rat hearts. The precise mechanism underlying this effect is uncertain. It seems reasonable to postulate, however, that antioxidant therapy would be more effective in hearts with decreased endogenous antioxidant capacity. This will  106 attenuate or prevent subsequent cellular damage resulting from ROS-mediated membrane lipid peroxidation. On the other hand, propofol may inhibit rat cardiomyocyte calcium channels at a concentration as low as 6 u M .  This inhibition increases in a  2 4 5  concentration-dependent fashion when propofol concentration exceeds 50 u M .  2 4 5  This  property of propofol could have contributed, in part, to the reduction or abolition of ischemic contracture in the propofol treatment groups. It has been shown that contracture development in rat cardiomyocyte is potentiated by a rise in intracellular calcium concentration. ' 246  2 4 7  In addition, propofol may attenuate contracture development by  enhancing energy preservation during myocardial ischemia-reperfusion. Energy depletion may play an important role in the acceleration of contracture development, contracture has been initiated in the presence  2 4 6 ;247  o r absence  2 4 8  248  once the  of a rise in intracellular  calcium. The strong negative correlation between 15-F -isoP generation during ischemia 2t  and post-ischemic myocardial function recovery highlights the role of ROS in mediating cellular injury early during ischemia. Susceptibility of heart tissue to ex vivo T B A R S formation in the presence of the peroxidizing agent r-BHP provided a functional measure of tissue antioxidant capacity. The significant correlation between tissue T B A R S formation and 15-F2 -isoP generation during ischemia but not during reperfusion t  indicates that the reduction in tissue antioxidant capacity occurred primarily during the ischemic phase in this experimental model. This would suggest that antioxidant interventions aimed at protecting against myocardial IRI are likely to be most effective i f undertaken immediately prior to the ischemic insult. The propofol concentration (67 u M or 12ug/ml) used in the present study is high, but still clinically achievable, based on our previous study.  125  A recent study from our  107 laboratory has shown that the cardiac protective effect of propofol was concentrationdependent, being greater at 12 ug/ml, when used primarily before and during ischemia as well as during the early phase of reperfusion, than at 5 u,g/ml in this experimental 213  model.  Therefore, only the highest concentration of propofol was used in the current  study before and during ischemia and in the early phase of reperfusion. Propofol significantly reduced 15-F2 -isoP generation during ischemia and reperfusion in both t  young and middle-aged rat hearts. It is noteworthy that, during early reperfusion, 15-F 2t  isoP levels decreased more rapidly in the C - Y than in the C - M group (Figure 4.1 A). This is coincident with a more profound percentage decrease of L V D P from Re-60 to Re-90 in the C - M group. This is of clinical significance. We have recently shown that the percentage changes o f p lasma free 1 5-F -isoP concentration during the early phase o f 2t  myocardial reperfusion inversely correlates with post-operative cardiac index.  43  A recently published study has shown that propofol may offer myocardial protection by inhibiting the mitochondrial permeability transition ( M P T ) , major cause of reperfusion injury, in ischemic-reperfused rat hearts.  249  another  at concentrations low as 2 to 4 ug/mL (11 to 22 uM) 249  Inhibition of the M P T by propofol, when applied at  low concentrations, is likely attributable to the well-known membrane stabilizing action of lipophilic anesthetic molecules 2  ug/m did not  mitochondria.  249  and the antioxidant properties of propofol. Propofol  inhibit M P T when applied directly to isolated  de-energised  Indeed, accumulating evidence strongly suggests oxidative stress as the  link between excessive mitochondrial calcium overload and M P T (for a review, see ref. ). 11 h as b een p roposed t hat M PT i s n ot a consequence o f t he o pening o f a p re252  formed pore, but the consequence of oxidative damage to pre-existing membrane  108  proteins. uM.  252  Propofol, however, may directly inhibit rat heart M P T at concentrations > 5 0  This is of importance. Clinically, propofol failed to offer appreciable protection  against myocardial IRI  1 3 6  when administrated using a target-controlled infusion system  with propofol plasma concentrations between 2 to 4 ug/mL (concentration provided by author in response to letter-to-editor by Ansley and X i a ) .  124  Interestingly, propofol, when  used at a high concentration of 67 u M before and during ischemia as well as during the early phase of reperfusion, enhanced myocardial function recovery in middle-aged rat hearts 9 0 min after reperfusion compared to untreated control and young rat hearts. Clinically, the application of high-concentrations of propofol (average 1 1 |j.g/mL) before and during myocardial ischemia in a patient population 3 5 years of age or older resulting in better myocardial function recovery 1 2 to 2 4 hours post-operatively comparing to 1 9S  application of propofol at lower concentrations.  Improved cardiac functional recovery  by propofol 9 0 min after reperfusion in the middle-aged rat hearts and 1 2 - 2 4 hours after cardiac surgery in patients is similar in effect to the "first window" and "second window" of protection phenomena observed after ischemic or pharmacological preconditioning.  254  The proposed "preconditioned state" in the heart induced by propofol, when applied at high concentrations as aforementioned, is unlikely to be mediated by the activation of mitochondrial  KATP  (HIKATP)  channels, a mechanism by which volatile  anesthetics are claimed to mimic cardiac preconditioning. Propofol, at concentrations 255  between  10  to  200  u M , did not affect  IIIKATP  isolated from male Sprague-Dawley rat hearts.  256  channel activity in cultured myocytes We propose that propofol, primarily at  high concentrations, may "precondition" the heart via mechanism(s) downstream of mK TP A  channel activation. Indeed, accumulating evidence supports  mKATP  as a trigger  109 and/or a mediator rather than an end-effector in myocardial preconditoning and protein kinase C (PKC) is likely one of the kinases downstream from  mK TP A  that may be  involved (for a review, see ref. ). Propofol stimulated purified rat brain P K C activity in 254  257  vitro,  and attenuated isoproterenol-stimulated increases in intracellular calcium via  activation of P K C activity in rat cardiomyocytes. Therefore, activation of P K C could 258  be a potential signal pathway through which propofol may "precondition" the heart. Also, it is important to note that inhibition of the M P T in early reperfusion could represent a distal effector mechanism of myocardial "preconditioning" with m K as a trigger or an intermediate step.  259  A  TP  activation acting  Taken together, propofol cardiac protection might  involve the activation of P K C activity before ischemia, and the inhibition of the M P T , directly  2 5 3  or indirectly through reducing oxidative stress,  2 5 2 ; 259  during reperfusion.  4.6. Conclusion  In summary, our present study suggests adequate antioxidant therapy with propofol as a potentially useful means to reduce myocardial IRI under clinical conditions (e.g. coronary revascularization and heart transplantation), especially in the more vulnerable population of the middle-aged patients.  234  Propofol may mediate cardiac  protection through a variety of mechanisms or signal pathways, with inhibition of ROSmediated lipid peroxidation likely being one of the major mechanisms of protection.  110  1.  CHAPTER 5: Effects of 15-F -isoP on myocardial IRI in 2t  isolated rat hearts: potential mechanism of 15-F -isoP 2t  action  5.1. Preface  A  manuscript  entitled  "15-F2 -isoprostane t  exacerbates myocardial  ischemia-reperfusion injury of isolated rat hearts: effects on endothelin-1 generation" reporting results of studies presented in this Chapter has been submitted for publication and is co-authored with D. M . Ansley, K . H . Kuo, D. V . Godin, M . J. Walker and M . Tao.  K . H . Kuo provided technical assistance with the myocardial infarct size  measurements. M . J. Walker provided valuable suggestions and M . Tao provided assistance in collecting and concentrating the coronary effluent samples. We thank Drs E. Puil and T. K . Chang for kindly allowing us to use their laboratory facilities.  5.2. Introduction  Myocardial ischemia-reperfusion injury (IRI) and its sequelae, cardiac depression and arrythmogenesis, have been shown experimentally to result, at least in part, from the disruptive action of reactive oxygen species (ROS) on membrane lipids and intracellular proteins required for cellular integrity and function.  "  The release of high levels of  ROS during ischemia-reperfusion can overwhelm endogenous antioxidant defenses, a crucial event determining the onset of irreversible cellular necrosis secondary to extensive lipid peroxidation.  40  Ill  A recent advance in free radical biology has been the discovery of isoprostanes, stable in vivo end products of arachidonic acid peroxidation. isoprostanes detected,  5 6 ;  6 2  6 3  Of the variety of  15-F -isoprostane (15-F t-IsoP) has been found to be a 61  2  2t  specific, reliable marker of oxidative stress. This has facilitated investigation of the role of ROS in a variety of disease states, most notably cardiovascular disease. O f interest, 15-F IsoP p ossesses p otent b iological activity, i ncluding v asoconstriction and p latelet 2r  activation under pathophysiological conditions.  71  15-F -IsoP has no effect on coronary 2t  flow in the absence of ischemia in the isolated rat heart (up to a concentration of 256 nM), but significantly reduces coronary flow in the hypoxic or post-ischemic reperfused rat heart (at 30 n M ) .  77  Clinically we identified an inverse correlation between the speed of decay of plasma 15-F -IsoP concentrations during early phase of reperfusion and post-operative 2t  cardiac functional recovery in patients undergoing coronary artery bypass surgery utilizing cardiopulmonary bypass (CPB).  4 3  We also have found that 15-F -IsoP 2t  generation during myocardial ischemia and reperfusion in an isolated rat heart model is inhibited by treatment with 2,6 diisopropylphenol (propofol), an anesthetic with powerful antioxidant properties.  Propofol administration is associated with improved post-  ischemic cardiac functional recovery  227  These studies have prompted us to postulate that  15-F -IsoP, in addition to being a marker of oxidative stress, may also play a major role 2t  in mediating myocardial IRI.  112 The characteristics of 15-F2 -IsoP production and its effects under conditions of t  ischemia and reperfusion are quite similar to those of endofhelin-1 (ET-1).  Endothelin-1  is one of the most potent vasoconstrictors known, and it has been postulated to contribute to post-ischemic myocardial dysfunction during and after myocardial ischemia  2 6 3  '  2 6 5 ; 2 6 6  2 6 4  . ET-1 release has been shown to increase  and its vasoconstrictor effect appears to be  increased (potentiated) during post-ischemic reperfusion in isolated hearts.  2 6 7  '  2 6 8  We recently determined that antioxidant therapy during cardiac surgery utilizing CPB significantly reduces plasma malondialdehyde ( M D A ) levels as well as ET-1 concentrations, and this is associated with improved post-operative cardiac function  2 6 9  . It  is plausible that antioxidant therapy reduced ET-1 release during myocardial ischemiareperfusion by reducing 15-F2 -IsoP production. t  We hypothesize that 15-F2 -IsoP can exacerbate myocardial ischemia-reperfusion t  injury and that the mechanism of 15-F2 -IsoP action may involve the release and/or t  enhancing the production of ET-1 during cardiac ischemia and reperfusion. Our hypothesis was tested in an isolated rat heart model, using SQ 29548, a thromboxane A receptor  (TXA2)  antagonist used to abolish the vasoconstrictive actions of 15-F2 -IsoP. t  200  2  113 5.3. Materials and methods  5.3.1. Heart preparation  This study was approved by the Committee of Animal Care of the University of British Columbia. Animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. Male Sprague-Dawley rats (280~320g) were anesthetized with pentobarbital (70mg/kg intraperitoneally) and heparinized with sodium heparin (1000 IU/kg, intraperitoneally). After  median  thoracotomy, hearts were quickly excised and immersed in ice-cold Krebs-Henseleit (KH) solution to stop contractions.  Hearts were gently squeezed to remove residual  blood to prevent clot formation. Hearts were retrogradely perfused via the aorta in a nonworking "Langendorff' preparation at a constant flow rate of lOml/min using a peristaltic pump. The perfusion fluid (pH 7.4; temperature, 37°C) was K H solution that contained (in mM): NaCl 118; N a H C 0 24; KC1 4.63; M g C l 3  2  1.2; CaCl 1.25; K H P 0 2  glucose 11. The perfusate was bubbled with a mixture of 95% 0 perfusate  2  2  4  1.17;  and 5% C 0 . The 2  solution and the bath temperature were maintained at 37°C using a  thermostatically controlled water circulating system. Coronary perfusion pressure (CPP) was measured via a side arm of the perfusion cannula connected to a pressure transducer (Statham p23 ID, Gould Electronics, Cleveland). A latex water-filled balloon fixed to a pressure transducer was inserted through the mitral valve into the left ventricle for the determination of left ventricular (LV) developed pressure (LVDP), which was calculated by subtracting end-diastolic pressure (LVEDP) from L V peak systolic pressure (LVSP). L V E D P was adjusted to approximately 5 mmHg before the start of the experiment by  114 adjusting the volume in the intraventricular balloon. Exclusion criteria included heart preparation times longer than 60 seconds and/or L V S P lower than 65 mmHg after 10 min of equilibration.  5.3.2. Experimental Protocol  A l l hearts were initially equilibrated for 10 min (BS10).  They then were  randomly assigned to a sham group or one of the four experimental groups (n=7 per group): ischemia-reperfusion untreated control (control), 15-F2 -IsoP (IsoP), 15-F -IsoP t  2t  plus SQ 29548 (IsoP-SQ) and SQ 29548 (SQ) alone groups. After BS 10, 15-F ,-IsoP 100 2  n M (IsoP), SQ 29548 l u M (SQ) or 15-F IsoP 100 n M plus SQ 29548 l u M (IsoP+SQ) 2r  were applied for 10 min, respectively in the corresponding groups, before global ischemia (40 min) was induced by stopping perfusion flow. Control hearts underwent an additional 10 min period of equilibration before global ischemia was induced. During ischemia, saline (control), 15-F -IsoP 100 n M (IsoP), SQ 29548 l u M (SQ) or 15-F -IsoP 100 n M 2t  2t  plus SQ 29548 l u M (IsoP+SQ) in saline was perfused through the aorta at 60 p-l/min using a mini-pump. K H was perfused during 60 min of reperfusion in the control group. Either 15-F -IsoP, SQ 29548 or 15-F -IsoP plus SQ 29548 in K H was perfused for the 2t  2t  first 15 min of reperfusion.  The perfusion flow rate (10 ml/min) was based on the result of a pilot study which showed  that  hearts  sham-perfused  without  ischemia  beat  well  and  remain  hemodynamically stable for 120 min (the duration of the experiment) in our experimental set-up. The reagent concentrations utilized were chosen based on findings in the  115 literature, demonstrating that: 1) 15-F2 -IsoP 100 n M had no effect on coronary flow in t  sham-perfused rat hearts but significantly reduced coronary flow in ischemic-reperfused rat hearts  2 0 0  ; 2) SQ 29548, a thromboxane A 2 (TXA2) receptor antagonist , abolished 85  15-F2 -IsoP (56 nM)-induced reduction in coronary flow in ischemic-reperfused rat hearts t  at a concentration of 0.1 u M ° ; and 3) SQ 29548, at l u M , abolished 15-F IsoP (>300 20  2r  nM) -induced reduction in coronary flow in isolated perfused guinea pig heart . 76  Baseline effluent perfusate was sampled at BS10.  Effluent samples during  ischemia w ere c ollected d uring t he first 3 0 m in o f i schemia (isch). A Iso, e ffluent w as sampled at 1 (Re-1), 5 (Re-5), 30 (Re-30) and 60 (Re-60) min of reperfusion in the four experimental groups or at the corresponding time points in the sham group. Aliquots of the effluent samples were immediately stored at -70 °C until analysis for cardiac specific creatine kinase (CK-MB) in all study groups and for 15-F2 -IsoP in the sham, the control t  and the SQ groups. Another portion of the effluent sample was initially concentrated (see below) and then stored at -70 °C for the analysis of ET-1 concentration. At the end of the 60 min of reperfusion, 37°C 1% 2,3,5-triphenyltetrazolium in buffer (0.1 M phosphate buffer adjusted to pH 7.4) was pumped into the heart at 1 ml/gm/min for 15 min until the epicardial surface became deep red. The hearts were then stored in 10% formaldehyde for later analysis of myocardial infarct size.  5.3.3. Measurement of Endothelin-1  Measurement of ET-1 concentrations in the coronary effluent was performed using a commercially available human ET-1 enzyme immunometric assay kit (human  116 ET-1 EIA kit 900-020, Assay Designs, Inc. Ann Arbor). Because ET-1 concentrations in the samples were often below the ET-1 sensitivity of this assay (0.14pg/ml) based on a pilot study, collected effluent samples were concentrated 4-fold by evaporation of solvent (i.e. the K H solution) at room temperature under a stream of dry nitrogen. Subsequently, the actual ET-1 concentration was calculated as l/4 of the measured ET-1 level in the th  concentrated sample. This concentration procedure did not diminish the accuracy of the ET-1 measurements, as tested by using known concentrations of ET-1 standards and control measurements without ET-1.  Enzyme immunoassays (EIA) were performed in duplicate by adding 100 uL of human ET-1 standards or concentrated samples onto 96-well microplates. After 1 hour of incubation at 37°C, the m icroplates were washed seven times with wash solution. The microplates were further incubated at 37°C for 30 min after the addition of 100 uL labeled antibody to each well, except the blank. The microplates were then washed nine times before adding 100 uL of the substrate solution to each well. Thereafter, the microplates were incubated for 30 min at room temperature, followed by the addition of 100 uL stop solution to each well. The plates were read at 450 nm and the values of the unknowns were expressed as picograms ET-1 per milliliter effluent. The samples were coded (as was the case for all assays in this study) and the investigator responsible for ET-1 assays was blinded until the completion of the assay.  117  5.3.4. Measurement of C K - M B  Measurement of C K - M B was performed using a commercially available enzyme immunoassay kit (Catalog number: BC-1121, BioCheck, Inc, Burlingame, CA). The EIA measurements were performed in duplicate by dispensing 20 uL of human C K - M B standard or samples into appropriate wells of a microplate followed by the addition of 200 uL enzyme conjugate reagent into each well. The plate was incubated at 4 °C for 20 hours and was then washed five times with double-distilled water. After the addition of 100 uL T M B reagent into each well, the plate was incubated at room temperature for 20 min. The reaction was terminated by adding 100 uL of stop solution to each well. The plate was read within 15 min at 450 nm and the values of the unknowns were expressed as nanograms C K - M B per milliliter effluent.  5.3.5.15-F -IsoP Assays 2t  Enzyme immunoassay of free 15-F2 -IsoP was performed according to the t  methods provided by the manufacturer (Cayman Chemical, Ann Arbor) as previously 213  described  . In brief, 50 uL standards and samples were added in duplicate to the 96-  well plate provided in the kit, followed by addition of 15-F2t-IsoP acetycholinesterase tracer and antibody. The prepared plates were then incubated overnight at room temperature. On the next day, the plates were washed 5 times with wash buffer, followed by addition of Ellman's reagent. After optimal color development, the plates were read at  118 405 nm, and the values of the unknowns were expressed as picograms 15-F2t-IsoP per milliliter effluent.  5.3.6. Myocardial Infarct Size Measurement  The measurement of infarct size was essentially identical to that described by Downey  2 7 0  except for the method of quantification. After the 2,3,5-triphenyl-tetrazolium  chloride (TTC) reaction, the hearts were sectioned transaxially, and size of infarct was evaluated as percentage of sectional area of infarcted tissue to the sectional area of the whole heart in 1 mm layers (five layers, L G scanner). Morphometric measurements of infarct size were performed with a L G scanner and 6.0 C E software. The histogram counts of the red (viable tissue) and white (infarcted tissue) were recorded. The percent infarction was calculated as white counts divided by the sum of the red plus white counts.  5.3.7. Statistical Analysis  A l l data are presented as means ± S E M . Hemodynamic variables and chemical assay parameters were compared by two-way analysis of variance (ANOVA) with repeated measures. One-way A N O V A was used to test for differences in infarct size between groups. The correlation was evaluated by the Pearson test. PO.05 was considered statistically significant.  5.4. Results  5.4.1. Endothelin-1 Release and its Relation with 15-F -IsoP 2t  119  Baseline effluent ET-1 concentrations did not differ among the experimental groups (Fig 5.1 A). Effluent ET-1 did not significantly change over time in the sham group. ET-1 increased in the control group during ischemia (Fig 5.1A, P O.OOl vs baseline) and increased further in thelso-P group compared to control (PO.05). ET-1 increased approximately 20% at Re-1 and 32.8±26.9% at Re-30 compared to baseline (BS10) in the control group. These increases did not reach statistical significance (P>0.1). Effluent ET-1 concentration in the IsoP group was significantly higher than that in the control group (PO.05) at reperfusion 60min (Re-60). Effluent ET-1 concentrations in both the IsoP-SQ and the SQ groups did not differ from those found in the control during ischemia and reperfusion. A weak but significant positive correlation (r = 0.77, P =0.04, Fig 5.IB) was noted between effluent concentrations of 15-F2 -IsoP and ET-1 during t  ischemia, but not during reperfusion, in the control (i.e., untreated) group.  5.4.2.15-F2t-IsoP Generation During Ischemia-reperfusion  Effluent 15-F2t-IsoP release in the sham group did not change over time during the 120 min perfusion period (data not shown). As shown in Fig 5.2, effluent 15-F2 -IsoP t  levels increased during ischemia (P < 0.001 vs BS10) and remained elevated at Re-1 (PO.05 or P O . O l vs BS10) in the control and the SQ groups. Effluent 15-F -IsoP 2t  release during early reperfusion (Re-1 and Re-5) in the SQ group tends to be higher than that in the control group, but the difference did not reach statistical significance (P>0.2).  120  1.00-1  ^^Sham I I Control ^•IsoP  0.75H  KSSSS IsoP.SQ  B S Q  0.50  0.25H  BS10  isch  Re-1  Re-5  Re-30  Re-60  Time during ischemia-reperfusion  Figure 5.1. A . Effluent Endothelin-1 (ET-1) concentrations during myocardial ischemiareperfusion. BS10 and isch indicate 10 min after stabilization and 30 min during global myocardial ischemia, respectively; Re-1, Re-5, Re-30 and Re-60 indicate 1, 5, 30 and 60 min after reperfusion, respectively. * PO.001 vs BS10; PO.05 or PO.01 vs control. #  (n=7 for each group). B. Relationship between 15-F -isoprostane (15-F -isoP) and ET-1 2t  2t  concentration during the first 30 min of ischemia in the control group. ET-1 release is positively correlated with 15-F -isoP concentration (r = 0.7695, 95% CI: 0.0389 2t  0.9640, P (two-tailed) = 0.043).  121  /ml)  125n  H Control  3SQ  100-  o> Q. Q. 7 5 O  .2  C M Ii. in  I I  50-  .1  I  250-  BS10  isch  Re-1  Re-5  ^ ni  Re-30  Re-60  Time during ischemia-reperfusion  Figure. 5.2. Effect of SQ 29548 (SQ) on 15-F -isoprostane (15-F -isoP) release during 2t  myocardial ischemia-reperfusion.  2t  BS10 and isch indicate 10 min after stabilization and  30 min during global myocardial ischemia, respectively; Re-1, Re-5, Re-30 and Re-60 indicate 1, 5, 30 and 60 min after reperfusion, respectively. * PO.001 or P< 0.05 vs BS10. P >0.05 SQ vs control.  122  5.4.3. C K - M B Release During Ischemia-reperfusion  Baseline C K - M B release was detectable in this model and did not differ among groups (Fig 5.3). Effluent C K - M B release did not significantly change over time in the sham group.  During ischemia, C K - M B increased relative to its baseline value in control hearts, but the increase did not reach statistical significance (P>0.05). C K - M B release was significantly higher than its baseline value (PO.05) during ischemia in the IsoP group, but unchanged in IsoP-SQ and SQ (P>0.05).  During reperfusion, C K - M B in the control group increased gradually and was significantly higher than the baseline value at Re-30 (PO.Ol). Effluent C K - M B concentration in the IsoP group increased more rapidly during reperfusion and was significantly higher than its baseline value at Re-5 (PO.05). The C K - M B level at Re-5 was greater in the IsoP group than in controls (PO.05). C K - M B levels in IsoP-SQ did not differ from untreated control during ischemia and reperfusion. C K - M B level in SQ was higher than in the control group and the IsoP group at Re-1 (O.05). It decreased quickly thereafter and was lower than the corresponding values in control and IsoP groups at Re-30. A positive correlation (r = 0.89, P =0.02) existed between effluent 15-F -IsoP and 2t  C K - M B levels measured in the control group during ischemia.(data not shown).  123  BS10  isch  Re-1  Re-5 Re-30 Re-60  Time during ischemia-reperfusion  Figure 5.3. Effluent C K - M B concentration during myocardial ischemia-reperfusion. BS10 and isch (ischemia) indicate 10 min after stabilization and 30 min during global myocardial ischemia, respectively; Re-1, Re-5, Re-30 and Re-60 indicate 1, 5, 30 and 60 min after reperfusion, respectively. IsoP and SQ indicate 15-F2 -isoprostane and SQ t  29548, respectively. *P< 0.05 vs BS10; P<0.05 vs control; P<0.05 vs isoP group. (n= 7 #  for each group)  +  124  A 30-,  Control  IsoP  IsoP-SQ  Group  SQ  Figure 5.4. A : Development of left ventricular end-diastolic pressure (LVEDP), reflecting myocardial contracture, during ischemia. B: Ischemic contracture onset time. The onset of contracture is defined as elevation of L V E D P > 2.5 mmHg vs baseline value.  #  P O . 0 5 vs control; P O . 0 5 or P O . O l vs IsoP (15-F2 -isoprostane) group; +  *PO.05 or P O . O l vs ischemia 30 min within the same group.  t  125  80-  ^Sham I Control I IsoP I IsoP-SQ ISQ  X 60H  E E Q. Q UJ >  40 204  B +  BS10  Pre-isch Re-10  Re-30  Re-60  Time points before and after reperfusion  Figure 5.5. Variations of left ventricular end-diastolic pressure (LVEDP), reflecting myocardial stiffness, during reperfusion. BS10 and Pre-isch indicate 10 min after stabilization and the time immediately prior to ischemia, respectively; Re-1, Re-5, Re-30 and Re-60 indicate 1, 5, 30 and 60 min after reperfusion, respectively. *P<0.05 or PO.01 vs BS10; * P O . 0 5 or PO.01 vs control; P O . 0 5 or PO.01 vs IsoP (15-F -isoprostane) +  2t  group.  126  5.4.4. Contracture Development during Ischemia  The L V E D P increased progressively during ischemia in the control group (Fig 5.4A). L V E D P in the IsoP group increased more quickly than that in the control group. At 30 and 35 min of ischemia, the magnitude of L V E D P in the IsoP group was significantly higher than that in the control group (PO.05). SQ 29548 attenuated the effect of 15-F2 -IsoP in augmenting L V E D P . The magnitude of L V E D P in the IsoP-SQ t  group was significantly lower than that in the IsoP group at ischemia 30 min and onwards.  The magnitude of L V E D P in the IsoP-SQ and the SQ group did not differ  from that in the control group during ischemia.  Time to the onset of ischemic contracture, defined as elevation of L V E D P >2.5 mmHg vs baseline value, was significantly shorter in the IsoP group (11.4±1.9 min) than in the control group (17.4±1.6 min, P O . 0 5 , Fig 5.4B). The latency to ischemic contracture in the IsoP-SQ (20.0±1.5 min) and the SQ (18.111.5 min) groups was significantly increased as compared to that in the IsoP group (PO.01 or PO.05), but did not differ from that in the control group (P>0.05, Fig 5.4B).  5.4.5. Functional Response to Ischemia-reperfusion  During reperfusion, L V E D P in the control group was significantly higher than that at baseline (Fig 5.5). 15-F2t-IsoP augmented the increase of L V E D P during  127 reperfusion. At Re-30 and Re-60, L V E D P values in the IsoP group were higher than those in the control group (PO.Ol). SQ 29548 attenuated the 15-F -IsoP-induced 2t  increase in L V E D P . The magnitude of L V E D P in the IsoP-SQ and the SQ groups did not significantly differ from that in the control group during reperfusion.  The L V D P in the sham group did not change significantly over time during the experimental period. The L V D P in the control group recovered to a maximum of 87.0±11.6, % of its baseline value at Re-30 (P>0.05 vs BS10, Fig 5.6) and decreased thereafter. The L V D P in the IsoP group recovered to a maximum of 56.5±13.5% of its baseline value at Re-30 (PO.05 vs BS10) and decreased quickly thereafter. At Re-60, L V D P in the IsoP group was lower than that in the control group. The L V D P values in the IsoP-SQ and the SQ group did not differ from those in the control group at Re-60. SQ 29548 exacerbated 15-F -IsoP induced reduction in L V D P relative to control group at 2t  Re-10.  128  ^^Sham r z n Control I IsoP mm IsoP-SQ B S Q  #  lllx  I 1  1: •I BS10  Pre-isch  Re-10  Re-30  #  Re-60  Time during ischemia-reperfusion  Figure 5.6. Recovery of left ventricular developed pressure (LVDP), reflecting effective myocardial contractility, during reperfusion. BS10 and Pre-isch indicate 10 min after stabilization and the time immediately prior to ischemia, respectively; Re-1, Re-5, Re-30 and Re-60 indicate 1, 5, 30 and 60 min after reperfusion, respectively. *P<0.05 vs BS10; #  P O . 0 5 vs control; P O . 0 5 or P O . O l vs IsoP (15-F -isoprostane) group. +  2t  129  Control  IsoP  IsoP-SQ  SQ  Figure 5.7. Myocardial infarct size. Top: representative images showing myocardial infarction (white) in the control (A), 15-F -isoprostane (IsoP, B), 15-F -isoprostane plus 2t  2t  SQ 29548 (IsoP+SQ, C) and SQ 29548 (SQ, D) groups. Bottom: Percentage infarction (Mean ± SEM): *P<0.05 vs control; P<0.05 or P O . O l vs IsoP group. +  130  5.4.6.  Coronary Perfusion Pressure  Neither 15-F -IsoP, SQ 29548, nor their combination affected CPP before 2t  ischemia. CPP did not increase significantly until after 60 min of reperfusion in the untreated control group (80.4±11.0 mmHg at Re-60 vs 51.3+1.1 mmHg at BS10, P<0.05). CPP in the IsoP group increased more quickly during reperfusion relative to the control group. At Re-30, the CPP value in the IsoP group (100.4±13.9 mmHg) was higher (PO.05) than its baseline value (52.1±3.9 mmHg) and higher (PO.05) than the corresponding value in the control group (65.6±4.6 mmHg).  SQ 29548 did not  significantly affect CPP as compared to the control group. At Re-60, CPP values did not significantly differ among the control (80.4111.0 mmHg), the IsoP (110.4114.9 mmHg), the IsoP+SQ (115.4114.9 mmHg) and the SQ (84.8113.5 mmHg) groups (P>0.05).  5.4.7.  Myocardial Infarct Size  As shown in figure 5.7, myocardial infarct size in the IsoP group is significantly larger than that of the control (untreated) group (PO.05). The myocardial infarct sizes in the IsoP-SQ and SQ groups are significantly smaller than those in the IsoP group (PO.05 or PO.01). Infarct sizes in the SQ group and IsoP-SQ groups were somewhat smaller than those in the control group, but the differences did not attain statistical significance.  131  5.5. Discussion  To our knowledge, this is the first study providing evidence that 15-F2 -IsoP may t  play a causative role in exacerbating myocardial IRI in the isolated perfused rat heart. Our findings include the following: (1) 15-F -IsoP(100 nM) did not affect pre-ischemic 2t  cardiac mechanics and coronary perfusion pressure but did reduce cardiac tolerance to ischemic insult, as manifested by an early onset and higher magnitude of ischemic contracture; (2) 15-F -IsoP stimulated the release and/or production of ET-1 during 2t  ischemia which was accompanied by increased severity of myocardial cellular damage evidenced by increased C K - M B release; (3) 15-F -IsoP increased myocardial infarct size 2t  and exacerbated post-ischemic myocardial dysfunction, which may be attributable, in part, to stimulation of ET-1 production and/or release during reperfusion.  Endothelin-1 has potent vasoconstrictor properties and is known to reduce myocardial contractility and contribute to the progression of the heart failure process '. 263  Plasma levels of ET-1 increase during cardiac operations requiring cardiopulmonary bypass ( C P B )  2 0 5  '  2 7 1  . A high plasma ET-1 level during the early postoperative period has  been associated with prolonged pharmacologic m anagement, longer intensive care unit stay, and complicated recovery ' . The present study clearly demonstrates that 15-F 205  272  2t  IsoP, whose formation increased in the myocardium and coronary artery during CPB surgery , can increase the release and/or production of ET-1 during myocardial 19  ischemia-reperfusion.  This might be a mechanism whereby 15-F -IsoP exacerbates 2t  myocardial IRI. The positive correlation between effluent concentrations of 15-F -IsoP 2t  132 and ET-1 during ischemia in the control (untreated) group suggests that endogenous 15F2 -IsoP may act to stimulate increased ET-1 release during ischemia. t  We observed a reduction in ET-1 concentration at Re-30, but a significant increase b y R e-60 c ompared t o control i n IsoP group (Fig 1 A). 1 5-F -IsoP m ay h ave 2t  triggered an increased formation of ET-1 during late reperfusion. In the IsoP group, the infusion of 15-F2 -IsoP was terminated at 15 min of reperfusion. Sequestration of a t  significant amount 15-F -IsoP in the heart tissue 45 min after the termination of 2t  exogenous 15-F -IsoP infusion is unlikely in this study, since the 15-F -IsoP decay half2t  2t  life in this model is about 4 min, as we observed in a preliminary study. 15-F -IsoP 2t  triggered increased formation of ET-1 during late reperfusion could represent an important mechanism responsible for post-ischemic myocardial dysfunction in the clinical setting. Whereas we previously found that plasma free 15-F -IsoP levels 2t  increased during ischemia-reperfusion for approximately 30 min during cardiac surgery, the 15-F -IsoP decay pattern during early reperfusion correlated with early postoperative 2t  cardiac recovery. cardiac surgery.  43  2 6 9  Plasma ET-1 levels may remain elevated at least 24 hours after We postulate that high levels of 15-F -IsoP during ischemia and/or 2t  early reperfusion induces ET-1 gene expression resulting in increased ET-1 production during late reperfusion.  272  Our study allows the postulation that 15-F -IsoP may increase the secretion of 2t  ET-1 into the coronary circulation relative to the myocardial tissue during ischemia. The study o f i solated p erfused r at h earts h as s hown t hat t he r atio o f E T-1 secretion t o t he interstitial transudates versus secretion to coronary effluent is about 6.6 at baseline.  2 6 5  However, the ratio of ET-1 secretion to the interstitial transudates versus coronary  133 effluent is reduced to about 2.5 during the period of low-flow ischemia and the first 30 min of reperfusion.  The relative reduction of ET-1 concentration observed in the IsoP  group at 30 min of reperfusion, 15 min after the termination of 15-F2 -IsoP infusion, t  indicates 15-F2 -IsoP may have primarily stimulated ET-1 release rather than its t  production during ischemia and early reperfsuion.  Despite 15-F2 -IsoP's bioactivity as a vasoconstrictor ' , reduction of coronary 71 74  t  flow is not likely a major mechanism of 15-F -IsoP action during myocardial IRI, at least 2t  in this model. In the current study, hearts were perfused at a constant flow rate. In addition, CPP at Re-60 did not differ significantly among experimental groups although L V D P in the IsoP group was significantly lower than that in the control, IsoP-SQ and the> SQ groups.  It is possible that 15-F2 -IsoP aggravates myocardial IRI by a complex t  mechanism involving the activation of N a - H exchange indirectly through the action of +  +  273* 274  ET-1  '  . Alternatively, 15-F2 -IsoP may act by reducing the intrinsic activity of nitric t  275  oxide  , an endogenous vasodilator. This may explain why the CPP value at Re-30 was  higher in the IsoP group relative to control irrespective of the similar effluent levels of ET-1 at this time point. Despite abolishing the deleterious effects of high concentration exogenous 15-F2 t  IsoP, SQ 29548 did not confer any beneficial effect in attenuating myocardial IRI compared to the control in this model. This is in keeping with previous findings describing the effect of exogenous 15-F -IsoP on the isolated guinea pig heart. 2t  7 6  The  relatively high concentration of C K - M B at Re-1 in the SQ group is likely due to rapid release of C K - M B from the ischemic tissue rather than the result of more intense tissue damage, since the infarct size of the SQ group is comparable to that in controls. The  134 inability of SQ 29548 to attenuate myocardial IRI in the isolated perfused heart model may suggest the following: (1) 15-F2 -IsoP production in the myocardium during t  ischemia and reperfusion is relatively low, and it is mainly a marker rather than a mediator of oxidative damage; (2) T X A may play little role in myocardial IRI in rat, a 2  finding similar to that found in gene knock-out m i c e .  276  The low L V D P at Re-10 in the IsoP-SQ group, although transient, is possibly a consequence of concomitant antagonism of the action of T X A by SQ 29584. ET-1 can 2  exert positive inotropic effect in the isolated rat heart. This effect could be potentiated via the action of T X A . The SQ 29548 blockade on T X A action may be enhanced in the 2  2  presence of 15-F -IsoP, an alternative ligand of the T X A receptor. 2t  2  Our finding that 15-F -IsoP can increase myocardial infarct size and exacerbate 2t  myocardial IRI may have important clinical implications. During cardiac surgery, systemic production of ROS occurs during CPB and may exceed production arising from reperfusion of the ischemic heart. Recent studies have found that the plasma level of 15F -IsoP dramatically increased shortly after the start of C P B . 2t  66  These high levels of 15-  F -IsoP could enter the heart either before aortic cross-clamping (the beginning of global 2t  myocardial ischemia) or at the time of aortic declamping, triggering and/or exacerbating myocardial IRI. The findings of the current study combined with our previous work on the effect of antioxidant supplementation with propofol suggest that combined therapy with antioxidant and 15-F -IsoP antagonism during ischemia and early reperfusion could 2t  offer a promising approach to attenuate myocardial IRI.  135  5.6. Conclusion  In summary, our study has demonstrated that 15-F2 -IsoP, a specific maker of t  oxidant damage, can exacerbate myocardial IRI as measured by elevated myocardial enzyme release, increased infarct size, and concomitant cardiac dysfunction. 15-F2t-IsoP, applied before, or present in high concentration during, ischemia produced an increase in effluent ET-1 concentration during reperfusion in the isolated rat heart. This may provide a mechanism whereby 15-F -IsoP mediates myocardial IRI. The 15-F -IsoP - ET-1 2t  2t  relationship in the pathogenesis of IRI requires further evaluation in the laboratory and clinical setting.  136  CHAPTER 6  G E N E R A L S U M M A R Y AND CONCLUSIONS  6.1. Summary  Ischemic heart disease is a major cause of death and/or disability for adult men and women living in industrialized societies. Its morbidity and mortality increase with aging, a process known to be associated with decreases in endogenous antioxidant capacity.  Myocardial ischemia-reperfusion injury (IRI) is a major pathophysiologic factor contributing to post-operative cardiac dysfunction in patients undergoing coronary artery bypass surgery utilizing cardiopulmonary bypass (CPB). Reactive oxygen species (ROS)mediated lipid peroxidation plays a critical role in mediating myocardial IRI. Using 15F2 -isoprostane as reliable measure of lipid peroxidation, we have demonstrated for the t  first time that significant in vivo lipid peroxidation occurs early during myocardial ischemia and continues during reperfusion (as described in Chapter 2) rather than primarily during reperfusion in patients undergoing cardiac surgery utilizing CPB. This finding is confirmed by r esults of a concomitant study by U lus and colleagues  6 6  who  submitted their work for publication in December 2002, shortly after the submission of our work in October 2002.  43  Our results provide direct evidence that effective  antioxidant intervention should be initiated and reinforced early during myocardial ischemia in order to maximally attenuate post-ischemic myocardial injury.  137  In Chapter 3, a unique therapeutic regimen of propofol was developed to best utilize its antioxidant properties in order to attenuate ROS generation during myocardial ischemia and early reperfusion using an isolated heart model. Propofol provides better cardiac protection when applied at the clinically achievable high concentration of 67 u M for 10 min before ischemia, during global myocardial ischemia, and continued during early reperfusion, followed by a relatively lower concentration during the later phase of reperfusion. This specific propofol treatment regimen has proven to be clinically promising (effective) in facilitating post-operative cardiac functional recovery in adult patients undergoing C A B G surgery or heart valve(s) replacement surgery and in pediatric patients undergoing open heart surgery for congenital ventricular septum defect repair (personal communication from Dr. David M . Ansley, Clinical Associate Professor of Anaesthesiology, Vancouver General Hospital, The University of British Columbia, Canada; Dr. Zhiyong Hunag, Clinical Associate Professor of Anesthesia, Sun Yat-sen Cardiovascular Hospital, Shenzhen, China; and Dr. Jiazhen Gu,  Professor of  Anesthesiology, Renmin Hospital, Wuhan University, Wuhan, China). Of particular relevance is our identification, using the isolated perfused heart model, that 15-F2r isoprostane is produced in situ during global myocardial ischemia. This finding provides evidence to support the use of antioxidant intervention during ischemia which would target the coronary endothelium and/or the cardiomycytes.  The study in Chapter 4 investigated whether or not aging could be a factor adversely affecting the cardiac protective effect on myocardial IRI. The results showed  138 that propofol, when applied at 67 u M before, during ischemia and during early reperfusion, equally preserved myocardial endogenous antioxidant capacity in the young and middle-age rat hearts and, more significantly, enhanced post-ischemic myocardial functional recovery in the middle-aged rat hearts relative to young rat hearts. This finding provides evidence to support the notion that drug(s) with antioxidant properties (such as propofol, in the our study) could be more effective in attenuating myocardial IRI in populations suffering from insufficient or decreased endogenous antioxidant capacity, such as the elderly. In our study, we identified a strong inverse correlation between myocardial 15-F2 -isoprostane and post-ischemic cardiac function in the isolated rat heart. t  This is similar in nature to the finding described in Chapter 2 showing an inverse relation between the decay pattern of plasma 15-F -isoprostane during early reperfusion and post2t  operative cardiac function, suggesting 15-F -isoprostane. itself could be a factor 2t  mediating myocardial IRI.  During myocardial ischemia and early reperfusion, apoptosis of coronary endothelial cells precedes myocyte apoptotic cell death in ischemia/reperfusion injury. Apoptosis spreads radially to the surrounding cardiac myocytes.  5 3  53  To investigate  whether propofol's protective effect against myocardial IRI involves the attenuation of vascular endothelial cell apoptosis, we examined the effects of propofol on tumor necrosis factor-alpha (TNF )-induced human umbilical vein endothelial cell ( H U V E C ) a  apoptosis (Appendix I). We found that propofol, in the concentration range from 12.5 to 100 u M , reduced TNF -induced ITTJVEC apotosis i n concentration-dependent fashion. a  The most apparent reduction in apoptosis being seen at propofol concentrations > 50 u M .  139 This finding is in keeping with the results described in Chapter 3 concerning the dosedependent protection by propofol against myocardial IRI and provides the first direct evidence that vascular endothelial cells are an important target of propofol action.  The strong correlation between plasma or coronary effluent 15-F -isoprostane 2t  and post-ischemic myocardial function as described in Chapters 2 and 4 suggests (but does not prove) a causative effect of 15-F2 -isoprostane in mediating myocardial IRI. t  Taken together with the fact that propofol attenuated, but did not completely prevent, the increase of 15-F2 -isoprostane during myocardial ischemia and early reperfusion, it would t  be important to know i f 15-F -isoprostane can directly mediate myocardial IRI and i f 152t  F2 -isoprostane antagonism could be a potential adjunct therapy. In the study described in t  Chapter 5, we found that 15-F -isoprostane, when applied during myocardial ischemia 2t  and early reperfusion, exacerbated myocardial IRI as evidenced by the increased myocardial infarct size, cellular damage and reduced post-ischemic myocardial function. 15-F2t-isoprostane antagonism with SQ 29548 abolished 15-F2 -isoprostane deleterious t  effects. We also found evidence that 15-F2 -isoprostane may mediate myocardial IRI, at t  least in part, by enhancing the release of ET-1 during ischemia and increasing ET-1 production during later reperfusion.  It is hoped that the studies described in the thesis have enhanced knowledge concerning the role of 15-F -isoprostane in the pathogenesis of myocardial IRI, from its 2t  mechanism(s) of action to its clinical relevance. It is hoped that our findings can aid in the development of novel and effective therapeutic interventions that would favourably  140 influence the unacceptably high mortality and morbidity associated with myocardial ischemia-reperfusion injury.  6.2. Conclusions  Role of  15-F2risoprostane in the pathogenesis of myocardial IRI  (1) 15-F2 -isoprostane release is increased during myocardial ischemia and t  reperfusion in isolated perfused rat hearts and in clinical settings using C P B , indicating increased lipid peroxidation during both myocardial ischemia and reperfusion.  (2) 15-F2 -isoprostane is a potential mediator of myocardial IRI, its mechanism of t  action appears to involve the stimulation of ET-1 release during ischemia and ET1 production during reperfusion. 15-F -isoprostane antagonism can abolish the 2t  deleterious effects of 15-F -isoprostane. 2t  Protective effect of propofol against myocardial IRI  (1) The concentration-dependent protection by propofol against myocardial IRI is associated with a reduction of ROS-mediated lipid peroxidation and attenuation of 15-F2t-isoprostane production during ischemia and early reperfusion.  141 (2) Propofol may exert its cardiac protection by attenuating vascular endothelial cell apoptosis induced by noxious stimuli.  (3) Propofol, at clinically relevant (high) concentrations (up to 67 uM), cannot completely prevent  the increased production of 15-F2 -isoprostane t  during  myocardial ischemia and reperfusion in the isolated rat heart model in the current study.  6.3. Future directions for research  Ischemic  preconditioning  or  anesthetic  preconditioning-mediated  cardiac  protection appears to require the generation of ROS prior to ischemia; however, it is not known whether or not 15-F2 -isoprostane could be acting as a mediator downstream of t  ROS generation. Understanding this would be important with regard to the development of optimal combination therapy to limit the severity of myocardial IRI.  15-F2 t  isoprostane antagonism should be most effective i f applied during ischemia and reperfusion i f its potential in mediating ischemic preconditioning is confirmed.  It is yet to be established i f 15-F2 -isoprostane primarily stimulates the release of t  ET-1 during myocardial ischemia, or i f it also stimulates ET-1 production, or increases ET-1 gene expression. Studies incorporating exogenous 15-F -isoprostane and specific 2t  endothelin-converting enzyme inhibitor(s) may help further address this question. Although vascular endothelium is considered the major source of ET-1, ET-1 can also be 977' 978  produced by cardiac myocytes  '  . Therefore, it would be of interest to investigate  142 whether or not 15-F2 -isoprostane-stimulated ET-1 release/production during myocardial t  ischemia-reperfusion  originates  primarily from  coronary  endothelium  or  from  cardiomycytes, or both.  Mechanistically, it would be meaningful to address whether 15-F2 -isoprostane t  can directly affect myocardial contractility under ischemic/hypoxic conditions. In addition, given the fact that the decay patterns of plasma 15-F2 -isoprostane predict the t  recovery of post-operative cardiac function as described in Chapter 2, it would be interesting to know i f 15-F2 -isoprostane can adversely affect human vascular endothelial t  cell integrity during reperfusion. Using the established human umbilical vein endothelial cell culture model as described in appendix I, we may investigate whether 15-F 2t  isoprostane can directly, or indirectly by affecting the secretion of ET-1, induce umbilical vein endothelial cell death. One could further explore the molecular signaling pathway(s) (i.e., specific G-protein-coupled receptors or different protein kinase C isomers) whereby 15-F2 -isoprostane exerts its actions. t  Endothelin-1 is one of the most potent vasoconstrictors thought to contribute to post-ischemic myocardial dysfunction. A recent study has shown that endothelin-1 can stimulate arachidonic acid release by cytosolic phospholipase A2 activation in rat vascular smooth muscle  1 1  . This would increase the substrate (i.e., arachidonic acid) for  15-F -isoprostane production. If this is the case during myocardial ischemia-reperfusion, 2t  then 15-F2 -isoprostane-induced ET-1 production during the late phase of reperfusion t  could in turn promote the formation of 15-F2 -isoprostane. This vicious cycle could t  potentially affect the patency of the grafted vessels after C A B G surgery. A high plasma  143 ET-1 level during the early postoperative period has been associated with prolonged pharmacologic management, longer intensive care unit stay, and complicated recovery. 205,272 j  n e r e  f  o r e j  m  e  interplay between 15-F -isoprostane and ET-1 during myocardial 2t  ischemia-reperfusion would be an important issue to address in the future studies.  Following these studies, a larger scale randomized clinical trial utilizing a combination of propofol and 15-F2 -isoprostane antagonism during ischemia and early t  reperfusion and/or ET-1 antagonism during late reperfusion is clearly merited.  144  References 1. 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Valgimigli M , Agnoletti L , Curello S, Comini L, Francolini G, Mastrorilli F, Merli E, Pirani R, Guardigli G, Grigolato PG, Ferrari R: Serum from patients with acute coronary syndromes displays a proapoptotic effect on human endothelial cells: a possible link to pan-coronary syndromes. Circulation 2003;107:264-70  192  APPENDIX 1:  Propofol  endothelial cell apoptosis:  inhibition  of  TNF-alpha-  induced  effects on Bcl-2 and Bax protein  vascular  expression  Introduction  The vascular endothelial monolayer serves as a barrier between the bloodstream and the vascular wall. Apoptotic endothelial cell death may critically disturb the integrity of endothelial monolayer and thereby contribute to vascular injury and atherosclerosis.  280  Apoptosis has become increasingly recognized as a mechanism of cell death during myocardial ischemia reperfusion injury (IRI),  281  although the relative contribution of  necrosis and apoptosis to total cardiac cell loss during IRI remains controversial. Recently, endothelial cell apoptosis was shown to precede myocyte cell apoptosis in the setting of myocardial IRI.  53  The latter study suggests that circulatory pro-apoptotic  inflammatory cytokines (such as tumor necrosis factor-alpha, TNF-alpha) and reactive oxygen species (ROS), that are elevated during myocardial IRI and atherosclerosis, promote myocyte apoptosis subsequent to the induction of endothelial cells apoptosis.  Propofol, 2,6-diisopropylphenol, an intravenous anesthetic agent frequently used during cardiac surgery and in postoperative sedation, tissue antioxidant capacity both in vitro and in vivo.  '  enhances red blood cell and We recently demonstrated that  propofol enhances myocardial antioxidant capacity and results in improved post-ischemic cardiac function in the isolated rat heart, in a dose-dependent manner.  213  In addition,  193 recent studies show that aging enhances the sensitivity of human endothelial cells toward 9X"i  apoptotic stimuli.  Interestingly, propofol, when applied at a clinically achievable high  concentration, enhances the ischemic tolerance of middle-aged rat hearts.  227  This finding  prompted us to postulate that propofol may produce a cardioprotective effect that is attributable to its ability to enhance endothelial cells resistance toward apoptotic stimuli. We hypothesized that propofol could inhibit TNF-alpha induced human umbilical vein endothelial cells (HUVECs) apoptosis by resuming a proper ratio of the antiapoptotic Bcl-2 protein over the pro-apoptotic Bax protein expression and that the propofol anti-apoptotic effect is related to its antioxidant capacity and its ability to enhance the generation of nitric oxide (NO), an important endothelial cell survival factor. • 284;285  Methods  Cell Culture Human umbilical vein endothelial cells (HUVECs) were isolated according to the method of Jaffe et a l .  286  Cells were cultured in a medium of D M E M (Gibco)  supplemented with 20% bovine calf serum (Sigma), maintained at 37 °C in 5% C 0 2 , and used at passage 2-3 to avoid "age-dependent" variations in levels of apoptosis.  287  Study 1. Dose-dependent effect of propofol on TNF-alpha induced H U V E C s apoptosis  When the cells were at 70% confluence, the cultured H U V E C s were divided into seven groups: H U V E C s in untreated group (control)  and propofol treatment control  (P25) group were further cultured at 37 °C for 24 hours, respectively, in the absence  194 (control) or presence of 25 u M propofol (Zeneca, Ltd.) in the medium; H U V E C s in the TNF-alpha (TNF) group and TNF-alpha plus propofol treatment groups were initially cultured for 30 min in the presence of zero (TNF), 12.5 (P . +TNF), 25 (P +TNF), 50 12  5  25  (P50+TNF) and 100 (P100+TNF) u M propofol, respectively. Cells were then cultured for 24 hours with TNF (40 ng/mL). The concentration of TNF used to induce apoptosis in the present study was chosen on the basis of previously published literature  2 8 8  in addition  to preliminary studies.  Study 2. Synergistic effect of H 0 and TNF in inducing H U V E C s apoptosis 2  2  When the cells were at 70% confluence, the cultured H U V E C s were divided into three treatment groups: (1) H U V E C s were cultured in the presence of 10 u M / L H 0 2  2  (H), (2) 10 u M IL H 0 plus 40 ng/mL TNF (TNF+H), or (3) 10 u M IL H 0 plus 40 2  2  2  2  ng/mL TNF and 50 u M propofol (TNF+H+P ) in the medium, respectively, at 37 °C for 50  24 h. Study results were compared with those obtained from the control and TNF-alpha groups of study 1.  Detection of Apoptosis Apoptosis was detected using D N A in situ terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL) staining a s p er t he m anufacturer' s p rotocol (Boshide B iotech Ltd, W uhan, C hina). In brief, after equilibration, end-labeling with digoxigenin-ll-dUTP by TdT enzyme in buffer was carried out for 1 hour at 37°C in a humidifying chamber. After treatment with stop/wash buffer, sections were incubated with anti-digoxigenin antibody-peroxidase  195 conjugate, rinsed, and stained with diaminobenzidine tetrahydrochloride. Negative controls were incubated with PBS instead of TdT enzyme, and positive controls were treated with DNasel. Sections were counterstained with Mayer's hematoxylin and mounted. A l l experiments were repeated on at least six independent occasions with consistent results.  Immunocytochemistry  Cells were washed in D M E M without BSA, and cytospins were performed (650 rpm for 6 minutes) on saline-coated slides at 1 x 10 cells/mL. Slides were fixed in 2% 6  paraformaldehyde for 15 minutes at room temperature and washed 5 times in PBS. Cells were permeabilized for 10 minutes at room temperature in blocking buffer (3% B S A i n PBS) plus 0.1% Triton X-100 followed by blocking of nonspecific binding in blocking buffer for 1 hour at room temperature. They were then incubated with the primary antibody (anti-Bcl-2 l:50)(Boshide Biotech Ltd, Wuhan); anti-nitrotyrosine 1:1000 diluted in phosphate-buffered saline (PBS) containing 1.5% goat serum(Boshide Biotech Ltd, Wuhan). Following overnight incubation at 4°C, cells were washed with PBS and incubated for 30 min at RT with the biotinylated secondary antibody. After washing, the cells were incubated for 30 min in Vectastain Elite A B C reagent (Boshide Biotech Ltd, Wuhan). After another wash, cells were incubated in peroxidase substrate solution until desired stain intensity is developed. The cells were then washed, dehydrated in increasing concentrations of ethanol, and cover-slipped using permount. Control sections were either incubated with the secondary antibody alone or immunoabsorbed with an excess of blocking peptide. Random fields (20-30 per slide) were examined at a high magnification  196 (x400) to calculate the prevalence of D N A fragmentation and Bcl-2/Bax expression. The percentage of T U N E L positive cells (termed apoptotic index, AI) was determined by dividing the number of positive-staining nuclei by the total number of nuclei of the cell and multiplying that value by 100. The percentage of Bcl-2/Bax expression was also determined. The density of Bcl-2 and Bax protein expression were determined using an automatic computer-assisted image analyzing system (IBAS-2000 Kontron, Germany), which automatically measure the density of 100 H U V E C s from 4 to 6 random fields. The density of Bcl-2 and Bax protein expression were expressed in arbitrary units.  Electron Microscopy Electron microscopy was performed to confirm that the ultrastructural features of apoptosis w ere p resent i n c ells e xposed t o T NF in t he p resent s tudy. E ndothelial c ells exposed to the conditions outlined above were fixed in 2.5% glutaraldehyde (pH 7.3) buffered with 0.1 mol/L sodium cacodylate overnight at 4°C and then washed with 0.1 mol/L sodium cacodylate buffer for 15 min before post-fixation with 1% osmium tetroxide buffered with 0.1 mol/L sodium cacodylate for 1 h on ice. After another wash with 0.1 mol/L sodium cacodylate buffer for 15 min, cells were dehydrated with increasing concentrations of alcohol. Next, cells were infiltrated with propylene oxide for 15 min, followed by 1:1 propylene oxide:epoxy resin for 1 h, 1:2 propylene oxide:epoxy resin for 2 h, and finally 100% epoxy resin for 2 h. Cells were embedded with fresh epoxy resin into molds and placed in a 60°C oven for 2 h. Ultrathin sections were stained with uranyl acetate and lead citrate and were examined with the use of a Hitachi H-600 electron microscope (Hitachi, Japan).  197  Nitric Oxide (NO) content Media from study 1 was collected 24 h after their respective treatments. The concentration of nitrites (NO2") and nitrates (NO3), stable end products of nitric oxide (NO), was determined by the Griess reaction, as follows. After deproteination by a solution of zinc sulfate, samples were incubated with cadmium granules to reduce nitrate to nitrite. The total nitrite was measured at 540 nm absorbance by diazotization with Griess reagent (Boshide Biotech Ltd, Wuhan). Nitrite concentrations were calculated by comparison with a standard calibration curve with sodium nitrite.  Statistical Analysis Results are expressed as mean ± S E M . Significance was evaluated using A N O V A followed by Tukey's post test. The correlation relationships were evaluated by the Pearsons test. P O.05 was considered significant.  Results Endothelial  cell apoptosis  T U N E L staining was rare in control (3.1±0.5%) and propofol (P25) treated H U V E C s (3.0±0.6%) (Fig. A - l , A , B). Stimulation of H U V E C s with TNF resulted in a dramatic increase in the A l to 45.5±1.2% (Fig.A-l,C, E). Propofol dose-dependently reduced TNF-induced apoptosis. More profound reduction in A l was seen in P25 (35.0±0.7%, Fig.A-l, D,E) and P  5 0  (25.2±0.8%). P i  decrease A l (22.6±0.5%) compared to that of P50.  0 0  did not significantly further  198 Immunohistochemical analysis of Bcl-2 and Bax protein expression As shown in figure A-2 and A-3, stimulation of H U V E C s with TNF leads to a significant reduction in Bcl-2 protein expression and a significant increase in Bax protein expression as compared to untreated controls. Propofol (25 uM) did not affect either Bcl2 or Bax protein expression in the absence of TNF stimulation. However, propofol, at >12.5 u M , significantly and dose-dependently attenuated TNF induced reduction in Bcl-2 protein expression (Fig.A-2, E). The maximal effect was seen at Pioo- The Bcl-2 density in the Pioo plus TNF (Pioo+TNF) group was significantly higher than that in the P50 plus TNF (P50+TNF) group. Propofol also significantly attenuated TNF induced elevation in Bax protein expression in a dose-dependent manner at the range 12.5 to 50 u M . There was no difference in effect between P  5 0  and Pioo- TNF reduced the ratio of Bcl-2  expression over Bax expression (Bcl-2/Bax) as compared to control (Fig.A-4, A). Propofol dose-dependently attenuated the TNF induced reduction in Bcl-2/Bax ratio (Fig. A-4, A). NO production As shown in figure A-5, stimulation of H U V E C s with TNF leads to significantly increased production of N O as compared to untreated control. Interestingly, addition of propofol (at 25 uM) to the culture medium also results in a significantly increased production of N O in the absence of a noxious stimulus (such as TNF). P12.5 and P25, in a dose-dependent manner, further increased TNF induced release of NO. However, P o and 5  Pioo did not result in more profound increase in H U V E C s N O release in the presence of TNF, as compared to P25.  199 Electron Microscopy  By qualitative electron microscopic analysis, typical features of apoptosis could hardly be seen in H U V E C s from control and P25 groups (Fig. A-6), but they were apparent in H U V E C s from TNF group (Fig A-6, C). Apoptotic morphologic changes can also be seen in H U V E C s from TNF plus propofol treatment groups (Fig A-6, D shows a typical endothelial cell from P25+T group), but to a much less degree in terms of severity.  Effect of H2O2 on TNF induced endothelial cell apoptosis, Bcl-2 and Bax expression  We then investigated whether H2O2 at a relatively low concentration (10 umol/L), could further augment the potential of TNF to increase apoptosis in HUVECs. As shown in Fig.A-7, H2O2 itself could independently increase the H U V E C apoptotic index (approximately 2-fold as compared to untreated c ontrol), but to a much less degree as compared to T N F which increased H U V E C apoptotic index by approximately 14-fold. Stimulation of H U V E C s concomitantly with H2O2 and T N F lead to about 20-fold increase in H U V E C s apoptotic index comparing to control (AI =62.7+1.4% in T+H group, vs 3.1+0.5% in control, Fig.A-7, F), showing apparent synergistic effects. Coincidently, cellular morphological changes are more severe in the T+H group than in the TNF group (data not shown).  As expected, stimulation of H U V E C s concomitantly with H2O2 and TNF led to a further significant decrease in Bcl-2 protein expression (Fig.A-8, A). It is of interest that H20 did not exaggerate TNF induced increase of Bax protein expression (Fig.8B). 2  200 Propofol (50 uM) significantly attenuated the joint effect of  H2O2  and TNF in  increasing FfUVECs apoptotic index and in decreasing Bcl-2 protein expression (Fig A-7 and A-8).  Correlation analysis  A tight inverse correlation exists between the ratio of Bcl-2/Bax protein expression and apoptotic index in untreated control HUVECs, and TNF treated H U V E C s with or without concomitant administration of propofol (r=-0.9520, P=0.0009, Fig.A-4, B). There was no relation between N O production and apoptotic index in H U V E C s from control and the propofol (25 uM) treated groups. This is because propofol increased N O production without affecting the A l in the absence of TNF stimulation. A trend of inverse relation between N O production and A l was seen in H U V E C s stimulated with TNF alone or T N F with varying concentrations of propofol, but this is not statistically significant (r =-0.85, P =0.07). However, a weak but significant positive correlation exists between N O production and the rate of Bcl-2/Bax protein expression (r=0.93, P =0.02) in H U V E C s stimulated with TNF with and without propofol treatments.  201  Conlroi  P25  T  P12 5+T P25+T  P50+T P10O*T  Groups  Figure A - l . TNF-induced apoptotic cell death in cultured H U V E C s is confirmed with T U N E L staining. T U N E L positive (TP) cells are stained brown. The percentage of TP cells is termed apoptotic index (Al). Representative photomicrographs of H U V E C s from untreated control (column A), propofol (25 p M , P25) treated (column B), TNF (T) treated (column C), and (column D) TNF and P25 co-treated (P25+T) groups. Original manifestations: ><200. Column E summarized the average A l of H U V E C s from control, P25  or  TNF (T) treated groups, or H U V E C s co-cultured with T plus varying  concentrations of propofol ranging from 12.5 (P25), 25 (P25), 50 (P50) and 100 (P100) p M . TUNEL-positive nuclei were counted and expressed as the percentage of total nuclei. A total of 1000 nuclei were counted in 10 random fields (n=10) on slides from each group. * PO.001 vs control; PO.001 vs T ; PO.001 or P O . 0 5 vs P50+T. +  #  202  Figure A-2. The expression of Bcl-2 protein evaluated by immunoperoxidase technique (PAP). Bcl-2 immunostaining positive cells display brown to deep brown particles (stained proteins) in the cytoplasm. Columns A through D show representative photomicrographs of H U V E C s from untreated control (A), propofol (25 u M , P25) treated (B), TNF (T) treated (C), and (D) TNF and P25 co-treated (P25+T) groups. Original manifestations: x200. Column E summarized the Mean (and SEM) levels of Bcl-2 protein (in arbitrary densitometry units) of H U V E C s from control, P25 or TNF (T) treated groups, or H U V E C s co-cultured with T plus varying concentrations of propofol ranging from 12.5 (P25), 25 (P25), 50 (P50) and 100 (P100) u M . * PO.001 vs control; PO.001 vs T; PO.001 or +  PO.05 vs P50+T. (n=100 cells per group).  #  203  Figure A - 3 . The expression of Bax protein evaluated by immunoperoxidase technique (PAP). Bax immunostaining positive cells display brown to deep brown particles (stained proteins) in the cytoplasm. Columns A through D show representative photomicrographs of H U V E C s from untreated control (A), propofol (25 u M , P25) treated (B), TNF (T) treated (C), and (D) TNF and P25 co-treated (P25+T) groups. Original manifestations: x200. Column E summarized the Mean (and SEM) levels of Bax protein (in arbitrary densitometry units) of H U V E C s from control, P25 or TNF (T) treated groups, or H U V E C s co-cultured with T plus varying concentrations of propofol ranging from 12.5 (P25), 25 (P25), 50 (P50) and 100 (P100) u M . * PO.001 vs control; PO.001 vs T; PO.001 or +  PO.05 vs P50+T. (n=100 cells per group).  #  204  Control  P25  T  P12.5+T P25+T P50+T P100+T  Groups  Figure  A - 4.  A . Bcl-2/Bax ratio. The Bcl-2/Bax ratio is significantly decreased in  endothelial cells treated with TNF (T). Propofol at 12.5 (P12.5), 25(P25), 50 (P50) and 100 u.M (PI00) dose-dependently increased Bcl-2/Bax ratio when administered with TNF.  *P<0.001 vs control; P O.001 vs T; P < 0.001 or P< 0.01 vs P50+T. B. A n +  #  significant inverse correlation exists between endothelial cell apoptotic index (Al) and the ratio of bcl-2 over Bax proteins expression (r=-0.9520, 95% CI: -0.9931 to -0.7029; P =0.0009).  205  Figure A-5. Nitric oxide (NO) concentration in the culture medium. Cultured endothelial cells were either untreated (control), treated with propofol (25 u M , P25) alone, with TNF (T) or T plus varying concentrations of propofol ranging from 12.5 (P25), 25 (P25), 50 (P50) and 100 (P100) u M . * PO.001 vs control; P O.001 vs T; P O.001 vs P50+T. +  (n=6 measures per group).  #  206  Figure A - 6. Representative electron microscopy of endothelial cells (ECs). A (control) and B (ECs treated with propofol alone at 25 uM): Normal ECs. C (EC treated with TNF): a typical apoptotic E C from TNF group. Local cytomembrane break, cytoplasm condensation and vesicle formation, chromatin condensation and margination, nucleolus can not be seen. D (EC treated with TNF and propofol at 25 uM): a typical E C in TNF+P25 group undergoing apoptosis. Cytomembrane intact, localized cytoplasm vesicle formation, nucleolus margination. Original magnification: ><5000.  E . a typical E C from TNF + P50 group.  207  208  Figure A-7. H2O2 and TNF synergistically induced apoptotic cell death in cultured HUVECs. T U N E L positive cells are stained brown. Representative photomicrographs of H U V E C s from untreated control (column A ) , H202 (H) treated (column B), T N F (T) treated (column C), H202 and TNF co-treated (H+T), H202 and TNF and propofol (50 u M , P) co-treated (T+H+P) groups. Original manifestations: x200. Column E summarized the average apoptotic index (AI) of the individual group.  TUNEL-positive nuclei were  counted and expressed as the percentage of total nuclei. A total of 1000 nuclei were counted in 10 random fields on slides from each group. * P<0.001 vs control; P<0.001 vs +  T; * PO.001 vs T+H.  209  210  *+#  *+#  1 Control !H F2221T BT+H+P50  Control  T  T+H  T+H+P50  Groups  Figure A-8. Effects of H 0 2  2  on TNF-mediated changes in Bcl-2 (A) and Bax (B)  expression. The expression of Bcl-2 and Bax protein were evaluated by immunoperoxidase technique (PAP). Levels o f B cl-2 a nd B ax p rotein e xpression (in a rbitrary d ensitometry units) were expressed as mean ± S E M . H U V E C s cultures was either untreated (control), treated with H 0 (H)25 or TNF (T), co-cultured with T plus H , or co-cultured with T+H 2  2  plus propofol at 50 u M (T+H+P50). * PO.001 vs control; PO.001 vs T; PO.001 vs +  T+H. (n=100 cells per group).  #  211  Discussion  TNF stimulation resulted in a reduced Bcl-2/Bax ratio in H U V E C s . Propofol dose-dependently enhances the ratio of the anti-apoptotic Bcl-2 protein over the proapoptotic Bax protein expression in this system. This was associated with graded suppression of TNF-induced apoptosis as assessed by T U N E L assay and confirmed by characteristic apoptotic morphologic changes.  At a dose range from 12.5 to 50 u M ,  propofol enhancement of Bcl-2/Bax ratio is achieved through an increase in expression of Bcl-2 and a decrease in the expression of Bax. However, this effect appears dose limited since the highest concentration of propofol (Pioo) does not significantly reduce Bax expression more than P o. However, Pioo did increase Bcl-2 expression more than P50. 5  This could be explained on the basis that Pioo completely abolished TNF-induced increases in Bax expression while maintaining baseline levels (Fig. A-3, E). This is likely an important mechanism of protection. Like other important molecules, notably nitric oxide (NO), Bax also has a dual role. Under pathological conditions, Bax overexpression may induce mitochondrial depolarization and cytochrome c release, downstream activation of executioner caspases  2 9 0  289  resulting in the  to augment apoptosis. The formation  Bax-Bax homodimer serves to induce apoptosis, while the Bax-Bcl-2 heterodimer formed under physiological conditions, is an important inhibitor of apoptosis.  291  It is noteworthy that propofol treatment primarily restores the expression of Bcl-2 and the Bcl-2/Bax ratio towards normal values and does not result in an overexpression of the anti-apoptotic Bcl-2 protein. This effect is not what we expected to see, but may represent a promising therapeutic approach. Bcl-2 is localized to intracellular sites of  212 ROS generation including mitochondria and may function in an antioxidant pathway to 909  prevent apoptosis. peroxidation.  Following an apoptotic signal, cells sustain progressive lipid  Overexpression of Bcl-2 functions to suppress lipid peroxidation.  292  However, in lymphocytes the level of Bcl-2 expression may determine the balance between apoptosis and necrosis, but does not prevent cell death induced by oxidized low density lipoproteins (oxLDL).  293  In cells expressing relatively high levels Bcl-2, oxLDL  induced mainly necrosis. In cells expressing relatively low levels Bcl-2, the rate of oxLDL-induced apoptosis was higher than that of primary necrosis.  293  Recent studies  demonstrate that overexpression of Bcl-2 paradoxically exerted a pro-apoptotic effect in the reperfused liver.  294  Taken together, these studies, together with ours, suggest that  stabilizing Bcl-2 and Bax expression, rather than induction of the "anti-apoptotic" Bcl-2 and/or suppression of the "pro-apoptotic" Bax protein, represents a more meaningful approach. Propofol (P25) treatment, in the absence of TNF, enhances the production and release of N O from H U V E C s . This is similar in nature to a previous report that application of propofol stimulates the production of N O from cultured porcine aortic endothelial cells.  295  Interestingly, TNF enhances the production of N O to a similar degree  as propofol (Fig. A-5), but, in contrast, this is accompanied by an increase in apoptosis in H U V E C s . It seems plausible that the TNF-induced N O overproduction relative to control levels (in the absence of antioxidant intervention) would result in increased production of peroxynitrite which may promote apoptosis by increasing Bax expression.  296  Co-culture  of H U V E C s with TNF and propofol led to profound overproduction of N O compared to TNF or propofol alone. This is associated with reduced Bax expression and enhanced  213 Bcl-2 production compared to the T N F group. This suggests that overproduction of N O by endothelial cells in response to TNF stimulation is initially aimed to protect the cells, rather than produce cell injury. This is manifested by the significant positive correlation between N O production and the rate of Bcl-2/Bax protein expression in FIUVECs stimulated with TNF, with and without propofol in increasing concentrations. Indeed, study shows that N O confers resistance to apoptosis in H U V E C s ,  297  likely secondary to  the attenuation of caspase activity by nitrosylating caspase 3 and stabilizing B c l - 2 .  298  The question arises "How does propofol confer its protective effect on endothelial cells?"  TNF stimulates upregulation of N O synthase activity and N O production in HUVECs  2 9 9  which can be accompanied by a burst in production (three- to fourfold  increase) of intracellular ROS including superoxide anion and H2O2.  300  The reaction of  N O with superoxide anion can increase the generation of peroxynitrite (ONOO"). Exposure of eNOS to oxidants (including peroxynitrite) causes increased enzymatic uncoupling and the generation of superoxide anion rather than N O , '  resulting in  increased oxidant stress and a net decrease in N O production. Propofol can directly scavenge ROS including O N O O " , ' 141  303  therefore, blocking the vicious cycle. Our finding  that the addition of H2O2 exaggerates TNF induced apoptosis supports the interplay between ROS and TNF. The H2O2 concentration (10 umol/L) used in this study is in the lowest range that induces endothelial cell apoptosis but not necrosis. a direct source of ONOO" (which increases Bax expression  2 9 6  304  Since H2O2 is not  ) , its use in this study helps  to elucidate the relative roles of ROS in inducing endothelial cell apoptosis.  The release of cytochrome c from mitochondria into the cytosol is considered as an i mportant e vent 1 eading t o i rreversible c ell d eath a nd t he o pening o f m itochondrial  214 transition pore (MPTP) may be an important mechanism for mitochondrial cytochrome c release.  Inhibition of MPTP  cytochrome c in H U V E C s .  reduced T N F induced release of mitochondrial  Further, it has been suggested that propofol may offer  myocardial protection by inhibiting M P T P ,  249  at concentrations low as 11-22 u M (2-  4ug/ml), likely acting indirectly through its antioxidant properties since ROS is a major mediator of MPTP opening. at concentrations >50 u M .  2 5 2  253  Propofol, however, may directly inhibit myocyte MPTP  Interestingly, the most profound inhibition of TNF induced  H U V E C s apoptosis is manifested at propofol concentrations >50 u M ( P  50  and  Pioo)  in  this study, suggesting that inhibition of MPTP in H U V E C s as one of the mechanisms of propofol protection.  Conclusions and clinical implications  Our s tudy p rovides e vidence for t he first t ime t hat p ropofol, a c ommonly u sed anesthetic agent for cardiac surgery and for postoperative sedation, attenuates T N F induced H U V E C s apoptosis in a dose-dependent manner. Given the fact that the release of inflammatory cytokines (including TNF) is increased during and after cardiac surgery and that serum from patients with acute coronary syndromes displays a pro-apoptotic effect on endothelial cells,  307  application of propofol, in high concentrations (-50 to 60  125  uM),  may prove to be a promising approach in reducing peri- and post-operative IRI  and related cardiovascular symptoms.  

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