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Potential deleterious effect of [Beta]-adrenergic stimulation during warm-blood cardioplegia in rabbit… Cook, Richard Chung-Sop 2002

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POTENTIAL DELETERIOUS EFFECT OF p-ADRENERGIC STIMULATION DURING WARM-BLOOD CARDIOPLEGIA LN RABBIT HEARTS By RICHARD CHUNG-SOP COOK B.Sc, The University of Alberta, 1988 M.D., The University of Alberta, 1992 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES (Department of Surgery) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA April 2002 © Richard Chung-sop Cook, 2002 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Surgery The University of British Columbia Vancouver, Canada April 25, 2002 ABSTRACT We hypothesized that p-adrenergic stimulation with isoproterenol during continuous normothermic cardioplegic arrest would enhance the regenerative and regulatory function of the myocardium, resulting in improved cardiac function. We studied isolated rabbit hearts paced at -200 beats/minute and cross-circulated by a support rabbit. We measured ventricular pressure over a range of ventricular volumes to determine maximal elastance at baseline and 20 and 45 minutes after discontinuation of cardioplegia. Myocardial oxygen consumption measurements were performed simultaneously and during cardioplegic arrest. Hearts were prospectively randomized to receive either isoproterenol 0.1M or control in blinded fashion for 10 minutes during a 1 hour continuous warm blood cardioplegic arrest. Compared to control hearts, isoproterenol-treated hearts had trends towards longer time to first spontaneous heart-beat (control 141 ± 43 vs isoproterenol 200 ± 74 seconds, p = 0.07), and longer time to capture of atrial pacing (control 214 ± 52 vs isoproterenol -288 ± 91 seconds, p = 0.06). There was no difference observed in the myocardial oxygen consumption between isoproterenol-treated and control groups of hearts. Myocardial oxygen consumption decreased during administration of cardioplegia (p<0.01) but there was no significant change in myocardial oxygen consumption during isoproterenol infusion during cardioplegic arrest. There was a significant reduction in maximal elastance compared to baseline 20 minutes after discontinuation of cardioplegic arrest in both groups (control 7.3 ± 1.7 mmHg/uL vs 9.0 ± 1.7 irimHg/uL, p = 0.02, isoproterenol-treated 6.8 ± 2.8 mmHg/uL vs 8.2 ± 2.6 mmHg/pL, p = 0.01, respectively) with recovery of maximal elastance by 45 minutes in control hearts only. We conclude that exposure of hearts to isoproterenol during warm cardioplegic arrest has a deleterious effect which may be mediated through mechanisms independent of increased myocardial oxygen consumption. TABLE OF CONTENTS Abstract i i Table Of Contents iv List Of Abbreviations vii List Of Figures ix Preface x CHAPTER I. INTRODUCTION 1 1.1 Myocardial Protection With Cardioplegic Arrest 1 1.2 p-Adrenergic Stimulation: Effects 2 1.3 P-Adrenergic Stimulation: Mechanisms 3 1.4 Hypothesis And Objectives 4 CHAPTER II. METHODS 5 2.1 Preparation Of The Support Rabbit 5 2.2 Preparation Of The Isolated Heart 6 2.3 Preparation Of Cardioplegia 7 2.4 Measurements 7 2.5 Experimental Protocol 8 r 2.6 Data Analysis 9 CHAPTER III. RESULTS 10 CHAPTER IV. DISCUSSION 12 4.1 Discussion Of Results 12 4.2 Potential Negative Effects Of P-Adrenergic Stimulation 15 4.3 Cardiac Metabolism 16 (i) Background 16 (ii) Glycolysis And Glucose Oxidation 16 (iii) Pyruvate Dehyrogenase Regulation 17 (iv) Fatty Acid Metabolism 17 (v) Effect Of Isoproterenol On Cardiac Metabolism 18 4.4 Emax, And Determination Of Left Ventricular Contractility 20 CHAPTER V. UNANSWERED QUESTIONS 22 CHAPTER VI. POTENTIAL FUTURE STUDIES: EFFECT OF VASOPRESSIN ON , CARDIAC M E T A B O L I S M 23 6.1 Introduction 23 6.2 Mechanism Of Action 23 6.3 Evidence Of Benefit After Cardiopulmonary Bypass 24 6.4 Evidence Of Benefit In Brain-Dead Organ Donors (Hearts) 25 6.5 Effect On Pyruvate Dehydrogenase 27 6.6 Evidence Of Benefit With Glucose-Insulin-Potassium In Human Hearts During And After Cardiopulmonary Bypass, And Possible Benefit In Heart Transplantation 28 6.7 Potential Deleterious Effects 30 CHAPTER VII. EXPERIMENTAL PROPOSAL 31 7.1 Introduction 31 7.2 Purpose 31 7.3 End Points 32 7.4 Methods 32 CHAPTER VIII. S U M M A R Y Figure Legends Bibliography LIST OF ABBREVIATIONS ATP Adenosine tri-phosphate BP Blood pressure bpm Beats per minute C a + + Calcium C A B G Coronary artery bypass graft cAMP Cyclic adenosine mono-phosphate Ca0 2 Arterial oxygen content Ccv0 2 Coronary venous oxygen content CI Cardiac index CoA Coenzyme A CPB Cardio-pulmonary bypass CT Computed tomography D C A Dichloroacetate ddAVP Desmopressin dp/dt Change in pressure over change in time, Ees Elastance at end-systole Emax Maximal elastance ESPVR End-systolic pressure-volume relationship FA Fatty acid FFA Free fatty acid G6P Glucose-6-phosphate GIK Glucose-insulin-potassium H + Hydrogen ion Hb Hemoglobin IABP Intra-aortic balloon pump K + Potassium KC1 Potassium chloride L V Left ventricular L V A D Left ventricular assist device M A P Mean arterial pressure M g + + Magnesium M g S 0 4 Magnesium sulphate M V 0 2 Myocardial oxygen consumption NAD+ Nicotinamide adenine dinucleotide NS Not significant O2 Oxygen Pa0 2 Arterial partial pressure of oxygen P.CO2 Partial pressure of carbon dioxide Pcv0 2 Coronary venous partial pressure of oxygen PDH Pyruvate dehydrogenase P 0 2 Partial pressure of oxygen SBP Systolic blood pressure SEM Standard error of the mean SIRS Systemic inflammatory release syndrome LIST OF FIGURES Figure 1. "Effect of mechanical activity and temperature on MVO2" . P- 48 Figure 2. "Effect of isoproterenol on MVO2 of arrested and beating hearts". P- 49 Figure 3. "Mechanism and actions of P-adrenergic stimulation". P- 50 Figure 4. "The perfusion apparatus". P- 51 Figure 5. "Example of approximation of Emax". P- 52 Figure 6. " M V 0 2 " . P- 53 Figure 7. "Emax". P- 54 Figure 8. "Diastolic pressure-volume relationship". P- 55 Figure 9. "Peak systolic pressure". P- 56 Figure 10. "Time to first beat and return to paced rhythm". P- 57 Figure 11. "Glycolysis". P- 58 Figure 12. "Aerobic glucose oxidation". P- 59 Figure 13. "Regulation of pyruvate dehydrogenase (PDH)". P- 60 Figure 14. "Fatty acid oxidation". P- 61 Figure 15. "Pressure-volume relationships in the working heart". P- 62 Figure 16. "Time-varying elastance". P- 63 Figure 17. "Pressure-volume relationships during isovolumetric contractions". P- 64 Figure 18. "Approximation of Emax". P- 65 P R E F A C E The experimental work described in this thesis was published in the Journal of Investigative Surgery. Cook RC, Goddard C M , Ashe K A , Chen K , Lichtenstein SV, Walley KR. Potential deleterious effect of p-adrenergic stimulation during warm-blood cardioplegia in rabbit hearts. Journal of Investigative Surgery 2001;14(4):213-220. CHAPTER I. INTRODUCTION 1.1 Myocardial Protection With Cardioplegic Arrest Performance of complex cardiac surgical procedures has become almost commonplace since the development of cardiopulmonary bypass (CPB) and cardioplegic arrest of the heart. Before the availability of CPB, the only way to perform intracardiac surgery was to immerse the patient into ice water in order to reduce the core temperature. This concept carried through into the development of cardioplegic arrest. After initial studies with potassium citrate demonstrated significant myocardial damage, the preferred methods for inducing cardiac arrest became ischemic arrest, fibrillation, and hypothermic arrest. Eventually, hypothermic coronary perfusion and perfusion with potassium chloride became the predominant methods of achieving cardiac arrest because of the observation that these methods were associated with complete recovery of the heart, along with preservation of high-energy phosphate stores in these hearts'. At that time, it was felt that hypothermia and potassium chloride had additive protective effects, with hypothermia being necessary to reduce the metabolic rate. Buckberg et al demonstrated that at normothermia, fibrillating hearts had a higher mean myocardial oxygen consumption (MVO2) than empty beating hearts; however, at temperatures of 32°C and less, fibrillating hearts had a lower mean MVO2 than that of empty beating hearts. At all temperatures, from 22°C to 37°C, arrested hearts had significantly lower M V 0 2 (Figure 1). A similar observation had been made by Bernhard et al in the early sixties. Both studies demonstrated that the majority of the myocardial oxygen consumption is due to mechanical activity - ie. the M V 0 2 of arrested hearts is -20% of that seen in the working heart. Furthermore, in arrested hearts, reducing the temperature from 37°C to 11°C only decreased the MVO2 slightly (from 2.2 to 1.6 cc CVlOOg whole heart/minute). Armed with this evidence, Lichtenstein et al 4 introduced the concept of warm blood cardioplegia to the modern era of cardiac surgery. One theoretical advantage of warm blood cardioplegia was the avoidance of the negative effects of hypothermia. These include enzymatic inhibition with blocking of glycolysis at temperatures < 10°C, protein denaturation, ion pump inactivation, and leftward shift of the oxygen dissocation curve of hemoglobin (ie. higher affinity of hemoglobin (Hb) for oxygen (O2) at lower temperatures). It was felt that continuous warm blood cardioplegia would avoid a period-of hypothermic ischemia, and would therefore minimize reperfusion injury. In addition, it was felt that maintenance of the basal metabolic activity of the heart would allow for continued adenosine tri-phosphate (ATP) production during cardioplegic arrest. In their clinical trial, it was shown that warm blood cardioplegia was associated with significantly fewer peri-operative myocardial infarctions and occurrences of low output syndrome, and a significantly lower requirement for intra-aortic balloon pump (IABP) support: Additional evidence of superior metabolic activity with normothermic cardioplegic arrest comes from a recent study of adenylate cyclase activity in human atrial biopsy specimens5. In this study, adenylate cyclase activity was induced by isoproterenol to a greater degree in tissue from normothermic hearts than atrial specimens from hypothermic he arts. 1.2 P-Adrenergic Stimulation: Effects The concept that P-adrenergic stimulation could perhaps increase the basal non-mechanical metabolic activity of the myocardium was explored by Klocke et al 6 in 1965 using an isolated canine heart preparation. At that time, it was as yet unclear how much of the myocardial oxygen demand was due to mechanical vs metabolic activity. The canine hearts were exposed to isoproterenol while beating, and again during a period of normothermic arrest, with the MVO2 measured before and after increasing doses of isoproterenol. They demonstrated that the majority of isoproterenol's effect on MVO2 was due to an increase in mechanical activity, however, there was also a 32% change in MVO2 in arrested hearts in 2 response to a lOug bolus of isoproterenol (Figure 2). The question which was not addressed, however, was whether this change in metabolic activity by isoproterenol could improve myocardial function after a period of cardioplegic arrest. 1.3 p-Adrenergic Stimulation: Mechanisms The inotropic effect of beta-adrenergic stimulation on cardiac myocytes is well understood and has been thoroughly characterized7 (Figure 3). Beta agonists bind to p receptors on the cell surface, thereby triggering a complex sequence of intracellular events. Upon coupling of the P-agonist and receptor, the a subunit of the G-protein switches from its inactive to its active state, and dissociates from the G-protein complex, thereby stimulating adenylate cyclase. Adenylate cyclase then converts ATP into cyclic adenosine mono-phosphate (cAMP), the second messenger of P-adrenergic stimulation. Protein kinase A is then stimulated by cAMP, which in turn phosphorylates other key intracellular enzymes, thereby converting them from their inactive into their active forms. One such protein is a sarcolemmal protein p27 which allows for increased Ca"1-1" entry into the cell through increased opening of the voltage-dependent L-type calcium channels. This resultant influx of C a + + causes a larger release of C a ^ from the sarcoplasmic reticulum, culminating in the positive inotropic effect of p-adrenergic stimulation. Other enzymes activated by protein kinase A are responsible for the metabolic effects of p-adrenergic stimulation, such as increased lipolysis and glycogenolysis. In the case of lipolysis, protein kinase A activates triacylglycerol lipase, which liberates fatty acids from triaclyglycerol. Glycogenolysis is mediated by phosphorylase a, which is responsible for the liberation of glucose-1-phosphate from glycogen. Activation of phosphorylase a thereby increases the rate of glycolysis. The conversion of phosphorylase b to phosphorylase a depends upon the action of phosphorylase kinase a, which itself is derived from phosphorylase kinase b via the action of protein kinase A. . The p i receptors are found primarily in cardiac tissue, and when stimulated, cause increased inotropy and tachycardia. They are also present in adipocytes and cause lipolysis when stimulated. The p2 receptors are found primarily in smooth muscle, and cause relaxation of smooth muscle fibers when stimulated, resulting in vasodilation (in blood vessels), bronchodilation (in bronchi), and urinary retention (genito-urinary tract). The p2 receptors in . muscle and liver are also involved in the stimulation of glycogeholysis and gluconeogenesis. 1.4 Hypothesis And Objectives Although the effects of isoproterenol on contracting hearts is well understood, the effect of p-adrenergic stimulation on arrested, warm, well-oxygenated, and perfused myocardium has not been extensively studied. We hypothesized that isoproterenol administered during continuous warm blood cardioplegia could increase the basal metabolic rate, thereby enhancing the regenerative and regulatory function of the myocardium, and resulting in an improvement in cardiac function following cardioplegic arrest. Our objectives were to determine whether isoproterenol delivered at the end of a period of continuous warm blood cardioplegia could (1) increase the metabolic activity of the myocardium as measured by the M V 0 2 , and (2) improve cardiac function afterwards as measured by the maximal elastance (Emax), time to first spontaneous heart beat, and time to capture of atrial pacing. > 4 C H A P T E R II. M E T H O D S This was a randomized, placebo-controlled, double-blind study of cross-circulated rabbit hearts exposed to isoproterenol or normal saline during a period of continuous warm-blood cardioplegic arrest. The surgical and experimental protocols used in the study were approved by the University of British Columbia Animal Care Committee, and adhered to the Canadian guidelines on animal experimentation. 2.1 Preparation Of The Support Rabbit Sixteen 3.7 ± 0.7 kg female New Zealand White rabbits were initially anaesthetized using isoflurane 5% by mask. Marcaine 0.75% was used to infiltrate the skin at all incision sites. Anaesthesia of the rabbit was maintained by inhaled isoflurane 1.5 - 2%. Adequacy of anaesthesia was confirmed by the absence of withdrawal reflex after painful stimulus of pressure applied to a hind toe. After tracheostomy, rabbits were ventilated and oxygenated with supplemental oxygen to maintain arterial partial pressure of 0 2 (PO2) at ~ 400 mmHg, and partial pressure of carbon dioxide (PCO2) at ~ 30 mmHg. A polyethylene catheter was inserted into the right internal carotid artery to supply oxygenated blood to an extracorporeal circuit to perfuse an isolated heart8 (see below), for continuous blood pressure monitoring, and for periodic arterial blood sampling. A second polyethylene catheter was inserted into the left common external jugular vein to allow venous return of deoxygenated blood from the isolated heart. Rabbits were anticoagulated with 1,000 IU/kg heparin intravenously, with subsequent doses administered every 2 hours during the experimental protocol. Normal saline was infused at a rate of 1 mL/minute and additional intermittent boluses of 10 - 20 mL of normal saline were administered via the left common external jugular vein line to maintain mean arterial 5 blood pressure above 60 mmHg. A rectal temperature probe was inserted, and core body temperature of the rabbit was maintained ~ 37.5 °C using a heating blanket. 2.2 Preparation Of The Isolated Heart Sixteen 2.3 ± 0.4 kg New Zealand White female rabbits were anaesthetized and ventilated using the same technique as for support rabbits. A midline sternotomy was performed, and a ligature was passed around the inferior vena cava. After identification and ligation of the descending thoracic aorta, ice cold high-potassium normosol (2 mEq KC1/20 mL normosol) was then injected via the carotid artery catheter until the heart arrested (< 20 mL). The inferior vena cava was then ligated, and the heart-lung block was rapidly removed and placed in cold normal saline. After separation of the heart from the lungs, the heart was weighed and affixed by the aorta onto the base of a perfusion column of an extracorporeal circuit (Figure 4). Arterial blood from the support rabbit was delivered using a roller pump to a water-jacketed glass heating chamber maintained at 37°C, through an in-line flow probe (Transonics systems), and then to the perfusion column. The perfusion column maintained coronary perfusion pressure at 80 mmHg at the level of the aortic valve. Anticoagulation of the support rabbit and perfusion apparatus was maintained by addition of heparin 1000 IU/kg to the venous return line every 2 hours. Once affixed to the perfusion column a polyethylene catheter was placed into the right ventricle of the isolated heart, through the tricuspid valve via the superior vena cava and right atrium. This catheter was used to obtain samples of coronary venous blood. A temperature probe was placed into the right ventricle through a small incision in the proximal pulmonary artery. A small incision was made in the free wall of the left atrium, and a 7 french single lumen pressure transducer catheter with a side port (Millar Instruments, Houston, TX) surrounded by a latex balloon was inserted into the left ventricle. The balloon did not develop measurable transmural pressure until it had been inflated to a volume of 784 ±175 uL, which defines the unstressed volume. At all times during the experiment ventricular volume was well below balloon unstressed volume so that measured pressure was due to ventricular forces and not due to the elastance of the balloon. The balloon was inflated with water to maintain a . resting diastolic pressure of 0 mmHg. Atrial pacing at a rate of ~ 200 bpm was achieved using pacing leads attached to the right and left atrial appendages. The isolated heart was kept at ~ 37.5°C by a water-jacketed glass heating chamber. 2.3' Preparation Of Cardioplegia Blood (124 ±11.7 mL) was collected through a polyethylene catheter placed into the right internal carotid artery of a third rabbit, anaesthetized in the same fashion as the support rabbit. Blood cardioplegia was prepared according to the recipe for cardioplegia used in human hearts at our institution, and consisted of a 4:1 ratio of heparinized blood to diluent. The composition of the diluent was as follows: normosol 100 mL, KC1 10 mEq, magnesium sulphate (MgS04) 250 pg, and nitroglycerine 150 pg. Prior to perfusion of the isolated heart, the cardioplegia was oxygenated by bubbling 95%02/5%C02 with the blood cardioplegia in a vertical glass column. Using a separate roller pump, oxygenated cardioplegia (P0 2 ~ 400 mmHg) was delivered to the isolated heart via a 3-way stopcock in the arterial line from the support rabbit, proximal to the water-jacketed glass heating chamber. • 2.4 Measurements Left ventricular (LV) contractility was determined using the slope of the end-systolic pressure-volume relationship (ESPVR), or Emax. Emax was chosen as the measurement of L V contractility because it is largely independent of ventricular preload and afterload9. The ESPVR 7 was determined by increasing the volume of the intraventricular balloon by 50 uL increments at 30 second intervals, and recording the systolic pressure just prior to each incremental increase in ( volume. The ESPVR was the slope of the best fit line for the recorded systolic pressures-volume points (Figure 5). Arterial blood samples and coronary venous blood samples were obtained for calculation of MVO2. Blood was analyzed for oxygen partial pressure (Px0 2) and content (CXO2) using a blood gas analyzer (ABL30; Radiometer, Copenhagen, Denmark) and a co-oximeter (IL-482, Instrumentation Laboratories, Lexington, M A ) . MVO2 was calculated as: M V 0 2 per g heart = flow x [(Ca0 2 - Ccv0 2 ) + (0.003 x (Pa0 2-PcvG 2)] / initial heart weight Where subscript "a" refers to arterial and subscript "cv" refers to coronary venous. 2.5 Experimental Protocol The experiment was blinded, randomized, and placebo-controlled. Approximately one hour after mounting the heart on the perfusion column, baseline M V 0 2 and Emax were measured prior to cardioplegic arrest. Then arterial blood from the support rabbit was diverted directly back to the support rabbit, and one hour of cardioplegia to the isolated heart was started. A bolus of high-potassium (K + ) normosol (2 mEq KC1 in 20 mL normosol, 0.1 - 0.5 mL ) was used to arrest the heart within 1 minute of starting cardioplegia. Effluent from the heart was collected and returned to the cardioplegia reservoir for re-oxygenation. M V 0 2 and Emax were measured 45 minutes after starting cardioplegia. Then isolated hearts were randomized to receive either 0.1 umol (0.25 ug) isoproterenol dissolved in normosol or normosol (control) as a constant infusion over 10 minutes directly into the cardioplegia in the arterial line using a separate pump. M V 0 2 and Emax were measured again at the end of this 10 minute experimental period. For the last 5 minutes of the hour of cardioplegia both groups again received drug-free cardioplegia. Cardioplegia was then discontinued, and the isolated heart was returned to perfusion from the support rabbit. Atrial pacing was resumed at the same rate used before cardioplegic arrest. After discontinuation of cardioplegia, the time to the first spontaneous heartbeat, and time to capture of atrial pacing were measured. MVO2 and Emax were determined at 20 and 45 minutes after discontinuation of cardioplegia. Perfusion of the heart was then discontinued, and the heart was weighed. 2.6 Data Analysis We tested for differences in MVO2 and Emax over time and between isoproterenol-treated versus control hearts using 2-way repeated measures analysis of variance. When a significant difference was observed, we identified specific differences using a sequentially rejective Bonferroni test procedure. Time to first heartbeat and capture of atrial pacing were analyzed using unpaired, 2-tailed Student's t-tests. For all statistical analyses, significance was defined by p < 0.05. A l l results are presented as mean ± standard error of the mean (SEM). 9 CHAPTER III. RESULTS As illustrated in figure 6 there was a significant change in MVO2 over time (p < 0.0001) due to the decrease in MVQ2 during cardioplegic arrest observed in both groups, however, there was no significant change in MVO2 after infusion of isoproterenol or normosol during cardioplegic arrest (p = 0.81). Furthermore, there was no difference observed in the MVO2 between isoproterenol-treated and control groups of hearts (p = 0.30) at any time-point. There was a significant change in Emax over time in both groups (p < 0.05) (Figure 7). Emax was significantly lower than baseline measurements 20 minutes after discontinuation of cardioplegia in both the control and isoproterenol-treated hearts (control 7.3 ±1 .7 rnmHg/uL vs 9.0 ± 1.7 mmHg/uL; p = 0.02 & isoproterenol 6.8 ± 2.8 mmHg/uL vs 8.2 ± 2.6 mmHg/pL, p = 0.01, respectively). Between 20 and 45 minutes post-discontinuation of cardioplegia, there was a significant improvement in Emax in the control group (7.3 ± 1.7 mmHg/uL @ 20 minutes vs 8.5 ± 2.4 mmHg/uL @ 45 minutes, p = 0.03). Conversely, in the isoproterenol-treated hearts, there was no improvement in Emax observed between 20 and 45 minutes after discontinuation of cardioplegic arrest (6.8 ± 2.8 mmHg/pL @ 20 minutes vs 7.0 ±2.1 mmHg/u.L @ 45 minutes, p = NS). Other measurements of ventricular function performed include the diastolic pressure-volume relationship and maximum systolic pressure. Figure 8 appears to demonstrate a progressive increase in the diastolic pressure-volume relationship in both control and isoproterenol-treated hearts after re-institution of cross-circulation. However, by R M A N O V A analysis, there are no significant changes in the diastolic pressure-volume relationship over time, and no differences between groups at any of the time points. Figure 9 shows the change in the peak systolic pressure over time. As with the diastolic pressure-volume relationship results, there appeared to be a change in the maximum systolic 10 pressure after 60 minutes of continuous warm blood cardioplegic arrest, however, there was no significant change over time, and no differences between control or isoproterenol-treated hearts. Figure 10 summarizes the time to the first spontaneous heartbeat, and time to capture of atrial pacing. There was a trend towards a longer time to first spontaneous heart-beat (isoproterenol-treated 200 ± 74 vs control 141 ± 4 3 seconds, p = 0.07), as well as a trend towards a longer time to capture of atrial pacing (isoproterenol-treated 288 ± 91 vs control 214 ± 52 seconds, p = 0.06) in the isoproterenol-treated hearts. 11 CHAPTER IV. DISCUSSION 4.1 Discussion Of Results In a rabbit heart model of continuous normothermic blood-based cardioplegia, we hypothesized that exposure to isoproterenol would increase MVO2 during cardioplegic arrest and thereby improve post-arrest cardiac function as measured by the Emax, time to first heart beat, and time to capture of atrial pacing. We found that isoproterenol did not significantly increase MVO2 during cardioplegic arrest, did not increase post-arrest Emax, and did not decrease time of electrical recovery. Instead, there were trends towards a longer time to first spontaneous heartbeat and capture of atrial pacing in the isoproterenol-treated group. Most importantly, both groups of hearts had a significantly lower Emax 20 minutes after discontinuation of cardioplegia. Although this was a temporary, effect in the control hearts, there was no significant recovery of Emax between 20 and 45 minutes after discontinuation of cardioplegia in the isoproterenol-treated hearts. Another potential indication of myocardial damage would have been an increase in the diastolic pressure-volume relationship, implying diastolic dysfunction or a decrease in compliance. In our experiments, the diastolic pressure-volume relationship appeared to increase in both groups of hearts after re-institution of cross-circulation, however this change was not significant when compared to baseline. Furthermore there were no differences between the control and isoproterenol-treated hearts. The majority of MVO2 of the myocardium is a result of contractile activity1 0. With cessation of contractile activity in normothermic hearts 76% of the MVO2 of empty beating hearts was eliminated. Hypothermia only results in a small further decrease in MVO2. Thus in warm arrested hearts MVO2 represents the basal metabolic activity of the myocardium which maintains organelle structure, ion cycling, and energy production. 12 In arrested hearts, the effect of P-stimulation has not been fully defined. In a study of normothermic arrested dog hearts, Klocke and colleagues6 observed that the MVO2 fell to 66% of the MVO2 of beating hearts. However, after the administration of a bolus of 3 ug of isoproterenol, there was a 17% change in the MVO2 in the absence of any mechanical activity. The investigators did not evaluate contractility, and concluded that the observed change in M V 0 2 was due to increased oxidative metabolism. Since normothermic blood cardioplegia.provides a metabolically active myocardium with a continuous supply of oxygenated blood, we hypothesized that it might be possible to enhance the basal metabolic activity of the myocardium during normothermic blood cardioplegic arrest using p-adrenergic stimulation, and thereby improve post-cardioplegic contractile function, potentially through an increase in energy stores. For,our experiments isoproterenol, a synthetic p i and p2 adrenergic amine, was used. A dose of 0.1 uM isoproterenol was administered for 10 minutes at the end of a 60 minute period of cardioplegic arrest. This dose was chosen because higher doses repeatedly stimulated ventricular contractions during cardioplegia in pilot experiments. We did not observe a change in M V 0 2 in hearts exposed to 0.1 uM (0.25 ug total dose) isoproterenol during cardioplegic arrest, and no difference in M V O 2 between isoproterenol-treated and control hearts. It is unclear why our observation in rabbit hearts was different from the 17% change in M V 0 2 observed in Klocke's experiments in dog hearts. One explanation . may have been the larger doses of isoproterenol were used in other experiments. In particular, Klocke's experiments of dog hearts required a bolus dose of 3ug before a change of 17% was observed in the baseline M V 0 2 . However, it should be noted that the results from Klocke's work were based on only 3 experimental animals, and the statistical significance of the change in MVO2 was not tested. Also, determination of MVO2 may have been less accurate in our 13 rabbit hearts due to their small size (relative to dog hearts) which made direct cannulation of the coronary sinus impractical. It may be that excessively high levels of isoproterenol are necessary to produce a change in M V O 2 during cardioplegic arrest, however we were limited in the amount of isoproterenol which we could administer while maintaining electrical silence during cardioplegic arrest. One other study of P-adrenergic stimulation during cardioplegic arrest also failed to show an increase in M V O 2 . In this study, Nozawa et a l 1 1 determined which dose of dobutamine produced at 50 -100% increase in Emax, and then administered the same dose of dobutamine 60 minutes after the hearts were arrested with KC1. Resting M V O 2 did not change significantly (from 1.01 to 1.07 cc 02/1 OOg ventricle/ min) with continuous infusion of dobutamine. As with Klocke's study, ventricular function was not assessed. The composition of the cardioplegia solution may have impacted the degree to which the myocardium was able to alter its metabolism. Although there were no significant changes in pH of the venous blood during cardiplegic arrest (data not shown), we did not measure levels of substrates in the cardioplegic solution during.the one hour long period of cardioplegic arrest. The cardioplegia solution was recycled several times during this period of cardioplegica arrest during which time it was re-oxygenated; however, the substrate components (eg. glucose, fatty acids, amino acids, etc.) were not monitored or altered. If there were significant changes in the substrate composition of the blood cardioplegia, this could have affected the metabolic behavior of the myocardium by determining which metabolic pathways were used preferentially. We did not observe a beneficial effect with isoproterenol. In fact exposure to r isoproterenol was found to be deleterious, with Emax significantly lower than baseline after discontinuation of cardioplegic arrest, and with trends towards longer time tofirst spontaneous 14 heartbeat and time to capture of atrial pacing in isoproterenol-treated hearts compared to control hearts. 4.2 Potential Negative Effects Of p-Adrenergic Stimulation In fact, there are several potential mechanisms by which isoproterenol could exert a negative effect, and it has been observed that isoproterenol at large doses is able to produce myocardial necrosis 1 2 . However, these observations have been derived primarily from experiments in working or beating hearts. This is of importance because the initial postulated mechanism of isoproterenol-induced myocardial necrosis was that it was mediated by excessive intracellular C a + + . O f the many activities involved in basal cellular metabolism, active ion (e.g. C a + + ) transport is one of the more significant ones, and has been found to use up to 30% of the 13 A T P consumed by the myocardial cell. Iri fact, in a study of isolated cardiomyocytes , M V O 2 was found to increase out of proportion to the increase in contraction amplitude induced by isoproterenol. Therefore, P receptor-mediated increases in intracellular Ca** causes increased consumption of A T P by processes other than those involved in contractility. The same effect was seen with administration of C a + + alone. However, because of the observation that p receptor-mediated influx of C a ^ is dependent on electrical excitation iri intact cultured rat myocytes 1 4 , Ca + + -mediated damage after exposure to isoproterenol during cardioplegic arrest may not have been a factor in our experimental model. Since the work of Fleckenstein, et al, it has become apparent that there must be other, Ca + +-independent mechanisms of myocardial damage secondary to excess catecholamine stimulation because inhibition of Ca4"* influx into the cell does not completely protect against isoproterenol-induced myocardial necrosis 1 5. Hence, it is possible that an increase in fatty acid metabolism might be partially responsible for the apparent deleterious effect of isoproterenol observed in our experiments. In order to better understand the possibility of such an effect, it is > 15 important to study what is known about cardiac metabolism, some of which has only come to light in recent years. 4.3 Cardiac Metabolism (i) Background "The heart is a metabolic "omnivore"16. This statement arises from the observation that the heart normally derives 60 - 70% of its ATP from fatty acid oxidation. The balance is derived from glucose oxidation. Other substrates that the heart is able to use include amino acids, ketone bodies, as well as lactate. Interestingly, glucose oxidation appears to be a more efficient 'means of energy production since glucose oxidation yields 3.17 ATP/oxygen atom compared to 2.80 ATP/oxygen atom with palmitate oxidation17. In conditions of ischemia and myocardial hypertrophy glucose metabolism becomes more important, particularly since [3-adrenergic stimulation seems to increase rates of glycolysis and stimulates pyruvate dehydrogenase (PDH) via increased intracellular Ca + + . Thus it is important to understand the metabolism of both fatty acids and glucose. (ii) Glycolysis And Glucose Oxidation Glucose metabolism begins with glycolysis (Figure 11), and thus occurs as the first step of glucose oxidation. This process can also occur in the absence of oxygen, and therefore is of importance in conditions of ischemia. Unfortunately, glycolysis itself only yields a net'of 2 ATP/glucose since the 1st step in glycolysis involves the conversion of glucose to glucoses-phosphate (G6P), and utilizes 1 ATP (as well as magnesium, Mg"1"1"). This step is felt to be irreversible, and because the glucose is now phosphorylated, it must remain intracellular from this point onwards. G6P is then phosphorylated again (using 1 ATP) to produce fructose 1,6-bisphosphate, which is then split to produce 2 triose phosphates. Each of these tripse phosphates is then oxidized to pyruvate (note that conversion of glyceraldehyde 3-phosphate to 16 1,3-bisphosphate is dependent on a supply of oxidized nicotinamide adenine dinucleotide, NAD+), and in the process yields a total of 4 ATP (1,3-bisphosphate to 3-phosphoglycerate, and phosphoenolpyruvate to pyruvate). Under anaerobic conditions, pyruvate is then converted by lactate dehydrogenase to lactic acid in order to regenerate the NAD+ necessary to allow glycolysis to continue. When the ATP generated by glycolysis is utilized in an anaerobic environment, 2 hydrogen ions (H +)/ATP are produced as ATP is hydrolyzed (ie. converted to ADP by adenosine triphosphatases), resulting in acidosis. Furthermore, in an acidotic environment, lactic acid is more likely to be converted into lactate anion + H ^resulting in further acidosis . In the presence of an adequate supply of oxygen, pyruvate is converted by pyruvate dehydrogenase (PDH) to Acetyl-Coenzyme A (CoA) or by pyruvate carboxylase to oxaloacetate, both of which then enter the citric acid cycle. When metabolized in this fashion, 1 glucose yields a net of 38 ATP (Figure 12). Hence the importance of PDH can be appreciated. (iii) Pyruvate Dehydrogenase Regulation In fact, PDH is such an important enzyme that its regulation has been well-described19 (Figure 13). There are 2 forms of PDH; active and inactive. Production of the active form is catalyzed by PDH phosphatase. PDH phosphatase is stimulated by insulin, M g + + , and Ca + + , which may explain how catecholamines stimulate PDH activity. Conversion of active PDH to inactive PDH is catalyzed by PDH kinase, and utilizes 1 ATP. PDH kinase is stimulated by fatty acid oxidation via its end-products (Acetyl CoA and NADH), and is inhibited by pyruvate, Ca + + , and dichloroacetate (DCA). (iv) Fatty Acid Metabolism Acetyl-CoA and the citric acid cycle comprise the final common pathway for oxidative metabolism of both glucose and fatty acids (Figure 14). The difference between glucose and fatty acids is the way in which they arrive in the form of Acetyl-CoA. With free fatty acid 17 metabolism, the first step is again one which requires a high-energy phosphate in order to produce an intermediate which can be catabolized by the cell. This step also involves CoA and results in the formation of an Acyl-CoA (active fatty acid, eg palmitoyl-CoA). Acyl-CoA is then converted into acylcarnitine, in order for the fatty acid to be able to penetrate the mitochondrial membrane. Once inside the mitochondrion, it is again combined with CoA in exchange for carnitine, and Acyl-CoA is reformed where it can undergo p-oxidation (ie. cleaving off of Acetyl-CoA units). The oxidation of each palmityol-CoA produces 129 ATP [(5 ATP/p cleavage x 7)+(12 ATP/Acetyl-CoA x 8)]. Fatty acids can be produced from Acetyl CoA via the action of Acetyl CoA carboxylase. This enzyme catalyzes the first step in fatty acid (FA) synthesis from Acetyl CoA, namely the formation of malonyl CoA. . (v) Effect Of Isoproterenol On Cardiac Metabolism Isoproterenol is a non-selective P-adrenergic agonist, and therefore can be expected to stimulate both p i and p2 receptors. It has usefulness both as an inotropic as well as a chronotropic agent. In these circumstances, isoproterenol clearly increases the ATP demands of the myocardium. In response, it has also been demonstrated that P-adrenergic stimulation of cardiac myocytes increases the activity of PDH, and is thus able to meet the increased demand for high-energy phosphates by increasing the production of ATP via glycolysis and oxidative phosphorylation17'20. However, its ability to modulate metabolic activity in the arrested heart has not been extensively studied. • • x Isoproterenol-stimulated conversion of PDH to active form by PDH phosphatase appears to be Ca^-dependent, and therefore might not occur in the quiescent, cardioplegic arrested myocardium. Evidence for this comes from studies of isolated rat cardiac myocytes in which intramyocardial C a + + increased only in conjunction with both isoproterenol and electrical stimulation21. This is quite logical since catecholamine-induced increases in ATP coincide with 18 increased demand caused by the positive inotropic effect of P-adrenergic stimulation. This observation is the foundation of the most compelling explanation of why isoproterenol administered during cardioplegic arrest failed to have a salutary effect. Although stimulation of PDH by p-adrenergic agents appears to depend on depolarization, the stimulation of F A oxidation may not. In fact, lipolysis and increased F A metabolism are felt to have significant importance in the development of isoproterenol-induced myocardial necrosis. Even though fatty acids are an important source of metabolic substrate for the heart, excessive accumulation of fatty acids can have deleterious effects. One important way in which fatty acids can have a negative effect on myocardial metabolism is through the stimulation of PDH kinase (conversion of active PDH to inactive PDH). Furthermore, it is known that protein kinase A converts Acetyl CoA carboxylase from its active form to its inactive form. This would result in the accumulation of Acetyl CoA, further stimulating the% action of pyruvate dehydrogenase kinase. In conditions of ischemia or p-adrenergic stimulation when glycolytic rates are increased, this can result in accumulation of lactic acid secondary to over-accumulation of pyruvate. Direct toxicity of fatty acids on plasma membranes have also been identified, especially in combination with lipoprotein lipase, which is also produced by myocardial endothelium15'22. Rat heart experiments have also demonstrated that high FA/albumin ratios can induce arrhythmias in non-ischemic hearts. Another possible negative effect of excess F A metabolism is the inhibition of ATP transfer from the mitochondria to the 23 contractile apparatus by excess Acyl-CoA . Because increased F A levels are known have toxic effects, it is important to note that p-adrenergic stimulation can cause increased liberation of lipids from adipose tissue via the action of triacylglycerol lipase (as mentioned previously), and can increase uptake of F A into cardiac myocytes23. These processes are important even in isolated heart preparations, such as the one 19 in our experiments, because adipocytes are present within the myocardium (endogenous lipolysis) 2 4. It is especially interesting to note that both ischemia and isoproterenol have been demonstrated to increase the rate of lipolysis in isolated cardiac myocytes (non-contracting) from rat hearts25. Another important finding from that study was the observation that p-blockade was able to prevent the increased lipolysis with isoproterenol, but not the increase in lipolysis secondary to ischemia. Another possible mechanism of isoproterenol-induced myocardial injury includes direct toxicity via formation of radical species such as hydrogen peroxide. At this time there is only indirect evidence for this possible mechanism, and comes from a study of rat hearts exposed to isoproterenol with and without vitamin E 2 6 . In the rat hearts pre-treated with vitamin E, levels of high-energy phosphates were preserved after exposure to isoproterenol. Conversely, hearts without vitamin E pre-treatment had a significant decline in high-energy phosphate levels after injection of 80mg/kg of isoproterenol. It should be noted, however, that the dose of isoproterenol administered was extremely high. It is possible that exposure to isoproterenol for longer than 10 minutes, or at a different point during cardioplegic arrest would have affected MVO2 differently, however a deleterious effect was seen with exposure to isoproterenol even without an observed change in MVO2. 4.4 Emax And Determination Of Left Ventricular Contractility In our study, one of the primary end-points was left ventricular function. The determination of left ventricular contractility is perhaps most accurately achieved through the measurement of the left ventricular end-systolic pressure volume relationship (LVESPVR), as 27 * described by Sagawa, et al., through the use of pressure-volume loops . The L V E S P V R is otherwise described as the elastance at end-systole (Ees), which is derived from regression analysis of the change in pressure per change in volume. An approximation of this is the Emax, : - ' 20 however, the 2 terms have been used interchangeably, even though they are measured at slightly different points on the pressure-volume loop (Emax being measured at the point of maximal systolic pressure, and Ees being measured just shortly after that point, at the true end-systolic point) (Figure 15). Regardless of whether the Ees or Emax is measured, the value obtained is relatively independent of changes in preload and afterload. It is this characteristic which makes Ees and Emax superior to other measurements of contractility, such as change in pressure over change in time (dp/dt), which can be affected by preload. In order to understand the concept of the LVESPVR, it is important to understand that the elastance of the myocardium varies throughout the cycle of contraction (Figure 16). It is therefore said to be a time-varying elastance because the elastance increases as the myocardium contracts. The maximal elastance occurs at the end of systole, however, there is a slight drop in elastance after the peak point just before closure of the aortic valve. If one measures the developed pressure at the point of maximal time-varying elastance for different ventricular volumes, a slope can be determined, and this is the Emax for that ventricle. If the developed pressure at end-systole is measured for increasing ventricular volumes, the slope calculated is then the Ees. In the case of our experiments, pressures for isovolumic contractions were measured in the left ventricle of each of the hearts studied using a Millar pressure transducer catheter within a home-made balloon whose volume was known and could be accurately manipulated. This method of calculating the elastance was necessary as we were not utilizing a working heart model. Therefore the "Emax" presented is an approximation of the elastance of the ventricle. However, it has been shown that both methods (ie. isovolumic contraction vs working heart) yield similar values as long as the measurements from isovolumic contractions are obtained at steady state (Figures 17 & 18). 21 CHAPTER V. UNANSWERED QUESTIONS Clearly, the results of this study were inconclusive due to several factors. In order to more conclusively explain why and how isoproterenol administered during cardioplegic arrest had its deleterious effect oh the heart, several other experiments would be needed. The first question to be answered is whether the functional changes reflected structural changes, and as such, histology and electron microscopy of the hearts would have been instructive. Furthermore, levels of creatine kinase or troponin from the venous effluent of the heart might have been indicative of myocardial necrosis. In order to determine whether or not the decreased left ventricular contractility was due to deleterious metabolic changes, several metabolic measurements would have been useful. The first of these would be levels of high-energy phosphates from myocardial biopsies to determine if ATP levels were affected by isoproterenol. Another useful parameter from myocardial biopsies would have been levels of PDH activity. Serum levels of pyruvate, lactic acid, and lipids would have been useful to further delineate the metabolic effects of isoproterenol during cardioplegic arrest. Finally, measurements of Ca"1^ flux would have been instructive, as many of these processes appear to be dependent on Ca4"1" influx. .22 CHAPTER VI. POTENTIAL FUTURE STUDIES : EFFECT OF VASOPRESSIN ON CARDIAC METABOLISM 6.1 Introduction The greater understanding of cardiac metabolism afforded by these experiments provides exciting opportunities for potential clinical research, especially in light of recent findings regarding the importance of PDH and pharmaceutical agents which might be able to manipulate its activity. As a continuation of my research, I would like to lay thefoundation for a possible • grant application to explore the effect of one such potential agent - vasopressin. 6.2 Mechanism Of Action Of Vasopressin Vasopressin is a hormone which is released from the posterior pituitary in response to various stimuli 2 8. The most potent stimuli are elevated plasma osmolality and hypovolemia/hypotension (decreased intravascular volume). Stimulation of a l adrenoceptors can also cause release of vasopressin. Although appropriate stimuli can cause a release of up to 20% of the total pool of vasopressin into the circulation, continued stimulation causes only slow continued release of vasopressin. This may be the reason why certain physiologic, states, such as septic shock, syndrome of inflammatory release syndrome (SIRS), and brain-death are associated with relatively low serum levels of vasopressin. "Normal" levels differ for different physiologic conditions: fasting hydrated people have levels < 4 pg/mL; water deprivation causes increases in levels to ~ 10 pg/mL; patients in early cardiogenic shock have levels of >20 pg/mL. The actions of vasopressin are dependent on the vasopressin receptors found in various tissues. V I receptors (aka Via) are located in vascular smooth muscle and result in vasoconstriction when stimulated, including efferent renal arterial vasoconstriction. The V1 receptors are also present in myocardium and may have a weakly positive inotropic effect at 23-lower doses, possibly through a Ca^-mediated mechanism29. Clinically, the bulk of evidence suggests that the overall effect of vasopressin is to leave cardiac output unchanged, or slightly decreased. The decreased cardiac output sometimes observed with vasopressin administration may be due to an increase in venous resistance, and subsequent decrease in venous return (ie. decreased pre-load)29. V2 receptors are present in the renal collecting duct system and are responsible1 for the anti-diuretic effect of vasopressin. Paradoxically, vasopressin causes increased urine output in certain pathologic states, such as septic shock, SIRS, and milrinone-induced hypotension. Although vasopressin has almost no effect on BP in healthy hemodynamically stable individuals, patients in septic shock or other states of vasodilatory shock are very sensitive to low doses of parenterally administered vasopressin. Doses of 0.4 - 2 units/hour of vasopressin have been used to successfully treat patients with vasodilatory shock of various etiologies, resulting in plasma levels of vasopressin > 100 pg/mL in some studies. 6.3 Evidence Of Benefit After Cardiopulmonary Bypass Similar to sepsis, CPB has been implicated in the development of a SIRS-like syndrome in certain groups of patients. Those most susceptible seem to be patients with bacterial endocarditis and those receiving a ventricular assist device (LVAD). The Columbia University 30 group has been a leader in investigating the utility of vasopressin in these patients . They first performed a randomized placebo-controlled study in patients who had undergone placement of a ; L V A D because of end-stage heart failure. Ten patients with mean arterial pressure < 70mmHg (mean 60 mmHg) with norepinephrine > 8ug/min (mean 19.7 ug/min) and with LVAD-assisted CI > 2.5 L/min/m 2 (mean 2.9 L/min/m2) were randomized to receive placebo vs 0.1 U/min IV vasopressin or placebo. Within 15 minutes, vasopressin increased mean arterial pressure from 57 to 84 mmHg and norepinephrine dose was reduced from a mean of 27 to, 11 ug/min, with 24 norepinephrine discontinued in 4 of 5 patients within the first 15 minutes. CI did not change significantly. There were no adverse reactions to the vasopressin which was continued for an average of 36 hours. The same group published a series of 3 patients who developed hypotension after CPB and were receiving inotropic support with milrinone3 1. A l l 3 patients were already receiving dopamine, epinephrine, and/or norepinephrine in order to maintain systolic blood pressure > 90 mmHg. Doses of 0.033 to 0.067 units/min vasopressin markedly increased SBP within minutes, and significantly improved urine output and serum creatinine levels without changing cardiac output. — A fourth case of post-CPB shock treated with vasopressin was presented by Overand, et al . Their case is interesting in that the patient's cardiac output increased from 3.5 to 4.8 L/min with vasopressin. In fact, there is animal research evidence which does suggest that vasopressin does in fact have a positive inotropic effect via the V1 receptor . There has also been one case report of the use of vasopressin during CPB in a patient who had refractory hypotension while on CPB despite several boluses of phenylephrine and norepinephrine34. Two boluses of 1 unit of vasopressin were administered approximately 8 minutes apart, each of which normalized mean arterial pressure to 80 mmHg and allowed weaning of the norepinephrine and phenylephrine. Interestingly, none of these investigators has examined the effect of vasopressin on cardiac metabolism, or mentioned the possibility that the positive effect of vasopressin may partially be due to an increase in the rate of aerobic glucose oxidation. 6.4 Evidence Of Benefit In Brain-Dead Organ Donors (Hearts) More compelling evidence for a possible beneficial effect of vasopressin over and above its ability to provide vasopressor support and thereby reduce the need for catecholamines comes from research involving brain-dead organ donors. Yoshioka et al studied 16 brain-dead 25 patients, all of whom had a major intracranial lesion on computed tomography (CT) scan and had a hypotensive episode with a drop in BP of at least 40 mmHg. Ten of the patients were supported with epinephrine alone, with cardiac arrest occurring in all of these patients within 48 hours. In contrast, 6 patients were treated with vasopressin at a dose of 1-2 units/hour with small doses of epinephrine (majority < 0.5 mg/hr) to maintain SBP > 90 mmHg. These patients had stable hemodynamic function up to 54 days after brain death, with the mean time of preserved cardiac function being 23.1 days after brain death. In addition, kidney and liver function remained normal during this extended period of time as well. The same group then published a second study on 25 brain-dead patients who were divided into 3 groups according to dose of vasopressin administered36. The first group received no vasopressin and all succumbed to cardiac arrest by 48 hours. The second group received 0.1 - 0.4 units/hour of vasopressin, however, 48 and 72 hours after brain death, the need for epinephrine to maintain stable blood pressure increased sharply, with cardiac arrest occurring by the end of the 3' d day. The third group received 1-2 units/hour of vasopressin, and all had stable circulation for more than 17 days, when the vasopressin and epinephrine were discontinued. It is interesting to note that cardiac arrest seemed to be associated with a need for large doses of epinephrine, and that vasopressin had an epinephrine-sparing effect. It is also of interest that epinephrine arid vasopressin had synergistic effects on mean arterial pressure (MAP), with either agent alone producing a M A P of 60 - 70 mmHg and both agents together producing a pressure of-100 mmHg. In the discussion of this paper, they speculate that cardiac metabolism is impaired in brain-dead patients, but do not elaborate on this idea. Renal function was again noted to be normal while hemodynamic stability was maintained regardless of the dose of vasopressin. More recently, the Columbia group37 has corroborated the Japanese observations in 12 hemodynamically unstable organ donors (MAP < 70 mmHg despite dopamine and/or 26 norepinephrine) who had not been exposed to vasopressin. Eleven of these donors were given 0.04 - 0.1 units/hour vasopressin. The mean M A P increased to 89 mmHg, with doses of dopamine and norepinephrine being reduced significantly from > 10 ug/min each to < 3 ixg/min each. There were no differences noted in the myocardial contractility of donor organs from vasopressin treated and untreated donors. 6.5 Effect On Pyruvate Dehydrogenase The probable importance of changes in cardiac metabolism in the above patients populations (ie. post-CPB, post-LVAD placement, brain-dead organ donors) has been elucidated by groups who have demonstrated the beneficial effect of glucose-insulin-potassium (GIK) -administration in similar situations. The use of GIK infusions is justified primarily on the observation that PDH activity is inhibited during early reperfusion after a period of global ischemia38, and the knowledge that insulin stimulates PDH phosphatase. Further evidence of the importance of PDH comes from studies of hypertrophied rat hearts exposed to a period of ischemia, in which cardiac function was improved in hearts exposed to dichloroacetate, a potent inhibitor of PDH kinase39. ' -These observations are potentially relevant in the case of vasopressin because the beneficial effect of vasopressin after CPB, in patients receiving a L V A D , and in brain-dead transplant donors has not been fully explained. The possibility that vasopressin's beneficial effects are mediated by changes in,cardiac metabolism arises from experiments on animal adipocytes and livers. In a study of rat adipocytes, vasopressin was found to greatly increase the activity of adipocyte P D H 4 0 . It has also been shown that vasopressin activates PDH in the rat liver, doubling the proportion of PDH in its active form after administration of vasopressin41. However, vasopressin's effect on cardiac PDH activity has not been examined. 27 6.6 Evidence Of Benefit With Glucose-Insulin-Potassium In Human Hearts During And After CPB, And Possible Benefit In Heart Transplantation Interest in the role of PDH in cardiac metabolism and cardiac function has developed in a parallel fashion in the animal lab and in clinical cardiac surgical research. Using a working perfused rat heart model, Mike Allard's lab has demonstrated that after a period of ischemia, . stimulation of PDH using D C A results in better left ventricular contractile function. This was hypothesized to be secondary to a reduction in the rate of glycolysis (in hypertrophied hearts only), and an increase in the rate of glucose oxidation, resulting in less H + production. Although this effect was observed in normal rat hearts, the effect was more pronounced in hypertrophied rat hearts39. However, it should be noted that the combination of D C A and insulin given prior to a period of ischemia has a detrimental effect on post-ischemic recovery4 2. With clinical cardiac surgical trials, several groups have demonstrated that the combination of glucose, insulin, and potassium can reduce mortality and improve cardiac function after CPB in patients undergoing surgical revascularization. In Toronto, the Insulin Cardioplegia Trial Investigators randomized 56 pts undergoing elective isolated coronary artery bypass graft (CABG) surgery to receive either regular or high glucose and no or 10 units regular insulin per litre of cardioplegic solution (4 groups). They found that the groups who received insulin had a significantly higher cardiac index and lower pulmonary capillary wedge pressure than those who did not38. Although hemodynamic parameters were not evaluated in an earlier study, Svensson et a l 4 3 found that administration of insulin after CPB resulted in significantly lower serum levels of free fatty acids (FFA) and greater glucose' uptake by the myocardium. In this same paper, the authors suggested that perhaps catecholamines (stress hormones) may have been responsible for insulin-resistance in the control group. Furthermore, they state that their observation of decreased substrate uptake by the myocardium (and therefore greater reliance on stored substrates) in the 28 absence of exogenous insulin after CPB resembles septic shock (in which vasopressin has been shown to be somehow beneficial)/ At the Texas Heart Institute44, 22 consecutive pts with poor L V function and post-CPB L V failure (CI < 2.1) were randomized to IABP + inotropic support or the same + GIK x 48hrs post-CPB. There was a "dramatic" decrease in FFA levels, and 40% increase in CI in the first 12 hrs in pts receiving GIK. Most importantly, their time on the IABP was 39hrs vs 64hrs, and the 3 0 day survival was 10/11 patients (91 %) vs 7/11 patients (64%). Similarly, Lazar et a l 4 5 at the Boston University Medical Center randomized 31 elective C A B G pts to receive placebo vs GIK pre-op, and then immediately after removal of the crossclamp x 12 hours. They observed significantly higher cardiac indices in pts who received GIK, as well as fewer atrial and ventricular arrhythmias, and a lower requirement for inotropic support and shorter times in the. intensive care unit and hospital. The question which remains to be answered is whether vasopressin's intrinsic activity is responsible for the beneficial effects of vasopressin, or whether it is vasopressin's catecholamine-sparing effect which provides the greatest benefit. There are several reasons why vasopressin can be hypothesized to be a superior stimulant of PDH than other potential PDH stimulants. Although P-adrenergic stimulation can cause stimulation of PDH via influx of Ca + + , it would appear that this comes at a cost of increased myocardial oxygen demand, increased arrhythmogenicity, and increased lipolysis and glycolysis. Alpha-adrenergic stimulation also can cause stimulation of PDH, however, this appears to be at the cost of increased renal vasoconstriction, resulting in decreased renal perfusion and poorer renal function. Compared to GIK infusion,' vasopressin may be superior in vasodilatory shock syndromes, because in addition to its potential ability to stimulate PDH, it is a potent 29 vasopressor, which is a feature that GIK infusion does not possess. Finally, dichloroacetate may riot be applicable to humans because of the possibility of vascular toxicity. 6.7 Potential Deleterious Effects Of Vasopressin There are 2 possible mechanisms whereby vasopressin might have a deleterious effect, especially in patients undergoing CABG. The first is obviously its vasoconstrictive effect, which could theoretically cause vasospasm in the conduits used in CABG, particularly the mammary and radial artery grafts. The saphenous vein grafts should not be as problematic since the mechanical vasodilation used to dilate the veins at the time of procurement probably renders them non-contractile. However, the problem of vasospasm has not been reported as a concern in the published reports of the use of vasopressin in pts undergoing CABG. Secondly, vasopressin is known to have procoagulant effects by stimulating the release of vonWillebrand factor and factor VIII from the vascular endothelium, thereby increasing platelet adhesion and decreasing bleeding times! These effects are mediated via the V2 receptors, and the vasopressin analogue, desmopressin, has been synthesized to maximize this effect46. Whether this effect would result in significant graft thrombosis after CABG has not been determined. 30 CHAPTER VII. EXPERIMENTAL PROPOSAL 7.1 Introduction ' 1 In order to further evaluate the exact mechanism of the beneficial effect of vasopressin, 2 studies are necessary. The first study which should be performed is an animal study of vasopressin's effect on cardiac PDH activity. As such, the isolated working hypertrophied rat heart model established by Allard et al would be ideal. In addition, the efficacy of vasopressin could be directly compared to that of dichloroacetate. If vasopressin is in fact found to produce an increase in cardiac PDH activity, a second study could be performed on hemodynamically unstable organ donors. These are patients who are already receiving inotropic support, and in whom, there has been a demonstrated benefit on survival with vasopressin therapy without any deleterious effects on either renal or liver function. Furthermore, desmopressin (ddAVP) has been used in many of these donors in order to treat an apparent diabetes insipidus. Because ddAVP is selective for the V2 receptor, it has none of the vasopressor effects of vasopressin. Conversely, vasopressin affects both V I and V2 receptors. -None of the previous investigators has studied the effect that vasopressin has on cardiac metabolism, specifically its effect on PDH. Furthermore, none of the published studies has evaluated clinical outcomes in hearts from donors treated with vasopressin, except for the Columbia study in which they stated that there was not difference in contractility without presenting any data. 7.2 Purpose It is my hypothesis that part of the beneficial effect of vasopressin is its ability to enhance the effect of PDH and thereby increase oxidative metabolism of glucose in the hearts of brain-dead patients. In addition, because vasopressin drastically reduces the need for inotropic and vasopressor support with traditional alpha and beta-adrenergic agents, brain-dead patients 31 treated with vasopressin can be expected.to have less fatty acid metabolism, less production of lactate, and therefore, less metabolic acidosis. Therefore, the purpose of this randomized placebo-controlled prospective trial would be to determine whether vasopressin has a positive effect on cardiac metabolism as determined by its effect on PDH. Secondary end-points of the trial would be clinical parameters to determine whether vasopressin has a positive effect on clinical outcomes after transplantation of hearts from donors treated with vasopressin. 7.3 End points 1. Metabolic parameters: pH, lactate, pyruvate, 0 2 extraction, PDH activity. 2. Clinical parameters: pulmonary capillary wedge pressure,.CI, L V stroke work index, survival, ejection fraction, need for inotropic support. 7.4 Methods • ' • The experimental protocol would involve randomization of brain-dead organ donors with a need for inotropic support in order to maintain a MBP > 70 mmHg. Equipment required would include: coronary sinus catheter (possibly) pulmonary artery catheter dry ice for blood samples 1 - liquid nitrogen for snap-freezing of biopsy samples (best site is probably left atrial appendage tissue) - randomized bags of vasopressin or 5% dextrose in water Measurements would consist of: Metabolic parameters immediately before vasopressin administration, and again 15 minutes later. - 3 0 32 Metabolic parameters at removal of crossclamp after the donor heart has been placed into the recipient and again 12 hours later. , Clinical parameters after coming off of bypass, and again 3,6, and 12 hours afterwards. - PDH activity in the lab from atrial tissue biopsy samples. 33 CHAPTER VIII. SUMMARY In summary, in a rabbit heart model of continuous normothermic blood-based cardioplegia, exposure to isoproterenol during cardioplegic arrest did not result in an increase in MVO2. Emax was significantly depressed after exposure to isoproterenol and there were trends towards a longer time to first spontaneous heartbeat and capture of atrial pacing in the isoproterenol treated hearts. We therefore conclude that exposure of hearts to isoproterenol during warm cardioplegic arrest has a deleterious effect which may be mediated through mechanisms independent of increased myocardial oxygen consumption. Recent investigations demonstrating the importance of PDH in the control of cardiac metabolism during CPB suggest that optimizing cardiac metabolism during cardiopulmonary bypass is best achieved by mechanisms not involving P-adrenergic stimulation. 34 FIGURE LEGENDS Figure 1. "Effect of mechanical activity and temperature on MVO2" . Histogram comparing mean myocardial oxygen consumption (MVO2) in beating empty hearts (green), fibrillating hearts (dark blue), and arrested hearts (light blue) at 37, 32, 28, and 22°C. Modified from Buckberg GD, et al. Journal of Thoracic and Cardiovascular Surgery 1977; 73(1): 87-94. Figure 2. "Effect of isoproterenol on MVO2 of arrested and beating hearts". Effect of increasing doses of isoproterenol on myocardial oxygen consumption ( M V 0 2 ) of beating (red line) and arrested (blue line, n = 3) dog hearts. The statistical significance of the change in MVO2 in the arrested hearts was not determined. Modified from Klocke FJ, et al. American Journal of Physiology 1965; 209: 913-918. Figure 3. "Mechanism and actions of P-adrenergic stimulation". Schematic of mechanism of P-adrenergic stimulation. Stimulation of the p-receptor (P R) by isoproterenol results in the activation of the G-proteina (yellow crescent) which then activates adenylate cyclase, (red oblong). The activation of adenylate cyclase catalyzes the formation of cyclic adenosine mono-phosphate (cAMP); the second messenger of p-adrenergic stimulation. cAMP then activates various protein kinases. One of the most important protein kinases, protein kinase A , phosphorylates the L-type calcium (Ca -^) channels (purple tube). These voltage-gated Ca** channels allow for the rapid influx of C a ^ upon depolarization (yellow lightning bolt) of the cell. Other protein kinases activated by cAMP are involved in lipolysis and 35 glycolysis, which explains in part the increased lipolysis and glycolysis observed with P-adrenergic stimulation. r "The perfusion apparatus". Line diagram of the perfusion apparatus. Red arrows indicate direction of flow of arterial (oxygenated) blood or cardioplegia, blue arrows indicate direction of flow of venous blood, and the green arrow indicates the direction of flow of isoproterenol or control. See text for details. "Example of approximation of Emax". Typical left ventricular (LV) pressure-volume data are shown from one rabbit heart at baseline and 20 minutes after discontinuing cardioplegia.' These end-systolic pressure-volume points fall along a line with the slope being an approximation of the maximal elastance (Emax), which is a load-independent measure of contractility. Figure 6. "MVO2" . Summary of changes in mean myocardial oxygen consumption (MVO2) in control (n=8, open blue circles connected by dashed lines) and isoproterenol-treated (n=8, solid red squares connected by solid lines) hearts. Error bars indicate standard error of the mean (control, vertical blue T and inverted T; isoproterenol, vertical red lines). Measurements of MVO2 were obtained prior to initiation of cardioplegic arrest ("Baseline"), 45 minutes after initiation of cardioplegic arrest ("Before drug"), after 10 minutes of exposure to control or isoproterenol ("After drug"), and 20 and 45 minutes after re-institution of normal blood perfusion ("20 min post", and "45 min post"). * p=0.0002 vs baseline. . Figure 4. Figure 5. 36 Figure 7. "Emax". Summary of changes in mean maximal elastance (Emax) in the control (n=8, open blue circles connected by blue lines) and isoproterenol-treated (n=8, . solid red squares connected by red lines) hearts is illustrated. Error bars indicate standard error of the mean (control, vertical blue T's; isoproterenol, vertical red lines). * p < 0.03, 20 minutes after reversal of cardioplegic arrest ("20 min post") vs baseline; ** p = 0.03, 45 minutes after re-institution of cross-circulation ("45 min post") compared to 20 min post in the control hearts only. Figure 8. "Diastolic pressure-volume relationship". Summary of changes in the diastolic pressure-volume relationship in control (n=8, open blue circles connected by blue lines) and isoproterenol-treated (n=8, solid red squares connected by red lines) hearts is illustrated. Error bars indicate standard error of the mean (control, vertical blue inverted T's; isoproterenol, vertical red lines). No significant change over time, and no differences between control and isoproterenol-treated hearts at any time point. Figure 9. "Peak systolic pressure". Summary of changes in the peak systolic pressure in control (n=8, open blue circles connected by blue lines) and isoproterenol-treated (n=8, solid red squares connected by red lines) hearts is illustrated. Error bars indicate standard error of the mean (control, vertical blue inverted T's; isoproterenol, vertical red lines). No significant change over time, and no differences between control and isoproterenol-treated hearts at any time point. 37 "Time to first beat and return to paced rhythm". Histogram of mean time to first spontaneous ventricular contraction and time to capture of atrial pacing following cardioplegic arrest and re-institution of normal blood perfusion of isolated hearts. Isoproterenol-treated hearts (n=8, solid red bars) versus control hearts (n=8, ( speckled blue bars). Error bars indicate standard error of the mean. "Glycolysis". Schematic of the process of glycolysis. See text for details. G6P = glucose-6-phosphate, NAD+ = nicotinamide adenine dinucleotide (oxidized), N A D H = nicotinamide adenine dinucleotide (reduced). "Aerobic glucose oxidation". Schematic of the process of glucose oxidation in an aerobic environment. G6P = glucose-6-phosphate, NAD+ = nicotinamide adenine dinucleotide (oxidized), N A D H = nicotinamide adenine dinucleotide (reduced), F A D H = flavin adenine dinucleotide (reduced). Note that pyruvate is not converted to lactate since regeneration of NAD+ is not necessary in an aerobic environment. Instead, pyruvate is transported to the mitochondria and converted by pyruvate dehydrogenase (PDH) to Acetyl-coA, which then enters Kreb's cycle, ultimately yielding 3 N A D H 2 and 2 F A D H 2 . N A D H 2 and F A D H 2 are then oxidized by the respiratory chain, yielding 3 ATP/ N A D H 2 and 2 ATP/ F A D H 2 . Lactate can also be a source for more pyruvate under aerobic conditions. "Regulation of pyruvate dehydrogenase (PDH)". PDH phosphatase catalyzes the formation of the active form of PDH, and is stimulated by magnesium (Mg + + ) , insulin, and calcium (Ca + +). The latter is probably the mechanism by which 0-adrenergic stimulation results in increased PDH activity. PDH kinase converts PDH to its inactive form. PDH kinase is stimulated by the products of fatty acid metabolism: Acetyl-CoA and reduced nicotinamide adenine dinucleotide (NADH 2 ) . PDH kinase is inhibited by dichloroacetate (DCA). Modified from Depre C, et al. Circulation 1999: 99: 578-588. Figure 14. "Fatty acid oxidation". Schematic of the process of fatty acid oxidation. See text for details. G6P = glucose-6-phosphate, N A D H = nicotinamide adenine dinucleotide (reduced), F A D H = flavin adenine dinucleotide (reduced). The light gray items indicate the pathway for glucose oxidation, demonstrating that both . glucose and fatty acid oxidation culminate in the final common product of Acetyl-CoA, which is then oxidized in the Kreb's cycle. Figure 15. "Pressure-volume relationships in the working heart". P = pressure, V = volume, open circle indicates peak systolic pressure, closed circle indicates end-systolic pressure, Ees = elastance at end-systole, Emax = maximal elastance. Top left diagram shows the changes in pressure over time during a single cardiac cycle. The bottom left diagram shows the changes in volume over time during a single cardiac cycle. The diagram on the right shows the changes in the pressure-volume relationships over a single cardiac cycle. Note that Ees and Emax occur at different points in the cardiac cycle, resulting in slightly different values for Ees (black.line) and Emax (blue line). Modified from Sagawa K. , et al. Cardiac 39 contraction and the pressure-volume relationship. Oxford: Oxford University Press; 1988. p.39 Figure 16. "Time-varying elastance". Pressures measured for increasing volumes at the same time in the cardiac cycle (msec = milliseconds, time from onset of systole) fall along a regression line, indicating the elastance at that moment (E t = time-varying systolic elastance) (blue circles = E t @ 160 msec, white circles = E t @ 100 msec). The E t increases (slope increases) throughout the systolic contraction up to the point of maximal elastance (Emax). Modified from Modified from Sagawa K., et al. Cardiac contraction and the pressure-volume relationship. Oxford: Oxford University Press; 1988. p.64. Figure 17. "Pressure-volume relationships during isovolumetric contractions". P - pressure, V = volume, closed circle indicates peak and end-systolic pressure, Emax = maximal elastance. Top left diagram shows the changes in pressure over time during a single cardiac cycle. The bottom left diagram shows the changes in volume over time during a single cardiac cycle. Note that there is no change in volume over time. The diagram on the right shows the changes in the pressure-volume relationships over a single cardiac cycle. The blue line represents an approximation of Emax. Modified from Sagawa K., et al. Cardiac contraction and the pressure-volume relationship. Oxford: Oxford University Press; 1988. p.39 Figure 18. "Approximation of Emax". Comparison of maximal elastance (Emax, dashed lines) determined by isovolumetric contractions (top diagram) and contractions 40 from a working heart (bottom diagram). Note, that the Emax determined by both methods are superimposable. P = pressure, V = volume. Modified from Sagawa K. , et al. Cardiac contraction and the pressure-volume relationship. Oxford: Oxford University Press; 1988. p.59. 41 BIBLIOGRAPHY 1 Hearse DJ, Stewart DA, Braimbridge M V . 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Vasopressin VI-receptor stimulation produces a positive inotropic response without affecting pHi in guinea pig papillary muscles. Japanese Journal of Pharmacology 1995;68:217-221. 34 Talbot MP, Tremblay I, Denault A Y , Belisle S. Vasopressin for refractory hypotension during cardiopulmonary bypass. Journal of Thoracic and Cardiovascular Surgery 2000;120:401-402. 35 Yoshioka T, Sugimoto H, Uenishi M , Sakamoto T, Sadamitsu D, Sakano T, Sugimoto T. Prolonged hemodynamic maintenance by the combined administration of vasopressin and epinephrine in brain death: A clinical study. Neurosurgery 1986;18:565-567. 36 Iwai A, Sakano T, Uenishi M , Sugimoto H , Yoshioka T, Sugimoto T. Effects of vasopressin and catecholamines on the maintenance of circulatory stability in brain-dead patients. Transplantation 1989;48:613-617. 45 37 Chen JM, Cullinane S, Spanier TB, Artrip JH, John R, Edwards N M , Oz M C , Landry DW. Vasopressin deficiency and pressor hypersensitivity in hemodynamically unstable organ donors. Circulation 1999; 100(supp II):II244-II246. 38 Rao V , Borger M A , Weisel RD, Ivanov J, Christakis GT, Cohen G, Yau T M . Insulin cardioplegia for elective coronary bypass surgery. Journal of Thoracic and Cardiovascular Surgery 2000; 119:1176-1184. 39 Wamboldt RB, Lopaschuk GD, Brownsey RW, Allard MF. Dichloroacetate improves postischemic function of hypertrophied rat hearts. Journal of the American College of Cardiology 2000;36:1378-1385. - . - ' 40 Cheng K, Larner J. Unidirectional actions of insulin and Ca2+-dependent hormones on adipocyte pyruvate dehydrogenase. The Journal of Biological Chemistry 1985:260:5279-5285. . . 41 Hems DA, McCormack JG, Denton R M . Activation of pyruvate dehydrogenase in the perfused rat liver by vasopressin. Biochemical Journal 1978;176:627-629. 42 Lopaschuk GD, Wambolt RB, Barr RL. A n imbalance between glycolysis and glucose oxidation is a possible explanation for the detrimental effects of high levels of fatty acids during aerobic reperfusion of ischemic hearts. Journal of Pharmacology & Experimental Therapeutics 1993;264:135-44. 43 Svensson S, Svedjeholm R, Ekroth R, Milocco I, Nisson F, Sabel K G , William-Olsson G. Trauma metabolism and the heart: Uptake of substrates and effects of insulin early after cardiac operations. Journal of Thoracic and Cardiovascular Sugery 1990;99:1063-1073. 44 Taegtmeyer H, Goodwin GW, Doenst T, Frazier OH. Substrate metabolism as a determinant for postischemic functional recovery of the heart. American Journal of Cardiology 1997;80:3A-10A. 46 45 Lazar HL, Philippides G, Fitzgerald C, Lancaster D, Shemin RJ. Apstein C. Glucose-insulin-potassium solutions enhance recovery after urgent coronary artery bypass grafting. Journal of Thoracic and Cardiovascular Surgery 1997;113:354-362. 46 Jackson EK. Vasopressin and other agents affecting the renal conservation of water. In: Hardman JG, Limbird L E , Molinoff PB, editors-in-chief. Goodman and Gilman's The Pharmacological Basis of Therapeutics, 9th ed. New York: McGraw-Hill Health Professions Division; 1996.p.715-731. 47 a • —H CD o CN o > o o a CD VH CD O CD CD —^> c/2 CD % £ r/) • —H C+H o o • • CN CD • —H ^ ^ ^ ^ WD c a © © CM © © OAIAI 49 r/3 3 S3 a o • —H C/3 CD Q i CD 43 H • • CD 3 X CD £ 73 00 CO '—' as CL) ^ a o o u O fj CN o =3 o co co CO -tn CO CH 0) o a B CH •I—c a -3 3 co ^ s s 7 3 I_l co w S o a _ CO o oo CO s-T CO cu 43 *0 o > ^ CO cT^ to 00 to j-T 2 o3 C co g 43 o o IS. s m o o +-» S X o I-H OH o O H m • • i n 3 OQ CO ^ 1 a . CD o CD o CL o CD TJ X i— CD E O E CD CJ) i t CD CO CO CD C II CD <— CD O E Q. o O II CM CO o IN. 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