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The role of ICAM-1 in myocardial dysfunction during sepsis and ischemia-reperfusion injury Yousefzadeh-Davani, Ehsan 2005

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T H E R O L E O F I C A M - 1 I N M Y O C A R D I A L D Y S F U N C T I O N D U R I N G S E P S I S A N D I S C H E M I A - R E P E R F U S I O N I N J U R Y by EHSAN Y O U S E F Z A D E H - D A V A N I M.D., Isfahan University of Medical Sciences, 1993 M.Sc , The University of British Columbia, 2002 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE F A C U L T Y OF G R A D U A T E STUDIES (Experimental Medicine) THE UNIVERSITY OF BRITISH C O L U M B I A July 2005 © Ehsan Y . Davani, 2005 The role of ICAM-1 in myocardial dysfunction during sepsis and ischemia-reperfusion injury Abstract Intramyocardial inflammation occurs in various types of cardiovascular diseases including ischemia-reperfusion, myocarditis, during orthotopic heart transplant rejection, and during sepsis-induced myocardial dysfunction. Intramyocardial inflammation results in increased expression of I C A M - 1 on cardiac tissue. I C A M - 1 can interact with its ligands including CD11/CD14 receptors on inflammatory cells, and fibrinogen leading to activation of intracellular signaling cascades. To test this hypothesis that I C A M - 1 activation on cardiac tissue can trigger signaling which leads to decreased cardiac contractility we used three different in vitro (cardiomyocytes, polymorphonuclear leukocytes ( P M N ) and fibrinogen co-culture), in vivo (cardiac contractility measurement using micro-catheter) and ex vivo (reperfusion o f isolated heart) methods. Cardiomyocyte I C A M - 1 -binding by activated P M N , by activated and kil led P M N s , and by I C A M - 1 cross-linking antibodies decreased cardiomyocytes contractility. In vivo, inducing severe cardiac inflammation by L P S injection decreases cardiac contractility by 58 ± 4% (using end systolic elastance) and increases I C A M - 1 expression on cardiac tissue by 2.35 fold compared to control. Morphometery of cardiac vessels indicated increased intravascular P M N s after L P S injection but the number of interstitial P M N s were not different compared to control excluding the role of P M N adhesion in I C A M - 1 activation in sepsis. This led to the next hypothesis that fibrinogen can interact with I C A M - 1 after L P S injection. Immunohistochemistry staining indicates increased interstitial fibrinogen infiltration six hours after L P S injection supporting the interaction of I C A M - 1 and i i fibrinogen during sepsis. Incubation of cardiomyocytes with fibrinogen decreased cardiomyocytes contractility. This effect of fibrinogen was abolished in the presence of anti ICAM-1 antibody. Treatment of isolated heart with IGF-1 decreases ICAM-1 expression by 2 fold (p<0.01), increases cardiac contractility and heart rate, and decreases CPK during reperfusion compared to TNFa. We conclude that PMN and fibrinogen interact with cardiomyocytes ICAM-1. Fibrinogen infiltrates into the myocardial interstitial space during sepsis and decreases cardiac contractility through adhesion to ICAM-1 receptor on the cardiomyocyte membrane. IGF-1 can be used as a therapeutic intervention to decrease ICAM-1 expression thereby protecting the heart from further injury during cardiac inflammation. iii Tables of contents Title page. i Abstract ii Table of contents iv List of tables vii List of figures viii List of abbreviations ix Acknowledgement ." xi Co-authorship statement xii 1.0 Chapter 1: Overview 1 1.1 Sepsis and myocardial ischemia are important health problems 1 1.2 The importance of cardiac physiology in cardiovascular diseases. 4 1.2.1 Cardiac function .4 1.2.2 Ca2+ receptor and excitation-contraction coupling 5 1.2.3 Cardiodynamics 8 1.3 Myocardial dysfunction in sepsis and ischemia reperfusion injury 12 1.4 Molecular mechanism of cardiac inflammation 28 1.4.1 ICAM-1 interaction initiates intracellular signaling 28 1.5 General Hypothesis 35 1.6 Final conclusion 36 2.0 Chapter 2: Novel regulatory mechanism of cardiomyocytes 39 contractility involving ICAM-1 and the cytoskeleton 2.1 Abstract 39 2.2 Introduction .41 2.3 Material and Methods .43 2.3.1 Isolation of rat ventricular myocyte 43 2.3.2 Cardiomyocyte ICAM-1 protein expression. .44 2.3.3 Cardiomyocyte ICAM-1 mRNA expression. .44 2.3.4 Measurement of cardiomyocytes fractional shortening 45 2.3.5 Isolation of peripheral blood PMN 46 2.3.6 CD1 lb expression on PMN .46 2.3.7 Co-culture of PMN and cardiomyocytes .46 2.3.8 ICAM-1 cross-linking .47 2.3.9 Immunoflurescent imaging of Focal adhesion kinase (FAK) .47 2.3.10 Cardiomyocyte Ca2+ transient .48 2.3.11 Data analysis .49 2.4 Results 50 2.4.1 Uniform co-culture conditions. 50 2.4.2 Adherent PMN reduce cardiomyocytes fractional shortening. 53 2.4.3 ICAM-1 binding mediates decreased cardiomyocytes contractility. 57 2.4.4 ICAM-1 cross-linking alters the cortical cytoskeleton. 59 2.4.5 Functional role of the cardiomyocytes actin cytoskeleton 59 2.4.6 Possible downstream signaling pathways 62 2.4.7 ICAM-1 activation changes the pattern of Ca 2 + release 64 2.5 Discussion 65 3.0 Chapter 3: Cardiac ICAM-1 mediates leukocyte-dependent 72 decreased ventricular contractility in endotoxemic mice 3.1 Abstract 72 3.2 Introduction .73 3.3 Material and Methods 75 3.3.1 Experimental preparation 75 3.3.2 Left ventricular contractility and cardiac function 78 3.3.3 The role of Leukocytes 78 3.3.4 Chimeric models 80 3.3.5 Cardiac ICAM-1 expression 81 3.3.6 Cardiac leukocyte infiltration 81 3.3.7 Statistical analysis 82 3.4 Results 83 3.4.1 Endotoxemic C57B6 wild type mice 83 3.4.2 Effect of increased and decreased leukocyte count 87 3.4.3 Endotoxemic ICAM-1 knock out mice 87 3.4.4 Endotoxemic chimeric mice 88 3.4.5 Morphometery of coronary vascular space 93 3.5 Discussion 95 4.0 Chapter 4: The role of fibrinogen and ICAM-1 interaction in 100 decreased cardiac contractility 4.1 Abstract 100 4.2 Introduction 102 4.3 Material and Methods 104 4.3.1 Induction of sepsis 104 4.3.2 Immunohistochemistry and Immunoflurescent study 104 4.3.3 Reperfusin of Alexa-488 labeled fibrinogen into the hearts 104 4.3.4 Incubation of soluble fibrinogen with isolated rat cardiomyocytes 105 4.3.5 Coating of fibrinogen to polystyrene beads 105 4.3.6 Blocking of cardiomyocytes ICAM-1 106 4.4 Results 107 4.4.1 Fibrinogen infiltration into the interstitial space 107 4.4.2 Fibrinogen effect on cardiomyocytes contractility 113 4.4.3 Fibrinogen induces decreased contractility through ICAM-1 116 4.5 Discussion 118 5.0 Chapter 5: IGF-1 protection of ischemic murine myocardium 121 from ischemia-reperfusion associated injury 5.1 Abstract 121 5.2 Introduction 123 5.3 Material and Methods 127 v 5.3.1 Ischemia-reperfusion model. 127 5.3.2 Histological evaluation 127 5.3.3 ICAM-1 expression of cardiac tissue 128 5.3.4 Ventricular function assessment 128 5.3.5 Detection of C P K 129 5.3.6 Mitochondrial DNA: nuclear D N A assay 129 5.4 Results 132 5.4.1 IGF-1 decreases ICAM-1 expression of cardiac tissue 132 5.4.2 Perivascular interstitial edema and tissue lattice integrity 132 5.4.3 IGF-1 improvement in myocardial performance during reperfusion 135 5.4.4 Low creatine phosphokinases level in IGF-1 treated hearts 136 5.4.5 Ratio of mitochondrial to nuclear D N A 136 5.5 Discussion 141 6.0 Chapter 6: Final conclusion 148 7.0 Reference 154 8.0 Appendix: Copyright permission 192 List of tables 3.1 Hemodynamic and ventricular function measures 84 3.2 Peripheral blood cellular components 86 3.3 Hemodynamic and ventricular function measures in chimeric mice 90 3.4 Peripheral blood cellular components in chimeric mice 92 vii List of figures 1.1 Schematic pathways involved in cardiac inflammation 37 1.2 schematic pathways involved in ICAM-1 presentation and activation 38 2.1 ICAM-1 protein expression on cardiomyocytes 51 2.2 P M N and cardiomyocytes co-culture conditions 52 2.3 Fractional shortening in the presence of normal or fixed PMNs. 54 2.4 Flow cytometery of CD1 lb expression on PMN. 56 2.5 ICAM-1 binding decreases cardiomyocytes contractility. 58 2.6 Focal adhesion kinase (FAK) presentation in cardiomyocytes. 61 2.7 ICAM-1 cross-linking decreases cardiomyocytes contractility. 63 2.8 C a 2 + release and reuptake in cardiomyocytes 66 3.1 Pressure volume loops. 77 3.2 The ration of ICAM-1 /Hoechst. 85 3.3 Stylized pressure volume loops 91 3.4 Number of neutrophils/area 94 4.1 Immunohistochemistry of LPS or saline treated C57B6 hearts 108 4.2 Immunohistochemistry of LPS or saline treated ICAM-1 K O hearts 109 4.3 A Retrograde reperfusion of Alexa-488 labeled fibrinogen 111 4.3B The intensity of fibrinogen staining 112 4.4 Dose response curve of soluble fibrinogen 114 4.5 Adhesion of fibrinogen coated beads on the cardiomyocytes. 115 4.6 Polystyrene beads coated with fluorescent fibrinogen 115 4.7 Fibrinogen coated beads decreases cardiomyocytes contractility 117 5.1 ICAM-1 protein and mRNA expression during ischemia reperfusion. 133 5.2 Immunohistochemistry of heart sections 134 5.3 Determination of cardiac performance. 13 8 5.4 C P K measures in ischemia reperfusion injury. 139 5.5 Determination of mitochondrial/nuclear D N A 140 6.1 Electron microscopy of RyR and DHPR receptors juxtaposition 151 List of Symbols and Abbreviations A H A American Heart association ARDS Acute respiratory distress syndrome ASPG Accessory subunit of the murine mitochondrial D N A polymerase y ATP Adenosine triphosphate B S A Bovine serum albumin C57B6 C57 black 6 mice C a 2 + Calcium ion C A B G Coronary artery bypass grafting cGMP Cyclic guanosine monophosphate C O X Cytochrome oxidase subunit 1 C P K Creatine phosphokinase C V D Cardiovascular diseases C Y Cyclophosphamide D A B Diaminobenzidine tetrahydrochloride D A G Diacylglycerol DHPR Dihydropyridine calcium receptors D N A Diamino nucleotide acid DP Systolic - diastolic pressure dP/dt Alteration of pressure in time E C G Electrocardiogram Ees End systolic elastance EF Ejection fraction Emax Maximum elastance EPO Erythropoietin F A K Focal adhesion kinase Fib/Nuc Fibrinogen to nuclear staining ration GCSF Granulocyte colony stimulating factor GFR Glomerular filtration rate G H Growth hormone H & E Hematoxylin and eosin H+/M- ICAM-1 competent heart/ICAM-1 knock out bone marrow derived cells H I F - l a Hypoxia inducible factor-1 HR Heart rate HRP Horse radish peroxidase H U V E C Human umbilical vascular endothelia cell ICAM-1 Intercellular adhesion molecule-1 ICU Intensive care unit IGF-1 Insulin-like growth factor-1 IHC Immunohistochemistry IL- lp Interleukin-1 beta IL-6 Interleukin-6 IP3R Inositol triphosphate receptors K O Knock-out LPS Polymorphonuclear leukocytes L V Left ventricle L V E D P Left ventricular end-diastolic pressure L V E D V Left ventricular end-diastolic volume L V E S V Left ventricular end-systolic volume M H C Major histocompatibility complex Mit Mitochondrial M K Modified Kreb's Henseleit working solution N C X Sodium-calcium exchange channels N F K B Nuclear factor kappa-B N K Sodium-potassium channels NO Nitrite oxide NOS Nitrite oxide synthesize NS Non significant P K C Protein kinase C PTA Percutaneous transluminal angioplasty PV Pressure-volume R N A Ribonucleotide acid ROS Reactive oxygen species R V Right ventricle RyR Ryanodine receptor SR Sarcoplasmic reticulum SV Stroke volume T N F a Tumor necrosis factor-alpha V C A M - 1 Vascular cellular adhesion molecule-1 V E G F Vascular endothelial growth factor V W Ventricular work vWf von-Willebrand factor Who World health organization X Acknowledgement This thesis is the result of the extensive effort of some of my best friends that I have ever had. I personally believe without their cooperation I could not have finished these projects. I first thank Maryam, my wife, for her patience, encouragement, and support while I was working on these projects. I thank Yingjing Wang who worked with me for the last five years. His patience and skills in animal surgeries and procedures made these investigations possible. M y friends and colleagues, Teresa Wood, Treena McDonald, Shelly Dai, Arnrite Samra, Kathy Grek, and Gurpreet Singhera were involved in different parts of this work. I have been trained by them and I always appreciate their cooperation. Dr. Thoma Kareko came as a postdoc in our laboratory. He dedicated his time to support others including me with his knowledge in physics and math. I always remember his extensive work on image deconvolution. I thank Dr. Ryon Bateman, and Hon Leong for their work on confocal microscopy. I thank Drs. Chan Hang Lee and Casey Van Breeman for cooperation in calcium influx measurement in their laboratory. I also thank Dr. Helen Cote, Zebrina Brumme and Dr. Richard Harrigan for using their method (mitochondrial/nuclear DNA) in chapter 5. M y deep appreciation to Drs. James Hogg, Peter Pare, Bruce McManus, Mike Allard, David Walker, Stephan Van Eeden, Blair Walker, Tom Podor, David Granville, and Bob Schellenberg and all people at iCAPTURE center for their valuable suggestions. I would like to thank research students, Shane Olaleye, May Tee, Karen Reevere, and Alan Ng. M y special thanks to Anna Meredith and Edmond Chau for their great works. I thank Dr. Delbert Dorscheid, my co-supervisor, who has been supporting me since he came to our laboratory. He gave a great opportunity to his graduate students to train summer research students in our laboratory. He also created a free and friendly space in his research laboratory for everyone which, I think, is the most important part of the research environment that can create trust and future cooperation. He was leading the ischemia reperfusion projects and his critical suggestions and comments were essential parts of the other projects in this thesis. I thank Dr. Keith Walley, my supervisor, for his persistent work, knowledge and involvement in the preparation of this thesis. His tremendous work on this projects resulted in supporting fund from CIHR. I always appreciate his great support especially in the first two years of my work at iCAPTURE center. M y sincere appreciation to CIHR, and Heart and stroke foundation for supporting grant to these projects. I also thank U B C department of medicine, American Lung Association, CFIC and CIHR for supporting travel funds to present my data. xi Co-Authorship Chapter 2 of this thesis was published as a manuscript in American Journal of Physiology. The authors are; Ehsan Y Davani, Delbert R Dorscheid, Chan Han Lee, Casey Van Breeman, and Keith R Walley. Dr. Chan Han Lee and Casey van Breeman were involved in Ca2+ measurement. Dr. Dorscheid partially supervised this project and Dr. Keith Walley supervised and leaded this project. Chapter 3 is in submission. Authors are Ehsan Davani, Delbert Dorscheid, Yingjing Wang, Anna Meredith, Edmond Chau, Gurpreet Singhera and Keith Walley. Yingjing Wang performed most of the animal surgeries. Anna Meredith worked on the immunohistochemistry and morphometery of the hearts slides. Gurpreet Singhera was responsible for some of the data collection and protein assays. Dr. Keith Walley was leading this project. In chapter 4, I have done immunohistochemistry, Immunoflurescent studies, confocal microscopy and mice studies based on the generated hypothesis from my previous works. I supervised Edmond Chau during his two months summer research studentship. Edmond Chau's tremendous work during this period resulted in important observations. He has done all of cardiomyocytes co-culture experiments in this chapter. Dr. Ryon Batemen has performed co-localization study of this chapter. Chapter 5 is the results of cooperation of Dr. Gurpreet Singhera, who measured the C P K level and Shane Olaleye who did some ex-vivo experiments during his summer research studentship. Drs. Helen Cote, Richard Harrigan, and Zebrina Brumme performed the mitochondrial/nuclear D N A ratio. Dr. Delbert Dorscheid supervised and led this project. xii To Dr. James Hogg, who dedicated his life time to improve and to support research in medicine. Chapter-1: Overview 1.1 Sepsis and myocardial ischemia are important health problems. The heart contracts continuously to prepare and facilitate life for other organs. It has been named the center of love, and of hate. Ancient Egyptian physicians believed that heart could speak to other parts of the body since they heard the sound of pulsation (Ebner Papyrus) and assumed the heart was the center of management for other organs. The heart beats 2.5 billion times during an average life of 70 years. It supplies energy for all organs and when it dies none of the other organs can survive. During past centuries cardiovascular diseases have been progressively affecting human life, especially in western countries. It has been estimated that cardiovascular diseases (CVD) will be the most common human health problem until 2020 (AHA). Sepsis and myocardial infarction are important health problems. Cardiovascular diseases are one of the most common problems in health care in our new world, resulting in more than 17,000,000 death annully worldwide (www.who.int). Each year millions of people die because of myocardial infarction. Many of these patients need cardiac transplantation, cardiac bypass, and invasive therapeutic interventions, with their consequent side effects and disabilities, direct either because of procedures or indirect because of using various types of drugs. C V D are the leading causes of mortality in North America resulting in the death of more than 432,000 males and 499,000 females annually (AHA). The majority of these deaths are a result of coronary vascular atherosclerosis and myocardial infarction. It has been estimated that C V D drains more that 329.2 billion dollars annually in North 1 America. Tissue damage due to myocardial infarction involves the innate immune system triggering the responses of defensive cells and proteins against the infarcted myocardium, thereby inducing further damage to the remaining non-infarcted myocardium. Sepsis also involves the innate immune system in our body. It is a severe inflammatory response of the body against bacterial and other infectious pathogens. Sepsis syndrome occurs as a result of primary infection and also as one of the consequences for trauma and post surgery patients. It is the leading cause of death in non-coronary ICUs. Sepsis affects more than 750,000 people annually in North America and results in 215,000 deaths. The average health care cost of a septic patient is about $22,000 in North America and results in more than $18 billion of expense annually Most septic patients die of severe hypotension and myocardial depression, leading to multiple organ hypoperfusion and dysfunction 2 . Although the reported number of cases of sepsis is about 1,800,000 per year worldwide 3 , it has been estimated that the actual number of reported and non-reported cases of sepsis could be around 18,000,000 per year worldwide, based on an observed incidence of 3 cases per 1000 patients. Given the 30% mortality rate, this would mean that sepsis is the leading cause of death worldwide. Epidemiological investigations of sepsis have defined a numbers of issues, including an increasing incidence of sepsis, and the lack of a unique definition to diagnose sepsis syndrome which results in high numbers of non-registered cases worldwide. According to recent clinical investigations, the stability of hemodynamic state is one of the most important factors required to survive from sepsis 4 . 2 This thesis is focused on one of the intracellular mechanisms of myocardial dysfunction during sepsis and ischemia-reperfusion injury. Here I explain the importance of cardiac contractility in various diseases including sepsis. 3 1.2 The importance of cardiac physiology in cardiovascular diseases 1.2.1 Cardiac function The main cellular component of the heart is the cardiomyocyte. It is the contractile part and forms the majority of heart mass and volume. A cardiomyocytes in vivo is approximately 20 um wide, 100 um long and 5 to 7 um thick. The contractile component of each cardiomyocyte is a sarcomere with a length of 2.5 to 3 um. The sarcomere is connected to the cardiomyocyte membrane (sarcolemma) through cytoplasmic cytoskeletal proteins, mainly microfilaments (actin) and intermediate filaments 5 . Each sarcomere has longitudinal networks of actin and myosin filaments with a specific arrangement. The contractile force is the product of interaction and sliding of sarcomeric actin filaments and myosin heads on each other. Each sarcomere is connected to the neighboring sarcomere through accumulation of a-actinin protein in a special area called the Z line which is visible by light microscopy. Light microscopy of the sarcomere shows light areas on either sides of Z lines, named I bands. I bands are fractions of sarcomeric actin filaments, tropomyosin and troponin proteins. Thick filaments (myosin) are interspersed among sarcomeric actin filaments (A band). They are divided in two sections in the middle by the H band. The H band is also divided in two parts by the M line. Various proteins are located in the M line including myomesin and creatine phosphokinase (CPK). C P K is an enzyme with various isotypes that maintains enough adenosine triphosphate (ATP) for muscular contraction. C P K is released into the plasma during muscular injury and death, and it is used as a diagnostic test to measure muscular or myocardial injury. Thick filaments are also connected to the Z lines and each other by titin and myomesin respectively. 4 Cardiomyocyte membrane is invaginated into the cytoplasm creating transverse tubules (T tubules). Many types of ion channels are located on the T tubule including dihydropyridine calcium receptors (DHPR), sodium-calcium exchange channels (NCX) and sodium-potassium channels (NK). Other important micro-organelles in cardiomyocytes are mitochondria and the sarcoplasmic reticulum (SR). Mitochondria are important organelles because they are the main source of energy production and occupying 40% of the total volume of each cardiomyocyte. SR is important due to its role as the source ofCa 2 + (100umol/L). 1.2.2 Ca 2 + receptor and excitation-contraction coupling Voltage dependent channels control cardiomyocyte sarcoplasmic C a 2 + concentration. The most acceptable theory for C a 2 + influx into the cardiomyocyte is C a 2 + induced C a 2 + release. The two major types of voltage sensitive C a 2 + channels are L and T type channels on the cardiomyocyte membrane 6 ' 7 . T type channels have a negligible role in regulating the C a 2 + gradient in cardiomyocytes. The L type C a 2 + channel is also called the DHPR receptor. Alteration in C a 2 + influx is the basic pathophysiological cause of cardiac arrhythmia and contractility dysfunction. Resting intracellular C a 2 + concentration is less than 0.1 jomol/L. Depolarization of the membrane activates DHPR and induces C a 2 + entry resulting in an increase in the C a 2 + concentration around ryanodine receptors (RyR) 8 . This event changes the protein configuration of the RyR receptor in 2 to 3 milliseconds and triggers C a 2 + release from the SR. This alteration in turn changes the intracellular C a 2 + concentration to 10 to 12 (imol/L. The raise in sarcoplasmic C a 2 + concentration 5 changes the DHPR receptor structure and negatively influences Ca entry so that the speed of Ca entry is reduced by 50% . Alteration in intracellular C a 2 + concentration during the cardiac excitation contraction period is mainly regulated by RyR and inositol triphosphate receptors (IP3R) on the SR 9 . It has been shown that IP3 receptors are involved in C a 2 + release from the SR in heart failure. It is worth noting that the IP3 receptors can be activated by endothelin-1, angiotensin, and a-adrenergic stimulation 1 0 , 1 \ RyR receptors control C a 2 + release, and interestingly, the tertiary structures of RyR receptors are also controlled by Ca concentration. T tubules in cardiomyocytes and skeletal muscle have special localization close to the SR projection (terminal cisterha). The space between T tubules and the terminal cisterna is about 10 to 20 nm and called the couplon space. This space is very important in maintaining the juxtaposition between RyR on the terminal cisterna and DHPR on T tubules. DHPR in the T tubule contains four similar proteins, and in skeletal ft 19 muscle it is aligned with one RyR on the SR ' .In cardiomyocytes one DHPR is aligned with 5 to 10 RyRs. This position ensures the opening of all RyR in one couplon space at once 1 3 . It is worth noting that in skeletal muscle most RyR isoforms are type 1 (RyRl) while in cardiomyocytes the majority is type 2 isoform (RyR2). The head of RyR is a large protein which is located in the couplon space. The function of RyR is dependent on the activation of voltage-dependent C a 2 + receptors or DHPRs 1 4 . Depolarization of T tubules leads to changes in tertiary structure of DHPRs and results in the opening of DHPR channels. During cardiac excitation-contraction, thousands of Ca sparks synchronize in time along the membrane 6 . Entry of 2 to 4 C a 2 + ions is sufficient to 6 activate the RyR channel and the whole couplon space . Each Ca spark is the function of a cluster of 6 to 20 activated RyR receptors increasing the C a 2 + influx. If these sparks are not synchronized in time, it causes wave shape propagation and non-harmonic sarcomere contraction leading to a reduction of cardiomyocyte contractility. Release of C a 2 + in the cytosol removes the inhibition of the actin myosin interaction 1 4 . This is the predominant position for cardiomyocytes at rest. The binding of four C a 2 + ions to troponin, located on the actin filament, changes the conformation of troponin, removes tropomyosin from the contractile part of myosin and exposes the binding site of myosin to actin. Using ATP, the cross-bridge between actin and myosin creates tension along the sarcomeric structure, generating enough force to fold the membrane and other organelles in the cardiomyocyte during contraction. The next step is dissociation of actin from myosin which requires ATP 1 3 . The ability of the sarcomere to generate force decreases 9+ when sarcomeric length reaches 2 to 2.2 um. Ca decay occurs during this time by activation of N C X , C a 2 + pumps on the membrane, SR and mitochondria 6 . The sum of the force of all the cardiomyocytes contracting generates enough force for ventricular contraction. Any type of influx alteration can change cardiomyocyte contraction. Alteration in C a 2 + sparks, juxtaposition of RyR and DHPR receptors, and propagation of C a 2 + ions can be the source of cardiac dysfunction. These above mechanisms have been postulated to describe myocardial dysfunction in sepsis and ischemia reperfusion injury. 7 1.2.3 Cardiodynamics The heart is the organ responsible for pumping blood into the conductive system, arteries and capillaries. Veins are the reservoirs of blood. The right ventricle (RV) is responsible for pumping non-oxygenated blood into the lungs. The left ventricle (LV) is the main pump in normal physiologic circulation and it distributes nutrients and oxygen to all organs. Thus the L V is responsible for maintaining systemic blood pressure. The power of the L V pump is directly related to its cone shape structure and the volume of blood which can be pumped out of the left ventricle. Since during L V (cone shape) contraction the L V height is almost constant, the L V ejects blood by reducing its volume through a reduction in radius. Since volume is directly related to the squared radius of L V (r2), the ejection of blood is directly related to r 2 of L V . L V output is an important factor in measuring workload of the left ventricle and maintaining systemic blood pressure in normal range. L V output is related to heart rate (HR) and the volume of ejected blood in each beat (stroke volume, SV). Thus the L V output = S V ' HR. The cardiac cycle starts by atrial contraction, a function that is especially important when the heart beats faster than normal. In fact 75% of the left ventricular blood volume comes from the pre-atrial contraction and atrial contraction is just responsible for the ejection of the remaining blood volume. However when heart beats faster, the diastolic phase decreases and atrial contraction plays its major function. The volume at the end of the diastolic phase is called the left ventricular end-diastolic volume (LVEDV) and in turn it makes a pressure gradient which is called the left ventricular end-diastolic pressure 8 (LVEDP). Ventricular contraction starts at the end of diastole. First it increases the intraventricular chamber pressure without changing the intraventricular volume, which is called isovolumic contraction. When left ventricular pressure reaches the pressure of the aorta, it causes opening of the aortic valve so that the blood is pushed by left ventricle contraction into the aorta until the end of contraction. The diastolic phase starts by closure of the aortic valve and relaxation of the left ventricle causing isovolumic relaxation. This cycle repeats in each beat. The plot between L V pressure and volume in each cardiac cycle generates a loop. Obtaining of number of loops with different L V volumes generates a tremendous amount of information about the function of L V (refer to Figure 3.1). The pressure-volume (PV) loops of L V contractility provide various types of cardiac parameters including end systolic elastance (Ees), maximum elastance (E m a x ) and ventricular work (VW). Ees and E m a x are from the slope of the end systolic PV loops and they are load independent cardiac parameters. V W is reflected by the size of the above loops and it is proportional to the pressure and stroke volume. The ventricular work also reflects oxygen consumption by ventricular muscles and vice versa because V W is related to ventricular wall tension. Normal L V output is around 5 liters per minute in humans. The L V output is not independent from R V and it is also related to the R V output. This phenomenon has been named Frank-Starling mechanism. According to Frank-Starling mechanism the energy produced by L V is a function of length of muscle fibers in L V , considering the radius of L V is adjusted by length of muscle. In fact the length of muscle fibers determines the preload. The maximal volume of L V is achieved at the end-diastolic time point exactly 9 before L V contraction when the muscle fibers have the highest normal length. This volume is called L V end diastolic volume (LVEDV). In humans L V E D V is about 120 to 140 mL. The tension and length of muscle fibers is also related to the L V end-diastolic pressure (LVEDP) because LVEDP is proportional to L V E D V . The L V E D P in human is around 12-14 mmHg. Thus an increase in L V E D V or LVEDP mirrors increment in L V preload which is related to R V function. Frank-Starling's law explains the balance between R V and L V function. A n increase in muscle fiber length in L V (preload) up to the optimal point (before disruption) induces more probability of cross bridging of actin and myosin in sarcomeric structure of L V muscle fibers because of an increase in tension. The more interaction between actin and myosin enhances the force of cardiomyocyte contraction. Eventually this results in increased in L V contraction and depletion of blood to the aorta so that the ventricular volume reaches the minimum level which is called left ventricular end-systolic volume (LVESV). The fraction of the blood that has been pushed into the aorta is called ejection fraction (EF%) which is stroke volume divided by L V E D V . It is practically important to measure changes in ventricular pressure and volume that can be used as a source of information about ventricular function. According to the Laplace law ventricular wall tension is directly related to pressure and radius of the left ventricle and inversely related to thickness of the left ventricle. Left ventricular contractility is not only affected by preload but it is also influenced by afterload or the resistance of the blood vessels. The L V contractility is related to both L V 10 force generation and the velocity of contraction. The velocity of L V contraction is affected by afterload. In any type of muscle contraction the velocity of contraction is inversely related to the force development. Hence in muscular contraction both force generation and velocity of shortening are important factors. L V shortening velocity is not affected by any changes in preload parameters such as venous pressure, LVEDP and L V E D V . This notes the importance of other cardiac parameters including alteration of pressure in time (dP/dt). This section explained that L V contractility is not only depends on the cardiomyocyte contractility and interaction between myosin and actin filaments but also is affected by external parameters including the preload and afterload which are also the results of contractility. The cardiomyocyte shortening is the main source of force generation of the L V . However L V force generation is under the influence of the other hydrodynamic parameters such as systemic or pulmonary blood pressure, and the volume of the blood in the venous lake. Heart function is also influenced by other organ functions such as liver, kidney and gastrointestinal systems thereby looking at the function of the heart in a patient should always be accompanied with consideration of other external factors. 11 1.3 Myocardial dysfunction in sepsis and ischemia reperfusion injury Myocardial dysfunction is an important feature of sepsis and cardiac ischemia-reperfusion injury. It is one of the most common causes of death in septic patients 1 6 . In fact during severe sepsis and septic shock, and in the presence of refractory hypotension, the only compensation to maintain sufficient blood flow in the vital organs is left ventricular contractility. Animal and human studies have shown early myocardial dysfunction during sepsis 1 1 . Myocardial dysfunction during sepsis is biventricular and biphasic. It occurs within day 2 to 4 of early presentation of sepsis and returns to normal within 7 to 10 days in survivors . Hypotension is the main characteristic of septic shock. Regardless of the cause of hypotension in septic shock, the mean arterial pressure is decreased because of reduction in vascular tone. This also increases the importance of cardiac output in saving organs from severe hypoperfusion. It has been shown that infusion of endotoxin in healthy individuals induced vasodilation follow by high cardiac output both of which are characteristic of sepsis 1 9 . During sepsis, the higher cardiac output results from a combination of tachycardia and the reduction in vascular resistance. Tachycardia is a cardiac reflexive defense for coping with hypotension, however the response of the heart rate to the degree of hypotension in sepsis is not as beneficial as in other types of 90 hypotensive crisis such as in severe bleeding . In septic patients, continuous inflammatory processes cause fever, diarrhea, perspiration and edema, leading to a reduction in fluid and preload. The reduction in cardiac preload causes decreased L V E D V and eventually falling cardiac output. 12 It is important to note that cardiac output is not predictive of the severity of sepsis. In fact most of patients who survive during and after the recovery phase have lower cardiac 91 output than non-survivors . It is only at the late stage of septic shock (near death), that non-survivors have very low cardiac output. Global circulation and the blood flow of different organs can change during sepsis 1 8 . Visceral blood flow is different in septic patients. Several studies have shown an increase 22 23 in hepatic blood flow in the hyperdynamic phase of sepsis compared to controls ' . Fong et al. have shown that low dose injections of endotoxin in normal individuals increases splanchnic blood flow and oxygen uptake, probably because of an increase in 9^ protein synthesis and gluconeogensis in the acute phase response of the liver . The splanchnic oxygen extraction ratio is also higher than the systemic one, possibly because of increased oxygen demands. Another study has shown a reduction in gastric mucosal pH indicating existence of hypoperfusion and ischemia 2 4 . In fact one of the speculated mechanisms of endotoxemia and sepsis syndrome in critically i l l patients was intestinal ischemia and translocation of bacterial endotoxin into the blood stream through the injured epithelium. This mechanism has been excluded in human septic shock. Renal blood flow increases during the early phase of sepsis. However renal blood flow usually declines during the course of sepsis leading to decreased glomerular filtration rate (GFR), and in severe cases results in renal failure. Most of the time septic patients present 9S with oliguria, and diminished creatinine clearance . 13 Cerebral blood flow is preserved during sepsis indicating normal global flow for the brain, however it does not exclude regional ischemia, especially because most septic 96 98 patients have symptoms of brain involvement such as lethargy and stupor " . Furthermore it is not clear i f the reduction in brain activity is because of reduced oxygen uptake or supply. Various studies have shown different conclusions on skeletal muscle perfusion during sepsis. Early studies indicated a higher fraction of cardiac output goes into the skeletal muscle while Neviere et al. have shown that skeletal muscle blood flow was decreased 90 during severe sepsis . Administration of endotoxin to normal individuals did not change lower limb blood flow and animal studies have also indicated conflicting results. Many different mechanisms have been considered to explain the pathogenesis of myocardial dysfunction during sepsis 1 6 . One of the manifestations of myocardial dysfunction in sepsis is pre-terminal or end stage fall in cardiac output and EF%. During sepsis and in the presence of normal pulmonary capillary wedge pressure (an indicator of left ventricular preload), left ventricular stroke volume decreases even in resuscitated patients 3 0 . It has been shown that the ratio of left ventricular stroke work and pulmonary capillary wedge pressure is decreased during sepsis and septic shock, indicating disturbance of left ventricular performance 3 0 . Raper et al. have shown that the slope of L V stroke work against L V E D V is significantly lower compared to other types of hypotension 3 1 . Injection of LPS in normal individuals depressed the above parameter 3 2 . One of the best characteristic parameters for measuring L V contractility during sepsis is 14 the slope of left ventricular end systolic pressure volume relation which is also called end-systolic elastance (Ees). Similarly the maximal elastance (E m a x ) of the left ventricle provides another index of the L V contractility. Ees and E m a x describe cardiac contractility without consideration of left ventricular loading. It has been shown that even in the early phase of sepsis and in the absence of shock, Ees and Emax can change 3 3 ' 3 4. Animal studies have also confirmed the efficiency of E m a x in predicting cardiac performance during sepsis. One of the postulated mechanisms of myocardial dysfunction during sepsis is global ischemia because of the presence of severe hypotension and presumably a reduction in coronary blood flow, which finally results in decreased EF%. However several studies 1 ft 1 R ^7 have shown different changes in EF% during sepsis and septic shock ' ' " . In fact most non-survivors of sepsis have a higher left ventricular EF% than survivors. It is also important to note that EF% is affected by vessels wall resistance or afterload. EF% is a load dependent cardiac parameter and this has been confirmed by several studies that explained different changes in left ventricular EF% of septic patients . According to the Frank-Starling Law the rise in left ventricular EF% can be accomplished by increased L V E D V . Several studies confirmed the increase in L V compliance during sepsis however there is good evidence of the opposite result, showing no difference in EF% between survivors and non-survivors of patients with ARDS, though ventricular compliances in survivors was higher than non-survivors 3 9 . Based on previous studies L V E D V can differ from the high volumes seen in the early phase of sepsis to the very low volumes that has been documented in the end-stage of septic shock 1 8 ' 4 0 , 4 1 . Moreover 15 it can also differ based on previous heart function and the nature of cardiovascular tissue. Taken together, it seems that L V E D V and EF% are not good indicators of depressed cardiac function during sepsis and septic shock, though they may change in a manner consistent with myocardial depression in some patients. Another speculation of myocardial dysfunction in sepsis is right ventricle stress. It has been shown that pulmonary arterial blood pressure was increased during sepsis because of the presence of various types of mediators, leading to a fall in right ventricular contractility and a rise in right ventricle volume, especially in volume resuscitated patients 4 2 . The high volume of the right ventricle shifts the interventricular septum to the left side and in turn reduces left ventricular motion 4 3 . However further studies using angiography excluded the above claim and indicated that right ventricular depression results from left ventricular dysfunction 4 4 . Other studies have suggested that the left ventricular depression during sepsis occurs because of a lack of response to endogenous inotropics substances such as cathecholamines. Low adernergic responses may be one of the reasons for lower cardiac output in septic patients 4 5 . Defects in cAMP production and downstream signals of R -receptors have also been suggested 4 6 . It has been shown that the high activity of inhibitory G protein in septic patients is involved in the mechanism of hypotension and low cardiac output 4 7 ' 4 8. 16 Disturbed coronary circulation has been suspected in septic patients and it raises many questions in describing ventricular dysfunction in sepsis. Coronary blood flow in septic patients is higher compared to controls, both in survivors and non-survivors 4 9 . Cunnion et al. have explained that the cardiac dysfunction in humans with severe septic shock is independent of myocardial blood flow and metabolism in most patients 5 0 . During septic shock myocardial blood flow is preserved or even increased in experimental animals 5 1 . Cunnion et al. concluded that coronary sinus blood flow is higher in most patients with septic shock compared to normal subjects, especially in non-survivors. 5 0 Bloos and Herbertson groups have shown that the septic heart has a higher global perfusion compared to controls. The above observations can be explained by vasodilative mechanisms such as NO production in the septic heart 5 1 , 5 2 . Dhainaut's study revealed that global ischemia is not responsible for the cardiac dysfunction in septic shock because there is no change in net lactate production and low lactate extraction by myocardium in the septic heart. However this does not exclude possible focal ischemia in the septic heart. They concluded that myocardial contractility depression is independent from blood flow and metabolic changes. In fact myocardial blood flow is often preserved in patients with septic shock . The septic heart is overperfused, however, oxygen extraction is less than in normal subjects . In the normal heart, coronary blood flow is highly different in myocardial regions over time and space. In fact even in the normal myocardium, blood flow is heterogenous because of differences in myocardial segmental force. This is directly related to the local force of contraction that is cardiomyocyte shortening force. This mechanism seems to be 17 necessary to adjust the demand and supply in various regions of the heart. This means that the septum of L V (highly active) has higher blood flow compared to the basal part of L V (less active) at the same time point 5 3 . It has been suggested that the differences in regional blood flow may be higher in septic heart and may affect the corresponding regional supply. Although workload and demands for one specific region of the heart in septic shock may be lower than in a normal heart because of the lower afterload (lower resistance), the septic heart does not have sufficient supply because of increased heterogeneity of blood flow resulting in focal ischemia 51,54,55 On the other hand, during sepsis and septic shock, oxygen metabolism is changed and the relation between oxygen delivery and uptake is interrupted. In normal individuals the critical oxygen delivery level is about 400 mL/min/m 2 of body surface with almost 100 mL/min/m 2 of oxygen uptake. During sepsis these levels are changed to significantly higher oxygen delivery (about 700-800 mL/min/m ) because of impaired oxygen extraction, and higher oxygen uptake because of increased demand 3 9 . In fact it has been suggested that the circulatory changes during septic shock are associated with impaired oxygen extraction, indicating that abnormal circulation rather than changes in cellular metabolism that causes impaired oxygen uptake . However there is still debate about the impact of impaired oxygen extraction versus defects in sufficient oxygen supply 3 5. 18 The endothelium functions as the first barrier for separating the vascular space from surrounding tissues. Endothelial damage and leakage has been mentioned as a reason for myocardial edema in septic shock that leads to leakage of coronary capillaries and finally vascular compression, suggesting more defects in the supply/demand of the heart during sepsis 5 6 . On the other hand endothelial dysfunction may occur because of intravascular coagulation or NO production 5 1. Other factors may be involved in the mechanism of myocardial depression including a hypercoagulative state, microembolisms, contractile-metabolic mismatch, and ion channel defects 3 7 ' 5 6 ' 5 8 . One of the most important components of the inflammatory response is the function of leukocytes. Our laboratory has shown the direct effects of inflammatory cells (macrophages and neutrophils) on the contractility of the cultured cardiomyoctes 5 9 ' 6 0 . Botha et al. have shown that leukopenia is a sign of progression of the inflammatory response and shock, to multi-organ dysfunction in septic and traumatic patients, suggesting neutrophil involvement 6 1 . Chiba's group studied the effect of leukocyte depletion on patient survival after cardiopulmnary bypass. They found that depletion of leukocytes and platelets protects cardiac function after cardiopulmonary bypass . This may suggest that leukocytes, especially neutrophils, play important roles in cardiac dysfunction during systemic inflammatory responses syndrome. Based on these observations, different anti P M N receptors blocking antibodies have been used. Study of the lung has shown that P M N number is significantly higher in the right atrium compared 19 to the left atrium of septic patients after cardiopulmonary bypass. Although leukocytes can infiltrate into the tissue in various types of inflammation including ischemia-reperfusion injury, there is no strong evidence for the presence of leukocytes in the myocardium of non-survivors of sepsis. However it has been documented that during sepsis, neutrophils and inflammatory cells release significant amount of toxic substances including oxygen free radicals and elastase " . Cytokines have always been suspected in many types of inflammatory responses. T N F a 6 6 ', IL- ip , and IL-6 are the major cytokines affecting myocardial contractility 6 7 . Although there is some strong evidence about the cardiac depressive effects of the above cytokines, some investigators did not find independent effects of the cytokines in vitro 5 9 ' 6 0 . The idea that T N F a and other cytokines are not specifically important in septic patients was strengthened by the failure of a clinical trial of anti-cytokines therapies 6 8 . Instead it is believed that these cytokines are very important in the presence of inflammatory cells which then exaggerate inflammatory responses69. Nitrite oxide (NO) is another factor that has been postulated to contribute to myocardial depression during sepsis 7 0 . Arginine is converted to NO under the effect of various types of NOS. NO increases cGMP concentration leading to modulation of contraction and finally more relaxation 7 1. It is worth noting that inhibition of NOS in a selective or non-selective way did not improve cardiac dysfunction 7 2 " 7 5 , however other investigations 1(\ 77 demonstrated the protective effects of NO inhibition with various methods ' . 20 Metabolic acidosis almost always accompanies sepsis, leading to increased potassium channel activation at the vascular level and vasodilation 7 8 . In cardiomyocytes acidosis interferes with intracellular C a 2 + concentration and the interaction between actin and myosin 1 9 . Reduction in intracellular pH first increases ionized C a 2 + concentration in the 9+ sarcolemma, however the total stored Ca concentration decreases over time. This may also affect cardiomyocyte metabolism and finally contraction. Hormonal alteration is one of the characteristic changes in sepsis. Growth hormone (GH) fluctuates in the first week of the sepsis state . It has been shown that the serum insulin Q 1 growth factor-1 (IGF-1) level is significantly lower in septic patients . The plasma insulin level is initially low however this level tends to increase rapidly after 24 hours o n O ' J ^ SI A ' . There is strong evidence of benefit of IGF-1 injection in various types of injury 8 6 . IGF-1 is involved in different pathways including anti-apoptotic signaling through the on protection of mitochondria . Erythropoietin (EPO) secretion is also affected by sepsis. It has been shown that the level of EPO is significantly lower in critically i l l patients and/or the response of target cells to EPO is impaired in septic patients . Recent studies on the effect of EPO in various models showed novel protective effects of EPO against cellular injury including anti-apoptotic and anti-oxidant effects. Interestingly recent clinical data have shown negative results to blood transfusion in critically i l l patients and conversely beneficial results after EPO injection, suggesting a more protective effect of EPO in those patients . 21 Although coronary blood flow is higher in septic patients, there is some evidence that may confirm the existence of focal ischemia in the cardiac tissue of these patients. However this theory needs to be investigated using new techniques. Furthermore ischemia, especially in cardiac tissue, is accompanied by a number of symptoms and signs including electrocardiogram (ECG) changes, alteration in ischemic area motion, arrhythmia and permanent sequelaes. Conversely in septic patients there is no strong evidence of such changes. The most common alteration in E C G of septic patients is supraventricular tachycardia which itself could be contradict the above theory. Autopsies of septic patients have shown evidence of necrosis only in those patients who had myocardial infarction during sepsis syndrome, probably because of reflex tachycardia 9 0 . Although most septic patients have myocardial dysfunction, there is no evidence of myocardial necrosis in the majority of these cases 9 0 . Additionally myocardial depression during sepsis is a reversible event. The myocardium regains its normal contractility by the end of 14 days in survivors However we cannot ignore the possibility of a type of ischemia that is not severe enough to induce myocardial sequelaes. This could be explained by stunned myocardium which can be seen in brief myocardial ischemia and more importantly it is also a reversible event which occurs in the prone regions of the injured myocardium after ischemia. Myocardial stunning occurs in the surrounding viable tissue around necrotic parts after myocardial infarction, cardiopulmonary bypass, and exercise induced ischemia. It is demonstrated by a persistent defect in regional myocardial motion even in the presence of normal blood flow. Within a few days to several weeks the stunned myocardium assumes 22 its normal function which was demonstrated by positron emission tomography scanning in the above patients 9 1 . Myocardial ischemia contributes to the pathophysiology of many different types of conditions in cardiovascular diseases including angina, infarction, cardiovascular surgery, angioplasty, transplantation, shock and sepsis. It was described as the imbalance between myocardial oxygen supply and demand. The duration of ischemia is also an important factor in the myocardial survival rate 9 2 . While prolonged ischemia is seriously harmful for the affected myocardium 9 3 brief periods of ischemia could result in different pathogenesis which also affects cardiac function in a reversible manner 9 1 . These reversible phenomena can be generated by the reperfusion of acutely ischemic myocardium and chronic myocardial ischemia. The two types of reversible myocardial injury are myocardial stunning and hibernation. Myocardial stunning is characterized by persistent and reversible systolic and diastolic depression of myocardial wall motion which is affected by previous ischemia, reperfusion or both injuries. The section of ischemic hearts which is affected by the above mechanisms is viable and it is capable of restoring its function gradually (days to weeks). The main mechanisms of myocardial stunning are not clear yet, however ROS formation, alteration in calcium homeostasis and low responds of contractile apparatus to calcium have been considered as the major mechanisms 9 1 ' 9 4 . Hibernating myocardium is similar to stunned myocardium with the exception that recovery occurs after removal of the causes of ischemia. The blood flow in hibernating myocardium is adequate to maintain its basic function and viability of at risk cardiomyocytes, however it is not sufficient to maintain optimal ventricular contractility 23 . Myocardial dysfunction in the hibernated or stunned myocardium responds to p4-stimulators. The function of stunned myocardium improves over time, whereas hibernating myocardium needs revascularization to recover function. This reversible myocardial dysfunction in ischemia-reperfusion injury is very similar to that of sepsis. In fact we can see these phenomena even during prolonged exercise and in the absence of coronary occlusion 9 5 . There are two major hypothesis to explain this type of myocardial dysfunction: first the role of oxygen radicals and secondly Ca involvement . Non-reversible myocardial ischemia results in cellular changes from metabolism to contraction. It changes oxidation phosphorylation and results in less ATP and creatinine phosphate production 9 3 . ATP is the main source of energy for activation of ATP dependent ion channels and actin-myosin interaction. The lack of channel activity during prolonged myocardial ischemia results in accumulation of intracellular Ca ions and an increase in cellular osmolar pressure which results in higher intracellular water, higher membranous tension, and finally cardimyocyte sacomeric fragility . Reactive oxygen species (ROS) are the products of the prolonged myocardial ischemia. They are also the main causes of progression of injury started by ischemia. During ischemia because of the failure in oxidation phosphorylation, ADP cannot be converted to ATP 9 8 . In fact it progresses to more dissociation of Phosphorus from ADP and creation of A M P which results in higher catabolism of adenosine nucleotides and accumulation of hypoxanthine. On the other hand because of ischemia, hypoxanthine dehydrogenase, an important enzyme which is responsible for converting hypoxanthine 24 to xanthine and finally uric acid, is changed to hypoxanthine oxidase leading to excessive production of ROS " . Other potential sources of ROS are semi-ubiquinone of mitochondria, cytochrome C450, cycloxygenase and N A D P H oxidases 1 0 ° . NO can also be a source of ROS formation when eNOS is not working properly under the influence of hypoxia. Oxygen free radicals interact with NO to create peroxynitrate, one of the most harmful ROS in cardiomyocyte physiology 9 3 . It has been shown that ROS can initiate cell signaling cascades via changes in regulatory proteins. Accumulation of ROS in Escherichia Coli changes the thiol group (H-S group) of regulatory proteins into the disulfide (S-S) leading to signal transduction of the membrane and activation of various types of transcription factors such as superoxide dismutase, catalase and glutathione reductase 1 0 1 . The role of ROS has been investigated extensively during the last 5 years in various types of cells. The results suggest that ROS can initiates some signals including activation of receptor tyrosine kinases, insulin receptors, increased intracellular Ca concentration, JNK and P38 M A P K pathways stimulation, and activation of N F K B and P K C - a 1 0 1 . Defects in ATP production also affect sodium-potassium channels, resulting in higher concentrations of N a + and C a 2 + ions. Lack of energy changes the oxidation phosphorylation pathway to glycolysis and production of acid. The depressed 9-f-intracellular pH leads to an increase in ionized Ca concentration and eventually a reduction in C a 2 + storage 1 0 2 . 25 Collectively, the defect in ATP production during myocardial ischemia increases cytoskeletal fragility, accelerates intracellular edema, changes membrane electro-potential and intracellular Ca concentration, and produces ROS which then promote further injury to cardiomyocytes during reperfusion . However all of the above alterations may be a signaling mechanism to save the myocardium from further injury by reducing cardiac contractility and demands. Indeed i f ischemia is severe enough, the cardiomyocyte goes into a rapid process of death such as apoptosis and or necrosis. Reperfusion, although necessary to restore blood flow into the myocardium and to save myocardium from severe ischemia, is another type of damage to the cardiac tissue. Many mechanisms are involved in reperfusion injury. Accelerated ROS formation that was started during ischemia leads to persistent damage to the coronary endothelial layer and cardiomyocyte membrane, action potential prolongation, early membrane depolarization and contractility failure. These events finally result in activation of N F K B transcription factor and translocation of this factor into the nucleus, followed by expression of different inflammatory proteins and cytokines including IL-6, IL-8, TNFa, tissue factors, iNOS 1 0 4 and adhesion molecules such as selectins, V C A M - 1 and ICAM-1 A rapid rise in intracellular Ca 2 + , higher numbers of open L-type channels or dihydropyridine receptors (DHPR), free release of C a 2 + from ryanodine receptors (RyRs), membrane leakage to C a 2 + and failure to clear C a 2 + from intracellular space are all 103 suggested mechanisms for further cardiac injury during the reperfusion period . It is important to note that during reperfusion white blood cells, especially polymorphonuclear 26 cells (PMN), support further injury by releasing free radicals, proteolytic enzymes, superoxide anions and hypochlorus acid into the injured area 6 1 ' 1 0 5 . Complement cascades also exaggerate reperfusion injury by activation of classic or alternative pathways to create a membrane attack complex. This process is continued by opsonization of target cells with iC3b and the chemo-attractive effect of C3a and C5a. Interstitial edema during reperfusion rapidly progresses because of the leaky endothelial layer causing lymphatic drainage failure and vicious cycle of edema 9 2>1 0 6. Taken together, during reperfusion fast recovery of extracellular osmolality is achieved while intracellular osomolarity is still higher than normal because of deficiencies in ion channel function suggesting increased cellular swelling and cellular vulnerability. Also cytoskeletal filaments are fragile because of the existence of intracellular ROS formation and low pH, while extracellular pH returns to normal faster indicating fast re-energization in the presence of high intracellular C a 2 + levels and membrane fragility that is also a cause of hypercontracture and disruption of sarcolema 9 2 , 1 0 6 . There are different theories and results explaining myocardial dysfunction during sepsis and ischemia reperfusion injury. There is one common belief that, myocardial dysfunction during these events is an inflammatory response which has intracellular consequences. 27 1.4 Molecular mechanism of cardiac inflammation 1.4.1 ICAM-1 interaction initiates intracellular signaling Regardless of the causes of inflammation in cardiac tissue, including endothelial cells or cardiomyocytes, the final pathway is the activation of intracellular signaling. These intracellular signaling pathways can be initiated by ROS formation (ischemia), LPS or toll like receptor activation on inflammatory cells, and triggered cytokines production (sepsis). They induce production of transcription factors such as N F K B and or HIF- la . These transcription factors are translocated into the nucleus and activate specific genes including adhesion molecule such as selectin, VCAM-1 and ICAM-1 on the endothelial cells. Additionally they can directly activate translation of different inflammatory proteins (Figure 1.1). The cardiomyocyte has various types of adhesion receptors including ICAM-1. ICAM-1 (CD-54), a 76-114 kDa glycoprotein, is expressed in both normal and activated cardiomyocytes 5 9 - 6 0 ' 1 0 7 . it is a member of the immunoglobulin gene superfamily, which includes other adhesion molecules such as ICAM-2, ICAM-3, V C A M - 1 , PECAM-1 , neural crest adhesion molecule-1 (NCAM-1) and myelin adhesion glycoprotein ICAM-1 and ICAM-2 are constitutively expressed on endothelial cells. Cytokines and LPS treatments increase endothelial ICAM-1 expression while the level of ICAM-2 is not changed after endothelial cells activation 1 0 9 , 1 1 0 . 28 ICAM-1 contains five extracellular Immunoglobulin domains, a transmembrane domain, and a short cytoplasmic tail at its C terminal end. A soluble form of ICAM-1 (s ICAM-1) has been found in normal human plasma at levels of 100-200 ng/mL U 1 " 1 1 4 . It is speculated that the levels of sICAM-1 is under the control of matrix metalloproteinases (MMP) and higher levels of sICAM-1 modulate inflammatory responses 1 1 5 , 1 1 6 . The sICAM-1 level increases markedly in many inflammatory reactions including atherosclerosis, ischemia reperfusion injury, transplant rejection and chronic inflammatory diseases 1 0 9> 1 1 7 ' 1 1 8. Lower levels of ICAM-1 expression on tumor cells is associated with increased metastasis and invasiveness of cancerous cells u 9 . ICAM-1 is a ligand for p2-integrins on leukocytes. This interaction between ICAM-1 and P2-integrins (LFA-1) was prevented using an anti-ICAM-1 monclonal antibody as reported by Dustin et al. 1 2 ° . ICAM-1 is widely distributed in different cells including endothelial cells 1 2 1 , 199 fibroblasts, dendritic cells, keratinocytes , mesenchymal cells, monocytes, i ^ i 11*7 so AH lymphocytes, epithelial cells , smooth muscle cells and cardiomyocytes ' . Expression of ICAM-1 on non-hematopoietic cells is low, but surface expression is markedly up-regulated by various types of inflammatory mediators including LPS, IL-1P, TNFoc, and IFNy 60>110>124>125. Cytokines can increase ICAM-1 production after 2-4 hours and maximal production occurs 18 to 24 hours after stimulation. Upregulation of I C A M -1 by cytokines or LPS also facilitates neutrophil binding and migration through a cultured endothelial cell monolayer in vitro, a process involving the interaction of ICAM-1 with LFA-1 (CD 11 a/CD 18) and Mac-1 (CDl ib /CD 18). Immunofluorescence studies have shown a punctuated distribution of ICAM-1 on endothelial cells and cardiomyocytes 29 ' . Localized distribution of ICAM-1 may facilitate its interaction with LFA-1 and Mac-1 1 2 6 . ICAM-1 on the cell surface presents as a homodimer, which may facilitate high-affinity binding to LFA-1 1 2 6 " 1 2 8 . ICAM-1 can interact with various ligands including fibrinogen 1 2 9 , Rhinoviruses 1 2 5 , Coxsackie viruses 1 3 0 , 1 3 1 5 and Plasmodium falciparum The relation between adhesion molecules such as Mac-1 and LFA-1 has been shown by Smith and Lo in different studies. They have shown that the adhesion of P M N to ICAM-1 on stimulated endothelial cells is largely Mac-1 dependent but in the un-stimulated cell it is exclusively dependent on LFA-1 ' . The above interaction can be inhibited using an anti-ICAM-1 antibody, however only some kinds of ICAM-1 antibodies inhibited the interaction between ICAM-1 and integrin receptors on leukocyte membranes. The extracellular domains of ICAM-1 are important for interaction with their ligands. This part is highly glycosylated indicating high solubility of ICAM-1. A flexible hinge between domains 2 and 3 creates a good position for domains 1 and 2 to interact with their ligands 1 3 3 . The binding site for LFA-1 on the extracellular domains of ICAM-1 is on the edge of domain 1 and at the center of C and D strands. Maximal production of ICAM-1 is reached within 18 to 24 hours and its expression is maintained about 48 hours after first activation. However the level of ICAM-1 expression can be high as long as the inflammatory process is active or around ten days after initiation of inflammation on coronary endothelial cells as reported by Bevilacqua and his 30 colleagues n 0 . LFA-1 (CD1 la/18) binds to the first and second domains and Mac-1 (CD1 lb/18) binds to the third domain of ICAM-1. Fibrinogen is also an important ligand for ICAM-1 1 3 4 , 1 3 5 . ft is a 340 kDa plasma protein involved in inflammation and the coagulation cascade. It has been shown that fibrinogen can facilitate the interaction of vascular endothelium and leukocytes 1 3 6 . The fibrinogen molecule (composed of three a, (3, and y chains) has three domains, a central E domain and two lateral D domains. Fibrinogen can infiltrate into the tissue during inflammation, ischemia, trauma and cancer. The association of fibrinogen with a higher risk of 1 ^7 myocardial infarction and atherosclerosis has been reported in several studies . The presence of thrombin affects peptides A and B of the central part of the a and P chains resulting in dissociation of the above two peptides from the related chains, polymerization of fibrin molecules together and formation of fibrin networks . Plasmin cleaves fibrin networks and separates the two D domains from the central E domain resulting in formation of fragment D and E in the serum. Fragment D contains a, [3 and y chains 1 3 9 . It has been shown that amino acid sequence of 117-133 of the y chain is the location for adhesion to the first domain of ICAM-1. Hence both fibrinogen and fragment D can attach to ICAM-1 1 3 9 ' 1 4 0 . Fibrinogen can increase endothelial leakiness and enhance angiogenesis on endothelial cells 1 4 M 4 3 . it is able to attach to ICAM-1 through its 117-133 amino acid sequence located on its y chain 1 3 4 , 1 3 5 . There is controversy about the location of fibrinogen adhesion on ICAM-1. It seems that aspartic acid and proline residues and amino acid 8-31 21 of domain-1 of ICAM-1 are very important for the above interaction. However those amino acids are not on the 8-21 amino acid sequence of ICAM-1, which were previously shown to be important for the above interaction 1 4 4 ' . Upregulation of ICAM-1 in cardiovascular diseases is also accompanied by high expression of fibrinogen and vice versa 1 4 5 , 1 4 6 . Fibrinogen and ICAM-1 interaction promotes platelet and leukocyte adhesion on endothelial cells 1 4 7 . It has been shown that fibrinogen deposition on endothelial cells was significantly reduced using ICAM-1 knock out mice in a model of intestinal ischemia reperfusion injury 1 4 8 . Hicks et al. have shown that fibrinogen deposition on endothelial cells induced vasodilation, however when they increased fibrinogen concentration this effect was converted to vasoconstriction 1 4 9 ' . Fibrinogen deposition upregulated ICAM-1 expression through activation of NFK(3 and IL-8 production 1 4 6 ' 1 5 0 . The short intracellular domain of ICAM-1 is an important part for initiation of signals. Conformational change in tertiary structure of the ICAM-1 molecule induces phosphorylation of different amino acids including tyrosine residues leading to activation or inactivation of other downstream signals. It is evident that the cortical cytoskeleton is an important regulator for the above function. Carpen et al. have shown that a specific region of ICAM-1, 478-505-peptide, interacted with alpha-actinin in Chinese ovary hamortoma cells 1 S 1 . This region is close to the cell membrane spanning region and contains several positively charged residues, and appears to mediate a charged interaction with alpha-actinin, which is not highly dependent on the order of the residues. They also showed that ICAM-1 activation is through homo-oligomerization. This needs a substrate 32 from PI pathway (PIP2) to promote conformational change in tertiary structures of intracellular domain of ICAM-1 1 5 1 . In another study, Vogetseder and his colleagues have shown that the intracellular domain of ICAM-1 is associated with actin filaments in endothelial cells 1 5 2 . Cross-linking of ICAM-1 on synovial cells induced IL- lp transcription and AP-1 activation. ICAM-1 functions not only as a glue for integrin binding, but also as a transducer for AP-1 activation signals that are important for IL-1 (3 gene transcription 1 5 3 . ICAM-1 cross-linking initiated production of oxidative metabolites and oxygen free radicals on monocytes 1 5 4 . It has been shown that cross-linking of ICAM-1 on H U V E C cells can increase V C A M - 1 expression and exacerbate the inflammatory reaction 1 2 1 . A study on brain microvascular endothelial cells reported by Durieu-Trautmann et al. has shown the tyrosine phosphorylation of the cytoskeleton-associated protein, cortactin, after ICAM-1 activation suggesting interaction of ICAM-1 with cytoskeletal proteins 1 5 5 . Activation of ICAM-1 receptors on Burkitt cell lymphoma increased C a 2 + concentration 1 5 6 . Etienne et al. have also found that ICAM-1 cross-linking on brain endothelial cells participated in cellular shape changes and gene 157 regulation Interaction between fibrinogen and ICAM-1 induces tyrosine phosphorylation at locations 474 and 485 of cortactin which is also associated with the intracellular domain of ICAM-1 1 5 5 . The above interaction also increased phosphorylation of cortactin on endothelial cells 1 4 5 , 1 5 8 . Holland et al. have shown adhesion of fibrinogen on ICAM-1 phosphorylated Erkl/2 which is through the interaction of sequences 117-133 of fibrinogen and 8-21 of ICAM-1 1 2 9 , 1 5 9 . 33 ICAM-1 is not only responsible for interaction of leukocytes and proteins with the target cells, but is also involved in antigen recognition through major histocompatibility complex (MHC-II) interaction. Exogenous antigens are engulfed and processed in antigen presenting cells followed by presentation of the above antigens to the CD4 T lymphocytes. Interaction between T cell receptor complexes such as CD3, CD4/CD8 with M H C - restricted antigen presenting cells initiates intracellular signaling. ICAM-1 acts as an accessory molecule in strengthening the interaction between CD4 and MHC-II to initiate an immune response once the T cells has interacted with exogenous antigens. Hence ICAM-1 plays a major role after exogenous antigens fragments have interacted with T cell receptors. In the next chapters I will discuss the novel finding of cardiomyocyte ICAM-1 activation in regard to cardiomyocyte contractility and finally cardiac contractility. The possible pathways which may be involved in ICAM-1 activation and intracellular signaling will be studied. The possible ligands for cardiomyocyte ICAM-1 were considered and studied in the in vitro and in vivo models in chapters 2, 3 and 4. Finally the similar ex vivo model for high expression of ICAM-1 on cardiac tissue was used to find a new therapeutic intervention to inhibit intracellular signaling. 34 1.5 General Hypothesis In this thesis my general hypothesis is that the expression and activation of cardiomyocyte ICAM-1 is responsible for myocardial depression during sepsis. I also hypothesize that the expression of ICAM-1 can be modulated by intervention in intracellular signaling cascade of ICAM-1 for example PI3K pathways (Figure 1.2). In order to test this hypothesis, in an in vitro model I first show that cardiomyocyte ICAM-1 expression increases after cytokine treatment and activation of ICAM-1 by adherent P M N and cross-linking of ICAM-1 decreases cardiomyocyte contractility. Using an in vivo model I then show that the absence of ICAM-1 on cardiac tissue is protective against the effect of LPS on cardiac contractility. I also investigate the presence of inflammatory cell in the cardiac tissue after LPS injection. Next I show that the interaction of ICAM-1 with fibrinogen results in a decrease in cardiomyocyte contractility and fibrinogen can infiltrate into the cardiac tissue after LPS injection thereby fibrinogen can trigger cardiomyocyte ICAM-1 activation. Using an ex vivo model of myocardial ischemia reperfusion injury, I show that ischemia-reperfusion injury is associated with high expression of ICAM-1.1 also show that IGF-1 therapeutic intervention can prevent ICAM-1 expression during reperfusion period resulting in better cardiac function and less cardiac injury. This latter effect of IGF-1 is partly because of less ICAM-1 expression on cardiac tissue. 35 1.6 Conclusions The final conclusions of this thesis are; 1- Cardiomyocyte ICAM-1 activation decreases cardiomyocyte contractility. 2- Cross-linking of IC AM-1 or adhesion of P M N activates IC A M - 1 . 3- ICAM-1 activation is associated with cortical cytoskeletal assembly and disruption of cortical actin filaments inhibits this effect of ICAM-1 on the cardiomyocyte contractility. 4- ICAM-1 activation alters intracellular C a 2 + propagation. 5- The lack of ICAM-1 on cardiac tissue is protective against the effect of LPS. 6- Fibrinogen adhesion to ICAM-1 can activate this receptor and decrease cardiomyocyte contractility. 7- Ischemia reperfusion injury is associated with an increase in cardiac ICAM-1 expression. IGF-1 prevents the membranous presentation of ICAM-1 thereby protects cardiac tissue from further injury. 36 ( ^ C a r d i a c inflammation^) (Q2^ expressbn^) ' "IT CjT} ( P M N ) C^ICAM-1 ac t ivat ion^ Endothelial cells Loosening gap junction f Interstitial edema C^T^egranulatk^n^^) ( J L J ) (l^6) ® \ Fibrinogen infiltration | Cardiac contractility Figure 1.1 Schematic pathways involved in cardiac inflammation. 37 Figure 1.2 Schematic pathways involved in ICAM-1 presentation and activation and the possible effect of IGF-1 on ICAM-1 expression. 38 Chapter-2 Novel regulatory mechanism of cardiomyocyte contractility involving ICAM-1 and the cytoskeleton *2.1 Abstract ICAM-1 mediates interaction of cardiomyocytes with the extracellular matrix and leukocytes, and may play a role in altering contractility. To investigate this possibility rat ventricular cardiomyocytes were activated using TNFct, IL- ip or LPS, washed, co-cultured with quiescent rat polymorphonuclear leukocytes (PMN) for 4 hours, and electrically stimulated to determine fractional shortening. P M N co-cultured with activated cardiomyocytes reduced control fractional shortening of 20.5±0.7% by -2.8±0.3% per adherent P M N (p<0.001). Fixing P M N with paraformaldehyde or gluteraldehyde did not prevent PMN-mediated decreases in cardiomyocyte fractional shortening. However, P M N adherence and decreased fractional shortening were prevented by anti-ICAM-1 and anti-CD 18 antibodies. Reduced fractional shortening was reproduced in the absence of P M N by ICAM-1 binding using cross-linking antibodies (reduced by 36±3% from control, p<0.01). Immunofluorescent staining demonstrated increased cortical cytoskeleton-associated F A K expression following ICAM-1 cross-linking, suggesting involvement of the actin cytoskeleton. Indeed, disruption of F-actin filament assembly using cytochalasin D or latrunculin A did not prevent P M N adherence but prevented decreased fractional shortening. Inhibiting the cytoskeleton-associated Rho kinase pathway using HA-1077 prevented ICAM-1 mediated decreases in cardiomyocyte contractility, further suggesting a central role of the actin cytoskeleton. 1 This chapter has been published in American Journal of Physiology (Heart and Circulatory) 2004 Sep;287(3):H1013-22. 39 Importantly, ICAM-1 cross-linking did not alter the total intracellular Ca transient during cardiomyocyte contraction but greatly increased heterogeneity of intracellular C a 2 + release. Thus, we have identified a novel regulatory mechanism of cardiomyocyte contractility involving the actin cytoskeleton as a central regulator of the normally highly coordinated pattern of sarcoplamic C a 2 + release. Cardiomyocyte ICAM-1 binding, by P M N or other ligands, induces decreased cardiomyocyte contractilty via this pathway. Key words: ICAM-1, Intracellular signaling, F A K , Rho, Calcium. 40 2.2 Introduction Intramyocardial inflammation, for example following ischemia-reperfusion, in inflammatory cardiomyopathy, during orthotopic heart transplant rejection, and during sepsis-induced myocardial dysfunction, results in increased expression of ICAM-1 on cardiomyocytes 9 2 , 1 6 0 - 1 6 2 a n d accumulation of intramyocardial leukocytes 9 2 , 1 6 0 " 1 6 2 . Adhesion of polymorphonuclear leukocytes (PMN) or macrophages to cardiomyocytes is mediated in part by binding to ICAM-1 on cardiomyocytes. If PMNs are highly activated they kil l cardiomyocytes within minutes by release of reactive oxygen intermediates ioo,i63,i64 However, ICAM-1 mediated adhesion of relatively quiescent macrophage/monocytes to cardiomyocytes causes a reduction in contractiltiy without cardiomyocyte killing 5 9 and a recent report implicates ICAM-1 in mediating reduced cardiomyocyte contractility independent of P M N accumulation 1 6 0 . ICAM-1 is fundamentally important in linking extracellular mechanical signals to regulation of intracellular processes ' . ICAM-1 is closely associated with the actin cytoskeleton 1 2 3 , 1 6 5 and ICAM-1 binding increases linkage between the cell surface ICAM-1 receptor and the cytoskeleton 1 5 2 , 1 6 6 . Whether the cytoskeleton plays a role in modulating cardiomyocyte contractility is unknown and potential signaling pathways and mechanisms involved have not been identified. Accordingly, we postulated that ICAM-1 and the cardiomyocyte cytoskeleton may be involved in a novel pathway leading to cardiomyocyte contractile dysfunction. While many mediators (e.g., cytokines, leukocytes, reactive oxygen intermediates, NO) 1 6 7 - 1 6 9 and pathways (e.g., apoptosis, adrenergic signaling) 1 6> 1 8 , 4 5> 4 6 , 6 9 , 1 7 0 ^ involved in myocardial dysfunction during 41 intramyocardial inflammation, here we focus on the potential role of ICAM -1 binding and its relationship to the cardiomyocyte cytoskeleton. 42 2.3 Materials and Methods This study was approved by the University of British Columbia Animal Care Committee and adheres to Canadian and National Institutes of Health guidelines for animal experimentation. 2.3.1 Isolation of rat ventricular myocytes: Male Sprague-Dawley rats (250-300 g) were anesthetized using 3% halothane and the heart was excised and mounted on a modified Langendorff apparatus and perfused with oxygenated (95% O2, 5% CO2) HEPES-Joklik modified M E M buffer (Gibco BRL, Grand Island, N Y ) at 37°C for 2 minutes. Then the perfusate was changed to 30 mL recirculating calcium free M E M containing 236 U/mL collagenase (Worthington Biochemical, Freehold, NJ). At 15 and 20 minutes, the calcium concentration was increased, stepwise to 0.025 and 0.075 mmol/L. After 30-40 minutes the ventricles were removed from the perfusion system and gently teased apart and agitated for 2-3 minutes at 37°C. The tissue and dispersed cells in solution were then filtered through a 200 \im nylon mesh. The cells were then washed three times at 37°C in M E M containing increasing C a 2 + concentrations (200 | i M , 500 [iM and 1 | iM) , allowing the cells to settle by gravity for 10 minutes between each wash. Cells were resuspended in 37°C HEPES-modified M l 9 9 buffer (Gibco BRL) with 1% BSA. The cells were diluted to a final concentration of 50,000 cells/mL, 100 \iL loaded into each well of 96 well plates, and incubated at 37°C in 95% 0 2 , 5% C 0 2 . At 90 minutes, the medium was changed to fresh M l 9 9 with B S A and the cardiomyocytes were incubated for 24 hours to allow them to become relatively quiescent. After 24 hours cells 43 were considered viable i f they demonstrated a characteristic rod shape without cytoplasmic blebbing. This morphometric assessment of viability was confirmed in a subset of experiments with trypan blue exclusion. We have found that the fraction of viable cardiomyocytes is always greater than 85%. 2.3.2 Cardiomyocyte ICAM-1 protein expression: We and others have shown that activation of cardiomyocytes by inflammatory cytokines increases cardiomyocyte expression of ICAM-1 5 9> 1 6 0 ' 1 7 1. To confirm this we measured ICAM-1 protein expression on cardiomyocytes incubated with TNFct (20 ng/mL) for 4 hours and then fixed using paraformaldehyde 3% for 30 minutes. Cardiomyocytes were incubated with mouse anti-rat ICAM-1 antibody (1A29, BD Pharmingen) 10 |j,g/mL for 3 hours, washed, and incubated with a fluorescent secondary anti-mouse antibody (Alexa-Fluor 488,1/500, Molecular Probe, Eugene, OR) for 3 hours and imaged using confocal microscopy (LEICA DIMRE2, Exton, PA). The mean of intensity / pixel of each cardiomyocyte was measured using Image Pro-Plus {Cybernetics, 1998}. 2.3.3 Cardiomyocyte ICAM-1 mRNA expression: Cardiomyocytes (5 x 105) were cultured in 60 mL laminin coated dishes and incubated with or without TNFa. Cells were then lysed and frozen at -70°C. R N A was extracted using standard technique of Qiagen R N A kit (Qiagen Inc. Mississauga, ON, Canada): 1 uL eluted R N A was added to 999 uL of 0.1 M NaOH solution in diethyl pyrocarbonate-water; 3 (ig of R N A were taken as constant template for RT cycle to make cDNA; 2 uL of cDNA were made from total R N A and used as template to amplify rat ICAM-1 and rat G A P D H using the following 44 primer sequence: 5' A G G T C A GGG GTG T C G A G C (forward ICAM-1 primer) and 5' C A A G G A G A T C A C ATT C A C G G (reverse ICAM-1 primer). The PCR conditions for Perkin Elmer 9700 model were 95°C for 15 minutes (Qiagen Taq Polymerase enzyme Activation), denaturing at 94°C for 30 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 1 min. The whole cycle was repeated 40 times. PCR products were separated on 3% agarose gel stained with ethedium bromide and visualized using Eagle eye U C scanner. The ratio of ICAM-1 signal was normalized to G A P D H signal for each time point sample. The final results of T N F a treated cardiomyocytes were compared to controls. 2.3.4 Measurement of cardiomyocyte fractional shortening: Fifteen minutes before measuring fractional shortening 2 (iL of 0.5% trypsin was added to each well. Preliminary experiments demonstrated that this concentration of trypsin did not alter cell viability and cleaved greater than 95% of the adherent cardiomyocytes from their attachments to the bottom of the well. Specially designed platinum electrodes were then lowered in each well in the 96-well plate, and the cardiomyocytes were electrically stimulated (Grass S48 stimulator, West Warwick, RI; 45V, 2.2 ms duration, 25-Q resistance) while being recorded by video-microscopy (Sony SLV-760HF) at 400X magnification. This electrical stimulus was chosen from preliminary threshold experiments, as two times the minimum electrical stimulus required to maximally contracting the cardiomyocytes. Still frames of systolic and diastolic cardiomyocytes were captured using Scion Image PC (Scion, Frederick, MD) and cardiomyocyte 45 fractional shortening was calculated as the difference between diastolic and systolic length divided by diastolic length. 2.3.5 Isolation of peripheral blood PMN: Male Sprague-Dawley rats (250-300 g) were anesthetized as above and, via cardiac puncture, 8 mL of blood was drawn into a syringe containing 2 mL anticoagulant citrate dextrose solution (Baxter, Deerfield, IL). Leukocyte rich plasma was obtained from the blood using dextran sedimentation. Red blood cells were lysed using sterile water and P M N were purified using Histopaque 1077 (Sigma-Aldrich Canada, Oakville, ON) to remove the remaining erythrocytes and mononuclear cells. Purified P M N were resuspended in M l 9 9 and used immediately. In further experiments P M N were fixed by incubation in paraformaldehyde 3% for 15 minutes or, alternatively, by incubation in glutaraldehyde 0.025 % for 30 minutes. 2.3.6 CDl lb expression on PMN: To confirm that this very low concentration of gluteraldehyde preserves the extracellular domains of P M N C D l l b 1 7 2 freshly isolated P M N and gluteraldehyde-fixed P M N were incubated with mouse anti-rat C D l l b R-PE conjugated antibody (Biosource, Camarillo, CA) or non-specific mouse antibody (IgG negative control, Biosource) 30 uL/mL for 30 minutes. Cells were washed with PBS, fixed with paraformaldehyde 3%, and read using a flow cytometer (EPICS X L - M C L , Beckman Coulter, Miami, FL). 2.3.7 Co-culture of PMN and cardiomyocytes: After 24 hours of cardiomyocyte incubation 50,000 freshly isolated P M N were added to each well of cardiomyocytes 46 (ratio of 10 P M N per cardiomyocyte) in the 96 well laminin-coated plates for co-culture experiments. During off-line analysis of captured video sequences, we viewed all cardiomyocytes throughout a contraction. To be counted as adherent, P M N had to move with the contracting cardiomyocyte and maintain a contact relative location on the cardiomyocyte membrane during contraction. 2.3.8 ICAM-1 cross-linking: Following previously reported methods of cross-linking ICAM-1 and initiating intracellular signaling 1 5 3 ' 1 7 3 ' 1 7 4 5 w e incubated cardiomyocytes with mouse anti-rat ICAM-1 1 ug/mL or control mouse non-specific IgG 1 ug/mL (Dako, Carpinteria, CA) for one hour. Cells were washed and then incubated with goat anti-mouse IgG 10 ug/mL (Transduction Laboratories, San Jose, CA) for 4 hours to cross-link the bound anti-ICAM-1. 2.3.9 Immunoflurescent imaging of Focal Adhesion Kinase (FAK): Cardiomyocytes were cultured on 8 well laminin coated slides (LAB-TEK, Naperville, IL). After 24 hours, ICAM-1 on cardiomyocytes was cross-linked as above. Cells were fixed and permeabilized with paraformaldehyde 3% for 20 minutes and Triton X-100 0.5 % for 5 minutes in Microtubule Stabilizing Buffer (MES 0.1 M , E G T A 2mM, M g C l 2 2mM, and 4% polyethylene glycol 8000, Sigma-Aldrich). After blocking with PBS + 0.5% B S A for 60 minutes, rabbit anti-FAK 40 ug/ml (UBI, Lake Placid, NY) was added and incubated overnight at 4°C. After washing, Alexa fluor 488 labeled secondary goat anti-rabbit 10 jxg/mL (Molecular Probes, Eugene, OR) was added for 45 minutes at room temperature. 47 Images were captured using a Noran Oz laser scanning confocal microscope with a high power objective lens (Nikon, Plan Fluor, 100X oil, N A : 1.30) and slit width of 10 um. For visualizing the Alexa 488-labelled specimens, the sample was illuminated using 488 nm light from an Argon-Krypton laser. A high-gain photomultiplier tube collected the emission after it had passed through a 525/52 nm band pass filter. A l l parameters (laser intensity, gain, etc.) were left unchanged during the experiment and were set so that the level of photo-bleaching was negligible. To obtain high resolution images within the cortical cytoplasm, multiple images were acquired along the Z-axis at 0.5 |im steps. Z-stack images were deconvolved using X C O S M 1 7 5 and a reconstructed image 2 | im from the top surface of each cardiomyocyte was evaluated. Fluorescence intensity of F A K staining was then measured as mentioned. 2.3.10 Cardiomyocyte Ca 2 + transient: Cardiomyocytes (104) were cultured in each well of 8 well laminin coated slides. After ICAM-1 cross-linking or control, cardiomyocytes were incubated with 2 | i M Fluo-4 (F14201, Lot 28A2-5, Molecular Probes) for 10 minutes, washed 3 times, and electrically stimulated as above. One hundred and fifty time series images were captured at 33 millisecond intervals during cardiomyocyte contraction using laser scanning confocal microscopy. To quantify coordination of simultaneity of intracellular C a 2 + release, three regions of interest were selected at the center and at each end of each cardiomyocyte. The mean intensity/pixel in each region of interest was measured from the beginning of contraction to the end of relaxation in each cardiomyocyte. The relative dispersion between the three regions of 48 interest was used to quantify heterogeneity. The time integral of Ca intensity curve was used to quantify total Ca release. 2.3.11 Data analysis: We tested for differences in fractional shortening and number of adherent P M N between control and treated groups of cardiomyocytes using A N O V A , choosing p<0.05 as significant. When a significant difference was found we identified specific differences between groups using a sequentially rejective Bonferroni test procedure. Data are expressed as means ± standard error throughout. 49 2.4 Results 2.4.1 Uniform co-culture conditions: We conducted preliminary experiments to establish uniform co-culture conditions for the experiments in this study. Preliminary experiments demonstrated that, after 24 hours incubation, cardiomyocyte fractional shortening was 20.5±0.7 % (n=36). In the absence of P M N , fractional shortening did not significantly change following activation of quiescent cardiomyocytes using a 2 hour incubation with T N F a 20 ng/mL (BD Pharmingen, San Jose, CA) (91.3±4% of control, p=NS), interleukin IL-1(3 20 ng/mL (Sigma-Aldrich) (91.1 ±6% of control, p=NS), or lipopolysaccharide 10 H-g/mL (Sigma-Aldrich) (86.5±5% of control, p=NS). One hour after T N F a activation, the ratio of cardiomyocyte ICAM-1 to G A P D H mRNA increased from 0.091±0.08 to 0.361± 0.10. Similarly, following T N F a activation, ICAM-1 protein expression significantly increased (Figure 2.1). When non-activated cardiomyocytes were co-cultured with freshly isolated P M N for 4 hours, fractional shortening did not change (20.7±1.2 %, n=36) (Figure 2.2A). However, TNFa-activated cardiomyocytes in co-culture with freshly isolated P M N did decrease fractional shortening. Based on the percentage of cardiomyocytes with normal morphology, the number of P M N adherent to each cardiomyocyte, and maximal contractile dysfunction (Figure 2.2) all subsequent experiments were conducted following a 4-hour co-incubation of TNFa-activated cardiomyocytes and freshly isolated P M N . 50 Figure 2.1 ICAM-1 protein expression on cardiomyocytes increases 4 hours after activation with T N F a (B) compared to non-activated controls (A), which was significantly different (*, p<0.01) (bottom panel). 51 0-1 . • . , . . . . . 1-0 0.5 1 2 4 8 Time in Co-culture (hours) Figure 2.2 Preliminary experiments were used to determine the conditions for all further experiments. Panel A shows that PMN in co-culture with quiescent cardiomyocytes (Triangle) did not alter fractional shortening while PMN in co-culture with activated cardiomyocytes (which induces cardiomyocyte ICAM-1 expression) significantly reduces fractional shortening by 4 hours of co-culture (Squares). Panel B shows that the number of PMN adherent to activated cardiomyocytes increases to a maximum at 4 hours and cardiomyocyte viability remains high until 4 hours in co-culture. Based on these results further experimental measurements used PMN in co-culture with TNFa-activated cardiomyocytes for 4 hours. 52 2.4.2 Adherent PMN reduce cardiomyocyte fractional shortening: When activated cardiomyocytes were co-cultured with freshly isolated P M N , fractional shortening decreased significantly in T N F a (-34.2±5%, p<0.001), IL- lp (-48.6±3.2%, p<0.001) and LPS (-23.1±3.9%, p<0.001) treated cardiomyocytes. Average P M N adhesion increased from 1.1±0.2 P M N per cardiomyocyte in the non-activated cardiomyocytes to 2.7±0.2, 1.4±0.1, 1.9±0.2, in the TNFa, IL- lp , and LPS treated cardiomyocytes respectively (p<0.05 for each). Fractional shortening of cardiomyocytes in co-culture with P M N decreased by -2.8±0.3% per adherent P M N (p<0.001) (Figure 2.3A). Adherent P M N may have contributed to decreased cardiomyocyte contractility simply by release of reactive oxygen intermediates or other mediators in close proximity to the cardiomyocyte 1 6 4 . Alternatively, adhesion itself may have contributed. To help distinguish between these two possibilities we fixed P M N before co-culture. First, P M N were fixed with paraformaldehyde 3% for 15 minutes. After washing five times, fixed P M N were co-cultured with cardiomyocytes. Paraformaldehyde-fixed P M N decreased fractional shortening by 27±2% compared to control (p<0.05), which is comparable to the effect of freshly isolated P M N . Gluteraldehyde-fixed P M N similarly decreased cardiomyocyte fractional shortening by 25±4% (p<0.05) compared to control. The relationship between the number of adherent P M N versus decrease in fractional shortening was preserved (Figure 2.3B) suggesting that this effect was due to adhesion of P M N to cardiomyocytes. To exclude the possibility that gluteraldehyde fixation may have prevented binding by altering the 53 Gluteraldehyde-fixed PMN/ Cardiomyocyte co-culture Paraformaldehyde-fixed PMN/cardiomyocyte co-culture B 0 1 2 3 4 Number of attached PMN / Cardiomyocyte Figure 2.3 Panel A illustrates that fractional shortening of cardiomyocytes decreased as the number of attached PMN per cardiomyocyte increased. Panel B illustrates that this same effect was present even when PMN were first killed and fixed using either gluteraldehyde or paraformaldehyde. These results implicate PMN-cardiomyocyte binding as a key step in mediating decreased cardiomyocyte fractional shortening. Numbers within columns indicate the number of cardiomyocytes measured. 54 extracellular domain of CD1 lb on P M N we used a low concentration of glutaraldehyde and confirmed unchanged CD1 lb expression on P M N using flow cytometry (Figure 2.4). 55 Freshly isolated PMNs Gluteraldehyde-fixed PMNs B D J \ J Fluorescent intensity Figure 2.4 Flow cytometery of freshly isolated (left hand panels) and gluteraldehyde-fixed (right hand panels) PMN is shown. Panel A shows results for freshly isolated PMN (identified from side scatter -back scatter plots) incubated with non-specific IgG. Panel C shows results for freshly isolated PMN, incubated with antibody to CD l ib . Increased fluorescent intensity specific for CD l i b is seen. Panel B shows results for gluteraldehyde-fixed PMN incubated with non-specific IgG. In comparison panel D demonstrates the same increase in fluorescent intensity specific for CD l i b as seen with freshly isolated PMN (panel C). 56 2.4.3 ICAM-1 binding mediates decreased cardiomyocyte contractility: Antibody to ICAM-1 and antibody to CD-18 (Pharminogen), to block this important PMN-expressed ligand of ICAM-1, prevented the PMN-induced decrease in cardiomyocyte fractional shortening (Figure 2.5A). The average number of P M N adherent to each TNFa-activated cardiomyocyte was also reduced by antibody to ICAM-1 and antibody to CD-18 (control 2.7±0.2, antibody to ICAM-1 1.2±1.0, antibody to CD-18 l . l i l . O adherent P M N per cardiomyocyte). Thus, the contractile effect of P M N binding involves binding to cardiomyocyte ICAM-1. To demonstrate that P M N binding, and the decrease in contractility, in co-culture with gluteraldehyde-fixed P M N were still ICAM-1 - dependent, we repeated these experiments with and without antibody to ICAM-1. The number of adherent gluteraldehyde-fixed P M N was reduced from 1.8 ± 0.2 to 0.58 ± 0 . 1 by antibody to ICAM-1 and the decrease in fractional shortening was prevented (fractional shortening in the presence of P M N without anti-ICAM-1 16.8 ± 1.1%, with anti-ICAM-1 22.0 ± 1.1%, p<0.05). To determine whether ICAM-1 binding itself can cause decreased contractility we used cross-linking antibodies to ICAM-1, in the absence of P M N . ICAM-1 cross-linking decreased fractional shortening by 36±3% compared to control groups at 4 hours (Figure 2.5B). In contrast, replacement of anti-ICAM-1 antibodies with nonspecific IgG, followed by addition of cross-linking antibodies, did not change fractional shortening compared to control. 57 ^ 25 .E 20 c 03 2 15 OT m c o 10 u <0 36 36 _I_ 36 24 Non-activated Non-activated TNF-activated anti-ICAM-1 + anti-CD-18 + cardiomyocytes cardiomyocyte- cardiomyocyte- TNF-activated TNF-activated PMN co-culture PMN co-culture cardiomyocyte- cardiomyocyte-PMN co-culture PMN co-culture _ ^ 25 • B O) .E 20 c 0) t £ 15. (0 re c .2 10 • o re 46 18 37 11 37 IgG Control ICAM-1 cross-linked ICAM-1 cross-linked + Cytochalasin D ICAM-1 cross-linked + Latrunculin A Figure 2.5 Panel A shows that the decrease in cardiomyocyte fractional shortening in the TNF-activated cardiomyocyte-PMN co-culture group (*, p<0.05) was prevented by either antibody to ICAM-1 or antibody to its important ligand, CD 18. Panel B demonstrates that, even in the absence of PMN, ICAM-1 cross-linking reduces cardiomyocyte fractional shortening (*, p<0.05). This effect is prevented by disruption of the actin cytoskeleton, either by cytochalasin D or latrunculin A. 58 2.4.4 ICAM-1 cross-linking alters the cortical cytoskeleton: ICAM-1 is connected to cytoskeletal proteins ' ' ' . To test for involvement of the cortical cytoskeleton following ICAM-1 cross-linking we examined the distribution of the actin cytoskeleton-associated protein F A K within cardiomyocytes. Immunofluorescent imaging of F A K at 2 um below the cardiomyocyte surface demonstrated increased cytoskeleton-associated F A K staining in ICAM-1 cross-linked cardiomyocytes compared to controls (Figure 2.6). 2.4.5 Functional role of the cardiomyocyte actin cytoskeleton: To test the hypothesis that the actin cytoskeleton is involved in signaling pathways that lead to decreased cardiomyocyte contractility induced by ICAM-1 binding, we preincubated cardiomyocytes with cytochalasin D 10 uM, an actin depolymerizing agent, for 2 hours. Control cardiomyocyte fractional shortening was not altered by this concentration of cytochalasin D (106±10% of control fractional shortening, n=39 vs. n=60 controls) confirming previous observations that these concentrations of cytochalasin D did not have a measurable effect on the function of sarcomeric F-actin 1 7 6 . Cytochalasin D prevented the decrease in cardiomyocyte contractility due to ICAM-1 cross-linking (Figure 2.5B). We repeated these experiments, disrupting the actin cytoskeleton in an independent way using Latrunculin A (Calbiochem, San Diego, CA) 10 uM, an agent 177 178 that sequesters monomeric actin and inhibits elongation of actin filaments ' . Latrunculin A by itself did not significantly alter cardiomyocyte fractional shortening (111±10% of control fractional shortening, n=29 vs n=22 controls). Again, latrunculin A prevented the decrease in cardiomyocyte contractility due to ICAM-1 cross-linking (Figure 2.5B). 59 Similarly, in experiments of P M N co-cultured with activated cardiomyocytes, cytochalasin D and latrunculin A prevented the PMN-induced decrease in cardiomyocyte fractional shortening (control 20.5±0.6%, cardiomyocyte-PMN co-culture 13.6±0.8%, cytochalasin D 17.5±1.2% and latrunculin A 19.9±2.7%) but did not alter the number of P M N adherent to cardiomyocytes (1.6±0.4, 1.8±0.5, and 1.3±0.8 in control, Cytochalasin D, and Latrunculin A respectively). 60 Control cardiomyocyte % ICAM-1 cross-linked cardiomyocyte 10 in CN 6 re u OT >1 2 O tn (A <D OQ 40 35 30 25 20 15 Control * - T T • ICAM-1 cross-linked Figure 2.6 Top panels show example FAX staining in control (top left hand panel) and ICAM-1 cross-linked cardiomyocytes (top right hand panel). The bottom panel shows average results for all cardiomyocytes (n=64 for each group from 6 experiments) indicating that ICAM-1 cross-linking increases FAK expression associated with the cortical actin cytoskeleton 2 um below the cardiomyocyte surface (*, p<0.05). 61 2.4.6 Possible downstream signaling pathways: Rho family proteins (small G proteins) are associated with the actin cytoskeleton and regulate both cytoskeletal reorganization and gene expression. To investigate the role of these pathways we used Fusadil (HA-1077, Sigma-Aldrich), an inhibitor of Rho-kinase, in ICAM-1 cross-linked cardiomyocytes. HA-1077 50 u M completely prevented ICAM-1 binding-induced decreases in cardiomyocyte fractional shortening (Figure 2.7) suggesting that this actin cytoskeleton-associated signaling pathway mediates ICAM-1 binding-induced decreases in cardiomyocyte fractional shortening. F A K activation leads to activation a series of proteins and pathways, including M A P K pathways in other cell lines 1 7 9 ' . To investigate the role of these pathways on contractility of ICAM-1 cross-linked cardiomyocytes, we treated ICAM-1 cross-linked cardiomyocytes with the ERK1/2 inhibitor, PD98059 10 u M (New England BioLabs Ltd. Mississauga, OA, Canada) 20 | i M . ERK1/2 inhibition had no effect on the ICAM-1-induced decreases in cardiomyocyte fractional shortening (Figure 2.7) suggesting that this signaling pathway was not directly involved. 62 25 n ^ 20 H O _c 'E <D 15 O CO 75 1 0 c o (0 68 _I_ 68 31 47 Control ICAM-1 cross-linked ICAM-1 cross-linked + HA-1077 ICAM-1 cross-linked + PD98059 Figure 2.7 The effect of ICAM-1 cross-linking on fractional shortening (*, p<0.05 compared to Control) is prevented by the Rho A inhibitor (HA-1077) but not by the MAPK inhibitor (PD98059). 63 2.4.7 ICAM-1 activation changes the pattern of C a z + release: To better understand how ICAM-1 binding could alter contractility we measured the cardiomyocyte intracellular calcium transient. ICAM-1 cross-linking changed the pattern of Ca2 +release in contracting cardiomyocytes. Ca release changed from homogenous and synchronized in control cardiomyocytes to a focal starting point propagating wave in ICAM-1 cross-linked cardiomyocytes (Figure 2.8). The focal starting point propagating wave occurred in 86±3% of ICAM-1 cross-linked cardiomyocytes compared to 31 ±7% of control cardiomyocytes (p<0.05). As a result, ICAM-1 cross-linking increased the duration of the C a 2 + transient (Figure 2.8A). The slope over the most linear middle 50% of the decay curve was decreased to 1.53 ± 0.14 X IO"4 in ICAM-1 cross-linked cardiomyocytes from 2.94 ± 0.24 X 10"4 in control cardiomyocytes (p<0.0001). Heterogeneity of C a 2 + release increased in the ICAM-1 cross-linked group as indicated by an increase in relative dispersion in ICAM-1 cross-linked cardiomyocytes (Figure 2.8B). Total C a 2 + release (integral of C a 2 + release in Figure 2.8A) did not differ significantly in ICAM-1 cross-linked cardiomyocytes versus controls. 64 2.5 Discussion The key finding of this study is that ICAM-1 binding decreases cardiomyocyte contractility. ICAM-1 binding by freshly isolated P M N , by P M N fixed in either paraformaldehyde or gluteraldehyde, and ICAM-1 binding using cross-linking antibodies, all caused a significant decrease in cardiomyocyte contractility. The actin cytoskeleton plays a central role. Evidence for this includes increased immunofluorescent staining of cortical actin cytoskeleton - associated F A K (2 um below the cell surface) after ICAM-1 cross-linking. The decrease in cardiomyocyte contractility due to ICAM-1 cross-linking could be prevented by cytochalasin D and, using an alternative strategy, by latrunculin A . Furthermore, inhibition of the cytoskeleton - associated Rho kinase pathway also prevented decreased contractility due to ICAM-1 cross-linking. The ultimate mechanism of loss of contractility due to ICAM-1 binding appears to be loss of the normal coordination of intracellular C a 2 + release. Even though total intracellular C a 2 + release during a single contraction did not change significantly, the release did not occur in the normal coordinated manner throughout the entire cell over a short period of time, so that fractional shortening decreased. 1 (YI 1 Si.(\ 1 SI 1 ICAM-1 expression on activated cardiomyocytes ' ' plays an important role in ICAM-1 - CD 18 dependent adhesion of P M N to cardiomyocytes 1 0 7 ' 1 6 4 ' 1 7 1 . m a whole animal model, antibody to CD 18 protects against myocardial damage and dysfunction 1 T 7 1 3 0 following ischemia-reperfusion ' and reduces P M N adherence within the heart. ICAM-1 and CD 18 deficient mice demonstrate a marked 65 • • • 0 ms 33 ms 100 ms 200 ms 400 ms 500 ms 1000 ms 1500 ms 2000 ms Control cardiomyocyte 2500 ms H P 0 ms 33 ms 100 ms 200 ms 400 ms D 500 ms 1000 ms 1500 ms 2000 ms 2500 ms ICAM-1 cross-linked cardiomyocyte 0-1 , , , , r-0 0.5 1 15 7 2.5 Time(s) 0 05 1 1 5 2 2.5 Time (s) Figure 2.8 The top panels illustrate in typical examples that Ca 2 + release and reuptake in control cardiomyocytes is rapid whereas Ca 2 + release in ICAM-1 cross-linked cardiomyocytes is much slower, with the appearance of a wave of Ca 2 + release moving along the length of the cardiomyocyte. Average results for all cardiomyocytes are shown in the bottom panel for control cardiomyocytes (n=37, solid line) and for ICAM-1 cross-linked cardiomyocytes (n=67, dashed line) demonstrating increased dispersion, or heterogeneity, of calcium release in ICAM-1 cross-linked cardiomyocytes. The coefficient of variation is particularly increased in ICAM-1 cross-linked cardiomyocytes for the first 1.5 seconds after cardiomyocyte electrical stimulation. 66 reduction in P M N accumulation and myocardial necrosis after acute ischemia-reperfusion 1 8 3 . Activated P M N in vitro can cause dysfunction and kil l cardiomyocytes within minutes via production of reactive oxygen intermediates l 6 3 , 1 6 4 . However, this short time course and the cardiomyocyte necrosis caused by highly activated P M N are not fully consistent with in vivo observations of the prolonged time course of ischemia/reperfusion-induced myocardial stunning 1 0 3 ' 1 8 4 and of sepsis 38'55>66>185. it is useful to note that P M N adhesion to human umbilical vein endothelial cells following anoxia/reoxygenation demonstrate both early effects (minutes) mediated by reactive oxygen intermediates and late effects (hours) involving other pathways including the actin cytoskeleton 1 8 7 . In these cells PMN-induced cytoskeletal changes are inhibited by antibodies to ICAM-1 1 7 3 . Thus, our results are consistent with, and extend, previous studies. This novel mechanism of decreased cardiomyocyte contractility implicates the cytoskeleton in maintaining normal coordination of intracellular C a 2 + release and, hence, 9-f- 9+ normal contractility. Ca -induced Ca release from the sarcoplasmic reticulum triggered by C a 2 + influx via dihydropyridine receptors is the main regulator of cardiomyocyte contractility 1 8 8 , 1 8 9 . The novel finding that impaired coordination of C a 2 + -induced C a 2 + release is an additional mechanism regulating cardiomyocyte contractility may have broad implications in myocardial processes involving inflammation and up-regulation of ICAM-1 expression on cardiomyocytes. It may play a role in disease processes which involve leukocyte infiltration in the heart including ischemia reperfusion, inflammatory cardiomyopathy, and septic myocardial dysfunction. 67 Furthermore, disease and repair processes that alter extracellular matrix constituents so that increased I C A M binding occurs, may also be implicated. For example, changing extracellular matrix composition and ICAM-1 expression also occur during normal embryogenesis 1 9 0 ' 1 9 1 . P M N and macrophages have previously been shown to bind ICAM-1 expressed on cardiomyocytes 5 9 ' 1 0 7> 1 6 4. in many of the studies of the interaction between highly activated P M N and cardiomyocytes, ICAM-1 mediates binding and activated neutrophils cause cardiomyocyte cell death by release of reactive oxygen intermediates. The effect of non-activated neutrophils and ICAM-1 binding by itself has not previously been examined. However, Simms et al. 5 9 reported that ICAM-1 mediated the effects of macrophage/monocyte binding on decreased cardiomyocyte contractility. Recently, Raeburn, et al. 1 6 0 report that ICAM-1 and V C A M - 1 mediate endotoxemic myocardial dysfunction independent of neutrophil accumulation in the myocardium. While the earlier results implicate ICAM-1 largely as a mediator of tethering of cytotoxic neutrophils to cardiomyocytes 1 0 7 ' 1 6 4 , these recent studies 5 9 ' 1 6 0 suggest a potential alternative role for ICAM-1 binding. Our current results identify and illuminate this potential alternative role. Binding of freshly isolated P M N to cardiomyocytes causes a decrease in contractility without cardiomyocyte cell death, presumably because the P M N are not highly activated and releasing reactive oxygen intermediates and other damaging mediators. To confirm this, and to exclude the possibility that P M N release damaging mediators as a result of P M N 68 activation during the isolation procedure, we repeated these experiments in both paraformaldehyde and gluteraldehyde - fixed P M N . Surprisingly, even when P M N are killed and fixed in two different ways, to prevent further activation and release of mediators, P M N still cause a dose-dependent decrease in cardiomyocyte contractility (Figure 2.3). The importance of ICAM-1 binding by itself in causing decreased contractility was then confirmed in a leukocyte-free condition where ICAM-1 cross-linking antibodies were applied - which results in a decrease in cardiomyocyte contractility. This decrease in contractility clearly involves the cortical actin cytoskeleton. First, utilizing 3D reconstruction techniques to examine the cortical region of cardiomyocytes, we found that cytoskeletal-associated F A K expression increases following ICAM-1 binding. Furthermore, inhibition of normal cytoskeletal function using either cytochalsin D or latrunculin A prevented ICAM-1 binding-induced decreases in cardiomyocyte contractility. Finally, inhibition of the actin cytoskeleton-associated Rho kinase signaling pathway also prevented ICAM-1 binding induced decrease in contractility. We found that the decrease in contractility was due to loss of simultaneity of intracellular C a 2 + release. One key coordinating mechanism of Ca2+-induced C a 2 + release is colocalization of dihydropyridine receptors on external or T-tubule sarcolemma with ryanodine receptors on the sarcoplasmic reticulum . This geometric arrangement is instrumental in allowing the relatively small intracellular C a 2 + flux introduced via dihydropyridine receptors to trigger Ca2+-induced C a 2 + release by ryanodine receptors. 69 We postulate that disruption of this coordinating mechanism would result in increased heterogeneity of C a 2 + release by the sarcoplasmic reticulum. That is, i f dihydropyridine receptors do not cause coordinated induction of Ca2+-induced C a 2 + release by ryanodine receptors, then simultaneous contraction throughout the cardiomyocyte would be impaired. In the absence of normal coupling, a stimulus sufficient to cause local sarcoplasmic release of C a 2 + would then cause further Ca2+-induced C a 2 + release by adjacent sarcoplamic reticulum ryanodine receptors. If true, this would take the form of a wave of contraction travelling from the initial site along the cardiomyocyte. Indeed, this is what we observed with ICAM-1 cross-linking (Figure 2.8). Thus, we think that it is reasonable to postulate that the actin cytoskeleton plays a key role in maintaining coordination of cardiomyocyte contractility, possibly by contributing to maintainance of normal co-localization of dihydropyridine receptors and ryanodine receptors. Identification of this novel pathway, which may contribute to myocardial dysfunction during intramyocardial inflammation (e.g., following ischemia-reperfusion, in inflammatory cardiomyopathy, during orthotopic heart transplant rejection, and during sepsis-induced myocardial dysfunction), is clearly only one of many pathways involved. Indeed, ICAM-1 binding maybe a component of some of these alternative pathways such as apoptosis 1 9 3 and reactive oxygen intermediate production 1 9 4 ' . It is interesting to speculate when and why such a mechanistic pathway may confer an evolutionary advantage. Is this mechanism of regulation of cardiomyocyte contractility just pathological loss of normal coordination or is there some benefit? This novel 70 pathway would be evoked during inflammation and subsequent resolution and repair or conceivably even during embryogenesis. We speculate that it may be beneficial to down-regulate contractility for a period of time during these processes. This ICAM-1 and cytoskeleton-mediated pathway could provide this mechanism. In summary, we have identified a novel pathway and mechanism of regulation of cardiomyocyte contractility. While we have demonstrated that this pathway plays a role in neutrophil-cardiomyocyte interaction, it may play a role in the interaction of the cardiomyocyte with other inflammatory cells and potentially even with the extracellular matrix. Thus, this novel mechanism of decreased contractility may have much broader implications. In the next chapter I will investigate the role of ICAM-1 in an in vivo model of endotoxemia to test the importance of ICAM-1 presentation and activation in vivo. I will also investigate the presence of inflammatory cells in the septic heart to test this hypothesis that whether inflammatory cell including PMNs can infiltrate and attach to the cardiomyocyte ICAM-1 receptors using their CD11/CD18 receptors. 71 Chapter-3 Cardiac ICAM-1 mediates leukocyte-dependent decreased ventricular contractility in endotoxemic mice. 3.1 Abstract Binding of ICAM-1 expressed on isolated cardiomyocytes decreases cardiomyocyte contractility by altering the intracellular C a 2 + transient. To test the hypothesis that signaling via ICAM-1 contributes to decreased left ventricular contractility in an in vivo model of sepsis, C57B6 wild-type mice and ICAM-1 knock out mice were treated with intraperitoneal lipopolysaccharide (LPS) then left ventricular contractility was measured 6 hours later using a volume-conductance micromanometer catheter. In C57B6 wild-type mice LPS injection significantly increased cardiac ICAM-1 expression and decreased left ventricular contractility (end-systolic elastance, Ees decreased 58 ± 4%, p<0.05, [dP/dtm ax]/EDV decreased 60 ± 6%, p<0.05). Cyclophosphamide pretreatment to decrease leukocyte count prevented the LPS-induced decrease in contractility. In ICAM-1 knock out mice LPS did not decrease any measure of contractility. We produced chimeric mice lacking ICAM-1 expression in bone marrow-derived cells (M-) and/or lacking ICAM-1 expression in the heart and other tissues (H-). LPS did not decrease left ventricular contractility in M+/H- mice but decreased contractility in M+/H+ and M-/H+ mice to the same extent as in C57B6 wild-type mice implicating the importance of cardiac ICAM-1. We conclude that signaling via cardiac ICAM-1 is necessary to mediate leukocyte-dependent decreases of left ventricular contractility in endotoxemic mice. 72 3.2 Introduction: Sepsis is responsible for more than 200,000 deaths annually in North America Myocardial dysfunction, caused by the systemic inflammatory response of sepsis, contributes to multi-organ dysfunction and death in these patients 1 9 5 . When the degree of myocardial dysfunction is substantial, cardiac output cannot be increased sufficiently to maintain an adequate systemic arterial pressure in the face of the decreased systemic vascular resistance of septic shock. Indeed high versus low cardiac output during septic shock distinguishes survivors from non-survivors 1 6 . Leukocytes are a component of the systemic inflammatory response of sepsis and have been implicated in contributing to myocardial dysfunction in experimental models both in vivo 6 5 ' 1 9 6 and in vitro 59>60>107'197. Leukocytes decrease isolated cardiomyocyte contractility in vitro by binding to ICAM-1 expressed on the cardiomyocytes5 9'6 0'1 0 7 and by release of reactive oxygen intermediates from the activated leukocytes , 0 7 ' 1 6 3 . in addition, even non-activated leukocytes or ICAM-1 cross-linking antibodies can decrease contractility by binding ICAM-1 expressed on cardiomyocytes 5 9 ' 6 0 . ICAM-1 binding then mediates decreased cardiomyocyte contractility by signaling via the cortical actin cytoskeleton, which leads 2+ to increased heterogeneity of intracellular Ca release and decreased cardiomyocyte contractility 6 0 . Whether ICAM-1 binding plays an important role in development of myocardial dysfunction in vivo has not been demonstrated. Accordingly, we first determined the extent of increased cardiac ICAM-1 expression using a lipopolysaccharide (LPS) injection model of systemic inflammation in mice. 73 Next, we tested the hypothesis that myocardial dysfunction occurs in this model and, further, we determined whether the degree of myocardial dysfunction is prevented by decreasing the peripheral blood leukocyte count. We then determined whether ICAM-1 expression is also necessary for the development of myocardial dysfunction in this model, using ICAM-1 knock-out mice. Finally, to determine i f cardiac ICAM-1 expression is necessary for myocardial dysfunction (versus ICAM-1 expressed on bone marrow-derived cells), we developed chimeric mouse models lacking ICAM-1 expression in bone marrow-derived cells and/or lacking ICAM-1 expression in the heart . and other tissues. We found that activation of cardiac ICAM-1 plays a key role in mediating decreased left ventricular contractility in this whole animal model of systemic inflammation. 74 3.3 Material and Methods This study was approved by the University of British Columbia Animal Care Committee and adheres to Canadian and U.S.A. National Institutes of Health guidelines for animal experimentation. 3.3.1 Experimental preparation: Mice, C57B6 wild-type and C57B6/J-ICAM t m l j c g r knock-out (ICAM-1 knock out, Jackson Laboratory, Bar Harbor, ME) weighing 25-30 g, were given intraperitoneal injection of purified lipopolysaccharide (LPS, E. coli strain 01 II: B4, Sigma, St. Louis, MO) 40 mg/kg or the same volume of normal saline. Six hours after LPS injection, mice were anesthetized using ketamine (75 mg/kg) and xylazine (10 mg/kg); total volume 0.2 mL, injected subcutaneously. Anesthesia effectiveness was confirmed throughout the procedure. Tracheal intubation was performed through a 1 cm midline neck incision and mice were ventilated (Mouse Ventilator model 687, Harvard Instruments, Holiston, M A ) at 120 breaths/min with a 200 uL tidal volume. A substernal transverse incision was made to expose the apical portion of the heart and inferior vena cava. The left ventricle was punctured using a 27 gauge needle approximately 1 mm lateral to the interventricular septum. Then a number 2 French volume-conductance micromanometer catheter (Mikro-tip SPR-838, Millar Instruments Inc., Houston, TX) was inserted into the left ventricle. Correct placement of the catheter was determined by checking pressure-volume loops for vertical isovolumic phase traces and characteristic diastolic and systolic trajectories. Pressure-volume data were recorded at steady state and the inferior vena cava was occluded for 3-5 seconds while pressure-volume data were 75 again recorded. The heart was excised and frozen (isopentane in liquid nitrogen) or fixed (10% formalin) for further histopathology and morphometry measurements. 76 100 2.5 5 7.5 Volume (uL) 10 12.5 Figure 3.1 Pressure-volume measurements of multiple heart beats during a vena-caval occlusion are illustrated. A best fit line to the end-systolic points of these beats (red line) is the end-systolic pressure-volume relationship. The slope of the end-systolic pressure volume relationship is end-systolic elastance, Ees; a preload and afterload independent measure of left ventricular contractility. 7 7 3.3.2 Left ventricular contractility and cardiac function: Left ventricular contractility and other measures of ventricular function were determined from pressure-volume measurements using Pressure-Volume Analysis software ( P V A N 2.9, Millar Instruments Inc., Houston, TX). Six to ten pressure-volume loops during a vena cava occlusion were sampled and used to measure end-systolic elastance (Ees), which is the slope of the end-systolic pressure-volume relationship (Figure 1). Ees is an ejection phase measure of left ventricular contractility which, among many others, is least sensitive to changes in preload and afterload 1 9 8 anticipated to occur in this model of sepsis. The volume axis intercept, Vd, was considered zero volume for the steady-state measurement of end-diastolic volume (EDV) and end-systolic volume (ESV). Pressure-volume loops measured during steady-state conditions were used to measure the maximum rate of change of intraventricular pressure during isovolumic systole divided by EDV, [dP/dtm a x]/EDV, which is a sensitive isovolumic phase measure of left ventricular contractility 1 9 9 . Steady state pressure-volume loops were also used to calculate ejection fraction as the difference between E D V and ESV divided by EDV, which is a further measure of cardiac function. End-systolic pressure during steady state was used as a measure of systemic arterial pressure afterload. 3.3.3 The role of leukocytes: To determine whether leukocytes contributed to decreased left ventricular contractility and function in this animal model we increased leukocyte count by pretreating C57B6 wild-type mice with GCSF and, alternatively, we decreased leukocyte count by pretreating C57B6 wild-type mice with cyclophosphamide. 78 Mice were then injected with LPS and left ventricular contractility and function were measured as above and compared to C57B6 wild-type mice treated with LPS. To increase leukocyte count, mice received GCSF (Filgrastim 300 Ug/ml vial, Neupogen) 120 Ug/kg subcutaneously twice daily for four days and were then rested for 3 days. This time course was determined from preliminary experiments where we measured the ratio of C D l l b expression to total actin content as an indicator of bone marrow cell maturation. Compared to non-treated controls we found a significant decrease in this ratio immediately after the 4 day GCSF treatment. Following 3 days of rest, the ratio of C D l l b expression to total actin content returned towards normal in the GCSF-treated mice indicating that 3 days of rest after GCSF injection was necessary to approximate normal bone marrow cell maturation. To decrease leukocyte count, mice received cyclophosphamide (Bristol Laboratories, Montreal, Canada) 150 mg/kg intraperitoneal daily for 2 days and were then rested for 3 days. This time course was determined from preliminary experiments and the literature which indicated that the nadir of the peripheral blood leukocyte count occurred 3 days 200 202 after the last cyclophosphamide injection " . To confirm the effect of GCSF and cyclophosphamide, we measured total leukocyte count and polymorphonuclear leukocyte (neutrophil) count in peripheral blood and in bone marrow using a Cell-Dyn 3700 counter (Abbott Diagnostic Division, Abbott, IL). 79 3.3.4 Chimeric models: We reasoned that any difference in LPS effect found in ICAM-1 knock-out mice could either be due to loss of cardiac ICAM-1 expression or due to loss of ICAM-1 expression on blood borne bone marrow-derived cells transiting the heart. To distinguish between these two possibilities, and thereby specifically implicate cardiac ICAM-1 expression, we designed three different chimeric mouse models; I C A M -1 expressed on marrow-derived cells and on heart cells (M+/H+), ICAM-1 expressed on marrow-derived cells but not on heart cells (M+/H-), and ICAM-1 not expressed on marrow-derived cells but expressed on heart cells (M-/H+). We developed these chimeric mice as follows. To increase the number of progenitor cells in donors, C57B6 wild-type and ICAM-1 knock out mice were first pretreated with GCSF as described above. Bone marrow from femurs and tibias of 8 to 10 GCSF-treated mice (donors) was extracted and separated in PBS plus A C D 10% (C3821 Acid Citrate-Dextrose solution, Sigma), washed 4 times in PBS plus A C D and filtered. After a final wash in PBS, cells were suspended in normal saline. In this bone marrow concentrate the total leukocyte count was 12.2 ± 1.2 x 109 /L , the fraction of granulocytes was 94.8 ± 0.3%, and the fraction of lymphocytes was 3.5 ± 0.1%. To suppress inflammatory cell progenitors in recipient mice, C57B6 wild-type and ICAM-1 knock out mice were first pretreated with cyclophosphamide as described above. Bone marrow cells concentrate (300 uL) from the donor mice were injected into recipient mice through a tail vein. Bone marrow from C57B6 wild-type mice injected into C57B6 wild-type recipients gave M+/H+ chimeric mice. Bone marrow from C57B6 wild-type 80 mice injected into ICAM-1 knock out recipients gave M+/H- chimeric mice. Bone marrow from ICAM-1 knock out mice injected into C57B6 wild-type recipients gave M -/H+ chimeric mice. A l l groups of chimeric mice were then treated with LPS or saline and 6 hours later left ventricular contractility and function were measured and hearts were harvested as described above. 3.3.5 Cardiac ICAM-1 expression: To measure ICAM-1 protein expression, 10 um thick frozen sections were prepared and fixed using 3% paraformaldehyde for 20 minutes and then incubated with Universal Blocking Agent (Dako, Carpinteria, CA) for 2-3 hours. Sections were treated with rat-anti mouse ICAM-1 antibody (1/500) (BD Pharmingen) at 4°C overnight followed by incubation with goat anti-rat fluorescently labeled antibody (1/ 1000) (Alexa-fluor 594, Molecular Probes, Eugene, OR) and Hoechst nuclear stain (1/1000) (H3570, Molecular Probes) for 3 to 4 hours at room temperature. Images were captured at 400 X using a confocal microscope (SP-2, Leica Corporation, Exton, PA). The ratio of mean intensity of ICAM-1 staining (red) to mean intensity of nuclear staining (blue) was measured (Fluocytogram, Leica Corporation, DIMRE2, Exton, PA). 3.3.6 Cardiac leukocyte infiltration: Sections (6 um) were prepared from formalin fixed hearts and stained with hematoxylin and eosin. Images were captured at 400 X using a spot camera and measurements were made using Image-Pro Plus software (Media Cybernetic Inc., Silver Spring, MD). The number of neutrophils within coronary arteries, the number of neutrophils adherent to the endothelial surface, and the number of 81 neutrophils within the perivascular space (defined as an area of double the largest radius of the corresponding coronary artery) were measured. 3.3.7 Statistical analysis: We tested for differences in Ees, [dP/dtm ax]/EDV, and ejection fraction between groups using analysis of variance, choosing p<0.05 as significant. When a significant difference was found we identified specific differences between groups using a sequentially rejective Bonferroni test procedure. Data are expressed as mean ± standard error throughout. 82 3.4 Results 3.4.1 Endotoxemic C 5 7 B 6 wild-type mice: Immunofiuorescent staining of frozen sections from LPS-treated and saline-treated C57B6 wild-type mice demonstrated that ICAM-1 expression in heart sections significantly increased 6 hours after LPS (Figure 2). The ratio of the mean intensity of ICAM-1 staining to the mean intensity of nuclear staining was greater in the LPS-treated mice (4.59 ± 0.32) than in control saline-treated mice (1.96 ± 0.16, pO.OOl). The intensity of ICAM-1 staining was heterogeneously distributed within each LPS-treated mouse heart section. We then determined whether this endotoxemic murine model of sepsis resulted in decreased left ventricular contractility and function comparable to large animal models ' and human sepsis ' . Six hours after LPS injection in C57B6 wild-type mice, heart rate increased by 72 ± 6% (p<0.01) and end-systolic pressure decreased by 12 ± 4% compared to saline-treated controls (Table 1). Left ventricular contractility decreased after LPS injection as indicated by a 58 ± 4% decrease in Ees (p<0.05; Figure 3A) and a 60 ± 6% decrease in [dP/dtm ax]/EDV. Ejection fraction decreased by 14 ± 10% despite the decrease in end-systolic pressure (Table 1). 83 Table 3.1 Hemodynamics and ventricular function measures C57B6 wild-type Saline C57B6 wild-type LPS C57B6 GCSF Saline C57B6 GCSF LPS C57B6 CY Saline C57B6 CY LPS ICAM-1KO Saline ICAM-1KO LPS Number 8 8 6 8 6 6 8 8 Heart rate (bpm) 280 ±41 481±18 * 303 ± 32 450 ± 16* 181 ±7 347 ± 29 * 340 ± 17 388 ±13 * End Systolic Pressure (mmHg) 77 ±7 68 ± 3 81 ± 6 62 ± 7 58 ± 6 76 ±5 103 ± 15 78 ±7 End Diastolic Pressure (mmHg) 4.7 ± 0.9 3.7 ±0.6 5.3 ±2.4 2.7 ±0.7 4.6 ±0.7 5.5 ±0.8 3.7 ± 1.3 3.2 ±0.4 End Systolic Volume (|rL) 6.4 ± 1.1 14.5 ±2.2 * 7.5 ± 1.3 13.9 ±2.2 * 25.7 ±5.5 10.4 ±3.1 7.4 ± 1.1 9.2 ±2.3 End Diastolic Volume ((XL) 12.9 ±2.3 24.7 ±2.7 * 16.6 ±2.7 20.4 ±2.7 48 ±7 18.2 ±5.8 13.9 ± 1.0 15.1 ±2.8 Ejection Fraction 48 ±4 41 ±5 54 ±5 34 ±5 46 ± 3 39 ±7 48 ±5 46 ±6 C57B6 GCSF: GCSF pretreated mice. C57B6 CY: cyclophosphamide pretreated mice. ICAM-1 KO: ICAM-1 knock out mice. * p<0.05 compared to controls. B T " • r C 5 7 B 6 C 5 7 B 6 + LPS ICAM-1 KO ICAM-1 KO + LPS Figure 3.2 Representative immune-fluorescent staining of myocardial sections from A) C57B6 saline-treated mice, B) C57B6 LPS-treated mice are shown in the top panel. The ratio of ICAM-1 to nuclear staining is shown for all mice in the above groups in the bottom panel. This ratio is significantly greater in C57B6 LPS-treated mice compared to saline-treated control mice (p<0.01). This ratio is greatly decreased in ICAM-1 knock out mice and is not altered by LPS. 85 Table 3.2 Peripheral blood cellular components CO C57B6 wild-type Saline C57B6 wild-type LPS C57B6 GCSF Saline C57B6 GCSF LPS C57B6 CY Saline C57B6 CY LPS ICAM-1 KO Saline ICAM-1 KO LPS WBC X 10 9 /L 3.6 ±0.2 0.5 ±0.1 4.3 ±0.6 1.78 ±0.3 0.1 ±0.02 0.02 ±0.01 4.7 ±0.8 0.9 ±0.2 Neutrophil X 10 9 /L 0.4 ± 0.04 0.4 ±0.1 0.7 ±0.3 1.7 ±0.3 0.04 ±0.01 0.004 ±0.001 1.3 ±0.3 0.7 ±0.1 Lymphocyte X 109/L 2.9 ±0.2 0.1 ±0.06 3.3 ±0.4 0.1 ±0.03 0.05 ±0.01 0.007 ± 0.002 3.1 ±0.6 0.3 ±0.1 Red blood cell X 10 1 2 /L 8.6 ±0.1 9.4 ± 0.2 8.4 ± 0.32 8.5 ±0.52 7.42 ±0.16 7.51 ±0.11 8.5 ±0.2 9.5 ±0.1 Platelet X 10 9 /L 913 ± 21 513 ± 90 354 ±23 85 ±5 771 ±29 306 ± 43 735 ±48 304 ±37 C57B6 GCSF: GCSF pretreated mice. C57B6 CY: cyclophosphamide pretreated mice. CAM-1 KO: ICAM-1 knock out mice. 3.4.2 Effect of increased and decreased leukocyte count: To test the hypothesis that leukocytes contribute to the early (6 hours) decrease in left ventricular contractility in vivo in this intact whole animal model of sepsis, we increased leukocyte counts using GCSF injection or decreased leukocyte counts using cyclophosphamide injection. The GCSF injection increased total peripheral leukocyte count by 31 ± 10% and increased peripheral neutrophil count by 20% over untreated control C57B6 wild-type mice (Table 2). The GCSF increased bone marrow leukocyte count 3.5 fold and increased bone marrow neutrophil count 4 fold. In GCSF-treated mice LPS injection resulted in an 59 ± 15% reduction in Ees (p<0.05), a 21 ± 19% decrease in [dP/dtm a x]/EDV, and an 36 ± 9% decrease in ejection fraction compared to controls (p<0.05; Table 1). Cyclophosphamide treatment decreased the total leukocyte count by 97 ± 1% to 0.08 ± 0.01 x 109/L, and decreased the fraction of neutrophils to 34.7 ± 0.9% (Table 2). Cyclophosphamide pretreatment abolished the effect of LPS, which did not decrease Ees, [dP/dtm a x]/EDV, or ejection fraction (Table 1) in these mice. 3.4.3 Endotoxemic ICAM-1 knock out mice: The ratio of ICAM-1 to nuclear immunofluorescent staining was greatly decreased in ICAM-1 knock out mice (0.15 ± 0.09) compared to C57B6 wild-type mice (1.96 ±0.16, p<0.05) and did not increase with LPS injection (0.18 ± 0.13) (Figure 2). In contrast to C57B6 wild-type mice, left ventricular contractility and function in ICAM-1 knock out mice, as measured by Ees (16.1 ± 3.6), [dP/dtm a x]/EDV (518 ± 146), and ejection fraction (46 ± 6%), was not 87 decreased (p=NS; Table 1 and Figure 3B). Similarly, six hours after LPS injection heart rate changed less in ICAM-1 knock out mice (increased 14%) compared to C57B6 wild-type mice (increased 72%) while end-systolic pressure decreased to the same extent in ICAM-1 knock out mice (by 24%) compared to C57B6 wild-type mice (by 23%). 3.4.4 Endotoxemic chimeric mice: LPS injection decreased left ventricular contractility of M+/H+ and M-/H+ chimeric mice. Specifically, Ees decreased by 43 ± 7% (p<0.05) in M+/H+ chimeric mice and Ees decreased by 56 ± 13% (p<0.05) six hours after LPS injection in M-/H+ chimeric mice. These changes in left ventricular contractility in M+/H+ and M-/H+ mice were not different from the effect of LPS injection in C57B6 wild-type mice (Table 1 and Figure 3). In contrast, LPS injection did not decrease left ventricular contractility of M+/H- chimeric mice. Indeed, in M+/H-chimeric mice LPS injection resulted in increases in contractility as measured by Ees (24 ± 3.7 in LPS group vs 13 ± 2.2 in controls), [dP/dtm a x]/EDV (1012 ± 178 in LPS group vs 500 ± 90 in controls) and ejection fraction (54 ± 8% in LPS group vs 45 ± 4% in controls, p=NS, Table 3). Taken together, the lack of LPS-induced myocardial dysfunction in H -mice compared to the significant LPS-induced myocardial dysfunction in both M+ or M -/ H+ mice indicate that cardiac ICAM-1 expression is an important factor leading to decreased left ventricular contractility following LPS injection. Injection of the bone marrow concentrate into the chimeric recipient mice significantly increased the peripheral blood total leukocyte count compared to non-injected cyclophosphamide-treated mice, confirming successful bone marrow transplantation. In 88 M+/H- mice the peripheral blood total leukocyte count was 1.2 ± 0.04 x 109/L, the fraction of neutrophils was 29.8 ± 3.7%, and the fraction of lymphocytes was 23.3 ± 3.6%. In M+/H+ mice the peripheral blood total leukocyte count was 1.2 ± 0.05 x 109/L, the fraction of neutrophils was 46.8 ± 5.6%, and the fraction of lymphocytes was 18.9 ± 5.3%. 89 Table 3.3 Hemodynamics and ventricular function measures in chimeric mice Chimera Saline Chimera M + / H + LPS Chimera M7H + LPS Chimera M +/H" LPS Number 5 4 6 6 Heart rate (bpm) 290 ± 17 414 ±12 * 384 ±27 * 382±18 * End Systolic Pressure (mmHg) 80 ±4 61 ±8.2 52 ±6.5 73 ± 10 End Diastolic Pressure (mmHg) 4.5 ±0.8 5.2 ±0.7 4.9 ±0.6 2.5 ±0.9 End Systolic Volume (uL) 6.4 ±0.8 15.5 ±2.9 4.2 ± 1.0 * 2 ± 0.4 * End Diastolic Volume (uL) 11.5 ±0.8 33 ±8 * 6.2 ± 1.5 * 4.1 ±0.6 * Ejection fraction 45 ±4 29 ±8 * 49 ± 8 54 ± 8 M + : ICAM-1 competent bone marrow cells M": ICAM-1 knock out bone marrow cells Ft: ICAM-1 competent heart H": ICAM-1 knock out heart * p<0.05 compared to slaine treated chimera. 90 Volume (uL) Figure 3 Stylized pressure volume loops drawn using average end-systolic elastance (Ees), end-diastolic volume (EDV), end-diastolic pressure (EDP) and end-systolic pressure (ESP) as reported in Table 1. End-systolic volume (ESV) derived from these plots corresponds closely (but not exactly due to averaging) to the measured ESV values reported in Table 1. A) In C57B6 wild-type mice, LPS results in a significant decrease in left ventricular contractility as measured by Ees. This is accompanied by an increase in ESV and EDV. B) In ICAM-1 knock out mice, LPS does not result in a decrease in left ventricular contractility measured by Ees. Figure 3.3B Volume (uL) 91 Table 3.4 Peripheral blood cellular components in chimeric mice Chimera M + / H + Saline Chimera M+/¥t LPS Chimera M + / H " Saline Chimera M7H" LPS Chimera MVtf LPS W B C X 109/L 1.2 ± 0.1 0.05 ±0.01* 1.2 ±0.04 0.06 ±0.01 * 0.05 ±0.01 * Neutrophil X 10 9 /L 0.6 ±0.1 0.04 ±0.01 * 0.4 ±0.1 0.04± 0.001* 0.03 ±0.01 * Lymphocyte X 109/L 0.2 ± 0.07 0.01 ±0.003 * 0.3 ±0.1 0.01 ±0.006* 0.01 ±0.001 * Red blood cell X 10 1 2/L 7.0 ±0.2 7.4 ±0.2 7.4 ± 0.2 8.3 ±0.5 7.9 ± 0.4 Platelet X 109/L 405 ± 18 109 ±9 * 466 ± 79 157 ± 6 4 * 131 ± 1 6 * M + : ICAM-1 competent bone marrow cells M": ICAM-1 knock out bone marrow cells H + : ICAM-1 competent heart Ff: ICAM-1 knock out heart * p<0.05 compared to all saline treated groups. 3.4.5 Morphometery of coronary vascular space: The LPS injection in C57B6 wild-type mice increased coronary intravascular neutrophil concentration from 2.5 ± 1.9 x 10 _ 5 /um 2 in control to 137 ± 22 x 10 _ 5 /um 2 in septic mice. LPS injection also increases the ratio of adherent neutrophil to endothelial surface from 3.2 ± 2.3 x 10 /um in control to 99 ± 18 x 10 " 5/um 2 in endotemic mice (p< 0.05). However LPS injection did not increase extracellular neutrophil infiltration into the cardiac tissue (Figure 4). 93 0.0018 CM 0.0016 ^ 0.0014 CD « 0.0012 0.0010 O i— "3 0.0008 CD ^ 0.0006 -| CD _Q 0.0004 E Z 0.0002 0.0000 • £ iijp J n Intravscular Neutrophi ls Adherent Neutrophils Extravascular Neutrophi ls J F i = C57B6 C57B6 + LPS ICAM-1 KO ICAM-1 KO + LPS Figure 4 Formalin fixed heart sections were stained with DAB and Hematoxyline & Eosin to measure the ratio of the number of inflammatory cells per surface area of intravascular and perivascular spaces. LPS injection significantly increased this ratio in the intravscular space (*p<0.01 compared to saline treated C57B6). The number of adherent inflammatory cells on endothelial surface is also significantly increased in LPS-treated mice (*p<0.01 compared to saline treated ICAM-1 KO). However this ratio is not significantly different in the perivascular space in LPS-treated mice. 94 3.5 Discussion LPS injection reduced left ventricular contractility in this whole animal model of systemic inflammation, similar to previous measurements in a variety of animal models of sepsis, and similar to the decrease in left ventricular function observed during human septic shock. Decreasing leukocyte count using cyclophosphamide abrogated the effect of LPS, confirming previous reports that leukocytes contribute to the development of decreased left ventricular contractility in sepsis. ICAM-1 knock out mice were resistant to the effect of LPS on left ventricular contractility indicating that ICAM-1 plays an important causal role in mediating the effect of LPS. Chimeric mouse constructs further implicate cardiac ICAM-1 expression, versus ICAM-1 expressed on bone marrow-derived cells transiting the heart. Thus, signaling via cardiac ICAM-1 is necessary for the development of leukocyte-dependent decreases of left ventricular contractility in this whole animal model of systemic inflammation. Many mechanisms contribute to myocardial depression during sepsis 5 4 . In sepsis, release of pathogenic toxins such as LPS from gram negative bacteria, glycolipid from gram positive bacteria, or mannan from fungal wall initiate a systemic inflammatory response. Pro-inflammatory cytokines trigger increased NO production by NOS III within minutes, and greatly increased NO production by NOS II within hours 2 0 4 ' . NO is an important mediator of myocardial dysfunction in sepsis 7 1 > 2 0 4 ' 2 0 5 . Reactive oxygen intermediates contribute directly and via formation of peroxynitrite radicals, to myocardial damage and dysfunction 1 6 3 , 2 0 5 . Coronary capillary endothelial activation, damage and dysfunction also contribute, in part by impaired regulation of coronary 95 microvascular blood flow, which impairs myocardial oxygen extraction 3 1 . Leukocytes also contribute. Leukocytes are slowed and retained within the coronary vasculature after endotoxin infusion 2 0 3 , predominantly within coronary capillaries. Leukocyte retention is associated with myocardial morphometric damage and decreased ventricular contractility 1 9 6 . In an ex vivo preparation, filtering leukocytes from coronary blood perfusing the heart prevented the decrease in contractility within the first 6 hours of endotoxin infusion 1 9 7 '. Activated neutrophils appear to be a particularly important leukocyte subset involved in early myocardial depression 6 5 . Our current results support these previous observations by demonstrating that the LPS-induced decrease in left ventricular contractility is leukocyte dependent - being abrogated by a decreased peripheral blood leukocyte count. Part of the leukocyte effect may be due to binding of leukocytes to cardiac ICAM-1. Pro-inflammatory cytokines, including IL-6 generated by the myocardium following ischemia-reperfusion, result in CD 18 - ICAM-1 dependent adhesion of P M N to cardiomyocytes 1 7 1 . Adhesion of highly (unphysiologically) activated P M N to cardiomyocytes results in cardiomyocyte cell death in vitro, possibly due to reactive oxygen intermediates 5 9 ' 1 0 7> 1 6 3 ' 1 6 4 . Co-culture of macrophages or neutrophils with cardiomyocytes resulted in ICAM-1 dependent adhesion and decreased cardiomyocyte fractional shortening 5 9 ' 6 0 . Adhesion alone was a fundamentally important aspect of this interaction because the same effect was reproduced by co-culture with killed and fixed neutrophils or simply by ICAM-1 cross linking antibodies60. 96 ICAM-1 is a 76 - 110 kDa glycoprotein from the immunoglobulin gene family ' . ICAM-1 expression by a variety of different cell types, including on cardiomyocytes, increases during inflammation 5 9 - 6 0 ' 1 0 7 , Activated coronary endothelial cells in cell culture increase ICAM-1 expression in 2 to 4 hours and maximum ICAM-1 expression is reached in 6 to 8 hours. High levels of ICAM-1 expression are maintained as long as the inflammatory response is active 1 0 9 . Activation of ICAM-1 stimulates further production and expression of vascular cellular adhesion molecule-1 (VCAM-1) and ICAM-1 Activation of isolated cardiomyocytes increases ICAM-1 mRNA production within 1 to 4 hours 6 0 and expression of ICAM-1 protein within 4-6 hours 5 9 . Maximum production of ICAM-1 occurs 18 to 24 hours after cytokine stimulation 1 0 7 . Ligands for ICAM-1 include CD 11/CD 18 receptors 2 0 6 ' 2 0 8 ' 2 0 9 , fibrinogen 1 3 5 , rhinovirus receptors 2 1 0 and 911 Plasmodium falciparum Our current findings extend these in vitro observations and demonstrate that binding of ICAM-1 expressed on cardiomyocytes reduced left ventricular contractility in a whole animal model of systemic inflammation. Using isolated rat cardiomyocytes we have shown that activation of ICAM-1 using cross-linking antibodies can initiate intracellular signals which cause discoordinate calcium influx leading to reduction in cardimyocyte contractility 6 0 . This effect of ICAM-1 cross-linking mediated via the cytoskeleton 6 0 . The presence of cardiac contractile dysfunction after LPS injection in M+/H+, and M -/H+ chimera mice, and absence in M+/H- chimera mice demonstrates the importance of ICAM-1 expression within cardiac tissue. Lack of ICAM-1 on bone marrow-derived inflammatory cells did not prevent the effect of LPS on cardiac contractility. 97 Several potential ligands may be involved in ICAM-1 binding and activation in this whole animal model of systemic inflammation, which results in decreased left ventricular contractility. Since we 1 9 7and others 1 9 6 have demonstrated that the LPS-induced decrease in left ventricular contractility is, in part, leukocyte dependent, it is reasonable to postulate that CD11/CD18, or other leukocyte expressed ICAM-1 ligands, bind and activate ICAM-1 expressed on cardiomyocytes in vivo. However, we did not find many neutrophils or other leukocytes within the cardiac parenchyma in this and other models of sepsis 6 5 . It is conceivable that leukocytes enter cardiac parenchyma but undergo apoptosis or other forms of cell death quickly and leave CD11/CD18 to bind to ICAM-1 expressed on cardiomyocytes. However, this has not been demonstrated. We propose an alternative mechanistic pathway to account for the observation of leukocyte-dependent decreases in left ventricular contractility mediated by cardiomyocyte expressed ICAM-1 binding and activation. Current and previous observations demonstrate that leukocytes are retained within coronary capillaries in systemic inflammatory states 6 5 . These leukocytes damage the capillary endothelium 6 5 leading to interstitial edema. We postulate that the interstitial edema fluid components that are ICAM-1 ligands, including fibrinogen, could then lead to decreased ventricular contractility via the ICAM-1 binding. This postulated pathway is consistent with the observations of Neviere and his colleagues 196 In summary, we found that LPS injection in mice resulted in a substantial decrease in left ventricular contractility. This decrease was leukocyte dependent and also dependent on cardiac ICAM-1 expression. However, we did not observe a large number of leukocytes 98 within the cardiac parenchyma at the time of substantially decreased left ventricular contractility. Therefore, we postulate that this leukocyte-dependent decrease in left ventricular contractility involves at least two steps in series - a leukocyte dependent step and an ICAM-1 dependent step. Leukocyte retention leading to endothelial damage and leakage of ICAM-1 ligands, such as fibrinogen, into the cardiac interstitium is one explanation compatible with our findings. We conclude that signaling via cardiac I C A M -1 expression is necessary to mediate leukocyte-dependent decreases of left ventricular contractility in endotoxemic mice. 99 Chapter-4 The role of fibrinogen and ICAM-1 interaction in decreased cardiac contractility 4.1 Abstracts Fibrinogen is involved in coagulation and inflammatory responses. Interaction of I C A M -1 and fibrinogen promotes platelet and leukocyte adhesion onto endothelial cells. Cardiomyocyte ICAM-1 activation decreases cardiomyocyte contractility through an increase in heterogeneity of Ca release. We hypothesize that during sepsis fibrinogen can infiltrate into the myocardial interstitial space and decrease cardiomyocyte contractility through the activation of ICAM-1. To investigate whether fibrinogen can infiltrate into the myocardial interstitial space and affect ICAM-1 on cardiomyocyte membranes, C57B6 mice were injected with LPS (40 mg/kg) or normal saline. Six hours later hearts were excised and stained with horse radish peroxidase conjugated anti-mouse fibrinogen antibody or reperfused with fibrinogen-labeled Alexa-488 for 10 minutes using an ex-vivo model and Langendorff system. To test whether the fibrinogen binding to ICAM-1 decreases cardiomyocyte contractility, isolated rat cardiomyocytes were co-cultured with soluble fibrinogen or fibrinogen coated beads for 3 to 4 hours. Cardiomyocyte fractional shortening was measured in the presence or absence of anti-ICAM-1 antibody. Confocal microscopy of heart sections showed infiltration of the fibrinogen labeled Alexa-488 into myocardial interstitial tissue in septic mice. Immunohistochemistry of 100 heart sections indicated lack of fibrinogen infiltration into the myocardial tissue of septic ICAM-1 knock out mice. Incubation of activated cardiomyocytes with soluble fibrinogen decreased cardiomyocyte fractional shortening from 17.2 ± 0.8% to 14.4 ± 0.5%. Incubation of cardiomyocyte with fibrinogen coated beads decreased cardiomyocyte contractility 16.8 ± 1.0% to 10.7 ± 1.0%. This effect of fibrinogen was abolished in the presence of anti ICAM-1 antibody (17.7 ± 0.6%) indicating an interaction of fibrinogen and ICAM-1. We conclude that fibrinogen infiltrates into the myocardial interstitial space during sepsis and decreases cardiac contractility through adhesion to the ICAM-1 receptor on the cardiomyocyte membrane. 101 4.2 Introduction Fibrinogen, 340 kDa protein, is involved in coagulation and inflammatory responses. Fibrinogen adhesion to ICAM-1 increases tyrosine phosphorylation. Interaction of ICAM-1 and fibrinogen promotes platelet 1 4 7 and leukocyte adhesion 2 1 2 onto endothelial cells. Cardiomyocyte ICAM-1 activation decreases cardiomyocyte contractility through an increase in heterogeneity of C a 2 + release 6 0 . We hypothesize that during sepsis fibrinogen can infiltrate into the myocardial interstitial space and decrease cardiomyocyte contractility through the activation of ICAM-1. Fibrinogen contains a, (3 and y chains. It is involved in the pathogenesis of various types of diseases, including ischemia, trauma and neoplasm. Increased plasma fibrinogen level is associated with a higher risk of cardiovascular diseases, increased risk of myocardial infarction, stroke, peripheral arterial disease, atrial fibrillation, heart failure and post angioplasty restenosis 2 1 3 , 2 1 4 . Fibrinogen increases vascular permeability by detachment of endothelial cells from the basement membrane 1 4 2 . It has been shown that fibrinogen can enhance adhesion of inflammatory cells and facilitates platelet adhesion to the endothelial surface during arteriosclerosis thereby accelerating atherosclerotic plaque formation 2 1 5 . Adhesion of fibrinogen on CD11 receptors of P M N reduced caspase activation and apoptosis of P M N 2 1 6 . Saito and his colleagues have shown the extravasation of fibrinogen in rat plural cavity after histamine injection. This effect of fibrinogen is associated with increased P M N infiltration into the plural cavity suggesting the close relation between P M N infiltration and fibrinogen exudation 2 1 7. Fibrinogen can increase the expression of ICAM-1 through activation of N F K B on human synovial 102 fibroblasts . Languino et al. demonstrated that fibrinogen functions as an intermediate protein for adhesion of monocytes to the endothelial cells Fibrinogen contains two peripheral, one central and three global domains. A specific site of the y chain of the fibrinogen molecule, between amino acid 117-133, is responsible for adhesion to amino acid 8-22 of the first extracellular domain of ICAM-1. Our previous findings have indicated that P M N adhesion can decrease cardiomyocyte contractility and that this effect of P M N occurs through interaction of the ICAM-1 receptor on cardiomyocyte membrane and CD11/CD18 receptors on inflammatory cells. We subsequently found that activation of ICAM-1 on cardiomyocytes using cross-linking methods decreased cardiomyocyte contractility (refer to chapter 2). We also found that cardiac contractility of ICAM-1 knock-out mice was preserved after LPS treatment. This was resistant to the effect of LPS because of the absence of ICAM-1 receptors on cardiac tissue (refer to chapter 3). However we did not observe infiltration of PMNs into the cardiac interstitial space after LPS injection, suggesting that, PMNs are not directly involved in ICAM-1 activation in vivo. We tested whether fibrinogen can infiltrate into the myocardium after LPS injection and i f fibrinogen can induce decreased cardiomyocyte contractility. 103 4.3 Materials and Methods 4.3.1 Induction of sepsis: C57B6 wild-type and ICAM-1 knock out mice (25-30 g, Jackson Laboratory, Bar Harbor, ME) were injected with purified Escherichia Coli lipopolysaccharide (LPS, 40mg/kg, strain 01 II: B4) or normal saline via intraperitoneal injection. Six hours after LPS injection, mice were anesthetized using Isoflurane 3%. Anesthesia was tested by toe-pinch to ensure absence of withdrawal to pain throughout the procedure. The heart was excised, frozen or fixed (10% Formalin) for further immuno-fluorescent or immuno-histochemistry studies. 4.3.2 Immuno-histochemistry and Immuno-fluorescent study: Formalin fixed hearts were embedded and 6 urn sections were prepared. After de-parafinization, sections were treated with rabbit anti-mouse fibrinogen (Inovation Research Southfield, MI) overnight follow by HRP-conjugated anti- rabbit antibody (BD Pharmingen, San Jose, CA) for 2 hours. The slides were then treated with D A B for 30 minutes follow by hematoxylin staining. Images (X400) were captured using spot camera. 4.3.3 Reperfusion of Alexa-488 labeled fibrinogen into the LPS or saline treated mice: In the preliminary experiments using a mixed in-vivo and ex-vivo method, animals were treated with LPS or saline for 6 hours. Hears were excised and cannulated using a 20 G needle and reperfused using a Langendorff apparatus as described in chapter-5 section-3. To test whether fibrinogen can infiltrate into the cardiac interstitial space, cannulated hearts were reperfused with fibrinogen labeled Alexa-488 (Molecular probe) for 15 minutes while monitoring pulse pressure. The hearts were frozen with Isopentane 104 in liquid nitrogen. Sections (6 um) from frozen hearts were prepared and stained with anti-von Willebrand factor (BD Pharmingen) overnight at 4 °C to illustrate coronary endothelial surface. Anti nuclear staining (Hoechst) was used to visualize nuclei. Sequential images were captured using confocal microscopy (LEICA DIMRE2, Exton, PA). 4.3.4 Incubation of soluble fibrinogen with isolated rat cardiomyocytes: Male Sprague-Dawley rat cardiomyocytes were isolated and cultured in 96 well plates as previously described in chapter-2, section-3. To increase ICAM-1 presentation on cardiomyocyte membrane, cultured cardiomyocytes were activated with T N F a (20 ng/ml) for 3 to 4 hours. 25000 fibrinogen coated, B S A coated and plain non-coated beads were incubated with 5000 cardiomyocytes in each well (100 uL) for 4 hours. In a separate set of experiments, cardimyocytes were also co-cultured with different concentrations of soluble fibrinogen. Cardiomyocyte fractional shortening was measured as previously described in chapter-2, section-3. 4.3.5 Coating of Fibrinogen to Polystyrene Beads: Polystyrene Beads (Bangs Laboratories; 8 um in diameters) were washed twice (5 minutes per wash) with acetate buffer (pH 5.4) and mixed with rat fibrinogen (Enzyme Research Laboratories) (300000 beads/ug fibrinogen, 2% solid in buffer) in a 500 uL eppendorf tube. The mixture was gently stirred for 2 hours at room temperature to promote fibrinogen adhesion to the plain beads through hydrophobic interactions. The beads were then washed three times with 105 fresh acetate buffer. The fibrinogen coated beads were re-suspended by passing them through a syringe with a 27.5G needle and co-cultured with cardiomyocytes. 4.3.6 Blocking of Cardiomyocyte ICAM-1: After TNF-a activation, the cardiomyocytes were incubated with an anti ICAM-1 blocking antibody (BD Biosciences) or control non-specific mouse IgG (DAKO) for 2 hours. They were co-cultured with fibrinogen coated beads as described previously. 106 4.4 Results 4.4.1 Fibrinogen infiltration into the interstitial space (in-vivo): Immunohistochemistry (IHC) was used to assess whether plasma fibrinogen infiltrates into the myocardium during endotoxemia. Fibrinogen staining was observed on cardiac sections of LPS treated animals indicating the presence of fibrinogen in the coronary vascular, perivascular and cardiac interstitial space. Fibrinogen staining in control sections was limited to the endothelial and endocardial surface (Figure 4-1). In LPS treated hearts, deposition of fibrinogen was observed in the myocardium immediately surrounding the vascular space, whereas in saline treated hearts, fibrinogen was located at the edge of the blood vessels and coronary endothelial surface (Figure 4-1). IHC staining did not show infiltration of fibrinogen in the cardiac interstitial space of ICAM-1 K O mice confirming the above observation. However coronary endothelial cells of ICAM-1 K O mice were stained with fibrinogen antibody (Figure 4-2) indicating the presence of fibrinogen in the serum of ICAM-1 K O mice. 107 Figure 4.1 Immunohistochemistry of heart sections illustrate the presence of fibrinogen in the myocardial interstitial and perivascular spaces of LPS treated C57. In saline treated mice fibrinogen staining is limited to the vascular space. 108 1 K O saline Figure 4.2 IHC of the heart sections of ICAM-1 knock out mice indicate the presence of fibrinogen staining in the vascular space of LPS or saline treated animals. Fibrinogen staining was not observed in the myocardial perivascular and interstitial spaces of ICAM-1 knock out mice indicating the protective effect observed in ICAM-1 knock out mice against infiltration of fibrinogen into the myocardial perivascular or interstitial spaces. 109 Confocal images of LPS or saline treated hearts (reperfused with Alexa-488 labeled fibrinogen ex-vivo) illustrate the presence of Alexa-488 in the coronary perivascular spaces of LPS treated mice (Figure 4.3 A). This shows the presence of a leaky endothelial surface and the feasibility of infiltration of labeled fibrinogen into the cardiac interstitial space of LPS treated mice. In saline treated mice the presence of Alexa-488 is limited to the coronary vascular space (Figure 4.3A). The ratios of fibrinogen to nuclear staining (Fib/Nuc) and of fibrinogen to vonWillebrand factor (Fib/vWf) are higher in LPS treated hearts indicating wide distribution of fluorescent fibrinogen in the cardiac tissue (Figure 4.3B, p<0.05). 110 1 • | C57 saline C57 LPS * C57 saline j C57 LPS j Figure 4.3A Illustrates the retrograde reperfusion of fluorescent fibrinogen (green) into the coronary arteries of LPS or saline treated mice using an ex-vivo model of reperfusion. Vascular spaces were stained with anti-vonWillebrand factor antibody (red). Confocal images indicate the presence of Alexa-488 fibrinogen in the vascular space of saline treated mice. In the LPS treated mice fibrinogen infiltrates into the myocardial perivascular and interstitial spaces. I l l 1.0 C57 saline C57 LPS Figure 4.3B C57B6 mice were treated with saline or LPS. Six hours later the heart was excised and reperfused with fluorescent fibrinogen for 15 minutes using a Langendorff apparatus. The hearts were frozen and stained with anti-nuclear and vonWillebrand factor antibodies. The ratio of fluorescent fibrinogen (green) to the nuclear staining (blue) or vonWillebrand factor (red) were compared between saline or LPS treated groups (*p<0.01 and # p<0.05). 112 4.4.2 Fibrinogen effect on cardiomyocyte contractility (in-vitro): Four hour incubation of different concentrations of soluble fibrinogen (0.025, 0.05, 0.1 and 0.2 mg/mL) with activated cardiomyocytes, decreases cardiomyocyte contractility in a dose-response dependant manner (Figure 4.4). The maximum effect of fibrinogen was achieved with a 0.2 mg/mL concentration of fibrinogen (-26.5±7.9%, p<0.004 vs. control). To demonstrate that the decrease in cardiomyocyte contractility is due to the effect of fibrinogen, polystyrene beads were coated with fibrinogen and co-cultured with activated cardiomyocytes (Figure 4.5). Confocal microscopy confirmed the deposition of fluorescent labeled fibrinogen onto the beads, indicating the successful adhesion of fibrinogen to the beads (Figure 4.6). 113 0.00 0.05 0.10 0.15 0.20 0.25 Fibrinogen Concentration (u.M) Figure 4.4 Co-incubation of soluble fibrinogen with activated cardiomyocytes decreased fractional shortening of cardiomyocytes in a dose dependant response manner (EC50 = 0.468, Hill slope = -1.0, Min = 0.0, and Max = 4.56, pO.OOl). 114 Figure 4.5 Adhesion of Fibrinogen Coated Beads. Fibrinogen coated beads remained adherent to cardiomyocytes during diastole (A) and systole (B). Figure 4.6 Polystyrene beads were coated with Oregon Green labeled fibrinogen and viewed under the fluorescence microscope (A) and confocal microscope (B and C). 115 Attachment of fibrinogen coated beads to the activated cardiomyocytes decreased cardiomyocyte fraction shortening by 32.0 ± 3.8% (10.7 ± 1.0) compared to control ( 16.9 ± 0.8, pO.OOl). Plain beads and BSA coated beads (negative control) did not decrease the fractional shortening of activated cardiomyocytes (16.8 ±1 .0 and 17.5 ± 0.8 p=NS, Figure 4.7). The adhesion of fibrinogen coated beads to cardiomyocytes also induced a dose dependent decrease in cardiomyocyte fractional shortening, confirming the similar effect of soluble fibrinogen oh fractional shortening (Figure 4.7). 4.4.3 Fibrinogen induces decreased cardiomocyte contractility through ICAM-1 ICAM-1 antibody decreases the number of adherent fibrinogen coated beads on the cardiomyocyte membrane. Incubation of activated cardiomyocytes with anti-ICAM-1 abrogates the effect of fibrinogen on cardiomyocyte contractility, indicating that the effect of fibrinogen is mediated by the ICAM-1 signaling cascade. Incubation of activated cardiomyocytes with non-specific IgG did not prevent adhesion of fibrinogen coated beads or the effect of fibrinogen on cardiomyocyte contractility, confirming the interaction between fibrinogen and ICAM-1 in co-culture experiments, and fibrinogen ICAM-1 mediated decreased contractility (Figure 4.7). 116 22 20 H 6 J 1 . — i 1 1 Control 0 1 to 2 3 to 4 5 to 9 Number of adherent fibrinogen coated bead / cardiomyocyte Figure 4.7 The effect of fibrinogen coated beads on cardiomyocyte fractional shortening. Fibrinogen coated beads decrease cardiomyocyte fractional shortening in a dose dependent manner. Incubation of co-culture with anti-ICAM-1 antibody inhibits the above effect of fibrinogen coated beads on cardiomyocyte fractional shortening, indicating the essential interaction of ICAM-1 and fibrinogen for a decrease in fractional shortening. Nonspecific IgG did not prevent the effect of fibrinogen on cardiomyocyte fractional shortening. 117 4.5 Discussion Myocardial dysfunction is one of the major causes of death in septic patients. Cardiac inflammation is associated with increased ICAM-1 expression on the endothelial cells and cardiomyocytes 5 9 ' 6 0 ' 2 1 8 . ICAM-1 is a 114 kDa protein adhesion molecule, activated by binding to various types of ligands including CD 11/CD 18 receptors of inflammatory cells , fibrinogen ~ , Plasmodium falciparum and rhinoviruses. We have shown that ICAM-1 activation on cardiomyocytes initiates a signaling cascade and alters calcium propagation during cardiomyocyte excitation contraction which results in decreased cardiomyocyte contractility 6 0 . Although the number of cardiac intravascular inflammatory cells is significantly higher in septic animals than control we did not observe infiltration of inflammatory cells into the cardiac interstitial space thereby we concluded that cardiac ICAM-1 can not be activated through binding of CD11/CD18 receptors on inflammatory cells during sepsis. Fibrinogen is a 340 kDa protein involved in coagulation and inflammation. It has been shown that fibrinogen can attach to ICAM-1 919 910 and phosphorylates various intracellular tyrosine residues ' . We found that incubation of cardiomyocytes with both soluble fibrinogen and fibrinogen coated beads can decrease fractional shortening. This effect of fibrinogen occurs through the adhesion of fibrinogen to cardiomyocyte membranous ICAM-1. We also found that cardiac sections of LPS treated mice (in-vivo studies) were stained with anti-fibrinogen antibody indicating the availability of fibrinogen in the interstitial space of cardiac tissue for activation of cardiomyocyte ICAM-1. This finding is confirmed by the ex-vivo infusion of fibrinogen labeled Alexa-488 showing infiltration into the cardiac section of LPS treated mice. 118 The y chain of fibrinogen binds to domain-1 of ICAM-1 through its 117-133 amino acid sequence 1 3 4 , 1 3 5 . Fibrinogen increases endothelial leakiness and enhances angiogenesis on endothelial cells 1 4 M 4 3 . We have reconfirmed the leakiness of the endothelial surface of coronary arteries using fibrinogen labeled Alexa-488 in the ex vivo retrograde reperfusion and immunohistochemistry of in-vivo LPS injection models. Upregulation of ICAM-1 in cardiovascular diseases is also accompanied by high expression of fibrinogen 1 4 5 , 1 4 6 . Fibrinogen and ICAM-1 interaction promotes platelet and leukocyte adhesion to endothelial cells 1 4 7 . This effect of fibrinogen was inhibited using ICAM-1 knock out mice and a model of ischemia reperfusion injury 1 4 8 . Hicks et al. have shown that fibrinogen deposition on endothelial cells induced vasodilation, however when they increased fibrinogen concentration this effect was converted to vasoconstriction 1 4 9 . Fibrinogen deposition also upregulated ICAM-1 expression through activation of NFK(3 and IL-8 production 1 4 6 ' 1 5 0 . Regardless of the main cause of myocardial depression during sepsis, we postulated that during sepsis LPS and cytokine release upregulates the expression of ICAM-1 on coronary endothelial cells and cardiomyocytes. Acute phase response during sepsis also increases the level of intravascular fibrinogen, enhancing inflammation and the coagulation cascade. Fibrinogen increases the adhesion of inflammatory cells on coronary endothelial cells which is followed by adhesion of inflammatory cells to endothelial ICAM-1 and V C A M - 1 . In turn inflammatory cells, including PMNs, release cytotoxic materials onto the coronary endothelial cells which results in the loosening of endothelia cell tight junctions and increased leakiness. This is followed by infiltration of 119 soluble fibrinogen into the cardiac tissue increasing probability of interaction of ICAM-1 and fibrinogen in the cardiac tissue leading to activation of ICAM-1 and the initiation of intracellular signaling. Activation of ICAM-1 increases the heterogeneity of intracellular C a 2 + release which reduces cardiomyocyte contractility. Whether fibrinogen can attach to other inflammatory adhesion molecules such as V C A M - 1 remains to be determined. Whether other postulated ICAM-1 ligands such as soluble of CD 11/CD 18 can infiltrate into the cardiac tissue through the leaky endothelium should also be determined. 120 Chapter-5 2IGF-1 protection of ischemic murine myocardium from ischemia-reperfusion associated injury 5.1 Abstract Introduction: Ischemia-reperfusion injury occurs in myocardial infarction, cardiac dysfunction during sepsis, cardiac transplantation and coronary artery bypass grafting and results in injury to the myocardium. Although reperfusion injury is related to the nature of ischemia, it is a separate entity that may jeopardize viable cells and ultimately may impair cardiac performance one ischemia is resolved and the organ heals. Method: The present study was conducted in an ex vivo murine model of myocardial ischemia-reperfusion injury. After 20 minutes of ischemia, isolated hearts were perfused for up to 2 hours with solution (modified Kreb's) only, solution plus insulin growth factor (IGF-1), or solution plus tumor necrosis (TNFa). Cardiac contractility was monitored continuously during this period of reperfusion. Results: On the basis of histological evidence, IGF-1 prevented reperfusion injury as compared with TNFa; T N F a increased perivascular interstitial edema and disrupted tissue lattice integrity, whereas IGF-1 maintained myocardial cellular integrity and did not increase edema. Also, there was a significant reduction in detectable creatine phosphokinase in the perfusate from IGF-1 treated hearts. By recording transduced pressures generated during the cardiac cycle, reperfusion with IGF-1 was accompanied by markedly improved cardiac performance as compared with reperfusion with T N F a or modified Kreb's solution only. The histological and functional improvement generated 2 The major part of this chapter has been published in Journal of Critical Care. 2003 Dec;7(6):Rl 76-83. 121 by IGF-1 was characterized by maintenance of the ratio of mitochondrial to nuclear D N A within heart tissue. Conclusion: We conclude that IGF-1 protects ischemic myocardium from further reperfusion injury, and that this may involve mitochondria-dependent mechanisms. 122 5.2 Introduction Cardiovascular diseases are among the leading causes of death in North America. The most important presentation of cardiovascular disease is ischemia, which leads to tissue hypoxia, cellular necrosis and apoptosis and in severe situations, organ dysfunction. The main treatment of acute ischemic heart disease is early vascular reperfusion to restore balance to cardiac metabolic demands. Although reperfusion is the foundation of therapy, it may actually initiate further injury to the myocardium. Although the phenomenon of reperfusion injury is related to the duration of ischemia, it is a separate entity and may be more severe than ischemic injury alone 2 2 0 , 2 2 1 . Ischemia-reperfusion injury can be generated in various cardiovascular diseases or therapies including myocardial infarction, cardiopulmonary bypass, coronary bypass grafting, heart transplantation and coronary thrombolytic therapy. It has also been speculated that the mechanism of myocardial dysfunction during septic shock is related to segmental ischemia and reperfusion in the left ventricular wall, because the involvement of 50 222 persistent global ischemia has been disproved ' . Ischemia results from the absence of or sluggish blood flow in coronary vessels. This leads to a mismatch between cardiac metabolic supply and demand. Ischemia of short duration may contribute to stunned myocardium without tissue injury, but prolonged ischemia results in a deficiency in energy supplies and waste removal, with eventual 93 initiation of cellular necrosis and priming of the myocytes for apoptosis . Early restoration of blood flow or reperfusion reduces the extent of myocardium at risk for death from necrosis. However in the presence of prolonged ischemia, reperfusion itself 123 initiates mechanisms of injury that are fundamentally different and potentially more severe than those of ischemia. Reperfusion injury is mediated by inflammation and characterized by the production of reactive oxygen species (ROS). Production of ROS may be initiated during the ischemic phase generating "primed" myocardium. ROS activate transcription factors, such as N F K B , both in cardiac myocytes and the endothelium; in turn, this initiates transcription of genes including those encoding adhesion molecules, cytokines, coagulation mediators and proteolytic enzymes 2 2 3 . In coordination with the complement cascade, ROS can 994 disrupt the integrity of both cardiac myocyte and endothelial cell membranes . These events can change the intracellular ion homeostasis, resulting in the accumulation of calcium and metabolic by-products. These changes increase the activation of enzymes that are utilized in the processes of necrosis and apoptosis and that alter mitochondrial function 1 0 6 . At the tissue level, this is manifested by interstitial edema and disruption of the tissue lattice. Concomitantly, neutrophils and other inflammatory cells migrate into the injured zone using adhesion molecules such as ICAM-1 under the stimulation of secreted cytokines and chemotactic factors. Recruitment and infiltration of neutrophils into the injured tissue is accompanied by neutrophils degranulation and further injury to the border zone of viable cells. These late cellular events in the myocardium only occur after reperfusion 9 3 ' 1 0 3> 2 2 1 ' 2 2 5 , Our previous experiments demonstrated that expression and activation of ICAM-1 is associated with decreased cardiomyocyte contractility and cardiac depression (refer to Chapter 2 and 3) 6 0 . 124 Sustainable functioning of the myocardium is the central objective of therapeutic intervention in myocardial infarction. Cardiac function and contractility are closely related to cardiac metabolism and energy production. In cardiomyocytes energy production is related to the number of mitochondria, with this organelle occupying up to 40% of the cardiomyocyte cytoplasm. Hence the total number of mitochondria in the myocardial tissue can be used as a measure of cardiomyocyte activity and health " . In HIV-infected patients with symptomatic hyperlactemia receiving anti retroviral therapy, Cote and co-workers showed that the ratio of mitochondrial D N A (mtDNA) to nuclear D N A (nDNA) can be used as a marker of drug-induced mitochondrial toxicity 2 2 9 . During the last century, there have been major improvements in the strategies used to protect myocardial tissue from ischemia and reperfusion injury. These include thrombolytic therapies, percutaneous transluminal angioplasty (PTA), coronary artery bypass grafting (CABG) in the setting of acute occlusion of coronary artery and controlled reperfusion, reperfusion with modified solutions, experimental endothelial gene transcription during cardiac surgery and heart transplantation 2 3 0 - 2 3 4 . However, the mechanism of the ischemic-reperfusion injury remains unknown and our abilities to treat or prevent it are therefore limited. Using methods similar to those used by Cote and coworkers, we investigated changes in heart mitDNA:nDNA ratio during myocardial ischemia and reperfusion phases, and compared these levels to additional measures of tissue injury. New markers of myocardial injury may provide mechanistic insights and reveal therapeutic possibilities in reperfusion injury. Here, we propose a new method, using 125 IGF-1, of protecting cardiac tissue against ischemia-reperfusion injury in an ex vivo murine model. The mechanism underlying this protective effect remains unclear. However, the level of ICAM-1 protein expression is significantly lower in the presence of IGF-1. The integrity of the myocardial tissue lattice was preserved and the development of interstitial edema in myocardial tissue was inhibited. These effects were correlated with improved perfusion pressure and left ventricular compliance. We also demonstrated that this IGF-1 mediated protection was accompanied by preservation of mtDNA content. It has been shown that mutation in mtDNA promotes apoptosis in the presence of predisposing factors such as ultraviolet 2 3 5 . Intragastic administration of ethanol in mice decreases mtDNA level of skeletal muscles, liver, cardiac, and brain tissues. This effect of ethanol can be prevented using antioxidants 2 2 1 . The high level of ROS formation is associated with higher level of mutated mtDNA 2 2 8 . 126 5.3 Methods and Materials 5.3.1 Ischemia-reperfusion model: C57B6 mice 25-30 g (Jackson Laboratories, Bar Harbor, ME) were anesthetized using 3% isoflurane (Baxter, Toronto, ON, Canada) for one minute and maintained at 1% isoflurane for 3-5 min during cardiac excision. To prevent coagulation in coronary vessels 500 units Heparin-sodium (Organon Teknika Inc. Toronto, ON, Canada) was injected intra-peritoneally 10 min before induction of anesthesia. The heart was excised and assembled on a Langendorff apparatus and perfused with oxygenated (95% O2 + 5% CO2) modified Kreb's Henseleit working solution (MK) at 37°C for 3-5 min 2 3 6 - 2 3 8 until monitoring reveal that organ was stable. Transduced left ventricle and aortic pressures, and heart rate were monitored continuously using Power lab/8sp detector (AD Instruments Pty Ltd. Castle Hi l l , Australia). Retrograde perfusion was then stopped for 20 min to model of global ischemia (a period of 20 min of ischemia was found to be optimal for the ischemic phase in this model). The ischemic hearts were then reperfused with M K solution alone, M K solution plus IGF-1 (10 ng/mL), or M K solution plus TNF-oc (10 ng/mL) for l h or 2h. After completion of the reperfusion period, the hearts were divided into symmetrical halves. One half was frozen using 2-methylbutane (Isopentane, M E R K KgaA, Darmstadt, Germany) in liquid nitrogen for 80 sec for eventual sectioning and/or D N A isolation. Paraffin embedding of the other half followed fixation of the sample in 10% formalin. 5.3.2 Histologic evaluation: Slides of paraffin embedded tissues from the apex to the basal portion of the hearts were prepared and stained with hematoxylin and eosin (H&E). Serial 400x magnified images were captured using a Nikon E600 microscope and Spot 127 Advanced software (version 3.4.2 S. Leffler & Silicon Graphics Inc. Mountain view, CA). Image Pro-Plus software (Media Cybernetics, Carlsbad, CA) was used to evaluate the severity of interstitial edema around the perivascular spaces of coronary arteries and veins. Measures were taken in 10 sections from each of 4 hearts for all conditions and time points. The extent of interstitial edema was measured by selecting a circular area with a radius two times greater than the vascular space contained within the drawn circle. The total vascular (V) and perivascular areas (pV) were measured. "Non-tissue" area (NT) was determined by color segmentation images constructed by Image Pro-Plus. Total interstitial edema was determined by subtracting the vascular area from the non-tissue area and expressing this as a percentage of total perivascular area Percentage of edema = [NT-V]/pV x 100. 5.3.3 ICAM-1 expression of cardiac tissue: Frozen sections of the hearts were fixed and stained with Alexa 594 anti-ICAM-1 antibody, and Hoechst nuclear staining. Using confocal microscopy (400x) images were captured. The mean intensity of ICAM-1 and Hoechst staining were measured using fluo-cytogram software in gray scale (0-255) as previously mentioned in chapter-3 section. The ratio of ICAM-1 to nuclear staining was measured and compared to control groups (refer to chapter 3 section 3). The level of ICAM-1 mRNA was also measured (refer to chapter 2 section 3.3). 5.3.4 Ventricular function assessment: Heart rate and pressure generated during the cardiac cycle was obtained by transduction of the aortic cannula and recorded 128 continuously during ischemia and reperfusion using Power lab/8 sp detector. Using Powerlab software the difference between ex vivo systolic and diastolic pressure (AP Sys/Dia) at different time points was calculated to assess ventricular performance. Pressure measured during systole reflected contractility and diastolic pressure drops reflected relaxation of the ventricle. Thus greater AP s y s /Di a values indicate better overall performance of the left ventricle. 5.3.5 Detection of CPK: A 1 mL sample of myocardial perfusate was collected every 15 minutes during the reperfusion phase. The samples were frozen in a mixture of ethanol and dry ice 2 3 9 ' 2 4 0 . The level of creatine phospho-kinase (CPK) was measured using Vitros C K slides (Ortho-Clinical Diagnostics, Rochester, NY) . Briefly 11 uL of perfusate was deposited on the slide and evenly distributed. Samples were incubated for 5 minutes at 37°C. After final interaction, leuco-dye is oxidized by hydrogen peroxide in the presence of peroxidase to form an insoluble dye. Reflection densities are monitored during incubation and the rate of change in reflection density is then converted to enzyme activity by using 670 nm wavelength in the Vitros Chemistry 250 System. 5.3.6 MitDNA:nDNA assay: Frozen hearts initially embedded in opaque tissue-fixation material were thawed, cut into small pieces (~3 mg) and then placed into lysis buffer. D N A was extracted using the Qiagen D N A isolation kit, (Qiagen Canada QIAGEN Inc. Mississauga, Ontario, Canada) in accordance with the manufacturer's protocol. Extracts were then diluted 1/80 with buffer A E before performing the mtDNA assay, as reported in murine tissues as described below 2 2 6> 2 2 9- 2 4 1, 129 For each D N A extract, one murine nuclear gene (accessory subunit of the murine mitochondrial D N A polymerase y (ASPG), Genbank Accession number A F 177202) and one murine mitochondrial gene (cytochrome oxidase subunit 1 [COX], Genbank Accession number AB042432) were quantified separately by real-time, quantitative PCR, using the Roche LightCycler (Roche Diagnostics, Indianapolis, IN). For the mitochondrial (COX) gene, the forward primer m C O X l F (5'-TCGTTGATTATTCTCAACCAATCA-3*) and the reverse primer mCOX2R (5'-G C C T C C A ATT ATT ATTGGT ATT A C T A T G A-3') were used. The oligonucleotides 3'-fluorescein-mCOXPRl (5'-AACC A G G T G C ACTTTT A G G A G A T G ACC-F3') and 5*-LC Red 640 3'-phosphate-blocked-mCOXPR2 ( 5 ' - L - A A T T T A C A A T G T T A T C G T A A C T G CCCATGC-P3') were used as hybridization probes. For the nuclear (ASPG) gene, the forward primer mASPGlF (5 ' -GGAGGAGGCACTTTCTCAGC-3') and the reverse primer mASPG2R (5 ' -GAAGACCTGCTCCCTGAACAC-3 ' ) were used. The oligonucleotides 5'-flourescein-mASPGPRl (GCGCTTTGGACCTTTGGGTGTAG-F3') and mASPGPR25'L- G T T A C G A A A G A A C C T A G C C T C A C A G T G G T - P 3 ' ) were used as hybridization probes. PCR reactions and amplification cycles were performed as described 2 2 9 . A standard curve consisting of serially diluted mouse D N A (30 000, 6000, 1200, 240 and 48 nuclear genome equivalents) were included in each run. The same standard curve was used to quantify both the nuclear (ASPG) and the mitochondrial (COX) gene. mtDNA and nDNA genes were assayed in duplicate. Results of the quantitative PCR assay were 130 expressed as the ratio of the mean value of the duplicate mtDNA measurements to the mean value of duplicate nDNA measurements. As a further quality control, a mouse D N A extract with a mtDNA:nDNA, known to be high and an extract with a mtDNA/nDNA ratio known to be low were included in every run. Repeat sample and intra-sample variations were <5%. 131 5.4 Results 5.4.1 IGF-1 decreases ICAM-1 expression of cardiac tissue during reperfusion: Twenty minutes ischemia follow by 2 hours reperfusion with M K increased the expression of ICAM-1 on cardiac tissue compared to control ( 3.4 ± 0.2 vs 1.5 ± 0.1 p<0.001) as determined by the measured ratio of ICAM-1 to nuclear staining. Two hours reperfusion with T N F a significantly increases ICAM-1 expression relative to control, and M K (4.0 ± 0.2, p<0.05). Reperfusion in the presence of IGF-1 did not increase ICAM-1 protein expression on cardiac tissue (1.9 ± 0.1 vs 1.5 ± 0.1, p=NS). This indicates that suppression of ICAM-1 expression during reperfusion in the presence of IGF-1 (Figure 5.1 A) may contribute to better cardiac performance. However reperfusion with IGF-1 did not prevent the increased level of ICAM-1 mRNA suggesting that the inhibition of ICAM-1 is at the level of protein expression during reperfusion (Figure 5.1B). 5.4.2 Perivasular interstitial edema and tissue lattice integrity: The cellular integrity of the myocardium was well preserved in the tissue of hearts reperfused with IGF-1 (Figure 5.2). The area of interstitial edema in IGF-1 treated hearts was 21 ± 4% compared to 34 ± 6% and 49 ± 5% for reperfusions with M K only and M K with TNF-a respectively (p<0.05). Representative tissue histology images are presented in Figure 5.2 and were similar throughout the four hearts and in all conditions. Additional histology observations included an increased number of shrunken, contracted myocytes with dense 132 5 4 4 c c o o 5 < o 1 4 * P<0.07 _r_ Control Ischemia MK IGF-1 TNF Figure 5.1 A Reperfusion of ischemic hearts with M K only and M K with TNFa increased the expression of ICAM-1 protein on cardiac tissue (p<0.01). This effect can be prevented using IGF-1 in MK. Figure5.1BReperfusion of ischemic hearts with M K only, M K with TNFa, and MK with IGF-1 increased the expression of ICAM-1 mRNA of cardiac tissue compared to control group (p<0.001). 133 Control Ischemia Modified Kreb's T N F a IGF-1 1 h reperfusion 2 h reperfusion Figure 5.2 Representative images (x400) of hematoxylin and eosin stained sections from murine hearts subjected to ischemia/reperfusion. Images are of control, ischemia without reperfusion, and reperfusion with modified Kreb's Henseleit working solution (MK) alone, MK plus tumor necrosis factor (TNFa), and MK plus insulin-like growth factor (IGF-1), both at 1 and 2 hours. Note the preservation of cellular and structural elements and the lack of interstitial edema in IGF-1 reperfused heart. 134 pycnotic nuclei with M K plus TNF-a reperfusions as compared with perfusate containing IGF-1. 5.4.3 IGF-1 improvement in myocardial performance during reperfusion: Cardiac performance was determined by calculating the pressure difference between systole and diastole (AP s y s/di a) at set time points. The systolic and diastolic pressures were determined by taking the average values from a window around the respective time points. Performance is then a measure of both contractility (stroke volume and force of left ventricle contraction manifesting as systolic pressure) and diastolic function, or relaxation of the left ventricle (a reduction in diastolic pressures). Improved performance is manifested then by a widening in AP s y s /di a . Pressure monitoring demonstrated that cardiac performance increased from 0 to 40 min of reperfusion for all conditions (Figure 5.3). After 40 min of reperfusion, cardiac performance arrived at a plateau and became negative for the remaining minutes for the M K alone or M K plus TNF-a reperfusions. With reperfusion with M K plus TNF-a the AP s y s /di a initially increased to 6.8 ± 0.7 mmHg as compared with reperfusion with M K alone (5.1 + 0.6 mmHg). However, reperfusion with IGF-1 generated a AP s y s /di a that was significantly greater (13.8 + 1.2 mmHg) than with TNF-a (6.8 ± 0.7 mmHg) by 20 min. Heart rate was significantly increased in the presence of IGF-1 (172.4 ± 4.2) compared to M K (58.3 ± 2.8) and T N F a (101.7 ± 3.8). This gain in cardiac performance was maintained up to 120 minutes of reperfusion with IGF-1. The enhanced performance was reflected in improvements in both systolic and diastolic pressures and heart rate. The late descent in slop at 120 minutes of reperfusion with IGF-1 was similar to that occurring with reperfusion with M K alone or M K plus 135 TNF-a, but may relate to ex vivo conditions other than the ischemia time and the reperfusion solution. A paired two sample t-test for means between groups demonstrated a statistically significant difference between IGF-1 and M K alone and M K with TNF-a (p<0.005). 5.4.4 Low creatine phosphokinases level in IGF-1 treated hearts: Collected perfusate from hearts treated with IGF-1, at all reperfusion time points, contained significantly lower quantities of detectable C P K (34.6 U/L) than did perfusate from TNF-a treated hearts (113.6 U/L). This is shown as an average for all time points (Figure 5.4). Single factor A N O V A revealed a significant difference between groups (p<0.005). 5.4.5 Ratio of mitochondrial to nuclear DNA: IGF-1 maintained or improved the mtDNA:nDNA ratio during reperfusion of ischemic myocardium as compared with control reperfusion with M K alone. There was a significant difference between all test groups (baseline, ischemia, M K alone reperfusion, and M K with IGF-1 reperfusion) in the determined mtDNA:nDNA (p<0.05 A N O V A ) . Based on previous work, it was thought useful to test the utility of mtDNA:nDNA to assess the "cellular health" of fyr)(i 90*7 ischemic and reperfused myocardium tissues ' . How IGF-1 preserves mtDNA:nDNA and i f this also means intact oxidative mitochondrial function that promotes cellular viability remains to be investigated. We found that IGF-1 appeared to protect heart tissue against a reduction in the mtDNA:nDNA, which was accompanied by improved histological grading and improved organ function in terms of contractility. A reduction in this ratio may represent either necrosis of at risk tissue or a reduction in mitochondrial 136 number after the initial stimulus (ischemia) followed by subsequent reperfusion (Figure 5.5). Such a reduction was noted with reperfusion with M K alone after the initial increase in mtDNA:nDNA that occurred after ischemia alone without reperfusion. Because this model utilized a cell free perfusate the mtDNA:nDNA is not confounded by potential contributions from immune cells, a point that has been raised as a possible explanation for changes in mtDNA:nDNA. 137 0. < c 5 'S co i -O o o m (0 c/> ISP 140 -120-100-Modified Kreb's solution (MK) MK + TNFa •wr 140-120-100 - MK + IGF-1 0 20 40 60 80 Reperfusion time (min) 140 Figure 5.3 Determination of cardiac performance. (A) Tracing from continuous monitor recording obtained during ischemia and reperfusion. (B) Determination of cardiac performance is as described in the methods section and includes calculation of the pressure gradient between systolic and diastolic pressure transductions from the aorta and left ventricle. Reperfusion with IGF-1 generates a significant improvement in cardiac performance at all time points (p<0.05). 138 300 ^ 250 A c "53 > Q. O CD CO 3 t CD Q. 200 150 H 100 H 50 H ft Omin 15 min 30 min 45 min 60 min Time of reperfusion (min) Figure 5.4 Measured creatine phosphokinase (CPK) in perfused murine hearts. Hearts were prepared for ex-vivo reperfusion. Perfusate solution was collected (1 ml/4 min) around each time points. Reperfusion with TNFa generates a significant elevation in the detectable amount of CPK activity relative to that detected with IGF-1 (p<0.005). 139 5 0 J Lii£} I I I I , I I f 1 1 1 , 1 1 Controls Ischemia MK1h MK2h IGF1h IGF2h Figure 5.5 Determination of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA) ratio. The mtDNA:nDNA ration was determined for all conditions (p<0.05). The ratio of mtDNA/nDNA was determined for the following conditions: control, ischemia without reperfusion, modified Kreb's alone for lh (MK lh), modified Kreb's alone for 2h (MK 2h), modified Kreb's with IGF-1 for lh (IGF lh), and modified Kreb's with IGF-1 for 2h (IGF 2h). The number within each histogram represents the number of hearts processed for that condition. The values for mtDNA/nDNA in the controls, ischemic myocardial tissue and either reperfusion group (MK or IGF-1) were significantly different from each other (p<0.05). 140 5.5 Discussion Despite a range of clinical interventions, our ability to prevent reperfusion injury after disruption of blood flow to vascular beds remains disappointing. A n appreciation of the mechanism of ischemia/reperfusion injury is central to development of better treatments. In the present study we demonstrated that IGF-1 can lessen reperfusion injury following an initial ischemic insult. The anti-inflammatory effect of IGF-1 on cardiac and other tissues has been shown previously 8 5 ' 1 0 2> 2 4 2. We found that IGF-1 inhibits the expression of ICAM-1 protein on myocardial tissue after ischemia and during reperfusion. However IGF-1 cannot inhibit the presence of ICAM-1 mRNA suggesting the effect is on translation or transportation of ICAM-1 during reperfusion. The failure in translation or transportation of ICAM-1 protein to the membrane could be related to the activation of PI3-kinase pathways which is activated by IGF-1 8 7 , 2 3 1 . This effect of IGF-1 can be through usage of PIP2 2 4 3 which is also necessary for ICAM-1 dimerization 1 5 1 , 2 4 4 . ICAM-1 expression and activation is associated with decreased cardiomyocyte contractility which results in decreased cardiac contractility. IGF-1 decreases ICAM-1 expression on cardiac tissue and improves cardiac performance. This effect of IGF-1 on the ischemic myocardium was supported by histological evidence of improved tissue and cellular integrity, including markedly less interstitial edema around the perivascular spaces. In this model, ischemic myocardium treated with IGF-1 141 had significantly lesser amounts of detectable C P K than did myocardium treated with TNF-a, suggesting reduced cellular injury. This is also consistent with the cardiac performance and left ventricular contractility of IGF-1 treated hearts that exhibited a greater APsys/Dia and heart rate compared to control and TNFa treated groups. It should be noted that the detectable C P K levels would not be above the normal range as determined for human whole blood samples. However, there was considerable histological evidence of tissue damage in the TNF-a treated hearts, suggesting the relative insensitivity of C P K in detecting lesser myocardial injuries. A more sensitive marker would be valuable not only for studying the mechanism that underlies reperfusion injury but also for evaluating the efficacy of therapeutic interventions. This is particularly true when one considers the segmental and intermittent ischemic/reperfusion zones that characterize dysfunctional myocardium in sepsis. The initial improvement in contractility, observed under all conditions of reperfusion, was probably the result of a new supply of nutrients after the ischemic period, including oxygen. The addition of IGF-1 significantly augmented this improvement in left ventricular pressure generation and relaxation (thus increasing AP sys/Dia)- This improvement was maintained throughout the period of reperfusion. Myocardial performance at the cellular level is associated with the number or functional capacity of mitochondria. To investigate indirectly whether mitochondrial function may represent a maker of this beneficial effect of IGF-1, we determined the mtDNA:nDNA ratio in relation to myocardial function. Although not appreciated clinically, ischemia and reprefusion are two distinct periods 87>98>225>230>232- The ischemic period has been described as 'priming' cardiac myocytes for either a necrotic or apoptotic death. A marked increase 142 in mtDNA:nDNA ratio was detected in the ischemic myocardium relative to baseline control levels. Apoptosis has been found to be an event that requires energy 2 4 5 , 2 4 6 . This increased mtDNA:nDNA ratio may indicate an increase in the number of mitochondria per cell or an increase in the genome copy number per mitochondria. However the above effect of IGF-1 should be investigated because of the effect of IGF-1 on saving mutant forms of mtDNA 2 2 8 . Myocardial reperfusion injury, as a separate event, can increase the extent of injury beyond that caused by ischemia alone. It has been shown that modification of solutions or other conditions during the reperfusion phase can alter the extent of cellular and functional damage to the myocardium. We determined that the nature of the reperfusate can affect mtDNA:nDNA ratio. Reperfusion with M K alone resulted in a reduction, in mtDNA:nDNA ratio toward baseline values. This may reflect either mitochondrial mitoptosis in damaged and 'primed' tissues or the necrotic loss of similar cells that were 'primed', resulting in elevated mtDNA:nDNA ratio after ischemia. The net effect would be that the remaining tissue is spared and should reflect baseline tissue. However, a mtDNA:nDNA ratio that does not differ from baseline does not indicate that the tissue is working normally. In fact, histology and contractility determinations demonstrated that the heart had sustained significant tissue damage and was dysfunctional after M K reperfusion. With IGF-1 reperfusion this reduction in mtDNA:nDNA ratio was prevented, suggesting that the extent of injury is not associated with elevated mtDNA:nDNA ratio alone. In fact, after ischemia reperfusion, it was found a normal mtDNA:nDNA early after reperfusion predicted significant tissue injury. The patterns of 143 mtDNA:nDNA ratio, as seen in this model, may prove useful in future investigation of possible mitochondrial-related mechanisms of reperfusion injury. IGF-1 can affect cardiomyocyte contractility through its receptor - a heterotetrameric protein with intracellular tyrosine kinase activity 2 4 7 . Downstream signals after receptor activation include She, Crk and phospholipase C, and activation of phosphatidylinositol-3 (PI3) kinase. Guse and coworkers 2 4 3 demonstrated that IGF-1 can increase IP3 levels in rat cardiomyocytes. Through its action on PI3 kinase, IGF-1 can affect both contractility 8 5 contractility and apoptosis 2 4 8 . The action of IGF-1, as demonstrated in our myocardial ischemia/reperfusion model, may occur via PI3 kinase and/ or effects on mitochondria. Increases in cardiomyocyte calcium levels and cardiomyocyte sensitivity to calcium 2 4 9 have been demonstrated to effect cardiac performance. Alteration in calcium metabolism may interfere with the action of calcium because the filamentous network of cardiomyocytes and their contractile properties are extremely sensitive to even small fluctuations in calcium ion concentration 2 5 0 . ICAM-1 activation and hemodimerization is dependent on the one of the products of PI3 kinase pathways ie PIP2 1 5 1 , 2 4 4 . The decreased expression of ICAM-1 in the presence of IGF-1 can be explained by an increase level of IP3 which need consumption of PIP2. This may show that IGF-1 reduces the level of PIP2 to increase production of IP3 and calcium release from sarcoplasmic reticulum. However in this ex vivo model of ischemia reperfusion injury the protective effect of IGF-1 may not be related to the ICAM-1 activation because of lack of ICAM-1 ligands such as CD 11/CD 18 receptors on inflammatory cells. Fibrinogen, a ligand for ICAM-1, is present but not at the concentration that may be in the circulation. Thereby the 144 protective effect of IGF-1 may be more from the indirect effect of IGF-1 on ICAM-1 expression, i.e., PI3K pathway. Hence it is logical to speculate that IGF-1 can be used as a therapeutic intervention to inhibit ICAM-1 expression in various types of inflammatory responses including sepsis especially when we use it before the initiation of inflammatory responses as a preventive agent. It is important to note that IGF-1 per se can increase the survival of inflammatory cells and increase the degranulation ability of the inflammatory cells thereby using IGF-1 as a therapeutic agents in inflammatory response should be with a very careful assessment and evaluation and it needs more investigations. A similar result to that presented here for IGF-1 in myocardial ischemia/reperfusion has been demonstrated for vascular endothelial growth factor (VEGF), suggesting that a final common 'protective' pathway may exist 2 5 ° . Anwar and coworkers 2 5 1 showed that TNF-oc decreased IGF-1 mRNA and increased IGF-1 binding protein-3 mRNA expression in vascular smooth muscle cells. These actions of TNF-a effectively reduce free IGF-1 levels and activity, promoted endothelial instability. Infusion of the modified IGF-1 reduced the TNF-a induced apoptosis. A n interaction between V E G F and IGF-1 was characterized in retinal neovascularization in diabetic patients 8 6 ' 2 5 2 . The authors of that report described common mitogen kinase 44/42 pathways that may be related to the mitogenic effect of those two molecules. However the short time effect for both IGF-1 and V E G F in myocardial ischemia/reperfusion models is most probable through the Akt pathway 8 6 ' 2 5 2 . Akt activation can improve contractility through PI3 kinase signaling and 145 is also an initiator of protein kinase C activation upstream. Protein kinase C plays an important role in cardiac function, calcium metabolism, and contractility. Michell and coworkers showed that IGF-1 and V E G F both stimulate nitric oxide production from endothelial cells and that inhibition of PI3 kinase by wortmannin and LY29004 decreases nitric oxide production and reduces cardiac function 8 6 . Akt signaling has also been demonstrated to prevent apoptosis. Whether this pathway alters the expression of bcl-2 family members by IGF-1 exposure remains unknown. It has been shown that IGF-1 can protect myocardium and other tissues against apoptosis in various animal models 8 4 ^ 2 5 \ IGF-1 may also improve cardiac function in diabetic patients 8 4> 1 0 2 ' 2 5 4- 2 5 6 and r a t models of myocardial infarction and reperfusion87. It has been shown that IGF-1 can protect myocardium by regulating changes in proapoptotic and/or antiapoptotic molecules such as Bcl-2, B c l - X L and Bax. These are all related to the mitochondrial apoptotic pathway and mitochondrial energetics 8 7 . This may also explain, in part, how IGF-1 protects myocardium even in the later phase of reperfusion injury. In an ex vivo model of myocardial ischemia and reprefusion we demonstrated that IGF-1 protects against the reperfusion-associated injury. It can decrease the level of the inflammatory response by the reduction of membranous ICAM-1 presentation. We found this protective effect of IGF-1 to be correlated with elevated mtDNA:nDNA relative to baseline, and this may represent a marker for the preservation of mitochondrial function. This study provides new insight into ischemia/ reperfusion and possible mechanisms and treatment for the tissue injury and organ dysfunction associated with this process. The 146 eventual benefit of this to our understanding of myocardial dysfunction in sepsis awaits further study. 147 Chapter-6 Final conclusion A l l cardiac inflammatory diseases including ischemia reperfusion injury, sepsis, transplant rejection, myocarditis and cardiomyopathy contain a common pathway regardless of the main causes of myocardial inflammation. The inflammatory response of the heart is manifested by lowered cardiac pumping function. At the cellular level, myocardial inflammation is associated with increased cytokine release, recruitment and activation of inflammatory cells 5 9 , 6 0 , 1 0 7 ' 1 0 8 and activation of coronary endothelial cells 109,110 a D O v e e v e n t s increase the level of adhesion molecules on endothelial cells and cardiomyocytes 92>110'160>162 followed by an interaction between receptors of endothelial cells and activated leukocytes. One of the most important receptors in cardiac inflammatory responses is ICAM-1. ICAM-1 (CD-54), a 76-114 kDa glycoprotein, is expressed in both normal and activated cardiomyocytes 5 9 , 6 0 , 1 0 7 , 1 0 8 . Within 2 to 4 hours cytokines and LPS treatments increase endothelial ICAM-1 expression 1 0 9 , 1 1 0 . This upregulation of ICAM-1 facilitates neutrophil binding and migration which has been demonstrated using cultured endothelial cell monolayer in vitro, a process involving the interaction of ICAM-1 with LFA-1 (CD1 la/CD18) and Mac-1 (CD1 lb/CD 18) 1 2 6 " 1 2 8 . Furthermore, the extracellular domains 129 of the ICAM-1 molecule can interact with various ligands including fibrinogen , Rhinoviruses 1 2 5 , Coxsackie viruses 1 3 0 , 1 3 1 , and Plasmodium falciparum 1 3 2 . 148 Our experiments demonstrate that ICAM-1 is not only a receptor for adhesion of leukocytes or other proteins, but also functions as a regulatory protein, specifically in cardiomyocyte contractility. In vitro we have shown that cardiomyocyte ICAM-1 activation, through the binding of CD 11/CD 18 receptors of inflammatory cells, or by cross-linking, reduces cardiomyocyte contractility. This effect of ICAM-1 activation is associated with cardiomyocyte cortical cytoskeleton assembly and related proteins such as F A K and RhoA which result in alteration of C a 2 + propagation during excitation contraction cycles. Whether this change in calcium influx is due to the alteration in juxtaposition of cardiomyocyte C a 2 + ions should be investigated through electron microscopy. Our preliminary experiments, using nanogold labeled antibodies to detect RyR and DHPR juxtaposition, seem promising (Figure 6.1). Using an in vivo model of sepsis in mice we then demonstrated that LPS injection increases ICAM-1 expression of cardiac tissue and decreases cardiac function. LPS injection in mice changes the peripheral blood cell differentiation showing decreased the leukocyte count, increased the percentage of P M N , and decreased the platelet count. The effect of LPS on cardiac function is related to the number of inflammatory cells as demonstrated by impaired cardiac function in neutrophillic (GCSF treatment) mice and improved cardiac function in neutropenic mice (cyclophosphamide treatment). These findings indicate the importance of inflammatory cells in inducing cardiac dysfunction in the endotoxemia models. 149 Lack of the ICAM-1 gene (protein) protects murine cardiac function against the effect of LPS. By using a chimeric model we have found that the level of ICAM-1 expression on cardiac tissue is directly associated with lower cardiac function. Although the presence of ICAM-1 on inflammatory cells is an important co-factor for antigen recognition, there is no defect of inflammatory response in our chimeric model demonstrated by the same response of chimeric (H+/M") and wild type mice to the effect of LPS. Although the above effect of LPS (decreased cardiac function) is associated with recruitment of circulatory inflammatory cells to the left ventricle, which was demonstrated by a higher number of intravascular and adherent inflammatory cells, there is no evidence of the presence of inflammatory cells in the cardiac interstitial space, excluding the role of inflammatory cells in the activation of cardiomyocyte ICAM-1 through the interaction of their ligands (CD11/CD18). Furthermore investigations using BrdU staining of bone marrow did not show the presence of inflammatory cells in the cardiac interstitial space confirming the above results. Therefore CD 11 /CD 18 receptors on inflammatory cells may not be a reason for cardiomyocyte ICAM-1 activation after LPS injection. 150 Figure 6.1 Preliminary experiment using nanogold beads and electron microscopy to define the juxtaposition of RyR (< 5 nm) and DHPR (6 nm). 151 We then demonstrated that fibrinogen can decrease cardiomyocyte contractility. This effect of fibrinogen occurs via its activation of ICAM-1. We also observed that after LPS injection fibrinogen infiltrates into the cardiac tissue indicating that fibrinogen can enter into cardiac tissue in the presence of a leaky endothelial surface. The result of investigation of IGF-1 in the model of ischemia reperfusion injury demonstrated that IGF-1 can inhibit the expression of ICAM-1 in cardiac tissue. The mechanisms of cardiac dysfunction in the model of ischemia reperfusion injury, although is different from those of the sepsis model, have many common intracellular cascades including increased ICAM-1 expression. During ischemia reperfusion injury some part of myocardial dysfunction is irreversible because of myocardial necrosis, while viable and at-risk cardiac tissues accompanies with less injury and reversible dysfunction. The important step to save myocardium from further cardiac damage during reperfusion is to save at-risk and remaining myocardium. I believe that the at-risk area of myocardium has a similar mechanism of myocardial dysfunction to that of sepsis. Whether IGF-1 can be used as a therapeutic intervention to improve cardiac function during sepsis has yet to be investigated. Although the clinical trial of IGF-1 in septic patients failed to show the benefits of IGF-1 treatment in critically i l l patients, I believe the decrease in ICAM-1 expression in the presence of IGF-1 should be seen before initiation of the severe inflammatory process as we have also seen in chapter 5. In addition to the other benefits of IGF-1 on cardiac function which were mentioned in chapter 5, it is important to consider that IGF-1 can improve calcium release which may abrogate the final effect of ICAM-1 activation on cardiomyocyte Ca influx. 152 Taken together we have found that one of the most important causes of decreased cardiomyocyte contractility is a result of ICAM-1 activation. ICAM-1 activation changes C a 2 + influx during excitation contraction. The expression of ICAM-1 on cardiac tissues was increased after LPS injection and the presence and activation of ICAM-1 on cardiac tissue is necessary and sufficient to induce cardiac dysfunction. LPS injection increases intravascular and adherent inflammatory cells on the coronary endothelium. The number of interstitial inflammatory cells in the LPS treated hearts did not increase thereby excluding the role of CD11/CD18 receptors in ICAM-1 activation. 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