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Myogenic and endothelial properties of the mouse septal artery in health and disease Lui, Amy Hoi-Mel 2001

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MYOGENIC AND ENDOTHELIAL PROPERTIES OF THE MOUSE SEPTAL ARTERY IN HEALTH AND DISEASE. by Amy Hoi-Mel Lui B.Sc, The University of British Columbia, 1994  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in  THE FACULTY OF GRADUATE STUDIES (Department of Pharmacology & Therapeutics) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA May 2001 © Amy Hoi-Mel Lui, 2001  In presenting this thesis in partial fulfilment  of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his or  her  representatives.  It  is understood that  copying or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department The University of British Columbia Vancouver, Canada  DE-6 (2/88)  ii  ABSTRACT  In this thesis I examined vascular responses o f the mouse ventricular septal artery to increases  in intravascular pressure  and pertinent  vasoactive mediators  in two  contrasting models: health and cardiac disease. This work is pertinent because the mouse has been extensively used as a cardiovascular and genetically modified animal model, with the differences between human and mouse genetics not as great as one would expect. Thus it is important to understand murine pharmacology in the context o f its use as a tool to understand human pathology. In our first study, responses in the "normal", "healthy" mouse coronary artery were characterized and quantified. Pressure-constriction and concentration-response curves were constructed and compared to published human coronary pharmacology. It was found that myogenic tone was higher in mice than in humans  and rats, greatly  concentration-dependent  contributed  vasodilation,  to  by endothelin. Noradrenaline  mediated  by  endothelium-  produced  located  0.2-  adrenoceptors and smooth muscle-located p-adrenoceptors. Acetylcholine also produced vasorelaxation, which was abolished with L - N A M E . Interestingly, the endotheliumdependent vasodilators bradykinin and substance P produced no vasomotor effect on the coronary arteries, possibly indicating the absence o f specific corresponding receptors. In the  second  study,  a murine  coxsackie B3-induced myocarditis was  representative model o f cardiac disease. evaluated  in  immunostaining  this  pathological  confirmed  as  a  Again, myogenic and endothelial function was  condition.  extensive  used  Although  myocyte  hemotoxylin  damage  due  immunohistological methods did not reveal increased endothelin-1  to  and  eosin  infection,  levels near the  coronary vasculature. Our study found no difference in coronary arterial pressure-induced  Iiiii vasoconstriction between infected  and uninfected/sham-infected  mice  in the  physiological pressure range, as well as between time points in the infected group. However, at a lower pressure range (10-60 mmHg), data from the early time points (1,3 and 7 days post-infection) indicated enhanced vessel tone which could be the result of increased release of vasocontrictor factors. Endothelial dysfunction manifested as a diminished response to acetylcholine occurred at 28 days post-infection, which was recovered with pre-treatment using the mixed endothelin A/B receptor antagonist bosentan. The results in this thesis have allowed a further insight into the use of the mouse as a relevant human model of disease. In addition, information with regards to coronary microvascular function in myocarditis has been obtained and contributes to the overall understanding of the mechanism of pathogenesis.  iv  T A B L E OF CONTENTS  ABSTRACT T A B L E OF CONTENTS LIST OF FIGURES LIST OF A B B R E V I A T I O N S ACKNOWLEDGEMENTS  1. I N T R O D U C T I O N 1.1 P R E F A C E 1.2 M Y O G E N I C T O N E 1.2.1 B A C K G R O U N D 1.2.2 I S O M E T R I C R I N G P R E P A R A T I O N V E R S U S PRESSURE M Y O G R A P H Y 1.2.3 R O L E O F E N D O T H E L I U M 1.2.4 T R A N S D U C T I O N M E C H A N I S M S D E P O L A R I Z A T I O N O F V S M A N D ELECTROMECHANICAL COUPLING S T R E T C H - A C T I V A T E D C H A N N E L S K + C H A N N E L S . C l - C H A N N E L S V O L T A G E - G A T E D Ca2+ C H A N N E L S S E C O N D M E S S E N G E R S C A L C I U M G P R O T E P N S A N D P H O S P H O L I P A S E C ( P L C ) P R O T E I N K I N A S E C C Y T O S K E L E T O N A N D E X T R A C E L L U L A R M A T R I X I N T E G R I N S C Y T O S K E L E T O N 1.3 V A S O A C T I V E M E D I A T O R S 1.3.1 N O R A D R E N A L I N E S Y N T H E S I S , S T O R A G E A N D R E L E A S E N O R A D R E N A L I N E I N T H E C O R O N A R Y C I R C U L A T I O N 1.3.2 A C E T Y L C H O L I N E S Y N T H E S I S , S T O R A G E A N D R E L E A S E A C E T Y L C H O L I N E I N T H E C O R O N A R Y C I R C U L A T I O N 1.3.3 E N D O T H E L P N - 1 S Y N T H E S I S , S T O R A G E A N D R E L E A S E E N D O T H E L P N - 1 I N T H E C O R O N A R Y C I R C U L A T I O N 1.3.4 N I T R I C O X I D E S Y N T H E S I S , S T O R A G E A N D R E L E A S E N I T R I C O X I D E I N T H E C O R O N A R Y C I R C U L A T I O N 1.4 C O X S A C K I E V I R U S E S 1.4.1 D I S C O V E R Y , T A X O N O M Y A N D P R O P E R T I E S 1.4.2 C V B T R A N S M I S S I O N A N D I N F E C T I O N  11 iv vii viii x  1 1 2 2 3 4 5 5 5 6 7 7 7 7 8 9 9 9 10 10 11 11 11 12 12 12 13 13 14 15 15 15 16 16 17  V  1.4.3 C V B A N D H U M A N DISEASE 1.5 C O X S A C K I E B 3 - I N D U C E D V I R A L M Y O C A R D I T I S 1.5.1 INTRODUCTION 1.5.2 ENDOTHELIN-1 IN V I R A L M Y O C A R D I T I S 1.5.3 HOST DEFENCE: CELL-MEDIATED I M M U N E PATHOGENICITY 1.5.4 VIRUS DETECTION A N D SEROLOGICAL M A R K E R S FOR DIAGNOSIS 1.5.5 P E R S I S T A N C E OF V I R A L R N A 1.5.6 F R O M M Y O C A R D I T I S TO D I L A T E D C A R D I O M Y O P A T H Y 1.5.7 INITIAL SUPPORTIVE M E A S U R E S A N D F U T U R E THERAPEUTIC IMPLICATIONS 2.  S T U D Y 1- M Y O G E N I C A N D E N D O T H E L I A L R E G U L A T I O N O F T O N E IN T H E H E A L T H Y M O U S E S E P T A L A R T E R Y 2.1 H Y P O T H E S E S A N D O B J E C T I V E S 2.2 M A T E R I A L S A N D M E T H O D S 2.2.1 TISSUE ISOLATION, P R E P A R A T I O N A N D D I A M E T E R MEASUREMENT 2.2.2 EXPERIMENTAL PROTOCOL 2.2.3 E X P R E S S I O N OF R E S U L T S A N D S T A T I S T I C A L A N A L Y S I S 2.2.4 SOLUTIONS A N D C H E M I C A L S 2.3 R E S U L T S 2.3.1 B A S A L TONE 2.3.2 EFFECT OF A C E T Y L C H O L I N E 2.3.3 E N D O T H E L I A L oc -ADRENOCEPTORS A N D NORADRENALINE 2.3.4 SMOOTH M U S C L E p-ADRENOCEPTORS A N D NORADRENALINE 2.3.5 ENDOTHELIUM-DEPENDENT VASODILATORS  17 18 18 19 19 20 20 21 21  25 25 25 25 26 28 28 29 29 33  2  3.  STUDY 2- M Y O G E N I C AND E N D O T H E L I A L R E G U L A T I O N OF T O N E IN T H E S E P T A L A R T E R Y O F T H E C V B 3 - I N F E C T E D MYOCARDITIC MOUSE 3.1 H Y P O T H E S E S A N D O B J E C T I V E S 3.2 M A T E R I A L S A N D M E T H O D S 3.2.1 TISSUE ISOLATION, P R E P A R A T I O N A N D D I A M E T E R MEASUREMENT 3.2.2 STUDY PROTOCOL I N O C U L A T I O N E X P E R I M E N T A L P R O T O C O L 3.2.3 E X P R E S S I O N OF R E S U L T S A N D S T A T I S T I C A L A N A L Y S I S 3.2.4 SOLUTIONS A N D C H E M I C A L S 3.3 R E S U L T S 3.3.1 BASAL TONE 3.3.2 AGONIST-INDUCED R E L A X A T I O N RESPONSES  33 36 40  40 40 41 41 41 41 42 42 43 43 43 48  vi 4. DISCUSSION 4.1 STUDY 1- MYOGENIC AND ENDOTHELIAL REGULATION OF TONE IN THE MOUSE SEPTAL ARTERY 4.2 STUDY 2- MYOGENIC AND ENDOTHELIAL REGULATION OF TONE IN THE MYOCARDITIC MOUSE SEPTAL ARTERY SUMMARY AND MAJOR CONCLUSIONS 5. 5.1 STUDY 1 5.2 STUDY 2 6. FUTURE DIRECTIONS 7. REFERENCES  51 51 56 60 60 60 61 63  vii  LIST OF FIGURES INTRODUCTION 1.5.1 1.5.2  Schematic diagram depicting pathophysiology o f myocarditis. 23 Schematic diagram showing the involvement o f the vasoactive mediators of the coronary microcirculation in the progression o f myocarditis. 24  STUDY 1 2.3.1 A Trace recording o f active and passive arterial diameter in the pressurized mouse septal artery. 2.3.IB Pressure-diameter relationship in pressurized mouse septal arteries possessing myogenic tone. 2.3.2 Bosentan-induced inhibition o f myogenic tone in pressurized septal arteries o f pentobarbital-anesthetized mice. Myogenic tone in endothelium-denuded arteries in the absence and 2.3.3 presence o f bosentan. 2.3.4 Acetylcholine concentration-response curve. 2.3.5 Table showing the effects o f various a - and (^-adrenoceptor ligands on septal arteries pressurized at 70 mm H g . 2.3.6 Noradrenaline concentration-response curve in septal arteries. 2.3.7 Noradrenaline concentration-response curve in mesenteric arteries. 2.3.8 Trace recording o f the effects o f acetylcholine, B H T 920 and isoprenaline on endothelium-denuded septal arterial diameter.  30 30 31 32 34 35 37 38 39  STUDY 2 3.3.1 3.3.2 3.3.3 3.3.4 3.3.5  Myogenic tone in pressurized septal arteries o f uninfected mice anesthetized with carbon dioxide. Myogenic tone in pressurized septal arteries o f sham-infected mice. Myogenic tone in pressurized septal arteries o f CVB3-infected mice at 1,3,7 and 28 days post-infection. Acetylcholine concentration-response curve o f arteries isolated from uninfected mice. Effect o f bosentan on the acetylcholine concentration-response curve of arteries from CVB3-infected mice sacrificed at 28 days post-infection.  45 46 47 49 50  DISCUSSION 4.4.1  Table showing species comparison o f the effect o f noradrenaline on coronary arteries.  54  viii  LIST O F ABBREVIATIONS  ACh ANOVA ATP B H T 920 H C I  acetylcholine analysis o f variance adenosine triphosphate 6-allyl-2-amino5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin  KH2PO4  dihydrochloride large conductance (potassium channel) calcium ion calcium chloride cyclic adenosine monophosphate choline acetyltransferase cyclic guanine monophosphate constitutive nitric oxide synthase chloride ion carbon dioxide charybdotoxin coxsackievirus B 3 diacylglycerol dilated cardiomyopathy Delbecco's Modified Eagle's M e d i a endothelin-converting enzymes ethylenediaminetetraaceticacid ethyleneglycoltetraaceticacid endothelial nitric oxide synthase endothelin-1 endothelin receptor type A endothelin receptor type B interferon immunoglobulin M intraperitoneal interleukin inducible nitric oxide synthase inositol trisphosphate potassium ion calcium-dependent potassium (channel) potassium chloride potassium phosphate  L-NAME M MgS0 min mL  N -nitro-L-arginine methyl ester muscarinic "type 3" (receptor) magnesium sulfate minute millilitre  BK Ca CaCl cAMP CAT cGMP cNOS Cl" 2 +  2  co  2  CTX CVB3 DAG DCM DMEM ECE EDTA EGTA eNOS ET-1 ET-A ET-B IFN IgM i-PIL iNOS IPs K +  Kca  KC1  3  4  m  ix  mm H g mRNA NA Na NaCl NAHCO3 nM +  Na P0 2  NO NOS  o  2  PBS pfu PKC PLC PSS S.E.M. SNP TFN VGC VSM pm pM  4  millimeters o f mercury messenger ribonucleic acid noradrenaline sodium ion sodium chloride sodium bicarbonate nanomoles sodium phosphate nitric oxide nitric oxide synthase oxygen phosphate buffered saline plaque forming units protein kinase C phospholipase C physiological salt solution standard error o f the mean sodium nitroprusside transferrin voltage-gated channel vascular smooth muscle micrometer micromole  ACKNOWLEDGEMENTS  I am very thankful to have had the opportunity to work with Dr. Ismail Laher, my principle supervisor. H i s insight, guidance and vast knowledge has made him a great mentor to me. In addition, I am very grateful to have had D r . Bruce M c M a n u s serve as my co-supervisor. Bruce's always-encouraging words and relentless pursuit o f scientific excellence and integrity w i l l never be forgotten. Lastly, I would like to send out my sincerest and heartfelt thanks to all my fellow lab mates, with special recognition to Eugene L a m , all o f whom have helped me to arrive at where I am today.  1  1.  INTRODUCTION  1.1 PREFACE The vascular myogenic response, inherent to smooth muscle o f small resistance blood vessels, is independent o f neural, metabolic and hormonal influences. B l o o d vessels, especially arterioles and small arteries, respond to increases in transmural pressure with constriction, and conversely, to pressure reduction with dilation. This myogenic response has two main physiological functions: to provide basal vascular tone and to autoregulate blood flow and capillary hydrostatic pressure. Basal vascular tone is required in order for the vessels to respond to dilator influences. This inherent state o f constriction allows for the provision o f a "regional blood flow reserve", upon which other mechanisms may produce a vasodilation or vasoconstriction. The myogenic response also  maintains  constant blood flow, thus minimizing changes in capillary hydrostatic pressure. It is known that the vasculature comprises 35% o f the volume o f the myocardium (Weber et al., 1987), and that at the microvascular level shows a heterogeneous response to injury and disease (Hoffman, 1995). In a pathological state, the myogenic response may be altered or impaired, which may result in decreased  blood flow causing ischemic  conditions. For example, it is thought that in the case o f acute viral myocarditis, there is generalized inflammation o f the heart muscle accompanied by the over-constriction o f the coronary microvasculature. This process is associated with both necrosis degeneration  o f the  myocytes,  and  incidence  is associated  with viral  and  infection.  Myocardial tissue damage may result from direct destruction by the organism, anti-heart immunity, and microvascular disease (Rossi and Mengel, 1992). Previous studies have reported that in the acute phase o f viral myocarditis, physiological profiling o f resistance  2  coronary vessels shows that disturbances o f the coronary microcirculation occur, and are characterized by focal areas o f microvascular spasm and vessel stiffness (Dong et al., 1992). This thesis characterizes the mouse coronary artery using various pharmacological agents as well as the myogenic response. In addition, a murine model o f coxsackie B3-induced myocarditis w i l l also be studied and compared to as a relevant and important model o f cardiac disease.  1.2 MYOGENIC TONE 1.2.1 B A C K G R O U N D The discovery o f the myogenic response was made in 1902 by Bayliss. H e noticed a physiological response in which isolated carotid arteries constricted following sudden distension (Bayliss, 1902), and postulated that intravascular pressure contributed to vascular tone. Later, Folkow demonstrated that denervated tissue preparations developed pressure-dependent vascular tone, thus proving this response to be non-neural in origin (Folkow, 1949). This phenomenon was observed in both nonvascular and vascular smooth muscle, and the later development  o f isolated vessel techniques  enabled  quantification o f the myogenic response in separation from other influences (Davis and H i l l , 1999). The myogenic response is a unique property o f resistance vessels, which consist o f small arteries (70 - 200 pm lumen diameter) through to distal arterioles (10 pm lumen diameter). In contrast, non-resistance arteries, which and are larger in size and differ in function, possess little tone. These arteries are conductance vessels and are mainly  3  responsible for the carrying and transport o f blood. In addition, unlike resistance vessels, these conduit vessels contribute little to total peripheral resistance. Myogenic tone is one component o f the auto-regulation o f flow during variations in systemic arterial pressure. Other factors controlling blood flow include metabolic, neural and hormonal.  A l l these in synchronization produce the overall resistance in a given  vascular bed. T o study the myogenic reactivity in small blood vessels without the influence o f extrinsic factors, two in vitro techniques can be used: the isometric ring preparations or the pressure myograph (Halpern et al., 1984).  1.2.2 I S O M E T R I C R I N G P R E P A R A T I O N V E R S U S P R E S S U R E M Y O G R A P H Y The isometric ring protocol, performed on the wire myograph, was developed in 1972 by Bevan and Osher (Bevan and Osher, 1972). This technique consists o f the mounting o f vessel ring segments onto two fine stainless steel wires that are clamped at each end to keep the vessel isometric. In addition, the apparatus is fastened to a force transducer and micrometer (Mulvany and Halpern, 1976). Thus using this myograph, the myogenic response can be investigated by using stretch o f the vessel to assess force development. The pressure myograph serves to mimic the physiological state more closely than the ring preparation wire myograph. Essentially, the equipment consists o f a tissue bath where two axially aligned and apposed glass microcannulae (tip diameter = 30-50 pm) are immersed. Using fine silk thread, one end o f the tissue segment is tied to the end o f one cannula, and the other end to the other cannula. U p o n clamping the distal cannula, the intraluminal pressure can be increased or decreased using a pressure-servo controlled peristaltic pump. In addition, the intraluminal pressure can also be detected using a flow-  4  through pressure transducer. Vessel diameter is measured using a video edge-detection system. The experimental protocol consists o f changing the intraluminal pressure and observing the effect on vessel diameter. The isometric ring preparation o f quantitating the vascular myogenic response differs from pressure myography in several ways. Firstly, in the isometric preparation, stretch activation is represented by a lower, secondary increase in tension after stretch ("stretchactivation"). In contrast, using the pressure myography technique,  an increase in  intraluminal pressure results in a constriction that secondarily reduces total wall tension (i.e., passive wall tension > active wall tension). Secondly, isometric preparations show maximal stretch activation in response to large (unphysiological) changes in length, whereas isobaric preparations show maximal constrictions in response to much smaller length changes (Davis, 1993), even in the absence o f detectable distension (Davis and Sikes, 1990). Finally, similar vessels may possess different agonist sensitivities (Dunn et al., 1994) and differences in magnitudes o f agonist-induced vascular smooth muscle depolarization (Schubert et al., 1996) in different preparations.  1.2.3 R O L E O F ENDOTHDELITJM The endothelium, lining the inner surface o f blood vessels, is the main regulator o f vascular wall homeostasis. It secretes various factors in response to mechanical and hormonal stimuli, which, in turn, influence smooth muscle cell contractility, vascular structure, blood fluidity, and other cell-to-cell interactions (Thorin and Shreeve, 1998). Inclusively, the endothelium is important in regulating myogenic tone. Early studies  5  performed by Harder and co-workers indicated that the myogenic response to pressure elevation was dependent on an intact endothelium. Removing the endothelial layer by chemical reduced both pressure-induced depolarization and the myogenic response (Harder, 1987). However, it was later shown in various tissue preparations that mechanical removal of the endothelium did not abolish myogenic tone. At present, it is believed that the myogenic response occurs as a result of mechanical stimuli that act directly on the vascular smooth muscle cells to generate constriction. The endothelium exerts its influence by releasing vasoactive factors, but the presence of an intact endothelium is not a prerequisite for myogenic reactivity in blood vessels.  1.2.4 TRANSDUCTION MECHANISMS DEPOLARIZATION OF VSM AND ELECTROMECHANICAL COUPLING It has been shown extensively that membrane depolarization occurs as a response of the smooth muscle cells to a stretch stimulus (Coburn, 1987; Knot and Nelson, 1998). This depolarization is graded as pressure is increased. The result is always vessel constriction. However, it has been difficult to establish a definitive cause-and-effect relationship between membrane depolarization and myogenic responsiveness. Some hypotheses are mentioned below. STRETCH-ACTIVATED CHANNELS Stretch-induced  depolarization  could  be  explained  by  activation  of special  mechanosensitive non-selective ion channels on endothelial cells (Davis et al., 1992), which are sensitive to membrane deformation. Cation-permeable mechanosensitive  channels were first described in cultured skeletal muscle cells (Guharay and Sachs, 1985), and have since been found in other cell types including smooth muscle cells. In vascular smooth muscle cells o f freshly isolated porcine coronary arteries, Davis et al. found that patch pipette suction activated a non-selective cation channel that is permeable to K , N a and C a . Activation o f these mechanosensitive channels initiates mechano+  +  2 +  electric transduction, resulting in a membrane depolarization that remains elevated in spite o f reduced vessel diameter- characteristic o f myogenic response. K C H A N N E L S +  Inhibition o f K channels is hypothesized to be a mechanism involved in stretch-induced +  depolarization. Although a direct role by K channels in initiating the myogenic response +  has not been shown, a membrane depolarization that inhibits potassium currents flowing through these channels may result in vascular tone. It has been observed that three types of K channels do not play a role in myogenic tone: the inward rectifier K +  +  channel, the  ATP-sensitive K channel (Quayle et al., 1997), and the novel K channel (Evans et al., +  1996). However, the voltage-dependent K  +  +  channel and the Ca -activated K 2+  +  channel  types may be involved in membrane re-polarization. Blockers o f K c channels have been a  found to augment myogenic tone and further decrease luminal diameter (Nelson and Brayden, 1993). In particular, the involvement o f B K channels is likely, since block o f these channels by the specific B K channel inhibitor charybdotoxin ( C T X ) enhances vascular tone.  7 Cl" CHANNELS Chloride channel activation may be another mechanism by which stretch-activated membrane depolarization can be explained. It has been shown that Cl" channel inhibitors DUDS and indanyloxyacetic acid inhibit tone of pressurized cerebral arteries (Nelson et al., 1997). However, the lack of specific Cl" channel blockers makes the role of these channels in the myogenic response inconclusive. VOLTAGE-GATED Ca  2+  CHANNELS  Much evidence has confirmed the importance of VGC channels in the myogenic response. The possibility that activation of these channels may result in membrane depolarization has been suggested. Dihydropyridines have been shown to attenuate pressure- or stretch-induced intracellular calcium concentration, and VGC channel activators increase depolarization (Harder, 1984). However, it was shown that in rat cerebral arteries Ca  2+  channel inhibitors (diltiazem and nisoldipine) blocked calcium  influx and vasoconstriction but had no effect membrane depolarization (Knot and Nelson, 1998). SECOND MESSENGERS CALCIUM The importance of the role of C a in arteriolar tone was first noted by Uchida and Bohr 2+  (Uchida and Bohr, 1969). They observed the abolishment of tone upon perfusion of the vessel with a Ca -free solution. Laher et al. later reported that myogenic tone was 2+  dependent on Ca entry (Laher et al., 1988), as demonstrated by Ca influx (Cipolla and 2+  2+  8  Osol, 1998). At present, it is known that in vessels possessing myogenic tone, increases in transmural  pressure lead to increases  in intracellular calcium concentration  ([Ca ]i)(Boltz and Pohl, 1998; Knot and Nelson, 1998). This hypothesis has been 2+  confirmed by use of Ca -sensitive fluorescent dyes in conjunction with video-based 2+  imaging and photometer techniques (Meininger et al., 1991). Although small arteries and arterioles possess intracellular C a dependence on extracellular C a  2+  of the sarcoplasmic reticulum C a  2+  stores, these vessels seem to show greater  for contractile activity (Cauvin et al., 1984). Inhibitors 2+  ATPase (SERCA) has been shown to have no effect  on amplitude of myogenic constriction in the rat cerebral arteries (Watanabe et al., 1993), although rate of development of tone is reduced. G PROTEINS A N D PHOSPHOLIPASE C G proteins are activated in coronary smooth muscle cells in response to mechanical stimulation (Wierbitzky et al., 1994). In addition, a role for P L C in mechanotransduction has been suggested. Osol et al. observed the abolishment of myogenic tone in isolated cerebral arteries in the presence of the P L C inhibitor U-73122 (Osol et al., 1993), and Narayanan et al. measured production of inositol trisphosphate (IP3) and diacylglycerol (DAG) in renal arterial smooth muscle cells in response to increased intraluminal pressure (Narayanan et al., 1994). The production of IP3 may contribute to the rise in [Ca ]i by releasing stored C a 2+  2+  from the sarcoplasmic reticulum, and D A G can directly  activate protein kinase C (PKC).  9 P R O T E I N K I N A S E C Activation o f P K C has been suggested to play a possible role in the myogenic response. It has been shown that inhibition o f P K C by calphostin C attenuates tone in human coronary arteries (Miller et al., 1997). Staurosporine was shown to cause dose-dependent inhibition o f myogenic tone in isolated rat cerebral arteries (Osol et a l , 1993). P K C activators increase the level o f arteriolar tone without an increase in [ C a ] i levels. In 2+  addition, agonists such as noradrenaline that act in part through P K C activation enhance myogenic responsiveness (Faber and Meininger, 1990). M o r e specifically, P K C has been implicated to sensitize the contractile proteins to C a , 2 +  and is important  in the  maintenance o f constriction. However, the precise role o f P K C in myogenic signaling has been made difficult due to its numerous isozymes. C Y T O S K E L E T O N A N D E X T R A C E L L U L A R M A T R I X I N T E G R I N S Integrins are a class o f membrane-spanning heterodimeric receptors, found on most cells, including vascular smooth muscle cells (Platts et al, 1998). In vascular smooth muscle, at least 11 o f the 22 known integrin subunit combinations occur. The predominance o f specific integrin types suggest interactions with collagens and laminin in the basement membrane and the interstitial matrix (Glukhova et al, 1995). Integrins function as transducers o f a variety o f cellular signals, and thus may also interact with tyrosine kinases, phospholipase C , protein kinase C, as well as modulate membrane  ion  conductance (Clark and Brugge, 1995). Since integrins form an important mechanical link between the extracellular matrix and the vascular smooth muscle skeleton, and are  10 able to associate with both cytoskeletal (Shattil and Ginsberg, 1997) and signaling proteins (Hynes, 1992), they may play a role in regulating vasomotor function. M o r e specifically, integrins may be responsible in the transduction o f the physical stimulus o f intraluminal pressure to result in contraction via the cellular contractile apparatus (Wang et al., 1993). It has been suggested that the integrin-dense plaque region o f the vascular smooth muscle may be a likely site for a mechanosensitive element, consisting o f an assemblage o f proteins (Davis et al, 2001). This "mechanosensor" may also be involved in the sustenance o f vascular tone after initial vasoconstrictor response. C Y T O S K E L E T O N Cytoskeletal  stiffness  increases  in proportion to  stress applied through  integrin  attachments (Muller et al., 1997). A potential role o f the V S M cytoskeleton is in control of mechanosensitive ion channel gating, and both the microtubule system and the actin cytoskeleton may play a role in active force development.  1.3 VASOA CTIVE MEDIA TORS Several vasoactive factors, released as either neural- or endothelial-derived mediators, are important in influencing the vascular state o f resistance arteries. Below, the functional roles o f noradrenaline, acetylcholine, endothelin-1 and nitric oxide in the coronary circulation are briefly summarized.  11  1.3.1 N O R A D R E N A L I N E S Y N T H E S I S , S T O R A G E A N D R E L E A S E Noradrenaline is synthesized as a result o f a series o f steps beginning from tyrosine, and immediately from dopamine by the enzyme dopamine P-hydroxylase. The hydroxylation of tyrosine is regarded as the rate-limiting step in the biosynthesis o f catecholamines (Zigmond et a l , 1989). Noradrenaline is co-stored in two types o f regulated-release storage vesicles, namely the large dense core vesicles and small dense core vesicles. Release is from perivascular sympathetic nerves (Burnstock and Sneddon, 1985), and act on a - and P-adrenergic receptors in the membrane o f the postsynaptic cell. N O R A D R E N A L I N E I N T H E H U M A N C O R O N A R Y C I R C U L A T I O N The sympathetic nervous system is o f great importance in the regulation o f coronary resistance. The heart possesses extensive sympathetic innervation, and noradrenaline mediates vasoconstriction o f human coronary arteries via a-adrenoceptors  (Corr and  Burnstock, 1991). However, some studies have concluded that p-adrenoceptor mediated relaxation exceeds the constriction mediated by a - adrenoceptors (Ginsberg and Bristow, 1980). a-adrenoceptors on the smooth muscle mediate vasoconstriction via receptormediated events which increase IP3 formation, and in turn increase intracellular calcium. P-adrenoceptors mediate vasorelaxation via increase in c A M P concentration.  cAMP  activates a protein kinase, which phosphorylates and inactivates myosin-light-chain kinase, thus inhibiting contraction (Rang and Dale, 1995).  12  1.3.2  ACETYLCHOLINE S Y N T H E S I S , S T O R A G E A N D R E L E A S E Acetylcholine is synthesized from choline by the enzyme choline  acetyltransferase  ( C A T ) . The rate-limiting process o f acetylcholine synthesis is choline transport, the activity o f which is regulated according to the rate at which acetylcholine is being released. After synthesis, acetylcholine is transported into and stored in synaptic vesicles, with release into the synaptic cleft coinciding with the arrival o f the action potential at the motor-nerve terminal (Katz and Miledi, 1965). Post-synaptically, acetylcholine can act on nicotinic or muscarinic receptors. Acetylcholine is degraded by acetylcholinesterase. A C E T Y L C H O L I N E I N T H E C O R O N A R Y C I R C U L A T I O N Acetylcholine has action on both the endothelial and smooth muscle cells o f the coronary vessels. Acetylcholine may act on muscarinic receptors located on the endothelial cells to initiate vasodilation. This initiates inositol trisphosphate production, and C a  2 +  release  from the endoplasmic reticulum and activation o f N O synthase by Ca /calmodulin. In 2+  addition, acetylcholine can cause vasoconstriction o f coronary arteries via direct action on smooth muscle cells, in the absence o f an intact endothelium (Schipke et al., 1985). Correlatively, vascular damage and/or endothelium dysfunction may also result in acetylcholine-induced  vasoconstriction,  as  a consequence  o f the  involvement  vasoactive substances such as angiotensin II (Becker et al, 1991) and  of  prostenoids  (Shimizu et al, 1993) which likely increase calcium influx into smooth muscle cells.  13  1.3.3  E N D O T H E L I N - 1 (ET-1) S Y N T H E S I S , S T O R A G E A N D R E L E A S E The potent vasoconstrictor peptide E T - 1 is synthesized by cleavage o f big E T - 1 by endothelin-converting enzymes ( E C E ) . In humans, big E T - 1 and E C E - 1 are primarily localized in secretory and storage granules in endothelial cells (Davenport et al., 1998). ET-1 is produced in these cells, but may also be expressed in other cell types including vascular smooth muscle cells ( L i u et al., 1998). Under normal conditions, it is postulated that there is a low level o f basal endothelin production and release, which mediates vascular tone in vivo. In addition, various stimuli such as hypoxia, ischemia or shear stress may induce transcription o f E T - 1 m R N A and subsequent synthesis and secretion o f E T - 1 . Under these and other pathological conditions such as coronary artery disease and myocardial infarction, plasma levels o f E T - 1 are significantly elevated (Lerman et al., 1991; Tonnessen et al., 1995). Receptors for endothelin have been isolated and their genes cloned (Arai et al, 1990). The receptors are classified on the basis o f their affinity for the various endothelin isoforms. ET-1 may act on E T - A or E T - B receptors, with the receptor types in humans exhibiting significant sequence similarity with 63% amino acid identity (Sakamoto et al, 1991). However, each receptor type has differential distribution in various tissues. Binding o f endothelin to its receptor is very tight with slow dissociation, resulting in prolonged pharmacological action. E T - A receptors are prevalent in the heart and vascular smooth muscle cells, whereas E T - B receptors are more widely distributed and predominate in the kidney, uterus, central nervous system and endothelial cells (Tamirisa et al, 1995).  14 Receptor activation causes stimulation o f phospholipase C and subsequent hydrolysis o f phosphatidylinositol. The result is two products: IP3 and D A G . In addition to the stimulation o f calcium release from the sarcoplasmic reticulum by IP3, activated E T receptor also stimulates voltage-dependent calcium channels and probably the receptoroperated  calcium channels.  The global effect  is increased  intracellular calcium  concentration and sensitization o f the cellular contractile apparatus and contraction. E N D O T H E L I N - 1 I N T H E C O R O N A R Y C I R C U L A T I O N In the human coronary circulation, E T - 1 acts predominantly on E T - A receptors. It is unknown  whether  the  mouse  coronary  circulation  exhibits  similar  phenotypic  distribution. E T - 1 plays important short-term and long-term roles: short-term actions include contraction o f vascular smooth muscle by mobilization o f intracellular C a increased extracellular C a  2 +  2 +  and  influx (Levin, 1995). Under long-term (chronic) pathological  conditions, endothelin concentrations are elevated, as is coronary artery tissue endothelin immunoreactivity (Lerman et al., 1995). It is hypothesized that E T - 1 plays a significant role as a modulator o f coronary vascular reactivity, both in coronary artery disease as well as in endothelial dysfunction. With respect to endothelial dysfunction, an imbalance between E T - 1 and nitric oxide ( N O ) synthesis may be present; decreased N O and/or enhanced E T - 1 production may lead to potentiation o f vasoconstrictor effects  and  increased global vasoconstriction. Boulanger et al. demonstrated that E T - 1 attenuated nitric oxide-induced cyclic G M P ( c G M P ) generation in vitro (Boulanger and Luscher, 1991), and Lerman et al. confirmed that coronary c G M P concentrations were suppressed  15  in humans with endothelial dysfunction and elevated endothelin concentrations (Lerman et al., 1995).  1.3.4  NITRIC O X I D E (NO) S Y N T H E S I S , S T O R A G E A N D R E L E A S E Nitric oxide is synthesized by N O synthase ( N O S ) enzymes. There are several isoforms of N O S , including e N O S (endothelial), c N O S (constitutive) and i N O S (inducible). N O S catalyzes a reaction between molecular oxygen and L-arginine to form nitric oxide. Nitric oxide is produced in response to cytokine stimulation in macrophages, neutrophils, vascular smooth muscle and endothelial cells. In addition to the endothelium, vascular nitric oxide is also present in platelets (Rang and Dale, 1995). N I T R I C O X I D E I N T H E C O R O N A R Y C I R C U L A T I O N Nitric oxide in vascular regulation couples endothelial and smooth muscle cells. Nitric oxide diffuses to smooth muscle cells and activates guanylyl cyclase. c G M P activates cGMP-dependent protein kinase which phosphorylates myosin light chain and causes vascular relaxation. The physiological role o f nitric oxide includes control o f regional blood flow and blood pressure. Pathologically, nitric oxide may cause or contribute to atherogenesis,  thrombosis and vasospasm. Nitric oxide also acts as a host defense  mechanism against viruses and bacteria.  16  /. 4 COXSACKIE VIRUSES 1 A. 1 D I S C O V E R Y , T A X O N O M Y A N D P R O P E R T I E S Coxsackieviruses were first discovered approximately 50 years ago. Soon thereafter it was recognized that some o f the coxsackievirus isolates caused generalized skeletal muscle destruction in the mouse, a histopathology distinct from other coxsackievirus isolates. Thus the different viruses were placed into two groups, A and B . The group A viruses cause generalized myositis, whereas the group B viruses cause infection o f many tissues and organs which lead to a slower and more paralytic death with destruction o f cardiac myocytes, pancreatic cells and hepatocytes. There are six serotypes o f C V B viruses. Genetically, these non-enveloped viruses have a single "plus" strand R N A genome with VPg-linked to the 5' end o f the genome and a poly A tail at the 3' end, typical o f picornaviruses. The genome, approximately 7000-7500 bases in length, is translated into a large polyprotein, which is cleaved into functional proteins by virus encoded proteases. Mutations in the genome result in antigenic variations in the virus proteins. The R N A o f most C V B serotypes has been sequenced and c D N A molecules shown to be capable o f replication (Kandolf and Hofschneider, 1985; Chapman et al, 1994). Recently it has been found that C V B R N A genomes can remain latent in cells, especially heart tissue, where no intact virus or viral proteins can be detected.  17  1.4.2 CVB TRANSMISSION AND INFECTION Coxsackieviruses are ubiquitous and can spread rapidly within the community. The site of entry is believed to be the alimentary tract, but aerosol transmission may also be significant under sub-sanitary conditions. Incubation period is approximately one to two weeks, but may be longer. Infection is mediated by cell surface receptors, which facilitate binding and entry of CVB into susceptible host cells (Martino et al., 2000). Resistant to low pH, they replicate in the mucosal cells of the small intestine prior to entering the bloodstream and causing systemic infection.  1.4.3 CVB AND ITCJMAN DISEASE The CVB are known to produce a variety of human diseases. A high proportion of infections with these viruses is subclinical, exhibited by a mild, undifferentiated febrile illness or upper respiratory tract infection. Infection in neonates can be both systematic and fatal. Clinical manifestations include myocarditis, meningitis, encephalitis and pulmonary disease (Chonmaitree and Mann, 1995; Grist et al., 1978). General symptoms include fever, severe unilateral chest pain, pleural inflammation, headache and sore throat. There may be abdominal tenderness associated with pancreatitis and hepatitis. In addition, virus-induced heart disease may develop necessitating heart transplantation. The role of CVB in virus-induced insulin-dependent diabetes melitus (IDDM) has also been suspected. Receptors may have a major role in the determination of viral tropism (Martino et al., 2000).  18  1.5 CVB3-INDUCED VIRAL MYOCARDITIS 1.5.1 I N T R O D U C T I O N Viral myocarditis exhibits different clinical phenotypes depending on the age o f patient. In infants, it can be manifested as acute heart failure and cardiogenic shock, and in older patients, it often presents as a chronic, slowly progressive heart failure and dilated cardiomyopathy ( L i u and Opavsky, 2000). C V B 3 myocyte damage and degeneration,  produces  myocarditis by direct  as well as necrosis, with potential treatment  uncertain. Myocardial and endothelial damage in the acute phase may progress to slow chronic  myocyte  loss  and  myocardial  fibrosis,  eventually  resulting  in  dilated  myocardiopathy. A well-established animal model o f C V B 3 - i n d u c e d myocarditis is the mouse, from which much information has been gained. It has been demonstrated that early inflammatory infiltrates post-infection are associated with observed foci o f necrosis, where direct infection o f myocytes leads to cell death ( M c M a n u s et al., 1993). In addition to viral infection and immune activation, coronary microvascular spasm is also thought to be involved (Silver and Kowalczyk, 1989). This abnormality may be secondary to endothelial cell damage (Dong et al., 1992), and may lead to potential sites o f ischemia and reperfusion.  1.5.2 E N D O T H E L I N - 1 (ET-1) I N V I R A L M Y O C A R D I T I S In the acute phase o f C V B 3 myocarditis, prior to the development o f inflammatory infiltrates or significant hemodynamic compromise, infected myocytes develop increased de novo synthesis o f E T - 1 . It has been shown that in encephalomyocarditic mice, peak plasma concentrations  o f E T - 1 occurred at 5 days post-infection, and peak heart  19  concentrations at 7 days post-infection (Ono et al., 1999). It is hypothesized that this elevated level o f circulating E T - 1 , contributed to by myocytes, interstitial and endothelial cells, may cause microvascular spasm and focal vasoconstriction, resulting in altered myocardial blood flow. The later introduction o f inflammatory infiltrates such as macrophages may also contribute to increased E T - 1 . The result is a condition which may be conducive o f impaired systolic and diastolic performance (Pandey et al., 1998), left ventricle failure, myocardial injury and ischemia (Hasdai et al., 1994), silent infarction (Tonnessen et al., 1995) and ultimately heart failure (Stewart et al., 1992).  1.5.3 H O S T D E F E N C E : C E L L - M E D I A T E D I M M U N E P A T H O G E N I C I T Y The first wave o f infiltrating cells in the mouse heart, consisting mainly o f natural killer ( N K ) cells, occurs about 4-14 days post-infection (subacute phase). Various cytokines, including JJL-1 beta, TNF-alpha, IFN-gamma and I L - 2 are produced at this stage (Kawai, 1999). In addition, at this time, nitric oxide ( N O ) is thought to play both beneficial and detrimental roles (Beckman et al., 1990; Martino et al., 1995). Animal models with I F N element-1 gene and i N O S knockouts show a marked increase in mortality, with minimal ability to attenuate viral proliferation ( L i u et al., 1996). Although N O is a modulator o f immunological self-defense mechanisms and kills infectious agents, it is also associated with detrimental effects on the myocardial tissue (Ishiyama et al., 1997). The second wave o f infiltrating cells consists o f T lymphocytes, which coincides with the most severe acute pathological damage. B lymphocyte infiltrates also increase at a later time point, with levels showing reciprocal changes o f less intensity than those o f the T lymphocytes (Kawai, 1999).  20  1.5.4 VIRUS DETECTION AND SEROLOGICAL MARKERS FOR DIAGNOSIS Coxsackie B viruses are present in the pharyngeal washings of infected individuals for 12 weeks after infection and for a prolonged period of several weeks in the feces. This, however, is not of diagnostic significance in relation to heart disease, since detection of virus in the affected target organ is important. In contrast, serological markers are commonly used to examine and diagnose viral involvement in heart disease. Patients with myocarditis show a fourfold rise in neutralizing antibodies to CVB3 (Abelmann, 1971). CVB-IgM antibody titres are elevated for up to six months in some myocarditic patients (McCartney et al., 1986), and is also a prevalent symptom in patients with dilated myocardiopathy (Kitaura, 1981).  1.5.5 PERSISTANCE OF VIRAL RNA The viral mechanism not only contributes to the acute phase of myocarditis, but also to the evolution of ongoing heart disease. Persisting virus may interact with the immune system and play a role in the transition from myocarditis to dilated cardiomyopathy. It is hypothesized that in the chronic phase of myocarditis, T lymphocytes infiltrate the myocardium in response to viral RNA in myocytes. Viral RNA may therefore generate new antigenic molecules with can trigger the immune inflammatory reaction (Martino et al., 1994). There is evidence that viral RNA is capable of replication (Klingel et al., 1992).  21  1.5.6 F R O M M Y O C A R D I T I S T O D I L A T E D C A R D I O M Y O P A T H Y Dilated cardiomyopathy ( D C M ) ,  defined by the enlargement o f cardiac  chambers,  thinning o f ventricular walls and reduced contractility (Fenoglio et al., 1983; Keating and Sanguinetti, 1996), is a prevalent cause o f heart disease and sudden death (Penninger et al., 1997). Most patients with D C M have a history o f myocarditis. In addition, C V B picornaviruses can be detected in as many as 50% o f these patients. The progression from acute viral myocarditis to D C M has been investigated, and it is hypothesized that the virus not only initiates the disease by infection and invasion, but persists as a molecular pathogen, causing cardiac damage, degeneration and dysfunction (Martino et al., 1994). Phenotypically, myocardial fibrosis is also prominent, as are cardiac lesions. The exact transition from viral myocarditis to D C M is unknown, but since culturable virus and viral capsid proteins are absent after the initial phase o f myocarditis, it has been suggested that a cell-mediated autoimmune mechanism(s) may play a role in the pathogenesis (Kawai, 1999).  1.5.7 I N I T I A L  SUPPORTIVE  MEASURES  AND  FUTURE  THERAPEUTIC  IMPLICATIONS Bed rest and avoidance o f vigorous exercise is important for patients with severe heart failure, a i - and P-adrenoceptor blockers, including prazosin or doxazosin (Sole and L i u , 1993), have shown to be effective in the treatment o f viral myocarditis and D C M . Angiotensin-converting enzyme ( A C E ) inhibitors such as captopril are also effective in reducing myocardial injury, at least in part by abolishing microvascular spasm. Calcium channel blockers, for example verapamil (Dong et al., 1992) and amlodipine, have been  22  observed to prolong survival and reduce myocardial damage. This may be due to altered inflammatory responses or the immunomodulating effect by inhibiting NO production (Matsumori, 1997). Endothelin receptor antagonists such as bosentan have been reported to have a cardioprotective effect by increasing cardiac output as well as decreasing left ventricular dysfunction and myocardial injury (Ono, K. et al, 1999). In addition, antiviral and immunosuppressive agents, as well as immunomodulating therapy may be an important approach to successful long- term therapy.  23  Schematic Diagram Depicting Pathophysiology of Myocarditis  ENDOTHELIN' Viremia  ACUTE PHASE  Increased virus titre in myocytes  Decreased virus titre in myocytes  ECE-1 mRNA'  Low plasma ET-I levels  2  Low  Focal necrosis Virus-induced cytotoxicity  Peak  Destruction of cardiac tissue due to cell-mediated immune mechanisms  increasing Peak plasma ET-1 levels peak Decreasing plasma ET-1 levels  Aviremia  PHYSIOLOGY  stabilizing  decreasing slightly and stabilizing  Apoptosis Decreased heart weight Decreased heart weight/body weight ratio Myocardial fibrosis Cardiac chamber enlargement  28  Ventricular wall thinning Reduced contractility  Fig. 1.5.1 Schematic timeline showing the pathophysiological progression o f myocarditis. Adapted from ' O n o et al., 1 9 9 9 . K a w a i , C , 1999. 2  24  Vasomediators as Contributors to the Progression of Myocarditis  T ET-1  ISCHEMIA Vasospasm  "A  (^virus)  MYOCARDIAL DEPRESSION  Direct myocyte damage  REDUCED CONTRACTILITY  Fibrosis  SMC injury?  Cell-mediated autoimmune, response  j/'SMC receptor-mediated responses?  I L V FUNCTION •I N O  -* Endothelial dysfunction  N  Days p.i.  7  0 A C U T E  28  > 42  C H R O N I C  Fig. 1.5.2 Schematic diagram showing the involvement of the vasoactive mediators of the coronary microcirculation in the progression of myocarditis.  25  2. STUDY 1 - MYOGENIC AND ENDOTHELIAL REGULATION OF TONE IN THE HEALTHY MOUSE SEPTAL ARTERY  2.1 HYPOTHESES AND OBJECTIVES  HYPOTHESIS 1: The septal artery of the mouse is similar to that in the human with respect to basal myogenic tone and to drug-induced responses. O B J E C T T V E 1: To characterize the septal artery of the CD-I mouse with respect to myogenic tone as well as responses to pertinent pharmacological agents and compare responses to published results from human data.  2.2 MATERIALS AND METHODS  2.2.1 TISSUE ISOLATION, PREPARATION AND DIAMETER MEASUREMENT Male CD-I mice (5-6 weeks of age) were anesthetized using sodium pentobarbital (Somnotol, O.lmL/mouse i.p.) and heparin sulfate (Hepalean, O.lmL/mouse ip). The heart was removed and placed into ice-cold physiological salt solution (PSS) following decapitation. The right ventricle was cut open and the septal artery dissected and transferred to a pressure arteriograph vessel chamber (Living Systems, Vt., USA) containing oxygenated (95% 0 / 5% CO2) PSS at room temperature. The superfusing 2  solution was recirculatedfroma 100 mL external bath at a flow rate of 12.5 mL/ min. One vessel end was tied to one borosilicate cannula (tip diameter 50-80 pm) using a single 4-0 nylon threadfilament.The other vessel end was tied in a similar manner to the distal cannula. Under no flow conditions, the intraluminal pressure was incrementally increased from 0 mmHg to 40 mmHg using an electronic pressure servo system (Living  26  Systems, V t . , U . S . A . ) . The bath temperature was then heated to 37°C using an external heat exchanger. U p o n reaching this bath temperature, the intraluminal pressure was again incrementally raised to 70 mmHg. The artery was equilibrated for 60 min at this pressure during which time spontaneous myogenic tone developed. The vessel chamber was placed under an inverted video microscope, where the vessel image could be observed on a monitor. Vessel diameter was measured using a monochromatic video edge-detection system (Living Systems, V t . , U . S . A . ) . Intraluminal pressure and diameter were continuously recorded onto a personal computer using the software program D A T A Q .  2.2.2 E X P E R I M E N T A L P R O T O C O L In experiments designed to measure the spontaneous myogenic response,  pressure-  diameter relationships were determined by increasing the intraluminal pressure from 10 to 20 mmHg, and in 20 mmHg increments to 120 m m H g thereafter. A t each pressure, a 5 min stabilization period was allowed during which time the vessel typically achieved a steady state diameter. The same protocol was repeated using vessels pre-incubated with 1 pM  or 10 p M bosentan for 30 min. In separate experiments, the concentration  dependence  o f noradrenaline-induced  relaxation was  determined.  Following  the  development o f tone, cumulative additions o f noradrenaline (1 n M - 1 0 y M ) were made to thePSS. To  investigate  adrenoceptor  activity,  the  ai-adrenoceptor  selective  agonists  phenylephrine and methoxamine were applied to the coronary arteries.  The a.2-  27  adrenoceptor  selective agonists  B H T 920  and  clonidine were  used  in  separate  experiments. To elucidate which receptors mediate noradrenaline-induced relaxation, arteries were incubated with the ©^-adrenoceptor selective antagonist, yohimbine (10 p M ) , and/or the nonselective P-adrenoceptor antagonist, propranolol (20 p M ) for 30 min prior to a noradrenaline (10 p M ) challenge. Phenylephrine (10 p M ) , the a.2-adrenoceptor selective agonist  6-allyl-2-amino5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d]azepin-dihydrochloride  ( B H T 920, 3 p M ) , and isoprenaline (10 p M ) were used as controls before and after the application o f an antagonist. B H T 920 (3 p M ) and isoprenaline (10 p M ) were also applied separately to the endothelium-denuded coronary artery. T o determine whether nitric oxide is a possible effector o f a,2-adrenoceptor-mediated vasodilation, arteries were incubated with N°-nitro-L-arginine methyl ester ( L - N A M E , 200 p M , 30 min) followed by a B H T 920 (1 p M ) or noradrenaline (10 p M ) challenge. L - N A M E (200 p M ) and propranolol (20 p M ) were also applied in combination. Additionally, acetylcholine (1 n M - 1 0 p M ) , both in the presence and absence o f L - N A M E , was applied cumulatively to the P S S and arterial luminal diameter recorded. This protocol was repeated using endothelium-denuded coronary vessels. To study endothelium-dependent  vasodilation, bradykinin and substance P were also  applied to the septal arteries. At the end o f each experiment, normal Ca -containing P S S was substituted with C a 2+  free P S S to determine passive luminal diameter.  2 +  28  2.2.3 EXPRESSION OF RESULTS A N D STATISTICAL ANALYSIS Myogenic tone at each pressure was expressed as a percent constriction = 100% x [(D Ca  free  - D  PSS)  / D  ca-free],  where D is the diameter in calcium free  (Dca-free)  or Ca 2+  containing PSS (D s). Percent relaxation was calculated using the equation 100% x [(Dd PS  - D )/(D - free - D )], where D is the diameter upon stabilization after drug addition (d), b  Ca  b  baseline (b) or calcium free. All results are expressed as mean ± S.E.M. of n animals. One vessel segment was obtained from each animal. Statistical evaluation was made using analysis of variance (ANOVA), and means were considered significantly different when P< 0.05.  2.2.4 SOLUTIONS AND CHEMICALS The composition of the PSS was (in mM): NaCl 119, KC1 4.7, K H P 0 1.18, NaHC0 2  4  3  24, MgS0 7H 0 1.17, CaCl 1.6, glucose 5.5 and EDTA 0.026. Ca -free solution was a 4  2  2+  2  PSS solution containing no CaCl and 2.0 mM EGTA. The composition of 80mM high 2  K solution was: NaCl 2.279, KC1 5.965, K H P 0 0.1606, MgS0 0.1409, NaHC0 +  2  4  4  3  2.092, and 0.006 EDTA. BHT 920 HCI (6-allyl-2-amino5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin-dihydrochloride) was supplied by Boehringer Ingelheim Canada, Ltd. (Ontario). Bosentan (4-tert-Butyl-N-  [6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-  2,2'-bipyrimidin-4-yl] benzene sulfonamide monohydrate) was from Actelion Ltd., Switzerland. All other drugs were purchased from Sigma Chemical Company (St. Louis, MO).  29  2.3 RESULTS 2.3.1 B A S A L T O N E Septal arteries (150-180 pm in diameter) developed basal tone in normal P S S . Spontaneous tone developed upon equilibration at 70 m m H g for 1 h. Step increases in pressure from 10 m m H g to 20 mmHg, then from 20-120 m m H g in 20 m m H g increments led to the development o f myogenic tone at 60 m m H g (n=4), manifested by a pressureinduced decrease in luminal diameter (Fig. 2.3.1 A ) . Thus increases in pressure leads to constriction o f the arteries in normal P S S (Fig. 2.3.IB). Vessel tone was present at pressures as l o w as 20 mmHg. Between 60-120 mmHg, vessel constriction was between 61-65% (n=4). Spontaneous rhythmic activity was not seen in any o f the arteries. In the presence o f 1 p M bosentan, vessel tone decreased and vasoconstriction was calculated to be between 40-43% (n=3-4, F i g . 2.3.2). With incubation o f the vessels with 10 p M bosentan, myogenic tone was further decreased, with vessel constriction between 29-33% at 60-120 m m H g (n=4-5, Fig. 2.3.2). In endothelium-denuded vessels, incubation with 10 p M bosentan resulted in similar vessel constriction o f 20-26% at 80-120 m m H g (n=4) as in control tissues (19-26%>, n=4, F i g . 2.3.3). Vasoconstriction due to 80 m M K  +  solution  was used as a control for any non-specific effect o f bosentan (10 p M ) ; constriction to K  +  was unaffected by bosentan (unpublished data). Tone was abolished when normal C a 2 +  containing P S S was substituted with C a -free P S S , whence step increases in intraluminal pressure only dilated the vessels (n=4, Fig.2.3.1 A , B ) .  30  Trace Recording of Active and Passive Arterial Diameter in the Pressurized Mouse Septal Artery  BO | j m -l  6  m  i n  Pressure-diameter relationship in Pressurized Mouse Septal Arteries Possessing Myogenic Tone  0-| 0  1  1  1  1  1  1  20  40  60  80  100  120  Pressure (mmHg)  Fig. 2.3.1 A Trace recording of arterial diameter in normal Ca -containing PSS and Ca free PSS. In normal PSS, spontaneous myogenic tone develops at 60 mmHg. In Ca free PSS, tone is absent and the vessel displays passive behavior. 3.3.1B The relationship between intraluminal pressure and vessel diameter in normal PSS and Ca -free PSS. In normal PSS, increases in pressure lead to a decrease in vessel diameter. Between 60-120 mmHg, luminal diameter measured between 79.3±6.2 pm - 81.5±3.2 pm ( • , n=4). In Ca -free PSS, increases in pressure lead to vasodilation. Between 60-120 mmHg, luminal diameter measured between 217.9±3.3 pm - 233.5+3.2 pm (A, n=4). 2+  2+  2+  2+  2+  31  Bosentan-induced Inhibition of Myogenic Tone in Pressurized Septal Arteries of Pentobarbital-Anesthetized Mice  75-,  1—I—I  c E 25H  1—  20  —i—  40  60  ~80~  100  •  control  •  1 |jM bosentan  A  1 0 pM bosentan  1 120  Pressure (mmHg)  Fig. 2.3.2 Development o f spontaneous myogenic tone i n mouse septal arteries. Intraluminal pressure was increased from 10 m m H g to 20 m m H g , then from 20 m m H g to 120 m m H g i n 20 m m H g increments. The vessel was allowed to equilibrate for 5 m i n at each pressure. Between 60-120 m m H g , vessel constriction i n normal P S S was 61-65% (B, n=4). In the presence o f 1 p M bosentan ( T ) , vessel constriction within the same pressure range was 40-43% (n=3-4). Pre-treatment with 10 p M bosentan ( A ) resulted i n 29-33% constriction (n=4).  32  Myogenic Tone in Endothelium-denuded Arteries in the Absence and Presence of Bosentan  Fig. 2.3.3 Development of spontaneous myogenic tone in endothelium-denuded mouse septal arteries. Between 60-120 mmHg, vessel constriction in normal PSS was 19-26% (B n=4). In the presence of lOpM bosentan (A), vessel constriction within the same pressure range was 20-26%) (n=4).Tone was abolished when normal Ca -containing PSS was substituted with Ca -free PSS, whence step increases in intraluminal pressure only dilated the vessels (n=4, Fig. 2.3.1 A, B). 5  2+  2+  33  2.3.2 E F F E C T O F A C E T Y L C H O L I N E Acetylcholine (1 n M - 1 0 u M ) caused concentration-dependent  vasodilation in septal  arteries possessing basal tone (n=3-9, F i g . 2.3.4). A t the threshold concentration o f 1 n M acetylcholine, there was a detectable relaxation o f 0.2+0.1% (% maximum response, n=5). A t the highest studied concentration (10 u M ) , acetylcholine produced 65.3+10.3% relaxation (n=3). Relaxation induced by 10 \iM acetylcholine was abolished by pretreatment with 200 |xM L - N A M E (n=5). In endothelium-denuded arteries, acetylcholine (10 p M ) elicited no vasodilatator response.  2.3.3 E N D O T H E L I A L a - A D R E N O C E P T O R S A N D N O R A D R E N A L I N E 2  There was no vascular response to either o f the ai-adrenoceptor  selective agonists  phenylephrine or methoxamine (unpublished data). In comparison, both agents caused concentration-dependent  vasoconstriction in size-matched mouse mesenteric  arteries  (unpublished data). The ct2-adrenoceptor agonist clonidine (1 p M and 10 p M , respectively) caused 2 2 . 1 ± 3.1%  and 33.5+3.3% relaxation o f arteries possessing basal tone (n=4, F i g . 2.3.5).  Another ©^-adrenoceptor agonist, B H T 920 (1 p M ) , chosen near its EC50 in rabbit femoral arteries (Garcia-Villalon et al., 1992), caused 25.8±3.7% relaxation o f arteries (n=5, Fig. 2.3.5). This response to B H T 920 was abolished after incubation with either 10 p M yohimbine (n=7, Fig. 2.3.5) or 200 p M L - N A M E (n=5).  34  Acetylcholine Concentration-Response Curve  100-1  Log Concentration ACh (Log M)  Fig. 2.3.4 Acetylcholine (ACh)-induced concentration-dependent relaxation o f septal arteries pressurized at 70 mmHg. Acetylcholine was cumulatively applied to the bath. The vessel was allowed to reach steady-state within a 5 min period prior to subsequent acetylcholine addition.  Table Showing the Effects of Various a- and p-adrenoceptor Ligands on Septal Arteries Pressurized at 70 mmHg  Treatment  Mechanism of Action  Clonidine (1 p M ) Clonidine (10 u M )  0C2-specific agonist  B H T 920 B H T 920 + yohimbine B H T 920 + L - N A M E  a.2-specific agonist a -specific agonist 2  ct2 + a , P agonist a + N O S inhibitor 2  Relaxation (% maximum) 22.1+3.1 (n=4) 30.5+3.3 (n=3) 25.8+3.7 (n=5) N o response N o response 82.2±1.0 (n=5) N o response  Isoprenaline Isoprenaline + propranolol  P agonist  Isoprenaline + yohimbine Noradrenaline  77.3+10.2 (n=4) 86.5+3.0 (n=5)  Noradrenaline + yohimbine  P agonist a , P agonist a,p agonist + a antagonist  Noradrenaline + propranolol Noradrenaline+ prazosin + propranolol  a,p agonist + P antagonist a , p agonist + a i antagonist +  83.1+8.4 (n=5) 45.0 (n=2)  Noradrenaline + L - N A M E + propranolol  P antagonist oc,P agonist + N O S inhibitor +  N o response  Noradrenaline + yohimbine + propranolol  P antagonist a , P agonist + a antagonist +  N o response  P agonist  37.5+3.0 (n=5)  P antagonist  Fig. 2.3.5 Effect o f various a - and p-adrenoceptor ligands on septal arteries pressurized at 70 mmHg. Concentrations used: B H T 920 = 1 p M ; isoprenaline; noradrenaline; prazosin; yohimbine = 10 p M ; propranolol = 20 p M ; L - N A M E = 200 p M .  36  In septal arteries with basal tone, noradrenaline caused concentration-dependent (1 n M 10 p M ) relaxation (n=5, F i g . 2.3.6), with a maximum response o f 86.5+3.0% (n=5). In contrast,  noradrenaline  caused  concentration-dependent  vasoconstriction o f  mouse  mesenteric arteries (n=5, F i g . 2.3.7), with a maximum response o f 100.0+0.0% (n=4). In the coronary arteries, 10  yohimbine was partially effective in reducing the response  to a single noradrenaline (10 p M ) challenge (37.5±3.0%, n=5). Arterial incubation with L - N A M E (200 p M ) resulted in a slight inhibition o f noradrenaline (10 pM)-induced vasodilation (79.7±8.7%, n=3). In arteries pre-incubated with both prazosin (10 p M ) and propranolol (20 p M ) , noradrenaline (10 p M ) produced 4 5 . 0 ± 2 . 7 % relaxation (n=2, F i g . 2.3.5). In pressurized endothelium-denuded arteries, neither acetylcholine (10 p M ) nor B H T 920 (3 p M ) elicited vasodilatory responses (n=4, Fig. 2.3.8).  2.3.4 S M O O T H M U S C L E p - A D R E N O C E P T O R S A N D N O R A D R E N A L I N E  A 10 p M isoprenaline caused vasodilation (82.2+1.0%, n=5), which was antagonized by 20 p M propranolol (n=5), but unaffected by 10 p M yohimbine (77.3+10.2%, n=4, Fig. 2.3.5). In endothelial-denuded arteries, isoprenaline (10 p M ) also induced vasorelaxation (Fig. 2.3.5). In septal arteries pressurized at 70 mmHg, propranolol (20 (Jvl) did not significantly reduce the response to noradrenaline (10 ^ M , 83.1±8.4%, n=5).  Combination o f  propranolol (20 ^ M ) and yohimbine (10 ^ M ) abolished the response  to  10 p M  noradrenaline (n=6, F i g . 2.3.5). In addition, the combination o f propranolol (20 p M ) and L - N A M E (200 p M ) abolished the response to 10 p M noradrenaline (0.2+1.2%, n=4).  37  Noradrenaline Concentration-Response Curve in Septal Arteries  Fig. 2.3.6 Noradrenaline (NA)-induced concentration-dependent relaxation o f septal arteries pressurized at 70 m m H g (n=5). Noradrenaline was cumulatively applied to the bath. The vessel was allowed to reach steady-state within a 5 min period prior to subsequent noradrenaline addition.  38  Noradrenaline Concentration-Response Curve in Mesenteric Arteries  100  o E  £1  -8  -7  -6  Log Concentration N A ( L o g  M)  F i g . 2.3.7 Noradrenaline (NA)-induced concentration-dependent constriction of mouse mesenteric arteries pressurized at 70 mmHg (n=4). Noradrenaline was cumulatively applied to the bath. The vessel was allowed to reach steady-state within a 5 min period prior to subsequent noradrenaline addition.  39  Trace Recording of the Effects of Acetylcholine, BHT 920 and Isoprenaline on Endothelium-denuded Septal Arterial Diameter  Fig. 2.3.8 Trace recording o f endothelium-denuded septal arterial diameter. The vessel was pressurized at 70 mmHg. Both a 10 p M acetylcholine challenge and a 3 p M B H T 920 challenge produced no effect. A 10 p M isoprenaline caused vasodilation.  40  2.3.5 ENDOTBELIJJM-DEPENDENT VASODILATORS The endothelium-dependent vasodilators bradykinin and Substance P were applied on the pressurized arteries. Neither bradykinin (10 pM) nor Substance P (0.1 pM) produced any effect.  3.  S T U D Y 2 - M Y O G E N I C A N D E N D O T H E L I A L R E G U L A T I O N O F T O N E IN THE  SEPTAL  ARTERY  OF  T H E CVB3-INFECTED  MYOCARDITIC  MOUSE  3.1 HYPOTHESES AND OBJECTIVES H Y P O T H E S I S 1:  In CVB3-induced murine myocarditis, pressure-induced vascular tone  is augmented compared to sham-infected and uninfected mice. O B J E C T I V E 1:  To compare pressure-induced constriction (myogenic response) in  CVB3-infected versus sham-infected and uninfected mice. HYPOTHESIS  2:  In CVB3-induced murine myocarditis, endothelium-dependent  agonist-induced relaxation responses are impaired. O B J E C T I V E 2:  To compare the effect of acetylcholine in coronary vessels from CVB3-  infected versus sham-infected and uninfected mice. H Y P O T H E S I S 3:  Inhibition of ET receptors with bosentan will result in greater  reduction of myogenic tone in CVB3-infected versus sham-infected and uninfected mice. O B J E C T T V E 3:  To compare the degree of inhibition of tone by bosentan in CVB3-  infected versus sham-infected and uninfected mice.  41  3.2 MA TE RIALS AND METHODS 3.2.1 T I S S U E I S O L A T I O N , C A N N U L A T I O N A N D D I A M E T E R M E A S U R E M E N T A l l methods were performed similar to study 1 (see section 2.2.1. above) except that the mice were anesthetized using 100% C02gas. Each mouse to be sacrificed was placed into a plastic chamber with a gas line running into it. The chamber was then closed off, and the gas was turned on, filling the chamber with CO2. It was turned off when the animal was no longer conscious.  3.2.2 S T U D Y P R O T O C O L I N O C U L A T I O N Three groups o f male C D - I mice (6-7 weeks o f age) were purchased from Charles River (Quebec, Canada). The sham inoculated treatment group was inoculated intraperitoneally with 2 m L sterile phosphate buffered saline ( P B S ) and sacrificed at 1, 3, 7 and 28 days post-inoculation. The CVB3-infected treatment group was inoculated intraperitoneally with 1 m L o f the Charles Guantt strain o f C V B 3 (University o f Nebraska M e d i c a l Centre, Nebraska, U S A ) diluted in Delbecco's Modified Eagle's M e d i a ( D M E M ) to 1.75 x 1 0  10  plaque-forming units (pfuVmL, and 1 m L sterile P B S . These mice were also sacrificed at the same four time points as above. The third treatment group was a non-treatment (uninfected) that was sacrificed at age-matched time points.  42 E X P E R I M E N T A L P R O T O C O L In experiments designed to measure the spontaneous myogenic response, pressurediameter relationships were determined by increasing the intraluminal pressure from 5 to 10 to 20 mmHg, and in 20 m m H g increments to 120 m m H g thereafter. A t each pressure, a 5 min stabilization period was allowed during which time the vessel typically achieved a steady state diameter. The same protocol was repeated using vessels pre-incubated with 10 p M bosentan for 30 min. To investigate agonist-induced vasodilation, acetylcholine (1 n M - 10 p M ) was applied cumulatively to the bath at 5 min intervals. A diameter reading was taken after each 5 min incubation period. In experiments using the endothelin receptor antagonist, the vessels were pre-incubated for 30 min with 10 p M bosentan prior to adding acetylcholine cumulatively. To establish smooth muscle responsiveness to nitric oxide, a single challenge o f 10 p M sodium nitroprusside ( S N P , a donor o f N O ) was added to arteries taken from either uninfected or virus-infected mice and relaxation response was quantified. At the end o f each experiment, normal Ca -containing P S S was substituted with C a 2+  2 +  free P S S to determine passive luminal diameter.  3.2.3. E X P R E S S I O N O F R E S U L T S A N D S T A T I S T I C A L A N A L Y S I S Similar to the previous study, myogenic tone at each pressure was expressed as a percent constriction = 100% x [(D ca-free - D PSS) / D ca-free], where D is the diameter in calcium free (Dc -fr ) or Ca -containing P S S (Dpss). Percent relaxation was calculated using the 2+  a  ee  43  equation 100% x [(Dd - Db)/(Dc -free- Dt,)], where D is the diameter upon stabilization a  after drug addition (d), baseline (b) or calcium free. All results are expressed as mean ± S.E.M. of n animals. One vessel segment was obtained from each animal. Statistical evaluation was made using analysis of variance (ANOVA), and means were considered significantly different when P< 0.05.  3.2.4 SOLUTIONS AND CHEMICALS The composition of the PBS was (in mM): NaCl 137, KC1 3.1, Na P0 8.51, K H P 0 2  4  2  4  1.76. The PBS was made sterile by passing the solution through a 0.22 pm filter into a sterile 1 L bottle. The composition of the PSS and Ca -free solutions were made similar 2+  to that in study 1. Sodium nitroprusside was purchased from Sigma Chemical Company (St. Louis, MO).  3.3 RESULTS 3.3.1 BASAL TONE In the uninfected group, pressure-induced myogenic constriction was found to be between 27-31% in the 80-120 mmHg intravascular pressure range (n=5, Fig. 3.3.1). The sham-inoculated group also constricted to a similar extent over this pressure range (n=35, Fig. 3.3.2). In addition, there was no significant difference found between shaminfected and uninfected groups (10-120 mmHg). In the CVB3-infected group, myogenic tone was found to be between 22-35% constriction between 80-120 mmHg (n=3-5, Fig. 3.3.3). Thus there were no significant differences in pressure-constriction curves between  44  the uninfected, sham-infected and CVB3-infected groups in the higher pressure (physiological) range.  45  Myogenic Tone in Pressurized Septal Arteries of Uninfected Mice Anesthetized with Carbon Dioxide  Pressure (mmHg)  F i g . 3.3.1 Development o f spontaneous myogenic tone in septal arteries o f mice anesthetized with 100% CO2. Intraluminal pressure was increased from 10 m m H g to 20 mmHg, then from 20 mmHg to 120 mmHg in 20 m m H g increments. The vessel was allowed to equilibrate for 5 min at each pressure. Between 80-120 mmHg, vessel constriction in normal PSS was 27-32% ( H  ;  n=5).  46  Myogenic Tone in Pressurized Septal Arteries of Sham-infected Mice  50-,  \^r^^  •  •  1 day p.i.  A  3 days p.i.  T  7 days p.i.  •  28 days p.i.  120  Pressure (mmHg)  Fig. 3.3.2 Development of spontaneous myogenic tone in septal arteries of the shaminoculated mice at timepoints 1 ( •. n=5), 3 (A, n=3), 7 (T, n=5) and 28 (•, n=4) days post-infection. There was no significant difference at all pressures among all time points.  47  Myogenic Tone in Pressurized Septal Arteries of CVB3-infected Mice at 1,3,7 and 28 days Post-infection  50-,  .2  §  £ .1  30H  c E 204  5#  •  1 day p.i.  T  3 days p.i.  •  7 days p.i.  •  28 days p.i.  +  10H —i—  20  —I— 40  —i—  60  80  100  120  Pressure (mmHg)  Fig. 3.3.3 Development of spontaneous myogenic tone in septal arteries of C V B 3 infected mice at timepoints 1 ( • , n=4), 3 ( A , n=4), 7 ( T , n=5) and 28 (•, n=4) days post-infection. There was no significant difference at all pressures among all time points.  48  Pre-treatment of coronary arteries with 10 pM bosentan reduces pressure-induced constriction of uninfected animals to 22-24% (over 80-120 mmHg, n=3). In comparison, vessels taken from infected mice possessed lesser myogenic tone (10-18% constriction, n=4-5) in the presence of the ET-1 receptor antagonist. This difference was not found statistically significant.  3.3.2 AGONIST-INDUCED RELAXATION RESPONSES In the uninfected group, maximum relaxation response to acetylcholine (ACh) was 51.3±6.0% relaxation (n=5, Fig. 3.3.4). There were no significant differences in relaxation response curves between uninfected and sham-infected groups (1 nM- 10 pM). There were also no significant differences in acetylcholine concentration-relaxation response curves between uninfected and virus-infected groups at 1,3 and 7 days postinfection (1 nM- 10 pM). There was, however, a significant difference in relaxation response between the uninfected and virus-infected group at 28 days post-infection. At this time point, responses to all concentrations of acetylcholine were attenuated compared to controls. Acetylcholine (1-10 pM) induced a statistically significant diminished vasodilatory response (n=3-5). Maximum response of these vessels to the highest acetylcholine concentration was only 29.1±8.0% relaxation (n=4, Fig. 3.3.5). However, relaxation response to acetylcholine fully recovered with pre-incubation of these vessels with 10 p M bosentan, with maximum response to 10 p M acetylcholine producing 72.4±10.3% relaxation (10 pM, n=4, Fig. 3.3.5). SNP (10 pM) produced 77.7±4.8% relaxation in uninfected mouse septal arteries (n=3). In the infected counterparts, SNP (10 pM) produced 84.7+5.0% relaxation (n=5).  49  Acetylcholine Concentration-response Curve of Arteries Isolated from Uninfected Mice  100-1  04 -9  1  1  1  1  -8  -7  -6  -5  Log Concentration A C h (Log M)  Fig. 3.3.4 Acetylcholine (ACh)-induced concentration-dependent relaxation of septal arteries pressurized at 70 mmHg, retrieved from non-infected mice anesthetized with 100% CO2. Acetylcholine was cumulatively applied to the bath. The vessel was allowed to reach steady-state within a 5 min period prior to subsequent acetylcholine addition. Maximum dilation achieved with 10 pM acetylcholine was 51.2±6.0% (% maximum, n=5).  50  Effect of Bosentan on the Acetylcholine Concentration-response Curve of Arteries from CVB3-infected Mice Sacrificed at 28 Days Post-infection  Fig. 3.3.5 Acetylcholine (ACh)-induced concentration-dependent relaxation of septal arteries of CVB3-infected mice sacrificed 28 days post-infection, in the absence ( • , n=4) and presence ( T , n=4 ) of 10 p M bosentan. Arteries were pressurized at 70 mmHg.  51  4. DISCUSSION 4.1 STUDY 1 - MYOGENIC AND ENDOTHELIAL REGULATION OF TONE IN THE HEALTHY MOUSE SEPTAL ARTERY The mouse is routinely used as a model system to assess cardiovascular diseases, such as atherogenesis (Keidar et al., 1999), thrombosis (Fujihira et al., 1993), myocardial infarction (Harada et al., 1999), chronic heart transplant rejection (Koglin and Russell, 1999), myocarditis (Carthy et al., 1998) and cardiotoxicity (Rosenoff et al., 1975). The first successful genetic manipulation in the mouse in the early 1980's and the eventual ease o f recombinant D N A technology naturally made it an animal o f choice for modeling o f human disease. However, the validity o f the mouse model in assessing human pathophysiology has not been evaluated. Although mice and humans share highly conserved genes that regulate fundamental aspects o f cardiovascular morphogenesis (Chien, 1996), there may still be important variability in physiological regulation and responses. For example, it has been reported that the coronary circulation o f small animals may not be optimal in the study o f coronary angiogenesis and revascularization, due to extensive collateralization (Chien, 1996). Uncertainties about the vasoregulatory states o f murine intramural coronary arteries and the applicability o f the mouse for modeling abnormalities hypothesized in human hearts, served as a foundation for our observations. The blood pressure o f the mouse is reported to be in the vicinity o f 90-100 m m H g (Wang et al., 1997). Our study demonstrated that the mouse septal artery possessed a relatively high degree o f myogenic tone within this pressure range. In comparison to the male rat septal artery where maximal tone is between 30-40 % constriction (Wellman et  52  al., 1996), basal tone in the mouse septal artery is approximately two-fold greater. The blockade o f endothelin receptors by bosentan significantly decreased the extent o f basal tone, indicating that in the mouse coronary circulation, endothelin is an important regulator o f diameter and hence coronary flow. Addition o f bosentan to deendothelialized arteries did not alter the extent o f myogenic tone (compared to control vessels untreated with bosentan), indicating that basal release o f endothelin occurs from endothelial cells. The target for endothelin is endothelin receptors located in smooth muscle cells o f the media. Noradrenaline produced a a - and P-adrenoceptor-mediated, concentration-dependent 2  relaxation in the mouse coronary resistance artery. Inhibition o f nitric oxide synthase with L - N A M E attenuated the vasodilatory response to the selective a -adrenoceptor 2  agonist B H T 920, thus suggesting that this response is mediated by nitric oxide. These results are in agreement with Thorin et al. (1998) who reported that a -adrenoceptor 2  mediated relaxation in mesenteric arteries o f mice could be antagonized by nitric oxide synthase inhibition. Furthermore, since B H T 920 elicited no response in the endotheliumdenuded artery, it was concluded that the a -adrenoceptors o f the mouse coronary artery 2  were endothelium-located. Interestingly, propranolol alone did not significantly inhibit noradrenaline-induced relaxation. This suggested the presence o f a possible convergence o f intracellular signaling between the a - and P-adrenoceptor-mediated vasodilation pathways, where the 2  inhibition o f a -adrenoceptors would potentially affect the signaling pathway and thus 2  response to noradrenaline by P-adrenoceptors, but not vice versa.  53  The concentration o f noradrenaline in the coronary circulation at rest is approximately 1.2 n M (Hjemdahl et al., 1983). This same concentration o f noradrenaline is reported to cause oti- and a.2- adrenoceptor mediated constriction in the isolated human coronary artery, even in the absence o f 3-blockade (Saetrum Opgaard and Edvinsson, 1997)(Fig. 4.4.1). It should be noted that a direct comparison o f the effects o f noradrenaline on the same septal artery o f different species using the same technique could not be made, thus we presented, as accurately as possible, the effects of this drug on different coronary arteries. However, we demonstrated that in the isolated mouse septal artery, not only is there is a lack o f ai-adrenoceptor response, but noradrenaline (1 n M - 10 p M ) consistently caused vasodilation; both mark important phenotypic species differences in the pharmacology o f the coronary circulation.  54  Table Showing Species Comparison of the Effect of Noradrenaline Applied on Coronary Arteries  MEDIATED VIA RECEPTOR TYPE(S):  REFERENCE(S)  SPECIES  C O R O N A R Y ARTERY  TISSUE PREPARATION  EFFECT OF NA  HUMAN  EPICARDIAL  ISOLATED SEGMENTS  VASOCONSTRICTION A N D VASODILATION  ALPHA  SAETRUM OPGAARD A N D EDVINSSON  RAT  INTRAMYOCARDIAL  ISOLATED SEGMENTS  VASOCONSTRICTION  ALPHA  SJORGREN A N D EDVINSSON  PIG  RIGHT CORONARY ARTERY  ISOLATED  VASODILATION  BETA  COHEN ET AL.  DOG  LARGE CORONARY ARTERIES  ISOLATED RINGS  VASOCONSTRICTION  ALPHA-1, 2  A N G U S E T A L . , 1986  HORSE  UNSPECIFIED  ISOLATED RINGS  VASOCONSTRICTION  ALPHA-1, BETA-1  OBI E T A L .  RABBIT  LEFT ANTERIOR DESCENDING  ISOLATED  VASODILATION; VASOCONSTRICTION A T HIGH (NA)  ALPHA BETA  CORR& BURNSTOCK  SEPTAL  ISOLATED SEGMENTS  VASODILATION  ALPHA-2, BETA  LUI ET AL.  MOUSE  RINGS  RINGS  Fig. 4.4.1 Species comparison of the effect of noradrenaline on various coronary arteries.  55 Our study also demonstrated that the mediation o f acetylcholine-induced vasodilation in the mouse coronary. In comparison to the rat coronary artery, that o f the mouse did not fully dilate to 10 p M acetylcholine (unpublished data). However, the relaxation response to acetylcholine in both species occurred via endothelium-derived nitric oxide, as confirmed by the lack o f response to acetylcholine in the presence o f L - N A M E or absence of endothelium. This mechanism o f acetylcholine action is similar to that in the isolated human coronary artery. Paradoxically, in human coronary microvessels (Angus et al., 1991) (non-septal) and epicardial vessels (Kalsner, 1985), variable populations o f acetylcholine receptors may also exist on the medial smooth muscle, mediating vasoconstriction, countering the role o f those on the endothelium which mediate vasorelaxation. Lastly, the endothelium-dependent vasodilators bradykinin and substance P were found to be inactive in the mouse coronary arteries. In contrast, bradykinin has been reported to relax the coronary arteries o f various species including human and dog (Regoli and Barabe, 1980), and is due to both the generation o f nitric oxide as well as activation o f phospholipase A with release o f prostaglandin I . The absence o f a vasodilatory 2  2  response to bradykinin in mouse septal arteries may be an important aspect under pathological conditions in which inflammation occurs. Substance P is also a coronary artery vasorelaxant in the human (Tousoulis et al., 1999), as well as in swine (Uchida et al., 1999). L i k e bradykinin, substance P causes vasodilation and increased permeability of smooth muscle cells. The role o f substance P in the coronary circulation is not fully understood.  56  In conclusion, the present study elucidated responses of the pressure myograph-mounted mouse coronary artery to pharmacological agents of pertinence to those previously reported in the human coronary artery. Briefly, the isolated mouse septal artery possessed a high degree of myogenic tone to which endothelin contributed significantly. These coronary arteries relaxed to noradrenaline via (endothelium-located) 0C2- and (smooth muscle-located) (^-adrenoceptors, as well as to acetylcholine via endothelium-derived nitric oxide. However, cti-adrenoceptor selective agonists as well as bradykinin and substance P were found to have no vasoactive effect. Differences between human and murine responses illustrated by these findings may impact on the relevance of the mouse coronary artery as a potential model of human coronary vessel diseases.  4.2 STUDY 2 - MYOGENIC AND ENDOTHELIAL  REGULATION OF TONE IN  THE MYOCARDITICMOUSE SEPTAL ARTERY Although previous studies have reported focal coronary arterial narrowing in myocarditis, our present study did not detect enhanced pressure-induced vasoconstriction in the physiological range at all studied time points. Time points were chosen accordingly to represent the phases and progression of myocarditis. Endothelial dysfunction was detected at 28 days post-infection, manifested by a blunted response to the endotheliumdependent agonist acetylcholine. Interestingly, this response fully recovered in the presence of the mixed endothelin A/B receptor antagonist bosentan. Small vessel disease is thought to be an important aspect of myocardial injury; microvascular abnormalities exist and likely contribute to the progression of viral myocarditis. Myocarditis may later exacerbate into further complications associated with  57  congestive cardiomyopathy. Previous studies have shown that, in the acute phase, extensive damage to the aorta, smaller coronary arteries and capillaries could be identified (Burch, 1975). These vascular lesions appeared to be a result of direct viral damage, as viral crystals could be detected adjacent to areas of cytonecrosis. Although one study did not report significant stenosis in the coronary arteries of myocarditic patients (Hasumi et al, 1983), it is now generally accepted that the virus is able to invade the vascular endothelium leading to vascular spasm. In addition, studies have shown lumenal narrowing and endothelial cell swelling and damage, all of which appear noncontiguous and variable in degree. A marked increase in infiltrating mononuclear cells around the microvessels was also noted (Nakamura et al., 1996). A later study confirmed CVB-induced coronary microvascular narrowing (Silver et al., 1989) be treated and successfully reversed by verapamil (Dong et al., 1992). It has been hypothesized that this pattern of focal constriction may be the result of increased production and release of endothelin-1. As a result, this hyperreactive myocardial microcirculation has been suggested as the pathogenic mechanism responsible for ischemia and reperfusion injury, silent myocardial infarction, and ventricular dysfunction (heart failure). In this study, histological staining with hematoxylin and eosin revealed extensive myocyte damage as well as damage of the spleen, liver and lung. Hematoxylin stains acidic structures, such as nuclei, rough endoplasmic reticulum and ribosomes, a purplishblue. Eosin stains basic structures, like cytoplasmic proteins, red or pink. In general, when the H&E staining technique is applied to animal cells, nuclei stain blue and the cytoplasm pink or red. However, immunohistochemical methods did not reveal any  5 8  significant ET-1 localization around the infected coronary vessels at any studied time points (unpublished data). Septal vessels isolated from uninfected and sham-infected mice anesthetized with carbon dioxide (study 2) possessed lower basal tone than vessels from mice anesthetized with sodium pentobarbital (study 1). We hypothesized that this was likely due to the loss of autoregulation as a direct result of carbon dioxide. Arteries dissected from hearts of CVB3-infected mice did not possess significantly greater basal tone at any studied time point in the physiological blood pressure range as compared to its uninfected and sham infected counterparts. However, slight elevations in myogenic reactivity at the lower pressure points at 1, 3 and 7 days post-inoculation indicated the possible presence of increased levels of circulating vasoconstrictor(s). In addition, in the presence of bosentan, there was no significant difference in myogenic tone in vessels from uninfected versus CVB3-infected mice (60-120 mmHg), although constriction was globally decreased as compared to untreated vessels. These results suggest that, using our technique of isolation and myography of cardiac resistance arteries, there was no physiological indication of increased production of ET-1 that effectively contributed to greater myogenic reactivity. Interestingly, this study found a significantly decreased vasodilation response to sub-maximal and maximal acetylcholine concentrations in vessels 28 days post-infection as compared to control and shaminfected groups. This diminution, estimated to be approximately 40%, was hypothesized to be the result of endothelial damage and dysfunction. This was confirmed with the use of the nitric oxide donor SNP, SNP-induced vasorelaxation was not impaired in the infected vessels, indicating no smooth muscle dysfunction with respect to signaling and initiation of vasodilation. This abnormal endothelial response is a likely mechanism  59  involved in and contributing to impaired coronary hemodynamics, and in combination with increased global ET-1 levels, contributes to ischemic myocyte injury and necrosis, eventually leading to cardiac hypertrophy and failure. ET-1 itself also regulates cardiomyocyte growth during hypertrophy (Seta et al., 2000), thus further potentiating the problem. Although this study does not examine acetylcholine response past 28 days postinfection, it would be interesting to continue monitoring endothelial dysfunction to see if responses are further diminished in the chronic stage of disease. Interestingly, pre-treatment of these vessels with bosentan prior to acetylcholine challenge resulted in complete recovery of the vasodilatory response. ET-1 antagonists have been shown to reduce myocardial infarct size in the ischemic heart (Brunner, 1997). Increased ET-1 levels can lead to hypertrophy; thus, potential treatment using ET-1 antagonists may prove therapeutic. Ono et al. showed that treatment with bosentan resulted in the lowering of both heart weight/body weight ratio as well as histological scores for myocardial necrosis in myocarditic mice (Ono et al., 1999). This finding is in agreement with the hypothesis that ET-1 levels are elevated in myocarditis, likely near the vasculature, and partially contributed to by ET-1 released from endothelial cells, interstitial cells and nearby macrophages. In conclusion, the present study evaluated the myocarditic coronary artery for enhanced myogenic tone or indication of hyperconstriction. No significant difference in pressureinduced constriction was found between arteries from infected and uninfected mice. With respect to endothelial function, the endothelium-dependent agonist acetylcholine was found to produce a diminished response in vessels isolated 28 days post-infection, reversed by pre-treatment with bosentan. More detailed studies, perhaps with the  60  inclusion o f more immunohistochemical data, may prove beneficial in assessing this condition further.  5. SUMMARY AND MAJOR CONCLUSIONS 5.1 STUDY 1 This study focused on determining the relevance o f the mouse model for assessing cardiac disease. This aim was achieved by quantifying myogenic and receptor-mediated responses using the mouse septal artery.  These resistance vessels were observed to  possess a high degree o f pressure-induced constriction. Endothelin acting on endothelin receptors present on the smooth muscle cells contributed significantly to this basal tone. Noradrenaline produced a a.2- and P-adrenoceptor mediated,  concentration-dependent  vasodilation. Furthermore, the ct2-adrenoceptors were found to be located on the endothelium, with the P-adrenoceptors on smooth muscle cells. This response differs from the pharmacology in the human coronary artery, where noradrenaline induces otiand a,2-mediated vasoconstriction. However, similar to the human coronary artery, acetylcholine produced concentration-dependent  relaxation o f the coronary  arteries,  mediated by nitric oxide. Lastly, the endothelium-dependent vasodilators bradykinin and substance P were found to be inactive in this mouse model, indicating yet another significant difference in physiological responses between the human and mouse coronary vasculature.  61  5.2 STUDY 2 This study compared a model o f cardiac disease, namely C V B 3 - i n d u c e d myocarditis, to health. Focus was placed on determining and quantifying changes in vascular tone and endothelial function. Although it is hypothesized that increased endothelin-1 levels in myocarditis lead to microvascular narrowing and/or spasm, our technique o f myography did not detect changes in myogenic tone in diseased vessels at 1,3,7 and 28 days postinfection in the physiogical range. However, increased tone was present at early time points at lower pressures. In addition, although extensive myocyte damage was verified, immunohistochemical techniques did not detect high E T - 1 levels near the coronary microvasculature. However, endothelial dysfunction was detected in the chronic stage o f myocarditis (28 days post-infection). Response to the endothelium-dependent  agonist  acetylcholine was ameliorated by the endothelin receptor antagonist bosentan.  6. F U T U R E D I R E C T I O N S Future directions include extension o f this study to include data past 28 days postinfection. Since this study only identified a difference at one time point for only one response, it would be interesting to observe a longer time course to see whether response to acetylcholine is further blunted, and whether bosentan proves beneficial in recovering the response. This work is worthwhile since the involvement o f E T - 1 in chronic heart failure is important, as endothelin receptor antagonists have been reported to increase cardiac output, and decrease left ventricular hypertrophy and dilatation as well as cardiac  62  fibrosis. Thus bosentan and other similar agents may prove cardioprotective and beneficial in the human heart resulting in an increase long-term survival. In addition, further investigation to identify the mechanism(s) contributing to the increase in myogenic tone seen in the low pressure range (under 60 mmHg) in the early time points of myocarditis should be made, since this preliminary observation correlates with previous studies reporting peak ET-1 levels between 3-7 days post-infection. Finally, the regulation of N O production in this disease should also be characterized, as the balance between N O and ET-1 is all-important in affecting and determining the vascular state. 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