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Adrenergic protection of the fish heart under simulated exercise conditions Hanson, Linda Marie 2006

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ADRENERGIC PROTECTION OF THE FISH HEART UNDER SIMULATED EXERCISE CONDITIONS by L I N D A M A R I E H A N S O N B . S c , Simon Fraser University, 2003 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E STUDIES (Zoology) T H E U N I V E R S I T Y O F BRITISH C O L U M B I A April 2006 © Linda Marie Hanson,2006 Abstract Exercise is an integral part of the survival of vertebrates. However, intense exercise disrupts extracellular homeostasis, resulting in venous blood becoming hypoxic, hyperkalemic and acidotic. These changes challenge the heart, as the venous blood passing through the ventricular lumen is the sole source of myocardial support for most vertebrates. Consequently, during exercise when the heart is working its hardest it must ironically be supported by the hostile composition of venous blood. Rainbow trout (Oncorhynchus mykiss) and African catfish (Claris gariepinus) were used to examine the role of adrenergic stimulation in protecting cardiac performance during exercise-induced changes in venous blood composition. As cardiac performance is ultimately limited by oxygen supply, in situ perfused rainbow trout hearts were first used to determine the effect of adrenergic stimulation on the venous hypoxic threshold during simulated exercise conditions (5.0 m M K + , p H 7.5); where the hypoxic threshold is defined as the lowest level of venous oxygen tension (PVO2) that can support maximum cardiac performance. Hearts were tested under these relevant conditions with both tonic (5 n M adrenaline, A D ) and maximal (500 n M A D ) adrenergic stimulation. With 5 n M A D maximum cardiac performance in hearts at 10°C was significantly reduced even at normoxia. In contrast, 500 n M A D fully protected cardiac performance under hyperkalemia and acidosis to hypoxia of 2.0 kPa, a Pvc-2 close to routine values in vivo. Temperature acclimation alters the response of the rainbow trout heart to adrenaline such that an increase in acclimation temperature is associated with a decrease in adrenergic sensitivity. To determine how this reduction in adrenergic effectiveness alters the protection ii afforded to the heart during exercise the in situ hypoxic threshold was determined for perfused rainbow trout hearts at 18°C. The hypoxic threshold at 18°C under the combined hyperkalemic, acidotic exposure with 500 n M A D was 5.6 kPa. In rainbow trout, the loss of adrenergic sensitivity at high temperature is attributed to a decrease in cell surface (3-adrenoceptor density (Bmax). This does not seem to be a universal mechanism among fish as no evidence of Bmax modulation was found in the tropical African Catfish. iii Table of Contents Abstract i i Table of Contents iv List of Figures vi List of Abbreviations vii Preface viii Acknowledgements ix Co-authorship Statement x Chapter 1: Introduction and literature review 1 Rainbow trout cardiac physiology 1 The rainbow trout ventricle and its muscle composition 2 Cardiovascular control and cardiac work during exercise 3 Oxygen supply to the myocardium during exercise 5 Myocardial oxygen consumption 5 Venous oxygen tension vs. venous oxygen content 5 Venous oxygen tensions and threshold levels 6 The coronary circulation 8 Effects of hypoxia on performance 9 Plasma acidosis 10 The dual nature of exercise-induced acidosis 10 The effects of venous acidosis on cardiac performance 11 Exercise-induced hyperkalemia 12 The source of excess plasma iC 12 Effects of hyperkalemia on cardiac performance 12 The importance of adrenaline 13 Adrenergic stimulation of cardiac tissue 13 The /^-adrenoceptor signal transduction system 14 Hypoxia and adrenaline 16 Acidosis and adrenaline 17 Hyperkalemia and adrenaline 18 Temperature as a modifier of cardiac performance 19 Effects on oxygen availability 19 Direct effects on myocardial tissue 19 Temperature and adrenaline 21 Research Objectives and Hypotheses: 22 References 24 i v Chapter 2: The role of adrenergic stimulation in maintaining maximum cardiac performance in rainbow trout (Oncorhynchus mykiss) during hypoxia, hyperkalemia and acidosis at 10°C 34 Introduction 34 Materials and Methods 38 Results 45 Discussion 48 References 59 Chapter 3: The hypoxic threshold for maximum cardiac performance during simulated exercise conditions at 18°C 64 Introduction 64 Materials and Methods 67 Results 71 Discussion 71 References 80 Chapter 4: The effect of temperature acclimation on myocardial p-adrenoceptor density and ligand binding affinity in African Catfish (Claris gariepinus) 85 Introduction 85 Materials and Methods 86 Results 89 Discussion 90 References 96 Chapter 5: Major findings and conclusions 98 Objective 1 98 Objective 2 99 Objective 3 .' 99 Objective 4 100 Objective 5 100 Perspectives 101 References 103 v List of Figures Figure 2.1: The effects of hyperkalemia on maximum cardiac output and maximum cardiac power output of perfused rainbow trout hearts at 10°C, p H 7.9 55 Figure 2.2: The effects of hyperkalemia and acidosis on maximum cardiac output and maximum cardiac power output of perfused rainbow trout hearts at 10°C 56 Figure 2.3: The maximum cardiac output and maximum cardiac power output of perfused rainbow trout hearts at 10°C under hypoxic perfusate 57 Figure 2.4: The maximum cardiac output and maximum cardiac power output of perfused rainbow trout hearts at 10°C under hypoxic, hyperkalemia acidotic perfusate with both tonic and maximal adrenergic stimulation 58 Figure 3.1: The maximum cardiac output of perfused rainbow trout hearts at 18°C under hypoxic, hyperkalemia acidotic perfusate with maximal adrenergic stimulation 76 Figure 3.2: The maximum cardiac power output of perfused rainbow trout hearts at 18°C under hypoxic, hyperkalemic, acidotic perfusate with maximal adrenergic stimulation 77 Figure 3.3: Heart rate at maximum cardiac output of perfused rainbow trout hearts at 18°C under hypoxic, hyperkalemic, acidotic perfusate with maximal adrenergic stimulation 78 Figure 3.4: Cardiac stroke volume during maximum cardiac output of perfused rainbow trout hearts at 18°C under hypoxic, hyperkalemic, acidotic perfusate with maximal adrenergic stimulation 79 Figure 4.1: Specific binding of [ 3H]CGP-12177 to ventricular P-adrenoceptors in rainbow trout and African catfish 94 Figure 4.2: [ 3H]CGP-12177 dissociation constant and ^-adrenoceptor density in African catfish at three acclimation temperatures 95 v i List of Abbreviations p-AR P-adrenoceptor Bmax P-adrenoceptor density A T P adenosine triphosphate A D adrenaline Pao2 oxygen tension in arterial blood Pco2 carbon dioxide tension Q cardiac output PO cardiac power output c A M P cyclic adenosine monophosphate Hb haemoglobin P50 half-maximal haemoglobin-oxygen saturation h heart rate Pin input pressure K d ligand binding affinity Qmax maximum cardiac output PO maximum cardiac power output M02 myocardial oxygen consumption n A D noradrenaline Paul output pressure P02 partial pressure of oxygen Vs stroke volume CVO2 oxygen content in venous blood PVO2 oxygen tension in venous blood P 1 va ventral aortic blood pressure Preface This thesis is comprised of five chapters. Chapter one is an introduction to the current body of literature where the effects of exercise-induced conditions on cardiac performance and the role of adrenergic stimulation in protecting performance under otherwise detrimental conditions are both thoroughly discussed. Emphasis is placed on cardiac function and its regulation in rainbow trout, the model species used for this thesis. The hypotheses under study and overall objectives of the thesis are outlined in the concluding elements of Chapter one. The following three chapters specifically address the thesis objectives and are written as individual manuscripts. This format was preferred, as Chapters 2 and 4 have already been published as individual studies in peer-reviewed journals, and I intend to submit the study presented as Chapter 3 for publication as well. The final chapter is a concise summary of the major findings and conclusions of the thesis, as a whole. vin Acknowledgements First and foremost, I would like to thank my supervisor Dr. Anthony Farrell for his never-ending patience throughout the long, arduous journey I like to call grad school. Whenever I was feeling totally overwhelmed he always managed to reassure me and bring things back into perspective. I would also like to thank my committee members Dr. Colin Brauner and Dr. Jeff Richards for agreeing to be a part of this project post-move, and consequently taking on a thesis that was already in the latter stages of completion. A huge thank you to all of the fun, insightful and fantabulous people I have been fortunate to work with over the years, both at S F U and now here at U B C . Some of the key people include current and past Farrellites: Suzanne Clutterham, Erika Eliason, Louise Kuchel, Mik i Nomura, Jonathan Stecyk, Danielle Simonot, Steve Tang, and Kieth Tierney; and colleagues from the Department of Biological Sciences at S F U and the Department of Zoology at U B C : Rosalind Leggat, Michelle Morrow, Jodie Rummer, and Kat Salvante. I would also like to thank Caroline Churchland, David McKill ikan, Janet Mouniargi and Shannon Obradovich who assisted me with some of the perfused heart experiments. Finally, I must express my utmost thanks to my amazing family. Whether it was dyeing my hair blue or deciding to spend yet another three years in post secondary education, they have always been supportive and never questioned any of my decisions. Mom, Dad, Krista, Gloria and Andrew, thank you for your infinite love and encouragement over the past 26 years! ix Co-authorship Statement Two of the manuscripts contained in this thesis are co-authored. Nevertheless, the vast majority of the research and data analysis was conducted independently. Two research assistants (Shannon Obradovich and Janet Mouniargi) assisted with portions of the perfused heart experiments in Chapter 2. In addition, Dr. Yuen (Alex) Ip obtained the African Catfish used in Chapter 4 and acclimated them in his laboratory in Singapore before shipping the hearts to me for further analysis. Following the conclusion of individual experiments, all manuscripts were written solely by me in consultation with my senior supervisor. x Chapter 1: Introduction and literature review Rainbow trout cardiac physiology Exercise is necessary for survival in most vertebrates. Activities such as migration, reproduction, prey capture and predator avoidance all depend on some form of exercise. In most animals, exercise in working muscles is fuelled by aerobic respiration, and hence exercise success is reliant on oxygen delivery. The heart pumps blood throughout the body and therefore facilitates oxygen delivery and removal of metabolic waste products. During exercise, the heart must work harder in order to increase blood flow, and thus oxygen delivery, to working muscles. As the heart is also a muscle, its oxygen needs also increase during exercise in proportion to the increased workload. Intense exercise, however, disrupts extracellular homeostasis in an effort to maintain intracellular homeostasis, and therefore affects oxygen delivery and metabolic waste removal. Depending on an animal's aerobic threshold - the point at which anaerobic energy pathways start to operate - intense exercise can result in increases in plasma potassium (hyperkalemia), sodium, glucose, lactate, and osmolarity as well as a decrease in p H (acidosis) and oxygen level (hypoxia, especially in the venous blood) (Wood et al. 1983; Boutilier et al. 1986; Tufts et al. 1991; Wang et al. 1994; Brauner et al. 2000). These changes not only challenge the skeletal muscles that are metabolically active during this bout of exercise but also pose a great challenge to the cardiac muscle. Because the myocardium of most vertebrates is avascular, the venous blood passing through the ventricular lumen is the sole source of myocardial support. During exercise, when the heart is working its hardest, it must contend with, and ironically be supported by, the hostile composition of the 1 venous blood. Nowhere is this more pronounced than in teleost fish; -80% of teleosts possess hearts that rely solely on venous blood (Santer, 1985). The rainbow trout (Oncorhynchus mykiss Walbaum), whose heart utilizes both coronary (arterial) and venous blood, but relies mainly (60-70%) on venous blood (Santer and Greer Walker, 1980; Farrell et al, 1988b) is the model species used for the experiments in this thesis. Because so much information is known regarding both exercise performance and cardiovascular morphology and physiology of rainbow trout, this species makes an ideal model species for investigating cardiovascular performance during exercise. The rainbow trout is also a member of the economically important salmonid family, thus research on rainbow trout may provide insight into the physiology of other salmonid species. For example, it has been proposed that recent declines in the number of successful migrating salmonids may be related to the deleterious effects of rising temperatures (along migratory routes) on cardiovascular performance. Understanding the physiology and potential limitations on cardiovascular performance in rainbow trout under similar conditions could provide important implications for wild migrating salmonids and other teleosts. The primary objective of this thesis is to examine the role of adrenaline in maintaining cardiovascular performance by combating the adverse extracellular environment generated during intense exercise. The rainbow trout ventricle and its muscle composition The teleost heart, a series of four chambers, is first perfused via the hepatic veins and ducts of Cuvier, which empty into the sinus venosus, venous blood then passes sequentially through the remaining three chambers of the heart: the atria, the ventricle and the bulbus 2 arteriosus. Teleost hearts share a common architecture but can differ greatly in structure, especially in the muscle composition of the ventricle, which may consist of two distinct myocardial layers, an inner, spongy myocardium (endocardium), and an outer, compact myocardium (epicardium). As the name implies, the former is characterized by a loose, trabecular arrangement of myocytes and multiple invaginations into the lumen. Conversely, compact myocardium possesses myocytes arranged into closely-packed, overlapping sheets that encircle the outside of the ventricle. Teleost ventricles are classified into four types (I-IV) based on proportion and structure of the two tissue types (Tota et al, 1983). Rainbow trout possess a type II ventricle, which consists of an inner layer of spongy myocardium, bathed solely in blood from the cardiac circulation (venous blood), and an outer layer of compact myocardium, perfused by arterial blood via coronary circulation (Tota, 1983). Spongy myocardium accounts for approximately 60-70% of the total myocardium (Santer and Greer Walker, 1980; Farrell et al, 1988b). Consequently, oxygen needs are met largely via diffusion from venous blood that has already oxygenated other tissues, a potential problem when venous oxygen tensions (PV02) are reduced due to exercise or environmental hypoxia. Cardiovascular control and cardiac work during exercise During exercise, cardiac performance increases substantially. Maximum cardiac output (Qmax), the product of heart rate ifH) and stroke volume (Vs), increases 2-3 fold (from 17.6-26.6 ml min"1 kg"1 to 43.9-62.5 ml min"1 kg"1) (Stevens and Randall, 1967b; Kiceniuk and Jones, 1977; Thorarensen et al, 1996). Stroke volume, the volume of blood ejected by each heartbeat, is mainly influenced by ventricular filling time, atrial contractility, and, 3 because it is inversely related to ventricular fi l l ing,// / . In rainbow trout, pacemaker cells located at the sinoatrial junction generate the intrinsic fa (Santer, 1985); regulation is both cholinergic and adrenergic. A tonic, cholinergic tone from the cardiac branches of the vagus nerve (Laurent et al, 1983) depresses fa below the intrinsic pacemaker rate, while varying degrees of neural and humoral adrenergic stimulation act to offset cholinergic inhibition. Adrenergic innervation (neural control) is present in the sinus venosus, atrium and ventricle of the trout heart, although ventricular innervation may be confined to compact myocardium and coronary vasculature (Gannon and Burnstock, 1969). Ventricular adrenergic stimulation increases contractility and potentially mediates increases in coronary blood flow observed during exercise (Axelsson and Farrell, 1993; Gamperl et al, 1994b; Gamperl et al, 1994a; Gamperl et al, 1995). Additionally, tonic levels of circulating catecholamines (humoral control), both adrenaline (AD) and noradrenaline (nAD), are present in rainbow trout plasma (Milligan et al, 1989) and, during intense exercise, can increase from 5-11 n M to over 200 n M (Butler et al, 1986; Milligan et al, 1989) to compensate for the detrimental consequences of exercise-induced changes in venous blood composition. Thus, one of the objectives of this thesis is to determine the degree to which adrenergic stimulation can maintain maximum cardiac performance during hypoxia, hyperkalemia, and acidosis combined, an experimental setting meant to simulate the venous blood composition following intense exercise. This will be assessed by determining the hypoxic threshold for maximum cardiac performance during relevant hyperkalemic, acidotic conditions, both with and without maximal adrenergic stimulation. 4 Oxygen supply to the myocardium during exercise Myocardial oxygen consumption Myocardial oxygen supply, in most teleosts, depends on the amount of oxygen available via cardiac circulation and is therefore a product of cardiac output (Q) and venous oxygen content (CV02). In rainbow trout, and several other teleosts, arterial blood from the coronary circulation supplements myocardial oxygen supply but may be limited to the compact myocardium leaving Cvo2 to satisfy the oxygen demand of the spongy myocardium (-70% of the heart). Even though myocardial oxygen demand can vary greatly, Farrell et al. (1985) calculated that the heart uses less than 4% of Cvo2 and thus Cvo2 is likely more than adequate to support myocardial oxygen consumption (Mo2). Seemingly, Mo2 would depend on cardiac mass, i.e. larger hearts require more oxygen, but this idea may be countered if larger hearts have lower mass-specific Mo2 (Graham and Farrell, 1989). It is clear, however, that Mo2 increases in proportion with cardiac workload (Farrell et al, 1985; Graham and Farrell, 1990) and can increase over 4-fold during intense exercise (Farrell and Jones, 1992). Venous oxygen tension vs. venous oxygen content During intense exercise, cardiac workload increases, and Q is amplified to meet increasing oxygen demand. In skeletal muscle, this means an 80% decrease in Cvo2 (Kiceniuk and Jones, 1977), but in myocardial cells, even during intense exercise, oxygen demand is still met via venous blood (Farrell et al, 1985; Farrell and Jones, 1992). Myocardial cells will continuously utilize oxygen, leaving the venous oxygen tension (Pvo2) directly adjacent to the myocardium lower than the Pvo2 in the more centrally located luminal 5 blood. This reduces oxygen diffusion to the spongy myocardium, despite that fact that there is still plenty of oxygen remaining in the luminal blood; therefore, myocardial oxygen supply is limited by diffusion, which is directly related to Pvo2- Intense exercise can reduce Pvo2 by -50% (Kiceniuk and Jones, 1977) but also increases fH, As a result, not only is Pvoj low, but the blood residence time in the lumen is decreased, subsequently reducing the time available for oxygen diffusion, all of which make oxygen supply to the spongy myocardium more difficult during intense exercise, a time when it is most imperative. Venous oxygen tensions and threshold levels Few studies have measured Pvo2 in fishes (Stevens and Randall, 1967ra; Eddy et al, 1977; Kiceniuk and Jones, 1977; Perry and Reid, 1994; Steffensen and Farrell, 1998; Farrell and Clutterham, 2003) and some of the earlier studies may have been done using stressed fish as resting Pvo2 values of 2.5 kPa (Stevens and Randall, 1967a) and 2.7 kPa (Thomas et al, 1994) represent less than 50% haemoglobin-oxygen saturation at the particular temperatures under study (Nikinmaa and Soivio, 1979; Perry and Reid, 1992a). In addition, all studies, save one, have measured Pvo2 using intermittent blood sampling, which can result in misleading data if samples are drawn during non steady-state swimming. Erratic swimming behaviours can create abrupt changes in Pvo2 (Farrell and Clutterham, 2003). Farrell and Clutterham (2003) used a fibreoptic micro-optode to measure the Pvo2 of maximally exercising rainbow trout and discovered that even at the most severe exercise intensity, Pvo2 did not drop below 2.0 kPa, the estimate obtained in another study via intermittent blood samples from exercising fish (Kiceniuk and Jones, 1977). Environmental 6 hypoxia induces more drastic changes in Pvo2, (< 1 kPa at 8 ° C ) (Thomas et al, 1994), however, especially when combined with exercise (1.3 kPa) (Steffensen and Farrell 1998). Existing evidence suggests that venous oxygen thresholds are influenced by both absolute cardiac workloads (van Raaij et al, 1996) and the extracellular conditions experienced by the heart. This is further emphasized by the finding that isolated perfused hearts exposed to hypoxic saline (3.3 kPa) decreased Qmux and maximum cardiac power output {POmax) by -50% and -80% respectively at routine physiological workloads (Farrell et al, 1989), while isolated trout hearts at sub-physiological workloads could perform under near anoxic conditions with no effects on cardiac performance (Arthur et al, 1992). It follows that the minimum Pvo2 for hypoxia will be lower than that with exercise. Nevertheless, the minimum Pvo2 will be one at which diffusion rates match myocardial oxygen demand; below this Pvc-2, the diffusion gradient will be insufficient to support myocardial function and survival of the heart will be compromised. A venous oxygen threshold, proposed to range from 0.8 to 2.1 kPa (Jones, 1986; Davie and Farrell, 1991; Perry and Reid, 1994; Steffensen and Farrell, 1998; Farrell and Clutterham, 2003), must exist to preserve the necessary oxygen diffusion gradient, but because of contrasting experimental conditions in existing literature, comparisons between environmental and exercise-induced hypoxia thresholds are difficult. Fittingly, another goal of this thesis is to establish the venous oxygen threshold for maximum cardiac performance under both hypoxia alone, and in conjunction with other exercise-induced changes in venous blood composition. 7 The coronary circulation Approximately 20% of all teleosts, including the rainbow trout, utilize a combination of coronary and cardiac circulation (Santer and Greer Walker, 1980). Increased oxygen demand of the compact myocardium may be offset by increasing coronary perfusion; as coronary blood flow can increase by up to two-fold (Axelsson and Farrell, 1993; Gamperl et al., 1994b; Gamperl et al., 1994a; Gamperl et al., 1995). Coronary ablation experiments have demonstrated that the coronary circulation is not necessary to maintain routine cardiac performance (Daxboeck, 1982; Steffensen and Farrell, 1998), despite the fact that the coronary artery has a routine flow (Axelsson and Farrell, 1993; Gamperl et al, 1994b; Gamperl et al, 1995). This suggests, that during routine conditions, oxygen diffusion from venous blood is presumably sufficient to meet the oxygen needs of both compact and spongy myocardium for resting cardiac power output, likely reflected in the routine Pvo2 (3-4 kPa) measured in rainbow trout. However, during exercise when Pvo2 of the cardiac circulation is reduced, the increase in coronary blood flow potentially accommodates oxygen diffusion from venous blood across the spongy myocardium to the compact myocardium. In fact, without this coronary supply, Steffensen and Farrell (1998) found that coronary-ligated rainbow trout reduced cardiac workloads by an estimated 37% during a hypoxic swimming challenge, a percent change very similar to the percentage of compact myocardium. Hence, the importance of coronary circulation may increase during exercise, but the majority of myocardial oxygen needs can still be supplied via diffusion from venous blood. 8 Effects of hypoxia on performance Hypoxia exposure elicits compensatory ventilatory, cardiovascular and humoral responses in teleost fish. During environmental hypoxia, increases in the volume of water moved (Kinkead and Perry, 1991; Perry and Gilmour, 1996; Perry, 1999) and frequency of gill ventilation (Jones, 1952; Holeton and Randall, 1967a; Powell et al, 1998) occur in fish, all to ensure arterial oxygen tension (Paoi) approaches that of the inhaled water. Regardless, Pao2, arterial oxygen content [Caoi), Pvoj and Cvo2 all decrease with progressive hypoxia. During exercise-induced hypoxia, similar increases in ventilation volume and frequency occur (Saunders, 1962; Smith et al, 1967; Stevens and Randall, 1967b; Stevens and Randall, 1967a) to maintain Pao2 and Cao2, but Pvo2 and Cvo2 decrease due to increased oxygen extraction by the tissues. In fish, the major cardiovascular adjustments to environmental hypoxia include a reduction in heart rate (bradycardia or negative chronotropy) (Holeton and Randall, 1967a; Holeton and Randall, 1967b; Farrell, 1982; Perry, 1999) and a reduction in myocardial contraction force (negative inotropy) (Gesser, 1977; Gesser et al, 1982; Farrell et al, 1989; Arthur et al, 1992; Overgaard and Gesser, 2004). Hypoxia can decrease heart rate by almost 60% (Farrell, 1982), which can be linked to an increased cholinergic inhibitory tone from the cardiac branches of the vagus nerve (Laurent et al, 1983). Bradycardia will increase the residence time of venous blood in the heart, allowing more time for oxygen to diffuse from venous blood into myocardial tissues (Farrell, 1984), and decrease the rate of blood flow through the gills, resulting in increased O2 uptake (Holeton and Randall, 1967b). Severe hypoxia (< 1.6 kPa) decreases the isometric force of isolated cardiac muscle strips by 60-90% (Gesser, 1977; Gesser et al, 1982; Overgaard and Gesser, 2004) and has similar 9 negative inotropic consequences for perfused hearts (Farrell et al, 1989; Arthur et al, 1992). This negative inotropy is likely due to an inability of myocardial anaerobic A T P production to overcome the deficit between oxygen supply and oxygen demand as well as metabolic acidosis. Limitations in myocardial oxygen supply may restrict exercise performance (Farrell, 2002), an idea that has been well studied in vivo (Kiceniuk and Jones, 1977; Gamperl et al, 1995; Steffensen and Farrell, 1998; Perry et al, 2000); however, only a few in situ studies have directly examined the effects of hypoxia on maximum cardiac performance (Farrell et al, 1989; Gamperl et al, 1994b; Overgaard et al, 2004b). For this reason, a,secondary objective of this study is to further quantify the effects of various levels of venous hypoxia on maximum cardiac performance in situ. Plasma acidosis The dual nature of exercise-induced acidosis Aerobic cellular respiration generates CO2 as a by-product of A T P production, while anaerobic A T P production generates lactic acid, both of which are released during strenuous exercise from working muscles (including the heart; Overgaard et al., 2004a) into the plasma. Venous blood p H may decrease from 7.9 to 7.5 or even 7.3, an acidotic condition that can persist for up to 8 hours during recovery (Kiceniuk and Jones, 1977; Wood et al, 1977; Graham et al, 1982; Holeton et al, 1983; Turner et al, 1983). Respiratory acidosis, when CO2 is hydrated at a non-catalyzed rate to form (pK a = 6.37) bicarbonate and protons, is different from a metabolic acidosis where lactic acid dissociates (pK a = 3.86) into lactate and protons. Metabolic and respiratory factors contribute equally to post-exercise acidosis 10 (Turner et al, 1983; Milligan and Wood, 1986b); however, the metabolic component is prolonged but slow to develop, while the respiratory component is quick to develop and short lived. The respiratory acidosis begins to disappear immediately following exhaustive exercise and by ~2 hours post-exercise, a metabolic acidosis dominates (Turner et al, 1983; Milligan and Wood, 1986b; 1986a; McDonald et al, 1989); nevertheless, both similarly reduce blood pH. Due to the more rapid onset of, and recovery from, hypercapnic acidosis, all experiments in the following studies utilized CO2 to reduce pH. The effects of venous acidosis on cardiac performance The negative inotropic and chronotropic effects of hypercapnic acidosis have been well studied on isolated cardiac muscle strips (Gesser and Jorgensen, 1982; Gesser et al, 1982; Kalinin and Gesser, 2002) and working perfused heart preparations (Farrell et al, 1986; Farrell and Milligan, 1986; Farrell et al, 1988a). Kalinin and Gesser (2002) observed a 60% decrease in isometric force of rainbow trout ventricular strips exposed to hypercapnic acidosis (pH 6.9), while Farrell et al. (1986) observed a -10% decrease in maximum cardiac power output (POmax), with hypercapnic acidosis (pH 7.4) in perfused rainbow trout hearts, a consequence of decreasing fn and contractile force. Potentially, an acidosis competes with calcium-troponin binding (Williamson et al, 1976; Fabiato and Fabiato, 1978; Gesser and Poupa, 1978; Gesser and Jorgensen, 1982) and therefore effectively reduces myocardial intracellular C a 2 + concentration, the major requirement for cardiac muscle contraction. Specifically, cardiac contraction in the teleost heart is induced by an influx of C a 2 + across the sarcolemma (Tibbits et al, 1992). Once inside the cell, C a 2 + binds to troponin-C inducing muscle contraction, and if an acidosis affects Ca 2 +-troponin binding, myocardial contraction 11 can be adversely altered. Experiments with perfused canine hearts suggest that the negative chronotropic effects of acidosis are directly related to p H effects on pacemaker cells, possibly via decreased rates and strength of firing, and prolonged action potentials (Satoh and Hashimoto 1983). Exercise-induced hyperkalemia The source of excess plasma Another major exercise-induced change in venous blood composition is hyperkalemia, an increase in plasma [K + ] , which is normally tightly regulated because of its effects on membrane potential. During intense exercise and recovery, venous plasma [K + ] increases from 2.5 m M to 3.4-4.7 m M (Holeton et al, 1983; Turner et al, 1983; Perry et al, 1987; Thomas et al, 1987; Nielsen and Lykkeboe, 1992; Hoik and Lykkeboe, 1998) as skeletal myocytes rapidly and repeatedly contract. Repeated stimulation means that Na7K ATPase cannot fully restore intracellular K + between successive contractions. Excess K + accumulates in the skeletal muscle interstitial fluid and diffuses into the plasma leading to venous hyperkalemia. Post-exercise, skeletal muscle and liver uptake of K + (Elfellah and Reid, 1990; Kes, 2001) and elimination via the kidneys (Ahmed and Weisberg, 2001) restores plasma [K + ] to normal levels. Effects of hyperkalemia on cardiac performance The detrimental effects of hyperkalemia on cardiac performance in rainbow trout are not thoroughly studied, but it is known that hyperkalemia in isolated cardiac strips reduces 12 the contractive force by -50% at 10 m M (Kalinin and Gesser, 2002) and is lethal at 12.5 m M (Nielsen and Gesser, 2001). When myocardial K + gradients are disrupted, resting myocyte membrane potentials are reduced (Miura et al, 1977; Chapman and Rodrigo, 1987; Hove-Madsen and Gesser, 1989; Bouchard et al, 2004). Depolarization may reduce the strength of ventricular contractions by inactivating a proportion of ventricular N a + channels and therefore slowing cardiac conduction and reducing the action potential strength (Chapman and Rodrigo, 1987; Fozzard and Shorofsky, 1992). In mammals, hyperkalemia ( K + > 5.5 mM) results in ventricular arrhythmia (Paterson et al, 1992; Kes, 2001). Hyperkalemia-induced depolarization can also indirectly affect myocardial contractility by affecting voltage-gated C a 2 + channels (Bouchard et al, 2004). Since the effects of hyperkalemia on cardiac performance have only been examined on paced, isolated cardiac muscle strips, little is known regarding the chronotropic effects of hyperkalemic exposure. Consequently, another aim of this research is to quantify both inotropic and chronotropic effects of hyperkalemia in a perfused heart preparation. The importance of adrenaline Adrenergic stimulation of cardiac tissue In rainbow trout, the predominant source of catecholamine synthesis and storage is the chromaffin tissue located near the head kidney in the posterior cardinal vein (Nandi, 1961; Reid et al, 1998). Cholinergic nerve fibres innervate the chromaffin tissue, and thus catecholamine release is controlled by the sympathetic nervous system. The primary circulating catecholamines are A D and noradrenaline (nAD), with resting plasma concentrations of approximately 5-11 n M and 14-16 n M , respectively (Milligan et al, 1989). 13 Under stressful conditions, including intense exercise, stored catecholamines (mostly AD) are released into the plasma (Reid and Perry, 1994). As a consequence of intense activity, nAD concentrations increase 2-15 fold, while studies in rainbow trout have reported post-exercise [AD] as high as 212 nM ± 89 nM (Butler et al, 1986), a 40-fold increase. This discussion will focus on the humoral role of AD, as the rainbow trout heart is ~10 times more sensitive to A D than nAD (Gannon and Burnstock, 1969; Ask et al, 1981; Farrell et al, 1986), and A D is the principal catecholamine released by rainbow trout during intense exercise and recovery. The B-adrenoceptor signal transduction system Adrenaline is derived from the amino acid tyrosine, as such, it does not readily cross lipid membranes, and hence it must bind to specific cell-surface receptors, known as adrenoceptors, in order to exert an effect on target cells. There are two distinct categories of adrenoceptors, a and P (Ahlquist, 1948), with each category being further divided into subtypes. As each category of adrenoceptors activates a different signal transduction pathway, catecholamines can trigger diverse effects in a wide range of cell types. The P-adrenoceptor (P-AR) signalling pathway is known to mediate the important cardiac inotropic and chronotropic actions of A D and nAD in fish (Ask et al, 1981; Temma et al, 1986; Gamperl et al, 1994c). Currently, it is believed that rainbow trout possess three distinct subtypes of p-AR receptors, Pi, P2, and P3, although the main P-AR subtype mediating these cardiac actions in rainbow trout is a p2 subtype (Ask et al, 1980; 1981; Keen et al, 1993; Gamperl et al, 1994c). P-adrenoceptor subtypes differ in their relative affinities for AD and nAD. pi and P3-AR possess an almost equal affinity for A D and nAD, with only a slight preference for nAD, while P2-AR show a clear preference for A D (Hoffman and Lefkowitz, 14 1982; Nickerson et al, 2003). Nevertheless, both A D and n A D are capable of stimulating all (3-AR subtypes; consequently, as A D is the principal catecholamine released by rainbow trout during intense exercise and recovery, the following experiments utilized only A D to elicit (3-AR stimulation in rainbow trout hearts. Stimulation of myocardial cell-surface P2-AR activates a G-protein mediated signal transduction cascade. Briefly, ligand binding activates an intracellular stimulatory G-protein which stimulates adenylyl cyclase, adenylyl cyclase catalyzes the formation of cyclic adenosine monophosphate (cAMP) from A T P , and the resulting increase in [cAMP] activates the enzyme cAMP-dependant protein kinase A ; which can phosphorylate a variety of intracellular proteins to produce a specific response depending on the cell type involved. For example, activation of the myocardial P-AR signal transduction cascade is thought to phosphorylate sarcolemmal L-type C a 2 + channels increasing myocardial calcium influx (Tibbits et al, 1992; Shiels and Farrell, 1997; Vornanen, 1998). Additionally, stimulation of erythrocyte P-AR activates N a + / H + exchange which results in erythrocyte alkalization (Nikinmaa and Huestis, 1984; Tang et al, 1988; Perry and Reid, 1992b; Perry and Gilmour, 1996; Val et al. 1998) and an increase in haemoglobin-oxygen affinity. Secondary results of this cascade include cell swelling, a decrease in extracellular pH, an increase in intracellular CI" and K \ and an increase in extracellular Na and bicarbonate. Both the [catecholamines] and the density of cell surface p 2 - A R (B m a x ) influence myocardial adrenergic responsiveness, and both of these can vary dependant upon conditions. 15 Hypoxia and adrenaline Hypoxia elicits significant increases in circulating [AD] and [nAD], with the peak [AD] being 2-6 fold higher than that of n A D (Aota et al, 1990; Perry and Gilmour, 1996). Catecholamine release appears to be triggered by arterial P02 levels ranging from 50 to 60% haemoglobin 02-saturation (Perry and Reid, 1992a; Thomas and Perry, 1992; Perry and Reid, 1994) and leading to changes in the P-AR signal transduction system. Hypoxia causes significant (29% to 62%) down-regulation in Bmax in mammalian and avian ventricular tissue (Voelkel et al, 1981; Blake et al, 1982; Marsh and Sweeney, 1989; Rocha-Singh et al, 1991), but prolonged (6 h) hypoxia (Pvo 2 ~ 1.1 kPa) did not trigger P-AR down-regulation in rainbow trout ventricular tissue (Gamperl et al, 1998). Adrenergic stimulation may be preserved in myocardial tissue that is reliant upon the hypoxia-prone cardiac circulation; this may be further supported with the information that spongy myocardium possesses 14% more P-AR than compact myocardium (Gamperl et al, 1998). The increase in anaerobic respiration during hypoxic exposure results in an intracellular acidosis, which is counteracted via adrenergic stimulation. Adrenergic stimulation of pacemaker cells opposes hypoxic bradycardia by increasing pacemaker self-excitation rates (Tibbits et al, 1992) and intracellular [Ca 2 + ] , which help offset the deleterious effects of hypoxia-induced acidosis. In contrast to ventricular p-AR, trout erythrocytes show P-AR up-regulation and a corresponding increase in adrenergic responsiveness, following a 30-minute hypoxic exposure (Reid et al, 1993). p-adrenergic stimulation of trout erythrocytes appears to increase N a + / H + exchange (Perry and Reid, 1992b; Reid et al, 1993), resulting in erythrocyte alkalization. Alkalization counteracts hypoxia-induced acidosis and increases haemoglobin-oxygen affinity, resulting in more effective O2 uptake at the gills. Improving oxygen 16 transport and uptake at the gills is crucial in maintaining fully saturated arterial blood oxygen levels during environmental hypoxia; however, increased haemoglobin-oxygen affinity will have negative consequences for myocardial oxygen delivery. Acidosis and adrenaline Studies with perfused hearts (Farrell et al., 1983; Farrell et al., 1986; Farrell and Milligan, 1986) and isolated cardiac muscle strips (Gesser and Jorgensen, 1982; Gesser et al., 1982) reveal that adrenergic stimulation can counteract acidosis-induced chronotropic and inotropic effects. Acidosis (pH 7.4) can significantly reduce cardiac performance in perfused sea raven and ocean pout hearts; however, performance was fully restored at p H 7.4 with 1 u M A D (Farrell et al., 1983). Likewise, concurrent adrenergic stimulation (100 u M A D ) at p H 7.4 also maintained cardiac performance in isolated cardiac muscle strips, while acidosis alone reduced contractile force by 30% (Gesser et al., 1982). Adrenergic p -AR stimulation counteracts the acidotic impairment of calcium-troponin binding by increasing intracellular [Ca ]. Adrenaline also restores plasma and erythrocyte p H during exercise-induced acidosis (McDonald et al., 1989) via P3b-AR-mediated erythrocyte N a + / H + exchange (Tang et al., 1988; Perry and Gilmour, 1996; Nickerson et al., 2003), which results in erythrocyte alkalization and enhances haemoglobin-oxygen affinity (Thomas and Perry, 1992). Fish exposed to the p-AR antagonist propranolol required 4 times as long (4 hr vs. 1 hr) to restore plasma p H as opposed to untreated fish (Tang et al., 1988). It is believed that an acidosis-induced hypoxia is what triggers catecholamine release (Perry et al., 1989; Aota et al., 1990) and, at least at the erythrocyte, is in place to prevent the Root effect from occurring and therefore compromising oxygen uptake at the respiratory surface. Additionally, while 17 plasma [AD] was shown to increase almost 5-fold during hypercapnic exposure, there were no significant changes in [nAD] (Perry and Gilmour, 1996) implying that adrenergic stimulation is responsible for acidotic compensation. Hyperkalemia and adrenaline Similarly to acidosis, the negative inotropic effects of hyperkalemia can also be alleviated by adrenergic stimulation. Concurrent adrenergic stimulation (10 u M A D ) of isolated cardiac muscle strips counteracted the 50% decrease in contraction force associated with 10 m M K + (Kalinin and Gesser, 2002) and the complete loss of contractile force associated with 12.5 m M K + (Nielsen and Gesser, 2001). Perhaps the ameliorating effects of adrenaline during hyperkalemia are likewise mediated by P-AR as the effects can be counteracted in mammals with propranolol (Todd and Vick, 1971; DeFronzo et al, 1981). As mentioned previously, hyperkalemia-induced depolarization inactivates cardiac N a + channels, preventing the upstroke of the cardiac action potential (Chapman and Rodrigo, 1987; Fozzard and Shorofsky, 1992). Potentially, adrenergic stimulation restores the action potential upstroke by increasing C a 2 + influx (Paterson et al, 1992) and activates skeletal muscle and liver K + uptake (Elfellah and Reid, 1990) (Kes, 2001) via Na + /K + -ATPase , all to moderate plasma K + concentrations. 18 Temperature as a modifier of cardiac performance Effects on oxygen availability Rainbow trout can encounter a wide range of seasonal temperatures, ranging from 2 ° C to 19°C. Outside their preferred temperature range of 11 to 13°C (Brett, 1964; Taylor et al, 1996), conditions become less favourable, necessitating compensatory changes in physiological systems. In particular, temperature-dependant changes in oxygen solubility limit oxygen availability at high temperatures. Oxygen availability is further restricted by a temperature-induced right-shift in the haemoglobin-oxygen dissociation curve (Perry and Reid, 1994), therefore reducing the 0 2 carrying capacity of the blood (Eddy, 1971), which in addition to reducing O2 uptake at the gills, favours unloading at the skeletal muscles and reduces Cvc^. Moreover, routine O2 needs of the heart increase with temperature (Farrell et al, 1985; Mortensen and Gesser, 1999), further exacerbating the potential imbalance between myocardial O2 supply and demand. Exercise at high temperatures can pose additional challenges including larger increases in blood Pco2 and resulting larger decreases in haemoglobin-oxygen affinity when compared to similar changes in Pcoj at lower temperatures (Eddy, 1971). Consequently, one of the objectives of this thesis is to determine how the hypoxic threshold for maximum cardiac performance changes as acclimation temperature increases from 10°C to 18°C. Direct effects on myocardial tissue In rainbow trout, increased temperature tends to have positive chronotropic and negative inotropic effects on cardiac muscle. Accordingly, increases in acclimation temperature are 19 associated with increased fH (Heath and Hughes, 1973; Farrell, 1984; Graham and Farrell, 1989; Farrell and Jones, 1992; Farrell et al, 1996; Aho and Vornanen, 1999), which can be up to 2-fold (25.8 to 53.6 min"') when, temperatures in perfused trout hearts are increased from 5 ° C to 15°C (Graham and Farrell, 1989). Similar results were seen in vivo with acclimation temperatures of 4 ° C and 17°C (Taylor et al, 1996) and with isolated ventricular strips at acclimation temperatures of 4 ° C and 17°C (Aho and Vornanen, 1999). Chronotropic effects of temperature are believed to arise from direct effects on pacemaker cells; however, temperature compensation involves extrinsic factors such as circulating catecholamines. Increases in are necessary to support temperature-induced increases in metabolic rate (i.e., body O 2 demand) but decrease the time available for O 2 diffusion from the cardiac circulation to the spongy myocardium, posing problems for myocardial 0 2 delivery. The effects of increased temperature on Vs are not as straightforward. As temperature increases above the optimum temperature ( 1 1 - 1 3 ° C as mentioned previously), Vs decreases.(Farrell et al, 1996) but Vs increases at values below the optimum temperature. Taylor et al. (1996) found that when acclimation temp was increased from 11°C to 18°C, Vs decreased from 1.3 ml beat"1 to 0.4 ml beat"1, but when temperature was increased from 4 ° C to 11°C Vs increased over 6-fold from 0.2 ml beat"1 to 1.3 ml beat"1. In order to simplify matters, this discussion will focus on the effects of temperature increase above the preferred temperature, as this is representative of the temperature change utilized in Chapter 3 of this thesis. The relationship between fH and Vs may be the source of conflicting results seen for the effect of temperature on Qmax. Temperature increases above the optimum have been shown to result in a decrease in Qmax in vivo (Taylor et al, 1996); however, Farrell et al. (1996) found no change in Qmax with increased temperature, but this was likely due to the extreme 20 temperatures being studied ( 1 5 - 2 2 ° C ) . In contrast to Qmax, temperature increases from 11°C to 18°C had no effect on routine Q (Taylor et al, 1996), suggesting that the detrimental effects of temperature increase in proportion to cardiac workload. This negative inotropy may be due to temperature-induced changes in ventricular C a 2 + sensitivity (Harrison and Bers, 1989; 1990). Temperature and adrenaline Temperature compensation is accomplished through extrinsic factors, including circulating catecholamines. Temperature acclimation is known to alter the response of the rainbow trout heart to A D (Ask et al, 1981; Graham and Farrell, 1989; Farrell et al, 1996). Specifically, an increase in acclimation temperature decreases A D sensitivity (Graham and Farrell, 1989; Keen et al, 1993), and some of this change has been attributed to a temperature-dependent change in ventricular cell surface P-AR density. Keen et al. (1993) found that increasing the acclimation temperature of rainbow trout from 8 ° C to 18°C resulted in a -65% decease in myocardial P2-AR density. A comparison between rainbow trout acclimated at 8 ° C and 14°C yielded similar results (Gamperl et al, 1998). Both studies found B m a x to decrease by as much as 11% per 1°C increase in temperature. P-adrenergic stimulation is extremely important in preserving the L-type Ca current in isolated trout myocytes (Shiels et al, 2000; Shiels et al, 2003). Studies suggest that despite reduced A D sensitivity at high temperature, the positive inotropic effect of P-adrenergic stimulation increases in importance at higher temperatures (Graham and Farrell, 1989). P-A R plasticity is critical for maintaining cardiac performance in temperate species such as rainbow trout that experience a wide range of seasonal environmental temperatures. 21 Accordingly, one of the objectives of this thesis is to determine if a similar relationship between (3-AR density and acclimation temperature exists in tropical species where acclimation mechanisms, such as P-AR plasticity, may be unnecessary due to relatively stable environmental temperatures. Research Objectives and Hypotheses: 1. To quantify the venous oxygen threshold for maximum cardiac performance using in situ perfused hearts. It is hypothesized that the venous oxygen threshold for maximum cardiac performance during exercise-induced hypoxia will be higher than that previously reported for environmental hypoxia. 2. To quantify the inotropic and chronotropic effects of venous hyperkalemia on in situ perfused hearts. 3. To simulate the effects of post-exercise levels of hyperkalemia and acidosis in combination with hypoxia in situ and determine the hypoxic threshold for maximum cardiac performance with and without concurrent adrenergic stimulation. It is hypothesized that adrenergic stimulation will alleviate the detrimental effects of a combined hyperkalemic, acidotic and hypoxic exposure on maximum cardiac performance. It is further hypothesized that under these conditions the threshold for cardiac collapse will approximate that found in exercising fish in vivo. 4. To determine the effect of elevated temperature on the in situ hypoxic threshold for cardiac collapse associated with a hyperkalemic, acidotic and hypoxic exposure at 18°C. It is hypothesized that the hypoxic threshold for cardiac collapse under 22 hyperkalemic, acidotic, hypoxic conditions, with maximal adrenergic stimulation, will be higher at 18°C than at 10°C because P-adrenoreceptor (P-AR) density is known to decrease with increasing acclimation temperature in rainbow trout. To determine whether the inverse relationship between P-adrenoreceptor density and temperature found in rainbow trout, a temperate species, also exists in African Catfish, a tropical species. If so this would suggest the P-AR mediated response is similar across species. Accordingly, it is hypothesized that P-AR density will show an inverse relationship with acclimation temperature in African Catfish. 23 References Ahlquist, R. P. (1948). A study of the adrenotropic receptors. Am. J. Physiol. 153, 586-600. 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Preconditioning stimuli do not benefit the myocardium of hypoxia-tolerant rainbow trout (Oncorhynchus mykiss). J. Comp. Physiol. 174, 329-340. 29 Paterson, D. J., Blake, G. J., Leitch, S. P., Phillips, S. M. and Brown, H. F. (1992). Effects of catecholamines and potassium on cardiovascular performance in the rabbit. J. Appl. Physiol. 73, 1413-1418. Perry, S. F. (1999). The control of blood pressure during external hypercapnia in the rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 202, 2177-2190. Perry, S. F. and Gilmour, K. M. (1996). Consequences of catecholamine release on ventilation and blood oxygen transport during hypoxia and hypercapnia in an elasmobranch (Squalus acanthias) and a teleost (Oncorhynchus mykiss). J. Exp. Biol. 199, 2105-2118. Perry, S. F. and Reid, S. D. (1992a). Relationship between blood 0 2 content and catecholamine levels during hypoxia in rainbow trout and American eel. Am. J. Physiol. 263, R240-R249. Perry, S. F. and Reid, S. D. (1992b). 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Biol. 203, 1487-1495. Powell, M. D., Haman, F., Wright, G. M. and Perry, S. F. (1998). Response of rainbow trout (Oncorhynchus mykiss) to a graded hypoxia following repeated intermittent exposure to chloramine-T. Aquacult. 165, 27-39. Reid, S. G. and Perry, S. F. (1994). Storage and differential release of catecholamines in rainbow trout (Oncorhynchus mykiss) and American eel (Anguilla rostrata). Physiol. Zool. 67,216-237. Reid, S. D., Lebras, Y. and Perry, S. F. (1993). The in vitro effect of hypoxia on the trout erythrocyte p-adrenergic signal transduction system. J. Exp. Biol. 176, 103-116. Reid, S. G., Bernier, N. J. and Perry, S. F. (1998). The adrenergic stress response in fish: control of catecholamine storage and release, Comp. Biochem. Physiol. C 120, 1-27. 30 Rocha-Singh, K. J . , Honbo, N. Y. and Karliner, J . S. (1991). Hypoxia and glucose independently regulate the P-adrenergic cyclase system in cardiac myocytes. J. Clin. Invest. 88,204-213. Santer, R. M. (1985). Morphology and Innervation of the fish heart. Adv. Anat. Embroyl. Cell. Biol. 89, 1-102. Santer, R. M. and Greer Walker, M. (1980). Morphological studies on the ventricle of teleost and elasmobranch hearts. J. Zool. 190, 259-272. Satoh, H. and Hashimoto, K. (1983). Effect of p H on the sino-atrial node cells and atrial muscle of dog. Arch. Int. Pharmacodyn. Ther. 261, 67'-78. Saunders, R. L. (1962). The irrigation of gills in fishes. II. Efficiency of oxygen uptake in relation to respiratory flow, activity and concentrations of oxygen and carbon dioxide. Cdn. J. Zool. 40, 817-862. Shiels, H. A. and Farrell, A. P . (1997). The effect of temperature and adrenaline on the relative importance of the sarcoplasmic reticulum in contributing C a 2 + to force development in isolated ventricular trabeculae from rainbow trout. J. Exp. Biol. 200, 1607-1621. Shiels, H. A., Vornanen, M. and Farrell, A. P . (2000). 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Changes in blood pressure heart rate and breathing rate during moderate swimming activity in rainbow trout. J. Exp. Biol. 46, 307-315. Tang, Y., Nolan, S. and Boutilier, R. G. (1988). Acid-base regulation following acute acidosis in seawater-adapted rainbow trout, Salmo gairdneri: a possible role for catecholamines. J. Exp. Biol. 134, 297-312. 31 Taylor, S., Egginton, S. and Taylor, E. (1996). Seasonal temperature acclimatisation of rainbow trout: cardiovascular and morphometric influences on maximal sustainable exercise level. J. Exp. Biol. 199, 835-845. Temma, K., Hirata, T., Kitazawa, T., Kondo, H. and Katano, Y. (1986). Are P-adrenergic receptors in ventricular muscles of carp heart {Cyprinus carpio) mostly the p2 type? Comp. Biochem. Physiol. 83C, 261-263. Thomas, S. and Perry, S. F. (1992). Control and consequences of adrenergic activation of red blood cell N a + / H + exchange on blood oxygen and carbon dioxide transport in fish. J. Exp. Zool. 263, 160-175. Thomas, S., Fritsche, R. and Perry, S. F. (1994). Pre- and post- branchial blood respiratory status during acute hypercapnia or hypoxia in rainbow trout, Oncorhynchus mykiss. J. Comp. Physiol. 164B, 451-458. Thomas, S., Poupin, J., Lykkeboe, G. and Johansen, K. (1987). Effects of graded exercise on blood-gas tensions and acid base characteristics of rainbow trout. Respir. Physiol. 68, 85-97. Thorarensen, H., Gallaugher, P. and Farrell, A. P. (1996). Cardiac output in swimming rainbow trout, Oncorhynchus mykiss, acclimated to seawater. Physiol. Zool. 69, 139-153. Tibbits, G. F., Moyes, C. D. and Hove-Madsen, L. (1992). Excitation-contraction coupling in the teleost heart. In Fish Physiology, vol. 12A (eds. W. S. Hoar D. J. Randall and A . P. Farrell), pp. 267-296. New York: Academic Press. Todd, E. P. and Vick, R. L. (1971). Kalemotropic effect of epinephrine - Analysis with adrenergic agonists and antagonists. Am. J. Physiol. 220, 1964-&. Tota, B. (1983). Vascular and metabolic zonation in the ventricular myocardium of mammals and fishes. Comp. Biochem. Physiol. 76A, 423-437. Tota, B., Cimini, V., Salvatore, G. and Zummo, G. (1983). Comparative study of the arterial and lacunary systems of the ventricular myocardium of elasmobranch and teleost fishes. Am. J. Anat. 167, 15-32. Tufts, B.L., Lang, Y., Tufts, K. and Boutilier, R.G. (1991). Exhaustive exercise in "wild" Atlantic salmon {Salmo salar): Acid-base regulation and blood gas transport. Cdn. J. Fish. Aquat. Sci. 48, 868-874. Turner, J. D., Wood, C. M. and Clark, D. (1983). Lactate and proton dynamics in the rainbow trout {Salmo gairdneri). J. Exp. Biol. 104, 247-268. Val, A.L., de Menezes, G.C., and Wood, C M . (1998) Red blood cell adrenergic responses in Amazonian teleosts. J. Fish Biol. 52, 83-93. 32 van Raaij, M. T. M., Pit, D. S. S., Balm, P. H. M., Steffens, A. B. and van den Thillart, G. E. E. J. M. (1996). Behavioral strategy and the physiological stress response in rainbow trout exposed to severe hypoxia. Horm. Behav. 30, 85-92. Voelkel, N. F., Hegstrand, L., Reeves, J. T., McMurtry, I. F. and Molinoff, P. B. (1981). Effects of hypoxia on density of P-adrenergic receptors. J. Appl. Physiol. 50, 363-366. Vornanen, M. (1998). L-type C a 2 + current in fish cardiac myocytes: Effects of thermal acclimation and P-adrenergic stimulation. J. Exp. Biol. 201, 533-47. Wang, Y., Heigenhauser, G. and Wood, C.M. (1994). Integrated responses to exhaustive exercise and recovery in rainbow trout white muscle: Acid-base, phosphogen, carbohydrate, lipid, ammonia, fluid volume and electrolyte metabolism. / . Exp. Biol. 195, 227-258. Williamson, J., Safer, B., Rich, T., Schaffer, S. and Kobayashi, K. (1976). Effects of acidosis on myocardial contractility and metabolism. Acta. Med. Scand. Suppl. 587, 95-112. Wood, C. M., McMahon, B. R. and McDonald, D. G. (1977). A n analysis of changes in blood p H following exhausting activity in the starry flounder, Platichthys stellatus. J. Exp. Biol. 69, 173-185. Wood, C.M., Turner, J.D. and Graham, M.S. (1983). Why do fish die after severe exercise? Fish Biol. 22, 189-201.. 33 Chapter 2 : The role of adrenergic stimulation in maintaining maximum cardiac performance in rainbow trout {Oncorhynchus mykiss) during hypoxia, hyperkalemia and acidosis at 10°C Introduction The extracellular environment is constantly fluctuating, with the consequence that cells and organs, such as the heart, must be able to function under a variety of conditions. For instance, strenuous exercise in rainbow trout (Oncorhynchus mykiss), like many animals, greatly alters the composition of the venous blood. During strenuous exercise and recovery venous blood becomes acidotic, with pH decreasing from p H 7.9 to 7.3-7.5 (Kiceniuk and Jones, 1977; Graham et al, 1982; Holeton et al, 1983; Turner et al, 1983), hypoxic, with oxygen tension decreasing from 4.9 kPa to 2.1-1.1 kPa (Kiceniuk and Jones, 1977; Steffensen and Farrell, 1998; Farrell and Clutterham, 2003); and hyperkalemic, with venous plasma [K + ] increasing from 2.5 m M to 3.4-4.7 m M (Holeton et al, 1983; Turner et al, 1983; Perry et al, 1987; Thomas et al, 1987; Nielsen and Lykkeboe, 1992; Hoik and Lykkeboe, 1998). Such changes can be particularly taxing because the rainbow trout heart is nourished predominantly by venous blood (Santer and Greer Walker, 1980) and, despite exposure to the detrimental effects of hypoxia, hyperkalemia and acidosis, rainbow trout must maintain a high cardiac output (Q) during and after intense exercise. Therefore, to properly evaluate the detrimental effects of such changes in the extracellular environment in the context of fish exercise, one needs to know their combined effects on maximum cardiac A version of this chapter has been accepted for publication: Hanson, L. M , Obradovich, S., Mouniarg i , J. and Farrel l , A . P. (2006). The role o f adrenergic stimulation in maintaining maximum cardiac performance in rainbow trout {Oncorhynchus mykiss) during hypoxia, hyperkalemia and acidosis at 10°C. J. Exp. Biol, (in press). 34 performance. To the best of our knowledge, no one has yet examined the combined effects of exercise-induced hypoxia, hyperkalemia and acidosis on maximum cardiac performance. What has been well studied in this regard are the negative inotropic and chronotropic effects of acidosis alone on both isolated cardiac muscle strips (Gesser and Jorgensen, 1982; Gesser et al, 1982; Kalinin and Gesser, 2002) and working perfused heart preparations (Farrell and Milligan, 1986; Farrell et al, 1986; Farrell et al, 1988). Kalinin and Gesser (2002) observed a 60% decrease in isometric force of rainbow trout ventricular strips exposed to hypercapnic acidosis (pH 6.9), while Farrell et al. (1986) observed a -10% decrease in maximum cardiac power output (POmax), with hypercapnic acidosis (pH 7.4) in perfused rainbow trout hearts, a consequence of declines in both heart rate (fn) and contractile force. It is thought that acidosis exerts its detrimental effects by competitively interfering with calcium-troponin binding (Williamson et al, 1976; Fabiato and Fabiato, 1978; Gesser and Jorgensen, 1982) and effectively reducing the myocardial intracellular Ca concentration. Adrenergic stimulation can protect cardiac performance and counteract the acidosis-induced chronotropic and inotropic effects in both perfused hearts (Farrell et al, 1983; Farrell and Milligan, 1986; Farrell et al, 1986) and isolated cardiac muscle strips (Gesser and Jorgensen, 1982; Gesser et al, 1982). A n acidosis of p H 7.4 significantly reduced both Q and PO of perfused sea raven and ocean pout hearts; however, cardiac performance was fully restored when the acidosis was given in conjunction with 1 yxM adrenaline (AD) (Farrell et al, 1983). Concurrent adrenergic stimulation (100 u M A D ) also maintained cardiac performance in isolated cardiac muscle strips, while acidotic cardiac muscle strips not exposed to A D exhibited a 30% decline in contractile force (Gesser et al, 1982). 35 Adrenergic stimulation counteracts the acidotic impairment of calcium-troponin binding by increasing myocardial C a 2 + influx via the L-type C a 2 + channels (Shiels and Farrell, 1997; Vornanen, 1998). Adrenergic stimulation also activates erythrocyte N a + / H + exchange (Tang et al, 1988; Perry and Gilmour, 1996) which helps restore plasma and erythrocyte p H during exercise-induced acidosis (McDonald et al, 1989). The detrimental effects of hyperkalemia on cardiac performance in rainbow trout are known but less thoroughly studied. Hyperkalemia (10 m M and 12.5 m M K + ) reduces the contractive force of isolated heart strips by -50% (Kalinin and Gesser, 2002) to -100% (Nielsen and Gesser, 2001) respectively. Hyperkalemia reduces the resting membrane potential of myocardial cells (Chapman and Rodrigo, 1987; Hove-Madsen and Gesser, 1989), which in turn decreases the duration of the myocardial action potential, and thus the strength of myocardial contractions (Chapman and Rodrigo, 1987). Additionally, hyperkalemia in mammals ( K + > 5.5 mM) has been shown to result in ventricular arrhythmia (Kes, 2001). Similarly to acidosis, the negative inotropic effects of hyperkalemia can be alleviated by adrenergic stimulation. Concurrent adrenergic stimulation (10 u M A D ) of isolated cardiac muscle completely eliminated a 50% decrease in contraction force associated with 10 m M K + (Kalinin and Gesser, 2002), and the complete loss of contractile force associated with 12.5 m M K + (Nielsen and Gesser, 2001). Since the effects of hyperkalemia on cardiac performance have only been examined on paced, isolated cardiac muscle strips, little is known about the chronotropic effects of hyperkalemic exposure. Consequently, one of the aims of this study is to quantify both inotropic and chronotropic effects of hyperkalemia in a perfused heart preparation. 36 The third important change in the extracellular environment of the trout heart during exercise is the venous oxygen tension (Pvoj). During intense exercise, and as skeletal muscle oxygen demand increases, venous oxygen tension is reduced by -50% (Kiceniuk and Jones, 1977). This reduces the oxygen gradient (driving diffusion of oxygen from the cardiac circulation to the myocardial tissues) at a time when myocardial oxygen consumption is concurrently increasing in proportion to the increased cardiac work during swimming (Farrell, 1985; Graham and Farrell, 1990). Hence, oxygen supply to approximately 70% of the ventricle becomes precarious at a time when it is most needed, despite the fact that coronary blood flow increases during exercise to the remaining myocardium (Axelsson and Farrell, 1993; Gamperl et al, 1994a; Gamperl et al, 1995). Severe hypoxia (< 1.6 kPa) decreases the isometric force of isolated cardiac muscle strips by 60-90% (Gesser, 1977; Gesser et al, 1982; Overgaard and Gesser, 2004). Isolated perfused hearts can perform routine physiological workloads at 3.3 kPa, but this level of hypoxia decreases Qmax and POmax by -50%) and -80% respectively (Farrell et al, 1989), which seems a rather high threshold given that Pvo2 decreases to around 2 kPa during prolonged swimming (Farrell and Clutterham, 2003). Clearly, the exact threshold will be determined by both the cardiac workload and the extracellular conditions experienced by the heart. With regard to the former, perfused trout hearts can generate a routine cardiac output even under near anoxic conditions provided the workload is sub-physiological (Arthur et al, 1992; Overgaard et al, 2004a). Therefore, to properly evaluate the effects of hypoxia it is necessary to examine maximum cardiac performance, which is now possible in trout using in situ heart preparations. In addition, while it has been theorized that a limited myocardial oxygen supply restricts exercise performance (Farrell, 2002), no studies have yet considered the 37 combined effects of hypoxia, acidosis, and hyperkalemia that are known to occur in vivo. Consequently, this study was designed to examine the effects of various levels of hypoxia on maximum cardiac performance under conditions simulating exercise in vivo. Hypoxia was studied alone and in conjunction with hyperkalemia (5 mM), acidosis (pH 7.5) and elevated catecholamines. We tested the hypothesis that adrenaline is critical in maintaining maximum cardiac performance under these conditions, which were intended to simulate those during and immediately after intense activity. Materials and Methods Fish Rainbow trout (Oncorhynchus mykiss Walbaum) of both sexes (mass = 483 g ± 12 g; relative ventricular mass = 0.087% ± 0.002%) were obtained from a local fish hatchery (Richard Henley Farm, Langley, B C , Canada) and held indoors in 2000 L fibreglass tanks continuously supplied with dechlorinated tap water. The fish were maintained under a natural photoperiod at a temperature of 10°C ± 1°C. Water temperature was maintained throughout the experimental period by utilizing either an immersible chiller or by heating the inflowing water with a heat exchanger of local construction. Fish were acclimated a minimum of two weeks prior to experimentation during which time they were fed commercial trout pellets (Pro-form Aquaculture Feeds, Chilliwack, B C , Canada) ad libitum three times per week. 38 Surgical Procedures Fish were anaesthetized in an oxygenated solution of buffered tricaine methane sulfonate (MS-222) (0.1 g L" 1 MS222 & 0.1 g L" 1 N a H C 0 3 ) , weighed and placed on an operating table where their gills were continuously irrigated with chilled, oxygenated anaesthetic (0.05 g L" 1 MS-222) buffered with 0.05 g L" 1 N a H C 0 3 . They were then injected with 1 ml kg"1 of heparinized saline (100 IU ml"1) via the caudal vessels. A n in situ perfused heart preparation was prepared as described in Farrell et al. (1986) as modified by Farrell et al. (1989). Briefly, a shallow lengthwise incision was made from the anal opening to an area just posterior to the pectoral girdle and a stainless steel input cannula was introduced into the sinus venosus via a hepatic vein. Perfusion of the heart, via the input cannula, was immediately commenced with chilled freshwater trout saline (composition below) containing 5.0 n M adrenaline (adrenaline bitartrate salt = A D ) and 10 IU heparin per millilitre. A stainless steel output cannula was then secured into the ventral aorta at a point confluent with the bulbus arteriosus, and purse string sutures were used to occlude both ducts of Cuvier and destroy the cardiac branches of the vagus nerve. In addition, the spine was severed. The total time to prepare the perfused heart preparation was 15-20 minutes. A l l experimental procedures complied with the policies of the University Animal Care Committees of both Simon Fraser University and the University of British Columbia. Following surgery, the fish was transferred to a temperature-controlled, physiological saline bath (124.1 m M NaCl , 2.5 m M KC1, 11.9 m M N a H C 0 3 , 2.0 m M C a C l 2 - 2 H 2 0 , 0.2 m M N a H 2 P 0 4 - H 2 0 , 3.4 m M N a 2 H P 0 4 , 0.9 m M M g S 0 4 - 7 H 2 0 ; all chemicals from Sigma-Aldrich, Oakville, O N , Canada). The input cannula was immediately connected to an adjustable, constant-pressure reservoir, and the output cannula was connected to a separate 39 constant pressure head set at 4.9 kPa to mimic resting in vivo ventral aortic blood pressure. The height of the input pressure reservoir was adjusted to set routine cardiac output (Q) at approximately 17.0 ml min' 1 kg"1 (Kiceniuk and Jones, 1977). Input (Pjn) and output (P o u t) pressure were measured through saline filled side arms (PE50 tubing) connected to disposable pressure transducers (DPT 6100, Smiths Medical, Kirchseeon, Germany). Cardiac outflow was continuously measured through the output line with an in-line electromagnetic flow probe (SWF-4, Zepada Instruments, Seattle, W A , U S A ) that had been previously calibrated with known flow rates of perfusate. The experimental solutions (both the perfusate and the saline bath) were contained in water-jacketed glassware so that temperature could be maintained at 10°C (Brinkman Instruments Inc., Mississauga, O N , Canada). Hearts were allowed to equilibrate for 5-10 minutes while receiving normoxic perfusate (see below) before the experiment commenced. The coronary circulation was not perfused in this preparation. Test Conditions Cardiac performance was assessed under several different protocols using the test conditions defined below. A tonic level of adrenergic stimulation (5 n M A D ) , consistent with that found in resting rainbow trout (Milligan et al, 1989), was used in all situations except when the protective effect of A D was being evaluated. In this latter situation, 500 n M of A D was used in order to ensure maximum adrenergic stimulation, as studies in rainbow trout have reported post-exercise [AD] as high as 212 n M + 89 n M (Butler et al, 1986). 40 Control (normoxic) condition: A l l preparations started under this condition. Freshwater trout saline (124.1 m M NaCl , 2.5 m M KC1, 0.9 m M M g S 0 4 - 7 H 2 0 , 2.5 m M C a C l 2 - 2 H 2 0 , D-glucose 5.6 m M , 11.9 m M NaHCOs) was gassed with 0.5% C 0 2 (balance air) to achieve a p H of 7.9 and an oxygen level of 20.0 kPa. Therefore, preliminary experiments (n = 8, data not shown) were performed to show no significant difference in maximum cardiac performance between hyperoxic hearts (95.5% 0 2 , 0.5% C 0 2 ) and hearts perfused with air, which was the control level of oxygen for all experiments. Hyperkalemia: The composition of the hyperkalemic perfusate was the same as the control perfusate except additional KC1 was added to increase the [K +] to either 5.0 m M or 7.5 m M . Acidosis: To achieve a p H of 7.5, the concentration of NaHC03 in the control perfusate was decreased to 10.1 m M and the solution was equilibrated with a gas mixture containing 1.0% C 0 2 (balance air). Hypoxia: Control perfusate was made hypoxic by equilibrating it with 0.5% C 0 2 and an amount of oxygen corresponding to the desired level of hypoxia (balance nitrogen). The hypoxia levels used in kPa were 20, 12.6, 10, 6.7, 5.0, 3.3, 2.7, 2.0 and 1.3. Premixed, calibrated gases (Praxair, Vancouver, B C , Canada) and Wostoff gas pumps (M303/a-F, Bochum, Germany) were used to generate gas mixtures. Hyperkalemia and acidosis: Acidotic perfusate (to achieve a p H of 7.5), was made hyperkalemic by increasing the [K + ] to either 5.0 m M or 7.5 m M . Hyperkalemia, acidosis and hypoxia: A hyperkalemic (5.0 nM), acidotic (pH 7.5) perfusate (as above) was gassed with a mixture of 1.0% C 0 2 and various concentrations of 0 2 (balance nitrogen) in order to achieve particular levels of hypoxia as specified for the hypoxic perfusate. 41 Experimental Protocols Maximum cardiac performance was repeatedly assessed under 3-5 conditions. By initially measuring both maximum cardiac output (Qmax) and maximum cardiac power output (POmax) under normoxic, control conditions each heart acted as its own control. To determine Qmax, Pin was gradually increased in increments of -0.05 kPa until cardiac output reached a plateau (usually around 0.4 kPa). To assess POmax, Pj n was left at its maximum and Pout was increased in a stepwise fashion in -0.5 kPa increments until PO reached a plateau. After POmax was determined, P o u t and Pj n were returned to resting levels and the heart was allowed to recover (-5 min) before being exposed to the next perfusate. Hearts were exposed to each perfusate for a total of 15 minutes; this time period ensured continued viability of the photosensitive A D (which was renewed with each change in perfusate) while remaining physiologically relevant, as Pvo2 can take more than 20 minutes to return to normal following exhaustive exercise (Farrell and Clutterham, 2003). Under some extreme conditions individual hearts did not perform for 15 minutes, succumbing to a cardiac collapse (i.e. cardiac output approached zero). These hearts were terminated early, the duration noted and normoxic conditions restored. Hearts that were unable to complete both cardiac performance tests were considered to have failed under that test condition. The following sets of protocols were used, each with its own order and combination of test conditions. Series I (Hyperkalemia alone) The main purpose of this series (n = 9) was to define a level of hyperkalemia that was physiologically relevant but did not result in irreversible cardiac failure under normoxic 42 conditions. Exercise in vivo increases plasma K to -5.0 m M (Thomas et al., 1987) but previous studies done on isolated cardiac muscle strips have tested higher concentrations of 5.0 - 12.5 m M . The order of the test conditions was: (1) control, (2) 5 m M K + (3) 7.5 m M K + (4) control, and (5) 7.5 m M K + with 500 n M A D . Series II (Acidosis & Hyperkalemia) The purpose of series II was to quantify under normoxic conditions the (a) effects of a combined hyperkalemic, acidotic exposure on maximum cardiac performance, and (b) the ameliorative effects of adrenaline. Several levels of hyperkalemia were tested in order to determine the tolerance threshold for these conditions. Individual hearts (n = 8) were tested under the following conditions (1) control, (2) 5.0 m M K + , p H 7.5, (3) control, (4) 5.0 m M K + and p H 7.5 with 500 n M A D , and (5) 7.5 m M K + and p H 7.5 with 500 n M A D . In addition, three preliminary preparations were tested using 7.5 m M K + and 5 n M A D at a pH of 7.5 directly after the first control step. However, this exposure resulted in an almost immediate decrease in cardiac output leading to a rapid (< 5 min), irrecoverable cardiac collapse. In view of this, 5.0 m M K + was used for all subsequent combined hyperkalemic exposures. Series III The purpose of series III was to determine the hypoxic thresholds for maximum cardiac performance for hypoxia alone and in conjunction with hyperkalemic (5.0 mM) acidosis (pH 7.5). The levels of respiratory acidosis and hyperkalemia chosen mimic those found in the plasma of exercising rainbow trout in vivo (Milligan and Wood, 1987; Nielsen 43 and Lykkeboe, 1992). Hearts were exposed to the following sequence of test conditions (1) control (normoxia), (2) hypoxia, (3) control, (4) hypoxia, 5.0 m M K + and p H 7.5 with 5 n M A D , and (5) hypoxia, 5.0 m M K + and p H 7.5 with 500 n M A D . The specific hypoxia levels used were 12.6 kPa (n = 6 fish), 10 kPa (n = 10 fish), 6.7 kPa (n = 6 fish) and 5.0 kPa (n = 3 fish). At lower oxygen tensions the combined hypoxic, hyperkalemic, acidotic exposure with 5.0 riM A D (step 4) resulted in myocardial failure. As this appeared to be specifically related to the absence of maximal adrenergic stimulation, the protocol was modified for series IV and V to permit further exploration of the hypoxic thresholds. Series IV To preclude the problem of a heart receiving tonic [AD] not being able to tolerate the hyperkalemic, acidotic test condition at Pvo2 levels below 6.7 kPa, series IV studied hypoxic thresholds with an abbreviated series of test conditions. The following sequence of perfusates was used: (1) control (normoxia), (2) hypoxia, (3) control, and (4) hypoxia, 5.0 m M K + and p H 7.5 with 500 n M A D . The specific oxygen tensions were 5.0 kPa (n = 7 fish), 3.3 kPa (n = 8 fish), 2.7 kPa (n = 8 fish) and 2.0 kPa (n = 3. fish). Series V Because series IV revealed hearts receiving only tonic adrenergic stimulation could not tolerate hypoxia alone below 2.7 kPa, hearts in series V were subjected to a further abbreviated experimental protocol: (1) control, (2) hypoxia, 5.0 m M K + and p H 7.5 with 500 n M A D , and (3) control. The hypoxia levels tested were 2.7 kPa (n = 6 fish), 2.0 kPa (n = 6 44 fish) and 1.3 kPa (n = 4 fish). These test conditions best simulate the changes in venous blood pH, [K + ] , Pvc-2 and [AD] seen in vivo during intense activity and recovery. Calculations and Statistical Analysis A l l experimental data was collected using data acquisition software (Labview version 5.1, National Instruments, Austin, T X , U S A ) , which allowed for real-time measurements of fn, Pin, Pout, Q, and PO. Statistical differences within test groups were determined by one-way repeated measures analysis of variance ( A N O V A ) . When warranted, the Holm-Sidak procedure was used for post hoc multiple comparisons. Sigma Stat (3.0, SPSS Inc., San Rafael, C A , U S A ) was used for all statistical analysis. For statistical comparisons a = 0.05. Results Hyperkalemia alone Based on the experiments performed in series I, both levels of hyperkalemia significantly (P < 0.05) decreased cardiac performance in a dose-dependant manner when compared to control (Fig. 2.1). The 30% decrease in Qmax and POmax with 5.0 m M K + was caused by a 25% decease in fH and a 10% decrease in maximum stroke volume (Vs). Similarly, the 60% reduction in Qmax and POmax with 7.5 m M K + was caused by a 45% decrease in fn and a 25% decrease in maximum Vs. A noticeable arrhythmia also developed near the end of the 7.5 m M K + exposure. Despite these effects of hyperkalemia, maximum cardiac performance was fully restored when hearts were returned to control conditions (Fig. 2.1). Maximal adrenergic stimulation significantly improved maximum cardiac performance 45 of hyperkalemic (7.5 m M K + ) hearts, with increases in both Qmax and P0max to within 20% (P < 0.05) of their original performance under control conditions (Fig. 2.1). Hyperkalemia combined with acidosis The results for 5 m M hyperkalemia alone, and in combination with acidosis (pH 7.5), are presented in figure 2. Hyperkalemia and acidosis significantly decreased (P < 0.05) both Qmax (-65.9% ± 6.0%) and POmax (-55.2% ± 14.6%) from control, and to a greater (20-30%) degree when compared to 5.0 m M K + alone. The additional 20-30% decrease in Qmax was mainly due to a further decrease (-62.0% + 6.6%, P < 0.05) in fH as maximum Vs was still only depressed by 10% (9.73% ± 4.8%, P > 0.05). The combined hyperkalemic, acidotic condition also resulted in a pronounced cardiac arrhythmia. Nevertheless, hearts fully recovered when returned to control conditions (not shown). In contrast, exposure to acidosis and 7.5 m M K + resulted in rapid, irrecoverable cardiac collapse (associated with severe cardiac arrhythmia). Maximal adrenergic stimulation completely prevented the debilitating effect of 5 m M K + and acidosis, allowing the hearts to perform at control levels of Qmax and PO,nax (Fig. 2.2). Moreover, concurrent maximum adrenergic stimulation allowed hearts to perform under the previously lethal conditions of 7.5 m M K + and acidosis, but with both Qmax and POmax -35% lower than control (P < 0.05; Fig. 2.2). Hypoxic thresholds without hyperkalemia and acidosis The first three test conditions in series III and IV provided an assessment of the effects of hypoxia alone and these data are summarized in figure 3. Hypoxia at 12.6 kPa and 10 kPa had no significant effect on Qmax and POmax either during hypoxia or with subsequent 46 normoxic exposure (Fig. 2.3). However, hypoxic levels between 6.7 and 3.3 kPa not only significantly decreased Qmax and POmax by 10-25% (Fig. 2.3), maximum performance did not show any immediate recovery during subsequent normoxia from the level seen under hypoxia. At 2.7 kPa, 3 out of 8 hearts failed during the hypoxia treatment, and all hearts failed during the 2.0 kPa (n = 3) and 1.3 kPa (n = 3) treatments. Based on these results, it appears the hypoxic threshold for impairment of Qmax is between 10 and 6.7 kPa, and the threshold for complete cardiac failure under these conditions is between 2.7 and 2.0 kPa. Hypoxic thresholds with hyperkalemia and acidosis As expected (based on the results of series III for normoxic hearts) the combined effects of acidosis and hyperkalemia in series IV impaired Qmax and POmax by 38% to 66% (P < 0.05) when Pvo2 > 6.7 kPa (Fig. 2.4). Thus, hypoxia > 6.7 kPa had no additive effect on maximum performance when compared to hyperkalemia and acidosis alone. Similar to normoxia, adrenergic stimulation fully restored Qmax and POmax with 5 m M K + and acidosis at 12.6 kPa and 10 kPa, while the protective effect of A D was apparently lost at 6.7 kPa (Fig. 2.4), this result could have been due to poor recovery from prior exposures (hypoxia alone or hypoxia, hyperkalemia, and acidosis with tonic [AD]) in this series of experiments. Therefore, series V was designed to eliminate this possibility. In series V , concurrent adrenergic stimulation protected maximum cardiac performance under hyperkalemia and acidosis down to hypoxia levels of 2.0 kPa, because neither Q,nax nor POmax were significantly different from control (Fig. 2.4). At hypoxia levels of 1.3 kPa, however, maximal adrenergic stimulation only partially protected cardiac performance, as Qmax and POmax were reduced by 29.4 ± 3.3% and 43.6% ± 2.8% 47 respectively (Fig. 2.4). Moreover, following the hypoxic, hyperkalemic, acidotic exposure, with 500 n M A D , hearts exposed to 2.7, 2.0, and 1.3 kPa did not recover from these effects when returned to normoxic conditions (data not shown, P < 0.05). Therefore, this suggests that under these hyperkalemic, acidotic conditions with adrenergic stimulation the hypoxic threshold for maximum cardiac performance is 2.0 kPa, but in the absence of adrenergic stimulation, there is no refuge from cardiac impairment. Discussion The in situ perfused heart preparation utilized in this study can perform at levels approximating maximum in vivo cardiac performance, and remain stable for several hours (Farrell et al., 1986; Farrell, 2002; Overgaard et al., 2004b). Correspondingly, our values for maximum cardiac performance under normoxic conditions (Qmax = 51.3 ± 1.7 ml min' 1 kg"1; POmax = 5.9 ± 0.2 m W g ventricle"1) are similar to previous in vivo and in situ studies done in rainbow trout at 10°C. Reported maximum cardiac output values in previous studies ranged from 43.9 to 62.5 ml kg"1 min"1, while maximum power output values ranged from 5.1 to 6.9 mW g ventricle"1 (Kiceniuk and Jones, 1977; Farrell et al., 1991; Faust et al., 2004; Gamperl et al, 2004; Overgaard et al, 2004b). Potential variations among studies due to fish stocks, fish size and level of adrenergic stimulation are not accounted for in these comparisons. As our longest experiments lasted for only 100 minutes, we are confident that time-induced deterioration was not significant. The goal of this experiment was to examine consequences on maximum cardiac performance of the venous extracellular conditions experienced during and after exhaustive exercise because the combination of factors has not been studied previously. The 48 hyperkalemia, acidosis and hypoxia conditions that we used were intended to simulate those seen in the plasma of maximally exercising rainbow trout in vivo (Milligan and Wood, 1987; Nielsen and Lykkeboe, 1992). Our results confirmed that acidosis and hypoxia decrease both the force (Farrell et al., 1983; Driedzic and Gesser, 1994) and frequency (Gesser and Poupa, 1983) of myocardial contractions. In addition, we confirmed that hyperkalemia has a detrimental effect on contraction force (Kalinin and Gesser, 2002) and negatively affects contraction frequency (this study). A novel finding is that without the chronotropic and inotropic protection provided by maximum adrenergic stimulation, physiologically relevant acidosis and hyperkalemia prevent maximum cardiac performance even under normoxic conditions. In fact, with only tonic adrenergic stimulation, complete cardiac collapse occurred at a Pvo2 level below 6.7 kPa. However, when hearts were maximally stimulated with adrenaline, the hypoxic threshold for maximum cardiac performance under physiologically relevant hyperkalemic and acidotic conditions was lowered to less than 2.0 kPa. This finding is consistent with earlier work that had shown that isolated perfused hearts with tonic adrenergic stimulation performed routine physiological workloads to 3.3 kPa, but this level of hypoxia decreased Qmax and POmax by -50% and 80% respectively (Farrell et al., 1989). Consequently, this study has clearly demonstrated for the first time that maximum adrenergic stimulation is necessary for maximum cardiac performance to occur at the levels of venous hypoxia, hyperkalemia and acidosis seen during intense activity and recovery in vivo. The importance of adrenergic stimulation for the hypoxic myocardium corresponds well with what is known about the role of hypoxia in releasing catecholamines into the circulation of rainbow trout. Perry and Reid (1992) found that rainbow trout exposed to a 49 graded hypoxia challenge released adrenaline when arterial blood oxygen tension (arterial P02) fell below 3.4 kPa. Here we found that hearts exposed to hypoxia < 3.3 kPa with only tonic adrenergic stimulation lasted less than 5 minutes before undergoing a catastrophic cardiac collapse. In contrast, hearts exposed to a hyperkalemic, acidotic perfusate in conjunction with maximal adrenergic stimulation were able to function maximally at levels of hypoxia as low as 2.0 kPa. We have shown that adrenergic stimulation can counteract the negative chronotropic and inotropic effects of hypoxia, hyperkalemia and acidosis. In fish adrenergic stimulation of cardiac tissue is mediated by the P-adrenoceptor (P-AR) signalling pathway (Ask et al, 1981; Temma et al, 1986; Gamperl et al, 1994c). P-AR mediated increases in myocardial C a 2 + influx help offset the deleterious effects of both hyperkalemia and acidosis. C a 2 + influx restores the action potential upstroke lost during hyperkalemia (Paterson et al, 1992), and counteracts the acidotic impairment of calcium-troponin binding. P-AR stimulation also helps restore plasma and erythrocyte p H by activating erythrocyte N a + / H + exchange (Tang et al, 1988; Perry and Gilmour, 1996). In addition, direct adrenergic stimulation of pacemaker cells opposes hypoxic bradycardia by increasing pacemaker self-excitation rate (Tibbits et al, 1992). One major difference between the present study and in vivo is the lack of a coronary circulation. The coronary circulation provides arterial blood to the 30% of the ventricle that comprises the compact myocardium; spongy myocardium which receives oxygen solely from the cardiac circulation (venous blood) comprises the remaining 70% (Santer and Greer Walker, 1980; Tota, 1983; Davie and Farrell, 1991). In vivo, the coronary circulation is not necessary to maintain routine cardiac performance, as demonstrated by coronary ablation 50 experiments (Daxboeck, 1982; Steffensen and Farrell, 1998), although routine flow does occur in the coronary arteries (Axelsson and Farrell, 1993; Gamperl et al, 1994a; Gamperl et al, 1995). Thus, during routine conditions, in vivo oxygen diffusion from venous blood is presumably sufficient to meet the needs of both compact and spongy myocardium, and presumably this would reflect the routine Pvc^s found in trout of around 3-4 kPa. When Pvo2 of the cardiac circulation is reduced, as happens during exercise or environmental hypoxia, coronary blood flow increases (Gamperl et al, 1994a; Gamperl et al, 1994b) up to 2 fold (Gamperl et al, 1995), reflecting the increased oxygen needs of the compact myocardium and the fact that oxygen diffusion from the lumen to the compacta becomes limited relative to this demand. Indeed, without this coronary supply, Steffensen and Farrell (1998) found that coronary-ligated rainbow trout reduced cardiac workloads by an estimated 37% during a hypoxic swimming challenge. Hence, while the coronary circulation increases in importance during exercise, the majority of the ventricular myocardial oxygen supply comes from venous blood, and so a venous oxygen threshold must exist below which the spongy myocardium fails. Thus, the lack of a coronary circulation in the present study will tend to overestimate the hypoxic thresholds for maximum cardiac performance. However, the coronary artery is difficult to cannulate while maintaining the integrity of the pericardium, which is integral to maximizing PO of the heart (Farrell et al, 1988). Agnisola et al. (2003) found that coronary perfusion in isolated trout hearts at 10°C can increase cardiac stroke work by 12%>, from 3.36 to 3.77 mJ g"1. Even with coronary perfusion, the POmax was only 56% of the control value obtained here (we assumed fa = 60 min"1 since the information was not provided). 51 A n additional factor that sustains lower Pvo2 thresholds in vivo is the oxygen buffering capacity of haemoglobin. Specifically, unloading of oxygen that occurs on the steep portion of the dissociation curve will have little effect on Pvo2, and thus the oxygen diffusion gradient to the myocardium can remain high. In contrast, the linear nature of oxygen solubility in saline and its lower oxygen capacitance means that cardiac oxygen removal from saline decreases P02 more so than oxygen extraction from blood. Although the oxygen buffering capacity of haemoglobin and the presence of a coronary circulation allow for lower P02 thresholds in vivo than we measured here, neither of these factors are likely to affect the main finding that adrenaline protected the heart under adverse conditions. Farrell and Clutterham (2003) used a fibreoptic micro-optode to measure the Pvo2 in vivo during maximal exercise in rainbow trout and discovered that at even the most severe exercise intensity Pvo2 did not drop below 2.1 kPa, a value that corresponds closely to the present study in which maximum cardiac performance at 2.0 kPa was not significantly different from that observed under control conditions. At the next lowest tested hypoxia value in this study (1.3 kPa), POmax decreased by 43.6%. The correspondence of these findings suggests that although the in vivo venous oxygen threshold may be lower than that determined here, the difference may not be that great. Any hypoxic threshold will be influenced by the absolute level of cardiac work (van Raaij et al., 1996), as shown by the ability of rainbow trout hearts to perform sub-physiological workloads under near anoxic conditions (Arthur et al., 1992). Therefore, comparisons of hypoxic thresholds in vivo need to incorporate the work load of the heart, as shown below. A situation comparable to the absence of coronary perfusion of the perfused heart is coronary ligation in vivo. Steffensen and Farrell (1998) swam coronary-ligated 52 rainbow trout in hypoxic water (5.2 kPa) and this resulted in a Pvo2 threshold of 10 torr (1.3 kPa). From their data, we can estimate that POmax was 4.1 m W g"1 ventricle for coronary-ligated fish at this Pvo2 threshold. . [Ventral aortic blood pressure (Pva) was -50 cm H 2 0 (-4.9 kPa). We assume that Qmax was 50 ml min"1 kg"1 (as above) in coronary-ligated and non-ligated fish (as suggested by the work of Gamperl et al, 1994a), and that rainbow trout have a ventricular mass of -1 g kg"1] By comparison, perfused hearts subjected to an acidotic, hyperkalemic challenge at a comparable Pvo2 level of 1.3 kPa generated a POmax of only 2.6 mW g"1 ventricle. Since POmax was 5.9 m W g"1 ventricle at 2.0 kPa, this comparison not only reemphasizes the importance of the coronary circulation in maintaining maximum cardiac performance during intense exercise, but reemphasise that the difference between the in vivo and in vitro hypoxic thresholds may not be that great. Rainbow trout hearts have a limited glycolytic potential and a PO of 1.5 mW g ventricle"1 could be maintained for 20 minutes during anoxia at 10°C (Overgaard et al, 2004a). While this PO is well below the POmax here, the possibility still exists that a small component of maximum cardiac performance near the hypoxic threshold could have been supported by glycolysis during the short-term hypoxic exposures used here. If this is the case, we would have underestimated the hypoxic threshold. Implicit with this possibility is that if Pvo2 does fall below the hypoxic threshold in vivo, a component of post-exercise cardiac performance could be briefly fuelled by glycolysis. Previously, Gamperl et al. (2001) found that rainbow trout hearts were stunned when exposed to extreme hypoxia (Po2 < 5 mm Hg) at sub-physiological workloads, with Qmax decreasing by 23-38% upon return to control conditions. Similarly, we found that cardiac recovery was compromised by some of the hypoxic conditions. This was true for hypoxia 53 alone, as well as the combination of hypoxia, hyperkalemia, and acidosis. The majority of hearts exposed to hypoxia alone at levels below 10 kPa experienced impaired recovery, and this may have led to an underestimation of the protection afforded by the maximum adrenergic stimulation treatment that followed. The converse may also be true since prior exposure to hypoxia may confer a protective advantage. Hypoxic pre-conditioning has been indirectly shown to confer a protective advantage in some (Gamperl et al, 2001) but not all strains of rainbow trout (Gamperl et al, 2004; Overgaard et al, 2004b). Nevertheless, hypoxia of 2.7 kPa did not result in preconditioning here as hearts pre-exposed to hypoxia experienced a larger reduction in Qmax compared to hearts with no previous hypoxic exposure. In summary, we conclude that adrenaline is critical in maintaining maximum cardiac performance during conditions that simulate those observed in venous blood during and following intense activity. Adrenergic stimulation, when administered in conjunction with hypoxia, hyperkalemia and acidosis, was found to lower the hypoxic threshold for cardiac collapse from 5.0 kPa to less than 1.3 kPa, a value that corresponds closely to Pvc-2 levels found in maximally exercising rainbow trout. 54 60 2.5 5.0 7.5 Concentration of K (mM) Figure 2.1: The effects of hyperkalemia on maximum cardiac output (A) and maximum cardiac power output (B) of perfused rainbow trout hearts at 10°C, p H 7.9. Values are reported as mean ± S E M . Individual hearts (n = 9) were exposed to the following sequence of perfusates: (1) control (normoxia), (2) 5 m M K + , (3) 7.5 m M K + , (4) control (recovery), and (5) 7.5 m M K + with 500 n M epinephrine (AD). Repeated measures one-way A N O V A and a Holm-Sidak multiple comparisons test were used to compare treatment means. Dissimilar letters denote significant differences at a = 0.05. 55 I**'- -l hyperkalemia & acidosis with 500 nM AD 5.0 7.5 Concentration of K + (mM) Figure 2.2: The effects of hyperkalemia and acidosis (pH = 7.5) on maximum cardiac output (A) and maximum cardiac power output (B) of perfused rainbow trout hearts at 10°C. Values are reported as mean ± SEM. Individual hearts (n = 8) were exposed to the following sequence of perfusates: (1) control (normoxia), (2) 5.0 m M K + , pH 7.5, (3) control (recovery), (4) 5.0 mM K + and pH 7.5 with 500 nM A D , and (5) 7.5 mM K + and pH 7.5 with 500 nM A D . Values from series I are presented for comparison purposes (see Fig. 2.1). One-way A N O V A and a Holm-Sidak multiple comparisons test were used to compare treatment means. *denotes a significant difference from control while J denotes a significant difference from pH 7.9 at that particular level of hyperkalemia, a = 0.05. Three preliminary preparations were exposed to 7.5 m M K + and 5 nM A D at a pH of 7.5 directly after the first normoxia step. However, this exposure resulted in an almost immediate decrease in cardiac output leading to a rapid (< 5 min), irrecoverable cardiac collapse. 56 Figure 2.3: The maximum cardiac output (A) and maximum cardiac power output (B) of perfused rainbow trout hearts at 10°C under hypoxic perfusate. The grey line indicates the level of recovery during a subsequent exposure to normoxic conditions. Individual groups of hearts were exposed to specific hypoxia levels as indicated on the x-axis. Results shown are from two different series of experiments, as indicated by the separate (and discontinuous) line segments; however, experimental protocols were identical up until this point. The number of hearts used for each experiment was series III: 12.6 kPa (n = 6), 10 kPa (n = 10), 6.7 kPa (n = 6), and series IV: 5.0 kPa (n = 7), 3.3 kPa (n = 8), 2.7 kPa (n = 5). Values are reported as percent change from an original assessment under normoxic conditions. One-way repeated measures A N O V A and Holm-Sidak multiple comparisons tests were used to compare treatment means and each heart acted as its own control. Values shown are means ± S E M . * Denotes a significant difference from normoxia (a = 0.05). 57 20% o •3 !-a o B 3 o o B 3 0% -20 % H & -40 % -\ 3 -60 % H -80 % H 3 o CD O 0> o 100% 0% -20 % H -40 % H -60 % H -80 % H -100% normoxia B • • • • — hyperkalemia & acidosis — hyperkalemia & acidosis with 500 nM AD H 1 1 normoxia • • • • 10 15 20 partial pressure o f oxygen (torr) Figure 2.4: The maximum cardiac output (A) and maximum cardiac power output (B) of perfused rainbow trout hearts at 10°C under hypoxic, hyperkalemic (5 mM), acidotic (pH 7.5) perfusate with both tonic (5 nM) and maximal (500 nM) adrenergic stimulation. Individual groups of hearts were exposed to specific hypoxia levels as indicated on the x-axis, and results are shown from several series of experiments, each indicated as a separate line segment. 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Overgaard, J., Stecyk, J. A . W., Gesser, H., Wang, T . and Farrell, A . P. (2004a). Effects of temperature and anoxia upon the performance of in situ perfused trout hearts. J. Exp. Biol. 207, 655-665. Overgaard, J., Stecyk, J. A . W., Gesser, H., Wang, T . , Gamperl, A . K. and Farrell, A . P. (2004b). Preconditioning stimuli do not benefit the myocardium of hypoxia-tolerant rainbow trout (Oncorhynchus mykiss). J. Comp. Physiol. 174, 329-340. Paterson, D. J., Blake, G. J., Leitch, S. P., Phillips, S. M. and Brown, H. F. (1992). Effects of catecholamines and potassium on cardiovascular performance in the rabbit. J. Appl. Physiol. 73, 1413-1418. Perry, S. F. and Reid, S. D. (1992). Relationship between blood 0 2 content and catecholamine levels during hypoxia in rainbow trout and American eel. Am. J. Physiol. 263, R240-R249. Perry, S. F. and Gilmour, K. M. (1996). Consequences of catecholamine release on ventilation and blood oxygen transport during hypoxia and hypercapnia in an elasmobranch (Squalus acanthias) and a teleost (Oncorhynchus mykiss). J. Exp. Biol. 199, 2105-2118. Perry, S. F., Malone, S. and Ewing, D. (1987). Hypercapnic acidosis in the rainbow trout (Salmo gairdneri): 1. Branchial ionic fluxes and blood acid-base status. Cdn. J. Zool. 65, 888-895. Santer, R. M. and Greer Walker, M. (1980). Morphological studies on the ventricle of teleost and elasmobranch hearts. J. Zool. 190, 259-272. 62 Shiels, H. A. and Farrell, A. P. (1997). The effect of temperature and adrenaline on the relative importance of the sarcoplasmic reticulum in contributing Ca to force development in isolated ventricular trabeculae from rainbow trout. J. Exp. Biol. 200, 1607-1621. Steffensen, J. F. and Farrell, A. P. (1998). Swimming performance, venous oxygen tension and cardiac performance of coronary-ligated rainbow trout, Oncorhynchus mykiss, exposed to progressive hypoxia. Comp. Biochem. Physiol. 119A, 585-592. Tang, Y., Nolan, S. and Boutilier, R. G. (1988). Acid-base regulation following acute acidosis in seawater-adapted rainbow trout, Salmo gairdneri: a possible role for catecholamines. J. Exp. Biol. 134, 297-312. Temma, K., Hirata, T., Kitazawa, T., Kondo, H. and Katano, Y. (1986). Are (3-adrenergic receptors in ventricular muscles of carp heart (Cyprinus carpio) mostly the P2 type? Comp. Biochem. Physiol. 83C, 261-263. Thomas, S., Poupin, J., Lykkeboe, G. and Johansen, K. (1987). Effects of graded exercise on blood-gas tensions and acid base characteristics of rainbow trout. Respir. Physiol. 68, 85-97. Tota, B. (1983). Vascular and metabolic zonation in the ventricular myocardium of mammals and fishes. Comp. Biochem. Physiol. 76A, 423-437. Turner, J. D., Wood, C. M. and Clark, D. (1983). Lactate and proton dynamics in the rainbow trout {Salmo gairdneri). J. Exp. Biol. 104, 247-268. van Raaij, M. T. M., Pit, D. S. S., Balm, P. H. M., Steffens, A. B. and van den Thillart, G. E. E. J. M. (1996). Behavioral strategy and the physiological stress response in rainbow trout exposed to severe hypoxia. Horm. Behav. 30, 85-92. Vornanen, M. (1998). L-type C a 2 + current in fish cardiac myocytes: Effects of thermal acclimation and beta-adrenergic stimulation. J. Exp. Biol. 201, 533-47. Williamson, J., Safer, B., Rich, T., Schaffer, S. and Kobayashi, K. (1976). Effects of acidosis on myocardial contractility and metabolism. Acta. Med. Scand. Suppl. 587, 95-112. 63 Chapter 3: The hypoxic threshold for maximum cardiac performance during simulated exercise conditions at 18°C. Introduction Like many animals, prolonged exercise in rainbow trout (Oncorhynchus mykiss) greatly alters the composition of the venous blood, such that it becomes hypoxic (Kiceniuk and Jones, 1977; Steffensen and Farrell, 1998; Farrell and Clutterham, 2003), hyperkalemic (Holeton et al, 1983; Turner et al, 1983; Perry et al, 1987; Thomas et al, 1987; Nielsen and Lykkeboe, 1992; Hoik and Lykkeboe, 1998), and acidotic (Kiceniuk and Jones, 1977; Graham et al, 1982; Holeton et al, 1983; Turner et al, 1983). These changes can be even more extreme following exhaustive exercise (Wood et al, 1983; Milligan, 1996). Such changes in venous blood composition can detrimentally affect cardiac performance, and are particularly taxing for the rainbow trout whose heart is nourished predominantly by venous blood (Santer and Greer Walker, 1980; Farrell et al, 1988). Nevertheless, adrenergic stimulation of cardiac tissue can provide effective protection for all but the worst of these conditions. Adrenergic stimulation has been shown to individually counteract the negative inotropic and chronotropic effects of hypoxia, hyperkalemia and acidosis (Gesser et al, 1982; Gesser and Jorgensen, 1982; Farrell et al, 1983; 1986; Farrell and Milligan, 1986; Gamperl et al, 1994b; Nielsen and Gesser, 2001; Kalinin and Gesser, 2002). In addition, maximum adrenergic stimulation can protect maximum performance of the in situ heart at 10°C from the combined challenges of venous hypoxia, hyperkalemia and acidosis at the levels seen during intense activity and recovery in vivo (Hanson et al, 2006). In fact, these A version of this chapter will be submitted for publication: Hanson, L. M . and Farrell, A . P. (2006). The hypoxic threshold for maximum cardiac performance during simulated exercise conditions at 18°C. 64 experiments showed that without adrenergic stimulation, the simulated exercise conditions of acidosis and hyperkalemia will debilitate maximum cardiac performance of the rainbow trout heart even when oxygen is not limiting. While adrenergic stimulation of the heart clearly plays an important protective role, temperature acclimation is known to alter the response of the rainbow trout heart to adrenaline (Ask et al, 1981; Graham and Farrell, 1989; Farrell et al, 1996). This is significant because rainbow trout can experience a wide range of seasonal temperatures (2 °C to 19°C) . Specifically, an increase in acclimation temperature is associated with a decrease in adrenergic sensitivity (Graham and Farrell, 1989; Keen et al, 1993), with some of this change being attributed to a temperature-dependent change in ventricular cell surface p-AR density. In fish, cardiac adrenergic stimulation is mediated by the P-adrenoceptor (P-AR) signalling pathway (Ask et al, 1981; Temma et al, 1986; Keen et al, 1993; Gamperl et al, 1994c), and thus changes in acclimation temperature have the potential to alter P-AR mediated cardioprotective mechanisms. With adrenergic receptor density decreasing by ~11% for every 1°C increase in acclimation temperature (Keen et al, 1993; Gamperl et al, 1998), it begs the question how does this reduction in adrenergic effectiveness impact the protection afforded to the heart under the adverse venous conditions experienced by rainbow trout post-exercise? It has been suggested that in fish, high temperature tolerance may be limited by oxygen availability (Farrell, 2002; Portner et al, 2004). Increasing temperature induces a right-shift of the haemoglobin-oxygen equilibrium curve (Eddy, 1971; Vorger, 1986) and this decrease in haemoglobin-oxygen (Hb-02) affinity favours 0 2 uptake by tissues, impairs 0 2 uptake at the gills and reduces arterial 0 2 saturation (Heath and Hughes, 1973). This 65 decrease in arterial oxygen saturation, combined with a temperature-induced increase in whole body oxygen uptake, decreases venous oxygen tension (PVO2). Pvc>2 determines the diffusion gradient of oxygen from venous blood to myocardial tissue and thus it determines oxygen supply for - 7 0 % of the rainbow trout myocardium (Santer and Greer Walker, 1980; Farrell et al., 1988). Thus, while myocardial oxygen needs increase with temperature (Farrell et al., 1985; Graham and Farrell, 1990; Mortensen and Gesser, 1999), the diffusion gradient of oxygen from venous blood is decreasing. The balance between myocardial oxygen supply and demand becomes even more precarious during exercise when myocardial oxygen demand can increase as much as four-fold (Farrell and Jones, 1992) and Pvo2 is reduced by - 5 0 % through increased O2 uptake by skeletal muscles (Kiceniuk and Jones, 1977; Farrell and Clutterham, 2003). If oxygen demand during exercise were to exceed oxygen supply then myocardial hypoxia could ensue resulting in reduced cardiac performance. Furthermore, exercise at high temperature can pose additional challenges when compared to exercise at low temperature as increased temperature exacerbates the potential mismatch between myocardial oxygen supply and oxygen demand induced by exercise. Temperature-induced myocardial hypoxia can result in a more glycolytic heart. Previous studies (Overgaard et al, 2004a) suggest that although glycolytic capacity increases with temperature, the glycolytic potential of the rainbow trout heart is limited and thus myocardial failure during severe hypoxia (P02 < 0.5 kPa) is primarily due to insufficient anaerobic energy production. In addition, glycolysis results in intracellular acidosis which reduces myocardial contractility and debilitates cardiac performance. The cardiorespiratory adjustments induced by high temperature effect the venous oxygen reserve such that at high temperature the point at which myocardial oxygen supply 66 balances demand, i.e. the venous oxygen threshold, will be higher than that seen at low temperature. Furthermore, if myocardial oxygen availability is the only determinant of cardiac performance, one would predict that the venous oxygen threshold for cardiac collapse would increase proportionally to the increase in myocardial oxygen consumption. On the other hand, if adrenergic mechanisms are what fails then one would expect the threshold to increase to a greater extent. This study tests the hypothesis that maximum cardiac performance during prolonged exercise at high temperature is limited by oxygen delivery. It is predicted that the hypoxic threshold for cardiac collapse under hyperkalemic and acidotic conditions with maximal adrenergic stimulation will increase with temperature, and thus will be higher at 18°C than previously discovered at 10°C (Chapter 2). Materials and Methods Rainbow trout (body mass 476 g ± 19 g S E M ; relative ventricular mass 0.080% ± 0.002%) of both sexes (Oncorhynchus mykiss Walbaum) were obtained from a local fish hatchery (Richard Henley Farm, Langley, B C , Canada), held indoors in 1000 L fibreglass tanks and acclimated to a temperature of 18°C ± 1°C for a minimum of two weeks prior to experimentation. A n in situ perfused heart preparation was prepared as described in Farrell et al. (1986) and as modified by Farrell et al. (1989), in order to perfuse the venous circulation via an input cannula in the sinus venosus and an output cannula in the ventral aorta. The total time to prepare the perfused heart preparation was 15-20 minutes. Following surgery, the fish was transferred to a temperature-controlled, physiological saline bath (124.1 m M NaCl , 2.5 m M KC1, 11.9 m M N a H C 0 3 , 2.0 m M C a C l 2 - 2 H 2 0 , 0.2 m M N a H 2 P 0 4 - H 2 0 , 3.4 m M N a 2 H P 0 4 , 0.9 m M M g S 0 4 - 7 H 2 0 ; all chemicals purchased from 67 Sigma-Aldrich, Oakville, O N , Canada). The input cannula was immediately connected to an adjustable, constant-pressure reservoir, and the output cannula was connected to a separate constant pressure head set at 4.9 kPa to mimic resting in vivo ventral aortic blood pressure. The height of the input pressure reservoir was adjusted to set routine cardiac output (Q) at approximately 25.0 ml min"1 kg"1. Input (Pj n) and output (Pout) pressure were measured through saline-filled side arms (PE50 tubing) connected to disposable pressure transducers (DPT 6100, Smiths Medical, Kirchseeon, Germany). Cardiac outflow was continuously measured through the output line with an in-line electromagnetic flow probe (SWF-4, Zepada Instruments, Seattle, W A , USA) . Hearts were equilibrated for 5-10 minutes while receiving control perfusate (see below) before the experiment commenced. The coronary circulation was not perfused in this preparation. Maximum cardiac performance was assessed sequentially under normoxic (control) and simulated exercise conditions (defined below) in a manner similar to that described by Hanson et al. (2006). By initially measuring both maximum cardiac output (Qmax) and maximum cardiac power output (POmax) under normoxia, each heart acted as its own control. To determine Qmax, Pin was gradually increased in increments of -0.05 kPa until cardiac output reached a plateau (usually around 0.4 kPa). To assess POmax, Pin was left at its maximum and P o u t was increased in a stepwise fashion in -0.5 kPa increments until PO reached a plateau. After POmax was determined, P o u t and P i n were returned to resting levels and the heart was allowed to recover (-5 min) before being exposed to the next perfusate. Hearts were exposed to each perfusate for a total of 15 minutes; this time period ensured continued viability of the photosensitive adrenaline (which was renewed with each change in perfusate) while remaining physiologically relevant, as Pvo2 can take more than 20 minutes 68 to return to normal following exhaustive exercise (Farrell and Clutterham, 2003). Under some extreme conditions individual hearts succumbed (i.e. cardiac output approached zero) before the end of the 15-min period. In this case, the duration was noted and normoxic conditions restored. Hearts that were unable to complete both cardiac performance tests were considered to have failed under that test condition. The purpose of these experiments was to determine the hypoxic threshold for maximum cardiac performance under levels of respiratory acidosis (pH 7.5) and hyperkalemia (5.0 mM) similar to those found in the plasma of exercising rainbow trout in vivo (Milligan and Wood, 1987; Nielsen and Lykkeboe, 1992). Adrenaline was used to simulate tonic and maximal adrenergic cardiac stimulation. For the control perfusate, 5 n M A D was used to produce a tonic level of adrenergic stimulation consistent with that found in resting rainbow trout in vivo (Milligan et al, 1989). In addition, in vivo studies in rainbow trout have reported post-exercise [AD] as high as 186-212 n M (Butler et al., 1986; Milligan et al, 1989), and previous in situ studies at 18°C (Farrell et al, 1996) suggest that 200 n M A D produces maximal adrenergic stimulation of rainbow trout hearts. Consequently, 500 n M of A D was added to the perfusate to ensure maximal adrenergic stimulation during the simulated exercise conditions. The experimental protocol consisted of two different test conditions: (1) control (normoxia), and (2) the combined hyperkalemic, acidotic and hypoxic exposure. In order to determine the hypoxic threshold a series of experiments was done to test hearts at various levels of hypoxia. Regardless of the hypoxia level being tested, hearts were exposed to the following sequence of perfusates: (1) control with 5 n M A D , (2) hypoxia, 5.0 m M K + and pH 7.5 with 500 n M A D , and (3) control with 5 n M A D . 69 Control (normoxia): A l l preparations started under this condition. Freshwater trout saline (124.1 m M NaCl , 2.5 m M KC1, 0.9 m M M g S 0 4 - 7 H 2 0 , 2.5 m M C a C l 2 - 2 H 2 0 , D-glucose 5.6 m M , 11.9 m M NaHCOs) was gassed with 0.5% C 0 2 and balance air to achieve a p H of 7.9 and a Po2 of 20 kPa. Hyperkalemia, acidosis and hypoxia: The control perfusate was made hyperkalemic by adding additional KC1 to increase the [K + ] to 5.0 m M . To achieve a p H of 7.5, the concentration of NaHC03 was decreased to 10.1 m M and the solution was equilibrated with a gas mixture containing 1.0% C 0 2 . In addition, the perfusate was made acidotic and hypoxic by equilibrating it with 1.0% C 0 2 and an amount of oxygen corresponding to the desired level of hypoxia (balance nitrogen). Each heart preparation was tested at one hypoxic level and the levels of hypoxia in kPa were 10 (n = 4 fish), 8.3 (n = 5 fish), 6.7 (n = 8 fish), 5.6 (n = 5 fish), 5.1 (n = 3 fish), 4.0 (n = 5 fish). Premixed, calibrated gases (Praxair, Vancouver, B C , Canada) and Wostoff gas pumps (M303/a-F, Bochum, Germany) were used to generate gas mixtures. A l l experimental data was collected using data acquisition software (Labview version 5.1, National Instruments, Austin, T X , U S A ) , which allowed for real-time measurements of fn, Pin, Pout, Q, and PO. Statistical differences within test groups were determined by one-way repeated measures analysis of variance ( A N O V A ) . When warranted, the Holm-Sidak procedure was used for post hoc multiple comparisons. SigmaStat (3.0, SPSS Inc., San Rafael, C A , U S A ) was used for all statistical analysis (a = 0.05). 70 Results The combined hyperkalemic, acidotic exposure with maximum adrenergic stimulation did not significantly alter maximum cardiac output (Qmax) at any oxygen tension > 5.6 kPa as compared with control (Fig. 3.1). One of five preparations failed at 8.3 kPa, two of five preparations failed at 5.6 kPa, all four preparations tested at 5.1 kPa failed and all five preparations tested at 4.0 kPa failed (data not shown). Qmax was fully restored under subsequent control conditions >8.3 kPa but remained significantly (38.6% ± 9.2%) lower than control at 8.3 kPa (Fig. 3.1). Similarly, under the combined hyperkalemic and acidotic conditions with maximum adrenergic stimulation, POmax was significantly (43.3% ± 7.9%) lower than control at 6.7 kPa, and recovery was incomplete at both 6.7 and 8.3 kPa, as indicated by significant decreases in POmax under control conditions (Fig. 3.2). Cardiac failure was primarily due to significant decreases in fa. Heart rate was significantly reduced with the combined hyperkalemic and acidotic perfusate at 6.7 kPa (Fig. 3.3) and under the subsequent control conditions at both 6.7 kPa and 5.1 kPa. In contrast, there were no significant changes in cardiac stroke volume (Vs) at any of the hypoxia levels tested (Fig. 3.4). Discussion Previous studies have shown that the in situ perfused heart preparation utilized in this study can perform at levels approximating maximum in vivo cardiac performance and remain stable for several hours (Farrell et al, 1986; Farrell, 2002; Overgaard et al, 2004b). The values obtained in this study for maximum cardiac output (54.0 ± 4.7 ml min"1 kg"1) and 71 maximum cardiac power output (5.9 ± 0.06 m W g ventricle") under control conditions are comparable, albeit slightly lower, than previous in situ studies at 18°C where Qmax ranges from 63-78 ml min"1 kg"1 and POmax ranges from 8.8-9.3 m W kg ventricle"1 (Keen and Farrell, 1994; Farrell et al, 1996). Also, it should be noted that the success rate for preparations at 18°C (87%) is lower compared with previous work conducted at 10°C by the same investigators (100%). Consequently, the reported cardiac performance levels may reflect an overestimate due to the removal of poor performers, a problem noted earlier (Farrell etal, 1996). The goal of this experiment was to determine the hypoxic threshold for maximum cardiac performance under the conditions experienced during and after exhaustive exercise. The hypoxic threshold for cardiac collapse was found to be 5.1-5.6 kPa, while the hypoxic threshold for maximum cardiac performance was 5.1-5.6 kPa for Qmax and 6.7 kPa for POmax. At 10°C the hypoxic threshold for maximum cardiac performance is reported as 2.0 kPa for both Qmax and POmax (Hanson et al, 2006), which is very similar to previously reported Pvo2 values in vivo (Kiceniuk and Jones, 1977; Farrell and Clutterham, 2003) and lower than the half maximal haemoglobin-oxygen saturation (P50) value of 3.1 (Perry and Reid, 1992). As predicted, the hypoxic threshold for maximum cardiac performance increases with acclimation temperature. Specifically, the 8 ° C change in acclimation temperature between these two studies is associated with a 3.6-4.7 kPa increase in the hypoxic threshold. Moreover, at high temperature there is the additional concern of poor recovery following the subsequent return to control conditions, which is not seen at 10°C. Due to the oxygen buffering capacity of haemoglobin, it is to be expected that the hypoxic threshold for a saline-perfused heart would be higher than the Pvo2 threshold in vivo. 72 The sigmoidal shape of the haemoglobin-oxygen equilibrium curve and its high oxygen capacitance would maintain a relatively stable P02 over a wide range of oxygen extraction given the myocardial oxygen demand (Farrell, 1986). In contrast, due to the linear nature of oxygen solubility in saline and its lower oxygen capacitance, cardiac oxygen removal from saline will decrease P02 more so than oxygen extraction from blood leading to a reduced diffusion gradient. Consequently, the oxygen buffering capacity of haemoglobin will facilitate lower Pvo2 thresholds in vivo. Despite these considerations, the difference in hypoxic thresholds for 10°C and 18°C appears unusually large. At 18°C exercising rainbow trout quit swimming with a Pvo2 of 3.2-3.9 kPa (Farrell and Clutterham, 2003), a value less than the P50 value of 4.7 kPa at this temperature (Eddy, 1971). The difference is therefore about 2.4 kPa for Qmax and 2.8 kPa for POmax, when the 0 2 demand is somewhat higher. Such differences are unlikely to be a result of myocardial O2 extraction from saline reducing Po2. If changes in saline P02 due to myocardial oxygen extraction were the sole determinant of maximum cardiac performance, the venous oxygen threshold should increase with temperature in proportion to myocardial oxygen needs. Based on the data of Graham and Farrell (1989), myocardial oxygen needs increase by < 1 kPa for an 8°C temperature increase. However, over a temperature increase of the same magnitude the venous oxygen threshold increases 3.6 kPa, from 2 kPa at 10°C (Hanson et al, 2006) to 5.6 kPa at 18°C (this study). If myocardial oxygen extraction was indeed the culprit, the expected venous oxygen threshold would be ~3 kPa and not 5.6 kPa. Consequently, it can be concluded that limited myocardial oxygen supply from saline is not the sole determinant of the high hypoxic threshold at 18°C. Accordingly, a temperature-induced loss of adrenergic effectiveness 73 known to occur at 18°C (Graham and Farrell, 1989; Keen et al, 1993) seems like a significant contributing factor to making the rainbow trout myocardium more hypoxia sensitive at 18°C compared with 10°C under the hyperkalemic and acidotic conditions typically encountered during intense activity and recovery. Another possibility to account for the higher hypoxic threshold at 18°C could be an increased reliance on the coronary circulation at higher temperature, which would make sense if Pvc-2 is being compromised in vivo. The role and plasticity of coronary circulation is poorly understood. Nevertheless, previous studies on rainbow trout have shown that an increase in acclimation temperatures is associated with an increase in both the proportion of compact myocardium (Farrell et al, 1988; Simonot, 2006) and the density of coronary vasculature (Egginton and Cordiner, 1997). The coronary circulation provides arterial blood to 30% of the ventricle (Santer and Greer Walker, 1980; Tota, 1983; Davie and Farrell, 1991). Coronary blood flow increases during exercise (Gamperl et al, 1994b; 1994a) up to 2 fold (Gamperl et al., 1995); likely to reflect the increased oxygen needs of the compact myocardium. The importance of increased coronary circulation during exercise is highlighted by the 37% decrease in cardiac performance seen in coronary-ligated rainbow trout during a hypoxic swimming challenge (Steffensen and Farrell, 1998). Agnisola et al (2003) demonstrated that coronary perfusion significantly improved the performance of isolated saline perfused rainbow trout hearts at 10°C, such that their performance was not significantly different from that seen under control conditions; however, their performance was still 44% lower than the POmax generated under similar conditions by perfused hearts in situ with only cardiac circulation (Hanson et al, 2006). Blood perfusion of the coronary circulation can help improve cardiac performance by increasing myocardial oxygen 74 consumption (Agnisola et al, 2003), which may help support lower thresholds in vivo, however work in other species (eels) suggests that blood perfusion helps POmax more so than Qmax (Davie et al, 1992). Clearly, the lack of a coronary circulation in the perfused heart preparation will overestimate the hypoxic thresholds for maximum cardiac performance, but to what degree the dependence on the coronary circulation increases at high temperature will have to await in vivo experiments. In conclusion, during exercise at high temperature, the negative effects of hypoxia, hyperkalemia and acidosis that would be alleviated through adrenergic stimulation are probably compromised by a decrease in adrenergic effectiveness (Graham and Farrell, 1989; Keen et al, 1993). This study showed a temperature-induced loss of adrenergic protection and as a result, the venous oxygen threshold required to maintain maximum cardiac performance at 18°C was relatively high under the conditions associated with intense activity and recovery. 75 3 OH -4—» O o o X a B c c3 u 2 0 % 0% -20 % H -40 % H -60 % H -80 % H -100% 5.0 m M K + , pH 7.5, 500 n M A D recovery (1) i — 10 11 partial pressure of oxygen (kPa) Figure 3.1: The maximum cardiac output of perfused rainbow trout hearts at 18°C under hypoxic, hyperkalemic (5 mM), acidotic (pH 7.5) perfusate with maximal (500 nM) adrenergic stimulation. The grey line indicates the level of recovery during a subsequent exposure to control conditions. Individual groups of hearts were exposed to specific hypoxia levels as indicated on the x-axis. The number of hearts used at each level of hypoxia was 10 kPa (n = 4 fish), 8.3 kPa (n = 5 fish), 6.7 kPa (n = 8 fish), 5.6 kPa (n = 5 fish), 5.1 kPa (n = 3 fish), 4.0 kPa (n = 5 fish). Values are reported as percent change from an original assessment under control conditions. One-way repeated measures A N O V A and Holm-Sidak multiple comparisons tests were used to compare treatment means and each heart acted as its own control. Values shown are means ± S E M . *Denotes a significant difference from control while X denotes a significant difference between the hypoxic, hyperkalemic, acidotic exposure and recovery (a = 0.05). 76 20 % -T//~ t-i o o -a o s § >< s a o 0% -20 % H -40 % -60 % 4 -80 % H -100% - V / ^ control 10 11 partial pressure of oxygen (kPa) Figure 3.2: The maximum cardiac power output of perfused rainbow trout hearts at 18°C under hypoxic, hyperkalemic (5 mM), acidotic (pH 7.5) perfusate with maximal (500 nM) adrenergic stimulation. The grey line indicates the level of recovery during a subsequent exposure to control conditions. Individual groups of hearts were exposed to specific hypoxia levels as indicated on the x-axis. The number of hearts used at each level of hypoxia was 10 kPa (n = 4 fish), 8.3 kPa (n = 5 fish), 6.7 kPa (n = 8 fish), 5.6 kPa (n = 5 fish), 5.1 kPa (n = 3 fish), 4.0 kPa (n = 5 fish). Values are reported as percent change from an original assessment under control conditions. One-way repeated measures A N O V A and Holm-Sidak multiple comparisons tests were used to compare treatment means and each heart acted as its own control. Values shown are means ± S E M . *Denotes a significant difference from control while % denotes a significant difference between the hypoxic, hyperkalemic, acidotic exposure and recovery (a = 0.05). 77 20 % -r//~ partial pressure of oxygen (kPa) Figure 3.3: Heart rate at maximum cardiac output of perfused rainbow trout hearts at 18°C under hypoxic, hyperkalemic (5 mM), acidotic (pH 7.5) perfusate with maximal (500 nM) adrenergic stimulation. The grey line indicates the level of recovery during a subsequent exposure to control conditions. Individual groups of hearts were exposed to specific hypoxia levels as indicated on the x-axis. The number of hearts used at each level of hypoxia was 10 kPa (n = 4 fish), 8.3 kPa (n = 5 fish), 6.7 kPa (n = 8 fish), 5.6 kPa (n = 5 fish), 5.1 kPa (n = 3 fish), 4.0 kPa (n = 5 fish). Values are reported as percent change from an original assessment under control conditions. One-way repeated measures A N O V A and Holm-Sidak multiple comparisons tests were used to compare treatment means and each heart acted as its own control. Values shown are means ± S E M . *Denotes a significant difference from control (a = 0.05). 78 e ' o > O i-i +-> t/D o «3 (50 o 20% 0% -20 % -40 % -60 % -80 % i -100% r 5.0 mM K + , pH 7.5, 500 nM AD recovery 10 11 partial pressure of oxygen (kPa) Figure 3.4: Cardiac stroke volume during maximum cardiac output of perfused rainbow trout hearts at 18°C under hypoxic, hyperkalemic (5 mM), acidotic (pH 7.5) perfusate with maximal (500 nM) adrenergic stimulation. The grey line indicates the level of recovery during a subsequent exposure to control conditions. Individual groups of hearts were exposed to specific hypoxia levels as indicated on the x-axis. The number of hearts used at each level of hypoxia was 10 kPa (n = 4 fish), 8.3 kPa (n = 5 fish), 6.7 kPa (n = 8 fish), 5.6 kPa (n = 5 fish), 5.1 kPa (n = 3 fish), 4.0 kPa (n = 5 fish). Values are reported as percent change from an original assessment under control conditions. One-way repeated measures A N O V A showed no significant differences between treatment means. Values shown are means ± S E M . 79 References Agnisola, C , Petersen, L. and Mustafa, T. (2003). Effect of coronary perfusion on the basal performance, volume loading and oxygen consumption in the isolated resistance-headed heart of the trout Oncorhynchus mykiss. J. Exp. Biol. 206, 4003-4010. Ask, J. A., Stene-Larsen, G. and Helle, K. B. (1981). Temperature effects on the pY adrenoreceptors of the trout atrium. J. Comp. 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Cardiorespiratory performance in salmonids during exercise at high temperature: Insights into cardiovascular design limitations in fishes. Comp. Biochem. Physiol. 132, 797-810. Farrell, A. P. and Milligan, C. L. (1986). Myocardial intracellular p H in a perfused rainbow trout heart during extracellular acidosis in the presence and absence of adrenaline. J. Exp. Biol. 125, 347-359. Farrell, A. P. and Jones, D. R. (1992). The heart. In Fish physiology, vol. XIIA eds. D. J. Randall and A . P. Farrell), pp. 1-88. San Diego: Academic Press Ltd. Farrell, A. P. and Clutterham, S. M. (2003). On-line venous oxygen tensions in rainbow trout during graded exercise at two acclimation temperatures. J. Exp. Biol. 206, 487-496. Farrell, A. P., MacLeod, K. R., Driedzic, W. R. and Wood, S. (1983). Cardiac performance in the in situ perfused fish heart during extracellular acidosis: Interactive effects of adrenaline. J. Exp. Biol. 107, 415-429. 80 Farrell, A. P., Wood, S., Hart, T. and Driedzic, W. R. (1985). Myocardial oxygen consumption in the sea raven, Hemitripterus americanus - The effects of volume loading, pressure loading and progressive hypoxia. J. Exp. Biol. 117, 237-250. Farrell, A. P., MacLeod, K. R. and Chancey, B. (1986). Intrinsic mechanical properties of the perfused rainbow trout heart and the effects of catecholamines and extracellular calcium under control and acidotic conditions. J. Exp. Biol. 125, 319-345. Farrell, A. P., Hammons, A. M., Graham, M. S. and Tibbits, G. F. (1988). Cardiac growth in rainbow trout, Salmo gairdneri. Can. J. Zool. 66, 2368-2373. Farrell, A. P., Small, S. and Graham, M. S. (1989). Effect of heart rate and hypoxia on the performance of a perfused trout heart. Cdn. J. Zool. 67, 274-280. Farrell, A. P., Gamperl, A. K., Hicks, J. M. T., Shiels, H. A. and Jain, K. E. (1996). Maximum cardiac performance of rainbow trout (Oncorhynchus mykiss) at temperatures approaching their upper lethal limit. J. Exp. Biol. 199, 663-672. Gamperl, A. K., Pinder, A. W. and Boutilier, R. G. (1994a). Effect of coronary ablation and adrenergic stimulation on in vivo cardiac performance in trout (Oncorhynchus mykiss). J. Exp. Biol. 186, 127-143. Gamperl, A. K., Pinder, A. W. and Grant, R. R. (1994b). Influence of hypoxia and adrenaline administration on coronary blood-flow and cardiac performance in seawater rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 193, 209-232. Gamperl, A. K., Wilkinson, M. and Boutilier, R. G. (1994c). ^-adrenoceptors in the trout (Oncorhynchus mykiss) heart - Characterization, quantification, and effects of repeated catecholamine exposure. Gen. Comp. Endocrinol. 95, 259-272. Gamperl, A. K., Axelsson, M. and Farrell, A. P. (1995). Effects of swimming and environmental hypoxia on coronary blood-flow in rainbow trout. Am. J. Physiol. 38, R1258-R1266. Gamperl, A. K., Vijayan, M. M., Pereira, C. and Farrell, A. P. (1998). P-receptors and stress protein 70 expression in hypoxic myocardium of rainbow trout and chinook salmon. Am. J. Physiol. 43, R428-R436. Gesser, H. and Jorgensen, E. (1982). Phi, contractility and C a 2 + balance under hypercapnic acidosis in the myocardium of different vertebrate species. J. Exp. Biol. 96, 405-412. Gesser, H., Andresen, P., Brams, P. and Sundlaursen, J. (1982). Inotropic effects of adrenaline on the anoxic or hypercapnic myocardium of rainbow trout and eel. J. Comp. Physiol. 147, 123-128. 81 Graham, M. S. and Farrell, A. P. (1989). The effect of temperature acclimation and adrenaline on the performance of a perfused trout heart. Physiol. Zool. 62, 38-61. Graham, M, S. and Farrell, A. P. (1990). Myocardial oxygen consumption in trout acclimated to 5 ° C and 15°C. Physiol. Zool. 63, 536-554. Graham, M. S., Wood, C. M. and Turner, J. D. (1982). The physiological responses of the rainbow trout to strenuous exercise - Interactions of water hardness and environmental acidity. Cdn. J. Zool. 60, 3153-3164. Hanson, L. M., Obradovich, S., Mouniargi, J. and Farrell, A. P. (2006). The role of adrenergic stimulation in maintaining maximum cardiac performance in rainbow trout (Oncorhynchus mykiss) during hypoxia, hyperkalemia and acidosis at 10°C. J. Exp. Biol, (in press). Heath, A. G. and Hughes, G. M. (1973). Cardiovascular and respiratory changes during heat stress in rainbow trout (Salmo gairdneri). J. Exp. Biol. 59, 323-338. Holeton, G. F., Neumann, P. and Heisler, N. (1983). Branchial ion exchange and acid-base regulation after strenuous exercise in rainbow trout (Salmo gairdneri). Respir. Physiol. 51, 303-318. Hoik, K. and Lykkeboe, G. (1998). The impact of endurance training on arterial plasma K + levels and swimming performance of rainbow trout. J. Exp. Biol. 201, 1373-1380. Kalinin, A. and Gesser, H. (2002). Oxygen consumption and force development in turtle and trout cardiac muscle during acidosis and high extracellular potassium. J. Comp. Physiol. 172B, 145-151. Keen, J. E. and Farrell, A. P. (1994). Maximum prolonged swimming speed and maximum cardiac performance of rainbow trout, Oncorhynchus mykiss, acclimated to two different water temperatures. Comp. Biochem. Physiol. 108A, 287-295. Keen, J. E., Vianzon, D. M., Farrell, A. P. and Tibbits, G. F. (1993). Thermal acclimation alters both adrenergic sensitivity and adrenoreceptor density in cardiac tissue of rainbow trout. J. Exp. Biol. 181, 27-48. Kiceniuk, J. W. and Jones, D. R. (1977). The oxygen transport system in trout (Salmo gairdneri) during sustained exercise. J. Exp. Biol. 69, 247-260. Milligan, C. L. (1996). Metabolic recovery from exhaustive exercise in rainbow trout. Comp. Biochem. Physiol. 113, 51-60. Milligan, C. L. and Wood, C. M. (1987). Regulation of blood oxygen transport and red cell pHj after exhaustive activity in rainbow trout (Salmo gairdneri) and starry flounder (Platichthys stellatus). J. Exp. Biol. 133, 263-82. 82 Milligan, C. L., Graham, M. S. and Farrell, A. P. (1989). The response of trout red cells to adrenaline during seasonal acclimation and changes in temperature. J. Fish Biol. 35, 229-236. Mortensen, B. and Gesser, H. (1999). 0 2 consumption and metabolic activities in resting cardiac myocytes from rainbow trout. J. Exp. Zool. 283, 501-509. Nielsen, J. S. and Gesser, H. (2001). Effects of high extracellular [K + ] and adrenaline on force development, relaxation and membrane potential in cardiac muscle from freshwater turtle and rainbow trout. J. Exp. Biol. 204, 261-268. Nielsen, O. B. and Lykkeboe, G. (1992). Changes in plasma and erythrocyte K + during hypercapnia and different grades of exercise in trout. J. Appl. Physiol. 72, 1285-90. Overgaard, J., Stecyk, J. A. W., Gesser, H., Wang, T . and Farrell, A. P. (2004a). Effects of temperature and anoxia upon the performance of in situ perfused trout hearts. J. Exp. Biol. 207, 655-665. Overgaard, J., Stecyk, J. A. W., Gesser, H., Wang, T . , Gamperl, A. K. and Farrell, A. P. (2004b). Preconditioning stimuli do not benefit the myocardium of hypoxia-tolerant rainbow trout (Oncorhynchus mykiss). J. Comp. Physiol. 174, 329-340. Perry, S. F. and Reid, S. D. (1992). Relationship between blood O2 content and catecholamine levels during hypoxia in rainbow trout and American eel. Am. J. Physiol. 263, R240-R249. Perry, S. F., Malone, S. and Ewing, D. (1987). Hypercapnic acidosis in the rainbow trout (Salmo gairdneri): 1. Branchial ionic fluxes and blood acid-base status. Cdn. J. Zool. 65, 888-895. Portner, H. O., Mark, F. C. and Bock, C. (2004). Oxygen limited thermal tolerance in fish? Answers obtained by nuclear magnetic resonance techniques. Respir. Physiol. Neurobiol. 141, 243-260. Santer, R. M. and Greer Walker, M. (1980). Morphological studies on the ventricle of teleost and elasmobranch hearts. J. Zool. 190, 259-272. Simonot, D.L. (2006). Cardiac remodelling in diploid and triploid rainbow trout (Oncorhynchus mykiss Walbaum). M.Sc. Thesis, University of British Columbia, Vancouver, British Columbia. Steffensen, J. F. and Farrell, A. P. (1998). Swimming performance, venous oxygen tension and cardiac performance of coronary-ligated rainbow trout, Oncorhynchus mykiss, exposed to progressive hypoxia. Comp. Biochem. Physiol. 119A, 585-592. 83 Temma, K., Hirata, T., Kitazawa, T., Kondo, H. and Katano, Y. (1986). Are P-adrenergic receptors in ventricular muscles of carp heart (Cyprinus carpio) mostly the p 2 type? Comp. Biochem. Physiol. 83C, 261-263. Thomas, S., Poupin, J., Lykkeboe, G. and Johansen, K. (1987). Effects of graded exercise on blood-gas tensions and acid base characteristics of rainbow trout. Respir. Physiol. 68, 85-97. Tota, B. (1983). Vascular and metabolic zonation in the ventricular myocardium of mammals and fishes. Comp. Biochem. Physiol. 76A, 423-437. Turner, J. D . , Wood, C. M. and Clark, D . (1983). Lactate and proton dynamics in the rainbow trout (Salmo gairdneri). J. Exp. Biol. 104, 247-268. Vorger, P. (1986). The effect of temperature on oxygen equilibrium curves of trout blood (Salmo gairdnerii). Comp. Biochem. Physiol. 83A, 525-531. Wood, C. M., Turner, J. D . and Graham, M. S. (1983). Why do fish die after severe exercise? J. Fish Biol. 22, 189-201. 84 Chapter 4: The effect of temperature acclimation on myocardial p-adrenoceptor density and ligand binding affinity in African Catfish {Claris gariepinus) Introduction The (3-adrenoceptor (P-AR) signalling pathway is known to mediate important cardiac inotropic and chronotropic actions of adrenaline and noradrenaline in fish (Ask et al, 1981; Temma et al, 1986; Gamperl et al, 1994). The main P-AR subtype mediating these cardiac actions in rainbow trout (Oncorhynchus mykiss) is a p 2 subtype (Ask et al, 1980, 1981; Keen et al, 1993; Gamperl et al, 1994), although other sub-types have been identified in other fish species (see Nilsson, 1983; Farrell and Jones, 1992). Temperature acclimation can alter the response of the rainbow trout heart to adrenaline (Ask et al, 1981; Farrell et al, 1996) and some of this change has been attributed to a temperature-dependent change in cell surface P-A R density ( B m a x ) (Keen et al, 1993; Shiels et al, 2002). For example, the increase in adrenergic sensitivity with cold acclimation in rainbow trout hearts is associated with an increase in p 2 - A R density (Keen et al, 1993; Gamperl et al, 1998) and this adrenergic stimulation is extremely important in preserving the L-type C a 2 + current in isolated trout myocytes exposed to cold temperature (Shiels et al, 2000, 2003). The objective of this study was to determine whether a tropical species, African catfish (Clarias gariepinus), exhibits a similar intraspecific plasticity in P-AR density in response to acclimation temperature as A version of this chapter has been previously published: Hanson, L. M , Ip, Y . K . , and Farrell, A . P. (2005). The effect of temperature acclimation on myocardial ^-adrenoceptor density and ligand binding affinity in African Catfish (Claris gariepinus). Comp. Biochem. Physiol. A , 141:164-168. 85 rainbow trout. It was expected that P-AR density would be inversely related to acclimation temperature. Materials and Methods Fish African catfish (Claris gariepinus) were originally obtained from a live fish supplier in Singapore and held at National University of Singapore for >6 months (with temperature exhibiting the normal diurnal fluctuation between 2 3 - 2 8 ° C ) . For the temperature acclimation experiment, fish were transferred into small fiberglass aquaria (2 per temperature group) that were then gradually adjusted to the three experimental temperatures ( 1 5 ° C , 2 2 ° C , 3 2 ° C) over a period of 2-6 days (i.e. approximately 1°C change per day). Fish in all three acclimation groups were then held at experimental temperature for 28 days before sampling. The fish were quickly killed by a blow to the head, the ventricle was excised, rinsed in a 0.8% NaCl solution and frozen in liquid nitrogen. Ventricles were frozen at - 8 0 ° C before being shipped on dry ice to Simon Fraser University, where they were stored at -80°C for no more than 2 months prior to analysis. Body mass ranged from 296 to 707 g and relative ventricular mass from 0.05% to 0.11% Earlier studies of cardiac P-AR in fish have included rainbow trout, therefore they were used as a reference species to ensure consistency of experimental techniques. Rainbow trout (Oncorhynchus mykiss) were purchased from Sun Valley Trout Farms (Langley, B C , Canada) and held in an outdoor tank ( 1 4 ° C ) for at least 3 weeks prior to sampling, The fish were killed by a quick blow to the head after which the cardiac ventricle was quickly excised, rinsed in saline (composition in m M : NaCl , 124.1; KC1, 3.1; M g S 0 4 , 0.90; C a C l 2 , 2.5; T E S 86 free acid, 7.0; T E S Na salt, 3.3; p H 7.85 at 15°C) , gently massaged to remove blood and frozen in liquid nitrogen. Tissues were stored at - 8 0 ° C for no longer than three months prior to analysis. Body mass ranged from 338 to 476 g and relative ventricular mass from 0.08% to 0.13% Quantification of ^ -adrenoceptors Cell surface P2-adrenoceptor density (Bm ax) and binding affinity (Kd) were determined for ventricular punches, using the tritiated ligand technique of Watson-Wright et al. (1989), as modified for fish hearts by Gamperl et al. (1994). Frozen ventricles were first rinsed in saline (see above) several times to remove any remaining blood. Ventricles were then partially refrozen at -80°C before being sliced (350 (rm thickness) with a Mcllwain tissue chopper (Brinkman, Rexdale, O N , Canada). Tissue slices were placed in ice-chilled saline while ventricular tissue punches (2 mm diameter by 350 um thickness) were taken. For rainbow trout, the punches were taken almost exclusively from the compact myocardium, while for African catfish the compact myocardium was not as well defined and punches were taken from the outer regions of the ventricular wall. Gamperl et al. (1998) showed that there was no significant difference between K j for the compact and spongy myocardium of rainbow trout, but that B m a x was 14% higher in spongy myocardium. Single punches were placed in separate wells of a 24-well tissue culture plate (Flow Laboratories, McLean, V A ) , with each well containing 500 uL of saline and various concentrations (0.05-3.5 nM) of the hydrophilic P2-adrenoceptor ligand [3H] CGP-12177 (CGP specific activity 55 and 52 Ci/mol; Amersham Life Science). To determine non-specific binding, separate tissue punches were incubated at each concentration with the competitive p2-adrenoceptor 87 antagonist timolol (10 uM). A l l tissue punches were incubated at 0 ° C for 2 h, which was sufficient for equilibrium binding to occur (Gamperl et al, 1994). The culture plates were agitated frequently during incubation to prevent the formation of a depletion layer. Following incubation, punches were washed twice in ice-chilled saline, placed into 8 ml scintillation vials containing 2 ml of Ecolite scintillation fluid (ICN Canada Inc., Mississauga, Ont., Canada), incubated for 18 h and counted with a liquid scintillation counter (LS 6500, Beckman Instruments, USA) . Specific binding was calculated by subtracting radioactivity (disintegrations per minute) of the punches incubated with C G P and timolol (4 punches) from the activity of punches incubated with C G P alone (6-8 punches). A single ligand-binding curve consisted of 6-8 replicates for each ligand concentration. Ventricle size determined the degree of replication for the binding curves. Rainbow trout were large enough to obtain a sufficient number of tissue punches to construct a single binding curve from each ventricle, therefore, the binding curve was replicated six times using six individuals (i.e., N = 6; 6 fish). For the African catfish, however, the smaller ventricular size required the pooling of tissue punches from several individuals to construct a single binding curve. The number of replicate binding curves (N) was 3 and the number of fish used was 5 for all three acclimation temperatures (ie, N = 3; 5 fish). Quench tests performed with scintillation fluid and heart tissue revealed no significant quenching. Data analysis Binding parameters were determined using a Scatchard plot as described by Zivin and Waud (1982) (not shown). The linearity of the Scatchard plots (r2 = 0.80-0.88) indicates the presence of a single adrenoceptor population. Protein content of representative punches was 88 determined using a Bradford protein assay, using bovine serum albumin (Sigma Chemicals, St. Louis, Missouri) as the standard, so that B m a x could be expressed as fmol mg protein"1. A l l statistical analysis was performed after specific binding was expressed as fmol mg protein"1 using SigmaStat 2.03 (1997). A one-way A N O V A was used to compare B m a x and Kd values between the three groups of African catfish, with the Holm-Sidak multiple comparisons procedure being used to determine between group significance. A separate one-way A N O V A was used to compare B m a x and Kd values of rainbow trout with those of African catfish. The Holm-Sidak multiple comparisons procedure was used to test whether the B m a x and Kd of each subgroup of African Catfish differed significantly from rainbow trout. Results The r 2 value for individual CGP-binding curves was always > 0.90 (range = 0.90-0.96). Values are presented as mean ± S E M (Fig. 4.1). No statistically significant differences were found among pVadrenoceptor density ( B m a x ) values for African catfish acclimated at 15°C, 2 2 ° C and 3 2 ° C . Nevertheless, ligand binding affinity (Kd) was statistically significantly higher (p = 0.002) for the 3 2 ° C acclimation group (Kd = 0.88) compared with both the 15°C (Kd = 0.48) and 2 2 ° C acclimation groups (Kd = 0.46) (Fig. 4.2). There were no statistically significant differences in the protein content of punches from the three acclimation groups. Protein content among the temperature acclimation groups ranged from 123 ± 12 ( 3 2 ° C ) to 145 ± 13 pg mg tissue"1 ( 2 2 ° C ) and was 154 ± 13 ug mg tissue"1 for rainbow trout. For rainbow trout, B m a x (26.4 fmol mg protein"1) was 89 significantly higher (p < 0.05) and Kd (0.19 nM) significantly lower (p < 0.001) than the values for African catfish at all three acclimation temperatures. Discussion B m a x and Kd values for the (32-AR in cardiac tissue of rainbow trout were used as a reference to compare with earlier studies. Our results for rainbow trout at 14°C ( B m a x = 26.4 fmol mg protein"1 and Kd = 0.19 nM) compare favourably with those of Gamperl et al. (1998), who reported a B m a x of 25 fmol mg protein"1 and a Kd of 0.16 n M for rainbow trout at 14°C. Similarly, Olsson et al. (2000) reported a B m a x of 23 fmol mg protein"1 and a Kd of 0.13 n M for rainbow trout at 12°C. For rainbow trout acclimated to a colder (8°C) temperature, Gamperl et al. (1994) reported an almost 2-fold increase in B m a x to 40 fmol mg protein"1 and an unchanged ICj of 0.16 n M . In view of this concordance, we are confident in the methods used to characterize myocardial p 2 - A R density and binding affinity. Intraspecific plasticity in ventricular p 2 - A R density in temperate rainbow trout is inversely associated with acclimation temperature (Keen et al, 1993; Gamperl et al, 1998). These two independent studies found that B m a x could decrease by as much as 11% per 1°C increase in temperature. Our results for the tropical African catfish provide a sharp contrast, with B m a x ranging from 14.3 to 17.8 fmol mg protein"1 over a 17°C temperature range, which suggests that P-AR plasticity may be species specific. Perhaps stenothermal tropical species do not posses the intraspecific P-AR plasticity exhibited by temperate species such as the rainbow trout, which experience a wide range of seasonal environmental temperatures, and in which P-AR plasticity is critical for maintaining cardiac contractility (Graham and Farrell, 1989; Shiels et al, 2003). Accordingly, acclimation mechanisms, such as cold-induced 90 increases in (3-AR density (Keen et al, 1993; Gamperl et al, 1998), may be superfluous in tropical fish. Further studies are needed to ascertain the exact environmental and physiological triggers for changes in (3-AR density in order to determine why temperature-mediated regulation has to date only been observed in a temperate species. Nevertheless, the increase in Kd for African catfish with increasing acclimation temperature is consistent with a decrease in adrenergic sensitivity, as observed in rainbow trout acclimated to elevated water temperature (Keen et al, 1993; Farrell et al, 1996). Thus, it is possible that trout downregulate cardiac adrenergic sensitivity at extremely warm acclimation temperatures by internalizing p-AR, whereas tropical African catfish change binding affinity. Olsson et al (2000) suggested that across a number of fish species myocardial P-AR density was directly related to acclimation temperature. The present results do not conform with this idea because B m a x for African catfish was significantly lower than that measured here and elsewhere for rainbow trout. As a result, we cannot support the suggestion that interspecific differences in B m a x are related to temperature differences among species. Indeed, preliminary work in our laboratory shows a wide range for B m a x among tropical fish species, which spans that reported for rainbow trout [10.5 ± 0.9 to 47.5 ± 2.9 fmol mg protein"1 for snakehead (Channa micropeltes), African catfish (Clarias gariepinus), four-eyed sleeper (Bostrichthys sinensis), marble goby (Oxyeleotris marmorata), black eeltail catfish {Plotosus canius), Bengal sergeant (Abudefdel bengalensis), and blue spotted fantail ray {Taeniura lymma)]. While the lack of an effect of temperature acclimation on B m a x in African catfish was clear-cut, there is some concern that the difference between the assay temperature and the range of acclimation temperatures may result in artifacts in the measured Kd values. A l l of 91 the assays were conducted and incubated at 0 ° C to minimize tissue degradation, which is a standard practice for these types of ligand binding assays. Although an assay temperature closer to the acclimation temperature may have improved the assessment of Kd, it would have resulted in variable tissue degradation and subjected receptors to cellular modifications such as desensitization, internalization and degradation, that would have aversely affected Bmax' Similarly, it is unlikely that our inability to separate compact and spongy myocardium was a confounding factor in the low B m a x observed for African Catfish (as compared to rainbow trout and tropical fish of previous studies, Olsson et al, 2000) because only a 14% difference in B m a x has been observed for these two tissue types (Gamperl et al. 1998). In addition, spongy cardiac tissue is known to have a slightly higher B m a x than compact tissue, and so it is possible that we have overestimated B m a x for the species comparison. Nonetheless, this degree of variation would not account for the differences we observed between rainbow trout and African catfish. When fish are sampled, they are briefly exposed to hypoxia. Hypoxia influences B m a x in mammalian cardiac tissues, with significant (29%-62%) hypoxic downregulation occurring in rat and chick ventricular tissue (Voelkel et al, 1981; Blake et al, 1982; Marsh and Sweeney, 1989; Rocha-Singh et al, 1991). However, this type of P-AR down-regulation did not occur in rainbow trout ventricle tissue after a prolonged 6 h hypoxic exposure (Gamperl et al, 1998). In contrast, P-AR up-regulation occurred in trout red blood cells following a 30 minute hypoxic exposure (Reid et al, 1992). Moreover, adrenergic responsiveness was increased by a concomitant increase in plasma catecholamine levels under hypoxic, but not normoxic conditions (Reid et al, 1992). In view of this, and the fact 92 that resting adrenergic tonus to the fish heart certainly shows considerable interspecific variability (Axelsson et al, 1987; 1992; Keen et al, 1995), a possibility worth exploring is whether the level of adrenergic stimulation is an important determinant of cardiac (3-AR density. In summary, we suggest that P-AR plasticity in response to temperature acclimation is dissimilar for tropical African catfish and temperate rainbow trout. 93 30 + Rainbow Trout Oncorhynchus mykiss .S 20 +-» o a M 10 5 3 0 c o 20 'o <u w 10 max = 26.4 ±3.0 K d = 0.19 ±0.02 African Catfish 15°C Clarias gariepinus (r2 = .= 17.8 ±2.0 0.48 ±0.03 0.916) African Catfish 22°C Clarias gariepinus B m a x = 15.4 ± 1 . 0 Kd = 0.46 ±0.08 (r2 = 0.967) African Catfish 32°C Clarias gariepinus B max =14.3 ± 1 . 0 K d = 0.88 ±0.01 3 0 [CGP] (nM) Figure 4.1: Specific binding of [ H]CGP-12177 to ventricular P-adrenoceptors. Due to the small size of some African catfish ventricles they sometimes had to be pooled for a single binding curve. The number of fish used and the number of binding assays performed is as follows: rainbow trout (N=6 binding assays, 6 fish), African catfish 15°C (N=3 binding assays; 5 fish), African catfish 2 2 ° C (N=3; 5 fish), and African catfish 3 2 ° C (N=3; 5 fish). The P-adrenoceptor density ( B m a x , fmol mg protein"1), [ 3H]CGP-12177 dissociation constant (K<_, nM) and r values for each graph are indicated. Values are mean ± S E M . 94 20 + o & 15 a .S 1 0 PQ 5 + 0.8 0.6 •a ^ 0.4 0.2 0.0 J _ 15 22 32 Temperature (°C) Figure 4.2: [ 3H]CGP-12177 dissociation constant (Kd) and ^-adrenoceptor density (B m a x ) in African catfish acclimated to 15°C (N=3, 5 fish), 2 2 ° C (N=3, 5 fish), and 3 2 ° C (N=3, 5 fish). Values are means ± S E M . * denotes significant differences at P < 0.05 (one-way A N O V A ) 95 References Ask, J.A., Stene-Larsen, G., Helle, K.B. (1980). Atrial beta2-adrenoreceptors in the trout. J. Comp. Physiol. 139, 109-115. Ask, J.A., Stene-Larsen, G., Helle, K.B. (1981). Temperature effects on the p 2 -adrenoreceptors of the trout atrium. J. Comp. Physiol. 143, 161-168. Axelsson, M., Ehrenstrom, F., Nilsson, S. (1987). Cholinergic and adrenergic influence on the teleost heart in vivo. Exp. Biol. 46, 179-186. Axelsson, M., Davison, W., Forster, M.E., Farrell, A.P. (1992). Cardiovascular responses of the red-blooded Antarctic fishes, Pagothenia bernacchii and P. borchgrevinki. J. Exp. Biol. 167, 179-201. Blake, M.S., Johnston, K.H., Russell-Jones, G.J., Gotschlich, E .C. (1982). A rapid, sensitive method for the detection of alkaline phosphatase-conjugated anti-antibody on western blots. Anal. Biochem. 136, 175-179. Farrell, A. P. and Jones, D. R. (1992). The heart. In Fish physiology, vol. XIIA (eds. D. J. Randall and A . P. Farrell), pp. 1-88. San Diego: Academic Press Ltd. Farrell, A.P., Gamperl, A.K., Hicks, J.M.T., Shiels, H.A., Jain, K.E. (1996). Maximum cardiac performance of rainbow trout, Oncorhynchus mykiss, at temperatures approaching their upper lethal limit. J. Exp. Biol. 199, 663-672. Gamperl, A.K., Wilkinson, M., Boutilier, R.G. (1994). p-Adrenoreceptors in the trout (Oncorhynchus mykiss) heart: characterization, quantification, and effects of repeated catecholamine exposure. Gen. Comp. Endocrinol. 95, 259-272. Gamperl, A.K., Vijayan, M.M., Pereira, C , Farrell, A.P. (1998). P-Receptors and stress protein 70 expression in hypoxic myocardium of rainbow trout and chinook salmon. Am. J. Physiol. 21 A, R428-R436. Graham, M.S., Farrell, A.P. (1989). The effect of temperature acclimation and adrenaline on the performance of a perfused trout heart. Physiol. Zool. 62, 38-61. Keen, J.E., Vianzon, D.-M., Farrell, A.P., Tibbits, G.F. (1993). Thermal acclimation alters both adrenergic sensitivity and adrenoreceptor density in cardiac tissue of rainbow trout. J. Exp. Biol. 181, 27-47. Keen, J.E., Aota, S., Brill, R.W. (1995). Cholinergic and adrenergic regulation of heart rate and ventral aortic pressure in two species of tropical tunas, Katsuwonus pelamis and Thunnus albacares. Can. J. Zool. 73, 1681-1688. 96 Marsh, J.D., Sweeney, K.A. (1989). P-Adrenergic receptor regulation during hypoxia in intact cultured heart cells. Am. J. Physiol. 256, H275-H281. Nilsson, S., 1983. Receptors for Transmitter Substances. In: Autonomic nerve function in the vertebrates, Zoophysiology, vol. 13 (ed. S. Nilsson), pp. 68-84. Berl in: Springer-Verlag. Olsson, H.I., Yee, N., Shiels, H.A., Brauner, C , Farrell, A.P. (2000). A comparison of myocardial P-adrenoreceptor density and ligand binding affinity among selected teleost fishes. J. Comp. Physiol. 170, 545-550. Reid, S.D., Lebras, Y., Perry, S.F. (1992). The in vitro effect of hypoxia on the trout erythrocyte P-adrenergic signal transduction system. J. Exp. Biol. 176, 103-116. Rocha-Singh, K.J., Honbo, N.Y., Karliner, J.S. (1991). Hypoxia and glucose independently regulate the p-adrenergic cyclase system in cardiac myocytes. J. Clin. Invest. 88,204-213. Shiels, H.A., Vornanen, M., Farrell, A.P. (2000). Temperature-dependence of L-type C a 2 + channel current in atrial myocytes from rainbow trout. J. Exp. Biol. 203, 2771-2780. Shiels, H.A., Vornanen, M., Farrell, A.P. (2002). The force-frequency relationship in fish hearts - a review. Comp. Biochem. Physiol. 132A, 811-826. Shiels, H.A., Vornanen, M., Farrell, A.P. (2003). Acute temperature change modulates the response of Ica to adrenergic stimulation in fish cardiomyocytes. Physiol. Biochem. Zool. 76, 816-824. Temma, K., Hirata, T., Kitazawa, T., Kondo, H., Katano, Y. (1986). Are P-adrenergic receptors in ventricular muscles of carp heart (Cyprinus carpio) mostly the p2 type. Comp. Biochem. Physiol. 83C, 261-263. Voelkel, N.F., Hegstrand, L., Reeves, J.T., McMurtry, I.F., Molinoff, P.B. (1981). Effects of hypoxia on density of P-adrenergic receptors. J. App. Physiol. 50, 363-366. Watson-Wright, W.M., Armour, J.A., Johnstone, D.E., Wilkinson, M. (1989). Myocardial slice: a physiological approach to P-adrenergic ([ 3H]CGP-12177) receptor binding in hamster and guinea heart. J. Pharmacol. Methods 22, 37-47. Zivin, J.A., Waud, D.R. (1982). How to analyze binding, enzyme and uptake data: the simplest case, a single phase. Life Sci. 30, 1407-1422. 97 Chapter 5: Major findings and conclusions This thesis examined the role that adrenergic stimulation plays in protecting cardiac performance during exercise-induced changes in venous blood composition, namely hypoxia, hyperkalemia and acidosis. The objectives were five-fold: (1) to quantify the venous hypoxic threshold for maximum cardiac performance using in situ perfused hearts, (2) to quantify the inotropic and chronotropic effects of hyperkalemia on in situ perfused hearts, (3) to simulate the effects of post-exercise levels of hyperkalemia and acidosis in combination with hypoxia in situ, and determine the hypoxic threshold for maximum cardiac performance with and without concurrent adrenergic stimulation, (4) to determine the effect of elevated temperature ( 1 8 ° C ) on the in situ hypoxic threshold for cardiac collapse associated with a hyperkalemic, acidotic exposure, and (5) to determine whether P-adrenoreceptor density (Bmax) is affected by warm temperature in the tropical African catfish as it is in the temperate rainbow trout. Objective 1 I found that the venous hypoxic threshold for maximum cardiac performance in saline perfused rainbow trout hearts was 2.7 kPa. This threshold is similar to previously reported venous oxygen tension (PV02) values of 2.0-2.1 kPa observed during maximal exercise in vivo (Kiceniuk and Jones, 1977; Farrell and Clutterham, 2003), but higher than reported venous oxygen levels < 1.3 kPa in rainbow trout during environmental hypoxia (Thomas et al., 1994; Steffensen and Farrell, 1998). This difference likely occurs because without exercise under just aquatic hypoxia the heart may not be working as hard and therefore will have a lower cardiac oxygen demand. 98 Objective 2 M y thesis contains the first study to quantify the chronotropic effects of hyperkalemia on fish hearts. I found that with only tonic adrenergic stimulation a physiologically relevant level of hyperkalemia (5.0 mM) reduced cardiac performance by 30%, with the majority of this decrease being due to negative chronotropy. As previous studies have focussed their attention on isolated, paced cardiac tissue, the 25% decrease in heart rate I observed is something previously unreported. In addition, I found that both the negative chronotropic and inotropic effects of 5.0 m M hyperkalemia could be completely eliminated by maximum adrenergic stimulation. Objective 3 I discovered that if in situ perfused rainbow trout hearts were exposed to the hyperkalemic, acidotic conditions experienced in venous blood during intense exercise, they could not reach the maximum level of performance seen during intense exercise and recovery in vivo, even if they received fully oxygenated saline. However, maximum adrenergic stimulation of the in situ hearts counteracted the detrimental effects of hyperkalemia and acidosis such that hearts could perform maximally at Pvo2 levels as low as 2.0 kPa, a hypoxic threshold similar to the Pvo2 values reported for maximally exercising fish in vivo. This suggests that adrenergic stimulation is obligatory when these adverse conditions are present during intense exercise and recovery. 9 9 Objective 4 As acclimation temperature increases above the optimum, maximum cardiac performance is compromised. In rainbow trout this occurs at approximately 18°C (Farrell et al, 1996). In accordance with this observation I found that the hypoxic threshold for maximum cardiac performance shifted dramatically when I tested in situ perfused rainbow trout hearts under simulated exercise conditions at 18°C (as compared to my previous experiments at 10°C) . The hypoxic threshold increased from 2.0 kPa at 10°C to 5.6 kPa at 18°C. Unlike the situation at 10°C, the in situ hypoxic threshold at 18°C is much higher than the in vivo threshold of 3.2-3.9 kPa (Farrell and Clutterham 2003). Potential reasons for this discrepancy are discussed below. Objective 5 Unlike the situation in the temperate rainbow trout, I failed to find any evidence of ventricular Bmax modulation with acclimation temperature in the tropical African catfish. The Bmax for African catfish was not significantly different over a 17°C difference in acclimation temperature, whereas in rainbow trout Bmax varies by as much as 11 % for every 1 °C change in acclimation temperature (Keen et al, 1993; Gamperl et al, 1998), creating the potential for a three-fold change over this temperature range. M y results suggest that the control of ventricular P-AR activated mechanisms in response to temperature acclimation does not appear to be a universal mechanism among fish. 100 Perspectives B y comparing the results of chapters 2 and 3, a discrepancy in the hypoxic thresholds becomes apparent. At 10°C the in situ hypoxic threshold was very similar to in vivo values (Kiceniuk and Jones, 1977; Farrell and Clutterham, 2003) and lower than the half maximal haemoglobin-oxygen saturation (P50) value of 3.1 (Perry and Reid, 1992). In contrast, the in situ threshold of 5.6 kPa at 18°C was much higher than the in vivo threshold of 3.2-3.9 kPa previously observed in exercising rainbow trout (Farrell and Clutterham, 2003), and also higher than the calculated P50 value of 4.7 kPa at this temperature (as determined from the data of Eddy, 1971). There are two potential reasons for this discrepancy: (i) the use of saline as a perfusate as opposed to blood or haemoglobin solution and (ii) the loss of critical adrenergic protective mechanisms at 18°C due to the temperature-induced decrease in P-AR density. Due to the increase in myocardial oxygen extraction at 18°C, the linear nature of the saline-oxygen dissociation curve, and the low oxygen capacitance of saline, the reduction in P02 as saline passes through the heart may be greater at 18°C than at 1 0 ° C . If this prediction were true, saline perfused hearts at 18°C would require a higher venous oxygen threshold in order to maintain an adequate oxygen diffusion gradient to the myocardium. Due to the higher oxygen capacitance of blood and the sigmoidal nature of the haemoglobin-oxygen dissociation curve, this situation would not arise in vivo. To determine if saline perfusion contributed to the above discrepancy in hypoxic thresholds, I needed to find out if temperature affects the reduction in P02 as saline passes through the heart. Using myocardial oxygen extraction data from Graham and Farrell (1990) and saline-oxygen solubility coefficient (alpha) values from Graham (1987), I calculated the 101 change in Po2 that occurred as saline passed through the heart during several sets of experiments (representing 32 fish) at both 10°C and 18°C. I found that although the change in saline P02 was significantly higher at 18°C, the difference was only 0.7 kPa between the two acclimation temperatures. Consequently, it seems unlikely that the corresponding reduction in the oxygen diffusion gradient would be sufficient to explain the 1.7-2.4 kPa discrepancy between the in vivo and in situ venous oxygen thresholds at 18°C. As saline-oxygen extraction dynamics cannot explain this discrepancy between in situ and in vivo hypoxic thresholds, it appears that adrenergically activated protective mechanisms may be the key to sustaining lower venous oxygen thresholds in vivo. Nevertheless, the reduction in saline P02 due to myocardial oxygen extraction (2.97 ± 0.05 kPa at 10°C and 3.67 ± .01 kPa at 18°C) means that in vivo venous oxygen thresholds could be several kPa lower than those seen in situ. Future studies utilizing either haemoglobin solution or blood perfusion would be needed to determine a more exact threshold. In addition, by examining the difference between the hypoxic thresholds obtained with haemoglobin solution perfusion and blood perfusion, future studies could also discern the importance of erythrocyte P-AR activated compensation mechanisms. In conclusion, the experiments contained in this thesis demonstrated the vital role that adrenergic stimulation plays in stimulating and protecting maximum cardiac performance during intense activity and recovery. While these effects are acutely temperature sensitive in rainbow trout, this may not be the case for other species. 102 References Eddy, F. B. (1971). Blood gas relationships in the rainbow trout Salmo gairdneri. J. Exp. Biol. 55, 695-711. Farrell, A. P. and Clutterham, S. M. (2003). On-line venous oxygen tensions in rainbow trout during graded exercise at two acclimation temperatures. J. Exp. Biol. 206, 487-496. Farrell, A. P., Gamperl, A. K., Hicks, J. M. T., Shiels, H. A. and Jain, K. E. (1996). Maximum cardiac performance of rainbow trout (Oncorhynchus mykiss) at temperatures approaching their upper lethal limit. J. Exp. Biol. 199, 663-672. Gamperl, A. K., Vijayan, M. M., Pereira, C. and Farrell, A. P. (1998). Preceptors and stress protein 70 expression in hypoxic myocardium of rainbow trout and chinook salmon. Am. J. Physiol. 43, R428-R436. Graham, M. (1987). The solubility of oxygen in physiological salines. Fish Physiol. Biochem. 4, 1-4. Graham, M. S. and Farrell, A. P. (1990). Myocardial oxygen consumption in trout acclimated to 5°C and 15°C. Physiol. Zool. 63, 536-554. Keen, J. E., Vianzon, D. M., Farrell, A. P. and Tibbits, G. F. (1993). Thermal acclimation alters both adrenergic sensitivity and adrenoreceptor density in cardiac tissue of rainbow trout. / . Exp. Biol. 181, 27-48. Kiceniuk, J. W. and Jones, D. R. (1977). The oxygen transport system in trout (Salmo gairdneri) during sustained exercise. J. Exp. Biol. 69, 247-260. Perry, S. F. and Reid, S. D. (1992). Relationship between blood 0 2 content and catecholamine levels during hypoxia in rainbow trout and American eel. Am. J. Physiol. 263, R240-R249. Steffensen, J. F. and Farrell, A. P. (1998). Swimming performance, venous oxygen tension and cardiac performance of coronary-ligated rainbow trout, Oncorhynchus mykiss, exposed to progressive hypoxia. Comp. Biochem. Physiol. 119A, 585-592. Thomas, S., Fritsche, R. and Perry, S. F. (1994). Pre- and post- branchial blood respiratory status during acute hypercapnia or hypoxia in rainbow trout, Oncorhynchus mykiss. J. Comp. Physiol. 164B, 451-458. 103 

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