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Cardiac remodeling in diploid and triploid rainbow trout (Oncorhynchus mykiss Walbaum) Simonot, Danielle Laurette 2005

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CARDIAC REMODELING IN DIPLOID AND TRIPLOID RAINBOW TROUT (ONCORHYNCHUS MYKISS Walbaum) by DANIELLE LAURETTE SIMONOT B.Sc.(Hon.), University of Saskatchewan, 1997 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (ZOOLOGY) THE UNIVERSITY OF BRITISH COLUMBIA December 2005 © Danielle L. Simonot, 2005 Abstract The extent, time course and qualitative aspects of cardiac remodeling were studied in rainbow trout (Oncorhynchus my kiss Walbaum) using chemically-induced anemia as the induction method. Injection of phenylhydrazine hydrochloride (PHZ) was used to induce a transient hemolytic anemia which increased routine cardiac output (Q) by 20% when the hematocrit (Hct) was reduced from 23% to 10%. It was therefore hypothesized that prior to cardiac remodeling, rainbow trout may rely on venous oxygen stores rather than compensate with energetically-costly increases of cardiac output. Indeed, after Hct was held at an average Hct of 17% for up to 8 weeks, relative ventricular mass (VM) in diploid anemic rainbow trout increased significantly within two weeks and by 84% after eight weeks. Cardiac remodeling was discovered to be influenced by water temperature. While there was a disproportionate increase in % compact myocardium at 6°C (from 30% to 37%, P<0.05), a proportionate increase resulted at 17°C. Furthermore, radioisotope tracer analysis of coronary vascular volume supports coronary angiogenesis of the compact myocardium during cardiac remodeling, as a 37% increase in rVM doubled coronary vascular volume. The potential benefits of cardiac remodeling were tested in vivo by measuring maximum cardiorespiratory and swimming performance in post-anemic rainbow trout that had recovered their Hct, but retained cardiac hypertrophy. As expected, a 27% higher rVM significantly increased maximum Q and stroke volume by 60%, but without affecting swimming performance, maximal oxygen consumption, heart rate or venous oxygen tension, possibly because the hemoglobin concentration had been fully restored in PHZ-treated fish, despite an equivalent Hct. Triploid rainbow trout, which have enlarged cardiomyocytes, responded similarly to PHZ-treatment as diploids cohorts in terms of the rVM and hematological responses to PHZ, but displayed significant effects of handling stress. Their disproportionate increase in spongy myocardium and lack of an angiogenic response may have restricted oxygen diffusion to the myocardium, helping to explain the significant respiratory distress and mortality in response to handling and PHZ treatment at warm temperatures. Thus, anemia-induced cardiac remodeling in rainbow trout is rapid, substantial and plastic, where increased compact myocardium benefits blood supply to the ventricle via the coronary artery, resulting in improved blood pumping capacity of the heart. ii Table of Contents Abstract ii Table of Contents iii List of Tables vi List of Figures vii Acknowledgements x Chapter 1: Introduction to the teleost circulatory system : 1 • The coronary circulation 3 • Cardiac plasticity in rainbow trout 5 • Phenylhydrazine as an inducer of anemia 9 • The hypertrophy versus hyperplasia debate 10 • Triploid rainbow trout: A 'natural' model of cardiac hypertrophy 12 • Objectives 12 • Scientific Hypotheses 13 • Literature Cited 14 Chapter 2: Remodeling of the myocardium in rainbow trout (Oncorhynchus mykiss Walbaum) 18 • Introduction 18 • Materials & methods: 21 ^ Animal acquisition and care 21 ^ Injection and hematocrit-monitoring techniques 22 ^ Tissue sampling protocol 23 ^ Experimental Protocols 24 > Statistics 27 • Results 27 ^ Experiment 1: Effect of chasing on cardiac remodeling 27 ^ Experiment 2: The effect of handling stress on cardiac remodeling 30 ^ Experiment 3: The acute effect of anemia on Hct and cardiac output 31 • Discussion 31 ^ Anemia causes significant elevation of cardiac output 32 ^ Temperature has a significant modulatory effect on cardiac remodeling and erythropoiesis 34 ^ Conclusion 35 • Literature Cited- 44 Chapter 3: Quantification of the coronary vascular volume in normal and hypertrophied rainbow trout (Oncorhynchus mykiss Walbaum) ventricles by radiotracer tracer analysis 46 • Introduction 46 • Materials & methods 48 ^ Animal acquisition and care 48 ^ Drug treatment 48 ^ Radioisotope tracer analysis 49 ^ Calculations and statistical analyses 50 • Results 52 ^ Hematocrits and cardiac remodeling 52 ^ Vascular volume analysis 52 • Discussion 52 ^ Comparison of coronary vascular volume estimates 53 ^ The coronary artery - a prolific angiogenic candidate 55 ^* Conclusion 57 • Literature Cited 62 Chapter 4: Effect of cardiac remodeling on the cardiovascular and swimming performance during a U c r i t swimming challenge in rainbow trout (Oncorhynchus mykiss Walbaum) 65 • Introduction 65 • Materials & methods 67 ^ Animal acquisition and care 67 ^ Drug treatment 67 ^ Surgical procedure 68 ^ Ucri, swim test protocol 69 ^ Analysis of cardiovascular data 70 ^ Experimental control protocols 71 > Statistics 71 • Results 72 • Physical and hematological characteristics 72 ^ Recovery following surgery and the Ucril swim test 73 ^ Cardiorespiratory and Ucrit swimming performance were equivalent 73 ^ Effect of ventricular hypertrophy on cardiac performance during Ucri, 74 ^ Venous oxygen tension 75 • Discussion 75 ^ Hematological considerations 76 ^ A significantly increased maximum cardiac output did not improve swimming performance 77 ^ Are cardiorespiratory measurements representative? 78 ^ Benefits of cardiac remodeling 80 ^ Conclusion 81 • Literature Cited- 92 iv Chapter 5: Cardiac remodeling and ventricular vascular volume determination in triploid rainbow trout (Oncorhynchus mykiss Walbaum) 95 • Introduction 95 • Materials & methods 97 ^ Animal acquisition and care 97 ^ Cardiac remodeling procedure 97 ^ Radioisotope tracer analysis 98 ^ Calculations and statistical analyses 98 . Results 99 ^- Cardiac Remodeling Experiment 99 ^* Vascular volume experiment 100 • Discussion 101 Stress induces significant hypertrophy in triploid hearts 101 ^" Triploid rainbow trout may be angiogenically-challenged 103 ^ Triploids yield equivalent tracer estimates 104 > Conclusion 104 • Literature Cited 113 Chapter 6 - Discussion 116 ^ Anemia stimulates significant cardiac remodeling 116 ^ Phenylhydrazine as inducer of hemolytic anemia 119 ^ The triploid conundrum: impressive, but deadly cardiac remodeling 121 ^ Benefits of Cardiac Remodeling 122 . Literature Cited 124 V List of Tables Table 2.1 43 Summary of the effects of a four-week PHZ or sham saline treatment versus untreated fish on cardiac and hematological characteristics of rainbow trout (A^IO). Presented is the mean ± SEM, (*) indicates significant differences as determined by ANOVA and Dunnett's test. Table 2.2 43 Changes in cardiac output, hemoglobin and hematocrit (Hct) following injection with phenylhydrazine hydrochloride. Rainbow trout (N=5) were equipped with a flowprobe and dorsal aortic cannula to concurrently monitor the cardiovascular effects of anemia. The mean normocythemic Hct was 22.7 ± 1.4%; the severe anemia Hct was 3.6 ± 0.5%. Differing letters indicate significant differences as determined by repeated-measures ANOVA and Tukey-Kramer multiple-range test (P<0.001). Table 3.1 .• 61 Total vascular volume per gram ventricle of control and PHZ-treated rainbow trout, as determined by radioisotope tracer analysis with Tc"m and l 2 5I-BSA. Tissue hematocrit (Hct) provides the hematocrit of the coronary circulation and the hematocrit ratio a comparison to systemic Hct. Table 4.1 90 Comparison of the mean physical characteristics (± SEM) between treatment groups equipped with a flowprobe and microsensor oxygen probe (/v=8) and unprobed groups (unprobed sham control, iV=4, unprobed PHZ-treated group, N=3). Table 4.2 90 Summary of routine and maximal cardiovascular variables of sham and PHZ-treated groups measured during a Ucrj, swimming test; mean (SEM), N=8. Sham M02 values were measured in separate sham-operated fish (control sham ^ =4, PHZ sham A ^ ) . Table 4.3 91 Summary of the U c r i t swim characteristics (mean ± SEM) of sham and PHZ-treated groups equipped with a flowprobe and oxygen probe (N=8). Unprobed control values were measured in separate, sham-operated fish swum to U c r i t (unprobed sham N=4; unprobed PHZ N=3). Table 4.4 :.91 Comparison of cardiovascular measurements with literature values of natural, maturation-induced ventricular hypertrophy (*Franklin & Davie 1992). Table 5.1 112 Total ventricular vascular volume per gram ventricle of diploid and triploid rainbow trout determined by dual radioisotope tracer analysis with Tc"m and 1 2 5I-BSA. vi List of Figures C H A P T E R 2 Figure 2.1 : 37 Changes in hematocrit during the experimental period in response to four injections (1) at weeks 0, 2, 4 and 6 followed by a 4-week recovery. Figure 2.2 38 The change in relative ventricular mass (rVM) during the experimental protocol is represented in the top left figure (A), while the rVM calculated by dry mass determination (dry rVM) is presented in (B). The linear relationship between wet and dry rVM is presented in the bottom figure (C). Figure 2.3 39 Dry relative ventricular mass (N=6) during an 8-week treatment with either saline saline or phenylhydrazine hydrochloride (PHZ) injections, followed by a 4-week recovery (week 12). Figure 2.4 40 The change in relative ventricular mass (rVM) during the experimental protocol is presented in the top left figure (A), while the dry rVM calculated by dry mass determination is presented in the top right figure (B). The linear relationship between wet and dry rVM is presented in the bottom figure (C). Figure 2.5 41 Changes in dry ventricular mass (A^IO) following a 4-week treatment with sham saline or phenylhydrazine hydrochloride (PHZ) injections, followed by a 4-week recovery at 6°C. Figure 2.6 42 The acute effect of phenylhydrazine hydrochloride treatment on the hematocrit, cardiac output (Q) and hemoglobin concentrations. C H A P T E R 3 Figure 3.1 58 Experimental set-up for radioisotope tracer analysis. Once transported to the lab, fish were kept anesthetized throughout the procedure by a re-circulating, aerated and buffered solution of MS-222. Lead shielding contained all radioactive isotopes, surgical equipment and tissues. Figure 3.2 58 Following stannous priming and dual injection of radioisotopes, fish were prepared for tissue sampling by a ventral incision between the pectoral girdles and retraction of the body wall to expose the pericardium. Figure 3.3 59 Ventricular vascular volume (|il) of the coronary circulation per gram ventricle of sham control (N=5) or PHZ-treated rainbow trout (N=3) calculated with either erythrocyte-tracer technetium pertechnetate (Tc"m) or radio-iodinated bovine serum albumin ( l25I-BSA). Figure 3.4 60 Comparison of individual ventricular vascular volumes (ul) versus individual relative ventricular masses of rainbow trout treated with phenylhydrazine hydrochloride (PHZ) or sham-injected with saline for four weeks, as measured by radioisotope tracer analysis with technetium pertechnetate (Tc"m). V l l C H A P T E R 4 Figure 4.1 82 Picture of an anesthetized rainbow trout following the surgical implantation of a PreSens oxygen microsensor probe into the ductus Cuvier (probe lead shown exiting the top of the operculum) and placement of a Transonic flowprobe on the ventral aorta (larger lead exiting the bottom of the operculum). Figure 4.2 82 The Brett-type swimming tunnel used to assess U c rj t and cardiorespiratory performance. Figure 4.3 83 Hematocrits (Hct) of rainbow trout during treatment and recovery. Data points represent mean triplicate Hcts on three fish from each group, while swim values represent 8 fish per group. Figure 4.4 •. 84 Mean hematocrit (B) and hemoglobin (C) profiles (± SEM) of two groups of rainbow trout treated with PHZ or saline and periodically sampled during an 8-week recovery. Figure 4.5 85 Comparison of the oxygen consumption (Mo2) of the sham (7V=8) and PHZ (N=7) rainbow trout groups during a Ucrj, swim protocol. Figure 4.6 86 Mean cardiac output (Q) ± SEM of sham and PHZ-treated groups (V=8) swum to U c r i t , followed by a 2 h recovery. Figure 4.7 87 Cardiovascular variables of sham control and PHZ-treated rainbow trout swum to U c r i t . All groups have N=S, except Pvo2 (N=6). Figure 4.8 88 Individual cardiac output (ml min"1 kg"1) values were plotted versus its two primary determinants, stroke volume (ml kg"1) and heart rate (beats min"1). Figure 4.9 89 Individual routine and maximal cardiac output (Q) values (ml min"1 g VM"1) are plotted versus the relative ventricular mass of sham or PHZ-treated rainbow trout (N=8). C H A P T E R 5 Figure 5.1 106 Hematocrit and relative spleen mass of diploid (N=5; solid lines) and triploid (N=6, dotted lines) rainbow trout treated with PHZ or saline for two weeks. Figure 5.2 107 Relative ventricular mass (rVM) of control diploid and triploid rainbow trout for the cardiac remodeling experiment (^ =6) and radioisotope tracer analysis experiment (N=7). Figure 5.3 108 Effects of PHZ or saline treatment on the relative ventricular mass (rVM) of diploid and triploid rainbow trout (N=6). viii Figure 5.4 109 Changes in the dry myocardial mass of diploid and triploid rainbow trout (N=6) over a 2-week treatment period with PHZ or saline. Figure 5.5 110 Vascular volume per gram ventricle (ul gV"1) of the coronary circulation of diploid (N=5) and triploid (N=6) rainbow trout (mean ± SEM) using technetium pertechnetate (Tc"m) and radio-iodinated bovine serum albumin ( 1 2 5 I-BSA). Figure 5.6 I l l Individual comparison of the relative ventricular mass versus coronary vascular volume (ul) in ventricles of sham control diploid (N=5) and triploid (N=6) rainbow trout. ix Acknowledgements I will always be grateful to everyone who helped me complete this research, from the patient wisdom of my supervisor, Dr. Anthony Farrell, to my labmates who were beyond helpful and always imaginative. Thanks to all of my wonderful friends and family, and special thanks to Andrew for not only helping navigate this thesis through the turbulent seas of formatting, but for providing me endless motivation, drive and inspiration. Many thanks to Ken Scheer and the Fraser Valley Hatchery for supplying diploid and triploid fish towards this scientific enterprise, and to Jim Pound and the Nuclear Medicine Services of Burnaby Hospital for kindly contributing a plentiful supply of technetium. I dedicate this thesis to my grandmeres, Georgine Simonot and Helena Phaneuf, whose amazing perseverance, courage and grace are hopefully genetic. x Chapter 1: Introduction to the teleost circulatory system The teleostian circulatory system is identical to its mammalian counterpart in that its primary function is the transport of oxygen and nutrients from the external environment to the tissues and the concurrent removal of metabolic wastes and carbon dioxide. Teleosts share a basic cardiovascular design comprised of a pressure-generating cardiac pump and an intricate web of vessels which infiltrate the entirety of the body to circulate blood from the olfactory bulbs to the distal tips of fins. The model teleost species in this thesis is the rainbow trout, Oncorhynchus mykiss Walbaum, a member of the environmentally and culinarily-celebrated salmonid family, originally native to the Pacific coast of North America. While historically delimited by the Rocky Mountains to the eastern Pacific Ocean, rainbow trout are now a worldly fish with a global distribution ranging from aquaculture production to introduced endogeny. The cardiovascular physiology of rainbow trout makes them a particularly interesting subject of study. Rainbow trout hearts sit at an evolutionary crux of structural development, notably in the development of the coronary circulation on which we mammals critically depend. While primitive fish such as the hagfish have no coronary circulation to the myocardium and rely entirely on venous return through the lumen, mammalian myocardium relies nearly exclusively on the coronary artery for oxygenation. Rainbow trout hearts are therefore an evolutionary intermediate with a partial reliance on the coronary artery. An additional difference between teleost and mammalian hearts, and the main topic of research within this thesis, is the routine ability of some teleost hearts to quickly and dramatically alter ventricular mass and composition (termed 'cardiac remodeling') in response to seasonal and reproductive demands for increased cardiac output. The teleost heart pumps deoxygenated blood from the venous systemic circulation in a unidirectional circuit towards the gills. The muscular, thin-walled atrium collects deoxygenated venous blood and pumps it into the pyramidal ventricle. In rainbow trout, this ventricle is composed of two muscle types - the inner spongy myocardium comprises the majority of the ventricle, while the outer compact myocardium generally accounts for a third of the ventricle. The blood is pumped by the ventricle through the last 1 cardiac chamber, the bulbous arteriosus, into the gill vasculature for oxygenation before completing the systemic circuit through the body and returning to the heart. The great majority of teleost ventricles (80%; Santer 1985) lack compact myocardium altogether and are comprised solely of spongy myocardium, classified as 'Type I' by Tota (1983). Type I hearts are common in species characterized by sluggish activity and sedentary habits such as benthic species. Spongy myocardium, also known as endocardium, is composed of loosely-arranged myocytes which form sponge-like, trabecular projections into the lumen of the ventricle. These trabeculae increase the surface area of myocardium available to oxygenation by the blood. Spongy myocytes form a complex lumen with many niches and channels, yielding a formidable avenue of myocardial cul-de-sacs (Tota et al. 1983). Rainbow trout are amongst the minority of teleosts that have a ventricle composed of both compact and spongy myocardium which are separated by a thin fibrous membrane (Poupa & Carlsten 1973). The compact myocardium, also termed epicardium, is composed of overlapping sheets of parallel myocytes which wrap around the spongy myocardium on the outside of the ventricle. The amount of compact myocardium varies markedly between species, from 5% in ratfish to 76% in tuna (Santer & Greer-Walker 1980), and also varies ontogenically within individuals. Salmonids experience a life-long variation of compact composition, from an irregular and sparse presence in juveniles to a significantly elevated composition which plateaus in older fishes, though the increase is not isometric and is more pronounced at certain life stages (Poupa et al. 1974). One-year old rainbow trout generally have 30% compact myocardium, while its composition in four-year old fish increases to 45% (Farrell et al. 1988). Perhaps the most significant difference between compact and spongy myocardium is in their differential source of oxygen and nutrients. Spongy myocardium is exclusively dependent on systemic venous blood returning through the ventricular lumen where oxygen must diffuse from the venous blood to the entire spongy musculature, from the centre of its lumen to its outer periphery. As such, the thickness of trabeculae in spongy myocardium must balance between maximizing tension development of the myocardium and maintaining an appropriate diffusion distance (Davie & Farrell 1991). As the ventricle is 2 the last tissue to receive systemic blood prior to re-oxygenation at the gills, the oxygen supply will already have been depleted by the remainder of the body. Rainbow trout swum to exhaustion can experience significant drops in venous oxygen tension (Pvo2) values from routine values, from 44 -21 torr (Kiceniuk & Jones 1977) or from 3 6 - 7 torr, alongside progressive hypoxia (Steffensen & Farrell 1998). While Jones (1986) has suggested that 10 torr may be the absolute limit providing myocytes adequate oxygenation, a review of literature values by Davie & Farrell (1991) found the Pvo 2 range for exhaustive exercise to vary between 6 and 16 torr. As the myocardial oxygen demand is in direct proportion to cardiac power output (Steffensen & Farrell 1998), the extreme range of available oxygen creates a precarious situation for spongy myocytes when the fish is swimming maximally and increasing both systemic oxygen extraction and myocardial oxygen demands. Conversely, the compact myocardium presumably does not suffer comparable oxygen constraints due to its exclusive supply of oxygen-rich arterial blood via the coronary artery. The coronary circulation While fishes (elasmobranches in particular) were the first vertebrates to evolve a coronary circulation (Franklin & Axelsson 1994), its origin and distribution are so varied that it has likely evolved more than once during evolution (Tota et al. 1983). In rainbow trout, which represents an intermediate model of coronary dependence, the coronary circulation is limited exclusively to the compact myocardium (Type II; Tota 1983) which then drains into the atrium near the atrioventricular junction (Davie & Farrell 1991). The coronary circulation in rainbow trout originates from the cranial hypobranchial arteries and progresses caudally along the ventral aorta and the bulbous arteriosus before branching off and embedding into the compact myocardium. The coronary artery is composed of a medial layer of vascular smooth muscle surrounded by an outer parenchyma sheet and an inner lamina composed of a single layer of epithelial cells (Farrell 2002). The vascular smooth muscle is much thinner in fish than in mammals, as the passage of blood through the gills prior to infiltrating the ventricle generates but a fraction of the mammalian central arterial pressure (Farrell 2002). The blood flow through the coronary circulation is continuous and phasic throughout the cardiac cycle during routine conditions, although 75% of coronary flow occurs during 3 diastole (Axelsson & Farrell 1993, Gamperl et al. 1995). While resting coronary blood flow is only ~ 1 % of cardiac output, the coronary circulation can accommodate considerable increases of flow. To accommodate the proportional increase in myocardial oxygen demands which accompany increases in cardiac power output (Steffensen & Farrell 1998), the factorial scope for increase in coronary blood flow during exercise is two-fold (Axelsson & Farrell 1993, Gamperl et al. 1994). The importance of the coronary circulation in maintaining cardiac performance remains somewhat speculative. Experiments suggest that although the coronary circulation in Type II hearts is not mandatory at routine cardiac work levels, it is required for maximal cardiac performance, particularly in hypoxic conditions when both Pao 2 and Pvo 2 are decreased (Davie et al. 1992, Davie & Farrell 1991, Farrell 1987, Farrell et al. 1989). When the coronary artery has been experimentally ablated in vivo, the fish do not die but rather experience a reduced maximal prolonged swimming speed (Farrell et al. 1990, Farrell & Steffensen 1987), cardiac arrhythmias (Steffensen & Farrell 1998) and a significantly reduced ventral aortic blood pressure (Farrell & Steffensen 1987). In contrast, ablation of its coronary supply to the Type IV heart of the skipjack tuna, which suggests an obligatory dependence on the coronary artery similar to mammals, results in the complete collapse of routine cardiac performance (Farrell et al. 1991). A recent study suggests that the compromised cardiac performance associated with coronary ablation in rainbow trout may be due to a significant reduction of the cardiac contractility of the myocardium, which reduces cardiac output and power output of the heart (Agnisola et al. 2003). The requirement of an additional coronary supply to maintain maximal cardiac performance thus suggests that there may be a constraint on oxygen delivery to the outer myocardium during exercise and hypoxic conditions. As oxygen measurements of venous blood indicate that the venous oxygen content is more than sufficient to satisfy myocardial oxygen needs even at maximal exercise (Farrell 2002), potential oxygen constraints to the ventricle associated with exercise and hypoxia are likely diffusion limited, rather than a perfusion limitation. I propose that such oxygen-diffusion limitations to the rainbow trout myocardium may help explain patterns of cardiac plasticity observed in rainbow trout heart. 4 Cardiac plasticity in rainbow trout The rainbow trout heart is a plastic organ that can alter its mass, resulting in an increase in the ventricle mass to body mass ratio (termed relative ventricular mass or rVM). Increases in r V M are often referred to as ventricular hypertrophy or cardiomegaly, while additional changes of cardiac geometry alongside mass alteration, are referred to as cardiac remodeling (Smits et al. 1991). As a rule, ventricular hypertrophy leads to augmentation of ventricular working capacity, although the term 'hypertrophy' itself can be quite misleading as it describes changes at the organ level, but does not describe processes at the cellular or biochemical structures (Jacob et al. 1991). Biophysically, hypertrophy of the ventricle functions to increase the thickness of the myocardial wall in order to counterbalance increased wall stress associated with larger cardiac work (in accordance with LaPlace's law). Increased r V M is therefore hypothesized to ameliorate cardiovascular functioning by maintaining elevated levels of stroke volume, cardiac output and pressure development of the heart at high cardiac work demands. The ability of the heart to adjust its mechanical performance to changing volumes and pressure loads in order to maintain cardiovascular homeostasis makes cardiac plasticity "of prime physiological importance" (Tota 1983). While endothermic mammals need only maintain physiological and cardiovascular functioning to a highly restricted isothermic temperature, ectothermic rainbow trout are exposed to a comparably enormous range of temperatures and must adjust accordingly. Cardiac plasticity likely reflects a necessary adaptation such that trout can maintain physiological function and myocardial contractility through vastly fluctuating temperatures, extreme environmental conditions and life history phases. Ventricular hypertrophy in rainbow trout is generally thought to improve cardiovascular functioning by increasing the myocardial, metabolic and biochemical components necessary to maintain a functional ventricle at elevated work loads. Hypertrophy occurs by expansion of the contractile protein in muscle (Bailey et al. 1997), and given that myocardial contractile proteins such as actin and myosin represent a considerable portion of the total protein complement in cardiac muscle, it is likely that increases in weight-specific tissue protein accompanying ventricular hypertrophy specifically promote greater contractile function and performance of the larger myocardium (Rodnick & Williams 1999). There is also evidence 5 that when the biosynthetic machinery of the myocyte is activated, a proportionate growth of cell organelles maintains cellular symmetry (Tota 1993). For example, the content of mitochondria and myofibrils remains constant during hypertrophy, reflecting additional biogenesis of these components (Clark et al. 2004), such that the enlarged ventricle maintains contractile performance comparable to normal-sized hearts (Franklin & Davie 1992). Ventricular hypertrophy can be hemodynamically induced by volume- or pressure-loading of the myocardium due either to increased end diastolic volume or increased resistance to ejected blood volume, respectively (Clark & Rodnick 1999). The type of loading exerted on the myocardium results in differential hypertrophic responses. In mammals, volume overload is generally associated with increases in cell length of the myocytes (eccentric hypertrophy) and enlargement of both the ventricular mass and lumen size (Tota 1993). Pressure overload, conversely, results in concentric hypertrophy and an increase in the ventricle at the expense of the chamber volume (Tota 1993). While cardiac remodeling is a normal physiological process, teleosts appear to have maintained a greater degree of functional plasticity than mammals. In humans, cardiac hypertrophy is a major independent risk factor for cardiovascular morbidity and mortality, including fatal arrhythmias, myocardial ischemia and heart failure (Ruzicka & Leenen 1995). The progression of ventricular hypertrophy in mammals has been described in two phases: hemodynamic/acute hypertrophy and architectural/chronic hypertrophy (Katz 2002). These phases are also referred to as physiologically 'adaptive' and 'maladaptive'. Acute hypertrophy is caused by an increase in venous return leading to ventricular dilatation and volume-loading of the myocardium. The increased diastolic volume leads to an increased ability of the heart to do work, via Starling's law of the heart, which results from abnormal proliferative compensation to the increased wall stress and reduced mechanical efficacy caused by increased venous return. Chronic hypertrophy in mammals generally results in a plethora of detrimental cardiovascular effects that can reduce or even impair cardiac performance. Besides detrimental mechanical function of the myocardium, limited angiogenesis of the coronary circulation is also a likely contributing factor towards impaired cardiac function. As coronary growth during hypertrophy does not parallel myocardial growth (Tomanek 1999), 6 there can occur a mismatch between the capillaries and cardiomyocytes, resulting in increased diffusion distance and impaired nutrient and oxygen supply, which can be further aggravated during increased workload (Friehs et al. 2004). In contrast, cardiac hypertrophy is quite a routine and beneficial adaptation in salmonids and can be induced by a variety of stimuli, as described below. Cold-Temperature Hypertrophy - Cold temperature is a known inducer of hypertrophy in many tissues including the liver, heart and red muscle of ectotherms (Kent & Prosser 1985). Rainbow trout undergo significant hypertrophy of the ventricle in response to cold water temperatures, with increases ranging from 20 - 40% (Farrell et al. 1988, Graham & Farrell 1989). Due to the depressive effect of cold temperature on enzymatic processes, there is a reduced contractility and mechanical power output of aerobic muscle and a significant reduction of the scope for cardiac output and cardiac work (Taylor et al. 1996). The heart must also work harder in cold conditions due to increased viscosity of the blood, which produces an increase in afterload pressure and potentially limits perfusion at the tissues due to a decrease in erythrocyte deformability (Egginton & Cordiner 1997). Such temperature-related reduction in myocardial performance is therefore compensated by the increased cardiac mass induced by the hypertrophic cardiac response. Spawning-Induced Hypertrophy - Ventricular hypertrophy can also be induced by reproductive maturation, albeit in a gender-specific manner, whereby male salmonids show significant 2- to 3-fold increases in r V M during spawning (Franklin & Davie 1992, Clark & Rodnick 1998, Clark et al. 2004), while females do not. The migratory trek undertaken by andronomous salmonids, combined with the continuous activity of maintaining territory during spawning, are extreme stresses on the cardiovascular system and have been a proposed promoter of hypertrophy (Davie & Thorarensen 1997), possibly due to increased hemodynamic overload (Clark & Rodnick 1999). However, females would likewise also be expected to undergo significant hypertrophy were the hypertrophic signal solely related to migration and spawning behaviours. Rather, spawning hypertrophy has been demonstrated to be hormonally-stimulated by naturally elevated levels of androgens in the plasma of maturing trout, as increases in r V M have been experimentally induced 7 by administering testosterone to both immature males and females (Davie & Thorarensen 1997). Androgens regulate protein synthesis by binding cytosolic or nuclear protein receptors for steroids which modulate transcription (Fitzpatrick et al. 1994), although the exact mechanistic link between androgens and r V M has not been determined (Clark & Rodnick 1998). Clark & Rodnick (1998) report that the significant increase in r V M observed during sexual maturation is due to myocyte hypertrophy, with a disproportionate increase to the arterially-oxygenated compact myocardium of the ventricle (also demonstrated by Graham & Farrell 1992). Anemia-Induced Hypertrophy - While induction of experimental hypertrophy in mammals has usually not been successful in producing increases comparable to natural conditions of ventricular hypertrophy, chronic anemia has been the only trigger able to increase heart mass up to twice the normal values in mammals (Poupa et al. 1974, Ruzicka & Leenen 1995). In mammals, anemia therefore represents an easily reproducible model of volume overload-induced cardiac hypertrophy with virtually no mortality related to the experimental procedure (Ruzicka & Leenen 1995). Anemia has similarly been shown to induce 30% increases in r V M in rainbow trout with no reported mortalities (Davie & Thorarensen 1997, McClelland et al. 2005) and therefore appears to represent an equally effective model of cardiac remodeling for rainbow trout. In mammals, anemia triggers an immediate and direct elevation of cardiac output, the most important adaptive response to anemia (Hebert et al. 2004). Elevation of cardiac output appears to be the principle compensatory adjustment to reductions in the oxygen-carrying capacity of fishes as well (Cameron & Davis 1970). The resultant cardiac remodeling triggered by anemia is potentially stimulated by two mechanisms: firstly, anemia will result in a volume-loaded effect on the heart, increasing the end-diastolic volume and intra-lumen pressure within the ventricle. The resulting increase in ventricular wall stress and compensatory increase in wall thickness is described by La Place's principle. Secondly, anemia can decrease the arterial and venous oxygen content of blood, thereby potentially limiting oxygen availability to the spongy myocardium which relies exclusively on venous return for oxygen. While the arterial oxygen content will also be reduced, the arterial supply to the compact myocardium is not subject to further 8 systemic depletion by other tissues, as is the spongy. This may create an increased dependence on the arterial supply to the compact myocardium for maintenance of proper myocardial functioning and may preferentially promote its hypertrophy (a hypothesis previously discussed in the literature, i.e. Thorarensen & Davie 1997). Increasing the proportion of adequately oxygenated myocardium would thereby ensure a more powerful cardiac workload and stroke volume. However, like mammals (Ruzicka & Leenen 1995), there are no data for rainbow trout regarding the effects of anemia on cardiac remodeling, the threshold necessary to induce cardiac changes or how recovery from anemia affects anemia-induced cardiac alterations. As anemia has proven to be a highly effective and reproducible inducer of ventricular hypertrophy in both mammals and fish, with no known side-effects, and as it provides an excellent model of oxygen-dependent cardiac remodeling in rainbow trout, it was chosen as the experimental protocol to induce and study cardiac remodeling in this thesis. Phenylhydrazine as an inducer of anemia Phenylhydrazine hydrochloride (PHZ) is a drug with an extensive history in the study of erythropoiesis in a broad range of fishes, anurans and mammals because it is a highly effective hemolytic agent in the induction of experimental anemia. PHZ's mode of action, which involves a complex myriad of chemical reactions, has been most astutely described as "oxidative denaturation" or "oxidative hemolysis" (Itano et al. 1976). Its chemical activity appears to be two-fold in nature (as reported by Vilsen & Nielsen 1984): the primary reaction is the formation of denatured, oxidized hemoglobin (Heinz bodies) by the reaction of PHZ metabolites and erythrocytes. The secondary reaction is the destabilization and disruption of the mechanical integrity of the erythrocyte cell membrane by the formation of disulphide bridges between membrane proteins and precipitated hemoglobin. Both reactions lead to cellular alterations that may lead to direct cellular hemolysis or selective removal of damaged erythrocytes from the circulation by the reticulo-endothelial system. In teleosts, the primary organ of the reticulo-endothelial system is the spleen (Dornfest et al. 1986). 9 PHZ has been used on a multiplicity of species and studies, both acute and chronic, with no mention of side effects in fish species other than those directly related to the induced anemia itself (Smith et al. 1971; Chudzik & Houston 1983; Byrne & Houston 1988; Jacob et al. 2002; McClelland et al. 2005). PHZ is also particularly attractive as its effects are transient and there are no long-term impairments of erythropoietic capacity (Byrne & Houston 1988). Following PHZ treatment, three weeks has been found adequate for complete restoration of pre-treatment hemoglobin levels in goldfish (Byrne & Houston 1988) and four weeks for restoration of hematocrit in rainbow trout (McClelland et al. 2005). Despite the initial hemolytic action of PHZ treatment, the primary effect of PHZ reported in a study of Xenopus was the stimulation of erythrocyte proliferation (Widmer et al. 1983). PHZ has been found to stimulate the immune system and induce both erythropoiesis and lymphopoiesis, likely to compensate for the initial cell loss of anemia (Dornfest et al. 1990). Although the use of chemical interventions as an experimental procedure inherently introduces concern regarding potential side-effects and physiological complications, PHZ is generally regarded as free of any major side effects and it is a highly preferable method to others previously used to induce anemia, especially in regards to maintaining the health and well-being of the study animals, including hypophectomy (Slicher & Pickford 1968) and starvation (McLeodetal. 1978). The hypertrophy versus hyperplasia debate While no attempts were made to differentiate between these processes in the following thesis research, it is important to address the pertinent question of whether observed increases in ventricular mass are in fact due to hypertrophic or hyperplastic responses, as rainbow trout ventricles are suggested to undergo both processes (Farrell et al. 1988). While the term 'hypertrophy' is commonly used in scientific literature and within this thesis to refer to generic increases of the r V M , it specifically refers to increased tissue mass due to increased cell size, whereas hyperplastic increases which result from cell division and increased cell number. 10 The differentiation between hypertrophic and hyperplastic responses is nevertheless important due to the implications on oxygen diffusion to the cardiomyocytes. The ability to regenerate cardiac and skeletal muscle cells, an evolutionary adaptation long-ago relinquished by adult mammalian myocytes, has been proposed to be a normal component of cardiac growth in rainbow trout. Clark & Rodnick (1998) recently corroborated results of Farrell et al. (1988), who found that to account for the observed increases in r V M in normal rainbow trout populations, cardiac growth must occur through a combination of cardiomyocyte hyperplasia and hypertrophy. They further proposed that hypertrophy is responsible for the increase in r V M observed during gonadal maturation, but that hyperplasia and the generation of new myocytes is responsible for cardiac growth between reproductive cycles, potentially to maintain minimal capillary to fibre ratios in compact myocardium. Some fish also have an impressive ability to undergo inducible hyperplasia unrelated to the normal growth cycle, best exemplified in a stunning experiment whereby the distal tip of zebrafish ventricles were amputated by 20% and were found to fully regenerate within two months (Poss et al. 2002). While considering prior scientific studies of cardiac remodeling, it is also important to recognize that one of the principle methods used to differentiate between hypertrophic and hyperplastic growth in ventricles is also one of the most problematic: the quantification of D N A and its ratio to R N A or protein content. While myocytes account for the bulk of ventricular mass, they are not the most abundant cell in the heart (McLelland et al. 2004). Most methodologies do not isolate myocytes from these other various cell types or components during the isolation of genetic or protein material. It is therefore unclear whether reported changes in D N A , R N A or protein content are actually due to an increase in specific cardiomyocyte DNA, as cardiac growth can be induced in a variety of myocardial components including myocytes, sarcoplasmic reticulum, connective or vascular tissue (McClelland et al. 2005, .Shuling et al. 1990). For example, mitochondria - which normally account for over a third of total cardiac protein (West & Driedzic 1999) -have also been shown to undergo hypertrophy-induced biogenesis (Clark et al. 2004). The most reliable studies are therefore those that have undertaken morphometric study of the cardiomyocytes. 11 By measuring cardiomyocyte dimensions directly (length, cross-sectional area and volume), Clark & Rodnick (1998) determined that ventricular hypertrophy associated with sexual maturation is due to myocyte hypertrophy. Comparatively, utilizing a DNA/protein ratio determination of the whole ventricle, Bailey et al. (1997) concluded that ventricular hypertrophy in sexually-mature male rainbow trout is hyperplastic due to the increased ratio. However, as they did not isolate cardiomyocytes specifically, their conclusion remains dubious. Determination of the specific mechanisms involved in cold and anemia-induced increases in r V M currently are consistent with hypertrophy (Farrell et al. 1988) and hyperplasia (McClelland et al. 2005) respectively, although these have not been measured morphometrically and so therefore remain speculative. Triploid rainbow trout: A 'natural' model of cardiac hypertrophy Triploidy is a tool used in aquaculture to produce sterile fishes to stock waterways without reproductive dilution of the wild gene pool. The process to induce triploidy involves heat or pressure-shocking embryos to disrupt the meiotic process and create fish that possess three sets of chromosomes per cell rather than the typical two. Triploid fish grow into physically and physiologically-comparable specimens, except for a 50% increase in cell size to accommodate their 50% increased chromosomal content, which renders them reproductively sterile (Benfey 1999). The larger than average red blood cells (Benfey 1999) and myocytes of triploid fishes (Mercier et al. 2002) provides a 'natural' model of hypertrophy and of potential limitations to oxygen delivery to compact cardiac tissue. As the diffusion of oxygen is hypothesized to be an important factor in cardiac remodeling of diploid rainbow trout, triploids will be utilized as a natural control and comparison to the hypertrophic response of normal, diploid trout. Objectives The goals of this thesis are three-fold: 1) To explore the structural and temporal aspects of cardiac remodeling in rainbow trout (both diploid and triploid) by inducing and quantifying the onset and degree of ventricular hypertrophy in response to anemia; secondarily, to measure whether changes are transient. Further, to elucidate the link between anemia and cardiovascular compensation by measuring cardiac output 12 prior to and following injection of PHZ and determine at what hematocrit cardiac adaptation, and thus cardiac remodeling, becomes necessary. 2) To measure the ventricular vascular volume and determine how the capillarity of the coronary circulation responds to increases in r V M in diploid and triploid fish, using triploid fish as a comparative model with 50% enlarged cardiomyocytes. Answers were sought for the following questions: Is the coronary vascular volume different in triploids? If triploid cardiomyocytes are inherently in a compensatory state, is angiogenic remodeling still possible in the coronary circulation? 3) To study the in vivo cardiovascular consequences of a hypertrophied ventricle and determine whether the associated cardiac remodeling will improve the swimming performance of rainbow trout. The effects of temperature, physical activity and the handling stress of the induction procedure will also be examined in separate control experiments. Scientific Hypotheses 1) It is hypothesized that induction of cardiac remodeling by anemia will result in a larger ventricle and disproportionate increase in compact myocardial composition, to benefit cardiac function by an increased supply of arterial circulation. Cardiac remodeling in triploid fish is hypothesized to be limited due to their inherently 50% larger oxygen diffusion distance. 2) It is further hypothesized that disproportionate hypertrophy of the compact myocardium will necessitate a proportional increase of coronary blood flow to maintain adequate oxygenation of the cardiomyocytes. A concurrent angiogenesis of the coronary circulation would be required to increase the ventricular vascular volume and maintain the enhanced oxygen requirements of the increased myocardial tissue mass. 3) It is also hypothesized that anemia-induced cardiac hypertrophy will improve cardiac functioning and swimming performance once fish have returned to a normocythemic state and that if the compact myocardium is disproportionately larger, fish would exhaust at a lower venous oxygen partial pressure than normal due to a reduced reliance on the venous return to the spongy myocardium. 13 Literature Cited Adler C P . , Friedburg, H., Herget G.W., Neuburger M . & Schwalb H. 1996. Variability of cardiomyocyte D N A content, ploidy level and nuclear number in mammalian hearts. Virchows. Arch. 429: 159-64. Agnisola C , Petersen L. & 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: 4003-10. Axelsson, M. & Farrell A.P. 1993. Coronary blood flow in the coho salmon. Amer. J. Physiol. 264: R963-71. Bailey J.R., West J.L. & Driedzic W.R. 1997. Heart growth associated with sexual maturity in male rainbow trout (Oncorhynchus mykiss) is hyperplastic. Comp. Biochem. Physiol. 118B: 607-11. Benfey T.J. 1999. The physiology and behavior of triploid fishes. Rev. Fish. Sci. 7(1): 39-67. Byrne A.P. & Houston A .H . 1988. Use of phenylhydrazine in the detection of responsive changes in hemoglobin isomorph abundances. Can. J. Zool. 66: 758-62. Cameron J.N. & Davis J.C. 1970. Gas exchange in rainbow trout (Salmo gairdneri) with varying blood oxygen capacity. J. Fish Res. Bd Can. 27: 1068-85. Chudzik J. & Houston A .H . 1983. Temperature and erythropoiesis in goldfish. Can. J. Zool. 61: 1322-5. Clark J.J., Clark R.J., McMinn J.T. & Rodnick K.J. 2004. Microvascular and biochemical compensation during ventricular hypertrophy in male rainbow trout. Comp. Biochem. Physiol. 139B: 695-703. Clark R J . & Rodnick K.J. 1999. Pressure and volume overloads are associated with ventricular hypertrophy in male rainbow trout. Am. J. Physiol. 277: R938-46. Clark R.J. & Rodnick K.J. 1998. Morphometric and biochemical characteristics of ventricular hypertrophy in male rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 201: 1541-52. Davie P.S. & Farrell A.P. 1991. The coronary and luminal circulations of the myocardium of fishes. Can. J. Zool. 69: 1993-2001. Davie P.S. Farrell A.P. & Franklin C E . 1992. Cardiac performance of an isolated eel heart - effects of hypoxia and responses to coronary artery perfusion. J. Exp. Biol. 262(2): 113-21. Davie P.S. & Thorarensen H. 1997. Heart growth in rainbow trout in response to exogenous testosterone and 17-a methyltestosterone. Comp. Biochem. Physiol. 117A: 227-30. Dornfest B.S., Bush M.E., Lapin D.M., Adu S., Fulop A. & Naughton B.A. 1990. Phenylhydrazine is a mitogen and activator of lymphoid cells. Ann. Clin. Lab. Sci. 20(5): 353-70. Dornfest B.S., Lapin D.M., Naughton B.A. Adu S., Korn L. & Gordon A.S. 1986. Phenylhydrazine-induced leukocytosis in the rat. J. Leuk. Biol. 39(1): 37-48. Egginton S. & Cordiner S. 2002. Cold-induced angiogenesis in seasonally acclimatized rainbow trout (Oncorhynchus mykiss). J. Exp Biol. 200(16): 2263-8. Farrell A.P. 2002. Cardiorespiratory performance in salmonids during exercise at high temperature: insights into cardiovascular design limitations in fishes. Comp. Biochem. Physiol. 132A: 797-810. 14 Farrell, A.P. 1987. Coronary flow in a perfused rainbow trout heart. J. Exp. Biol. 129: 107-23. Farrell A.P., Hammons A .M . , Graham M.S. & Tibbits G.F. 1988. Cardiac growth in rainbow trout, Salmo gairdneri. Can. J. Zool. 66: 2368-73. Farrell, A.P., Johansen J.A., Steffensen J.F., Moyes C D . , West T.G. & Suarez R.K. 1990. Effects of exercise-training and coronary ablation on swimming performance, heart size and cardiac enzymes in rainbow trout, Oncorhynchus mykiss. Can. J. Zool. 68: 1174-9. Farrell, A.P., Small S. & Graham M.S. 1989. Effect of heart rate and hypoxia on the performance of a perfused trout heart. Can. J. Zool. 67: 274-80. Farrell, A.P. & Steffensen J.F. 1987a.Coronary ligation reduces maximum sustained swimming speed in chinook salmon {Oncorhynchus tshawytscha). Comp. Biochem. Physiol. 87A: 35-7. Fitzpatrick M.S., Gale W.L. & Schreck C B . 1994. Binding characteristics of an androgen receptor in the ovaries of coho salmon, Oncorhynchus kisutch. Gen. Comp. Endocrinol. 95(3): 399-408. Franklin C E . & Axelsson M. 1994. Coronary hemodynamics in elasmobranchs and teleosts. Cardioscience 5(3): 155-61. Franklin C E . & Davie P.S. 1992. Sexual maturity can double heart mass and cardiac power output in male rainbow trout. J. Exp. Biol. 171: 139-48. Gamperl A.K. , Axelsson M. & Farrell A.P. 1995. Effects of swimming and environmental hypoxia on coronary blood flow in rainbow trout. Am. J. Physiol. 38: R1258-66. Gamperl A. , Pinder A. & Boutilier R. 1994. Effect of coronary ablation and adrenergic stimulation on in vivo cardiac performance in trout {Oncorhynchus mykiss). J. Exp. Biol. 186(1): 127-43. Graham M.S. & Farrell A.P. 1992. Environmental influences on cardiovascular variables in rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish. Biol. 41: 851-8. 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. Hebert P . C , Van der Linden P., Biro G. & Hu L.Q. 2004. Physiologic aspects of anemia. Crit Care Clin. 20(2): 187-212. Itano H.A., Hosokawa K. & Hirota K. 1976. Induction of haemolytic anaemia by substituted phenylhydrazines. Br. J. Haematol. 32(1): 99-104. Jacob E., Drexel M. , Schwerte T. & Pelster B. 2002. Influence of hypoxia and of hypoxemia on the development of cardiac activity in zebrafish larvae. Am J. Physiol. Regul. Integr. Comp. Physiol. 283: R911-7. Jones D.P. 1986. Intracellular diffusion gradients of oxygen and ATP. Am. J. Physiol. 250: C663-75. Katz A . M . 2002. Maladaptive growth in the failing heart: the cardiomyopathy of overload. Cardiovasc. Drugs Ther. 16(3): 245-9. Kent, J.D. & Prosser C L . 1985. Protein hypertrophy in liver and heart following cold acclimatization and acclimation in channel catfish. Am. Zool. 25: 134. Kiceniuk J.W. & Jones D.R. 1977. The oxygen transport system in trout {Salmo gairdneri) during sustained exercise. J. Exp. Biol. 69: 247-60. 15 McClelland G.B., Dalziel A .C. , Fragoso N.M. & Moyes C D . 2005. Muscle remodeling in relation to blood supply: implications for seasonal changes in mitochondrial enzymes. J. Exp. Biol. 208: 515-22. McLeod T.V., Sigel M .M. & Yunis A.A. 1978. Regulation of erythropoiesis in Florida gar, Lepiosteus platyrhincus. Comp. Biochem. Physiol. 60A: 145-50. Poss K.D., Wilson L.G. & Keating M.T. 2002. Heart regeneration in zebrafish. Science. 298: 2188-90. Poupa O. & Carlsten A. 1973. Experimental cardiomyopathies in poikilotherms. Recent Adv. Stud. Cardiac Struct. Metab. 2: 321-51. Poupa O., Gesser H., Jonsson S. & Sullivan L. 1974. Coronary-supplied compact shell of ventricular myocardium in salmonids: growth and enzyme pattern. Comp. Biochem. Physiol. 48A: 85-95. Rodnick K.J. & Williams S.R. 1999. Effects of body size on biochemical characteristics of trabecular cardiac muscle and plasma of rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 122A: 407-13. Ruzicka M. & Leenen F.H.H. 1995. Renin-angiotensin system and volume overload-induced cardiac remodeling. In: Dhalla N.S., Beamish R.E., Takeda N. & Nagano M. (Eds). The Failing Heart. Lippincott-Raven Publishers, Philadephia. SanterR.M. 1985. Morphology and innervation of the fish heart. Adv. Anat. Embryol. Cell Biol.%9: 1-99. Santer R.M. & Greer-Walker M.G. 1980. Morphological studies on the ventricle of teleost and elasmobranch hearts. J. Zool. Lond. 190: 259-72. Slicher A . M . & Pickford G.E. 1968. Temperature-controlled stimulation of hemopoeisis in a hypophysectomized cyprinodont fish, Fundulus heteroclitus. Physiol. Zool. 41: 293-7. Smith C.E., McLain L.R. & Zaugg W.S. 1971. Phenylhydrazine-induced anemia in Chinook salmon. Toxicol. App. Pharm. 20: 73-81. Smits J.F., Cleutjens J.P., van Krimpen C , Schoemaker R.G. & Daemen M.J. 1991. Cardiac remodeling in hypertension and following myocardial infarction: effects of arteriolar vasodilators. Basic Res. Cardiol. 86: 133-9. Steffensen J.F. & 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. 199A: 585-92. Taylor S.E., Egginton S. & Taylor E.W. 1996. Seasonal temperature acclimatization of rainbow trout: cardiovascular and morphometric influences on maximal sustainable exercise level. J. Exp. Biol. 199: 835-45. Tota B. 1983. Vascular and metabolic zonation in the ventricular myocardium of mammals and fishes. Comp. Biochem. Physiol. 76A: 423-7. Tota B. 1993. Plasticity of the heart and hemodynamic loads: basic and comparative aspects. In: Bicudo J.P.W. (Ed.) The vertebrate gas transport cascade: adaptations to environment and mode of life. CRC Press, Inc., Boca Raton. Tota B., Cimini V., Salvatore G. & 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. 16 Vilsen B. & Nielsen H. 1984. Reaction of phenylhydrazine with erythrocytes: cross-linking of spectrin by disulfide exchange with oxidized hemoglobin. Biochem. Pharm. 33(17): 2739-48. West J.L. & Driedzic W.R. 1999. Mitochondrial protein synthesis in rainbow trout (Oncorhynchus mykiss) heart is enhanced in sexually mature males but impaired by low temperature. J Exp Biol. 202: 2359-69. Widmer H.J., Hosbach H.A. & Weber R. 1983. Globin gene expression in Xenopus laevis: anemia induces precocious globin transition and appearance of adult erythroblasts during metamophosis. Deyel. Biol. 99: 50-60. 17 Chapter 2: Remodeling of the myocardium in rainbow trout {Oncorhynchus mykiss Walbaum) Introduction Rainbow trout routinely alter ventricular mass and composition to compensate for changes in cardiac work demand associated with environmental or physiological cues. The degree of plasticity exhibited in rainbow trout cardiac remodeling apparently depends strongly on the stimulatory signal and its duration. Exercise-induced cardiac remodeling increases the relative ventricular mass (rVM) based on the intensity and duration of training, ranging from a 12% increase following 6 months of low-intensity training conditions up to 46% following 26 days of high intensity training (Hochachka 1961, Greer-Walker & Emerson 1978). Cold acclimation can increase r V M between 20 - 40%, with the greatest increases observed at the lowest temperatures (Farrell et al. 1988, Graham & Farrell 1989). Sexual maturation appears to impart the strongest remodeling signal with r V M able to double in sexually-mature males (Franklin & Davie 1992). Acute anemia has also been shown to increase r V M by 30% (McClelland et al. 2005). Besides alteration of the cardiac mass, the myocardial composition of rainbow trout also demonstrates considerable plasticity. While hypertrophy associated with sexual maturation preferentially increases the percentage of compact myocardium (Tota et al. 1983, Graham & Farrell 1992, Clark & Rodnick 1998), hypertrophy stimulated by cold acclimation results in preferential growth of the spongy myocardium (Farrell et al. 1988). Such differential changes to the myocardial layers have been attributed to compensatory mechanisms necessary to adapt to either temperature effects or the behavioural demands associated with spawning, but another factor that may influence patterns of cardiac remodeling is oxygen availability. The myocardium becomes prone to hypoxic damage as the capillary to myocyte fibre distance increases in hypertrophied conditions (Canby & Tomanek 1990). As coronary angiogenesis is quite limited in adult mammalian hearts (Tomanek 1999), the hypertrophic response can often be maladaptive and pathological. Conversely, rainbow trout appear to undergo angiogenic compensation when cardiac mass increases in response to certain stimuli, including cold-acclimation and sexual maturation (Clark & Rodnick 1999, 18 Egginton 2002, Clarke et al. 2004). Thus, in contrast to many instances of cardiac remodeling in mammals, hypertrophy of the myocardium in rainbow trout appears to be adaptive, with the metabolic requirements of the increased myocardial mass being met by angiogeriesis of the coronary circulation. Compact and spongy myocardial layers receive anatomically and qualitatively differing oxygen supplies. The outer compact myocardium is the exclusive recipient of richly-oxygenated arterial blood via the coronary artery, while spongy myocardium relies on depleted venous return through the lumen of the ventricle. Compared to the compact myocardium, the spongy myocardium is more susceptible to fluctuations in oxygen supply during exercise or physiological stress when the venous oxygen supply becomes increasingly depleted. It is therefore possible that cardiac remodeling strategies may be influenced by myocardial oxygen conditions and remodel accordingly, in order to ensure that the ventricle is operating with a maximal amount of adequately-oxygenated myocardium. During cold-temperature acclimation, cardiac remodeling occurs in an oxygen-rich environment alongside a general reduction in activity. Such cardiac remodeling may therefore be considered oxygen-independent remodeling as the myocytes are not constrained by oxygen limitations. Conversely, cardiac changes associated with sexual maturation occur in warm, summer conditions where the oxygen content of the water is reduced and the fish are engaged in the rigorous activities of migration and spawning. As myocytes will be exposed to much more constrained oxygen conditions, remodeling may be considered oxygen-dependent. If oxygen diffusion to the myocardium is indeed a selective force in cardiac remodeling, it might be expected that differential oxygen conditions and cardiac workload demands may result in differential hypertrophy of the compact or spongy myocardium. While the exact physiological triggers for myocardial remodeling remain largely speculative (beit temperature, physical activity, reproductive hormones or oxygen constrains), so too are many other aspects such as the temporal onset and degree of cardiac remodeling possible. Given the difficulties in quantifying and controlling for natural instances of cardiac remodeling such as sexual maturation or cold-acclimation, these questions may be best addressed using a reproducible, physiologically-defined and non-gender 19 specific method with documented scientific reference; chemically-induced anemia. Anemia is an effective inducer of cardiac remodeling as it promotes an immediate cardiovascular response to compensate for the decreased oxygen-carrying capacity of the blood (termed hypoxemia). Immediate elevation of the cardiac output is the principle compensatory adjustment to reduced oxygen-carrying capacity in fishes (Cameron & Davie 1970) and it is thought that ensuing cardiac remodeling is necessary to maintain the elevated cardiac workload associated the compensatory elevation of cardiac workload. Phenylhydrazine hydrochloride (PHZ) is commonly utilized to study experimental anemia in a wide range of species by causing oxidative hemolysis of red blood cells (Itano et al. 1976) and has no known incidences of side effects other than those directly related to the anemia itself (dating back to Smith et al. 1971). By selectively removing erythrocytes from the primary circulation, PHZ-induced anemia reduces the oxygen-carrying capacity of the blood and the resultant compensatory increase in cardiac output is suspected to trigger cardiac remodeling. A dose of 10 - 12.5 ug g B M " ' PHZ causes a well-characterized transient hemolytic anemia in many fish species, with a 75 - 80% decrease in hematocrit within 2 - 4 days (Smith et al. 1971, Chudzik & Houston 1983, McClelland et al. 2005). While carbon monoxide inactivation of hemoglobin has been shown to increase cardiac output in rainbow trout (Holeton 1977), the link between hematocrit, hemoglobin concentration and cardiac output during PHZ-induced anemia has never been directly established for rainbow trout. The primary purpose of this study was to determine the degree of cardiac remodeling possible during chronic anemia, whether remodeling is influenced by oxygen availability and to what degree these cardiac changes are transient. This will be accomplished by using PHZ to induce anemia and monitoring the cardiac changes of PHZ-treated fish compared to time-matched, sham-injected controls. The use of sham injections and young, sexually-immature fish will ensure a direct control for the effects of sexual maturation and the injection procedure. The relationship between hematocrit and cardiac output will also be examined by the use of dorsal aortic cannulation and a flowprobe on the ventral aorta to concurrently monitor both parameters following injection with PHZ to determine when, during anemia, cardiovascular compensation by elevation of cardiac output become necessary. 20 Materials & methods: Animal acquisition and care Rainbow trout (Oncorhynchus mykiss Walbaum) were obtained form the Sun Valley Trout Hatchery (Mission, British Columbia, Canada) and were transported to Simon Fraser University (Burnaby, BC , Canada) in June 2003. The fish were housed in 140 1 indoor fiberglass tanks with flow-through, de-chlorinated municipal ground water. They were fed a maintenance diet of trout pellets from Aquafeed Limited (Chilliwack, B C , Canada) and were kept on a 12L:12D light cycle. Two chronic and one acute studies of PHZ-induced anemia were conducted. One was performed in August through October of 2003 when mean water temperature was 17.3°C, but decreased seasonally from 18°C to 14°C during the experimental period. The mean body mass (BM) of these fish was 135.9 ± 2.9 g, fork length was 21.1 ±0.1 cm and the condition factor was 1.43 ± 0.02. A second experiment was conducted in February to April 2004 when the mean water temperature was 6.4°C (ranging between 6 and 7°C). The B M of the second experimental group was 96.6 ± 2.6 g, fork length was 19.1 ± 0.2 cm and the condition factor was 1.35 ± 0.02. Fish were separated into treatment groups two weeks prior to the experiment. A l l experimental protocols were approved by Simon Fraser University's University Animal Care Committee in accordance with the Canadian Council on Animal Care. A third study was conducted to consecutively monitor cardiac output (Q), Hct and hemoglobin (Hb) prior to and following an acute injection of PHZ. The experiment was conducted at the University of British Columbia (Vancouver, BC , Canada) in February and March of 2005 at a water temperature of 6°C. Rainbow trout (Oncorhynchus mykiss Walbaum) were obtained from Aquafarm JV (Fort Langley, British Columbia, Canada) and acclimated for several months prior to study. The average body mass was 1045 ± 113 g, the fork length was 40.1 ± 1.5 cm and the condition factor was 1.51 ± 0.07. The experimental protocol was approved by the University of British Columbia's Animal Care Committee in accordance with the Canadian Council on Animal Care. 21 Injection and hematocrit-monitoring techniques PHZ or sham saline treatments were administered to individually netted rainbow trout, anaesthetized in 0.1 g l"1 of buffered MS-222 (0.1 g l"1 sodium hydrogen carbonate and 0.1 g 1"' tricaine methanosulfonate). Fish were weighed and received either an intra-peritoneal injection of physiological saline (100 ul kg BM"') or PHZ (10 ug g BM"') dissolved in physiological saline. The injection site received a topical application of penicillin and the fish were placed in a recovery tank until injections were completed for all fish in their treatment group. Each treatment group was housed in a separate, proximate tank. To induce cardiac remodeling, I sought to maintain Hct below 20% for the duration of the treatment protocol. Rainbow trout have a pronounced intra-specific variability in Hct values, ranging from 17% to 44% (Wells & Weber 1991) and based on the relationship between Hct and swimming performance, Gallaugher et al. (1995) considered a Hct below 21.6% to be functionally anemic. A Hct below 20% was therefore considered severe anemia, below most natural Hct variation, without compromising the health of the fish. Due to the transient action of PHZ and the swift erythropoietic abilities of rainbow trout, a significant recovery from anemia can occur within a week of treatment at warm temperatures (Byrne & Houston 1988, McClelland et al. 2005). A pilot study conducted prior to experiments monitored the Hct of a small group of rainbow trout (N=3) and found that despite weekly injections of PHZ, fish were able to substantially recover from anemia between injections such that the Hct was not maintained at a steady 10%, but rather fluctuated from anemia to recovery values between injections. The pilot study also found that rainbow trout displayed decreased sensitivity following several PHZ injections, such that an increased dose was necessary to cause a comparable decrease in Hct, as described below. Hematocrits were measured periodically to minimize the stress of Hct sampling and preserve the well-being of anemic fish, despite precluding the ability to more closely monitor and maintain the Hct at a steady anemic state. The small body size and large number of experimental rainbow trout negated the use of less invasive techniques of monitoring the Hct, such as dorsal aortic cannulation. Instead, Hct monitoring of experimental subjects was conducted by caudal vein puncture 3 to 4 days following PHZ injection, when peak anemia has been noted to occur (Smith et al. 1971, Chudzik & Houston 1983, Byrne 22 & Houston 1988, McClelland et al. 2005), and then on a weekly basis. For each Hct check, three fish from each treatment group were netted, anesthetized and a minimal blood sample (200 ul) withdrawn from the caudal vein in a heparin-rinsed syringe to determine the Hct in triplicate. To prevent repeated Hct determination of individual fish, each Hct-sampled fish received a small identifying nick on the distal tip of the pectoral fin which was dabbed with penicillin. Blood samples were transferred to triplicate heparinized capillary tubes and centrifuged for Hct determination (Readacrit centrifuge, Becton Dickinson). Tissue sampling protocol On sampling days, six fish from each group were netted and anaesthetized with 0.1 g 1"' of buffered MS-222. A blood sample was withdrawn for Hct determination and 100 ul of heparinized saline (100 IU ml"') was injected into the caudal vein to prevent blood clotting during tissue sampling. The fish was then euthanized by cervical dislocation, weighed and measured for fork length, width and depth. The condition factor for each fish was calculated as body mass/length3 x 100, where body mass is in g and length in cm. The ventricle, spleen and gonads were removed through a mid-ventral incision along the abdomen between the pelvic to pectoral girdles. The ventricle was carefully cleared of atrium and bulbus arteriosus and was placed in chilled physiological saline for later mass determination (to a precision of 0.1 mg), after which the ventricle was placed into 70% ethanol for preservation and dissection. The spleens were weighed immediately and discarded, while gonadal masses were not recorded as fish were found to be either immature or extremely undeveloped. A l l ventricles were dissected by separation of the compact and spongy myocardial layers under dissection microscope. Separation of the myocardial layers is possible due to the presence of a thin fibrous membrane between the layers which makes the spongy layer easily distinguishable and separable from the compact layer (Poupa & Carlsten 1973). The dry mass was determined for each myocardial layer following dessication for 3 days at 65°C. This method of myocardial composition analysis is an easy method once practiced and provides otherwise unavailable information about dry mass and water content of the ventricle compared with digital methods of quantification, although it may introduce more inherent variability. A l l ventricular mass data reported, including wet or dry mass, are presented as ratios relative to the body mass, 23 to account for variability in body size. Dry mass data for compact or spongy myocardial layers are also reported relative to the body mass. Experimental Protocols Controls - Three experimental controls were used to quantify and control for potentially confounding variables associated with the experimental protocol. In all experiments, a control group of fish was injected with physiological saline as a time-matched, sham-injected control to the PHZ-treated group in order to control for potential effects caused by the injection procedure itself which, during the experimental period, involved repeated netting, anesthetization and handling. It is possible that rainbow trout may behaviourally compensate for their anemic state by becoming lethargic and diminish the need for cardiovascular compensation and cardiac remodeling, therefore in the first experiment, two groups of fish - a PHZ-treated group and a sham control group - were chased on a daily basis to introduce an oxygen challenge and negate potential behavioural compensations. Chasing was also an additional control for the effects of handling by the addition of a daily, stressful perturbation. In the second experiment, an additional control group of fish was maintained in a proximate tank and was not handled or subjected to injections and routine hematocrit monitoring. This untreated control group provided a measure of any effects caused by injection and Hct-testing of fish during the experimental protocol. Experiment 1: Effects of chronic anemia on cardiac remodeling - This study involved the serial injection of fish with either PHZ or saline over an 8-week period at 17°C. Two weeks prior to the experiment, fish were separated from a 1,000 1 holding tank into four proximate 140 1 tanks. Two groups were designated for PHZ treatment and the other two for sham saline injections. One group of each treatment was further designated for a daily chasing protocol. There were therefore a total of four experimental groups: PHZ-treated fish, sham fish, PHZ-treated chased fish and sham chased fish. Fish were injected on weeks 0, 2, 4 and 6. The first two injections were at a dose of 10 ug g B M ' 1 , while the last two were doubled to 20 ug g B M " 1 . Six fish randomly picked from the four groups were sampled at week 0 to obtain control pre-treatment values, while 6 fish from each group were sampled at weeks 2, 4, 6 and 8 of treatment, as described above. Following a 4-week recovery, fish were sampled at week 12. 24 Fish were chased on a daily basis, according to procedures described by Perry et al. (1996). As chasing was used to promote increased cardiac work and not necessarily exhaust the fish, the chasing period was typically 2 minutes in duration, approximately five times shorter than routine protocols to induce complete exhaustion (Milligan 1996). Due to their anemic status and reduced erythrocyte complement, PHZ-treated fish were chased first and the saline fish chased accordingly. If anemic fish became refractory to gentle prodding before the 2 minute chasing interval was completed, the chasing protocol would be considered completed for that fish. To ensure all fish were chased, fish were individually netted, chased in a separate tank, and then placed into a holding tank until the entire group was swum and finally returned to their respective tank. While the fish were initially chased with a net as per Perry et al. (1996), the fish quickly accustomed to the unthreatening daily jostling of the net and were eventually required additional chasing by manual prodding. There were a combined total of 13 mortalities in the two PHZ-treated groups within the first 3 weeks of the experiment; 6 PHZ-treated fish and 7 PHZ-treated chased fish. Many of the mortalities may have been a result of either initial overzealous chasing or handling stress, as most deaths occurred immediately following chasing or the injection procedure. There was only one control mortality when a sham fish jumped from the tank. The remainder of the PHZ-treated chased group (N=7) died during the recovery period due to a faulty water supply and thus no week 12 recovery values were obtained for this group. Experiment 2: Effects of handling stress on cardiac remodeling - Due to the potentially confounding drop in ambient water temperature during the first experiment and the possible effect of the injection procedure observed in sham fish, a second experiment was conducted to control for both of these factors. As 4 weeks of anemia induced a significant increase of r V M , this study was comprised of a single sampling period at a steady water temperature of 6°C, with the addition of an untreated test group to control for the handling stress of the injection procedure. This group, termed the untreated group, was held in a proximate tank and not handled whatsoever except for sampling. Treated fish were injected with PHZ or saline on weeks 0 and 2 and sampled (7V=10) on day 0 (to established pre-treatment control values), week 4 of treatment and on 25 week 8 following a 4-week recovery. Fish were injected and sampled as described above. There was one PHZ mortality following the initial treatment. Experiment 3: The acute effect of anemia on Hct cardiac output - Cardiac output, Hct and Hb were monitored in rainbow trout prior and following injection of PHZ. Q was measured by a Transonic flowprobe (Transonic Systems, Ithaca, N Y , USA) placed on the ventral aorta (according to procedures described in Farrell & Clutterham 2003), while a cannula inserted into the dorsal aorta allowed blood sampling for Hct determination. Fish (A^=5) were housed in 200 1 tanks in mesh tubes that restricted bursts of activity and prevented entangling of flowprobe and cannula leads. Following a 24 hr recovery, Q was recorded on Labview software (National Instruments, Ottawa, Ontario, Canada) for 20 minutes every two hours, from early morning until evening, to establish routine, normocythemic values. Post-recording blood samples were then taken to establish consecutive cardiovascular and hematological measurements. A blood sample of -200 ul was withdrawn from the cannula in a heparinized syringe, which was replaced with an equivalent volume of physiological saline. A Hct was determined for each blood sample and the remaining blood was frozen in heparinized vials for later Hb determination by standard cyanmethemoglobin assay (Total Hb assay kit, Sigma Diagnostics, St. Louis, USA) . Recordings and blood samples when the fish were unduly active were discarded. Following recording of routine values, intra-peritoneal injection of PHZ (20 ug g BM" 1 ) , fish were monitored for three days post-injection. Q was recorded every two hours and blood samples were taken three times daily (morning, noon and late afternoon). Q measurements were recorded every 10 sec, but averaged into 1 min blocks for statistical analyses. Heart rate (fH) was calculated from Q data also using Labview software and the stroke volume (SV) was calculated as the quotient of Q divided by fH. Following termination of the experiment, the fish were euthanized by cervical dislocation and the ventricle excised. The compact myocardial composition was determined by dissection as previously described. The r V M was found to be 0.146 ± 0.01 and the % compact myocardium was 40.1 ± 1.5%. 26 Statistics Al l data are reported as the group means ± standard error of the mean (SEM). In the cardiac remodeling experiments, changes to a variable over the course of each experiment were analyzed for each treatment group by A N O V A and Dunnett's test, where results were compared to a pre-treatment group of fish sampled prior to treatment on day 0 of the experiment (also designated the pre-treatment control group). As many variables can exhibit natural variation over time (i.e. seasonal effects on rVM) and given the extended length of the 8-week experiment, PHZ treatment groups were also compared with time-matched sham controls. Comparison between time-matched treatment groups were analyzed by either A N O V A or Student's t-test statistic. Changes following the injection of PHZ were detected by a repeated-measures A N O V A statistic and a Tukey-Kramer multiple range test. The minimum level of significance assigned for all statistical analyses was .P<0.05. A l l statistical analyses were conducted using JMP 5.0 software (SAS Institute Inc., Cary, N C , USA) , except repeated-measures A N O V A which was conducted on SigmaStat 3.0 software (SPSS, Chicago, IL, USA). Results Experiment 1: Effect of chasing on cardiac remodeling Hematological Characteristics - The overall mean Hct for the sham control group during the treatment period was 33.2 ± 1.1% and varied between 27% and 38% (Figure 2.1). Daily chasing had no significant effect on the Hct. The sham chased group had an overall mean of 31.1 ± 1.0%, which varied from 23% to 35% (Figure 2.1) As there was no difference between the two sham control groups, the data were pooled over time to yield an average control Hct of 32.1 ± 0.8%. During the 8-week anemic period, the overall mean Hct of PHZ-treated group was 17.7 ± 2.0%, and showed four oscillations between 6 and 29% that reflected each PHZ treatment. As with sham chased fish, chasing of the PHZ-treated group had no significant effect on the Hct (17.0 ± 2.1%), therefore data were pooled for the PHZ treatments yielding an average Hct of 17.4 ± 1.8% during the 8-week treatment period, a value significantly lower than the pooled control value (PO.0001). While the Hct of the PHZ-treated group recovered to 28.6 ± 1.7% by the end of recovery and although still significantly lower than the day 0 control value, it was not significantly different from the pooled time-matched control Hct (29.0 ± 1.6%). As previously reported by Smith et al. (1971), 27 PHZ-treated fish were also observed to display characteristic yellow discolouration of the body due to erythrocyte lysis and congestion of the tissues with hemoglobin-laden plasma. Each PHZ treatment produced a characteristic decrease and recovery in Hct (Figure 2.1). The first PHZ injection resulted in a 63% reduction from a control value of 34.7 ± 1.5% to 12.8 ± 1.0% (Figure 2.1), which had recovered to 24.1 ± 1.0% by week 2. A similar decrease was observed in the chased PHZ-treated group with a 71% reduction to 10.1 ± 0.9% at week 1 and a recovery to 24.9 ± 2.4% by week 2 (Figure 2.1). A second injection of PHZ again decreased the Hct in both test groups, however, the decrease was less in chased fish than the first injection and both Hct recoveries at week 4 were marginally higher than at week 2. Thus, the injected dose of PHZ was doubled for weeks 4 and 6. Four days following the third and fourth PHZ injections, the mean drop in Hct for both PHZ-treated groups was 5.2% and 9.8%, respectively. The relative spleen mass (rSM) of sham control fish remained constant throughout the experimental period, ranging from 0.072 to 0.010. The mean rSM of each PHZ-treated group was significantly higher than that of the sham control group at all weeks (P<0.0005), and by the end of PHZ treatment at week 8, the PHZ-treated group had a mean rSM of 0.277 ± 0.02 compared to 0.080 ± 0.01 for sham control fish. Chasing had no effect on the rSM in either treatment group. Following recovery, the rSM of sham control and PHZ-treated groups were statistically comparable. Cardiac Remodeling - The changes in r V M in all groups during the experimental period are presented in Figure 2.2A. As with Hct, chasing had no significant effect on the r V M in either group during the entire experimental protocol, therefore data for each chased treatment groups were pooled with non-chased data. r V M of the sham control group increased 20% from 0.086 ± 0.002 at week 0 to 0.103 ± 0.002 between weeks 2 and 4 and remained significantly elevated for the remainder of the experiment (Figure 2.2A). Regardless of the significant 20% increase in the r V M of control fish, PHZ treatment induced significantly larger increases in r V M compared to the sham control group during the treatment protocol (PO.0001). 28 Within 2 weeks, r V M of the PHZ-treated group was 35% greater than sham control fish, an increase similar to that reported by McClelland et al. (2005) for rainbow trout treated with PHZ at similar water temperature (30%). By week 8 of the PHZ treatment protocol, r V M had reached 0.158 ± 0.004 and was 84% higher than pre-treatment control fish and 68% higher than time-matched control fish. The mean PHZ-treated group r V M was significantly higher than time-matched control values at all weeks sampled during treatment (PO.0001). Although Hct was restored in the PHZ-treated group by week 12, r V M remained elevated from control and sham time-matched fish indicating a slower recovery for r V M than for Hct. Concurrent with wet r V M results, the dry r V M of sham control fish remained unchanged throughout treatment, with a significant increase in the sham chased group at week 8. In contrast, there was a significant increase from week 2 onwards in the PHZ-treated groups, reaching an increase of 40% over time-matched sham controls at week 8 (Figure 2.2B). Regression analysis of the r V M versus dry r V M showed a significantly proportional relationship (Figure 2.2C), indicating there was no effect of treatment or chasing on the ventricular water content. The pre-treatment control group had a mean water content of 87.6 ± 0.6%, while PHZ and sham-treated groups had mean values of 87.8 ± 0.2% and 87.2 ± 0.2%, during the experimental period. Compact myocardial analysis - The percentage of compact myocardium remained unchanged throughout treatment and recovery in all groups of sham control or PHZ-treated fish, despite an increase in dry r V M during recovery, therefore data was again pooled for each treatment protocol. Therefore despite significant increases in the compact, spongy and total ventricular dry masses of PHZ-treated fish by week 6 (Figure 2.3), the change in myocardial composition was proportional. While there were slight decreases in myocardial mass following recovery at week 12, the dry r V M of the PHZ-treated group remained significantly elevated from pre-treatment control values (P<0.0001) and from time-matched controls (PO.0001). In addition, the compact myocardial mass and dry r V M in control fish was significantly increased following recovery, during which the ambient water temperature continued to drop from 18°C to 12°C. 29 Experiment 2: The effect of handling stress on cardiac remodeling Hematocrits and relative spleen mass - Results for Hct, r V M and % compact composition are summarized in Table 2.1. There was no change in the Hct of untreated and sham group throughout treatment and recovery. The rSM increased significantly in both groups at week 4. Similar to experiments at 17°C, injection of PHZ at week 0 produced a 70% drop in Hct from 38.6 ± 1.1% to 12.0 ± 2% within three days. By week 8, the Hct remained significantly lower than both the untreated Hct at week 0 and the time-matched sham and untreated groups. The average Hct for the PHZ-treated group over the 4-week treatment was 8.8 ± 1.9% and ranged from 4-16%. The rSM of the PHZ-treated group doubled compared with untreated fish at weeks 4 and 8. Cardiac remodeling - The changes in r V M during the experimental period are presented in Figure 2.4. As indicated by both wet and dry r V M data, untreated fish experienced no change in either r V M or % compact throughout the treatment protocol (Figure 2.4A & B). Conversely, while sham injection resulted in an 18% increase in r V M , PHZ treatment resulted in a significantly higher increase of 35%, which remained significantly elevated following a 4-week recovery. Regression of the wet and dry r V M (Figure 2.4C) shows a significantly proportional relationship similar to that obtained in the previous experiment (Figure 2.2C), signifying no effect of treatment or handling on the ventricular water content. Pre-treatment control fish had a myocardial water content of 87.8 ± 0.9%, compared to 86.7 ± 0.3%, 86.3 ± 0.3% and 87.3 ± 0.2% for the untreated, saline and PHZ-treated groups, respectively. Dry mass determination of the r V M and compact dry mass shows significant increases of both at weeks 4 and 8 in the PHZ-treated group and week 4 of the sham control group (Figure 2.5). There was no change to the myocardial mass of the untreated group throughout the experimental protocol and no significant differences compared to the sham group. The disproportionate increase in compact myocardial mass of the PHZ-treated group lead to an overall significant increase in % compact composition from 29.4 ± 1.8% at week 0 to 37.0 ± 1.5% at week 4. There was no change in the spongy myocardial mass in any group. 30 Experiment 3: The acute effect of anemia on Hct and cardiac output The acute effect of a single PHZ injection in fish equipped with a flowprobe and dorsal aortic cannulation to simultaneously monitor Q and Hct is summarized in Table 2.2 and Figure 2.6. While increased cardiac workload is considered the trigger to initiate cardiac remodeling, cardiac output was not found to increase significantly (and only by 20%) until a Hct of 10% (P<0.005 by repeated measures ANOVA) . As significant cardiac remodeling has been found to occur by one week, but that peak anemia does not develop from PHZ treatment until 3-4 days following injection, it is possible that cardiac remodeling may be initiated prior to the elevation of Q, possibly due to a hypoxic signal due to anemia. Significant cardiovascular compensation does appear below a Hct of 10%, however, where Q increased as much as 100%). Hemoglobin and Hct were also found to decrease proportionally (Figure 2.6). Discussion Rainbow trout ventricles were shown to nearly double in mass in response to eight weeks of chronic anemia, a response that was first detected within two weeks of PHZ treatment and confirmed by both wet and dry mass analysis of the r V M . While the response of r V M to anemia was found to be transient, indicated by the decrease in r V M during the recovery to normocythemia, recovery was slight and lagged substantially behind erythropoietic recovery of the Hct. Following a 4-week recovery, the r V M remained significantly higher than time-matched controls at 6°C, while the r V M at 17°C remained 30% higher than time-matched control values, though not significantly different. While anemia-induced cardiac remodeling induced a disproportionate increase of % compact myocardium and dry compact myocardial mass at cold temperatures, similar to selective patterns of ventricular hypertrophy observed in spawning and cold temperature conditions (Graham & Farrell 1992, Davie & Thorarensen 1997, Clark & Rodnick 1998), a proportionate myocardial response occurred at warm water temperatures. A number of control experiments were performed to ensure that cardiac remodeling was a direct result of the reduced oxygen-carrying capacity and not procedural artifact. While untreated controls demonstrated that the injection procedure did result in slight, insignificant increases of r V M , PHZ treatment resulted in significantly higher increases. The lack of an additive effect of chasing on r V M of either group indicates that chasing stress itself does not appear to directly affect the r V M . While there was no effect of chasing 31 observed in sham normocythemic fish, control fish were chased only to the degree tolerated by their anemic counterparts and may therefore not have been challenged or stressed enough to require increased cardiac work. The lack of plasticity in the response of the chased versus non-chased PHZ-treated group suggests that the compensatory physiological processes associated with the cardiac remodeling response in anemic fish may either be routinely maximized, as the added stress of chasing had no additive effect on remodeling, or that the anemic state of PHZ-treated fish simply does not permit sufficient activity necessary to further augment cardiovascular compensation. Anemic mammals display the former strategy, whereby pronounced recruitment of cardiovascular regulation mechanisms at rest prevents additional cardiovascular compensation to support additional activity (Magosso & Ursino 2004). Therefore, if chasing did not stimulate cardiac remodeling in either group, the observed increase in both sham control groups may therefore be related to the actual injection procedure or the seasonal decrease in ambient water temperature during the experiment, a known trigger of ventricular hypertrophy. The use of a constant water temperature in the second experiment, combined with a similar increase in r V M of sham fish as the first experiment, demonstrated an effect of the injection procedure regardless of water temperature. While no such increase was reported for control fish in McClelland et al. (2005), control fish were only anesthetized, not sham injected, and the experimental protocol consisted of a single acute treatment, as opposed to chronic treatment in the current studies. Regardless, the effect of PHZ treatment at similar high water temperatures resulted in a nearly identical increase in r V M following two weeks in both studies; r V M increased 30% from pre-treatment values (McClelland et al. 2005), compared to 35% in the first experiment. Anemia causes significant elevation of cardiac output As expected, acute anemia caused a significant increase in Q, but the relationship between Q and Hct was not proportional as expected. Rather, Q was maintained at a fairly steady level until a Hct of ~10%, whereupon a further decrease in Hct resulted in an exponential increase in cardiovascular response. There are several alternative physiological strategies which may have precluded significant cardiovascular adjustment at intermediate levels of anemia. Firstly, anemic fish may have reduced overall oxygen 32 consumption either metabolically or behaviourally, thereby necessitating a lower routine arterial oxygen delivery (further facilitated by the placement of the fish within holding tubes). Secondly, fish may have increased oxygen extraction at the tissue level to compensate for decreased arterial oxygen availability. Finally, fish may have been stressed at the onset of the experiment resulting in an artificially-elevated routine cardiac output, resulting in underestimation of subsequent increases of Q. Comparison of routine Q measurements to known literature values for rainbow trout at similar water temperatures (17.6 - 18.0 ml min"1 kg"1; Kiceniuk & Jones 1977, Gamperl et al. 1994) suggests that routine Q was indeed comparable (18.3 ± 3.2 ml min"1 kg"1) and therefore not unduly elevated due to stress. As the Hct was found to be proportional to the concentration of Hb, changes in Hct therefore reflect changes to the arterial content of the arterial blood (assuming full oxygen saturation of the blood). Therefore, given that a 20% increase in routine Q accompanies a 50% reduction in Hct, it appears likely that fish are compensating for anemia by either increasing M o 2 or decreasing the venous oxygen content. Further, given the 100% increase in Q at the lowest anemic values, a Hct of 10% appears to be the minimal oxygen carrying-capacity value required to meet physiological needs barring major cardiovascular compensatory effort. It is therefore possible that prior to cardiac remodeling during chronic anemia, it is more physiologically-advantageous for rainbow trout to compensate for acute reductions in arterial oxygen-carrying capacity by utilizing venous oxygen stores, rather than increasing cardiac output. The observed changes in cardiovascular functioning do support current volume-loading hypotheses involved in the induction of anemia-induced cardiac remodeling. Increasing Q by stroke volume is a suspected key component to the hypertrophic response as dilatation of the ventricle results in myocardial stretch and the transient expression of heat-shock proteins, which are the putative signal between mechanical stretch and a cascade of adaptive myocardial changes (Delcayre et al. 1988). An interesting experiment would be to similarly monitor cardiac parameters following chronic PHZ treatment and cardiac remodeling to assess whether there is a differential cardiovascular compensatory response. 33 Hypoxia is an additional trigger known to influence both cardiovascular response and erythropoiesis, and a likely candidate to have a modulating role during anemia-induced cardiac remodeling. Mi ld hypoxia has been found to increase the venous pressure and cardiac pre-load resulting in both increases to stroke volume and cardiac output (Sandblom & Axelsson 2005). Additionally, hypoxia has emerged as a principle stimulus evoking erythropoiesis and hemoglobin accumulation in vertebrates - it is reasonably assumed that decreases in arterial oxygen tension play a major role in prompting erythropoiesis stimulating factor and release (Tun & Houston 1986). Therefore, as it appears that the immediate compensatory adjustment to acute, intermediate anemia may be at the level of venous oxygen content, in combination with a moderate increase in Q, cardiac remodeling is likely influenced by multiple factors including anemia, hypoxia and myocardial stretch. Temperature has a significant modulatory effect on cardiac remodeling and erythropoiesis Temperature was found to have a significant modulatory effect on r V M of rainbow trout in two ways. The different acclimation temperatures of experiments 1 and 2 resulted in a significantly higher routine r V M off ish at 6°C, 27% higher than at 17°C (PO.05). This difference in r V M likely reflects naturally-occurring and well-characterized ventricular hypertrophy due to cold-acclimation (Farrell et al. 1988, Graham & Farrell 1989). In addition, the decrease in water temperature in experiment 1 saw a concurrent 16% increase in sham r V M when the temperature decreased from 17°C to 12°C during a 4-week period. A similar 24% increase in the r V M of control fish was observed over 10 weeks when the temperature dropped from 12 °C to 2 °C (McClelland et al. 2005). A third and novel effect of temperature on cardiac growth was the finding that anemia-induced hypertrophy was enhanced at warm temperatures. In the first experiment conducted at 17°C, the PHZ-treated group experienced a 26% increase in r V M compared to time-matched controls within 4 weeks, while fish at 6°C experienced just a 15% increase in 4 weeks. The r V M was also observed to decrease faster at warm temperature following return to normocythemia. Following a 4-week recovery at 17°C, fish experienced a 10% decrease in r V M , whereas a similar recovery period at 6°C yielded a decrease of only 4%. There were also notable differences in the erythropoietic response to PHZ treatment at each temperature. At 17°C, 34 there was a rapid and substantial recovery of the Hct following the first two injections of PHZ. Injection of the same dose at 6°C did not induce a comparable erythropoietic recovery. A similar temperature-dependent effect in the erythropoietic response to PHZ has also been demonstrated in goldfish where PHZ treatment at 30°C resulted in a complete Hct recovery in several days, but with no recovery demonstrated at 7.5°C following several months (Chudzik & Houston 1983). While rainbow trout were observed to have a substantial recovery of Hct following four weeks regardless of temperature, recovery occurred more rapidly at warm temperatures, and necessitated increased treatment dosages to induce comparable reductions of Hct. Temperature may also affect the metabolic processes involved in the detoxification and metabolism of PHZ, resulting in a prolonged chemical effect at cold temperatures. The significant effect of temperature on the metabolic response and erythropoietic abilities of rainbow trout may result in differential stimulatory signals for cardiac remodeling and should be considered an important discovery in these experiments, as temperature was observed to influence cardiac remodeling. Preferential increase of compact myocardium occurred at cold temperatures, while proportional myocardial growth occurred at warm temperatures. A slower recovery to normocythemia at cold temperature may result in an increased dependence on arterially-oxygenated compact myocardium, thus resulting in a significantly increased compact myocardium. The enhanced metabolic response at higher temperatures, conversely, promotes a more rapid erythropoietic recovery, which may result in diminished hypertrophic response and reduced dependence on arterial oxygenation, hence the lack of a disproportionate myocardial response. Conclusion Rainbow trout were shown to exhibit considerable cardiac plasticity. Both r V M and myocardial composition responded to anemia-induced hypertrophic induction and to temperature. The acute effect of anemia on routine Q did not appear to be proportional to the Hct. The primary response may therefore have been to decrease the venous oxygen content (Cvo2) rather than increasing Q, and only at a threshold Cvo 2 was a significant increase in Q observed. A preliminary reliance on existing oxygen stores (i.e. venous reserve), rather than increasing Q, could be energetically conservative until the heart is remodeled. More research is needed to examine these dynamics, including the physiological triggers and thresholds of 35 cardiac remodeling. Due to the exclusive arterial coronary supply to compact myocardium, the primary anemic response of decreasing Cvo 2 could also explain why compact myocardial hypertrophy appears to be the favourable adaptation to chronically-decreased oxygen availability, as it reduces the proportion of ventricular myocardium dependent on variable venous oxygen through the lumen. However, a caveat of this hypothesis is that there is a concurrent increase in arterial coronary vascularity accompanying compact myocardial hypertrophy. This hypothesis will be examined in the following chapter. 36 Figure 2.1: Changes in hematocrit during the experimental period in response to four injections (1) at weeks 0, 2, 4 and 6 followed by a 4-week recovery. Fish were treated with phenylhydrazine hydrochloride (PHZ) to induce anemia, while sham control fish received sham saline injections. One group of each treatment was chased daily to induce an exercise challenge. Mean values for weeks 0, 2, 4, 6, 8 and 12 have an N=6; all others N=3. 37 Time (weeks) Figure 2.2: The change in relative ventricular mass (rVM) during the experimental protocol is represented in the top left figure (A), while the r V M calculated by dry mass determination (dry rVM) is presented in (B). The dashed line connecting weeks 8 and 12 represents the 4-week recovery. Dissimilar letters indicate significant differences from pre-treatment control values, as detected by A N O V A and Dunnett's test. Underlined letters represent chased groups, while black letters represent a significant difference in both groups of a treatment. The linear relationship between wet and dry r V M is presented in the bottom figure (C). Significant differences between time-matched PHZ and sham groups were determined by Student's t-test statistic. Black (*) indicate significant different of the PHZ-treated group versus sham and untreated group. 38 F i g u r e 2 .3 : D r y re la t i ve ven t r i cu l a r mass (d ry r V M ) d u r i n g a n 8 - w e e k t reatment w i t h e i ther sa l ine or p h e n y l h y d r a z i n e h y d r o c h l o r i d e ( P H Z ) i n jec t i ons (N=6), f o l l o w e d b y a 4 - w e e k r e c o v e r y ( w e e k 12). S i g n i f i c a n t d i f f e rences f r o m pre- t reatment con t ro l s are i nd i ca ted b y d i s s i m i l a r let ters, as detec ted b y A N O V A a n d Dunne t t ' s test. B l a c k letters represent s i gn i f i can t changes i n d r y r V M , w h i l e y e l l o w letters denote s i gn i f i can t changes i n c o m p a c t m y o c a r d i a l mass . S o l i d l i nes denote the m e a n pre- t reatment m a s s , w h i l e do t ted l i nes represent ± S E M . 39 0.130 r 0.120 V 0.110 r 0.100 0.090 r 0.080 0.000 0.024 JE 0.020 0.016 0.012 r 0.008 0.000 1 Time WeekO Week 4 Week 8 WeekO Week 4 Week 8 PHZ treatment group Sham control group Unperturbed control group B * R =0.736, P<0.001 © y=0.1145x +0.0015 • PHZ treated fish © Unperturbed control fish © Control fish • Untreated control fish 0.016 0 2. a> 0.014 t' < (D 3 0.012 c 0.010 0.000 3 0) (A (fl 0.060 0.080 0.100 0.120 0.140 0.160 0.180 0.200 Relative ventricular mass Figure 2.4: The change in relative ventricular mass (rVM) during the experimental protocol is presented in the top left figure (A), while the dry r V M calculated by dry mass determination is presented in the top right figure (B). Groups were treated for a 4-week period with phenylhydrazine hydrochloride (PHZ) or sham saline injection, then allowed a 4-week recovery period. The linear relationship between wet and dry r V M is presented in the bottom figure (C). Dissimilar letters indicate significant differences as detected by A N O V A and Dunnett's test. Significant differences between time-matched PHZ and control groups were determined by A N O V A and are indicated by (*). 40 0.018 0.016 0.014 3 0.012 | V) in E 0.0101 0) 0.008 | > «| 0.0061 0.004 0.002 b T X Pre-treatment total myocardial mass Pre-treatment compact myocardial mass 0.000 1 Week 4 (Treatment) Week 8 (Recovery) PHZ compact myocardium PHZ spongy myocardium Control compact myocardium Control spongy myocardium Untreated compact myocardium Untreated spongy myocardium Figure 2.5: Changes in dry ventricular mass (7v=10) following a 4-week treatment with saline or phenylhydrazine hydrochloride (PHZ) injections, followed by a 4-week recovery. Significant differences from pre-treatment controls are indicated by dissimilar letters, as detected by A N O V A and Dunnett's test. Black letters represent significant changes in dry r V M , while yellow letters denote significant changes in compact myocardial mass. Solid lines represent the pre-treatment control mean, while dotted lines represent ± SEM. 41 Figure 2.6: The acute effect of phenylhydrazine hydrochloride treatment on the hematocrit, cardiac output (Q) and hemoglobin concentrations. A Transonic flowprobe was placed on the ventral aorta of rainbow trout and the Q measured for 10 minute intervals every 2 hours following an injection of PHZ. Concurrent blood samples were taken from a cannula in the dorsal aorta, from which the hematocrit and hemoglobin concentrations were determined. Each colour represents values for an individual rainbow trout (N=5). 42 Table 2.1: Summary of the effects of a four-week PHZ or sham saline treatment versus untreated fish on cardiac and hematological characteristics of rainbow trout (N=10). Presented is the mean ± SEM, (*) indicates significant differences as determined by A N O V A and Dunnett's test. Treatment Hematocrit (%) Relative ventricular mass Compact composition (%) Relative spleen mass WeekO Control 38.6 ±1.1 0.087 ± 0.003 29.4 ± 1.8 0.108 ±0.01 Week 4 Untreated Sham PHZ 38.6 ± 1.9 33.5 ± 3 . 4 15.6 ± 1.5* 0.095 ±0.003 0.103 ±0.005* 0.118 ±0.004* 31.1 ± 1.4 31.2 ± 1.2 37.0 ± 1.5* 0 .156±0.01* 0.159 ±0 .03* 0.199 ±0 .01* Week 8 Untreated Sham PHZ 34.1 ± 1.0 34.9 ± 1.3 28.1 ± 1.5* 0.088 ± 0.003 0.092 ± 0.003 0.113 ±0.005* 29.3 ± 1.4 30.3 ± 1.8 34.2 ± 1.4 0.110 ±0.01 0.068 ± 0.006 0.210 ±0 .04* */><0.05 Table 2.2: Changes in cardiac output, hemoglobin and hematocrit (Hct) following injection with phenylhydrazine hydrochloride. Rainbow trout (A/=5) were equipped with a flowprobe and dorsal aortic cannula to concurrently monitor the cardiovascular effects of anemia. The mean normocythemic Hct was 22.7 ± 1.4%; the severe anemia Hct was 3.6 ± 0.5%. Differing letters indicate significant differences as determined by repeated-measures A N O V A and Tukey-Kramer multiple-range test (PO.001). Cardiac Output Hematocrit Hemoglobin Heart rate Stroke volume (ml min"1 kg'1) (%) (gdl">) (min"1) (ml kg"1) Normocythemia 18.3 ± 3 . 2 a 22.7 ± 1.4" 6 . 6 ± 0 . 7 a 51 .0±3 .0 0.408 ± 0.09 ~10% anemia 21.7 ± 3.5 9.4 ± 0.6b 4 . 0 ± 0 . 5 b 47.9 ±4 .2 0.471 ±0 .10 Severe anemia 3 3 . 3 ± 3 . 9 b 3 . 6 ± 0 . 5 C 1.1 ±0.2° 53.2 ± 4 . 2 0.640 ±0 .10 43 Literature Cited-Byrne A.P. & Houston A .H . 1988. Use of phenylhydrazine in the detection of responsive changes in hemoglobin isomorph abundances. Can. J. Zool. 66: 758-62. Cameron J.N. & Davis J.C. 1970. Gas exchange in rainbow trout (Salmo gairdneri) with varying blood oxygen capacity. J. Fish Res. Bd. Can. 27: 1068-85. Canby C A . & Tomanek R.J. 1990. Regression of ventricular hypertrophy abolishes cardiocyte vulnerability to acute hypoxia. Anat. Rec. 226: 198-206. Chudzik J. & Houston A .H . 1983. Temperature and erythropoiesis in goldfish. Can. J. Zool. 61: 1322-5. Clark J.J., Clark R.J., McMinn J.T. & Rodnick K.J. 2004. Microvascular and biochemical compensation during ventricular hypertrophy in male rainbow trout. Comp. Bioch. Physiol. 139B: 695-703. Clark R J . & Rodnick K.J. 1999. Pressure and volume overloads are associated with ventricular hypertrophy in male rainbow trout. Am. J. Physio. 277: R938-46. Clark R J . & Rodnick K.J. 1998. Morphometric and biochemical characteristics of ventricular hypertrophy in male rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 201: 1541-52. Davie P.S. & Thorarensen H. 1997. Heart growth in rainbow trout in response to exogenous testosterone and 17-a methyltestosterone. Comp. Biochem. Physiol. 117A: 227-30. Delcayre C , Samuel J.L., Marcotte F., Best-Bellepomme C , Mercardier J.J. & Rapaport L. 1988. Synthesis of stress proteins in rat cardiac myocytes 2-4 days after imposition of hemodynamic overload. J Clin. Invest. 82(2): 460-8. Egginton S. 2002. Temperature and angiogenesis: the possible role of mechanical factors in capillary growth. Comp. Biochem. Physiol. 132A: 773-87. Farrell A.P. & Clutterham S.M. 2003. On-line venous oxygen tensions in rainbow trout during graded exercise at two acclimation temperatures. J. Exp. Biol. 206: 487-96. Farrell A.P., Hammons A .M . , Graham M.S. & Tibbits G.F. 1988. Cardiac growth in rainbow trout, Salmo gairdneri. Can. J. Zool. 66: 2368-73. Franklin C E . & Davie P.S. 1992. Sexual maturity can double heart mass and cardiac power output in male rainbow trout. J. Exp. Biol. 171: 139-48. Gallaugher P.E. 1994. The role of haematocrit in oxygen transport in swimming salmonid fishes. PhD thesis, 248 pp. Department of Biological Sciences, Simon Fraser University, Burnaby, B.C. Gamperl A., Pinder A. & Boutlier R. 1994. Effect of coronary ablation and adrenergic stimulation on in vivo cardiac performance in trout (Oncorhynchus mykiss). J. Exp. Biol. 186(1): 127-43. Graham M.S. & Farrell A.P. 1992. Environmental influences on cardiovascular variables in rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish. Biol. 41: 851-8. 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. Greer-Walker M. & Emerson I. 1978. Sustained swimming speeds and myotomal muscle function in the trout, Salmo gairdneri. J. Fish Biol.. 13: 475-81. 44 Hochachka P.W. 1961. The effect of physical training on oxygen debt and glycogen reserves in trout. Can. J. Zool. 127: 565-87. Holeton G.F. 1977. Constancy of arterial blood pH during CO-induced hypoxia. Can. J. Zool. 55(6): 1010-3. Itano H.A., Hosokawa K. & Hirota K. 1976. Induction of haemolytic anaemia by substituted phenylhydrazines. Br. J. Haematol. 32(1): 99-104. Jacob R., Brandle M. , Dierberger B. & Rupp H. 1991. Functional consequences of cardiac hypertrophy and dilatation. In: Smits J. , De Mey M., Daemen M . & Struyker Boudier H. (Eds) Pharmacology of cardiac and vascular remodeling. Steinkopff Verlag Darmstadt, Germany. Kiceniuk J.W. & Jones D.R. 1977. The oxygen transport system in trout {Salmo gairdneri) during sustained exercise. J. Exp. Biol. 69: 247-60. Magosso E. & Ursino M. 2004. Modeling study of the acute cardiovascular response to hypocapnic hypoxia in healthy and anaemic subjects. Med. Biol. Eng. Comput. 42(2): 158-66. McClelland G.B., Dalziel A .C. , Fragoso N.M. & Moyes C D . 2005. Muscle remodeling in relation to blood supply: implications for seasonal changes in mitochondrial enzymes. J. Exp. Biol. 208: 515-22. Milligan C L . 1996. Metabolic recovery from exhaustive exercise in rainbow trout. Comp. Biochem. Physiol. 113A: 51-60. Perry S., Reid S. & Salama A. 1996. The effects of repeated physical stress on the P-adrenergic response of the rainbow trout red blood cell. J. Exp. Biol. 199(3): 549-62. Poupa O. & Carlsten A. 1973. Experimental cardiomyopathies in poikilotherms. Recent Adv. Stud. Cardiac Struct. Metab. 2: 321-51. Sandblom E. & Axelsson M . 2005. Effects of hypoxia on the venous circulation in rainbow trout (Oncorhynchus mykiss). Comp. Bioch. Physiol. 140A:233-9. Smith C.E., McLain L.R. & Zaugg W.S. 1971. Phenylhydrazine-induced anemia in Chinook salmon. Toxicol. App. Pharm. 20: 73-81. Tomanek R.J. 1999. Angiogenesis in non-ischemic myocardium. In: Ware J.A. & Simon M. (Eds) Angiogenesis and cardiovascular disease. Oxford University Press, New York. Tota B., Cimini V. , Salvatore G. & 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. Tun N. & Houston A .H . 1986. Temperature, oxygen, photoperiod, and the hemoglobin system of the rainbow trout, Salmo gairdneri. Can. J. Zool. 64: 1883-88. Wells R.M.G. & Weber R.E. 1991. Is there an optimal haematocrit for rainbow trout, Oncorhynchus mykiss (Walbaum). An interpretation of recent data based on blood viscosity measurements. J. Fish Biol. 38: 53-65. 45 Chapter 3: Quantification of the coronary vascular volume in normal and hypertrophied rainbow trout {Oncorhynchus mykiss Walbaum) ventricles by radiotracer tracer analysis Introduction An effective and apparently complication-free method of studying cardiac remodeling is to induce anemia by the drug phenylhydrazine hydrochloride (PHZ). PHZ creates hemolytic anemia by the oxidative denaturation of hemoglobin (Itano et al. 1976) and the selective removal of damaged red blood cells by the erythropoietic system (Dornfest et al. 1986). The result of the chemical ablation of hemoglobin is a reduction in the arterial oxygen-carrying capacity of the blood, which potentially leads to tissue hypoxemia (Jacob et al. 2002) and triggers compensatory physiological adaptations including an increase of the cardiac output (Q). In the previous chapter, an acute reduction in hematocrit (Hct) to 10% was found to increase routine Q by 20%, whereas a Hct below 10% increased routine Q by 100%. Therefore, at least in the early stages of anemia, a decrease in Cvo 2 appeared to be compensated mainly by adjustments at the systemic venous level, with only a moderate increase in cardiac work. However, a chronic elevation in Q is a necessary compensatory response to stabilize the ventricular wall stress associated with increased cardiac workloads and has been proposed to trigger cardiac remodeling (McClelland et al. 2005). Anemia-induced cardiac remodeling can therefore also be associated with a disproportionate increase of compact myocardium, which receives an exclusive blood supply by way of the coronary artery unlike the spongy myocardium which relies on oxygen-depleted venous blood (Chapter 2). It is therefore hypothesized that disproportionate hypertrophy of the compact myocardium necessitates a proportional increase of coronary vascularity to maintain adequate oxygen diffusion to the cardiomyocytes. The angiogenic response in hypertrophied rainbow trout ventricles has previously been gauged morphometrically by measuring myocyte fibre dimension and capillary density of hypertrophied ventricles associated with sexual maturation and cold temperature acclimation in rainbow trout (Clark & Rodnick 1998, Clark et al. 2004, Egginton 2002). Clark & Rodnick (1998) found that capillary density, length 46 density and intercapillary distance were unchanged in the hypertrophied ventricles of sexually-mature males and concluded that vascular compensation was necessary during cardiac remodeling to maintain proportional myocyte oxygenation. Clark et al. (2004) also measured a proportional increase capillary density in hypertrophied ventricles of sexually-mature male rainbow trout, providing further support for compensatory capillary angiogenesis during cardiac remodeling. Egginton (2002) similarly found that cold temperature-induced cardiac remodeling also stimulated angiogenesis. Therefore, while there does appear to be recruitment of angiogenic processes during ventricular hypertrophy to supply adequate blood flow to the increasing myocardium, there has yet to be quantification of the efficacy of angiogenesis in terms of absolute blood flow or vascular volume to the myocardium. Another approach to estimating myocardial capillarity is radioisotope dilution analysis, a standard method to measure vascular volumes in teleosts (refer to Bri l l et al. 1998, Bushnell et al. 1998, Gingerich et al. 1987, 1990). Radioisotope tracer analysis operates on the principle of single-compartment isotope dilution within the primary circulatory system which, for the rainbow trout ventricle, is limited exclusively to the coronary circulation of the compact myocardium. Once the ventricular lumen is cleared of venous blood, radioisotope tracer analysis quantifies only the coronary system of the ventricle by comparing ventricular radioisotope count to a known volume of systemic blood. Vascular volume estimates provide but an index of capillarity and relies on a proportional relationship between capillarity and vascular volume. Angiogenesis and increased vascularity of the coronary circulation are often currently measured in mammals as changes in the coronary reserve (Dedkov et al. 2005, Lei et al. 2004, Davis et al. 1999), thereby providing a functional, volumetric index of changes to the ventricular microvasculature. As four weeks of PHZ treatment induces significant cardiac remodeling (Chapter 2), including a disproportionate increase in the composition of compact myocardium, a similar treatment protocol was utilized to induce cardiac remodeling prior to radioisotope tracer analysis. I sought to verify the hypothesis that a concurrent angiogenesis of the coronary circulation, measured as an increase in ventricular vascular volume, occurs with cardiac remodeling to maintain an adequate oxygen supply to the increased compact myocardial tissue mass. The radioactive tracers technetium pertechnetate (Tc"m) and radio-iodinated 47 albumin ( 1 2 5 I-BSA) were used to provide independent estimates of the erythrocyte and plasma volumes, respectively, in the coronary circulation of rainbow trout treated with PHZ to induce anemia and cardiac remodeling. Materials & methods Animal acquisition and care Rainbow trout (Oncorhynchus mykiss Walbaum) were obtained from the Fraser Valley Trout Hatchery (Langley, British Columbia, Canada) in August 2003 and transported to Simon Fraser University (Burnaby, BC , Canada). Fish were housed in 2,500 1 fibreglass outdoor tanks equipped with flow-through, dechlorinated municipal ground water and supplemental aeration. Fish were fed a maintenance diet of trout pellets from Aquafeed Limited (Chilliwack, B C , Canada). Seasonal water temperatures ranged 16°C to 18°C during the 4-week experimental period in August 2004. Fish had a mean body mass (BM) of 1.09 ± 0.06 kg, fork length of 39.8 ± 2.6 cm and a condition factor of 1.72 ± 0.06. Gonadal weights could not be obtained due to safety restrictions of the radioisotope protocol. A l l experimental protocols were approved by Simon Fraser University's University Animal Care Committee in accordance with the Canadian Council on Animal Care. Drug treatment A month prior to analysis with radioisotopes, fish were divided into two separate, proximate outdoor tanks - a sham, saline-injected group (N=l) and a PHZ-injected treatment group (7V=12). At the beginning of the four week treatment period, the fish were individually netted and anaesthetized by immersion into 0.1 g l"1 buffered MS-222 (0.1 g l"1 sodium hydrogen carbonate and 0.1 g l"1 tricaine methanosulfonate). The fish were weighed and the control group received an intra-peritoneal injection of physiological freshwater trout saline (100 ul kg BM" 1 ) , while the treatment group received injection of 10 ug g B M " 1 of PHZ in physiological saline to obtain a desired hematocrit of 10%. The injection site received a topical application of penicillin and the fish were returned to their respective tanks. There were no mortalities in the sham control group, but within a week of treatment, 5 mortalities occurred in the PHZ group. As a result, only a single PHZ treatment was used to induce anemia, unlike repeated 48 injections in Chapter 2. Triplicate hematocrit (Hct) determination was conducted on three fish from each group to monitor the hematocrit before, two weeks following injection, and at the end of the experiment to minimize additional stress to the PHZ fish. While previous studies have monitored the Hct more frequently (McClelland et al. 2005), the effects of PHZ are expected to be comparable to those in Chapter 2. Radioisotope tracer analysis Four weeks following PHZ treatment, ventricular vascular volume was measured using radioisotope tracer analysis. Analysis was conducted over a two-day period, with one treatment group analyzed each day. Fish were anaesthetized in 0.1 g l"1 buffered MS-222 and transported from the outdoor tanks to the laboratory where they were weighed and measured for fork length and width. Fish were then placed dorsally on a foam-lined surgical sling with the gills continually irrigated throughout the procedure with aerated, 0.05 g 1"' buffered MS-222 (Figure 3.1). The tracer analysis procedure was adapted from the methods described by Carati et al. (1988). Fish first received a 0.1 ml kg"1 B M injection of a stannous tin preparation (15 mg Na 4 P 2 O 7 10H 2 O and 3.8 mg SnCl 2 -2H 2 0 in 1.0 ml of sterile saline, kept under N 2 gas) into the caudal vein. Stannous tin priming is a necessary prelude to technetium tracer analysis, required to reduce T c " m from a stable but inert isotope (which freely diffuses in and out of cells and does not bind to Hb) into its reduced state, which can bind to the beta-chain of hemoglobin and be retained by the cell (Danpure & Osman 1994). Following 20 minutes, T c " m (80 uCi kg"1) and 1 2 5 I -BSA (5 uCi kg'1) were sequentially injected into the caudal vein and also allowed to circulate for 20 minutes. While erythrocyte-tracing isotopes have been shown to be adequately circulated in rainbow trout within 5 minutes (Duff et al. 1987), a longer circuit was allowed to account for any potential circulatory constraints caused by anaesthetization. A few minutes prior to the end of the 20 minute circulation period, the pericardium was carefully exposed by an incision along the mid-ventral aspect of the fish between the pectoral girdle and retraction of the body wall (Figure 3.2). At the 20 minute mark, the fish was euthanized with an injection of saturated potassium chloride into the pericardium. Duplicate 1 ml blood samples were then withdrawn from the caudal vein and each sample was placed into an individual heparinized scintillation vials. Once arrested, 49 the heart was immediately removed from the pericardium, the atrium and bulbous arteriosus were carefully cut away, and the ventricle lumen was rinsed with heparinized saline until clear of superficial blood. The ventricle was gently blotted, placed into a pre-weighed scintillation vial and weighed. One blood sample was used for a triplicate hematocrit determination (Readacrit centrifuge, Becton Dickinson) prior to being placed into a centrifuge for plasma isolation. Two 100 pl samples of plasma were withdrawn from the second blood sample and placed into two scintillation vials for duplicate radioisotope counting. A l l samples were then counted for T c " m in duplicate immediately following the experiment. Samples were counted in duplicate by a Gamble Technologies gamma counter (Model 2MW212) with Nucleus analyzer software (Oak Ridge, TN, USA). 125I samples were counted for 5 minutes (± 2% two sigma error), while T c " m samples were counted for 1 minute (± 2% two sigma error). Blood and plasma samples then received 1 ml of EcoLite scintillation cocktail (ICN Biomedicals, C A , USA) and were vortexed while the ventricles were digested with ammonium hydroxide for 1 h at 60°C prior to addition of scintillate. A l l samples were placed in a lead-lined cupboard for three days during T c " m decay and were then re-counted in duplicate for 1 2 5 I -BSA activity. Separate surgical tools were used for each fish to prevent cross-contamination of isotopes and all equipment was decontaminated and sterilized between sets of fish. The entire procedure for two fish lasted between 2 and 3 h. T c " m was supplied daily courtesy of the Nuclear Medicine Services of Burnaby Hospital (Burnaby, B C , Canada). 1 2 5 I -BSA was purchased from PerkinElmer L A S Canada, Inc. (Woodbridge, Ontario, Canada). Calculations and statistical analyses The total vascular volume of the ventricle was calculated for each isotope individually using the average of the duplicate radiation counts. The total blood volume of the ventricle was calculated as the quotient of isotope activity in the ventricle by the isotope activity per ml of systemic blood collected from the caudal vein. The ventricular vascular volume calculated by T c " m used erythrocyte quantification to directly determine the packed cell volume (PCV) and was calculated as the quotient of T c " m activity in the ventricle divided by the T c " m activity per ml of whole blood (1). The total ventricular vascular volume 50 (TVV) was then calculated by dividing the P C V by the hematocrit (2), and the plasma volume (PV) was measured by subtracting the P C V from the total vascular volume (3). To account for any unbound Tc"m, the P C V was corrected by the amount of T c " m activity present in the plasma fraction. Conversely, using 1 2 5 I -BSA to calculate the ventricular volume by plasma albumin quantification, the P V was calculated by dividing the ventricular activity of 1 2 5 I -BSA by that per ml of whole blood (4). The T W and P C V were then measured as below (5,6). (1) PCV = Tc"m activity per ventricle / Tc"m activity per ml whole blood (2) TVV = PCV /Hct (3) PV = T V V - P C V (4) PV = 1 2 5I-BSA activity per ventricle / 1 2 5I-BSA activity per ml whole blood (5) T W = (1 -PV)/Hct (6) PCV = T W - P V -where PCV = packed erythrocyte cell volume, T V V = total vascular volume, PV = plasma volume The tissue hematocrit of each ventricle was calculated as the quotient of the relative P C V of the ventricle divided by the combined total blood volume. The hematocrit ratio is the ratio of the tissue hematocrit divided by the whole blood hematocrit. The hematocrit ratio provides a reliable estimate of the relative volumes of the plasma and blood spaces in tissues relative to the distribution of each tracer in the general circulation (Gingerich & Pityer 1989). The relative ventricular mass (rVM) is presented as the ratio of wet ventricular mass to body mass to correct for variation in fish size. A condition factor was calculated for each fish as body mass/length3 x 100, where mass is in g and length in cm. Due to two mortalities during the tracer analysis procedure and discarded results due to low isotope binding efficiencies, vascular volume results are based on five sham control and three PHZ fish. Comparisons between sham control and PHZ fish were made using a Student's t-test statistic. A l l data presented are group means ± standard error of the mean (SEM). The minimum level of significance for all statistical analysis is P<0.05. A l l statistical analyses were conducted using IMP 5.0 software (SAS Institute Inc., Cary NC, USA). 51 Results Hematocrits and cardiac remodeling There were no significant differences in body mass, fish length or condition factor between sham and PHZ fish. Two weeks following PHZ injection, the PHZ group had a 27% lower hematocrit than control fish (25.9 ± 1.8% versus 38.2 ± 2.4%; P<0.05), indicating the expected induction of anemia. Four weeks following PHZ injection, erythropoiesis had ameliorated the difference in Hct, but sham and PHZ group Hcts remained significantly different (33.9 ± 2.8% versus 44.7 ± 1.7% respectively, P<0.01). The mean PHZ group r V M was 37% larger (Table 3.1, P<0.005). Vascular volume analysis Using Tc "m, the vascular volume of the coronary circulation per gram of ventricle in sham control fish was 42.4 ± 5.3 uL g V M ' 1 (Figure 3.3, Table 3.1). PHZ treatment doubled the volume of the coronary circulation, 92.0 ± 19.2 uL g V M " 1 (PO.05). The vascular volumes obtained with 1 2 5 I -BSA were more than double those obtained using T c " m in both sham and PHZ groups. Despite a difference between groups in 1 2 5 I -BSA volumes, 108.7 ± 5.33 uL g V M " ' for sham controls and 236 ± 88.0 uL g V M " 1 for PHZ fish, the difference only approached a statistical significance of P=0.1977 due to the unusually large variance in plasma volume of PHZ fish, a feature not observed in control fish or with erythrocyte volume estimates (Figure 3.3). Nevertheless, the ratio of vascular volumes between control and hypertrophied groups was identical for both isotopes, T c " m (2.56) and 1 2 5 I -BSA (2.56). Similarly, there were no significant differences between either the tissue Hcts of control and hypertrophied ventricles (21.0 ± 1.7% and 20.7 ± 7.9% respectively), or between the Hct ratios (0.48 ± 0.04 and 0.63 ± 0.24). When individual r V M were plotted against individual coronary volume estimates (Figure 3.4), the disproportionate effect of increasing r V M on vascular volume was evident. Discussion This study is the first to demonstrate significant plasticity of the coronary vascular volume in rainbow trout ventricles during experimentally induced cardiac remodeling. Cardiac remodeling doubled the volume of the coronary circulation. In the previous chapter, I demonstrated that an anemia-induced increase in r V M at high water temperatures is accompanied by a proportionate increase in the amount of compact 52 myocardium. Although it was not possible to determine the compact myocardial composition of the radioactive ventricles due to the necessary decay period for Tc "m, a similar result may be assumed for the present study. The doubling of the coronary volume accompanying a 37% increase in r V M indicates that there was a disproportionate increase in vascular volume, a conclusion supported when individual data were compared. Therefore, these results corroborate previous suggestions of a "powerful angiogenic response" alongside ventricular hypertrophy (Egginton 2002). Previous measurements of myocyte dimensions also support the idea that angiogenesis must accompany ventricular growth. Capillary density remained constant during ventricular hypertrophy in sexually-maturing males, whereas the capillary to fibre ratio increased with cold acclimation (Clark & Rodnick 1999, Clark et al. 2004, Egginton 2002). Thus, the present results for experimental anemia appear more similar to disproportionate angiogenic results obtained during cold acclimation. Comparison of coronary vascular volume estimates Prior estimates of coronary vascular volumes for rainbow trout ventricles are presented for comparison in Table 3.1. Two important points emerge from comparison of literature values with those obtained herein. These early studies present a combined estimate of an erythrocyte and plasma tracers (Gingerich et al. 1987, Gingerich & Pityer 1989, Gingerich et al. 1990). However, my data clearly show that ventricular vascular volume estimates obtained by albumin and erythrocyte tracers should not be combined, as the two volume estimates for control fish are statistically different and the tissue Hct is half the systemic value, as reflected in the Hct ratio being -0.5. This plasma marker consistently yielded vascular volume estimates two-times that of the erythrocyte marker, similar to the 0.39 Hct ratio observed by Gingerich & Pityer (1989). Based on other research, the finding that vascular volume estimates calculated with albumin tracers exceeded those calculated by erythrocyte tracers was also expected (Gingerich & Pityer 1989, Gingerich et al. 1987 & 1990, Bri l l et al. 1998, Bushnell et al. 1998). This difference is best described by the differential volume ratio between albumin and erythrocyte tracers, which was identical for each treatment group (2.56 for PHZ, 2.56 for control) and similar to the 3.5-fold difference reported by Gingerich & Pityer 53 (1989). The most likely explanation is that albumin overestimates vascular volume because albumin can leak from the primary circulation into tissue extravascular space or into the secondary circulation (Gingerich & Pityer 1989, Bri l l et al. 1998, Bushnell et al. 1998). In this regard it is interesting that while the erythrocyte to plasma radioisotope ratio remained constant between treatment groups, there was much greater variation of albumin estimates in the hypertrophied ventricles. This difference may arise because of the angiogenic processes themselves, as new capillaries may be more susceptible to albumin leak. Regardless, the erythrocytic volume estimate remains the more reliable and conservative estimate of the true coronary vascular volume as erythrocyte tracers remain exclusively within the primary circulation (Duff et al. 1987), and thus is the estimate used here for further comparisons. That the present study obtained a T c " m vascular volume at least 30 ul g V M " 1 higher than earlier work is not unexpected. There are two possible reasons why this difference may exist. Firstly, fish used in my experiments were over double the body mass of those in previous studies. As rainbow trout increase in body size, so too does the proportion of the compact myocardium - a correlation first described in 1974 by Poupa et al. "as a consequence for the need for greater wall tension to maintain pressure in a large chamber". Therefore, larger fish would be expected to have higher vascular volumes via ontogenic growth of the compact myocardium. The second reason a higher vascular volume is expected is related to the method used to arrest the heart. To obtain a consistent measure of the ventricular coronary volume of each fish, saturated potassium chloride solution (KC1) was utilized to induce hyperpolarized cardioplegic arrest. KC1 depolarizes the membrane potential and inactivates the fast, voltage-activated N a + channels, resulting in diastolic arrest of the ventricle (Kobayashi et al. 2004). As coronary blood flow is phasic and in concert with ventricular contraction compressing vessels (Axelsson & Farrell 1993), sampling the ventricle during diastole would have obtained an estimate of the maximal coronary reserve and I preferred to minimize the risk of sampling the ventricle in differing stages of the cardiac cycle with inconsistent filling of the coronary vasculature. Previous studies have generally euthanized the fish by cervical dislocation, a method which does not ensure arrest of the heart in diastole. 54 The coronary artery - a prolific angiogenic candidate The rainbow trout coronary artery is a highly responsive and prolific vessel, known to undergo inducible angiogenesis due to mechanical stimulation. Following its experimental ablation, several researchers have reported its re-growth or the appearance of new vessels around the ablation site within a few days to weeks (Daxboeck 1982, Steffensen & Farrell 1998, Farrell et al. 1989). In addition, the coronary artery is especially sensitive to both mechanical (stretch) and flow stimuli, showing significant increases in mitotic activity of the vascular smooth muscle within 2 days of mechanical abrasion (Gong & Farrell 1995). Following repeated stresses of a U c r i t swimming challenge, coronary blood flow has the potential of increasing as much as 200% as rainbow trout swim to exhaustion (Gong et al. 1996; Gamperl et al. 1994). Angiogenesis involves growth from pre-existing capillaries (Hudlicka & Tyler 1986) to form new vascular vessels and capillary beds and its primary stimulus is a mismatch between substrate delivery and the metabolic demand of a tissue (Hudlicka et al. 1992). Anemia-induced cardiac remodeling in mammals has been shown to result in angiogenic microvascular growth of the myocardium (Rakusan et al. 2001). In mammals, angiogenesis can occur by two distinct mechanisms and stimuli, which can operate additively in chronically stimulated muscular contraction (discussed in Egginton 2002). The intra-lumen stimuli of increased blood flow and shear stress causes inward division or splitting of the vessel (Zhou et al. 1998a) whereas abluminal/external stretch stimuli leads to sprouting of new capillaries (Zhou et al. 1998b). Both types of stimuli have been proposed to occur during ventricular hypertrophy in rainbow trout. Egginton (2002) proposes that cold-induced ventricular hypertrophy is resultant from stretch and not hemodynamic forces due to substantial cold-acclimation reductions of blood flow and pressure. Clark & Rodnick (1999) found that both hypertension and hypervolemia are associated with ventricular hypertrophy in sexually-mature male rainbow trout. As PHZ-induced anemia results in volume-loading of the ventricle and increased cardiac output (refer to Chapter 2), it is possible that either shear stress or myocardial stretch associated with increased coronary flow, or the reduced venous oxygen content caused by anemia, provides a stimulatory angiogenic signal. 55 Previous studies of sexually-mature male rainbow trout ventricles generally find a proportional increase of the capillary to myocardial fibre ratio such that the ratio is constant throughout various degrees of hypertrophy (Clark & Rodnick 1998, Clark et al. 2004). As the resultant increase in coronary vascular volume due to PHZ-induced hypertrophy is disproportionate, the increase may in fact be additive and stimulated by both the hypoxic conditions of the anemia itself, in addition to a hypertrophic response. Angiogenesis has also previously been linked to both hypoxemia and increased blood flow (Maxwell & Radcliffe 2002), while mild hypoxia has been shown to modulate cardiac work and stroke volume in rainbow trout ventricles by increasing pre-load or volume-loading the myocardium (Sandblom & Axelsson 2005). While there was a consistent relationship between the r V M and vascular volume in control fishes, fish treated with PHZ displayed exponentially increased vascular volumes. Whether this increase is due to hypoxia, increased cardiac work, or a combination of each remains undetermined. Without morphometric analysis of cardiomyocytes, changes to the vascular volume of the ventricle rely on the assumption that increases of the coronary vascular volume are proportional to its capillarity. The coronary circulation of the rainbow trout ventricle is comprised mainly of the coronary artery and capillary beds within the compact myocardium, with venous drainage limited to the atrioventricular region (Davie & Farrell 1991). Therefore, given the prior documentation of capillary angiogenesis in the compact myocardium of rainbow trout (Clark & Rodnick 1998, Clark et al. 2004, Egginton 2002), it is quite probable that expansion of the vascular volume in the current experiment is due to angiogenic increases of coronary vascular capillarity. Similar increases of coronary reserve in response to ventricular hypertrophy in mammalian hearts is attributed specifically to anemia-induced angiogenesis of the coronary micro vasculature (Rakusan et al. 2001) and angiogenic increase of vascularity in the coronary arteriolar system (Lei et al. 2004, Dedkov et al. 2005). 56 Conclusion I have demonstrated for the first time that there is considerable plasticity in the vascular volume of the rainbow trout coronary circulation. Anemia-induced hypertrophy of rainbow trout ventricles doubled the vascular volume of the coronary circulation. However, without information on blood flow rates during anemia, it is difficult to attribute angiogenesis as a singular response to either increased cardiac work, hypoxia or some combination. 57 Figure 3.1: Experimental set-up for radioisotope tracer analysis. Once transported to the lab, fish were kept anesthetized throughout the procedure by a re-circulating, aerated and buffered solution of MS-222. Lead shielding contained all radioactive isotopes, surgical equipment and tissues. Figure 3.2: Following stannous priming and dual injection of radioisotopes, fish were prepared for tissue sampling by a ventral incision between the pectoral girdles and retraction of the body wall to expose the pericardium. 58 350 2 > o U5 rs > 300 0> 250 c > E rs i-u> 1-o Q. 0i E o 1 0 0 > 200 150 50 h Tc m erythrocyte tracer 125I-BSA albumin tracer Sham control PHZ Figure 3.3: Ventricular vascular volume (ul) of the coronary circulation per gram ventricle of sham control (N=5) or PHZ-treated rainbow trout (/V=3) calculated with either erythrocyte-tracer technetium pertechnetate (Tc"m) or radio-iodinated bovine serum albumin (125I-BSA). The dashed line indicates the mean literature mean (^ =15) based on 5 1 Cr isotope determination by Gingerich et al. (1987) and Gingerich and Pityer (1989). (•&) indicates a significant difference between mean Tc"m and 1 2 5I-BSA, while differing letters indicate a significant increase from sham controls. 59 140 20 A 0 L i 1 1 1 1— 0.00 0.06 0.08 0.10 0.12 Relative ventricular mass Figure 3.4: Comparison of individual ventricular vascular volumes (ul) versus individual relative ventricular masses of rainbow trout treated with phenylhydrazine hydrochloride (PHZ) or sham-injected with saline for four weeks, as measured by radioisotope tracer analysis with technetium pertechnetate 60 Table 3.1: Results of ventricular vascular volume analysis of PHZ-treated rainbow trout, compared with sham control fish (present experiment and from literature) as determined by radioisotope tracer analysis with Tc"m and 1 2 5 I-BSA. Tissue hematocrit (Hct) provides the hematocrit of the coronary circulation and the Hct ratio a comparison to systemic Hct. Body mass (g) Relative ventricular mass Vascular volume (ul) per gram ventricle Tc"m/ 5 1Cr a' b 1 2 5I-BSA Systemic Hct (%) Tissue Hct (%) Hct Ratio PHZ treatment Sham control Control" 1014 ±64 .0 1132 ± 124 445.4 ± 2 5 . 0 0.107 ±0 .005* 0.080 ±0 .007 0.140 ±0 .01 92.0 ± 19.2* 236.0 ±88 .0 N=3 7V=3 42.4 ±5 .33 108.7 ±12 .8 N=5 N=6 18.6 ± 1.2 17.9 ± 3 . 2 63.3 ±10 .7 N=7 /V=7 33.9 ± 2 . 8 44.7 ± 1.7 20.7 ± 7.87 21.0 ± 1.66 0.63 ± 0.24 0.48 ± 0.04 Control" 465.5 ±83 .3 1 4 ± 6 0.39 ±0 .12 Source: Gingerich et al. 1987"; Gingerich & Pityer 1989b •Significance from sham control as assigned by Student's t-test statistic, (P<0.05) 61 Literature Cited Axelsson M. & Farrell A.P. 1993. Coronary blood flow in vivo in the coho salmon (Oncorhynchus kisutch). Am. J. Physiol. 264(5): R963-71. Bri l l R.W., Cousins K.L., Jones D.R., Bushnell P.G. & Steffensen J.F. 1998. Blood volume, plasma volume and circulation time in a high-energy-demand teleost, the yellowfin tuna (Thunnus albacares). J. Exp. Biol. 201: 647-54. Bushnell P.G., Conklin D.J., Duff D.W. & Olson K.R. 1998. Tissue and whole-body extracellular, red blood cell and albumin spaces in the rainbow trout as a function of time: a reappraisal of the volume of the secondary circulation. J. Exp. Biol. 201: 1381-91. Canby C A . & Tomanek R.J. 1990. Regression of ventricular hypertrophy abolishes cardiocyte vulnerability to acute hypoxia. Anat. Rec. 226: 198-206. Carati C.J., Rambaldo S. & Gannon B.J. 1988. Changes in macromolecular permeability of microvessels in rat small intestine after total occlusion ischemia/reperfusion. Microcirc. Endo. Lymph. 4(1): 69-86. Clark J.J., Clark R.J., McMinn J.T. & Rodnick K.J. 2004. Microvascular and biochemical compensation during ventricular hypertrophy in male rainbow trout. Comp. Bioch. Phys. 139B: 695-703. Clark R J . & Rodnick K.J. 1999. Pressure and volume overloads are associated with ventricular hypertrophy in male rainbow trout. Am. J. Physio. 277: R938-46. Clark R.J. & Rodnick K J . 1998. Morphometric and biochemical characteristics of ventricular hypertrophy in male rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 201: 1541-52. Danpure H.J. & Osman S. 1994. Radiolabelling of blood cells - methodology. In: Sampson C B . (Ed.) Textbook of Radiopharmacy - Theory and Practice. Gordon and Breach Science Publishers, Great Britain. Davie P.S. & Thorarensen H. 1997. Heart growth in rainbow trout in response to exogenous testosterone and 17-a methyltestosterone. Comp. Bioch. Phys. 117A: 227-30. Davie P.S., Wells R.M.G. & Tetens V. 1986. Effects of sustained swimming on rainbow trout muscle structure, blood oxygen transport, and lactate dehydrogenase isozymes: evidence for increased aerobic capacity of white muscle. J. Exp. Zool. 237: 159-71. Davis L.E., Hohimer A.R. & Morton M.J. 1999. Myocardial blood flow and coronary reserve in chronically anemic fetal lambs. Am. J. Physiol. 277(1): R306-13. Daxboeck C. 1982. Effect of coronary artery ablation on exercise performance in Salmo gairdneri. Can. J. Zool. 60: 375-81. Dedkov E.I., Christensen L.P., Weiss R.M. & Tomanek R J . 2005. Reduction of heart rate by chronic beta 1-adrenoreceptor blockade promotes growth of arterioles and preserves coronary perfusion reserve in postinfarcted hearts. Am. J. Physiol. Heart Circ. Physiol. 288(6): H2684-93. Dornfest B.S., Lapin D.M., Naughton B.A., Adu S., Korn L. & Gordon A.S. 1986. Phenylhydrazine-induced leukocytosis in the rat. J. Leuk. Biol. 39(1): 37-48. Duff D.W., Fitzgerald D., Kullman D., Lipke D.W., Ward J. and Olson K.R. 1987. Blood volume and red cell space in tissues of the rainbow trout, Salmo gairdneri. Comp. Biochem. Physiol. 87A: 393-8. 62 Egginton S. 2002. Temperature and angiogenesis: the possible role of mechanical factors in capillary growth. Comp. Biochem. Physiol. 132A: 773-87. Farrell A.P., Hammons A . M . , Graham M.S. & Tibbits G.F. 1988. Cardiac growth in rainbow trout, Salmo gairdneri. Can. J. Zool. 66: 2368-73. Franklin C E . & Davie P.S. 1992. Sexual maturity can double heart mass and cardiac power output in male rainbow trout. J. Exp. Biol. 171: 139-48. Gamperl A.K. , Axelsson M . & Farrell A.P. 1995. Effects of swimming and environmental hypoxia on coronary blood flow in rainbow trout. Am. J. Physiol. (Regulatory Integrative Comp. Physiol. 38): R1258-66. Gamperl A.K. , Pinder A. & Boutilier R. 1994. Effect of coronary ablation and adrenergic stimulation on in vivo cardiac performance in trout (Oncorhynchus mykiss). J. Exp. Biol. 186(1): 127-43. Gingerich W.H. & Pityer R.A. 1989. Comparison of whole body and tissue volumes in rainbow trout (Salmo gairdneri) with 125I bovine serum albumin and 51Cr-erythrocyte tracers. Fish Physiol. Biochem. 6(1): 39-47. Gingerich W.H., Pityer R.A. & Rach J.J. 1987. Estimates of plasma, packed cell and total blood volume in tissues of the rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. 87A: 251-6. Gingerich W.H., Pityer R.A. & Rach J.J. 1990. Whole body and tissue blood volumes of two strains of rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 97A: 615-20. Gong, B. & Farrell A.P. 1995. A method of culturing coronary artery explants for measuring vascular smooth muscle proliferation in rainbow trout: the effect of vascular injury. Can. J. Zool. 73: 623-31 Gong, B.Q., Farrell A.P., Kiessling A. & Higgs D. 1996. Coronary vascular smooth muscle responses to swimming challenges in juvenile salmonid fish. Can. J. Fish. Aquat. Sci. 53: 368-71. Graham M.S. & Farrell A.P. 1992. Environmental influences no cardiovascular variables in rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish. Biol. 41: 851-8. 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. Hochachka P.W. 1961. The effect of physical training on oxygen debt and glycogen reserves in trout. Can. J. Zool. Ill: 565-587. Hudlicka O., Brown M.D. & Egginton S. 1992. Angiogenesis in skeletal and cardiac muscle. Physiol. Rev. 72: 369-417. Hudlicka O. & Tyler K.R. 1986. Angiogenesis. Academic Press, London. Itano H.A., Hosokawa K. & Hirota K. 1976. Induction of haemolytic anaemia by substituted phenylhydrazines. Br. J. Haematol. 32(1): 99-104. Jacob E., Drexel M. , Schwerte T. & Pelster B. 2002. Influence of hypoxia and of hypoxemia on the development of cardiac activity in zebrafish larvae. Am. J. Physiol. Regul. Integr. Comp. Physiol. 283(4): R911-7. Kobayashi S., Yoshikawa Y . , Sakata S., Takenaka C , Hagihara H., Ohga Y. , Abe T., Taniguchi S. & Takaki M . 2004. Left ventricular mechanoenergetics after hyperpolarized cardioplegic arrest by nicorandil and after depolarized cardioplegic arrest by KC1. Am. J. Physiol. Heart Circ. Physiol. 287: H1072-80. 63 Lei L, Zhou R., Zheng W., Christensen L.P., Weiss R.M. & Tomanek R.J. 2004. Bradycardia induces angiogenesis, increases coronary reserve and preserves function of the postinfarcted heart. Circulation 110(7): 796-802. McClelland G.B., Dalziel A .C. , Fragoso N.M. & Moyes C D . 2005. Muscle remodeling in relation to blood supply: implications for seasonal changes in mitochondrial enzymes. J. Exp. Bio. 208: 515-22. Maxwell P.H. & Ratcliffe P.J. 2002. Oxygen sensors and angiogenesis. Cell Dev Biol.13(1): 29-37. Poupa O., Gesser H., Jonsson S. & Sullivan L. 1974. Coronary-supplied compact shell of ventricular myocardium in salmonids: growth and enzyme pattern. Comp. Bioch. Physiol. 48A: 85-95. Rakusan K., Cicutti N. & Kolar F. 2001. Effect of anemia on cardiac function, microvascular structure and capillary hematocrit in rat hearts. Am. J. Physiol. Heart Circ. Physiol. 280(3): H1407-14. Sandblom E. & Axelsson M . 2005. Effects of hypoxia on the venous circulation in rainbow trout (Oncorhynchus mykiss). Comp. Bioch. Phys. 140A: 233-9. Steffensen J.F. & 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. 199A: 585-92. Taylor S.E., Egginton S. & Taylor E.W. 1996. Seasonal temperature acclimatization of rainbow trout: cardiovascular and morphometric influences on maximal sustainable exercise level. / . Exp. Bio. 199: 835-45. Zhou, A.-L. , Egginton S., Hudlicka O. & Brown M.D. 1998a. Internal division of capillaries in rat skeletal muscle in response to chronic vasodilator treatment with a l antagonist prazosin. Cell Tissue Res. 293: 293-303. Zhou, A.-L. , Egginton S., Hudlicka O. & Brown M.D. 1998b. Capillary growth in overloaded, hypertrophic adult rat skeletal muscle: an ultrastructural study. Anat. Rec. 252: 49-63. 64 Chapter 4: Effect of cardiac remodeling on the cardiovascular and swimming performance during a U c r i t swimming challenge in rainbow trout (Oncorhynchus mykiss Walbaum) Introduction Cardiac remodeling is a necessary compensatory response in salmonids and mammals to sustained elevations in cardiac workload and the resultant increase in ventricular wall stress (Tota 1993, Friehs et al. 2004). Like mammals, the rainbow trout myocardium responds to changes in mechanical stress, such as pressure and volume overload, by hypertrophic growth of cardiomyocytes which results in ventricular hypertrophy (Clark & Rodnick 1999). Such remodeling is characterized by an overall increase in cardiac size compared to the rest of the body (termed the relative ventricular mass or rVM). Apart from the physiological triggers of cold water acclimation (Farrell et al. 1988) and reproductive increases in androgens (Davie & Thorarensen 1997), ventricular hypertrophy has also been found to be an inducible response to anemia (Davie & Thorarensen 1997, McClelland et al. 2005), which can result in a disproportionate increase to both the compact myocardium (Clark & Rodnick 1998, Graham & Farrell 1992, Chapter 2) and the coronary vascular volume (Chapter 3). There currently exist no studies on the in vivo consequences of ventricular hypertrophy during exercise and whether ventricular hypertrophy affects cardiovascular and swimming performance. Preferential hypertrophy of the compact myocardium has been hypothesized to provide the heart with stabilized wall stress and improved oxygenation via the coronary circulation, which exclusively supplies the compact myocardium (Davie & Thorarensen 1997, Farrell et al. 1988). A greater proportion of arterially-oxygenated musculature might therefore better support increases in cardiac work demand than the spongy myocardium, which relies on a potentially oxygen-limited spongy venous supply. If a disproportionately increased compact myocardium does indeed improve myocardial oxygenation and cardiac output during ventricular hypertrophy, it is hypothesized that it should also improve systemic oxygen delivery and swimming performance in normocythemic fish by delaying myocardial failure due to exercise-induced oxygen deprivation. 65 This hypothesis is based on the suggestion that prolonged swimming in salmonids is limited by maximum cardiac performance (Farrell 2002). As fish exercise, the oxygen requirements of the myocardium increase parallel the increasing cardiac workload. Cardiac output increases 3- to 5-fold during exercise (Farrell & Jones 1992) and myocardial oxygen consumption increases linearly with myocardial power output (Farrell et al. 1985, Farrell 1993, Graham & Farrell 1990). However, as venous oxygen tension (Pvo2) decreases by 2.1-fold at Ucrit (Kiceniuk & Jones 1977), the possibility exists that low Pvo2 limits oxygen delivery to the spongy myocardium. Besides the 2-fold increase in coronary blood flow during exercise (Axelsson & Farrell 1993), the increased reliance on myocardial oxygen delivery via the compact myocardium is also demonstrated by the critical dependence of the coronary circulation in maintaining maximal cardiac function (Davie et al. 1992, Davie & Farrell 1992, Farrell et al. 1990, Steffensen & Farrell 1998, Agnisola et al. 2003). The possibility therefore exists that there may be an inherent oxygen diffusion limitation in myocardium which, when exacerbated by exercise-induced increases in heart rate and systemic oxygen consumption, leads to cardiac dysfunction and swimming failure (Farrell 2002). Based on this potential relationship between swimming performance and myocardial oxygenation, I therefore hypothesized that an increased proportion of arterially-oxygenated compact myocardium would sustain cardiac function to a higher maximal level and result in enhanced swimming ability. Testing of my hypothesis requires that a hypertrophic cardiac state exists in normocythemic fish. This is feasible because of the previous demonstration that erythropoietic recovery following PHZ-induced anemia was significantly faster than the decay of cardiac hypertrophy, which persisted through recovery to normocythemia (Chapters 2 & 3). Thus, a group of rainbow trout was treated with chronic injections of phenylhydrazine hydrochloride (PHZ) to induce cardiac remodeling. When the mean hematocrit (Hct) of the PHZ-treated group had recovered to a level that was statistically comparable with the control group, fish were swum to Ucrit with concurrent measurement of the cardiac output (Q), venous oxygen pressure (Pvo2), heart rate (fH), stroke volume (SV) and oxygen consumption (Mo2). Post-experiment measurement of r V M confirmed that PHZ-treated fish were still hypertrophic. 66 Materials & methods Animal acquisition and care Rainbow trout (Oncorhynchus mykiss Walbaum) were obtained from Aquafarm JV (Fort Langley, British Columbia, Canada) and transported to Simon Fraser University (Burnaby, B C , Canada) in May 2004. Fish were housed in 2,500 1 outdoor fibreglass tanks supplied with dechlorinated, flow-through municipal groundwater and supplemental aeration. The fish were maintained at seasonal water temperatures and fed a maintenance diet of trout pellets from Aquafeed Limited (Chilliwack, B C , Canada). The average body mass was 823 ± 24 g, fork length was 37.4 ± 0.2 cm and condition factor was 1.57 ± 0.01. A l l experimental protocols were approved by Simon Fraser University's University Animal Care Committee in accordance with the Canadian Council on Animal Care. Drug treatment Two weeks prior to PHZ treatment, fish were separated into two groups, a PHZ treatment group (N=20) and a control sham group (N=20). Both groups were housed in proximate, outdoor tanks for the duration of the experimental period. PHZ treatment lasted six weeks during June and July of 2004 and was followed by an 8-week recovery period (refer to Chapter 2 for injection procedure). Six weeks of PHZ treatment has previously been shown to induce a 50% increase in r V M relative to a time-matched sham control (Chapter 2). An 8-week recovery period was used to ensure a similar Hct in both the PHZ-treated and control fish prior to the swimming challenge. To maintain an anemic Hct in the PHZ group, defined by Gallaugher (1995) to be < 20% based on swimming performance, the fish were re-injected on days 0, 17, 29, 39 and 49. Hematocrit was monitored by sampling three fish from each group three to four days post-injection and then on a weekly basis, by methods described in Chapter 2. As rainbow trout decreased their sensitivity to serial PHZ injections, the last three doses of PHZ were doubled (20 ug g BM" 1) . Following 6 weeks of PHZ-treatment, groups were left to recover unperturbed, except for weekly hematocrit checks. Swimming experiments were begun in October 2004, once hematocrits were statistically comparable. Water temperatures varied seasonally from 12°C at the beginning of treatment in June, peaked at 18.5°C during recovery in August and ranged from 12 - 14°C during the swimming experiment. There were no treatment-related mortalities in either group. 67 Surgical procedure The surgical procedure is adapted from methods described by Farrell & Clutterham (2003). Fish were dip-netted and anesthetized in 0.1 g l"1 buffered MS-222, weighed and the fork length, width and depth measurements were recorded. Condition factor was calculated for each fish as body mass/length3 x 100, with body mass in g and length in cm. Fish were placed dorsally on a foam-lined surgery table, covered with a wet cloth and maintained under anesthesia throughout surgery by irrigation of the gills (0.05 g l"1 buffered MS-222). The gills were carefully retracted to expose the cleithrum and protected by a plastic guard. The ventrai aorta, visible on the ventral edge of the cleithrum along the gill arch, was exposed and isolated by careful incision of the outer connective tissue. Once an area large enough to accommodate the flowprobe had been cleared and the aorta had been separated from underlying connective tissue, a 2.0 SB-type Transonic flowprobe (Transonic Systems, Ithaca, N Y , USA) was carefully placed around the aorta. Several silk sutures firmly anchored the probe lead to the skin without disturbing flow in the ventral aorta. To expose the ductus Cuvier, an incision was made on the cleithral surface of the gill arch in a dorso-ventral orientation angled towards the dorso-cranial edge of the gill arch. The ductus Cuvier was carefully cleared of connective tissue, avoiding adjacent nerves and blood vessels running along its caudal edge. A loose silk suture loop was placed over the exposed vessel and the ductus was then gently lifted through the suture center with fine forceps. The suture was securely tightened onto the vessel wall, which was then retracted vertically to minimize blood loss when opened. A small nick was made on the retracted ductus and the implantable, heparin-soaked microsensor fiber-optic oxygen probe (PreSens GmbH, Regensburg, Germany) was gently slid in an antegrade fashion into the ductus. The probe was then securely sutured in the ductus with several sutures and anchored to the inner cleithral surface. The incision was liberally dabbed with penicillin and the fish was then gently turned ventrally in the surgical sling to complete suturing of the lead to the body wall. The flowprobe and oxygen sensor leads were secured together onto the body wall with multiple sutures and led off the cranial edge of the dorsal fin to minimize interference of normal swimming activities, i.e. away from probe lead-entangling pectoral fins (Figure 4.1). The fish was then placed in a Brett-type swim tunnel 68 (as described in Jain et al. 1997) for a 2 h for recovery period prior to the U c r i , swim at a water velocity comfortable to the fish (10 cm s"', Figure 4.2). Hematocrit measurements taken prior to and following surgery confirmed that there was no significant blood loss associated with these procedures (hematocrits varied within 2%). The entire surgical procedure lasted approximately 1 h and the fish quickly recovered from anaesthetization, regaining buoyancy and position within minutes of placement into the swim chamber. Ucrit swim test protocol Each fish was subjected to a modified ramp-critical speed test (U c r i t) as described by Jain et al. (1997). The ramp phase of the swim test consisted of elevating the water velocity in five increments of 5 cm s"' every 5 min, beginning from the recovery velocity of 10 cm s"1, to bring to the fish to approximately 50% of the anticipated U c r i , value. Following the first ramp phase of the U c r i t , each subsequent velocity increment of 5 cm s"1 lasted 15 min or until the fish fatigued. Fatigue was gauged as the inability of the fish to continue swimming or failure to avoid contact with an electrified grid at the read of the tunnel for a maximum of 10 s. The grid provided mild electrical stimulation (2 - 3 V) to discourage the fish from resting. The U c r i t is calculated as: U c r i t = speed of penultimate step + (speed of final step)(proportion of final step completed) Prior to the experiment, the water velocity (cm s"1) in the swim chamber was calibrated to the frequency readings for the pump using a current meter (Valeport Marine Scientific Ltd., Dartmouth, UK) . Triplicate water velocities were measured both at the top and bottom of the tunnel, and were then averaged for each frequency reading. There was little variation between triplicate velocity measurements - readings at each frequency were on average 99.3% similar. Water velocities were adjusted for the solid blocking effect as described by Bell and Terhune (1970) using the length (1), width (w) and area (A) of each fish to calculate the fractional uncorrected swimming speed (Fs) = 0.5(l/w) and the proportional error due to solid blocking (Es) = 0.8(Fs)(A/324.3)''5. A l l water velocities were multiplied by (1 + Es) and standardized to body length per second (BL s"') for comparison and presentation. To allow for standardized comparisons between groups and individual fish, swimming velocities were converted to a percentage of the U c r i t . 69 Following exhaustion, the fish was allowed a two-hour recovery prior to removal from the tunnel and euthanization by anaesthetic overdose. The ventricle, spleen and gonads were weighed (the ventricle to a precision of O.OOOlg) and the ventricle preserved in 70% ethanol. A l l ventricles were dissected by separation of the compact and spongy myocardial layers under dissection microscope, as described in Chapter 2. Besides providing valuable information regarding myocardial composition of the ventricle, dry mass determination is an accurate method to confirm that any increase in the r V M measured by wet mass determination is not due to changes in the water content of the ventricle. To account for variability in body size, wet and dry ventricular mass data were presented as ratios relative to the body mass, as were compact and spongy myocardial masses. Percentages of compact myocardium were based on dry mass data. Of the 16 fish swum in the study, there were 4 males (one saline, three PHZ-treated) and 12 females (7 saline and 5 PHZ-treated). As sexual maturation has been shown to have a significant effect on the r V M of male salmonids (Davie & Thorarensen 1997), both gender and gonadal mass of males were correlated to r V M and found to have no significant confounding effect on r V M . Analysis of cardiovascular data The cardiac output (Q), venous oxygen tension (Pvo2) and heart rate (fH) were all recorded continuously throughout the recovery from surgery, U c r i , swim and recovery. Routine measurements of cardiovascular parameters and oxygen consumption (Mo 2) was measured immediately prior to the swim and during the last 20 minutes of the recovery period following U c r i t . M o 2 was also measured at each 15 minute increment phase during U c r i t . Q and fH were recorded using Labview software (National Instruments, Ontario, Canada) which recorded data in 10 s intervals. For statistical analysis, these variables were averaged into 1 minute blocks. Stroke volume (SV) was calculated as the quotient of Q divided by fH. Calculations and comparisons of Q and SV were normalized to body mass to allow for standardized comparison. To allow the swim data to be compared uniformly between fish, data were also presented as a % U c r j, for each fish. Pvo 2 data was collected using a Microx T X fibre-optic oxygen meter and accompanying software (PreSens GmbH, Regensburg, Germany) which measures oxygen as % air saturation on the principle of dynamic luminescence quenching. The oxygen sensor was calibrated daily using manufacturer-recommended two-70 point calibration with automatic temperature compensation. Data was recorded every 2.6 seconds as an average of 10 data points. To allow for comparable statistical analysis, Pvo 2 was also averaged into 1 minute blocks. Pvo 2 values were recorded as air saturation and were converted to torr with the following equation, where P a t m (mbar) is the atmospheric pressure and Tm (°C) is water temperature: Pvo 2 = [ ( P a t m - (7.16 x 10 ( T m / 1 7 09 )))(% air saturation/100)(0.2095)]/1.33322 Experimental control protocols Two sham fish and three PHZ fish underwent surgical procedures identical to those outlined above for probed fish, without actual probe implantation. As it was expected that the oxygen probe and flowprobe leads would increase drag experienced by fish during the swim, the unprobed fish were utilized as controls to quantify the effects of drag on U c r i t and M o 2 m a x . To ascertain potential effects of the surgical intervention, two additional sham fish were placed directly into the swim tunnel with no surgical intervention whatsoever. Following a 2 h recovery, all control fish were swum to U c r i t with M o 2 measurements using the same protocol as above. To monitor the recovery of the hemoglobin concentration ([Hb]) and Hct following PHZ treatment, and therefore assess the arterial oxygen-carrying capacity of the blood of the swum PHZ group, separate groups of fish (N=6) were injected with 10 pg g B M " 1 of PHZ or saline and the Hct and [Hb] were monitored on a weekly basis during an 8-week recovery. Each fish received an identifying nick on a particular fin to permit identification during the experimental protocol. [Hb] was quantified using the standard cyanmethemoglobin method (Total hemoglobin assay kit, Sigma Diagnostics). Statistics Al l reported data represent the mean ± standard error of the mean (SEM) for each treatment group where JV='8 (except for Pvo 2 where A?=6 as two oxygen probes from each group became nonfunctional during the swim). Statistical comparisons of swim cardiovascular variables between time-matched groups were conducted by A N O V A . Data points within a swim were compared by repeated-measure A N O V A and a Dunnett's test, i f significance was indicated. To ensure that body mass standardization did not result in correlary artifacts, non-normalized data were also confirmed statistically significant by correlating residuals 71 (as per Bennett 1987). A l l statistical analyses were conducted using JMP 5.0 software (SAS Institute Inc., Cary, N C , USA) , except repeated-measures A N O V A which used SigmaStat 3.0 software (SPSS, Chicago, IL, USA). The minimum level for assigned significance is P O . 0 5 . Results Physical and hematological characteristics PHZ treatment had no effect on the physical condition of PHZ-treated fish compared to sham fish. There were no significant differences in body mass, fork length, condition factor, Hct or relative gonadal mass (Table 4.1). The mean sham Hct during the experimental protocol was 34.7 ± 1.0%, ranging individually from 24 - 49%. The PHZ-treated group experienced a 70% decrease in Hct from 35.8 ± 1.6% on day 0 to 10.6 ± 1.5%) a week after PHZ treatment (Figure 4.3). The mean Hct for the PHZ-treated group during the 6-week treatment period was 16.3 ± 1.1%, with individual Hcts ranging from 8 - 30%. The Hct remained significantly lower (P<0.05) than the sham control group throughout the treatment. Following 8 weeks of recovery, there was no difference between the Hct of PHZ-treated and control groups. A separate control experiment assessed the relationship between Hct and [Hb] prior and following PHZ-induced anemia (Figure 4.4). While a proportional relationship was found between Hct and [Hb] (Figure 4.4A), where Hb correlated significantly with the Hct for both groups offish (PO.0001, Hb = 0.299(Hct) + 0.1594), Hb was found to recover more slowly than the Hct in both groups (Figure 4.4B & C). Four weeks following peak anemia, erythropoiesis had substantially increased the PHZ-treated group Hct, such that it was only 16% different from the time-matched sham group (Figure 4.4B). However, the Hb value of the PHZ-treated group remained 50% lower, and little increase occurred in either Hct or Hb during the remainder of recovery (Figure 4.4C). Following an additional four weeks of recovery with no intermittent Hct testing, the sham Hct increased from 27.7 ± 1.5% to 33.3 ± 1.0%, but with no concurrent increase of Hb. Therefore, although there remained a 33% difference in Hct and 38% difference in Hb between groups at the end of the experiment, this is likely due to a lag between Hct and Hb recovery in the sham group whereby despite a 20% increase in Hct between days 51 and 81, there was no change to the [Hb]. The lack of a significance difference between values during the last sampling period likely results from a reduced N value (A/=3 sham controls; N=4 PHZ treatment). 72 While PHZ treatment increased r V M by 27% (PO.005) and dry r V M by 30% (0.0161 ± 0.0009 versus 0.0121 ± 0.0008 respectively, PO.005), there was no significant difference between the % compact myocardium of sham and PHZ-treated groups (Table 4.1). The mean water content of the ventricle was identical between the PHZ-treated group and the sham control group (87.7 ± 0.8% and 87.6 ± 0.3% respectively) and the wet and dry r V M correlated linearly (R 2 = 0.761, PO.0001, r V M = 1.373(dry rVM) -0.0014). PHZ treatment also increased the relative spleen mass by 70% (PO.01). Recovery following surgery and the Ucri, swim test Both groups of fish were found to recover similarly following surgery. Pvo 2 levels rose in exponential fashion and a steady, routine state was reached after 35 minutes in the PHZ-treated group and after 25 minutes in the control group. Cardiac output also rose within minutes of the fish rousing in the tunnel, but was generally elevated for the first hour of tunnel acclimatization. Following a 2 h recovery from surgery, there was no difference between the fH, Pvo 2 , Q per gram of ventricle (g VM" 1 ) or SV of the PHZ-treated group and sham control groups (Table 4.2). However, while Q per kg B M " 1 and routine M o 2 of the PHZ-treated group were significantly elevated compared to sham control at the beginning of the swim, there was no difference in these or any other parameter at the end of the swim recovery. Following the U c r i t swim, control fish required 65 minutes for the mean Pvo 2 , Q and fH values to return within 10% of routine levels, compared to 80 minutes for the PHZ-treated group. This likely reflects a differential metabolic recovery due to the overall longer swims by the PHZ-treated group, rather than differences in the cardiovascular recovery response. Q returned to routine levels in comparable fashion - during the first 10 minutes of recovery, Q dropped 54% in the PHZ-treated group and 53% in the sham control group. Cardiorespiratory and UcrU swimming performance were equivalent There were no differences in the swim duration, distance swum, U c r i t values and M o 2 m a x between the two groups (Tables 4.2 & 4.3). Thus, the elevated routine M o 2 of the PHZ-treated group prior to the swim had no effect on M o 2 m a x . As with the probed fish, there were no significant differences in the size, condition and Hct between the unprobed PHZ-treated and the unprobed sham groups (Table 4.1). Probes resulted in significantly shorter and slower swim as unprobed sham fish swam 40% longer (PO.01) and 75% further than probed sham fish (PO.005), while unprobed PHZ-treated fish swam 38% longer (PO.01) and 62% 73 further (P<0.01) than probed PHZ-treated fish. The probes therefore reduced U c r i t by 23% in sham fish and 24% in PHZ fish. The effect of drag by the probe leads was also reflected by comparing the M o 2 values of probed and unprobed fish (Figure 4.5). Unprobed M o 2 values were lower for the majority of the swim in both treatment groups, indicating that the presence of the probes required 15 - 25% extra metabolic effect in the probed sham and PHZ-treated groups at most speeds. Nevertheless, M o 2 m a x values of the unprobed groups were no different than their probed counterparts. Therefore, all groups of fish swam to the same maximal metabolic capacity, indicating that the significantly decreased U c r i t values of probed fish can be attributed to drag created by the probe leads and that maximal cardiovascular responses were likely unaffected by the presence of probes. Effect of ventricular hypertrophy on cardiac performance during Ucrit Al l routine and maximal cardiovascular data are presented in Table 4.1. Both groups of fish responded similarly to changes in water velocity by increasing Q at each new ramp of the swim, often characterized by brief, compensatory 'overshoots' of Q during the second ramp phase of the swim (Figure 4.6). When compared uniformly as % U c r i t , the PHZ-treated fish increased Q significantly higher throughout the swim, doubling the Q m a x of sham fish (P<0.001, Figure 4.7A). The increase of Q by the PHZ-treated group was due almost exclusively to a higher S V m a x (PO.0001, Figure 4.7B). Linear regression of Q versus SV shows a highly significant correlation to SV (Figure 4.8). To ensure this correlation was not an artifact of the normalization to body mass (Bennett 1987), non-normalized Q and SV data were also correlated and similarly resulted in a highly significant relationship (R 2 = 0.964, PO.0001, Q - 82.92(SV) - 2.7626). PHZ treatment had no effect on the fH of PHZ fish (Figure 4.7C), where sham and PHZ-treated groups experienced similar increases of 24% and 20% respectively. There was no correlation between fH and Q in either group. Q was improved not only by an overall increased myocardial mass (Q per kg BM" 1 ) , but by enhanced myocardial functioning. When Q was also normalized to ventricular mass to account for difference in r V M between control and PHZ-treated groups and analyzed as a % U c r i t , the routine Q per g V M " 1 was identical in both PHZ and control groups (Table 4.2). However, at maximal values, the PHZ-treated group was able to 74 pump an extra 10.6 ml min"1 of blood per g V M " ' , almost 30% higher than control hearts. When maximal Q was plotted versus r V M (Figure 4.9), the relationship was best explained by a bell-shaped curve with maximal Q achieved with a r V M of 0.122. There was no significant relationship between routine Q and r V M . The PHZ-treated group also reached maximal Q significantly sooner during the U c r i t swim than did their control counterparts (Figures 4.6 and 4.7), and unlike control fish, experienced a significant decrease in Q from maximal values to exhaustion values. Thus, for the last 12% of the U c r j , swim, PHZ-treated hearts were not performing maximally. Venous oxygen tension No significant differences existed between PHZ-treated and sham control groups routine Pvo 2 values or at any time during the U c r i , swim and recovery (Figure 4.7D). Both groups experienced a 54 - 56% drop in Pvo 2 during U c r i t (Table 4.1). Therefore, while the PHZ-treated group maximally pumped a 63% larger Q per kg B M " 1 and 28% higher Q per g V M " 1 , they did so with a venous oxygen tension comparable to sham fish. Despite these similarities, while sham fish experienced a steady decline in Pvo 2 throughout the swim (Figure 4.7D), whereas the PHZ-treated group maintained a higher Pvo 2 throughout the swim prior to a 26% insignificant, decline in Pvo 2 around 83% of U c r i t , which corresponds to the significant collapse of Q at the end of the swim. Further, if [Hb] was comparable to the control experiment and remained 38% lower than sham controls, oxygen delivery to the myocardium of the PHZ-treated group might be even lower than reported (assuming an equivalent Hb-0 2 curve between groups). Discussion The protocol of this experiment was particularly challenging due to the necessity to induce cardiac remodeling, while restoring Hct and hopefully the arterial oxygen-carrying capacity. The lengthy recovery period required for a complete recovery of Hct sacrificed the peak period of cardiac hypertrophy (Chapter 2). Nevertheless, a significant 27% increased r V M did ensue in the PHZ-treated group and appeared to directly alter cardiovascular functioning. However, contrary to my hypothesis, there was no significant effect on either the U c r i t or Pvo 2 , nor was there cardiac remodeling with a disproportionately increased compact myocardium. Even so, cardiac remodeling in this experiment was consistent with experimental results obtained at a similar water temperature in Chapter 2, where a proportional increase of both the 75 compact and spongy myocardial layers was observed at 17°C. Cardiovascular results are also consistent with the demonstrated disproportionate increase in coronary vascular volume obtained at a similar water temperature 3 following a 35% increase in r V M (Chapter 3). Therefore regardless of a significant change in % compact, a 27% increased r V M (which increased compact dry mass by 43% and also likely disproportionately increased coronary vascular volume), clearly enhanced Q m a x per kg B M * 1 by 60% and Qmax per g V M " 1 by 30%, modulated by a 60% larger SV. Hematological considerations Rainbow trout demonstrated characteristic hematological responses to PHZ treatment. Hct and Hb both displayed the biphasic recovery response described by Houston & Murad (1995) and also observed in Chapter 2, whereby a rapid increase from anemia is followed by a slower, more gradual recovery. Also, the steady decline in Hct and Hb observed in sham fish following a series of saline injections is a characteristic response to handling stress and has been previously described in goldfish (Houston et al. 1988). As PHZ treatment destroys a great majority of the red blood cell contingent which is then replaced by new, younger cells, it introduces a substantial variability in the mean erythrocytic [Hb] and a likely overall reduction in the Hb content of the blood. In rainbow trout, older erythrocytes have been shown to have 20% more Hb per erythrocyte than younger cells (Phillips et al. 2000). Therefore, while the Hct was nearly identical between the groups swum to U c r i t and a proportional relationship existed between Hct and [Hb], the PHZ-treated group likely had a reduced arterial oxygen transport, as Hb recovery was demonstrated to lag significantly behind the Hct. In accordance with the Fick equation, a reduced oxygen-carrying capacity may explain why the Mo2m a x and Pvo 2 of the PHZ group remained the same as control, despite the fact that & S V m a x of PHZ-treated ventricles pumped 60% more blood than the control value (Mo 2 = Q (CAO 2 - Cvo2)). Multiplying the Hb data for each group following a 6-week recovery in the control experiment to the Q m a x data from the swim experiments results in a nearly proportional ratio (1.15), indicating that the significant increase of Q in the PHZ-treated group may indeed be compensatory for a reduced oxygen-carrying capacity. 76 Cardiac remodeling was therefore shown to significantly compensate for a reduced oxygen-carrying capacity and may therefore represent a chronic, compensatory adaptation to reduced oxygen availability, while allowing for comparable swimming and cardiorespiratory function. In an experiment measuring the acute 3-day cardiovascular response to anemia (Chapter 2), a significant compensatory increase of Q was not recorded until the Hct dropped to 10% or below. As significant increases in r V M were recorded within one week of PHZ injection (Chapter 2), but that the peak decrease in Hct is not obtained until 3-4 days following injection, it is possible that the initial cue to initiate cardiac remodeling may be hypoxia, rather than increased cardiac workload. As cardiac output was not significantly increased until very low Hcts, the acute compensatory response of rainbow trout to anemia may therefore be a reduction of venous oxygen stores, a response that may be physiologically beneficial prior to the onset of cardiac remodeling. While ventricular hypertrophy serves to normalize increased wall stress and myocardial energy demands associated with increased cardiac work demands (Li 1986), chronic increases of cardiac output in an unadapted ventricle can disproportionately increase myocardial oxygen consumption and reduce the mechanical efficiency and stroke volume of the heart (Hansen et al. 2002). Therefore despite a possible deficit in [Hb], rainbow trout in the current study were able to maintain Pvo 2 values equivalent to control fish (presuming an equivalent Hb -0 2 curve), by significantly increasing Q. These differential strategies in coping with increased oxygen demands may reflect an additional advantage of cardiac remodeling, by allowing the fish to maintain an adequate arterial oxygen transport capacity without compromising venous oxygen stores at the systemic level. A significantly increased maximum cardiac output did not improve swimming performance Contrary to the hypothesis, a significantly larger heart and compact myocardium did not improve swimming performance, despite significantly improving cardiovascular performance. Research suggests that improved cardiovascular performance may simply not translate into enhanced locomotory output. Increases of cardiac muscle mass have been found to have little overall change in the physiological performance of zebrafish (Pelster et al. 2003), while Gallaugher et al. (2001) found that high intensity exercise training improved M o 2 and Q in salmon without increasing U c r i t . Similar findings are reported for rainbow trout, where exercise-training resulted in a significantly elevated Q, SV and 25% greater maximum 77 power output generation, with no change to U c r i t (Farrell et al. 1991). Gallaugher et al. (2001) concluded that the benefit of increased Q may be to better "multi-task" other physiological functions while swimming, such as immune function, metabolism or osmoregulation. Improved Q might also be required to meet the elevated metabolic demands associated with the hypertrophic response, as both cardiac protein synthesis and maximal activities of cardiac enzymes associated with mitochondria and oxidative metabolism has been shown to increase in response to elevated cardiac work (Houlihan et al. 1988, Farrell et al. 1990, Farrell et al. 1991). The extent to which a decreased oxygen carrying-capacity would actually impair U c r i t performance remains speculative. Previous research comparing the U c r i t performance of salmon treated with sodium nitrite to induce methaemoglobinaemia found that there was no effect on U c r j t performance until the Hb concentration was below half of control value (Brauner et al. 1993). Additionally, Gallaugher (2001) found U c r i t performance was unaffected until Hcts decreased < 20%, i.e. a 50% reduction in arterial oxygen content. Therefore, while the benefit of cardiac remodeling on swimming performance remains unknown, it may also have gone undetected, given the possible Hb deficit of PHZ-treated fish. Are cardiorespiratory measurements representative? Results obtained in this experiment were comparable to cardiovascular measurements of natural conditions of hypertrophy. Table 4.4 shows the excellent agreement with those of Franklin & Davie (1992) who studied maximal in vitro cardiovascular parameters in a mixed population of rainbow trout with varying degrees of sexual maturation and ventricular hypertrophy (Table 4.4). Based on comparison with these measurements of Q m a x and S V m a X i we can be confident that the induced hypertrophy and ensuing cardiovascular measurements are indeed representative of naturally hypertrophied ventricles. Routine Q for control rainbow trout (13.7 ml min"1 kg"1) is slightly lower than other literature values (17.6 -18.0 ml min"1 kg"1 (Kiceniuk & Jones 1977; Gamperl et al. 1994), as was the Q m a x value (36.5 ml min' 1 kg"l versus 48.7 - 70.0 ml min"1 kg" 1; Thorarensen & al. 1996, Taylor et al. 1996). Nevertheless, the factorial scope for Q of a 2.6-fold is comparable to other studies reported in literature (2.5 to 3.3-fold; Taylor et al. 1996, Kiceniuk & Jones 1977, Thorarensen et al. 1996). With these comparisons, it must be remembered 78 that these fish had an unusually high condition factor (1.5), possibly contributing to low routine Q and Q m a x values, but not to the factorial scope. Routine heart rates for both groups (min"1) were higher than many reported values, which range from 54.2 - 68.7 min"1 (Conklin et al. 1997, Steffensen & Farrell 1998, Gamperl et al. 1995, Sandblom & Axelsson 2005, Gallaugher 1994). The sham routine M o 2 value (1.28 mg min"1 kg"1) was also comparable to literature values, which range from 1.0 - 1.2 mg min"1 kg"1 for rainbow trout (Gallaugher 1994, Henry & Houston 1984, Evans 1990). The elevated routine M o 2 of the PHZ-treated group could be due to many factors including agitation of the fish, an artifact of PHZ treatment or an inadequate recovery time from surgery, although several results suggest that the elevation is likely stress-related rather than physiological. Firstly, there was no difference in routine M o 2 values between sham PHZ-treated and control groups. Secondly, routine Q values for the PHZ-treated group temporarily decreased once the swim began. Despite these initial differences, there were no differences in any cardiovascular parameter at the end of swim recovery, suggesting initial differences were stress-induced. The low routine values of saline fish also nullify the possibility of insufficient surgery recovery time. As the PHZ-treated group was the first group to be swum and would have been exposed to less-practiced experimental protocols, it is quite probable that their initial agitation is due to procedural error. The mean U c r i t value obtained for the unprobed sham fish (1.90 ± B L sec"1) is within the range of reported U c r i , values for uncannulated salmonids of similar size (summarized by Thorarensen et al. 1996): 1.74 -2.65 B L sec"1. There is a much wider range of literature values for U c r i t with probed fish ranging from 0.50 - 2.35 B L sec"1, likely due to variable fish stocks and the extent of surgical interventions (Kiceniuk & Jones 1977, Thorarensen et al. 1996). The mean U c r i , value for control fish in this study, 1.57 ± 0.10 B L sec"1, is within the range of results obtained for rainbow trout swum within the same swim tunnel with a similar oxygen probe (1.32 - 1.98 B L sec"1; Farrell & Clutterham 2003) and was also demonstrated to be significantly reduced by 24% due to drag by the probe leads. 79 The routine Pvo 2 values (21.2 & 23.2 torr) reported here are in good agreement with values for an afferent gill artery (20 - 22 torr, Thomas et al. 1994) and a ventral aortic value of 19 torr (Stevens & Randall 1967). However, the majority of studies report higher Pvo 2 values between 30 - 40 torr (Kiceniuk & Jones 1977, Steffensen & Farrell 1998, Farrell & Clutterham 2003). Specifically, a previous experiment conducted with the same equipment and using similar oxygen probes reported Pvo 2 values of 36.9 torr for rainbow trout held at 6 - 10°C (Farrell & Clutterham 2003). It is possible that a 2 h recovery period was insufficient for the fish to fully recover from the stress of the surgical procedure, and this assumption is supported by a previous study. While the Pvo 2 of cold-acclimated fish were found to have recovered within an hour of a similar surgery (minus the flowprobe) and 2 hour recovery period, fish at warmer temperatures (13 - 15°C, similar to the 12 - 14°C of the current experiment) experience a much more protracted recovery, such that Pvo 2 values were significantly higher following an additional 24 hours. No such difference was observed in cold-acclimated fish. Benefits of cardiac remodeling Similar to results by Davie & Franklin (1992), cardiac remodeling was found to significantly increase S V m a x , resulting in a significantly increased Q m a x with no change to fH. Increasing SV, rather than fH, may be particularly advantageous to benefiting coronary flow, as discussed by Gamperl et al. (1995). Coronary blood flow is not continuous, but phasic to ventricular systole, therefore 75% of coronary blood flow occurs during diastole. Maintaining a low fH leads to longer diastolic periods and increases both the coronary flow to the compact myocardium and allow for longer periods of oxygen diffusion. This effect may have been demonstrated by the significantly improved Q per gVM" ' . While maximal exercise in a normal rainbow trout heart is characterized by the development of myocardial hypoxia and a decrease in the pumping efficiency of the heart due to decreases in the stroke volume (Farrell et al. 1989, Franklin & Davie 1992), the hypertrophied ventricles of the PHZ-treated group demonstrated a significant collapse of cardiac function compared to the control group characterized by drastic decline of Q prior to exhaustion. As well exemplified by the sham group, maximum Q and SV in rainbow trout typically occurs towards the end of U c r i t - for example, Q m a x values are typically obtained at 97.3% of U c r i t 80 (Thorarensen et al. 1996), 94% of U c r i , (Gallaugher et al. 2001) and 96% (present study). In the PHZ-treated group, however, there occurred a significant decline in the output of the ventricle at 88% of U c r j t , induced by a progressive decline in the SV beginning at 84% of U c r i t which also resulted in a precipitous drop in Pvo 2 and oxygen supply to the myocardium. As there was no significant increase in % compact myocardium and presumably of the coronary circulation, the observed cardiac collapse may be due to maladaptive cardiac remodeling, which is characterized by mismatch between increasing myocardium and angiogenesis of the coronary vasculature. In this study, a r V M of around 0.12 may therefore represent the optimal size for maximal oxygen diffusion and functioning of the myocardium, as values above and below this result in reduction of the cardiac scope. Conclusion A 27% increased r V M and proportional increase in compact myocardial mass significantly increased Q m a x and S V m a x by 60%, but failed to improve U c r i t or M o 2 m a x , possibly due to insufficient recovery of the arterial oxygen-carrying capacity in the PHZ-treated group. Although sham and PHZ-treated fish exhausted at the same Pvo 2 , PHZ-treated fish doubled and maintained a higher Pvo 2 during the majority of the U c r i t swim, prior to the cardiac collapse preceding exhaustion, for which a significantly increased ventricular mass and reduced arterial oxygen-carrying capacity may be responsible. Regardless, this experiment provided the first in vivo evidence that ventricular hypertrophy benefits SV and Q through increased mass and myocardial functioning. 81 Figure 4.1: Picture of an anesthetized rainbow trout following the surgical implantation of a PreSens oxygen microsensor probe into the ductus Cuvier (probe lead shown exiting the top of the operculum) and placement of a Transonic flowprobe on the ventral aorta (larger lead exiting the bottom of the operculum). Figure 4.2: The Brett-type swimming tunnel used to assess U c n t and cardiorespiratory performance. The plexiglass swim chamber is 97 cm long with a 21 cm diameter. Water current is produced by a 3-phase induction motor and a centrifugal pump attached to a pre-calibrated tachometer. 82 50 Mean sham group Hct Mean PHZ group Hct 0 20 40 60 80 100 Time (days) Figure 4.3: Hematocrits (Hct) of rainbow trout during treatment and recovery. Data points represent mean triplicate Hcts on three fish from each group, while swim values represent 8 fish per group. (J.) denotes times of injection. The dotted lines represent the mean Hct of both groups during treatment, while the dashed lines indicate Hct values during the recovery period. Dissimilar letters signify a significant difference from day 0 values (ANOVA and Dunnett's test). All points in the PHZ-treated group, prior to swim values, are significantly lower than both day 0 and time-matched sham control values. 83 Figure 4.4: Mean hematocrit (B) and hemoglobin (C) profiles (± SEM) of two groups of rainbow trout treated with PHZ or saline and periodically sampled during an 8-week recovery. N=6 for all data points except those on day 80, where N-3 for saline and N=4 for PHZ. A linear regression (A) between both groups yielded a significant correlation. (•&) denotes a significant difference (P<0.05) between treatment groups by A N O V A . Differing letters signify significant differences from pre-treatment controls as determined by A N O V A and Dunnett's test. 84 / c E E c >< X O 6 h 5 h c 4 > " o CL E 3 in c o o 3 h 2 h A PHZ vs sham control - • - PHZ treatment -®— Sham control B Unprobed PHZ vs Unprobed sham control 20 40 % u„ 60 80 20 40 % U„ 60 80 Figure 4.5: Comparison of the oxygen consumption (Mo 2) of the sham (A"=8) and PHZ (A"=7) rainbow trout groups during a U c r j t swim protocol. Groups were fitted with a flowprobe and microsensor oxygen probe. Unprobed fish underwent a sham surgical procedure without actual probe implantation and were subjected to the same swim protocol. Differing letters represent a significant increase from routine values (0% U c r i t ) ; (*) designate significant increases for sham control fish. 85 0 50 100 150 200 250 300 T i m e (minu tes) * — P H Z t rea tment •@— S h a m c o n t r o l •®— W a t e r ve l oc i t y F i g u r e 4.6: M e a n ca rd iac output ( Q ) ± S E M o f s h a m a n d P H Z - t r e a t e d g r o u p s (A"=8) s w u m to U c r i t , f o l l o w e d b y a 2 h r e c o v e r y . N u m b e r s ind ica te the c h a n g i n g N v a l u e o f the m e a n as i n d i v i d u a l f i sh cease s w i m m i n g . T h e wa te r v e l o c i t y represents the r a m p U c r i , p r o t o c o l fo r the s w i m . T h e m e a n P H Z Q is s i g n i f i c a n t l y h i ghe r f r o m the b e g i n n i n g o f the s w i m u n t i l i n d i c a t e d b y (ft). I n l a y e d is the Q pe r g r a m o f ven t r i c l e ( m l m i n " 1 g V M " 1 ) . 86 O) 'c I 3 a 3 o o re "a k. re O 70 60 50 40 30 20 10 0 " A Cardiac Output |Maximai Q| C Heart rate --Sn — 20 40 60 80 Sham control PHZ treatment 100 o % U 20 40 60 80 100 crit Figure 4.7: Cardiovascular variables of sham control and PHZ-treated rainbow trout swum to U c r i t . All groups have N-8, except Pvo 2 (N=6). Blue and red lines represent ± S E M of the mean, whereas solid green lines represent a significant difference between groups (P<0.05). 87 80 ® Sham routine 0.00 0.20 0.40 0.60 0.80 1.00 0 50 60 70 80 90 100 110 Stroke volume (ml kg1) H e a r t r a t e ( m i n - i ) Figure 4.8: Individual cardiac output (ml min"1 kg'1) values were plotted versus its two primary determinants, stroke volume (ml kg"1) and heart rate (beats min"1). The black dots in the stroke volume plot represent literature values for natural conditions of ventricular hypertrophy in rainbow trout (refer to Table 4.4, Franklin & Davie 1992). 88 70 60 50 40 2 > O) 'c E I £• 30 3 o u re •B re O 20 10 Routine Q • PHZ treatment © Sham control Maximal Q R2=0.290, P=0.1055 • • ~k Mean PHZ treatment t'r Mean sham control • • I ^ Y — I * % • • • • _t 1 1 1 1 1 ® _l< 1 1 0.00 0.08 0.10 0.12 0.14 0.00 0.10 0.12 Relative ventricular mass 0.14 Figure 4.9: Individual routine and maximal cardiac output (Q) values (ml min"1 g VM"1) are plotted versus the relative ventricular mass of sham or PHZ-treated rainbow trout (/V=8). Presented routine Q values are post-swim recovery values, due to elevated pre-swim values in the PHZ-treated group. (*) represent group means (± SEM). 89 Table 4.1: Comparison of the mean physical characteristics (± SEM) between treatment groups equipped with a flowprobe and microsensor oxygen probe (7v=8) and unprobed groups (unprobed sham control, N=4, unprobed PHZ-treated group, JV=3). Body Mass Condition Hematocrit Relative % Compact Relative Relative factor (%) ventricular mass myocardium spleen mass gonadal mass Control 814.0 ±21.8 1.59 ±0.07 35.5 ±2.5 0.099 ± 0.006 36.7 ± 1.86 0.092 ± 0.08 2.2 ± 1.1 PHZ 831.7 ±45.0 1.56 ±0.05 36.0 ±2 .0 0.126 ±0.003* 39.7 ± 2.05 0.154 ±0 .02* 2.3 ±0 .7 Control sham 855.1 ±86.7 1.61 ±0.1 39.9 ±4 .0 0.104 ±0.005 39.8 ± 1.3 0.110 ±0.02 7.6 ±2.3 PHZ sham 794.3 ± 84.8 1.58 ±0.1 36.0 ±4.5 0.121 ±0.008 36.1 ± 1.6 0.166 ±0.03 1.4 ±2.1 •Differs significantly from control group (PO.005). Significance assigned using a Student's t-test statistic. Table 4.2: Summary of routine and maximal cardiovascular variables of sham and PHZ-treated groups measured during a U c r i t swimming test; mean (SEM), A"=8. Sham M o 2 values were measured in separate sham-operated fish (control sham N=4, PHZ sham N=3). Routine values Maximal values Post-swim recovery Variable Sham PHZ-treated Sham PHZ-treated Sham PHZ-treated Cardiac Output 16.8 28.3* 36.5 59.4* 13.7 18.9 (ml min'1 kg'1) (1.40) (2.00) (1.48) (3.50) (0.07) (3.26) Cardiac Output 17.3 22.6 37.3 47.7* 14.2 14.0 (ml min' gV'1) (1.53) (1.45) (2.95) (2.90) (0.66) (1.78) Heart rate 68.2 68.7 84.5 82.7 68.2 66.9 (min1) (1.67) (4.44) (3.48) (2.40) (1.67) (4.42) Pvo2 21.9 21.9 9.3 10.6 21.2 23.2 (torr) (3.1) (2.9) (0.9) (1.5) (3.3) (2.2) Stroke Volume 0.24 0.42 0.46 0.76* 0.21 0.27 (ml kg1) (0.02) (0.06) (0.05) (0.04) (0.01) (0.04) M02 1.28 2.29+* 5.02 5.36 (mg min'1 kg1) (0.10) (0.46) (0.24) (0.24) Sham M02 1.74 1.71 4.31 5.91 (mg min"1 kg"1) (0.24) (0.38) (0.27) (0.88) ^ote: measured prior to swim; sham routine M02 measured at end of recovery •Differs significantly from control value (P<0.05) Significance between routine and maximal values was assigned by Student's t-test statistic. 90 Table 4.3: Summary of the U c r i , swim characteristics (mean ± SEM) of sham and PHZ-treated groups equipped with a flowprobe and oxygen probe (N=8). Unprobed control values were measured in separate, sham-operated fish swum to U c r i , (unprobed sham N=4; unprobed PHZ N=3). Ucrit Swim distance Swim duration (BL sec"1) (km) (min) Sham control 1.57 ± 0.10 2.7 + 0.3 110.3+9.0 PHZ-treated 1.68 + 0.07 3.4 + 0.3 128.7 + 7.7 Unprobed sham 1.91 ±0.07 4.2 ± 0.4* 154.7 ±7 .2* Unprobed PHZ 2.12 ± 0.13* 5 .7±0 .7* 177.7 ± 13.9* *Differs significantly from same-treatment probed group (P<0.05). Significance assigned using a Student's t-test statistic. Table 4.4: Comparison of cardiovascular measurements with literature values of natural, maturation-induced ventricular hypertrophy (*Franklin & Davie 1992). Treatment/Reproductive Status Relative ventricular mass Maximum Q (ml min'1 kg"1) Maximum SV (ml kg1) Sham control 0.099 ± 0.006 36.5 ± 1.5 0.46 ± 0.05 PHZ treatment 0.126 ±0.003 59.4 ±3.5 0.76 ± 0.04 Imm. males & females* 0.076 ± 0.003 36.3 ±2.7 0.53 ± 0.03 Mature males* 0.138 ± 0.011 54.1 ±3.9 0.77 ±0.04 ""refers to % increase of PHZ or mature males groups versus their respective controls. 91 Literature Cited-Axelsson M . & Farrell A.P. 1993. Coronary blood flow in vivo in the coho salmon (Oncorhynchus kisutch). Am J Physiol. 264(5): R963-71. Bell W.M. & Terhune L.D.B. 1970. Water tunnel design for fisheries research. Tech. Rep. Fish. Res. Bd Can. 195: 69. Bennett A.F. 1987. Inter-individual variability: an underutilized resource. In: Feder M.E., Bennett A.F., Burggren W.W. & Huey R.B. (Eds). New directions in ecological physiology. Cambridge University Press, Cambridge. Pp. 147-69. Brauner C.J., Val A .L . & Randall D.J. 1993. The effect of graded methaemoglobin levels no the swimming performance of Chinook salmon (Oncorhynchus tshawytscha). J. Exp. Biol. 185: 121-35. Clark J.J., Clark R.J., McMinn J.T. & Rodnick K.J. 2004. Microvascular and biochemical compensation during ventricular hypertrophy in male rainbow trout. Comp. Biochem. Physiol. 139B: 695-703. Clark R.J. & Rodnick K.J. 1999. Pressure and volume overloads are associated with ventricular hypertrophy in male rainbow trout. Am. J. Physiol. 277: R938-46. Clark R.J. & Rodnick K J . 1998. Morphometric and biochemical characteristics of ventricular hypertrophy in male rainbow trout (Oncorhynchus mykiss). J. Exp. Biol.lQl: 1541-52. Conklin D., Chavas A., Duff D., Weaver L., Zhang Y . & Olson K.R. 1997. Cardiovascular effects of arginine vasotocin in the rainbow trout Oncorhynchus mykiss. J. Exp. Biol. 200: 2821-32. Connaughton M.A. & Taylor M.H. 1994. Seasonal cycles in the sonic muscles of the weakfish, Cynoscion regalis. U.S. Fish. Bull. 92: 697-703. Davie P.S. & Farrell A.P. 1991. The coronary and luminal circulations of the myocardium of fishes. Can. J. Zool. 69: 1993-2001. Davie P.S. Farrell A.P. & Franklin C E . 1992. Cardiac performance of an isolated eel heart - effects of hypoxia and responses to coronary artery perfusion. J. Exp. Biol. 262(2): 113-21. Davie P.S. & Thorarensen H. 1997. Heart growth in rainbow trout in response to exogenous testosterone and 17-a methyltestosterone. Comp. Biochem. Physiol. 117A: 227-30. Davison W. 1989. Training and its effects on teleosts fish. Comp. Bioch. Physiol. 94A: 1-10. Evans D.O. 1990. Metabolic thermal compensation by rainbow trout: effect on standard metabolic rate and potential useable power. Trans. Amer. Fish. Soc. 199: 585-600. Farrell A.P. 2002. Cardiorespiratory performance in salmonids during exercise at high temperature: insights into cardiovascular design limitations in fishes. Comp. Biochem. PhysiolA32A: 797-810. Farrell A.P. 1993. Cardiac output: regulation and limitations. In: Bicudo E. (Ed.), The Vertebrate Gas Transport Cascade: Adaptations to environment and mode of life. CRC Press, Inc, Boca Raton. Pp. 208-14. Farrell A.P. & Clutterham S.M. 2003. On-line venous oxygen tensions in rainbow trout during graded exercise at two acclimation temperatures. J. Exp. Biol. 206: 487-96. Farrell A.P., Hammons A .M . , Graham M.S. & Tibbits G.F. 1988. Cardiac growth in rainbow trout, Salmo gairdneri. Can. J. Zool. 66: 2368-73. 92 Farrell, A.P., Johansen J.A., Steffensen J.F., Moyes C D . , West T.G. & Suarez R.K. 1990. Effects of exercise-training and coronary ablation on swimming performance, heart size and cardiac enzymes in rainbow trout, Oncorhynchus mykiss. Can. J. Zool. 68: 1174-9. Farrell A.P., Johansen J.A. & Suarez R.K. 1991. Effects of exercise-training on cardiac performance and muscle enzymes in rainbow trout, Oncorhynchus mykiss. Fish Physiol. Biochem. 9(4): 303-12. Farrell, A.P. & Jones D.R. 1992. The heart. In: Hoar W.S., Randall D.J. & Farrell A.P. (Eds). Fish Physiology, Vol . XIIA. Academic Press, San Diego, pp. 1-88. Farrell, A.P., Small S. & Graham M.S. 1989. Effect of heart rate and hypoxia on the performance of a perfused trout heart. Can. J. Zool. 67: 274-80. Farrell, A.P., Johansen J.A., Steffensen J.F., Moyes C D . , West T.G. & Suarez R.K. 1990. Effects of exercise-training and coronary ablation on swimming performance, heart size and cardiac enzymes in rainbow trout, Oncorhynchus mykiss. Can. J. Zool. 68: 1174-9 Farrell A.P., Wood S., Hart T. & 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. 117:237-50. Franklin C E . & Davie P.S. 1992. Sexual maturity can double heart mass and cardiac power output in male rainbow trout. J. Exp. BiolAll: 139-48. Friehs I., Moran A . M . , Stamm C , Choi Y . -H . , Cowan D.B., McGowan F.X. & del Nido P.J. 2004. Promoting angiogenesis protects severely hypertrophied hearts from ischemic injury. Ann. Thorac. Surg 11: 2004-11. Gallaugher P., Thorarensen H., Kiessling A. & Farrell A.P. 2001. Effects of high intensity exercise training on cardiovascular function, oxygen uptake, internal oxygen transport and osmotic balance in Chinook salmon (Oncorhynchus tshawytscha) during critical speed swimming. J. Exp. Biol. 204: 2861-72. Gamperl A.K. , Axelsson M. & Farrell A.P. 1995. Effects of swimming and environmental hypoxia on coronary blood flow in rainbow trout. Am. J. Physiol. 38: R1258-66. Gamperl A., Pinder A. & Boutlier R. 1994. Effect of coronary ablation and adrenergic stimulation on in vivo cardiac performance in trout (Oncorhynchus mykiss). J. Exp. Biol. 186(1): 127-43. Graham M.S. & Farrell A.P. 1992. Environmental influences no cardiovascular variables in rainbow trout, Oncorhynchus mykiss (Walbaum). J. Fish. Biol. 41: 851-8. Graham, M.S. & Farrell A.P. 1990. Myocardial oxygen consumption in trout acclimated to 5°C and 15°C Physiol. Zool. 63: 536-54. Hansen J. , Gesser H. & Altimiras J. 2002. Mechanical efficiency of the trout heart during volume and pressure-loading: metabolic implications of the stiffness of the ventricular tissue. J. Comp. Physiol. 172B: 477-84. Henry J.A.C. & Houston A .H . 1984. Asbsence of respiratory acclimation to diurnally cycling temperature conditions in rainbow trout. Comp. Biochem. Physiol. 11 A: 727-34. Houston A .H . & Murad A. 1995. Erythrodynamics in fish: recovery of the goldfish Carassius auratus from acute anemia. Can. J. Zool. 73: 411-18. Houston A .H. , Murad A, & Gray J.D. 1988. Induction of anemia in goldfish, Carassius auratus L., by immersion in phenylhydrazine hydrochloride. Can. J. Zool. 66: 729-36. 93 Jain, K.E., J.C. Hamilton & Farrell A.P. 1997. Use of a ramp velocity test to measure critical swimming speed in rainbow trout, Oncorhynchus mykiss. Comp. Biochem. Physiol. 117A: 441-4. Kiceniuk J.W. & Jones D.R. 1977. The oxygen transport system in trout (Salmo gairdneri) during sustained exercise. J. Exp. Biol. 69: 247-60. Lund S.G., Phillips M.C., Moyes C D . & Tufts B.L. 2000. The effects of cell ageing on protein synthesis in rainbow trout (Oncorhynchus mykiss) red blood cells. J. Exp. Biol. 203: 2219-28. McClelland G.B., Dalziel A . C , Fragoso N.M. & Moyes C D . 2005. Muscle remodeling in relation to blood supply: implications for seasonal changes in mitochondrial enzymes. J. Exp. Biol. 208: 515-22. Pelster B., Sanger A . M . , Siegel M. & Schwerte T. 2003. Influence of swim training on cardiac activity, tissue capillarization, and mitochondrial density in muscle tissue of zebrafish larvae. Am J Physiol Regul Integr Comp Physiol. 285(2):R339-47. Phillips M . C , Moyes C D . & Tufts B.L. 2000. The effects of cell aging on metabolism in rainbow trout (Oncorhynchus mykiss) red blood cells. / . Exp. Biol. 203: 1039-45. Sandblom E. & Axelsson M . 2005. Effects of hypoxia on the venous circulation in rainbow trout (Oncorhynchus mykiss). Comp. Bioch. Physiol. 140A:233-9. Steffensen J.F. & 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. 199A: 585-92. Stevens E.D. & Randall D.J. 1967. Changes of gas concentrations in blood and water during moderate swimming activity in rainbow trout. J. Exp. Biol. 46: 329-37. Taylor S.E., Egginton S. & Taylor E.W. 1996. Seasonal temperature acclimatization of rainbow trout: cardiovascular and morphometric influences on maximal sustainable exercise level. J. Exp. Biol. 199: 835-45. Thomas S., Fritsche R. & Perry S.F. 1994. Pre- and post-brachial blood respiratory status during acute hypercapnia or hypoxia in rainbow trout, Oncorhynchus mykiss. J. Comp. Physiol 164B: 451-8. Thorarensen, H., Gallaugher P.E. & Farrell A.P. 1996. Cardiac output in swimming rainbow trout, Oncorhynchus mykiss. Physiol. Zool. 69: 139-53. Tota B. 1993. Plasticity of the heart and hemodynamic loads: basic and comparative aspects. In: Bicudo J.P.W. (Ed.) The Vertebrate gas transport cascade: adapatations to environment and mode of life. CRC Press, Inc., Boca Raton. 94 Chapter 5: Cardiac remodeling and ventricular vascular volume determination in triploid rainbow trout (Oncorhynchus mykiss Walbaum) Introduction Triploidy is used in fisheries and aquaculture management to produce reproductively-sterile fish. The potential physiological implications of triploidy have been debated for many years and are well discussed in the literature (reviewed by Benfey 1999). Use of triploid salmonids in aquaculture is based on the assumption that sterile fishes would not be subject to production losses associated with the physical deterioration which accompanies sexual maturation (Thorgaard & Gall 1979). Sterile triploids would also help protect the genetic integrity of wild salmonid populations during stocking of waterways or in the event of accidental release. Despite the general consensus that diploid and triploid salmonids are physiologically equivalent in many capacities, triploids continue to harbour a poor reputation for viability, especially under stressful conditions such as cohabitation with diploids (Benfey 1999), high water temperatures (Ojolick et al. 1995, Hyndman et al. 2003) and bacterial gill disease (Yamamoto & Iida 1994). While seemingly comparable to diploids physiologically (Benfey 1999), the most striking morphological difference between diploid and triploid counterparts is the 1.5-fold larger cell size that accommodates the increased nuclear chromosomal content of triploid cells (Benfey 1999). The larger cell size is compensated by an overall reduction in cell number in many tissues, such that the tissues and organs remain the same relative morphological size to those in diploids (Benfey 1999). Despite this inherent difference in cellular morphology, many systematic physiological processes appear to function within normal diploid parameters including the critical swimming performance (Small and Randall 1989, Parsons 1993, Stillwell & Benfey 1996, Altimiras et al. 2002), stress responses (Benfey & Biron 2000, Sadler et al. 2000) and recovery from exhaustive exercise (Hyndman et al. 2003). There is also no difference of in situ maximal cardiac performance (Mercier et al. 2002). Nevertheless, some critical differences are reported in the literature. In particular, the oxygen carrying capacity and hematology characteristics of triploids vary considerably. Compared to diploids, triploids can 95 have a reduced aerobic capacity (Graham et al. 1985, Ojolick et al. 1995, Altimiras et al. 2002, Bernier et al. 2004), as well as a similar aerobic capacity (Small & Randall 1989, Stillwell & Benfey 1996 & 1997, Sadler et al. 2000). While the majority of research suggests there is a compensatory reduction in erythrocytes and total blood hemoglobin to accommodate larger blood cells (Benfey & Sutterlin 1984, Parsons 1993, Biron & Benfey 1994, Benfey 1999, Sadler et al. 2000), a few studies show no significant difference (Stillwell & Benfey 1995, 1996). With the effects of triploidy on the oxygen-carrying capacity of blood and the aerobic capacity of triploid fish yet to be firmly resolved, the situation for cardiac function is very interesting for two reasons: while Mercier et al. (2002) found that maximal cardiac parameters (including heart rate, stroke volume, cardiac output and myocardial power output) of triploid brown trout did not differ significantly from diploid rainbow trout, as measured under ideal perfusion conditions in vitro, they also discovered a heightened sensitivity to ryanodine which may benefit larger cardiomyocytes. In view of this potential cardiac adaptation, which may reflect diffusional limitations at the level of cardiomyocytes, the larger than average erythrocytes and muscle cells of triploids provide an inherently interesting model to study the potential limitations of oxygen delivery in cardiac tissue. Given the implicit differences of triploid cellular morphology, the extent to which cardiac and vascular remodeling may be affected is of particular interest. I was interested in addressing two hypotheses: 1) the coronary vascular volume is increased in triploids to compensate for larger cell size and possibly increased intra-myocardial diffusion distance from the cell membrane to the mitochondria, and 2) triploid cardiomyocytes, already in an inherently compensatory state, may not display comparable cardiac plasticity to diploids. These hypotheses were tested by subjecting diploid and triploid cohorts to a 4-week treatment of phenylhydrazine hydrochloride (PHZ), a protocol previously demonstrated to produce significant cardiac remodeling in diploids (Chapter 2). Control groups of each ploidy were also similarly treated with physiological saline to provide time-matched controls for the injection procedure. The vascular volume of the coronary circulation was then quantified in each group using dual radioisotope tracer analysis with the erythrocyte-tracer technetium pertechnetate (Tc"m) and the plasma-tracer radio-iodinated bovine serum albumin ( 1 2 5 I-BSA). 96 Materials & methods Animal acquisition and care Diploid and triploid rainbow trout (Oncorhynchus mykiss Walbaum) from the same genetic brood line and age class were kindly provided by the Fraser Valley Trout Hatchery (Langley, British Columbia, Canada). Fish of each ploidy were separately transported to Simon Fraser University (Burnaby, B C , Canada) in August 2003. Triploids were created by pressure treatment. Fish were housed in separate indoor 140 1 fibreglass tanks equipped with flow-through, dechlorinated municipal ground water with supplemental aeration to each tank. The fish were fed a maintenance diet of trout pellets from Aquafeed Limited (Chilliwack, B C , Canada) and maintained in a 12L:12D light cycle. A few physical abnormalities were observed in the triploid fish, including a few occurrences of reduced opercula and lordosis, features which have been previously reported for triploid salmonids (Sadler et al. 2000). These fish were included in the ventricular analysis experiment nevertheless as they were likely representative of the population. A l l experimental protocols and procedures were approved by Simon Fraser University's University Animal Care Committee in accordance with the Canadian Council on Animal Care. Cardiac remodeling procedure Cardiac remodeling was measured over a two-week period in January 2004 when the water temperature was 6°C. Triploid fish (N=10) had a mean body mass (BM) of 76.0 ± 3.2 g, a fork length of 17.1 ± 0.3 cm and a condition factor of 1.50 ± 0.02, while diploid fish (N=10) had a mean B M of 121 ± 7.5 g, a fork length of 19.8 ± 0.4 cm and a condition factor of 1.48 ± 0.02. Two weeks prior to the experimental period, fish were divided into two groups of each ploidy - a sham control group (N=20) and a PHZ treatment group (AT=20). On the first day of the experiment, six fish of each ploidy were sampled and the remaining fish were injected with PHZ (10 ug g BM" 1) or physiological saline (100 ul kg BM" 1 ) . Diploids received an additional injection of 5 ug g B M " ' PHZ after one week. However, as triploids displayed respiratory distress during anesthetization, they were not re-injected at week 1 to minimize stress. Six fish from each group were sampled at weeks 1 and 2. Complete sampling and injection procedures are presented in Chapter 2, although no gonadal weights were recorded as fish of both ploidies were sexually immature. 97 Radioisotope tracer analysis Ventricular vascular volume was measured at seasonal water temperatures of 15°C to 17°C during the 4-week experimental period in August 2004. Triploid fish had a mean body mass (BM) of 639 ± 72 g, length of 34.9 ± 1.0 cm and a condition factor of 1.47 ± 0.07, while diploid fish with a mean body mass (BM) of 1089 ± 61 g, length of 39.8 ± 2.6 cm and a condition factor of 1.72 ± 0.057. Diploid fish are the same as those described in Chapter 3. They were acclimated in outdoor 2,500 1 fibreglass tanks with flow-through, dechlorinated municipal ground water and were fed a maintenance diet of trout pellets from Aquafeed Limited (Chilliwack, B C , Canada). A month prior to analysis with radioisotopes, fish of each ploidy were moved into separate, proximate outdoor tanks to create two treatment groups per ploidy - a sham control group (7V=10) and a PHZ treatment group (/V=10). Fish were netted and placed in portable tubs of aerated water for relocation between tanks. Despite their minor handling stress, triploids displayed pronounced signs of respiratory distress including surfacing behaviours and loss of equilibrium, resulting in two triploid mortalities. Diploids displayed no comparable behaviour or mortalities. At the beginning of the 4-week treatment period, fish were injected with either PHZ or saline by methods described in Chapter 2. Within a week of PHZ treatment, there were nine triploid and five diploid mortalities. No mortalities occurred within the control saline-treated fish of either ploidy. Due to the precarious health of the anemic fish, hematocrit was determined during treatment only at the two-week period. After four weeks of treatment, ventricular vascular volume was measured using radioisotope tracer analysis methods described in Chapter 3. A triploid mortality occurred in the sham control group during this procedure. During the netting of the last two control triploids, following sampling of their cohorts from the same tank during the day, one fish lost equilibrium prior to netting and died during transport to the laboratory. Calculations and statistical analyses Calculations used in the determination of vascular volumes by T c " m and 1 2 5 I -BSA and calculation of the tissue hematocrit and hematocrit ratio are detailed in Chapter 3. Comparisons between time-matched 98 treatment and ploidy groups were analyzed using a Student's t-test statistic while changes over the course of an experiment were analyzed by A N O V A and Dunnett's test to compare to pre-treatment control values. A l l data presented in data tables, figures and text are group means ± standard error of the mean (SEM). The minimum level of significance for all statistical analysis is P O . 0 5 . A l l statistical analyses were conducted using IMP 5.0 software (SAS Institute Inc., Cary NC, USA). Results Cardiac Remodeling Experiment There was no difference in either the initial hematocrit (Hct) or relative spleen mass (rSM) of diploid and triploid fish (Figure 5.1). While the sham Hct remained unchanged throughout the experimental period in diploid fish, with a mean value of 32.5 ± 1.5%, the triploid sham group experienced a significant but minor reduction in Hct to 27.8 ± 1.9% by week 2. PHZ treatment caused a comparable decrease of Hct in both diploids and triploids at week 1; Hct dropped to 9.3 ± 1.4% following treatment in the diploid group and 7.3 ± 1.0% in the triploid group. Following a second PHZ injection, the diploid Hct dropped to 5.2 ± 2.2% at week 2. Lack of an additional PHZ injection did not preclude an anemic state in triploids, where Hct remained 7.6 ± 1.8% at week 2. The initial relative spleen mass (rSM) of diploid and triploid controls were similar (0.128 ± 0.03 and 0.106 ± 0.01, respectively). PHZ treatment also significantly increased (PO.0001) rSM by almost three-fold in both diploid and triploid fish following one week (Figure 5.1). While rSM remained elevated in diploids, there was a partial recovery of rSM in triploid by week 2. At the onset of the experiment, neither r V M nor % compact myocardium differed between diploid and triploid fish. In diploid fish, PHZ treatment significantly increased r V M by 8% at week 1 and 20% by week 2, whereas saline injections had no effect on the r V M of sham diploid fish (Figure 5.3). In contrast, saline injections in triploid sham fish caused a 20% increase in r V M by week 1, but no further change by week 2. PHZ treatment in triploids also significantly increased r V M at week 1 (27%) and week 2 (36%). Therefore, by the end of the experiment, both triploid groups had significantly higher r V M than their 99 diploid counterparts: triploid sham rVM was 20% higher than in diploids, while rVM was 16% higher in PHZ-treated triploids than diploids. While there was no difference in the initial dry rVM'or compact myocardial mass of diploid and triploid fish (Figure 5.4), triploids had a significantly higher spongy myocardial mass than diploids. Injection of saline to control diploid fish had no effect while in triploids, there was a significant 17% increase in dry rVM and 14% increase of spongy myocardial mass at week 1. Two weeks of treatment with PHZ in diploids resulted in a significant 17% increase in dry rVM and 16% increase in dry compact myocardium. Conversely, two weeks of PHZ treatment in triploids resulted in a 36% increased dry rVM and 40% increased compact myocardial mass, but no change to spongy myocardial mass. Vascular volume experiment The mean Hct for the diploid sham group during the 4-week post-injection period was 41.4 ± 2.0%, unchanged from the initial Hct of 39.9 ± 2.3%. The diploid PHZ-treated group had a significantly lower Hct than sham controls at both sampling periods; 25.9 ± 1.9% at week 2 and 33.9 ± 2.9% at week 4. The initial triploid Hct (40.3 ± 1.8%) did not differ from diploids, nor did the final Hct at week 4 (41.8 ± 3.7%) differ from diploids (44.7 ± 1.7%). The triploid sham Hct was not measured during the treatment period to prevent additional stress and mortality. Although there was no difference in rVM of diploid and triploid fish at 6°C, the rVM of sham control triploids at 17°C was significantly 20% higher than that of sham control diploids (0.095 ± 0.003 versus 0.080 ± 0.007, Figure 5.2). As the rVM of diploids and triploids were both significantly higher at 6°C than their same-ploidy counterparts at 17°C, it appears that triploids undergo similar cold-induced hypertrophy to diploids. Why the triploid hearts were significantly higher than diploids at 17°C remains unknown. Due to three mortalities during the tracer analysis procedure and discarded results due to low isotope binding efficiencies, vascular volume results are limited to five control diploids and six triploids. In diploids, ventricular vascular volumes estimated by Tc"m and 125I-BSA were 42.4 ± 5.3 ul g VM - 1 and 100 108.7 ± 12.8 ul g VM"1, respectively (Figure 5.5). In triploid rainbow trout, estimates of 69.7 ± 22.2 ul g VM"1 and 76.5 ± 22.2 ul g VM"1 were obtained by the same tracers. There were no significant differences detected between diploid and triploid ventricular vascular volumes by either isotope. When individual coronary vascular volumes were plotted against rVM (Figure 5.6), a significant positive correlation existed between vascular volume and rVM in control diploids (R2=0.970, P<0.005). The tissue Hcts of diploids and triploids differed significantly (P<0.05), 21.0 ± 1.7% versus 45.2 ± 8.7% respectively, as did the Hct ratio. The tissue Hct in triploids approached the true Hct, unlike diploids where the tissue Hct underestimated the systemic Hct (Table 5.1). Similarly, while there was a 2.6-fold difference in the albumin to erythrocyte estimate ratio calculated for diploids, the 1.1 ratio for triploids was a nearly proportional. Discussion Stress induces significant hypertrophy in triploid hearts Triploid rainbow trout were shown to undergo both cold-temperature and anemia-induced ventricular hypertrophy, similar to diploids. Triploids also had a comparable rVM, % compact composition and hematological characteristics to diploids at 6°C. However, despite research that suggests that diploid and triploid rainbow trout exhibit comparable stress responses (Benfey & Biron 2000, Sadler et al. 2000), the triploids in this study also demonstrated significant ventricular hypertrophy in response to handling stress, increasing rVM by 20% within one week with selective enlargement of the spongy myocardium. While diploids have demonstrated a similar but non-significant 20% increase in rVM in response to the injection procedure (Chapter 2), the stress effect in diploids took nearly three-times as long to develop, was in response to repeated PHZ treatment and occured concurrent a seasonal decrease in water temperature. Additionally, no such response was observed in this study, despite an additional injection to the diploids. The cardiac response of triploids to handling stress therefore differs both in its acute onset and degree of a compared with diploids. An additional finding not previously reported is that triploids also experienced significant ventricular hypertrophy at high water temperatures. While there was no difference in the rVM or myocardial composition of diploid and triploid rainbow trout at 6°C, triploid rVM was 20% higher than diploids at 17°C. Cold-induced cardiac remodeling has been hypothesized to compensate for reduced 101 muscle contractility and increased blood viscosity associated with cold water temperatures (Farrell et al. 1988, Graham & Farrell 1989). The requirement for increased cardiac mass, and presumably of increased Q, at warm temperatures may therefore reflect reduced oxygen availability or simply reflect reproductive remodeling cues associated with warm water temperatures. Despite the significant increase in rVM in sham triploid, the PHZ-treated triploid group was shown to undergo similar cardiac responses to anemia as diploids. When the increase in rVM in the PHZ-treated triploid group was corrected for the sham increase in rVM, the change in rVM was quantitatively, similar to that of diploids. This indicates that while the PHZ-treated triploid group demonstrated a significantly higher increase in rVM compared to diploid PHZ-treated fish, the majority of this response was unrelated to anemia. The attribution of greater handling stress sensitivity to triploids is supported by several behavioural observations. Triploids displayed considerable distress including loss of buoyancy, pronounced buccal ventilation and surfacing behaviours in response to transport, handling and sampling of cohorts. Chronic stresses have been hypothesized to unbalance a triploid's tolerance to stress and ultimately result in erythrocyte-limited oxygen delivery to the tissues (Ojolick et al. 1995). Therefore, the combination of a decreased oxygen-carrying capacity (due to high water temperatures, anemia or stress), coupled with potential hypertrophy of an already-enlarged cardiomyocyte, may contribute to the mortalities experienced at high water temperatures. While exposed to the same protocol, diploids showed no such responses. Nevertheless, while only one triploid survived the treatment with PHZ, the PHZ-treated diploid groups also suffered several mortalities, indicating that anemia at warm water temperatures was detrimental in both diploid and triploid rainbow trout. The consequence of larger cardiomyocytes on oxygen diffusion in triploids is debatable. While it is documented that triploids have larger overall muscle fibres (Greenlee et al. 1995, Suresh & Sheehan 1998, Johnston et al. 1999, Mercier et al. 2002), it is also hypothesized that triploid myocytes do not suffer limiting diffusive changes due to their larger size and that possible ploidy effects are reputedly more pronounced in round cells, not elongated ones such as heart cells (Hyndman et al. 2003). Despite the fact that fish cardiomyocytes are indeed smaller and longer in shape than skeletal muscle, their lack of a T-102 tubular network and centrally-located mitochondrion (Santer 1985) may in fact render them more susceptible to diffusional changes associated with alterations of the surface area to volume ratio incurred with cellular hypertrophy (Clark & Rodnick 1998). Seemingly contrary to the idea of oxygen limitations, research has shown that triploid muscle undergoes greater rates of hypertrophic growth than in diploids (Benfey 1999). As triploid muscle has fewer overall cells and thereby fewer satellite cells necessary to recruit new muscle fibres (Greenlee et al. 1995), there is a reduced rate of hyperplasia and significant compensatory increase in muscle fibre hypertrophy (Suresh & Sheehan 1998). Despite the significant differences between skeletal and cardiac muscle, the significant hypertrophic responses herein demonstrated in triploid hearts suggests that cardiac muscle may also undergo pronounced hypertrophy. Triploid rainbow trout may be angiogenically-challenged In addition to potential effects of stress on cardiac structure, triploids may also demonstrate reduced angiogenic tendencies. Despite a significantly higher rVM, triploids did not have a significantly higher vascular volume than diploids, whereas diploids with a 27% increased rVM were shown to have a disproportionately higher ventricular vascular volume compared to sham controls (Chapter 3). Additionally, while there is a significant correlation between rVM and coronary vascular volume in diploids, the same is not true for triploids - a finding likely related to the dissimilar composition of spongy myocardium and the myocardial response to stress. Triploids demonstrated a significant increase in spongy myocardial mass in response to stress, although responded similarly in myocardial compact development If coronary angiogenesis did not accompany concurrent increase of the compact myocardium, oxygen deprivation could occur in the expanding compact myocardium. In addition, as spongy myocardium relies on venous oxygen and maintaining an appropriate diffusion distance (Davie & Farrell 1991), excessive hypertrophy of this myocardial layer in oxygen-poor conditions may prove extremely precarious to the functioning myocardium and further limit oxygen availability. 103 Triploids yield equivalent tracer estimates Several results suggest that triploid hearts may vary structurally from diploids. Firstly, as well reported in literature and corroborated here, plasma tracers traditionally yield much higher vascular volume estimates than do erythrocyte tracers, though 5'chromium has traditionally be used in place of Tc"m (there are no other known measurements of vascular volume in teleosts using Tc m as a tracer). The 125I-BSA-calculated ventricular volume estimate for diploids in this study is 2.6-fold higher than the Tc"m estimate -well within the 3.5-fold differential range reported between erythrocyte and plasma tracer volumes in rainbow trout ventricles (Gingerich & Pityer 1989). However, the tissue Hct ratio in triploids was nearly equivalent, as opposed to the diploid tissue Hct which was nearly half that of the true value, suggesting an equal distribution of isotopes within the vasculature. Therefore, while triploid vascular volume estimates generally tend to be more variable than diploids, the mean isotope volumes calculated by erythrocyte or plasma volume were extremely comparable. The difference between volume estimates for diploids (63 pl g VM"1) is nearly 10 times that of triploids (7 pl g VM"1). The lack of a similar difference between tracer estimates in triploids could indicate an inherent difference in the vascular structure which causes either less 125I-BSA to be extravasated into the ventricular extravascular space or less plasma skimming to occur elsewhere in the vasculature. As albumin is known to lodge in small capillaries (Smith 1994), larger capillaries to accommodate larger triploidic erythrocytes may prevent such accumulation. While the erythrocyte tracer estimates are known to vary with Hct (Smith 1994), most studies report that triploids have equivalent or reduced erythrocyte Hb concentrations (Graham et al. 1985, Parsons 1993, Yamamoto & Iida 1994), therefore it is unlikely that the Tc"m volume is overestimated. Conclusion Triploids were shown to undergo cardiac remodeling in response to warm- and cold-temperature acclimation and anemia comparable to diploid rainbow trout. While triploids were found to have similar rVM and % compact to diploids at 6°C, triploids shams showed considerable cardiac remodeling in response to handling stress, characterized by a disproportionate increase of the spongy myocardium. Correspondingly, radioisotope tracer analysis found the ventricular vascular volume of triploids was not 104 significantly higher than diploids, despite a 20% higher r V M . Contrary to all reported diploid literature, triploids also demonstrated an equivalent ratio of albumin to erythrocyte tracer estimates, suggesting a difference in vascular structure. Therefore, despite some overall similarities to diploid hearts in the ability to remodel, triploids have some significant differences in cardiac remodeling strategies. 105 Figure 5.1: Hematocrit and relative spleen mass of diploid (^ =5; solid lines) and triploid (N=6, dotted lines) rainbow trout treated with PHZ or saline for two weeks; mean ± SEM. Different letters represent a significant difference from control values as determined by ANOVA and Dunnett's test (P<0.05); underlined letters distinguishes significance in triploids. (*) represents a significant difference between treatment groups of a particular ploidy as determined by Student t-test. 106 0.12 0.11 0.10 if> TO E 3 O •= 0.09 5 05 > (0 o 0.08 0.07 h 0.06 Cardiac remodeling experiment Radioisotope analysis experiment Diploid Triploid Diploid Triploid Figure 5.2: Relative ventricular mass (rVM) of control diploid and triploid rainbow trout for the cardiac remodeling experiment (^ =6) and radioisotope tracer analysis experiment (N=7); mean ± SEM. Significant differences between ploidies at different temperatures is indicated by (*), while (*) indicates a significant difference between ploidy groups at a given temperature. 107 Figure 5.3: Effects of PHZ or saline treatment on the relative ventricular mass (rVM) of diploid and triploid rainbow trout (N=6); mean ± SEM. Significant differences between treatment groups of each ploidy are designated with a ), while (*) indicates significant differences (P<0.005) from corresponding diploid groups. Differing letters represent significant increases from pre-treatment control values (ANOVA and Dunnett's test). 108 0.014 0.012 M in ro £ 0.010 0.008 h 0.006 h 0.004 h 0.000 A Spongy myocardium B Compact myocardium C Total ventricular mass —#— 2n sham control A 2n PHZ treatment —•— 3n sham control •A • 3n PHZ treatment 0.022 0.020 D 0.018 3 fi) < CD < <D 0.016 3 c 0.014 0.012 0.000 3 B> in in Time (weeks) Figure 5.4: Changes in the dry myocardial mass of diploid and triploid rainbow trout (A"=6) over a 2-week treatment period with PHZ or saline. Represented are the spongy myocardial mass (A), compact myocardial mass (B) and the total ventricular mass (C). Differing letters represent significant change from control values as determined by ANOVA and Dunnett's test; underlined letters designate PHZ-treated group, while non-underlined letters designate control groups. (*) represents significant differences between both PHZ-treated and control triploid groups compared to their treatment-matched diploid counterparts; (*) or (*) designate significant differences between treatments of a particular ploidy. 109 140 120 « . 100 O TZ +-> c > E DJ k. O a E 3 O > re 40 3 O (fl re > 20 80 60 • Tc m erythrocyte tracer l-BSA plasma tracer ti I Diploid Triploid Figure 5.5: Vascular volume per gram ventricle (pl gV"') of the coronary circulation of diploid (N=5) and triploid (JV=6) rainbow trout (mean ± SEM) using technetium pertechnetate (Tc"m) and radio-iodinated bovine serum albumin (125I-BSA). ( * ) represents a significant difference between the estimates calculated by each tracer. The dotted line represents the literature mean (^ =15) based on 5 1Cr isotope determination by Gingerich et al. (1987) and Gingerich & Pityer (1989). 110 60 © Diploid # Triploid R2=0.970 0 I II 1 1 1 1 0.000 0.060 0.080 0.100 0.120 Relative ventricular mass Figure 5.6: Individual comparison of the relative ventricular mass versus coronary vascular volume (ul) in ventricles of sham control diploid (N=5) and triploid (N-6) rainbow trout. A linear regression is presented for diploid data. Represented coronary vascular volumes were determined by technetium pertechnetate (Tc"m) tracer analysis. I l l Table 5.1: Total ventricular vascular volume per gram ventricle of diploid and triploid rainbow trout determined by dual radioisotope tracer analysis with Tc"m and 1 2 5 I-BSA. Relative Vascular volume (\iV) per gram Body mass ventricular ventricle Systemic Tissue Hct Ploidy (g) mass Tc"m/ 5 ,Cr a' b' c 1 2 5I-BSA Hct (%) Hct (%) Ratio Triploid Diploid Diploid" 639.0 ±71 .6 1132 ± 124 445.4 ± 25.0 0.105 ±0.003 0.080 ±0.007 0.14 ±0.01 69.7 ± 22.2 N=6 42.4 ± 5.3 N=S 18.6 ± 1.2 A"=8 17.9 ±3 .2 N=l 76.5 ± 22.2 N=6 108.7 ± 12.8 N=5 41.8 ± 3 . 7 44.7 ± 1.7 45.2 ± 8 . 7 21.0 ± 1.7 1.13 ±0.34 0.48 ± 0.04 Diploid1" 465.5 ± 83.3 63.3 ± 10.7 N=l 14 ± 6 0.39 ±0.12 References: Gingerich et al. 1987"; Gingerich & Pityer 1989" •Significance was assigned by Student's t-test statistic (P<0.05) 112 Literature Cited Aliah R.S., Inada Y., Yamaoka K. & Taniguchi N. 1991. Effects of triploidy on hematological characteristics and oxygen consumption of ayu. Nippon Suisan Gakkaishi 57: 833-6. Altimars J., Axelsson M., Claireaux G., Lefrancois C, Mercier C. & Farrell A.P. 1992. Cardiorespiratory status of triploid brown trout during swimming at two acclimation temperatures. J. Fish Biol. 60(1): 102-16. Benfey T.J. 1999. The physiology and behavior of triploid fishes. Rev. Fish. Sci. 7(1): 39-67. Benfey T.J. & Biron M. 2000. Acute stress response in triploid rainbow trout (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis). Aquaculture 184: 167-76. Benfey T.J. & Sutterlin A.M. 1984. The haematology of triploid landlocked Atlantic salmon, Salmo salar L. J. Fish Biol. 24: 333-8. Bernier N.J., Brauner C.J., Heath J.W. & Randall D.J. 2004. Oxygen and carbon dioxide transport during sustained exercise in diploid and triploid Chinook salmon (Oncorhynchus tshawytscha). Can. J. Fish. Aqu. Sci. 61: 1797-1805. Biron M. & Benfey T.J. 1994. Cortisol, glucose and hematocrit changes during acute stress, cohort sampling and the diel cycle in diploid and triploid brook trout (Salvelinus-fontinalis mitchill). Fish Phys. Bioch. 13(2): 153-60. Brill R.W., Cousins K.L., Jones D.R., Bushnell P.G. & Steffensen J.F. 1998. Blood volume, plasma volume and circulation time n a high-energy-demand teleosts, the yellowfin tuna (Thunnus albacares). J. Exp. Biol. 201: 647-54. Bushnell P.G., Conklin D.J., Duff D.W. & Olson K.R. 1998. Tissue and whole-body extracellular, red blood cell and albumin spaces in the rainbow trout as a function of time: a reappraisal of the volume of the secondary circulation. J. Exp. Biol. 201: 1381-91. Canby CA. & Tomanek R.J. 1990. Regression of ventricular hypertrophy abolishes cardiocyte vulnerability to acute hypoxia. Anat. Rec. 226: 198-206. Carati C.J., Rambaldo S. & Gannon B.J. 1988. Changes in macromolecular permeability of microvessels in rat small intestine after total occlusion ischemia/reperfusion. Microcirc. Endo. Lymph. 4(1): 69-86. Clark RJ. & Rodnick K.J. 1998. Morphometric and biochemical characteristics of ventricular hypertrophy in male rainbow trout (Oncorhynchus mykiss). J. Exp. Biol. 201: 1541-52. Davie, P.S. & Farrell A.P. 1991. The coronary and luminal circulations of the myocardium of fishes. Can. J. Zool. 69: 1993-2001. Duff D.W., Fitzgerald D., Kullman D., Lipke D.W., Ward J. & Olson K.R. 1987. Blood volume and red cell space in tissues of the rainbow trout, Salmo gairdneri. Comp. Biochem. Physiol. 87 A: 393-8. Egginton S. 2002. Temperature and angiogenesis: the possible role of mechanical factors in capillary growth. Comp. Biochem. Physiol. 132A: 773-87. Gingerich W.H. & Pityer R.A. 1989. Comparison of whole body and tissue volumes in rainbow trout (Salmo gairdneri) with 125I bovine serum albumin and 51Cr-erythrocyte tracers. Fish Physiol. Bioch. 6(1): 39-47. 113 Graham M.S., Fletcher G.L. & Benfey T.J. 1985. Effect of triploidy on blood oxygen content of Atlantic salmon. Aquaculture 50: 133-9. Greenlee A.R., Kersten C.A. & Cloud J.G. 1995. Effects of triploidy on rainbow trout myogenesis in vitro. J. Fish Biol. 46: 381-8. Hyndman C.A., Keiffer J.D. & Benfey T.J. 2003. The physiological response of diploid and triploid brook trout to exhaustive exercise. Comp. Bioch. Physiol. 134A: 167-79. Johnston I.A., Strugnell G., McCracken M.L. & Jonstone R. 1999. Muscle growth and development in normal-sex-ratio and all-female diploid and triploid Atlantic salmon. J. Exp. Biol. 202: 1991-2016. McClelland G.B., Dalziel A.C., Fragoso N.M. & Moyes CD. 2005. Muscle remodeling in relation to blood supply: implications for seasonal changes in mitochondrial enzymes. J. Exp. Biol. 208: 515-22. Mercier C , Axelsson M., Imbert N., Claireaux G., Lefrancois C, Altimiras J. & Farrell A.P. 2002. In vitro cardiac performance in triploid brown trout at two acclimation temperatures. J. Fish Biol. 60: 117-33. Ojolick E.J., Cusack R., Benfey T.J. & Kerr S.R. 1995. Survival and growth of all-female diploid and triploid rainbow trout (Oncorhynchus mykiss) reared at chronic high temperature. Aquaculture 131: 177-87. Parsons G.R. 1993. Comparisons of triploid and diploid white crappies. Trans. Am. Fish. Soc. 122(2): 237-43. Poupa O., Gesser H., Jonsson S. & Sullivan L. 1974. Coronary-supplied compact shell of ventricular myocardium in salmonids: growth and enzyme pattern. Comp. Biochem. Physiol. 48A: 85-95. Sadler J., Pankhurst P.M. & King H.R. 2001. High prevalence of skeletal deformity and reduced gill surface area in triploid Atlantic salmon (Salmo salar L.) Aquaculture 198: 369-86. Sadler J., Wells R.M.G., Pankhurt P.M. & Pankhurst N.W. 2000. Blood oxygen transport, theology and haematological responses to confinement stress in diploid and triploid Atlantic salmon, Salmo salar. Aquaculture 184: 349-61. Santer R.M. 1985. Morphology and innervation of the fish heart. Adv. Ana. Embryol. Cell Biol. 89: 1-99. Skov P.V. & Steffensen J.F. 2003. The blood volumes of the primary and secondary circulatory system in the Atlantic cod Gadus morhua L., using plasma bound Evans Blue and compartmental analysis. J. Exp. Biol. 206: 591-9. Small S.A. & Randal D.J. 1989. Effects of triploidy on the swimming performance of coho salmon (Oncorhynchus kisutch). Can. J. Fish. Aqu. Sci. 46(2): 243-5. Smith C.E., McLain L.R. & Zaugg W.S. 1971. Phenylhydrazine-induced anemia in Chinook salmon. Toxicol. App. Pharm. 20: 73-81. Stillwell E.J. & Benfey T.J. 1997. The critical swimming velocity of diploid and triploid brook trout. J. Fish Biol. 51: 650-3. Stillwell E.J. & Benfey T.J. 1996. Hemoglobin level, metabolic rate, opercular abduction rate and swimming efficiency in female triploid brook trout (Salvelinus fontinalis). Fish Physiol. Biochem. 15: 377-83. Stillwell E.J. & Benfey T.J. 1995. Hemoglobin level, metabolic rate and swimming performance in triploid brook trout (Salvelinus fontinalis). Aquaculture 137: 358-68. 114 Suresh A.V. & Sheehan R.J. 1998. Muscle fibre growth dynamics in diploid and triploid rainbow trout. J. Fish Biol. 52: 570-87. Thorgaard G.H. & Gall G.A. 1979. Adult triploids in a rainbow trout family. Genetics 93: 961-73. Yamamoto A. & Iida T. 1994. Oxygen consumption and hypoxic tolerance of triploid rainbow trout. Fish Path. 29: 245-51. 115 Chapter 6 - Discussion I have used anemia-induced cardiac remodeling to demonstrate that rainbow trout exhibit remarkable ventricular myocardial and angiogenic plasticity. While rainbow trout were well known to increase cardiac mass in response to certain physiological triggers, never before were they shown to undergo inducible hypertrophy with selective myocardial growth in response to anemia and handling stress (diploids preferentially increased compact at cold temperatures, while triploids selectively increased spongy in response to stress). I have also recorded the first measurements of vascular volume in either a hypertrophied or triploid ventricle, providing a volumetric index of the effectiveness of angiogenesis in increasing coronary vascular reserve. While the experiments presented in this thesis could not possibly explore every facet of cardiac remodeling, they did reveal several novel findings which could contribute to the understanding of how rainbow trout adapt so successfully to changes in cardiac workloads, especially in regards to triploid cardiophysiology. The exhibited plasticity of the compact myocardium and the coronary circulation make an especially important contribution to understanding how cardiac remodeling can maintain cardiovascular functioning through an extreme range of temperatures and activities and perhaps more importantly, to determine whether oxygen diffusion limitations are responsible for cardiac malfunction at high levels of activity or hypoxia. Anemia stimulates significant cardiac remodeling As hypothesized, and previously demonstrated by others, anemia-induced volume-loading of the heart was found to be a highly inducible stimulatory signal in rainbow trout, resulting in reproducible and significant increases to cardiac mass and structure. Given the 2 to 3-fold increases in rVM have been reported in spawning male salmonids (Franklin & Davie 1992) and the plateau in % rVM following 8 weeks of anemia, it seems unlikely that anemia induces comparable hypertrophy to sexual maturation. As the degree of cardiac remodeling is known to be dependent on the strength of the stimulus (i.e. exercise training effects, refer to Chapter 1), it could be that anemia induced in these studies was either too variable or not severe or prolonged enough to elicit a signal necessary to induce a similar increase in rVM. However, as the induction process by sexual maturation and anemia are physiologically distinct, so too may be their progression and outcomes. As stated by Tota (1993), "hemodynamic loads stimulate in the heart a complex 116 cascade of short- and long-term adaptive remodelings from molecular and subcellular to cell, tissue and whole organ levels". Differential stimuli could affect any step in the physiological cascade involved in cardiac remodeling, or stimulate different pathways altogether, therefore more controlled experiments would need be conducted to differentiate between the cardiac remodeling induced by each protocol. In addition, as I have studied remodeling at the whole organ and tissue levels, many of the precise mechanisms ongoing at the molecular and cellular levels remain entirely speculative. However, one of the most obvious follow-up experiments would be to conduct morphometric measurements of cardiomyocytes to definitely conclude whether hypertrophic or hyperplastic processes are involved with anemia-induced cardiac remodeling. It is an assumption in these experiments that the vascular volume is representative of the capillarity of the compact myocardium, such that an increased vascular volume is indicative of angiogenesis. While it is impossible to determine whether the increased vascular volume of hypertrophied ventricles resulted directly from the formation of new capillaries or from radial expansion of existing coronary vessels, evidence from prior experiments supports the hypothesis that anemia does trigger angiogenic processes. Firstly, angiogenesis has been demonstrated in rainbow trout during hypertrophy, induced by both cold and sexual maturation by morphometric measurement (Egginton 2002, Clark & Rodnick 1999). In addition, the coronary artery has been found to readily undergo angiogenesis following ablation (Daxboeck 1982, Steffensen & Farrell 1998, Farrell et al. 1989) and the vascular smooth muscle becomes mitotically active following both mechanical and flow stimulus (Gong & Farrell 1995, Gong et al. 1996). Mammalian experiments also use coronary reserve (a measurement of the capacity of the coronary circulation) as an index of angiogenic growth of the coronary capillarity (Dedkov et al 2005, Lie et al. 2004, Davis et al. 1999). Cardiac output was found to increase significantly at low hematocrits, providing stimulus for the long-standing hypothesis that volume-loading and myocardial stretch may trigger the cascade of physiological responses leading to cardiac remodeling, including angiogenesis. Stretch of the coronary epithelium, alongside increased coronary flow during volume-loading of the ventricle, is considered a primary stimulus 117 for coronary angiogenesis (Chen et al. 1994, Tomanek 1990). However, it has been shown that rainbow trout may have differential strategies to compensate for acute or chronic decreases in arterial oxygen-carrying capacity, choosing to negate cardiovascular compensation and rely on venous oxygen stores following an acute reduction in Hct, while significantly increasing cardiac output following chronic anemia. As larger hearts are able to pump higher cardiac workloads and maintain balanced cardiac energetics, PHZ-treated fish were able to not only to compensate for a potential 38% reduced [Hb], but to preserve venous oxygen stores and swim comparably to normocythemic fish. Thus, in addition to the benefits of cardiac remodeling in maintaining elevated levels of cardiac workload, cardiac remodeling may also help preserve systemic oxygen stores. Despite the measured increase in cardiac output following PHZ, and given the inherent reduction in oxygen-carrying capacity associated with anemia, one also cannot discount the possible role of hypoxia in initiating the hypertrophic response - especially in regulating the hematocrit. Hypoxia has emerged as a principle stimulus evoking erythropoiesis and hemoglobin accumulation in vertebrates — it is reasonably decreases in arterial oxygen tension likely play a major role in prompting erythropoiesis stimulating factor and release (Tun & Houston 1986). However, despite the oscillations in hematocrit (and possibly of the cardiac output) during treatment with PHZ, rVM increased over at least eight weeks of treatment. This would indicate that despite a sustained yet variable hypertrophic signal, there was no attenuation of the hypertrophic response. This also supports the idea that the initial mechanical stretch associated with volume-overload triggers a cascade of physiological responses. Similarly, the actual threshold for initiating the hypertrophic reaction was not determined in these experiments. An interesting experiment to determine this variable would be to inject a series of fish with varying concentrations of PHZ (or inducing variable degrees of anemia), to determine at what hematocrit and cardiac output remodeling is initiated. The differences increases observed in myocardial hypertrophy may result from differential stimulatory signals. Present data clearly show that the anemia-stimulated hypertrophy is not equivalent stimulation at different water temperatures. Volume-loading of the myocardium, through an increased cardiac output and venous return (i.e. an inotropic response), is considered the trigger for anemia-induced ventricular 118 hypertrophy and was measured to occur significantly at or below an acute decrease in hematocrit to 10%, once venous oxygen stores were depleted. However, this response was demonstrated at cold water temperatures - the response may not be the same at higher temperatures where trout typically exhibit a compensatory chronotropic cardiovascular response due to reduced water oxygen content (Taylor et al. 1993). It is therefore a noteworthy, but presumptive, possibility that the hypertrophic induction by anemia did not provide an equivalent stimulatory signal at both temperatures, resulting in a differential hypertrophic response. Phenylhydrazine as inducer of hemolytic anemia As previously documented, the chronic use of PHZ had no observable effect on the health of rainbow trout except at high temperatures, demonstrated by comparable physical characteristics between treatment groups in all experiments, although several mortalities occurred in anemic fish at higher temperatures. Perhaps the only side effect of major concern related to the use of PHZ is not related to the drug itself, but of the injection procedure and its associated stress. Besides the ethical duty to keep experimental animals as stress-free as possible, there is also the added concern of keeping anemic fishes stress-free due to their reduced Hct. Injection of PHZ to maintain chronic levels of anemia required each fish to be netted, anesthetized, weight and injected on a regular basis. It also required frequent hematocrit checks on fishes to assess hematocrit levels and determine re-administration of the drug. Such handling has been shown to produce increases in plasma C o r t i s o l and other established indicators of stress (Laidley and Leatherland 1988). Experiments were carefully controlled for possible stress effects of the injection handling. Despite some indication of possible handling stress on the control fish, their rVMs were not significantly different from unperturbed fish, indicating that any handling effect is relatively minor. Conversely, while the stress of daily chasing did not affect rVM whatsoever in either treatment groups, it is possible that chasing did not provide a stress response equivalent to the injection or hematocrit procedure, possibly due to the added protocol of anesthetization. In triploids, however, identical handling stress was demonstrated to be produce a significant increase of rVM, additive to the effects of PHZ-induced anemia. However, other considered 119 methods of PHZ administration were simply deemed not feasible. An alternative method proposed by Houston et al. (1988) involved immersion of goldfishes in dissolved PHZ solutions. While this method would have precluded much handling stress, the induction of anemia was much lengthier and was associated with considerable mortality (42%), not to mention necessitating a non-circulating water supply which was not amenable to the housing of rainbow trout. Therefore, despite the complications associated with injection, the provision of adequate time- and brood-matched controls should control for any confounding stress effect, which were found to be relatively minor in diploids. Interestingly, rainbow trout also demonstrated a consistent decreased sensitivity to PHZ at warm temperatures, necessitating a doubling of dose to elicit consistent decreases in hematocrit, though in a temperature-dependent manner. As most PHZ studies are acute, single injection protocols, this has not been previously reported in rainbow trout. The mechanism of this resistance is unknown but may be due to upregulation or priming of PHZ metabolism by the liver, the possible promotion of anti-oxidant metabolites in the circulation or upregulation of erythropoiesis. Genetic upregulation of compensatory or regulatory mechanisms may be an important factor. Anurans treated with PHZ have shown cytoplasmic inclusions of excess ribosomal RNA, normally synthesized in erythroid cells under physiological stress and thus providing a potential regulatory system for the production of hemoglobin (Cianciarullo et al. 2000). Similar genetic regulation of hemoglobin has also been recorded in polyploid fishes (Leipoldt & Engel 1983). Another major area in need of research is the effects of PHZ on myoglobin. Myoglobin is a respiratory pigment found in muscle, both skeletal and cardiac, which aids in oxygen diffusion to the myocytes. Myoglobin is necessary at low Po2 levels in some teleost hearts to maintain adequate oxygen metabolism (Legate et al. 1998). As it is structurally related to hemoglobin, myoglobin may be similarly denatured by PHZ, but as I was unable to find any research studying the effects of PHZ on myoglobin, its effects are entirely assumed. Any effects of PHZ on myoglobin would be especially important to the swimming challenge, where increasingly depleting Pvo2 levels would make myoglobin function imperative. 120 The triploid conundrum: impressive, but deadly cardiac remodeling Despite research that suggests that diploid and triploid rainbow trout exhibit comparable stress responses and cardiac performances (Benfey & Biron 2000, Sadler et al. 2000, Mercier et al. 2002), I found some significant and previously unreported differences in cardiac structure and stress effects between ploidies of rainbow trout. The principle finding was that triploid exhibited significant ventricular hypertrophy in response to handling stress and high water temperatures, both 20% higher than diploids cohorts at the same conditions. If triploids demonstrate significantly greater abilities to undergo cardiac remodeling - a vital, adaptive physiological process - why did they suffer such morbidity and mortality? Due to the triploid strategy of reducing cell number to accommodate increased cell size (Benfey 1999), triploid muscles have fewer satellite cells necessary to recruit new muscle fibres. In skeletal muscle, triploids therefore experience a reduced rate of hyperplasia, relying instead on a significant compensatory increase in muscle fibre hypertrophy (Greenlee et al. 1995, Suresh & Sheehan 1998). If hyperplasia is equally reduced in cardiac muscle, than any further compensatory hypertrophy of cardiomyocytes could result in even greater increases of the oxygen-diffusion distance, barring a compensatory angiogenic response to maintain diffusion distances. Oxygen availability to the myocardium could be even further jeopardized as triploids were observed to preferentially increase the spongy myocardium following stress, potentially increasing the already precarious oxygen balance between venous oxygen tension and spongy myocytes. Pathological hypertrophy in mammals is characterized by a mismatch between myocyte fibre hypertrophy and angiogenesis which impairs the nutritional and oxygen delivery to the muscle and can lead to eventual cardiac failure (Friehs et al. 2004). I would therefore suggest that in contrast to diploids, the triploid rainbow trout in this study demonstrated a maladaptive, pathological ventricular hypertrophy in response to stress. Nevertheless, radioisotope analysis of the triploid coronary vasculature did reveal another very interesting finding - that plasma and erythrocyte tracers yield nearly proportional estimates. Nearly all experiments measuring blood volumes in teleosts by both plasma and erythrocyte tracers, dating back five decades to the most recent research, report large differences between tracer estimates (Gingerich & Pityer 1989, 121 Gingerich et al. 1987 & 1990, Brill et al. 1998, Bushnell et al. 1998). Hypotheses to explain this trend centre variously on the permeability of fish epithelium and the accumulation of isotopes in the secondary circulation or extravascular space (Gingerich & Pityer 1989, Brill et al. 1998, Bushnell et al. 1998), especially since the mass of the principle plasma protein in trout is greater than that of BSA and could potentially be more susceptible to extravasation from the primary circulation (Ohkawa et al. 1987). Why triploids do not have similar overestimates remains unknown, but could be due to a less permeable vascular epithelium, reduced extravascular space or due to hematological considerations. A more complete hematological analysis of the triploids utilized in the radioisotope analysis would have ameliorated data interpretation of this matter. Benefits of Cardiac Remodeling The main and primary benefit of ventricular hypertrophy is improved cardiovascular performance following increased cardiac demand by maintaining constant systolic-wall stress and thus energy consumption per unit myocardial mass (Jacob et al. 1991). Wall tension is one of the most important parameters of myocardial oxygen consumption therefore as the wall tension increases with increased cardiac output, so too its oxygen consumption in disproportionate amount resulting is reduced mechanical efficiency and stroke volume (Hansen et al. 2002). Compensatory increases in myocardial mass and wall thickness that accompany an excessive hemodynamic load (i.e. volume loading of the ventricle caused by anemia) thus serve to normalize the increased wall stress and myocardial energetics, as described by La Place's law (Li 1986). Besides maintaining balanced cardiac energetics, cardiac remodeling also increases cardiac output and stroke volume by 60%. While the triploid strategy of selectively increasing the spongy myocardium appears contrary to the hypothesis that increasing compact myocardium would better benefit the heart by increasing the arterial oxygen content, there may be other benefits to a higher proportion of spongy myocardium. Spongy myocardium is architecturally designed to develop less transmural tension (Tota et al. 1983) and as wall stress is one of the most important parameters determining myocardial oxygen consumption (Gibbs 1978), a preferential increase of the spongy myocardium may therefore simply be a 122 means of reducing myocardial oxygen demands associated with wall stress and may exceed the benefits of expanding the compact musculature and its arterial coronary circulation. It has been suggested that the small lacunae of the spongy myocardium function as "many small hearts", generating considerable mechanical advantage compared to compact myocardium (Johansen 1965). In addition, spongy myocardium inherently has higher activities of oxidative enzymes than compact myocardium (Tota 1983, Gamperl et al. 1998) and peak activity of cytochrome oxidase in diploids occurs in non-active winter months, coinciding with increased spongy myocardial composition (Farrell et al. 1988). Therefore, while many of the whole organ and tissue characteristics of anemia-induced cardiac remodeling have been explored within this thesis, there remains much interesting research to be done, especially in the field of triploid cardiophysiology where much remains to be studied. By helping to determine how rainbow trout so successfully adapt cardiac function to extreme variations in environment and physiology, it is hoped that a broader understanding of the process can be applied not only to predicting how salmonids will cope with increasing environmental temperatures, but also to understanding why cardiac remodeling remains such a detrimental process in human cardiac disease. 123 Literature Cited Benfey T.J. 1999. The physiology and behavior of triploid fishes. Rev. Fish. Sci. 7(1): 39-67. Benfey T.J. & Biron M. 2000. Acute stress response in triploid rainbow trout {Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis). Aquaculture 184: 167-76. Brill R.W., Cousins K.L., Jones D.R., Bushnell P.G. & Steffensen J.F. 1998. Blood volume, plasma volume and circulation time in a high-energy-demand teleost, the yellowfin tuna (Thunnus albacares). The J. Exp. Biol. 201: 647-54. Bushnell P.G., Conklin D.J., Duff D.W. & Olson K.R. 1998. Tissue and whole-body extracellular, red blood cell and albumin spaces in the rainbow trout as a function of time: a reappraisal of the volume of the secondary circulation. J. Exp. Biol. 201: 1381-91. Chen Y., Torry R.J., Baumbach G.L. & Tomanek R.J. . 1994. Proportional arteriolar growth accompanies cardiac hypertrophy induced by volume overload. Am. J. Physiol. 267(6): H2132-7. Cianciarullo A.M., Bertho A.L. & Meirelles M.D. 2000. Mitochondrial kinetics during amphibian erythropoiesis related to haeme synthesis. Cell Biol. Int.. 24(3): 183-92. Clark R.J. & Rodnick K.J. 1999. Pressure and volume overloads are associated with ventricular hypertrophy in male rainbow trout. Am. J. Physiol. 277: R938-46. Davis L.E., Hohimer A.R. & Morton M.J. 1999. Myocardial blood flow and coronary reserve in chronically anemic fetal lambs. Am. J. Physiol. 277(1): R306-13. Daxboeck C. 1982. Effect of coronary artery ablation on exercise performance in Salmo gairdneri. Can. J. Zool. 60: 375-381. Dedkov E.I., Christensen L.P., Weiss R.M. & Tomanek R.J. 2005. Reduction of heart rate by chronic betal-adrenoreceptor blockade promotes growth of arterioles and preserves coronary perfusion reserve in postinfarcted hearts. Am. J. Physiol. Heart Circ. Physiol. 288(6): H2684-93. Egginton S. 2002. Temperature and angiogenesis: the possible role of mechanical factors in capillary growth. Comp. Biochem. Physiol. 132A: 773-787. Farrell A.P., Hammons A.M., Graham M.S. & Tibbits G.F. 1988. Cardiac growth in rainbow trout, Salmo gairdneri. Can. J. Zool. 66: 2368-73. Farrell, A.P., Small S. & Graham M.S. 1989. Effect of heart rate and hypoxia on the performance of a perfused trout heart. Can. J. Zool. 67: 274-80. Franklin CE. & Davie P.S. 1992. Sexual maturity can double heart mass and cardiac power output in male rainbow trout. J. Exp. Biol. 171: 139-48. Friehs I., Moran A.M., Stamm C, Choi Y.H., Cowan D.B., McGowan F.X. & del Nido P.J. 1994. Promoting angiogenesis protects severely hypertrophied hearts from ischemic injury. Ann Thorac Surg. 77(6): 2004-10. Gong, B. & Farrell A.P. 1995. A method of culturing coronary artery explants for measuring vascular smooth muscle proliferation in rainbow trout: the effect of vascular injury. Can. J. Zool. 73: 623-31. Gong B., Farrell A.P., Kiessling A. & Higgs D. 1996. Coronary vascular smooth muscle responses to swimming challenges in juvenile salmonid fish. Can. J. Fish. Aquat. Sci. 53: 368-71. 124 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. 174: R428-36. Gibbs CL. 1978. Cardiac energetics. Physiol Rev. 58(1): 174-254. Gingerich W.H. & Pityer R.A. 1989. Comparison of whole body and tissue volumes in rainbow trout (Salmo gairdneri) with 125I bovine serum albumin and 5lCr-erythrocyte tracers. Fish Physiol. Bioch. 6(1): 39-47. Gingerich W.H., Pityer R.A. & Rach J.J. 1987. Estimates of plasma, packed cell and total blood volume in tissues of the rainbow trout (Salmo gairdneri). Comp. Bioch. Physiol. 87A(2): 251-6. Gingerich W.H., Pityer R.A. & Rach J.J. 1990. Whole body and tissue blood volumes of two strains of rainbow trout (Oncorhynchus mykiss). Comp. Biochem. Physiol. 97A(4): 615-20. Greenlee A.R., Kersten CA. & Cloud J.G. 1995. Effects of triploidy on rainbow trout myogenesis in vitro. J. Fish Biol. 46: 381-8. Hansen J., Gesser H. & Altimiras J. 2002. Mechanical efficiency of the trout heart during volume and pressure-loading: metabolic implications of the stiffness of the ventricular tissue. J. Comp. Physiol. 172B: 477-84. Houston A.H., Murad A., & Gray J.D. 1988. Induction of anemia in goldfish, Carassius auratus L., by immersion in phenylhydrazine hydrochloride. Can. J. Zool. 66: 729-36. Jacob R., Brandle M., Dierberger B. & Rupp H. 1991. Functional consequences of cardiac hypertrophy and dilatation. Basic Res. Cardiol. 86(1): 113-30. Legate N.J.N., Bailey J.R. & Driedzic W.R. 1998. Oxygen consumption in myoglobin-rich and myoglobin-poor isolated fish cardiomyocytes. /. Exp. Biol. 280: 269-76. Lei L, Zhou R., Zheng W., Christensen L.P., Weiss R.M. & Tomanek R.J. 2004. Bradycardia induces angiogenesis, increases coronary reserve and preserves function of the postinfarcted heart. Circulation 110(7): 796-802. Leipoldt M. & Engel W. 1983. Hidden breaks in ribosomal RNA of phylogenetically tetraploid fish and their possible role in the diploidization process. Biochem Genet. 21(7-8): 819-41. Mercier C, Axelsson M., Imbert N., Claireaux G., Lefrancois C , Altimiras J. & Farrell A.P. 2002. In vitro cardiac performance in triploid brown trout at two acclimation temperatures. J. Fish Biol. 60: 117-33. Ohkawa K., Tsukada Y., Nunomura W., Ando M., Kimura I., Hara A., Hibi N. & Hirai H. 1987. Main serum protein of rainbow trout (Salmo gairdneri): its biological properties and significance. Comp. Biochem. Physiol. 88B: 497-501. Sadler J., Wells R.M.G., Pankhurt P.M. & Pankhurst N.W. 2000. Blood oxygen transport, theology and haematological responses to confinement stress in diploid and triploid Atlantic salmon, Salmo salar. Aquaculture 184: 349-61. Steffensen J.F. & 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. 199A: 585-92. Suresh A.V. & Sheehan RJ. 1998a. Muscle fibre growth dynamics in diploid and triploid rainbow trout. /. Fish Biol. 52: 570-87. 125 Suresh A.V. & Sheehan RJ. 1998b. Biochemical and morphological correlates of growth in diploid and triploid rainbow trout. J. Fish Biol. 52: 588-99. Tomanek RJ. 1990. Response of the coronary vasculature to myocardial hypertrophy. J. Am. Coll. Cardiol. 15(3): 528-33. Tota B. 1983. Vascular and metabolic zonation in the ventricular myocardium of mammals and fishes. Comp. Biochem. Physiol. 76A: 423-27. Tota B., Cimini V., Salvatore G. & 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. Tota B. 1993. Plasticity of the heart and hemodynamic loads: basic and comparative aspects. In: Bicudo J.P.W. (Ed.) The vertebrate gas transport cascade: adapatations to environment and mode of life. CRC Press, Inc., Boca Raton. Tun N. & Houston A.H. 1986. Temperature, oxygen, photoperiod, and the hemoglobin system of the rainbow trout, Salmo gairdneri. Can. J. Zool. 64: 1883-88. 126 

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