Science, Faculty of
Zoology, Department of
DSpace
UBCV
Lo, Wing Man Mandy
Master of Science - MSc
University of British Columbia
An entirely novel mechanism to modulate heart rate was recently discovered in the Pacific hagfish (Eptatretus stoutii): a soluble adenylyl cyclase (sAC)-mediated pathway that increases cyclic adenosine monophosphate (cAMP) production upon stimulation by HCO₃₋ to increase heart rate. However, still unknown is whether this cardiac control pathway is present in other species as well. The objective of my study was to determine the effects of increasing extracellular [HCO₃₋] on the in vitro cardiac function of other fish species and whether the sAC-mediated pathway is associated with recovery of cardiac function during debilitating conditions. Exposure to severe hypoxia (100% N₂) and hypercapnic acidosis (7.5% or 15% CO₂) significantly decreased the heart rate of isolated, freely beating hearts and reduced the isometric tension (contractility) of electrically paced ventricular strips from Pacific lamprey (Lampetra richardsoni), Pacific spiny dogfish (Squalus suckleyi), Asian swamp eel (Monopterus albus), white sturgeon (Acipenser transmontanus), zebrafish (Danio rerio), and starry flounder (Platichthys stellatus). Spontaneous recovery in heart rate or contractility was not observed during severe hypoxia or hypercapnic acidosis for any of the species tested. Addition of HCO₃₋ (up to 50 mM) was associated with a complete and dose-dependent recovery of control heart rate in lamprey, dogfish, and swamp eel hearts during severe hypoxia, and in dogfish, sturgeon, and swamp eel hearts during hypercapnic acidosis. A partial recovery of control heart rate was observed in lamprey and zebrafish hearts during hypercapnic acidosis. However, HCO₃₋ had no effect on the heart rate or contractility in flounder hearts and had little to no effect on restoring control contractility in dogfish, swamp eel, and flounder ventricular strips. The addition of KH7 (sAC blocker) abolished the HCO₃₋-induced recovery of heart rate during severe hypoxia only in the lamprey heart. Thus, the sAC-mediated pathway in cardiac control appears to be unique to the cyclostomes and not present in the other species tested. While the sAC-mediated pathway was associated with the recovery of heart rate in the lamprey heart, the specific mechanisms behind how HCO₃₋ was associated with the recovery of heart rate in the other species still needs to be determined.
EFFECTS OF BICARBONATE ON CARDIAC FUNCTION IN FISH by Wing Man Mandy Lo B.Sc. Hons., University of British Columbia, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Zoology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2017 © Wing Man Mandy Lo, 2017 ii Abstract An entirely novel mechanism to modulate heart rate was recently discovered in the Pacific hagfish (Eptatretus stoutii): a soluble adenylyl cyclase (sAC)-mediated pathway that increases cyclic adenosine monophosphate (cAMP) production upon stimulation by HCO3- to increase heart rate. However, still unknown is whether this cardiac control pathway is present in other species as well. The objective of my study was to determine the effects of increasing extracellular [HCO3-] on the in vitro cardiac function of other fish species and whether the sAC-mediated pathway is associated with recovery of cardiac function during debilitating conditions. Exposure to severe hypoxia (100% N2) and hypercapnic acidosis (7.5% or 15% CO2) significantly decreased the heart rate of isolated, freely beating hearts and reduced the isometric tension (contractility) of electrically paced ventricular strips from Pacific lamprey (Lampetra richardsoni), Pacific spiny dogfish (Squalus suckleyi), Asian swamp eel (Monopterus albus), white sturgeon (Acipenser transmontanus), zebrafish (Danio rerio), and starry flounder (Platichthys stellatus). Spontaneous recovery in heart rate or contractility was not observed during severe hypoxia or hypercapnic acidosis for any of the species tested. Addition of HCO3- (up to 50 mM) was associated with a complete and dose-dependent recovery of control heart rate in lamprey, dogfish, and swamp eel hearts during severe hypoxia, and in dogfish, sturgeon, and swamp eel hearts during hypercapnic acidosis. A partial recovery of control heart rate was observed in lamprey and zebrafish hearts during hypercapnic acidosis. However, HCO3- had no effect on the heart rate or contractility in flounder hearts and had little to no effect on restoring control contractility in dogfish, swamp eel, and flounder ventricular strips. iii The addition of KH7 (sAC blocker) abolished the HCO3--induced recovery of heart rate during severe hypoxia only in the lamprey heart. Thus, the sAC-mediated pathway in cardiac control appears to be unique to the cyclostomes and not present in the other species tested. While the sAC-mediated pathway was associated with the recovery of heart rate in the lamprey heart, the specific mechanisms behind how HCO3- was associated with the recovery of heart rate in the other species still needs to be determined. iv Lay Abstract Animals need to be able to modulate heart activity in order to adapt to changing environmental conditions. During conditions of low O2 or high CO2, heart activity typically declines. To compensate, the brain can communicate with the heart directly or hormones could be released to alter heart activity. This study found that bicarbonate is associated with recovery of heart activity during exposure to low O2 and during high CO2 in several fish species. While the mechanism for the bicarbonate associated recovery in heart rate in the hagfish and lamprey has been discovered, the specific mechanism for the other species remain unknown. A better understanding of how the heart functions and limitations on its performance will allow us to assess the possible impacts of climate change on the physiology, distribution, and survival of different fish species. v Preface This thesis is the original, unpublished, and independent research by the author, Mandy Lo. Jinae Roa from the University of California, San Diego assisted with immunofluorescence imagining of the lamprey heart. Arash Shahriari from the University of British Columbia assisted with some of the lamprey experiments. All other experimental work was conducted at the University of British Columbia and at the Centre for Aquaculture and Environmental Research. I designed and conducted all other experiments, carried out all analyses, and wrote the manuscript under the supervision of Dr. Anthony Farrell. vi Table of Contents Abstract .......................................................................................................................................... ii Lay Abstract ................................................................................................................................. iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables .............................................................................................................................. viii List of Figures ............................................................................................................................... ix List of Abbreviations .....................................................................................................................x Acknowledgments ....................................................................................................................... xii Chapter 1: Introduction ................................................................................................................1 1.1 Cardiac Control in Fish ..................................................................................................1 1.2 Extracellular Changes on Cardiac Function ..................................................................5 1.3 Acidosis..........................................................................................................................6 1.4 Hypoxia ........................................................................................................................13 1.5 Cardiac Support Mechanisms ......................................................................................16 1.6 Soluble Adenylyl Cyclase ............................................................................................17 1.7 Thesis Objectives .........................................................................................................19 1.8 Study Species ...............................................................................................................22 Chapter 2: Research Chapter .....................................................................................................25 2.1 Introduction ..................................................................................................................25 2.2 Materials and Methods .................................................................................................28 2.2.1 Animal Husbandry ........................................................................................28 2.2.2 Chemicals ......................................................................................................29 2.2.3 pH and [HCO3-] Measurements ....................................................................30 vii 2.2.4 Heart Extractions ..........................................................................................30 2.2.5 Heart Rate Measurements .............................................................................31 2.2.6 Contractility Measurements ..........................................................................31 2.2.7 Severe Hypoxia Experiments ........................................................................33 2.2.8 Hypercapnic Acidosis Experiments ..............................................................35 2.2.9 Adrenergic Stimulation Experiments ............................................................36 2.2.10 Immunofluorescence Staining ....................................................................36 2.2.11 Statistical Analysis ......................................................................................37 2.3 Results ..........................................................................................................................38 2.3.1 Severe Hypoxia .............................................................................................38 2.3.2 Hypercapnic Acidosis ...................................................................................40 2.3.3 Adrenergic Stimulation of Lamprey Hearts ..................................................42 2.3.4 Immunofluorescence Staining ......................................................................42 2.4 Discussion ....................................................................................................................50 2.4.1 sAC-mediated Control of Lamprey Heart Rate ............................................50 2.4.2 Adrenergic Stimulation of Lamprey Hearts ..................................................52 2.4.3 Effects of Severe Hypoxia ............................................................................54 2.4.4 Effects of Hypercapnic Acidosis ..................................................................55 2.4.5 Summary .......................................................................................................57 Chapter 3: Conclusion .................................................................................................................59 3.1 General Discussion ......................................................................................................59 3.2 Future Directions .........................................................................................................60 Bibliography .................................................................................................................................64 viii List of Tables Table 1. Experimental conditions during severe hypoxia ..............................................................34 Table 2. Experimental conditions during hypercapnic acidosis ....................................................33 ix List of Figures Figure 2.1. Effects of cumulative NaHCO3 additions on the normalized heart rate of isolated hearts during severe hypoxia in different species ..........................................................................43 Figure 2.2. Effects of cumulative NaHCO3 additions on the normalized contractility of cardiac strips during severe hypoxia in different species ...........................................................................44 Figure 2.3. Effects of cumulative NaHCO3 additions on the normalized heart rate of isolated hearts during hypercapnic acidosis in different species .................................................................45 Figure 2.4. Effects of cumulative NaHCO3 additions on the normalized contractility of cardiac strips during hypercapnic acidosis in different species ..................................................................46 Figure 2.5. Effect of increasing isoproterenol and propranolol concentrations on the normalized heart rate of isolated lamprey hearts during normoxia ..................................................................47 Figure 2.6. Effect of increasing forskolin concentrations on the normalized heart rate of isolated lamprey hearts during severe hypoxia ...........................................................................................48 Figure 2.7. Immunofluorescences staining of sAC in lamprey hearts ...........................................49 x List of Abbreviations ADP ANOVA ATP Ca2+ CaCl2 cAMP CO2 CP DMSO H+ HCN HCO3- If K+ KCl Mg+ MgSO4 N2 Na+ Na2HPO4 NaCl NaHCO3 NaOH NCX O2 Pi PO2 PCO2 Ammonium dihydrogen phosphate Analysis of variance Adenosine triphosphate Calcium ion Calcium chloride Cyclic adenosine monophosphate Carbon dioxide Creatine phosphate Dimethyl sulfoxide Hydrogen ion Hyperpolarization-activated cyclic nucleotide-gated channel Bicarbonate ion Funny current Potassium ion Potassium chloride Magnesium ion Magnesium sulfate Nitrogen gas Sodium ion Disodium phosphate Sodium chloride Sodium bicarbonate Sodium hydroxide Sodium/calcium exchanger Oxygen gas Inorganic phosphate Partial pressure of oxygen Partial pressure of carbon dioxide PKA Protein kinase A xi sAC s.e.m UBC tmAC TMAO SR Soluble adenylyl cyclase Standard error of the mean University of British Columbia Transmembrane adenylyl cyclase Trimethylamine N-oxide Sarcoplasmic reticulum xii Acknowledgements I would like to first express my gratitude to my supervisor, Dr. Anthony Farrell, for his supervision and never ending support through my research. His advice, wisdom, and patience has helped me tremendously and this thesis would not have been possible without him. I would also like to thank my committee members, Dr. Colin Brauner and Dr. Phil Matthews for offering their guidance and valuable feedback. I am also grateful to my fellow colleagues in the Department of Zoology and everyone in the Farrell Lab. Special thanks to Adam Goulding, Helen Drost, Yangfan Zhang, Rachel Sutcliffe, Matthew Gilbert, Zhongqi Chen, James Marchant, Arash Shahriari, Phillip Morrison, Mike Sackville, Kevin Stiller, Xiang Lin, Naomi Pleizier, and Graham Smyth for all their help. Finally, I would like to thank my parents for all their unwavering support and encouragement along the way. 1 Chapter 1: Introduction Understanding the mechanisms and adaptations that allow the heart to adjust to different environmental conditions has broad relevance to and implications for ecology and conservation. This is because the heart is a crucial part of the cardiovascular system that supplies oxygen and nutrients to, and removes metabolic wastes from all parts of the body. The heart must respond appropriately whenever organisms encounter new or debilitating environmental conditions, such as warmer temperatures when metabolic rate increases, during activity when their tissues need more O2, or when they encounter environmental hypoxia. Thus, cardiac output must be tightly regulated to maintain normal physiological function. To regulate cardiac output, the rate of cardiac beating (heart rate) can be changed as well as the volume pumped with each heartbeat (cardiac stroke volume), which is affected by the force of muscular contraction. The rest of this introduction will provide background information on how cardiac output can be regulated, the effects low O2 and high CO2 on cardiac output, and an entirely novel mechanism to modulate heart rate that was recently discovered in the hagfish. Thus, the objective of my thesis is to examine whether the same mechanism to increase cardiac output in the hagfish is present in other fish species as well. 1.1 Cardiac Control in Fish The beating of the heart is regulated by pacemaker cells, which spontaneously generate action potentials and set the intrinsic heart rate without any external signaling. The intrinsic heart rate is set by the speed with which the pacemaker potential depolarizes the pacemaker cell membrane to reach the threshold voltage to initiate an action potential. Autonomic nervous controls (excitatory sympathetic (adrenergic) and inhibitory parasympathetic (vagal) innervation 2 from the brain) can then modulate heart rate, while temperature, pH, concentrations of various ions, and oxygen availability can also directly modulate pacemaker rates (Randall 1970). Cardiac stroke volume is set intrinsically by the amount of cardiac filling (through the Frank-Starling mechanism; Starling and Visscher 1927), which sets end-diastolic volume, and by the strength of cardiac contraction (generally referred to as contractility), sets end-systolic volume. Similarly, contractility has an intrinsic capacity and is also under autonomic nervous control. As with heart rate, environmental conditions (hypoxia, acidosis, extracellular ionic concentrations) can also modulate the force of cardiac contraction by altering the ionic currents involved in cardiac excitation-contraction coupling (Vornanen et al. 2002). One control mechanism common to all vertebrate hearts is through β-adrenergic stimulation (Holmgren 1977, Nilsson 1981) by catecholamines (adrenaline and noradrenaline). Adrenaline is released into the blood stream from either the adrenal gland or other chromaffin tissues while noradrenaline is released from sympathetic nerve terminals in the heart (Burnstock 1969). These catecholamines then bind to β-adrenergic receptors on the cardiomyocyte cell membrane to activate a transmembrane adenylyl cyclase (tmAC) to increase heart rate and contractility (Drummond and Duncan 1970). tmAC stimulates cAMP production (Sutherland et al. 1962) and cAMP modulates heart rate by binding to hyperpolarization-activated cyclic nucleotide-gated (HCN) channels to alter their probability of opening (Wainger et al. 2001). HCN channels allow Na+ and K+ to slowly enter the cytoplasm (collectively termed the funny current, If), which progressively depolarizes the pacemaker cell membrane. By activating cAMP-dependent protein kinase A (PKA), HCN channels are phosphorylated to further increase their probability of opening (Chang et al. 1991; Wainger et al. 2001). This speeds up the rate of membrane depolarization and reduces the time needed until the cell membrane potential reaches 3 the threshold potential to open T-type Ca2+ channels, which allow Ca2+ to rush into the cell (Hagiwara et al. 1988, Bers 2002). Increased cytosolic [Ca2+] in the pacemaker cell then induces L-type Ca2+ channels to open, and even more Ca2+ enters the cytosol across the sarcolemma, thus generating the depolarization phase of the action potential (Hagiwara et al. 1988, Bers 2002). The action potential generated by the pacemaker cells then quickly spreads to the contractile cardiomyocytes, triggering the heart to beat in a coordinated fashion. T-type Ca2+ channels then begin to close and voltage-gated K+ channels open, repolarizing the pacemaker cell and completing the action potential cycle (Irisawa et al. 1993). This model is referred to as the membrane clock. A second model, referred to as the calcium clock, suggests that the spontaneous cycling of Ca2+ to and from the sarcoplasmic reticulum (SR) and the cytosol is responsible for initiating, sustaining, and regulating the rhythm observed in pacemaker cells (Lakatta 1992; Maltsev and Lakatta, 2007). The model suggests that Ca2+ stores are released from the SR before each action potential. The rhythmically released Ca2+ then activates the Na+/Ca2+ exchanger (NCX) inward current, depolarizing the pacemaker cell until the opening threshold potential for T-type Ca2+ channels is reached and an action potential is then generated. In this model, β-adrenergic stimulation would shorten the duration of each Ca2+ release and uptake cycle, thereby increasing the action potential firing rate via effects on SR Ca2+ cycling proteins (Maltsev et al. 2014). While debate exists over the relative importance of the two models in the rhythmic control of cardiac pacemaker cells, they likely interact with each other to determine the intrinsic pacemaker rate (Capel and Terrar 2015). In addition to modulating heart rate, increased cAMP production following β-adrenergic stimulation also activates more PKA to phosphorylate L-type Ca2+ channels, allowing for 4 increased entry of Ca2+ into the cytosol to increase the contractility of ventricular cardiomyocytes (Gao et al. 1997). More Ca2+ enhances myosin cross-bridge formation and contractility by increasing Ca2+ binding to troponin C (Vornanen et al. 2002). In mammals, the magnitude of Ca2+ influx across the sarcolemma (via L-Type Ca2+ channels) varies in different species, and the influx is believed to be insufficient to support contraction (Bers 1985). The influx of Ca2+ then induces the release of much more Ca2+ from the SR (via ryanodine receptors), and it has been proposed that the Ca2+ released from the SR is a major source of Ca2+ for contraction (Fabiato and Fabiato 1978; Tibbits et al.1991). However, the relative importance of the SR Ca2+ release compared to L-type Ca2+ influx in supplying cytosolic Ca2+ to bind to troponin C and to modulate cardiac muscle contraction appears to be very species dependent among fishes and even affected by temperature. In rainbow trout (Oncorhynchus mykiss) hearts, Ca2+ release from the SR appears to not contribute to pacemaker activity at low temperatures (<11 °C), but at 18 °C, inhibition of SR Ca2+ cycling with ryanodine and thapsigargin depressed heart rate by 44% (Haverinen and Vornanen 2007). More active fishes such as the yellowfin tuna (Thunnus albacares; Shiels et al. 1999) and skipjack tuna (Katsuwonus pelamis; Keen et al. 1992) appear to have a greater reliance on SR Ca2+ release compared to less active species such as the goldfish (Carassius carassius L.; Vornanen 1989), and in benthic and sluggish plaice (Pleuronectes platessa), the SR is entirely absent (Santer and Cobb 1972). However, in isolated hagfish hearts, the addition of ryanodine had no effect on their heart rate (Wilson 2014), suggesting that Ca2+ release from the SR is likely of little importance for their regulation of heart rate. Inhibition of heart rate and contractile force of the heart, in contrast, involves direct vagal stimulation and the release of acetylcholine, which bind to muscarinic receptors (Holmgren 5 1977). Muscarinic receptors are linked to an inhibitory G-protein that when stimulated inhibits tmAC, which decreases cAMP production (Jakobs et al. 1979). Acetylcholine also increases K+ permeability in the pacemaker cell, causing it to hyperpolarize and take longer to reach threshold potential, and decreases Ca2+ permeability, slowing the rate of depolarization. Both mechanisms slow intrinsic heart rate (DiFrancesco and Tromba 1988). In most teleosts, the heart receives dual (adrenergic and vagal) autonomic innervation, but the degree of adrenergic innervation varies among species (Taylor et al. 1999), and is even absent in the plaice (Pleuronectes platessa) (Cobb and Santer 1973). 1.2 Extracellular Changes on Cardiac Function Changes in external conditions can have different effects on cardiac function and thus the normal functioning of the heart can be disrupted. Acidosis, which can be brought on by exercise or high environmental CO2 levels, can reduce heart rate and contractility, while hypoxia can also have similar negative effects on the heart (Driedzic and Gesser 1994). Decreases in temperature can lower heart rate by hyperpolarizing the resting membrane potential, prolonging action potential duration and reducing ion channel activity (Cranefield and Hoffman 1958). Contractility can be lowered with decreases in temperature, due to decreases in Ca2+ sensitivity of the contractility system (Gillis et al. 2000), but increases in contractility has been seen in some other species (like in mammals and amphibians), due to changes in force generation, Ca2+ sensitivity, and Ca2+ availability (Driedzic and Gesser 1994). Ion balance is also crucial for maintaining proper cardiac function. An increase in extracellular [K+] depolarizes the resting membrane potential, decreases the rate of depolarization, and lowers the threshold potential of pacemaker cells. A decreased rate of 6 depolarization decreases the firing rate of the pacemaker, but a less negative resting membrane potential closer to the threshold potential may increase firing rate, offsetting one another to some degree (Surawicz 1966). Since the membrane is not normally very permeable to Na+, changes in extracellular [Na+] have almost no effect on the resting membrane potential, but increases in extracellular [Na+] can increase Na+ influx to increase the rate of depolarization and firing rate (Cranefield and Hoffman 1958). Reductions in extracellular [Na+] can increase cardiac contractility due to increased Ca2+ influx by a Na+-Ca2+ exchanger. (Tillisch and Langer 1974). High levels of [Ca2+] can cause the threshold potential to become less negative, and prolong the duration of the action potential and the absolute refractory period, decreasing firing rate (Surawicz 1966). 1.3 Acidosis Fish can experience a lower blood pH in two main ways: through either a respiratory or a metabolic acidosis. CO2 is produced by normal metabolism and its rate of production is usually balanced by its rate of excretion. However, an increased rate of CO2 production during activity can lead to a respiratory acidosis if an animal encounters difficulties excreting CO2. A respiratory acidosis can also be brought on by increased CO2 levels in the environment. Increased CO2 in the respiratory medium can, if uncompensated for, decrease both extracellular and intracellular pH. CO2 in the atmosphere has been rapidly increasing due to human fossil fuel combustion resulting in ocean acidification (Feely et al. 2009), which has been shown to have various detrimental physiological effects on aquatic animals (Doney et al 2009; Wittmann and Portner 2013; Heuer and Grosell 2014). Since pre-industrial times, the average ocean surface water pH has decreased by about 0.1 pH units, and a further decrease of 0.3–0.4 pH units is 7 expected by 2100 if atmospheric CO2 concentrations continue to increase at the current rate (Feely et al. 2009). A metabolic acidosis can be brought on by intensive or anaerobic exercise when skeletal muscle relies on glycolysis rather than oxidative phosphorylation. While anaerobic metabolism can produce ATP in the absence of O2, ATP yield is about 15-times lower than during oxidative phosphorylation. In addition, the generation of ATP through anaerobic or non-mitochondrial sources also causes an accumulation of lactate and H+ in the blood from the working muscle, causing pH to be lowered (Robergs et al. 2004; Richards 2011). Marine teleosts also need to continuously ingest seawater to replenish water that has been lost to the hyperosmotic environment. However, seawater is rich in Ca2+ and Mg2+. Continued ingestion of these ions could result in accumulation to toxic levels and would also produce an osmotic gradient that would be detrimental for fluid absorption in the intestines (Cooper et al. 2010). To overcome this problem, HCO3- is secreted from the blood into the intestines to react with Ca2+ and Mg2+, forming insoluble carbonate precipitates (CaCO3 and MgCO3) that are then excreted out. However, hydration of CO2 to form HCO3- also produces an H+, which acidifies the blood. Increasing amounts of Ca2+ and Mg2+ in seawater means that an individual has to produce more HCO3- to excrete these ions, and so the blood becomes more acidic (Cooper et al. 2010). A reduction in heart rate (bradycardia, also termed a negative chronotropic effect) in vertebrate hearts can be caused by acidosis, which affects the activity of HCN channels of the pacemaker cells. Increased [H+] slow down the speed of activation of HCN channels by shifting the voltage dependence of channel activation to more hyperpolarizing voltages, resulting in bradycardia (Biel et al. 2002). Thus, under acidotic pH conditions, HCN channel activity is reduced. Reductions in cAMP levels caused by acidosis will also reduce HCN channel activity, 8 which will then reduce heart rate and contractility (Chiappe de Mon et al. 1978; Nakanishi et al. 1987). While acidosis generally causes bradycardia, exceptions can be found in some species when heart rates were examined in vivo. When exposed to water bubbled with CO2, reductions in heart rate were observed in rainbow trout (Oncorhynchus mykiss; 18% reduction with 4.5% CO2; Perry et al. 1999), Pacific spiny dogfish (Squalus acanthia; 50% reduction with 5% CO2, Kent and Peirce 1978), and tambaqui (Colossoma Macropomum; 30% reduction with 5% CO2; Sundin et al. 2000), while no significant change in heart rate was observed in the brown bull head (Ameiurus nebulosus) and American eel (Anguilla rostrate) (with 1.5% CO2; Perry and Gilmour 2002). In contrast, white sturgeon (Acipenser transmontanus) exhibited an 8% increase in heart rate when exposed to water bubbled with 10% CO2 (Crocker et al. 2000). A reduction in cardiac contractility (termed a negative inotropic effect), can also be caused by acidosis. Contractility can be measured by the maximum isometric (constant length) force the cardiac muscle can generate. Usually, a strip of cardiac muscle is attached to an isometric force transducer and electrically stimulated to contract. Different environmental conditions would cause changes in the isometric force that can be generated by the heart. It is postulated that H+ competes with Ca2+ for binding sites on troponin C, thus having a negative effect on the muscle’s ability to form myosin cross-bridges necessary for contraction (Williamson et al. 1976). The binding of Ca2+ to troponin C and to the proteins of the SR is central to the regulation of cardiac contractility (Gesser and Poupa 1983). Indeed, increasing cytosolic Ca2+ concentrations in the isolated heart muscle not only increases cardiac force development (Langer 1973), but can also offset the negative inotropic effect of extracellular acidosis (Williamson et al. 1976). 9 The negative inotropic effect due to acidosis has been observed in all vertebrates tested, including many fish species (Driedzic and Gesser 1988; Tibbits et al. 1991; Shiels and White 2008) and acidosis has also been shown to have larger negative effects on fish than in the other vertebrate groups (Bennett 1978). In teleosts like the rainbow trout and Atlantic cod (Gadus morhua), exposure of strips of cardiac muscle to acidosis resulted in loss of force generated by the heart (Gesser and Johansen 1982). Bubbling with 13% CO2 for trout and 15% CO2 for cod reduced the saline bath from pH 7.6 to 6.9 and decreased the contractility of the cardiac muscle strips by 50% for both species after 30 min (Gesser and Johansen 1982). However, not all vertebrate species have a monophasic decrease in contractility with acidotic conditions. The cardiac response to acidosis seems to differ between water- and air-breathing vertebrates (Gesser and Poupa 1978; Salas et al. 2006; Joyce et al. 2015). An unusual biphasic response to acidosis has been observed in air-breathing fishes and vertebrates like in an air-breathing catfish (Pangasianodon hypophthalmus), the rat (Rattus noruegicus), and leopard frog (Rana pipiens) (Gesser and Poupa 1983; Joyce et al. 2015). The initial decline in contractile force was accompanied by a spontaneous recovery even with continued acidification and without addition of any drugs or chemicals to the saline bath, and isometric force generation may even exceed levels recorded prior to the introduction to acidosis. In P. hypophthalmus, 10% CO2 reduced the pH of the saline bath from 7.65 to 6.70 and decreased the contractility of the cardiac muscle strips by 20% after 5 min, but this was followed by a recovery in contractility to 120% of normocapnic levels after about 10 min in sustained 10% CO2 (Joyce et al. 2015). In the rat and leopard frog, 15% CO2 decreased the contractility of the cardiac muscle strips by 20% in rats and 10% in leopard frogs after 5 min, with recovery back to 100% of normocapnic levels within 20 min despite sustained CO2 levels (Gesser and Poupa 1983). This biphasic response has also been 10 seen in the flounder (Pleuronectes flesus), a benthic water-breathing fish that is frequently exposed to higher environmental CO2 levels due to being buried in sediment (Gesser and Johansen 1982). However, the biphasic response in all the species examined has only been observed when the acidosis was induced by hypercapnia, which also directly increases extracellular [HCO3-] (Gesser and Poupa 1983). In the turtle (Chrysemys picta bellii), the recovery in contractile force was also only seen during hypercapnic acidosis and not with lactic or hydrochloric acidosis (Yee and Jackson 1984). It has also been noted that in strips of cardiac tissue from plaice hearts, a greater amount of force could be spontaneously recovered with higher initial [HCO3-] in the saline (Poupa and Johansen 1975). The spontaneous recovery response is thought to be an adaptation to higher levels of blood and tissue CO2 associated with breathing air (Poupa et al. 1978). The observed difference in cardiac response to hypercapnic acidosis between water- and air-breathing vertebrates may be due to the evolutionary transition of vertebrates from water- to air- breathing. The transition had likely modified the sensitivity of the heart to CO2/HCO3-, since air-breathing vertebrates typically have higher blood and tissue CO2 and plasma [HCO3-] than water-breathing vertebrates (Poupa et al. 1978). Since O2 has a much lower solubility in water than air, water-breathing vertebrates must ventilate a larger amount of water to obtain the same amount of O2 as an air-breather. However, the solubility of CO2 in water and air are similar and thus water-breathers have relatively lower blood CO2 levels than air breathers. For air-breathing vertebrates, since O2 levels are higher in the air, a lowered ventilation rate is required to obtain sufficient O2. However, the lower ventilation rate leads to an increase in blood PCO2 and a respiratory acidosis if the [HCO3-] in the blood could not be increased or excess CO2 could not be removed. A major 11 difference in blood pH regulation between air- and water-breathing vertebrates is that water-breathing vertebrates typically regulate blood pH by changes in [HCO3-], while air-breathing vertebrates typically regulate blood pH by changing ventilation rate to change PCO2 (Ultsch 1996). Thus, the evolutionary transition from water-breathing to air-breathing for vertebrates was accompanied by a change in blood acid-base status, from low [HCO3-] and PCO2 for the water breathers, to high [HCO3-] and PCO2 for the air breathers (Ultsch 1996). The mechanism behind this spontaneous recovery response is still unclear. The ability to resist changes in intracellular pH likely depends on active pH regulation, and in cardiac muscle, it seems to rely primarily on the extrusion of H+ in exchange for Na+, on a Na+/HCO3- cotransporter or a HCO3-/Cl- exchanger (Madshus 1988; Lagadic-Gossmann et al. 1992; Liu et al. 1990). Intracellular stores of Ca2+ are present in three main sites: in the internal surface of the sarcolemma, SR, and mitochondria (Langer et al. 1982). Gesser and Poupa (1978) proposed that increased CO2 could cause a release of Ca2+ to explain the recovery of cardiac contractility, but provided no suggestions for a transduction mechanism. Increased levels of intracellular [Ca2+] were indeed observed in the rat, turtle, and flounder myocardium, but not in the trout myocardium which did not experience recovery in contractile force (Gesser and Jorgensen 1982). Gesser and Poupa (1978) observed spontaneous recovery of resting tension in viper (Vipera horus) cardiac strips during hypercapnic acidosis even in Na+- and Ca2+-free saline. They also found that if mitochondrial stores of Ca2+ were depleted (via anoxia and anoxia with oliomycin treatments) before exposing the viper cardiac strips to hypercapnic acidosis, there was a greater reduction in contractility due to acidosis and spontaneous recovery was substantially depressed. They suggested that hypercapnic acidosis triggers the release of intracellular Ca2+ stores, likely from the mitochondria, and that intracellular pH regulation by the extrusion of H+ in 12 exchange for Na+ cannot fully explain the spontaneous recovery (Gesser and Poupa 1978). Salas et al. (2006) observed an initial decline in contractility of toad (Buffo arenarum Hensel) ventricular strips during hypercapnic acidosis (due to a decreased Ca2+ myofilament responsiveness). This was followed by a spontaneous recovery in contractility that was independent of intracellular pH recovery, and instead was due to an increase in the influx of Ca2+. Additionally, plaice hearts do not contain any SR yet spontaneous recovery in cardiac force has been observed (Poupa and Johansen 1975). The addition of caffeine, which releases Ca2+ stores from the SR, had no effect on spontaneous force recovery in viper cardiac strips during hypercapnic acidosis (Gesser and Poupa 1978). These findings suggest that the SR may not serve an important role in the observed spontaneous recovery of cardiac force during hypercapnic acidosis. However, the perfused hearts of some species appear to be resistant to hypercapnic acidosis. Perfused hearts of armoured catfish (Pterygoplichthys pardalis) exposed to up to 5% CO2 had no effect on maximum cardiac performance and exposure to 7.5% CO2 only caused a 35% decrease in cardiac performance. White sturgeon (Acipenser transmontanus) perfused hearts exposed to 3% CO2 showed no significant decrease in maximum cardiac performance, and exposure to 6% and 8% CO2 only caused a 25% decrease in cardiac performance (Baker et al. 2011). Full cardiac performance could be recovered in both species upon return to control conditions (except in sturgeon hearts exposed to 8% CO2). This tolerance to high CO2 levels was believed to be due to preferential intracellular pH regulation of the heart (Hanson et al. 2009; Baker et al. 2011), in contrast to what was observed during spontaneous recovery in contractility for hearts of other species. 13 1.4 Hypoxia Low levels of environmental O2 are common in aquatic habitats due to the low solubility of O2 in water and is also becoming more common in estuarine and coastal marine ecosystems due to eutrophication, decomposition of organic material, and increased environmental temperatures (Diaz and Rosenberg 1995; Rabalais et al. 2014). However, the PO2 in the environment is also very important as it is the driving force for diffusion of O2 into the organism. In addition to low PO2 in the environment, hearts in an organism can be exposed to hypoxia if tissue O2 demand exceeds blood O2 supply, which can occur during intense exercise, temperature extremes, anemia, and changes to respiratory surfaces. Since a lack of O2 can pose severe physiological challenges, many animals have strategies in place to survive extended periods of exposure to low PO2 levels (hypoxia) or a total lack of PO2 (anoxia) in the environment. A major problem for organisms in O2 limiting environments is the reduced capacity for producing ATP via mitochondrial oxidative phosphorylation. Although energy production is possible in the absence of O2, the formation of ATP is most efficient via oxidative phosphorylation (Hochachka and Lutz 2001). In the absence of O2, the partial catabolism of 1 mole of glucose via anaerobic glycolysis produces 2 moles of ATP, whereas the complete oxidation of 1 mole of glucose via oxidative phosphorylation produces about 32 moles of ATP (Wegener 1992). Another advantage of generating ATP via oxidative phosphorylation is that the wastes generated (H2O and CO2) can be easily removed from the body. During glycolysis, the anaerobic metabolic wastes (lactate and H+) generated cannot be passively excreted from the body and can have detrimental effects on cell function with accumulation of these products (Wegener 1992). Thus, the survival time under anoxia can be limited by the size of glycolytic energy stores to generate ATP and by the ability to remove accumulated wastes. Insufficient O2 14 delivery to tissues causes organisms to rely on anaerobic metabolic instead, which produces CO2, lactate, and H+ that can cause blood acidosis if these waste products are not removed (Robergs et al. 2004; Richards 2011). Thus, hypoxia is often accompanied by a metabolic acidosis due to accumulation of acidic metabolic wastes. Organisms die when deprived of sufficient O2 in part because of cardiac failure, which can be attributed to an inadequate matching of cardiac ATP supply to ATP demand (Farrell and Stecyk 2007). Heart performance depends on Ca2+ in the cytosol interacting with contractile proteins, and the energy is derived from the hydrolysis of ATP. One key survival strategy is for an animal to reduce its metabolic rate so that the reduced ATP supply can be matched with the ATP demand (Boutilier 2001). Low O2 levels can also have negative inotropic (contractility) and chronotropic (rate) effects on the heart. However, the ability of the heart to maintain optimal performance under anaerobic conditions varies largely among different species (Gesser 1985). Most vertebrates have a limited ability to tolerate hypoxia, and for example, the Atlantic cod can only tolerate exposure to water with a PO2 of 10 mmHg at 10 °C for a matter of minutes (Gamperl and Driedzic 2009). Anoxia tolerant species, such as the hagfish, can survive anoxic waters at 5 ºC for at least 36 h (Cox et al. 2011), European eels (Anguilla anguilla L.) for a few hours at 15 ºC (van Waarde et al. 1983), goldfish for several days at 4 ºC (Walker and Johansen 1977), and crucian carp (Carassius carassius L.) for as long as 4.5 months at 2 ºC (Piironen and Holopainen 1986). Exposure to hypoxia is also often associated with bradycardia in a variety of fish species, and the decline in heart rate can also vary widely between species (Randall and Smith 1967; Gesser 1985; Farrell 2007). Hypoxic bradycardia is present in species like the rainbow trout (70% reduction after 1 h of progressive hypoxia; Marvin and Burton 1973) and Atlantic cod 15 (30%-50% reduction after 6 min; Fritsche and Nilsson 1988), while weak or absent in other species such as the sea raven (Hemitripterus americanus; not significantly different after 80 min of progressive hypoxia; Farrell et al. 1985), winter flounder (not significantly different after 1 h of progressive hypoxia; Cech et al. 1977), and some air-breathing fish, such as in two armored catfish species (Pterygoplichthys gibbiceps and Liposarcus pardalis; not significantly different after 2.5 h of progressive hypoxia; MacCormack et al. 2003). Hypoxia also frequently reduces cardiac contractility, and again, the magnitude of decline varies among species. In plaice, tension generated by cardiac strips was reduced by 50% after a 30 min exposure to anoxia, while in Atlantic cod, it only takes approximately 10 min for the same reduction to occur (Gesser 1985). The two major hypotheses to explain why cardiac contractility decreases during hypoxia is due to a decrease in the Ca2+ sensitivity of the myofibrils or due to an intracellular acidosis (Kentish 1986). Gesser (1977) observed a recovery in contractile force during hypoxia in the carp (Cyprinus carpio) myocardium, but this was only observed with higher CO2/HCO3- concentrations, and that there was a greater force recovery upon re-oxygenation with a higher CO2/HCO3- concentration in both the carp and trout myocardium. During anoxia, generation of ATP by anaerobic glycolysis will lead to the formation of lactic acid, which decreases blood pH if it cannot be excreted. The accumulation of H+ will have negative inotropic effects on the heart as described above during exposure to acidosis. Loss of cardiac contractility is mainly due to H+ competing with Ca2+ for binding sites on troponin C, thus having a negative effect on the ability of muscle to form myosin cross-bridges necessary for contraction (Williamson et al. 1976). While evidence suggests that intracellular acidosis seems to be a significant factor during anoxia in mammalian cardiac muscle (Matthews et al. 1981), there 16 is conflicting evidence as to whether it is significant in other vertebrate hearts. Evidence suggests that intracellular acidosis is not of importance in anoxic trout hearts as they show a non-significant decrease in intracellular pH from 7.36 to 7.27 (Nielsen and Gesser 1984). However, cardiac intracellular pH seems to decrease significantly in tilapia (Oreochromis niloticus × mossambicus × hornorum) (from 6.89 to 6.73; Speers-Roesch et al. 2010) and turtle (Chrysemys picta bellii), (from 7.37 to 7.22; Wasser et al. 1990) exposed to anoxia. While the intracellular pH of toad (Bufo marinus) ventricular preparations decreased significantly by 0.26 while exposed to hypoxia, the decline in contractility did not correlate with changes in intracellular pH, as the decline occurred before any pH changes could be detected (LaManna et al. 1980). Thus, there appears to be important differences between mammalian hearts and fish hearts, and even between fish species, as to the major cause of loss of cardiac contractility during anoxia. 1.5 Cardiac Support Mechanisms Different species have different strategies in place for the heart to deal with hypoxic and acidotic conditions. It appears that a hypoxic bradycardia provides a number of benefits to the heart when O2 supply is low. These benefits include increasing the diastolic residence time of blood in the lumen of the heart to increase the time for gas diffusion, increasing stroke volume to stretch the cardiac chambers, potentially reducing the diffusion distance for oxygen, improving cardiac contractility through the negative force-frequency effect, and reducing cardiac oxygen energy demand by reducing cardiac output (Farrell 2007). A reduction in force generation during hypoxia might also be beneficial since a high hypoxic force development is thought to be associated with higher formation rates of lactate and H+, contributing to an intracellular acidosis (Gesser 1977). 17 Hypercapnia exposure is often associated with bradycardia in a variety of fish species, but cardiac output is typically maintained by increases in stroke volume (Perry et al. 1999). Experiments have identified that CO2 chemoreceptors in the gills are responsible for initiating cardiorespiratory responses to changing water PCO2, rather than pH (Gilmour et al. 2005). It has been proposed that bradycardia, even when cardiac output is kept constant, could aid in CO2 release at the gills through lamellar recruitment due to higher pulsatile pressure, increased residence time of blood in at the gills, and increased gas permeability (Davie and Daxboeck 1982; Perry and Gilmour 2002). To maintain cardiac function in debilitating environmental conditions, heart rate and/or contractility can be increased. Thus, fish can either reduce inhibitory (vagal) nervous stimulation, increase stimulatory (adrenergic) nervous stimulation, or increase blood catecholamine levels to activate tmAC to increase cAMP production (Farrell 2007). 1.6 Soluble Adenylyl Cyclase In addition to increasing cardiac function via tmAC, a completely novel mechanism was recently discovered in Pacific hagfish (Eptatretus stoutii), a HCO3--mediated stimulation of soluble adenylyl cyclase (sAC) to increase heart rate. sAC has been found in a variety of cell types and works with carbonic anhydrase to act as a sensor of pH and CO2, detecting changes in the concentration of HCO3- (Chen et al., 2000). Carbonic anhydrase catalyzes the formation of HCO3- from CO2 (CO2 + H2O  H2CO3  HCO3- + H+). sAC has been discovered to play roles in various organs, including the kidney, eye, respiratory tract, digestive tract, and pancreas, and in the nervous and immune systems (Tresguerres et al. 2011), and found in many different animal groups, from cnidarians to bony fish to mammals (Tresguerres et al. 2014). In mammalian cells, sAC has been known to play a role in apoptosis of coronary endothelial cells 18 and cardiomyocytes (Chen et al., 2010). In sharks and a few teleosts, it has been established that sAC functions as a sensor for acid-base homeostasis and as a regulator of NaCl and water absorption in the intestines (Tresguerres et al. 2014). However, it was not until the work done by Wilson et al. (2016) that it was discovered that sAC also plays a unique role in influencing the heart rate of an animal. Ancestral chordates, as represented by modern day hagfish, lack cardiac innervation from the brain, which eliminates the normal vagal and direct sympathetic controls of the heart (Augustinsson et al. 1956). Yet, they can still modulate their heartbeat over a 4-fold range (Cox et al. 2010). Hagfish can modulate heart rate via the release of catecholamines from chromaffin tissue located within the heart (Ostlund et al. 1960; von Euler and Fange 1961). Like in other vertebrates, catecholamines stimulate production of cAMP by tmAC, which then binds to HCN channels to alter the probability of the channel opening to increase heart rate (Wainger et al. 2001). The addition of adrenaline only slightly increases heart rate in hagfish, while the addition of sotalol, a β-adrenoreceptor antagonist, greatly decreases heart rate, suggesting a tonic level of catecholamine stimulation even during normoxia (Axelsson et al. 1990). Injections of reserpine, which depletes catecholamine stores, decrease contractility and heart rate of hagfish hearts (Bloom et al. 1961). These findings suggest that catecholamine stores in the hagfish heart are important in cardiac control in lieu of innervation from the brain. An entirely novel mechanism to activate HCN channels and modulate heart rate has been recently proposed in hagfish, one mediated by a sAC-mediated pathway (Wilson et al. 2016). In addition to tmAC, cytoplasmic sAC can also increase cAMP concentration but in response to elevated [HCO3-] (Tresguerres et al. 2014). Both adrenergic stimulation of cAMP production through tmAC and HCO3- stimulation of cAMP production through sAC have been implicated in 19 influencing the heart rate of the hagfish (Wilson et al. 2016). During anoxia, hagfish heart rate declines about 50%, but addition of HCO3- can bring heart rate back to about 140% of the normoxic rate (Wilson et al. 2016). cAMP production in the heart was found to increase by 2-fold upon the addition of 60 mM HCO3- during anoxia, and both heart rate and cAMP production could be brought back to anoxic levels with the addition of KH7, a sAC antagonist (Wilson et al. 2016). The addition of forskolin, a tmAC agonist, to hagfish hearts during anoxia fails to increase heart rate beyond normoxic rates (Wilson et al. 2016). Since O2 is required during catecholamine synthesis to convert tyrosine to 3,4-dihydroxyphenylalanine (the rate-limiting step of catecholamine synthesis) and produce noradrenaline from dopamine (Levitt 1965), the sAC pathway may be a compensating mechanism for hagfish to regulate their heart rate during anoxia in lieu of neural innervation. Immunofluorescence staining of hagfish hearts with anti-dogfish sAC identified sAC throughout the atrium and ventricle, with strong staining evident in cardiac trabecula and in the atrium (Wilson et al. 2016). 1.7 Thesis Objectives It is currently unknown whether the role of sAC in cardiac control is unique to hagfish or if this cardiac control pathway is present in other species. Since sAC increases heart rate through increasing cAMP concentration, it is also unknown whether sAC increases cardiac contractility as well. My thesis will explore the role of sAC in cardiac function recovery during hypercapnic acidosis and hypoxia in different fish species. Despite sustained acidosis or hypoxia, the hearts of some species are able to maintain and even spontaneously recover cardiac function, but the associated mechanisms are unknown. The negative inotropic effects of acidosis (H+) are likely due to decreased Ca2+ sensitivity of the 20 contractile system and its ability to form myosin cross-bridges necessary for contraction. Experimental additions of extracellular Ca2+ or catecholamines increase cardiac contractility and rate, even during acidosis or anoxia (Hanson et al. 2006). In all previous experiments that examined the spontaneous recovery of cardiac force during hypercapnic acidosis in various species, the physiological saline bath had always contained some added HCO3-. I hypothesize that sAC could be present in these species and when stimulated by HCO3-, sAC triggers a signal transduction cascade to increase cAMP concentration that could explain the spontaneous recovert. The increased cAMP concentration can potentially increase heart rate by increasing the If current by binding to HCN channels and can increase the force of contraction by allowing increased entry of Ca2+ into the myocardium. Carbonic anhydrase catalyzes the formation of HCO3- and H+ from CO2 and H2O, and so any increase in CO2 could increase the concentration of HCO3- in the heart. The presence of sAC could explain why a recovery in cardiac contractility was only observed during hypercapnic acidosis and not during lactic or hydrochloric acidosis. The main questions being tested in my thesis are: 1) What are the effects of increasing extracellular [HCO3-] on in vitro cardiac function during severe hypoxia and hypercapnic acidosis? During anoxia, addition of HCO3- was associated with a total recovery of heart rate in the hagfish (Wilson et al. 2016), and a spontaneous recovery of ventricular contractility was observed in the flounder and air-breathing catfish heart (Gesser and Poupa 1978; Joyce et al. 2015). Thus, I hypothesize that addition of extracellular HCO3- during severe hypoxia and hypercapnic acidosis will recover heart rate and contractility in the lamprey (which are closely related to hagfish), flounder (spontaneous recovery in contractility was previously observed), 21 swamp eel (air-breathing fish), and sturgeon (tolerant of high environmental CO2) hearts. To test this hypothesis, isolated hearts and ventricular strips will be subjected to severe hypoxia or hypercapnic acidosis to impair cardiac function. Heart rate and contractility will then be measured upon addition of HCO3- to the saline bath to determine if any recovery in cardiac function occurs. If my hypothesis is correct, a recovery of heart rate and contractility back to control levels would be observed upon the addition of HCO3-. 2) Is sAC associated with cardiac recovery during severe hypoxia and hypercapnic acidosis? In hagfish, the recovery in heart rate during anoxia upon addition of HCO3- is associated with a sAC-mediated pathway, where HCO3- stimulates sAC to increase cAMP concentration to increase heart rate (Wilson et al. 2016). I hypothesize that any recovery in cardiac function observed upon addition of extracellular HCO3- will also be associated with the sAC-mediated pathway. To test this hypothesis, KH7, a sAC blocker, will be added to isolated hearts and ventricular strips from question 1. If my hypothesis is correct, addition of KH7 should be able to abolish any HCO3- associated recovery in cardiac function and return heart rate and contractility to severe hypoxia and hypercapnic acidosis levels. 3) Is the sAC-mediated cardiac control pathway unique to the hagfish or an adaptation to debilitating environmental conditions? I hypothesize that the sAC-mediated cardiac control pathway may be an adaptation for animals frequently exposed to debilitating environmental conditions. The sAC-mediated pathway in cardiac control has only been observed in the hagfish, which are tolerant of anoxic conditions (Wilson et al. 2016), while spontaneous recovery of cardiac contractility during hypercapnic 22 acidosis was observed in various hypoxia tolerant and air-breathing vertebrate species (Gesser and Poupa 1978; Salas et al. 2006; Joyce et al. 2015). To test this hypothesis, the cardiac responses from species from different phylogenetic groups and with different life histories will be compared to see which species the sAC-mediated cardiac control pathway is present in. 1.8 Study Species For this study, 6 fish species were chosen in part for their phylogenetic position and interesting life histories, which are described in more detail below. Fish species were chosen from 3 phylogenetic groups so that evolution of the sAC-mediated cardiac control pathway can be examined to determine where it was lost and no longer involved in cardiac control. Different life histories were also chosen to see if the sAC-mediated pathway is an adaptation for species living in debilitation environmental conditions. Cyclostomes The most obvious species to examine is the lamprey, which belongs to the same class as the hagfish, the cyclostomes. While both lampreys and hagfish possess cardiac chromaffin tissues that contain catecholamines, a major difference between the two species is that hagfish hearts are aneural while lamprey hearts are innervated by the vagus nerve (Augustinsson et al. 1956). In vertebrate hearts, stimulation of the vagus nerve slows heart rate via the release of acetylcholine, and while hagfish hearts do not react to acetylcholine, lamprey heart rates are increased instead (Augustinsson et al. 1956). Similar to hagfish, lamprey are known to burrow in soft sediments, which exposes them to hypoxic environments (Bartels et al. 2011). Thus, they must have physiological adaptations to withstand limited O2 availability in the environment. No 23 study yet has examined whether or not the sAC-mediated cardiac control mechanism is also present in the lampreys. Elasmobranchs The vertebrate lineage split into the jawless fish (Agnatha), which includes the hagfish and lampreys, and the jawed fish (Gnathostomata) around 540–505 million years ago during the Cambrian (Meyer and Zardoya 2003). The gnathostome lineage then split into the cartilaginous fish (Chondrichthyes), which includes sharks and rays, and the bony fish (Osteichthyes), which includes the teleosts and the tetrapods (Meyer and Zardoya 2003). Thus, the elasmobranchs are an intermediate phylogenetic group between the cyclostomes and teleosts. Similar to the lampreys but unlike the teleosts, elasmobranch hearts have inhibitory vagal innervations but lack stimulatory adrenergic innervations (Short et al. 1977). sAC has been found in the gills of dogfish and plays an important role as a blood acid-base sensor, which is activated by HCO3- (Tresguerres et al. 2010). It is not known if sAC has other physiological roles in the dogfish. Teleosts Sturgeon are phylogenetically positioned between the elasmobranchs and teleosts and are one of the most CO2 tolerant fish species. It has been observed that with exposure to 8% CO2, heart rate decreased by 25% while stroke volume was unchanged, which suggested that the high tolerance of sturgeon hearts to CO2 is associated with a protection in contractility rather than heart rate (Baker et al. 2011). Could their cardiac tolerance to CO2 be attributed to the presence of the sAC-mediated pathway? The swamp eel, while not a true eel, is a facultative air breathing 24 teleost that utilizes its buccal cavity for air-breathing when water becomes hypoxic (Iversen et al. 2012). It is unknown if they also exhibit the same spontaneous recovery in contractility as observed in other air-breathing vertebrates. Zebrafish, a common model species, will also be tested as they are not known to be especially tolerant to either hypoxia or hypercapnia. Flounder are often found in high salinity and hypoxic benthic waters, sometimes burying themselves as deep as 12-15 cm in the sand (Jorgensen and Mustafa 1980). Since flounder are found inshore, they will also be exposed to variations in abiotic conditions, including temperature, salinity, dissolved gases, and water pH (Weber and de Wilde 1975). Thus, flounder can be frequently exposed to waters that are low in O2 and high in CO2, leading to acidic waters. Burrowing in mud would also likely impede CO2 excretion, leading to a respiratory acidosis. A recovery in contractile force was observed during hypercapnic acidosis only in air-breathing vertebrates and in the flounder, but it is unknown if sAC plays a role in the recovery. Since acidotic conditions in the heart can have large negative inotropic effects, perhaps the sAC-mediated cardiac control pathway could be an adaptive advantage for the flounder to regulate the contractility of its heart since it is often exposed to high levels of CO2/HCO3- anyway. This would allow the heart to continue functioning in the face of ongoing environmental challenges. 25 Chapter 2: Research Chapter 2.1 Introduction The heart is a crucial part of the cardiovascular system that supplies O2 and nutrients to and removes metabolic wastes from all parts of the body. Physiological requirements change during warmer temperatures when metabolic rate increases, during activity when their tissues need more O2, or when they encounter environmental hypoxia. Thus, when encountering new or debilitation environmental conditions, the heart must be able to respond appropriately to regulate cardiac output to maintain normal physiological function. The heart can respond and modulate its cardiac output by changing the rate of cardiac beating (heart rate) and the volume pumped with each heartbeat (stroke volume), which is affected by the force of muscular contraction. Almost all vertebrate hearts are under dual sympathetic (stimulatory β-adrenergic) and parasympathetic (inhibitory vagal) innervation from the brain, both of which are involved in control of cardiac function (Nilsson 1983). Hagfish hearts lack both adrenergic and vagal innervations from the brain despite having a heart similar to other vertebrates (Augustinsson et al. 1956). In the vertebrate lineage, vagal innervation to the heart was first present in the lamprey heart, but excites rather than inhibits cardiac function (Augustinsson et al. 1956). Elasmobranchs also have vagal innervations but lack adrenergic innervations to the heart (Short et al. 1977). Dual adrenergic and vagal innervations to the heart are present in almost all other teleosts and vertebrates, but adrenergic innervation was found to be absent in some flatfish species (Cobb and Santer 1973). Even without cardiac innervation from the brain, hagfish can still modulate their heart rate over a 4-fold range (Cox et al. 2010), mainly via the release of catecholamines from 26 chromaffin tissue located within their heart that stimulates tmAC to increase cAMP production, which then increase heart rate (Ostlund et al. 1960; von Euler and Fange 1961; Perry et al. 1993). It was recently discovered that hagfish have another way in which they can modulate their heart rate: a sAC-mediated pathway that increases cAMP concentration to increase heart rate upon stimulation by HCO3- (Wilson et al. 2016). Although sAC has been found to have various functions in many different animal groups, from cnidarians to bony fish to mammals (Tresguerres et al. 2014), and present in coronary endothelial cells and cardiomyocytes in mammals (Chen et al., 2010), the role of sAC in the control of cardiac function had not been previously examined prior to the study done by Wilson et al. (2016), Bicarbonate is a major component of the extracellular buffering system, and it serves a critical role in pH homeostasis. Deviations in pH can have negative effects on protein structure, enzyme function, and ion transport, and thus HCO3- has a major role in maintaining whole animal performance. Deviations from the normal intracellular pH can be caused by acidosis and even hypoxia, which will then influence cardiac contractility and excitability (Orchard and Kentish 1990). In studies performed on Sprague Dawley rat and New Zealand albino rabbit whole hearts perfused with acidic solutions, cardiac force quickly declines, but the addition of HCO3- fails to increase ventricular contractility (Shapiro 1990; Sirieix et al. 1997). Yet, a spontaneous recovery after an initial decrease in contractility was observed by Gesser and Poupa (1983) with rat ventricular strips exposed to hypercapnic acidosis, where the elevation in CO2 directly increases extracellular [HCO3-] to some degree. The observed spontaneous recovery in contractility had also been observed in other air-breathing fishes and vertebrates like in an air-breathing catfish (Pangasianodon hypophthalmus), the rat (Rattus noruegicus), and leopard frog (Rana pipiens) (Gesser and Poupa 1983; Joyce et al. 2015). However, few studies have 27 examined the direct effects of extracellular HCO3- on the cardiac function of non-mammalian species and whether it has direct non-pH related effects on cardiac function. This present study will examine some of the effects of addition of extracellular [HCO3-] on the cardiac function of different fish species and whether the sAC-mediated cardiac control pathway is present in other fish species as well, acting as another mechanism to modulate cardiac function. I hypothesize that increasing extracellular [HCO3-] during severe hypoxia and hypercapnic acidosis will recover heart rate and contractility and will be associated with a sAC-mediated pathway, where HCO3- stimulates sAC to increase cAMP concentration to increase cardiac function. To test this, isolated hearts and ventricular strips will be exposed to severe hypoxia or hypercapnic acidosis to impair cardiac function. Heart rate and contractility will be measured upon addition of HCO3- to the saline bath to determine if any recovery in cardiac function occurs. Addition of KH7, a sAC blocker, should abolish any recovery if it is associated with a sAC-mediated pathway. Six fish species were chosen for this study in part for their phylogenetic position and differences in their life histories. The Pacific lamprey (Lampetra sp.) was chosen as a representative for the cyclostomes, the Pacific spiny dogfish (Squalus suckleyi) was chosen for the elasmobranchs, while the White sturgeon (Acipenser transmontanus), starry flounder (Platichthys stellatus), Asian swamp eels (Monopterus albus), and zebrafish (Danio rerio) were chosen for the teleosts. Sturgeon were chosen for their high tolerance to CO2, flounder for their benthic lifestyles, swamp eels for their dual water- and air- breathing ability, and zebrafish due to their use as a common model species. 28 2.2 Materials and Methods 2.2.1 Animal Husbandry Juvenile lampreys (0.31 ± 0.02 g; mean ± s.e.m.) were collected from the Fraser River near Langley, British Columbia, using dip nets. Lampetra richardsoni and Entosphenus tridentatus were likely used as both lamprey species were present at the collection site and visual distinction between juvenile of the two species was not possible (personal communication, Dr. R. Beamish). They were transported to the University of British Columbia (UBC) and held in 40 L glass tanks with recirculating and filtered water at 10 °C. Lampreys were fed baker’s yeast once per week. Spiny dogfish (Squalus suckleyi; 1.48 ± 0.06 kg) were caught using rod and reel from English Bay near West Vancouver, British Columbia. They were transported to the Centre for Aquaculture and Environmental Research at West Vancouver, British Columbia, and held in 2,000 L fiberglass tanks with flow-through, aerated seawater at 9-11 °C. Dogfish were fed squid until satiation three times per week. Juvenile white sturgeon (Acipenser transmontanus; 14.39 ± 0.71 g) were obtained from the Vanderhoof Hatchery in Vanderhoof, British Columbia. They were held at UBC in 400 L fiberglass tanks with recirculating and filtered water at 10 °C. Sturgeon were fed trout pellets until satiation three times a week. Starry flounder (Platichthys stellatus; 57.32 ± 4.13 g) were caught using seine nets from English Bay near West Vancouver, British Columbia. They were transported to UBC and kept in recirculating and filtered 400 L fiberglass tanks containing aerated artificial saltwater made up 29 using Instant Ocean Salt Mix (Aquarium Systems, Mentor, OH) at 30 ppt at 10 °C. Flounder were fed with shrimp until satiation three times a week. Asian swamp eels (Monopterus albus; 120.51 ± 10.42 g) were purchased from a local seafood market (Parker Seafood, Richmond, British Columbia). They were transported to UBC and held in 400 L fiberglass tanks with recirculating and filtered water at 15 °C. Eels were fed with shrimp until satiation three times a week. Zebrafish (Danio rerio; 0.32 ± 0.03 g) were purchased from a local pet store (Petsmart, Vancouver, British Columbia). They were transported to UBC and held in 40 L glass tanks with recirculating and filtered water at 20 °C. Fish were fed with commercial staple flake food until satiation three times a week. All experiments were approved by the Animal Care Committee of the University of British Columbia (A16-0038) and the Centre for Aquaculture and Environmental Research (16-0038-001A2) and conducted in accordance with the Canadian Council on Animal Care guidelines. 2.2.2 Chemicals KH7 and forskolin were dissolved in DMSO to their maximum solubility, 100 mM for KH7 and 25 mM for forskolin. These were then further diluted with the appropriate saline (see below) before being added during experiments. Isoproterenol and propranolol were freshly prepared and serially diluted in distilled H2O to the appropriate concentrations before being added during experiments. Isoproterenol, propranolol, NaHCO3, NaOH, and DMSO were purchased from Sigma Aldrich (St. Louis, Missouri), while KH7 and forskolin were purchased from Tocris Bioscience (Minneapolis, Minnesota). 30 2.2.3 pH and [HCO3-] Measurements pH measurements were made using a Mettler Toledo SevenEasy pH meter with an attached Mettler Toledo InLab 413 SG probe (Schwerzenbach, Switzerland). Total CO2 in the saline was measured using a Corning 965 Carbon Dioxide Analyser (Corning Ltd., Halstead, England). PCO2 was calculated from the measured pH and total CO2 using the Henderson-Hasselbalch equation (pH = pK` + log ([HCO3-] / αPCO2)] and [HCO3-] was calculated using total CO2 = αPCO2 + [HCO3-]. pK` and α were calculated from equations obtained from Kelman (1967), where pK` = 6.086 + 0.042 (7.4 – pH) + (38 – temp) [0.00472 + 0.00139 (7.4 – pH)], and α = 0.0307 + 0.00057 (37 – temp) + 0.00002 (37 – temp)2. This measurement was conducted to confirm that the HCO3- added to the saline (as 1 M NaHCO3) remained in the saline and was not bubbled off as CO2. 2.2.4 Heart Extractions All species were sacrificed with a swift blow to the head to ensure the animal was unconscious, followed by immediate pithing of the brain to destroy brain tissue. The heart was then quickly excised and placed in physiological saline bubbled with pure O2 at the experimental temperature (see below for a summary) to allow the heart to stabilize. The composition of the initial HCO3--free saline depended on the species. A freshwater fish saline was used for the lampreys, sturgeon, swamp eels, and zebrafish, and consisted of (in mM): 125 NaCl, 2.5 KCl, 0.9 MgSO4, 2.5 CaCl2, 5.6 glucose, 3.9 TES free acid, and 6.1 TES Na+ salt in distilled H2O. A saltwater fish saline was used for the flounder, with the same composition as the freshwater fish saline but with 180 mM NaCl. The saline used for dogfish consisted of (in mM): 260 NaCl, 5 31 KCl, 3 CaCl, 1.33 MgSO4, 5.6 glucose, 1 Na2HPO4, 350 urea, and 70 TMAO in distilled H2O. All salines were adjusted to a pH of 7.8 using 1 M NaOH. 2.2.5 Heart Rate Measurements Isolated, freely beating hearts were transferred to a counting dish with 20 mL of the appropriate saline at the temperature to which the fish were acclimated (see below for a summary). The experimental temperature was maintained in the dish by placing it in a water jacket connected to a programmable laboratory chiller (1160S, VWR International, USA). Hearts were allowed to stabilize to the conditions for 30 min, during which heart rate settled to a steady rate. The number of atrial beats was visually counted for one min using a dissecting microscope and the value was recorded as the heart rate. The whole hearts of lampreys, sturgeon, swamp eels, and zebrafish were used. However, due to limited numbers of dogfish and flounder, half of the ventricle was excised to be used for the contractility measurements. Atrial rates before and 30 min after the ventricle was excised were not statistically different and were assumed to represent the intrinsic heart rate. Each heart was only exposed to either the severe hypoxia or the hypercapnic acidosis treatment, not both. 2.2.6 Contractility Measurements Isometric force generation was measured in ventricular strips prepared similar that described in Shiels and Farrell (1997). Specifically, the excised ventricle was transferred to a bath of the appropriate saline at the experimental temperature. A razor blade was used to dissect four longitudinal myocardial strips from the ventricle of approximately equal size (<1 mm width, ~4 mm long). Preparations consisted of both compact myocardium and spongy myocardium. 32 Each myocardial strip was tied at each end with surgical silk and one end of the silk was looped around a threaded rod and secured between two stainless steel nuts while the other end of the silk was secured to the attachment site of an isometric force transducer (MLT0202, ADInstruments, Sydney, Australia). Preparations were lowered into a water-jacketed organ baths containing 20 mL of the appropriate saline at the experimental temperature. The bath was bubbled with pure O2 and the preparations were allowed to equilibrate for 10 min before stimulation began. Two silver electrodes, positioned on either side of the muscle, were connected to a Grass SD9 stimulator (Quincy, Massachusetts) that provided 10 ms pulses at 100% of the voltage necessary to elicit the maximum force of contraction. Experiments on four myocardial strips from the same heart were carried out at the same time and all preparations were stimulated at 0.2 Hz. Preparations were then stretched with a micrometer screw to reach maximum force of contraction and then left to stabilize at maximum force of contraction for 1 h before exposing the ventricular strips to either severe hypoxia or hypercapnic acidosis as described below. In addition, contractility was measured for the dogfish, flounder, and swamp eel hearts but could not be measured in juvenile lamprey, sturgeon, and zebrafish hearts due to their small size. Signals from the transducers were recorded by a data acquisition software (AcqKnowledge, Biopac Systems, Goleta, California). Force of contraction was calculated by the software as the change from minimum to maximum tension exerted by the myocardial strip on the transducer. The length of the myocardial strip was measured using a pair of digital calipers, while wet mass was also determined with a balance. Cross-sectional area of the myocardial strips was estimated from the measured length and weight, assuming a uniform thickness and a density of 1.06 g/cm3 (Layland et al. 1995). Force of contraction values were taken over 30 s intervals and was then expressed as mN/mm2. 33 2.2.7 Severe Hypoxia Experiments Severe hypoxia was achieved by bubbling the saline baths of the hearts and ventricular strips with 100% N2 for 30 min to 2 h (see below for a summary), until the heart rate or force of contraction decreased to a stable level. This was referred to as severe hypoxia because anoxia could not be achieved due to surface agitation of the saline when bubbling with N2, which introduced some O2 back into the saline. Then the hearts and ventricular strips were subjected to one of the following treatments: either recording the change in either heart rate or force of contraction 15 min after the addition of any drug. Each of four cardiac strip preparations from the same heart received a different treatment at the same time. Solutions of NaHCO3 and NaOH were bubbled with 100% N2 before an aliquot was added to the bath saline to prevent excess introduction of O2. 1) KH7 treatment NaHCO3 was added in 10 mM increments every 15 min until a final concentration of 50 mM in the saline bath. 50 μM of KH7 was then added to the saline bath. 2) DMSO treatment NaHCO3 was added in 10 mM increments every 15 min until a final concentration of 50 mM in the saline bath. Then the same volume of DMSO used to dissolve 50 μM of KH7 was added to the saline bath. This treatment was done to control for the effects of adding DMSO. The DMSO treatment was performed only if KH7 had an effect on heart rate or contractility. 34 3) NaOH treatment NaOH was added to the saline bath every 15 min until the same pH was reached as with the NaHCO3 additions. This treatment was done to control for the effects of pH and additional [Na+] being added during the other treatments. 4) Control treatment Nothing was added. This treatment was done to determine if any spontaneous recovery in heart rate or contractility occurred. Table 1. Experimental conditions during severe hypoxia. Dogfish Flounder Swamp Eel Lamprey Sturgeon Zebrafish Temperature 10 °C 10 °C 15 °C 10 °C 10 °C 20 °C Duration of Hypoxia 1 h 1 h 1 h 2 h 1 h 30 min Initial pH of Saline 7.8 pH of saline with N2 + 50 mM NaHCO3 8.7 Initial [HCO3-] (mM) 0.9 0.8 0.7 0.7 0.7 0.7 [HCO3-] (mM) with N2 + 50 mM NaHCO3 48.6 46.5 42.9 43.1 43.1 43.8 35 2.2.8 Hypercapnic Acidosis Experiments Hypercapnic acidosis was achieved by bubbling the saline baths of the hearts and myocardial strips with a mixture of CO2 and O2 for 30 min, either 7.5% CO2 : 92.5% O2 or 15% CO2 : 85% O2 until either heart rate or force of contraction decreased to a stable level. 15% CO2 was chosen to facilitate comparisons with previous studies that used a similar CO2 percentage. A lower CO2 percentage was used if the heart stopped beating before 30 min. Gas mixtures were obtained from a gas mixing pump (Wosthoff, Bochum, West Germany). Then the hearts and myocardial strip preparations were subjected to one of the same four treatments described for the severe hypoxia experiment, while heart rate or force of contraction were recorded every 15 min. Solutions of NaHCO3 and NaOH were not bubbled with the CO2/O2 gas mixture before being added. Table 2. Experimental conditions during hypercapnic acidosis. Dogfish Flounder Swamp Eel Sturgeon Lamprey Zebrafish Temperature 10 °C 10 °C 15 °C 10 °C 10 °C 20 °C % CO2 Used 15% 7.5% Initial of pH saline 7.8 pH of saline with CO2 6.0 5.9 5.9 6.1 pH of saline with CO2 + 50 mM NaHCO3 7.1 7.2 7.2 7.5 [HCO3-] (mM) with CO2 3.0 2.5 2.5 2.0 2.0 2.2 [HCO3-] (mM) with CO2 + 50 mM NaHCO3 49.2 49.5 49.7 49.1 48.1 48.4 36 2.2.9 Adrenergic Stimulation Experiments A separate set of lamprey hearts that had not previously been tested were used for the experiments with isoproterenol, a β-adrenergic agonist. Lamprey hearts were quickly excised and placed in 10 mL of freshwater saline at 10 °C oxygenated with 100% O2. The hearts were then left for 30 min to allow the heart rate to stabilize and reach steady state. Increasing concentrations of isoproterenol (1, 5, 10, 50, and 100 μM) were then added to the saline every 10 min and heart rate was recorded 10 min after each addition. The number of atrial beats was visually counted for 1 min using a dissecting microscope and the value was used as the heart rate. Increasing concentrations of propranolol (1 and 5 μM), a β-adrenergic antagonist, were then added to the same hearts every 10 min, and heart rate was recorded 10 min after each addition. A separate set of lamprey hearts that had not been previously tested were used for the forskolin experiments. Lamprey hearts were quickly excised and placed in 10 mL of freshwater saline at 10 °C oxygenated with pure O2. The hearts were then left for 30 min to allow heart rate to stabilize and reach steady state. The saline bath was then bubbled with pure N2 for 2 h for the heart to reach severe hypoxia heart rates. Increasing concentrations of forskolin (1, 10, 100 nM, 1, 10, 100 μM) was added to the saline bath every 15 min, and heart rate was recorded 15 min after each addition. 2.2.10 Immunofluorescence Staining Immunofluorescence staining was performed only on lamprey hearts since the sAC-mediated pathway in cardiac control was not observed in the hearts of the other species tested. Excised lamprey hearts were immediately placed in ice cold saline and rinsed of excess blood. Hearts were then fully submerged in fixative (3% paraformaldehyde, 3% glutaraldehyde, 0.35% 37 in 0.1 M Sodium Cacodylate, pH 7.4, Electron Microscopy Sciences) at 4 ºC for 5 h. Hearts were then rinsed with ice cold 50% ethanol, and fully submerged in 50% ethanol at 4 ºC for 5 h. Hearts were then rinsed with ice cold 70% ethanol and submerged in fresh 70% ethanol at 4 ºC overnight. Tissues were then stained and imaged as described in Wilson et al. (2016). Briefly, fixed hearts were the sectioned to a thickness of 7 μm using a rotary microtome and incubated in blocking buffer (PBS, 2% normal goat serum, 0.02% keyhole limpet hemocyanin, pH 7.8) for 1 h. Sections were then incubated in anti-sAC, the primary antibody, overnight at 4 °C. Slides were washed three times in PBS and sections were incubated in the secondary antibody at room temperature for 1 h, followed by incubation with the nuclear stain Hoechst 33342 (Invitrogen, Grand Island, NY, USA) for 5 min. Slides were then washed three times in PBS and sections mounted in Fluorogel with Tris buffer (Electron Microscopy Sciences, Hatfield, PA, USA). Immunofluorescence was detected using an epifluorescence microscope (Zeiss AxioObserver Z1) connected to a metal halide lamp and with the appropriate filters. Digital images were adjusted for brightness and contrast only, using Zeiss Axiovision software and Adobe Photoshop. 2.2.11 Statistical Analysis In order to test for statistical differences, comparisons among control and treatment values were tested using a one-way, repeated measures ANOVA followed by a Holm-Sidak post-hoc test. Comparisons between sets of treatments were tested using a one-way ANOVA also followed by a Holm-Sidak test. Data was transformed before analysis if it did not meet assumptions of normality and equal variance. Statistical analysis was done on the raw data but data were graphically displayed as normalized data to allow for direct comparison between 38 different species. All heart rate and contractility measurements were normalized and plotted as percent of initial value. Data are presented as mean ± s.e.m unless otherwise stated. Statistical significance was assessed as P <0.05. All statistical analysis was performed using SigmaPlot 12.0 (Systat Software Inc.; www.sigmaplot.com). 2.3 Results 2.3.1 Severe Hypoxia Heart Rate After exposure to severe hypoxia, hearts from all species tested experienced a significant decrease in heart rate (Fig. 2.1). Lamprey heart rates slowed by 32.8%, from 36.3 ± 1.2 min-1 to 25.5 ± 3.3 min-1, dogfish hearts by 34.0%, from 20.5 ± 0.9 bmp to 13.5 ± 0.6 min-1, sturgeon hearts by 43.1%, from 29.0 ± 2.4 min-1 to 16.5 ± 1.5 min-1, flounder hearts by 22.2%, from 64.7 ± 4.1 min-1 to 49.7 ± 3.2 min-1, swamp eel hearts by 28.9%, from 38.0 ± 2.2 min-1 to 27.5 ± 1.6 min-1, and zebrafish hearts by 44.8%, from 113.7 ± 9.9 min-1 to 64.0 ± 9.7 min-1. Cumulative additions of HCO3- up to 50 mM to the hypoxic hearts was associated with a dose-dependent and significant increase in heart rate in the lamprey, dogfish, and swamp eel. Normoxic heart rate in the dogfish and swamp eel hearts was restored with addition of 50 mM HCO3- (Fig. 2.1B, C), while lamprey heart rate increased to 115% of the normoxic rate with the addition of 40 mM HCO3- (42.0 ± 3.1 min-1; Fig. 2.1A). The addition of HCO3- had no significant effect on heart rate in the flounder heart, while heart rate continued to decrease further in the sturgeon and zebrafish hearts (Fig. 2.1D, E, F). Addition of NaOH to hypoxic hearts, to mimic the pH change associated with the addition of HCO3-, had no significant effect on the hypoxic heart rate in the 39 hearts of any species tested and also did not stop the continued decrease in heart rate of sturgeon and zebrafish hearts. Addition of KH7 blocked the increase in heart rate associated with the addition of HCO3- in the lamprey heart only, such that heart rate was not significantly different to that observed during severe hypoxia (Fig. 2.1A). Addition of DMSO, the carrier for KH7, had no effect on lamprey hearts during severe hypoxia (Fig. 2.1A). Contractility After exposure to severe hypoxia, cardiac strips from all species tested experienced a significant decrease in contractility (Fig. 2.2). Contractility in dogfish ventricular strips decreased by 63.5%, from 14.8 ± 0.8 mN/mm2 to 5.5 ± 0.6 mN/mm2, swamp eel strips by 71.0%, from 10.2 ± 0.6 mN/mm2 to 3.0 ± 0.6 mN/mm2, and flounder strips by 42.3% from 11.3 ± 0.9 mN/mm2 to 6.4 ± 0.4 mN/mm2. The effect of addition of up to 50 mM HCO3- to the hypoxic cardiac strips was species specific. Contractility in the swamp eel increased significantly, but modestly from the hypoxic value of 3.0 ± 0.6 mN/mm2 to 4.7 ± 0.7 mN/mm2 (Fig. 2.2B). However, contractility did not change over the course of the experiment for dogfish strips, whereas contractility decreased significantly in the flounder, from 6.4 ± 0.4 mN/mm2 to 4.0 ± 0.5 mN/mm2 (Fig. 2.2A, C). Additions of NaOH and KH7 had no effect on contractility in any of the species tested. 40 2.3.2 Hypercapnic Acidosis Heart Rate After exposure to hypercapnic acidosis, hearts from all species experienced a significant decrease in heart rate (Fig. 2.3). Lamprey heart rates slowed by 80.9%, from 27.0 ± 1.9 min-1 to 5.0 ± 0.9 min-1, dogfish hearts by 24.9%, from 20.8 ± 0.5 min-1 to 15.7 ± 0.9 min-1, sturgeon hearts by 36.6%, from 30.3 ± 2.4 min-1 to 18.7 ± 0.9 min-1, flounder hearts by 38.8%, from 49.5 ± 3.8 min-1 to 30.3 ± 4.3 min-1, swamp eel hearts by 41.6%, from 29.5 ± 0.7 min-1 to 17.2 ± 0.7 min-1, and zebrafish hearts by 33.8%, from 101.1 ± 2.9 min-1 to 67.2 ± 5.3 min-1. The percentage decrease in heart rate brought on by hypercapnic acidosis in the lamprey, flounder, and swamp eel hearts was greater than that brought on by severe hypoxia in these four species, but less in the dogfish, sturgeon, and zebrafish hearts. In sturgeon and zebrafish hearts, the larger reduction in heart rate during severe hypoxia may be in part due to a lower CO2 percentage (7.5% CO2) compared to the dogfish, flounder, and swamp eel hearts (15% CO2), but lampreys hearts (7.5% CO2) experienced a much larger percentage decrease in heart rate during hypercapnic acidosis (81%) than during severe hypoxia (33%). Addition of up to 50 mM HCO3- to the hypercapnic hearts was associated with a significant, dose-dependent increase in heart rate in all species with the exception of the flounder, which continued to decrease its heart rate over the course of the experiment (Fig. 2.3E). Normocapnic heart rate in the dogfish, swamp eel, and sturgeon hearts was restored with the addition of 50 mM HCO3- (Fig 3B, C, D). Lamprey and zebrafish heart rates were significantly increased but not fully restored to normocapnic levels with the addition of HCO3-. Lamprey heart rate increased from 5.0 ± 0.9 min-1 to 16.0 ± 1.3 min-1 with addition of 40 mM HCO3-. While zebrafish heart rate increased from 67.2 ± 5.3 min-1 to 87.5 ± 4.5 min-1 with 30 mM HCO3- (Fig. 41 2.3A, F), further addition of HCO3- resulted in the zebrafish hearts to stop beating. Addition of KH7 reduced heart rate only in the hypercapnic lamprey heart from 16.0 ± 1.3 min-1 to 9.2 ± 0.9 min-1, a level still significantly higher than the hypercapnic heart rate of 5.0 ± 0.9 min-1 (Fig. 2.3A). Addition of DMSO, the carrier for KH7, again had no effect on lamprey hearts during hypercapnic acidosis (Fig. 2.3A). Addition of NaOH, to mimic the pH change associated with the addition of HCO3-, significantly increased heart rate in dogfish and sturgeon to normocapnic levels, but had no effect on the hearts of the other species (Fig. 2.3B, D). No spontaneous recovery in heart rate was seen for any of the species tested when hearts were maintained under hypercapnic conditions (Fig. 2.3). Contractility After exposure to hypercapnic acidosis, cardiac strips from all species experienced a significant decrease in contractility (Fig. 2.4). Contractility in dogfish decreased by 68.0%, from 14.9 ± 1.4 mN/mm2 to 4.7 ± 0.8 mN/mm2, swamp eel by 34.2%, from 12.0 ± 0.8 mN/mm2 to 7.8 ± 0.5 mN/mm2, and flounder by 41.1%, from 10.7 ± 0.9 mN/mm2 to 6.2 ± 0.3 mN/mm2. The percentage decreases in contractility brought on by hypercapnic acidosis were similar to the decreases brought on by severe hypoxia in the dogfish and flounder cardiac strips, but less in the swamp eel strips. The effect of additions of up to 50 mM HCO3- during hypercapnia was species specific. Contractility in dogfish increased significantly from 4.7 ± 0.8 mN/mm2 to 9.2 ± 1.1 mN/mm2, which was still lower than the normocapnic level (Fig. 2.4A). Contractility did not change over the course of the experiment in swamp eel hearts, while contractility continued to decrease significantly in the flounder, from 6.2 ± 0.3 mN/mm2 to 4.2 ± 0.5 mN/mm2 (Fig. 2.4B, C). 42 Addition of KH7 had no effect on contractility in cardiac strips of any of the species. Addition of NaOH had no effect on swamp eel and sturgeon cardiac strips, but NaOH significantly increased contractility of dogfish cardiac strips back to normocapnic levels. No spontaneous recovery in contractility was seen in control cardiac strips in any of the species tested. 2.3.3 Adrenergic Stimulation of Lamprey Hearts During exposure to normoxia, addition of isoproterenol to lamprey hearts caused a dose-dependent increase in heart rate by 71.0%, from 33.2 ± 1.4 min-1 to 56.8 ± 2.9 min-1 with 100 μM isoproterenol (Fig. 2.5). Increasing concentrations of propranolol reversed the positive chronotropic effect of 100 μM isoproterenol, decreasing heart rate to 44.8 ± 2.8 min-1 with 1 μM propranolol and 25.6 ± 4.0 min-1 with 5 μM propranolol, 23.0% lower than the initial heart rate. After exposure to severe hypoxia, the significant decrease in heart rate of the lamprey heart (a decrease of 28.9%, from 31.5 ± 3.2 min-1 to 22.8 ± 3.7 min-1; Fig. 2.6) was fully reversed by the addition of 10 μM forskolin (29.8 ± 2.4 min-1; Fig. 2.6). However, heart rate significantly decreased (18.0 ± 1.4 min-1) back to the hypoxic level upon addition of 100 μM forskolin. 2.3.4 Immunofluorescence Immunofluorescence staining of the lamprey heart with anti-sAC identified sAC (in green) throughout the heart with co-staining of nuclei (in blue) (Fig. 2.7). 43 Figure 2.1. Effects of cumulative NaHCO3 additions on the normalized heart rate of isolated hearts during severe hypoxia in different species. (A) Lamprey. (B) Dogfish. (C) Swamp Eel. (D) Sturgeon. (E) Flounder. (F) Zebrafish. Black squares represent values obtained from the KH7 treatment, open circles represent the NaOH treatment, and open triangles represent the DMSO treatment. Data are presented as mean ± s.e.m. Different letters indicate statistical differences within the KH7 treatment while asterisks indicate significance of the NaOH or DMSO treatment with the highest [NaHCO3] (statistical analysis done on raw data, P<0.05, one-way repeated measures ANOVA within KH7 treatment, one-way ANOVA between treatments, n=6). 44 Figure 2.2. Effects of cumulative NaHCO3 additions on the normalized contractility of cardiac strips during severe hypoxia in different species. (A) Dogfish. (B) Swamp Eel. (C) Flounder. See Figure 2.1 for further details. 45 Figure 2.3. Effects of cumulative NaHCO3 additions on the normalized heart rate of isolated hearts during hypercapnic acidosis in different species. (A) Lamprey. (B) Dogfish. (C) Swamp Eel. (D) Sturgeon. (E) Flounder. (F) Zebrafish. 15% CO2 for dogfish, swamp eel, and flounder and 7.5% CO2 for lamprey, sturgeon, and zebrafish. Black squares represent values obtained from the KH7 treatment, open circles represent the NaOH treatment, open triangles represent the DMSO treatment, and open squares represent the control treatment. Data are presented as mean ± s.e.m. Different letters indicate statistical differences within the KH7 treatment while asterisks indicate significance of the NaOH, DMSO, or control treatment with the highest [NaHCO3] (statistical analysis done on raw data, P<0.05, one-way repeated measures ANOVA within KH7 treatment, one-way ANOVA between treatments, n=6). 46 Figure 2.4. Effects of cumulative NaHCO3 additions on the normalized contractility of cardiac strips during hypercapnic acidosis in different species. (A) Dogfish. (B) Swamp Eel. (C) Flounder. See Figure 2.3 for further details. 47 Figure 2.5. Effect of increasing isoproterenol and propranolol concentrations on the normalized heart rate of isolated lamprey hearts during normoxia. After the addition of 100 μM isoproterenol, 1 μM (black triangle) and 5 μM propranolol (black square) were the added. Data are presented as mean ± s.e.m. Different letters indicate statistical differences between treatments (statistical analysis done on raw data, P<0.05, one-way repeated measures ANOVA within KH7 treatment, one-way ANOVA between treatments, n=6). 48 Figure 2.6. Effect of increasing forskolin concentrations on the normalized heart rate of isolated lamprey hearts during severe hypoxia. Data are presented as mean ± s.e.m. Different letters indicate statistical differences between forskolin concentrations (statistical analysis done on raw data, P<0.05, one-way repeated measures ANOVA within KH7 treatment, one-way ANOVA between treatments, n=6). 49 Figure 2.7. Immunofluorescences staining of sAC in lamprey hearts. (A) sAC staining in green while co-staining of nuclei in blue. (B) Negative control stain with only secondary antibodies and nuclei staining. 50 2.4 Discussion This study investigated the effects of elevated extracellular [HCO3-] on the cardiac function of fish during severe hypoxia and hypercapnic acidosis to examine the potential role that might be played by sAC, which had previously been implicated in increasing the heart rate of the anoxic hagfish heart (Wilson et al. 2016). I discovered that sAC was also implicated in the control of heart rate in both the hypoxic and hypercapnic heart of the lamprey, another primitive cyclostome like the hagfish. In contrast, sAC was not implicated in the control of either heart rate or contractility of a representative elasmobranch species and representative teleost species. However, HCO3- was associated with influencing cardiac function via other mechanisms in some, but not all, of the species tested. Thus, beyond extending our knowledge on the role of HCO3- in influencing heart rate and contractility, the present work provides support that the sAC-mediated control of heart rate is a mechanism unique to the primitive cyclostome lineage and may have been lost in more modern fish lineages. 2.4.1 sAC-mediated Control of Lamprey Heart Rate In the lamprey heart, addition of extracellular HCO3- was associated with an increase hypoxic heart rate to 115% of the normoxic rate and hypercapnic heart rate to 83% of the normocapnic rate, and both of these increases in heart rate could be blocked by KH7, a sAC blocker. Addition of KH7 decreased lamprey heart rate to the same rate observed during severe hypoxia, which suggests that sAC stimulation of heart rate was not normally present during severe hypoxia. If there was a tonic level of sAC stimulation present, the addition of KH7 should have brought heart rate below the rate seen during severe hypoxia. The addition of DMSO, the 51 carrier for KH7, to the saline bath had no effect on heart rate, which suggests that the decrease in heart rate seen in lamprey hearts when KH7 was added was not due to DMSO. These results were consistent with findings obtained by Wilson et al. (2016) in the hagfish, where heart rate dropped to about 50% of the normoxic rate during anoxia and the addition of 60 mM HCO3- increased heart rate to about 140% of the normoxic rate. KH7 had also blocked the increase in the hagfish heart rate caused by the addition of HCO3- and returned heart rate back to the anoxic rate. Immunofluorescence staining of the lamprey hearts identified sAC throughout the heart, a distribution similar to what was also observed in the hagfish heart (Wilson et al. 2016). Similar increases in the lamprey heart rate was observed upon addition of forskolin to hypoxic hearts, but forskolin did not increase the hypoxic heart rate above 100% as observed during sAC stimulation. Since forskolin directly stimulates tmAC to produce cAMP, it seems likely that sAC stimulation by HCO3- also increases cAMP production to cause the increase in heart rate observed. Increases in heart rate seen upon the addition of HCO3- are consistent with HCO3- stimulating sAC to catalyze the conversion of ATP into cAMP, which, according to the membrane clock hypothesis for pacemaker control, would then bind onto HCN channels (Difrancesco and Tortora 1991; Wainger et al. 2001). This then causes the HCN channel’s probability of opening to increase, increasing the pacemaker cell’s permeability to Na+ and K+, and therefore increases the If current to increase heart rate (Wang et al. 2001). Under the calcium clock hypothesis, cAMP would then activate PKA, which would then phosphorylate SR Ca2+ cycling proteins to speed up Ca2+ release and uptake to increase heart rate (Lakatta et al. 2006). With the addition of KH7, it binds to sAC, preventing HCO3- stimulation and therefore preventing the further production of cAMP, reducing heart rate back to the severe hypoxia rate. However, direct cAMP measurements upon addition of HCO3- will be needed to confirm cAMP 52 involvement. In hagfish, cAMP production increases upon HCO3- stimulation of sAC, and the response could also be blocked upon the addition of KH7 (Wilson et al. 2016). During hypercapnic acidosis, addition of KH7 could not completely block the increase in heart rate associated with the addition of HCO3-. Heart rate decreased from 62% (with 40 mM HCO3-) of normocapnic rate to 37% (with KH7), but was still higher than the hypercapnic heart rate (19%). During severe hypoxia, however, KH7 completely blocked the increase in heart rate upon addition of HCO3-, from 115% of the normoxic rate back to the severe hypoxia rate (63%). Although the hypercapnic acidosis treatment results in a saline with a higher extracellular [HCO3-] than during the severe hypoxia treatment, the slight increase in extracellular [HCO3-] is unlikely to explain the incomplete blockade. The difference in extracellular [HCO3-] in the saline was only approximately 10%, while the difference between heart rates with and without KH7 during hypercapnia was approximately 20%. Also, addition of more KH7 (up to 100 μM, data not shown) was not able to further reduce heart rate from the 50 μM KH7 rate. The incomplete blockade of the heart rate back to the hypercapnic value may be associated with a combination of a sAC-mediated recovery pathway and a different HCO3- mechanism both being contributing factors in increasing heart rate in the lamprey. 2.4.2 Adrenergic Stimulation of Lamprey Hearts Isoproterenol, a β-adrenergic agonist, increased lamprey heart rate during normoxia in a dose-dependent manner, and heart rate increased to 170% of the initial heart rate. These results contrast to what had been observed in the hagfish, as their hearts were relatively insensitive to β-adrenergic stimulation (Fange and Ostlund 1954; Axelsson et al. 1990). The addition of propranolol, a β-adrenergic antagonist, diminished the effect of adrenergic stimulation, and 53 caused the heart rate to be 77% lower than the initial heart rate, and resulted in a similar heart rate seen during severe hypoxia. These findings support that tmAC stimulation is present during normoxia and stops under severe hypoxia, thereby slowing heart rate. This is not surprising since oxygen is required during catecholamine synthesis to convert tyrosine to 3,4-dihydroxyphenylalanine (the rate-limiting step of catecholamine synthesis) and produce noradrenaline from dopamine (Levitt 1965). Therefore, during severe hypoxia, catecholamines cannot be synthesized, and as a result β-adrenergic receptors cannot stimulate tmAC to produce cAMP to increase heart rate. The addition of forskolin, a tmAC agonist, to lamprey hearts during severe hypoxia only increased heart rate back to the normoxic rate, while addition of HCO3- was associated with an increase of heart rate to 115% of the normoxic rate. While tmAC stimulation may cease during severe hypoxia, sAC stimulation can continue to increase cAMP levels even during severe hypoxia. Heart rate returned to the severe hypoxia rate with higher forskolin concentrations and may potentially be attributed to limited ATP production and ATP availability due to cardiac demand, which would then limit ATP available to be converted to cAMP. These findings suggest that even with maximal tmAC stimulation, heart rate cannot increase beyond normoxic rates during severe hypoxia, and that multiple cardiac control pathways exist in the lamprey. However, a question still exists as to why sAC can stimulate lamprey heart rate above normoxic rates during severe hypoxia yet direct tmAC stimulation fails to do so, since β-adrenergic stimulation increases normoxic rate even higher than sAC stimulation. It has been well established that hagfish and lampreys can modulate their heart rate via the release of catecholamines from chromaffin tissue located within the heart (Ostlund et al. 1960; von Euler and Fange 1961). These catecholamines bind to β-adrenergic receptors, 54 stimulating tmAC to produce cAMP to increase heart rate. The finding that propranolol reduced lamprey heart rate to that of severe hypoxia even during normoxia in is agreement with previous studies, which suggested that paracrine release of catecholamines may provide tonic stimulation of heart rate, rather than circulating catecholamines providing that role (Ostlund et al. 1960; von Euler and Fange 1961). Therefore, the lamprey heart appears to act as an endocrine gland as in the hagfish, providing paracrine stimulation of heart rate (Axelsson et al. 1990; Wilson 2014). 2.4.3 Effects of Severe Hypoxia Severe hypoxia decreased heart rate in all species by 55% to 75% of their normoxic rate, which is similar to the observed reduction in heart rate by up to 50% in various other fish species (Randall and Smith 1967; Marvin and Burton 1973; Farrell et al. 1985; Gesser 1985; Fritsche and Nilsson 1988; MacCormack et al. 2003). While addition of HCO3- was associated with an increase in heart rate of dogfish and swamp eel hearts back to the normoxic rate, the addition of KH7 had no effect, implying no role of sAC and suggesting that the HCO3--mediated increases in heart rate in these two species occur via a different pathway from the lamprey. A direct effect of an extracellular pH on recovery was ruled out as the addition of NaOH to the saline bath to create the same pH observed with the addition of 50 mM HCO3- had no effect on heart rate in any of the species tested, even in the lamprey. During severe hypoxia, contractility of the cardiac strips in all species decreased by 42% to 71% of their initial value, greater than the 20%-50% loss in contractility reported in other fish species (Gesser 1977; Gesser 1985; Hartmund and Gesser 1992; Overgaard et al. 2007). Despite being air-breathers, the swamp eel does not appear to have a heart that is particularly tolerant to hypoxia. Swamp eel cardiac strips experienced a 71% loss in contractility under severe hypoxia, 55 similar to the 75% loss in contractility observed by Iversen et al. (2013) for the same species. In contrast, the cardiac strips of another air breathing catfish Pangasianodon hypophthalmus experienced a reduction in contractility of only 40% during anoxia (Joyce et al. 2015). While the addition of HCO3- to the saline during severe hypoxia was associated with a recovery of heart rate back to normoxic levels in the dogfish, and swamp eel hearts, it had little effect on contractility. The addition of HCO3- was associated with a slight increase in the contractility of the swamp eel cardiac strips from 29% to 45% but had no effect on the contractility of dogfish and flounder hearts. Whether the recovery of heart rate and contractility occurs via different mechanisms is currently unknown. NaOH had no effect on contractility, suggesting the increase in contractility seen in the swamp eel cardiac strips was not due to an extracellular pH effect. 2.4.4 Effects of Hypercapnic Acidosis During hypercapnic acidosis, the heart rate of all species tested decreased by 25% to 81% of their normocapnic rate, which was similar to decreases (20-65%) observed for other fish species (Perry et al. 1999; Sundin et al. 2000; McKendry et al. 2001; McKenzie et al. 2002). The addition of HCO3- was associated with an increase in the heart rate of dogfish, swamp eel, and sturgeon hearts back to the normocapnic rate, and an increased in zebrafish heart rate slightly to an intermediate rate, even during sustained hypercapnic acidosis. Under normal physiological conditions, the [HCO3-] in blood plasma is typically about 6 mM (Gesser 1977; Wood et al. 1982). However, under severe hypercapnic acidosis, [HCO3-] in plasma can reach up to 40 mM in some fish species (Clairborne and Heisler 1986; Iwama and Heisler 1991; Ishimatsu et al. 2004; Damsgaard et al. 2015). Any increase in intracellular HCO3- would require the conversion 56 of HCO3- into CO2 in the saline, the CO2 would then need to diffuse into the heart and be converted back to HCO3- by carbonic anhydrase inside the heart. Intracellular [HCO3-] would then likely be lower than the amount of HCO3- that has been added to the saline bath as not all of the HCO3- is likely to have moved intracellularly. Thus, intracellular [HCO3-] should be measured to determine if the concentration of HCO3- added is physiologically relevant. Addition of NaOH to mimic the pH change associated with the addition of HCO3- was associated with the same increases in heart rate in dogfish and sturgeon hearts, but had no effect on swamp eel and zebrafish hearts. Dogfish and sturgeon hearts may possibly be more tolerant of high CO2 levels than in the other species, and an extracellular pH increase back to physiological levels may have been enough to recover heart rate back to the normocapnic rate. It was not surprising that the sturgeon heart was able to recover heart rate during hypercapnic acidosis as they are able to tolerate high CO2 levels that are lethal to other fish species (Baker et al. 2011). During hypercapnic acidosis, contractility of cardiac strips in all species tested decreased by 34% to 68% of their initial value, similar to the decreases (20-60%) observed in other studies (Gesser and Jorgensen 1982; Gesser and Poupa 1983; Salas et al. 2006; Shiels et al. 2010; Joyce et al. 2015). The addition of HCO3- was associated with an increase in contractility of dogfish ventricular strips from 32% to 64% but had no effect on the contractility of swamp eel and flounder ventricular strips. Similar to what was observed in the severe hypoxia experiments, addition of HCO3- during hypercapnic acidosis was associated with recovery of heart rate in swamp eel hearts but was unable to recover contractility in their cardiac strips. Addition of NaOH was also associated with the same magnitude of increase in contractility in dogfish cardiac strips as HCO3-, suggesting that the increase in contractility of dogfish cardiac strips may also be due to a pH effect. 57 These results were not expected since Gesser and Jorgensen (1982) had observed spontaneous recovery in contractility for their flounder cardiac strips. Contractility for their flounder cardiac strips had initially experienced about a 35% loss in contractility but spontaneously recovered to about 130% of initial normocapnic values after a 20-min exposure to 15% CO2, without addition of any HCO3-. In this present study, the contractility of flounder cardiac strips continued to decline after an initial 40% loss in contractility even with the addition of HCO3-, and no spontaneous recovery was observed in any of the species tested. A spontaneous recovery in cardiac contractility of other air-breathing fish and vertebrate species had also been observed (Gesser and Poupa 1978; Salas et al. 2006; Joyce et al., 2015). However, in this present study, the swamp eel heart showed no spontaneous recovery even after the addition of HCO3-. 2.4.5 Summary This research has demonstrated that a sAC-mediated cardiac recovery pathway that is stimulated by HCO3- has also been observed in the lamprey heart. This sAC-mediated pathway was associated with full recovery of heart rate during severe hypoxia but only partial recovery of heart rate during hypercapnic acidosis, which suggests that lampreys may be more tolerant to low O2 than high CO2. With the discovery that the sAC-mediated recovery pathway in cardiac control was only observed in lampreys and hagfish (Wilson et al. 2016) and not in the other species tested, it suggests that this cardiac recovery pathway is unique to the cyclostomes. While the sAC-mediated recovery pathway may be unique to the cyclostomes, the sAC protein has been found to be present in the gill, rectal gland, cornea, intestine, white muscle, and heart of another elasmobranch, the leopard shark (Triakis semifasciata) (Roa and Tresguerres 2017). This 58 suggests that sAC could also be present in the hearts of other species as well, but may play other roles instead that may not be involved in cardiac control. While flounder heart rate and contractility were unresponsive to additions of extracellular HCO3- to the saline, the addition of HCO3- to the hearts of the other fish species during severe hypoxia and hypercapnic acidosis was associated with two recovery responses that were sAC-independent for heart rate and contractility. The first was a HCO3--mediated recovery in cardiac function, observed in the dogfish and swamp eel heart rate during severe hypoxia, swamp eel cardiac contractility during severe hypoxia, and swamp eel and zebrafish heart rate during hypercapnic acidosis. The second was a pH-mediated recovery in cardiac function, observed in dogfish and sturgeon heart rate, and dogfish cardiac contractility, but this recovery pathway was only observed during hypercapnic acidosis. Furthermore, HCO3- may have different effects on heart rate and contractility, as it was observed that increasing extracellular [HCO3-] was associated with a full recovery of heart rate back to initial levels during severe hypoxia and hypercapnic acidosis, but fails to fully recover contractility back to initial levels. Although other studies of cardiac contractility in hypoxia tolerant and air-breathing species have shown a biphasic and spontaneous recovery response during hypercapnic acidosis, the mechanism behind the spontaneous recovery in contractility is unlikely due to sAC as the addition of KH7 had no effect on heart rate or contractility in this study. While different mechanisms have been proposed, either by intracellular pH regulation or increasing Ca2+ influx, the exact mechanism behind how HCO3- is able to elicit a cardiac recovery response is still unclear. 59 Chapter 3: Conclusion 3.1 General Discussion 1) What are the effects of increasing extracellular [HCO3-] on in vitro cardiac function during severe hypoxia and hypercapnic acidosis? Increasing extracellular [HCO3-] during severe hypoxia and hypercapnic acidosis was associated with a recovery of heart rate and contractility for all species tested in this study except for the flounder. However, the magnitude of the recovery and mechanism of action varied for each species. Furthermore, it appears as though increasing extracellular [HCO3-] has different effects on heart rate and contractility. Thus, the results I observed in this study provide support for the hypothesis that the addition of extracellular HCO3- during severe hypoxia and hypercapnic acidosis is associated with a recovery in heart rate in some fish species, but appears to have little to no effect on contractility. It is still unclear the exact mechanism behind how the heart can recover cardiac function upon addition of HCO3- during ongoing severe hypoxia (only known in the cyclostomes) and hypercapnic acidosis and why HCO3- has different effects on heart rate and contractility. 2) Is sAC associated with cardiac recovery during severe hypoxia and hypercapnic acidosis? While the sAC-mediated pathway in cardiac control likely plays a role in the recovery of heart rate observed for the lamprey heart during severe hypoxia and hypercapnic acidosis, it does not explain the recovery in heart rate and contractility observed upon the addition of HCO3- for the dogfish, sturgeon, swamp eel, and zebrafish hearts. The addition of KH7 could only block the 60 increase in heart rate in the lamprey but not in the other species tested. Thus, three recovery responses were observed in this study, a sAC-mediated, a HCO3--mediated, and a pH-mediated recovery of cardiac function. The sAC-mediated pathway is also unlikely to explain the spontaneous recovery in cardiac contractility observed in other studies (Gesser and Poupa 1978; Yee and Jackson 1984; Salas et al. 2006; Joyce et al., 2015) and may be due to a HCO3-mediated pathway instead. Therefore, I reject the hypothesis that recovery in cardiac function observed upon addition of extracellular HCO3- during severe hypoxia and hypercapnic acidosis is always associated with the sAC-mediated pathway. 3) Is the sAC-mediated cardiac control pathway unique to the hagfish or an adaptation to debilitating environmental conditions? sAC has been found to be present in a variety of different animal groups, from cnidarians to bony fish to mammals species (Tresguerres et al. 2014) and even in leopard shark hearts (Roa and Tresguerres 2017), but based on findings obtained in this study, it appears as though sAC is only involved in the cardiac control of hagfish and lampreys only. Consequently, I reject the hypothesis that the sAC-mediated pathway in cardiac control is an adaptation for animals frequently exposed to debilitating environmental conditions as the pathway is likely to be unique to the cyclostomes. 3.2 Future Directions While the sAC-mediated recovery pathway was observed in the lamprey and hagfish, it is unknown if sAC plays other roles in cardiac control. In lamprey and hagfish hearts, sAC has been found through immunofluorescence staining to be expressed in both atrial and ventricular 61 myocardium and not just in the pacemaker region (Wilson et al. 2016). Therefore, sAC might possibly play a role in the regulation of cardiac contractility as well, but is presently untested in hagfish and lamprey hearts. sAC can increase cardiac contractility by increasing cytosolic Ca2+ influx through PKA phosphorylation of L-type Ca2+ channels (Gao et al. 1997). However, during severe hypoxia and hypercapnic acidosis, increasing extracellular [HCO3-] had different effects on heart rate and cardiac contractility in the species tested in this study. Thus, further experimentation will be required to discover the other roles of sAC in cardiac control. While the addition of extracellular HCO3- was associated with a recovery in heart rate and contractility in the hearts of some fish species in vitro, it is unknown if the same recovery will be observed and how important it would be in vivo. During exposure to anoxia, hagfish experienced a reduction of heart rate of about 55%, and following normoxic recovery after 1 h, heart rate increased by 50% of the routine rate (Cox et al. 2011). Would the recovery beyond routine heart rate be associated with the sAC-mediated pathway? Would the addition of HCO3- to the blood of the hagfish during anoxia be associated with the same recovery in heart rate in vivo as observed by Wilson et al. for hearts in vitro (2016)? What about for other species examined in this study? Another question of interest would be at what O2 saturation level catecholamine synthesis and tmAC stimulation resume, and whether the sAC-mediated pathway is only activated during low O2 saturation or if it is present even during normoxia. This study was unable to discover the exact mechanism behind how increasing extracellular [HCO3-] is associated with an increase in heart rate or cardiac contractility during severe hypoxia and hypercapnic acidosis. One possibility may be due to intracellular pH recovery due to active pH regulation, via a Na+/HCO3- cotransporter or a HCO3-/Cl- exchanger to increase intracellular [HCO3-] (Madshus 1988; Lagadic-Gossmann et al. 1992; Liu et al. 1990). 62 In addition, since NaHCO3 was added to the saline bath during the experiments, increases in intracellular [HCO3-] would require the conversion of HCO3- into CO2 in the saline. The CO2 would then diffuse into the heart and be converted back to HCO3- by carbonic anhydrase inside the heart to then stimulate sAC. In embryonic zebrafish 4 days post-fertilization, hypercapnia caused an increase in heart rate which was mediated by the interaction of catecholamines with β-adrenergic receptors (Miller et al. 2014). The tachycardia was prevented with atenolol, a β-adrenergic antagonist, and also reduced with acetazolamide, a carbonic anhydrase inhibitor, which suggests that the conversion of CO2 in the heart to HCO3- plays a role in increasing heart rate. Would the blockade of HCO3- transporters or carbonic anhydrase block the cardiac recovery response seen in this study? Intracellular pH measurements can also be carried out to see if recovery in cardiac function was associated with active intracellular pH regulation back to physiological levels instead. In addition, it would be interesting to determine if the sAC-mediated pathway could play a role in the observed increase of heart rate in embryonic zebrafish during hypercapnia (Miller et al. 2014). The cardiac response of embryonic zebrafish to hypercapnia appears to be different than the bradycardia that is typically observed in other adult fish (Perry et al., 1999; Sundin et al., 2000; McKendry et al., 2001; Gilmour et al., 2005). It has been suggested that the embryonic zebrafish fish heart is not yet innervated until about 4 to 7 days post fertilization (Schwerte et al. 2006; Miller et al. 2014), which would mean that the embryonic zebrafish heart would go through a functionally aneural stage, just like the hagfish heart. Could the sAC-mediated pathway be present during developmental stages when the heart has yet to be innervated but subsequently be absent during adult stages? Experiments could be carried out on embryonic 63 zebrafish and other species to see if increases in heart rate during hypercapnia could be blocked by addition of KH7. From the results observed in this study, it appears as though the sAC-mediated cardiac control pathway is not an adaptation for animals frequently exposed to debilitating environmental conditions and is unique to the cyclostomes instead. However, a more complete phylogenetic approach should be done to determine the origin of the sAC-mediated cardiac control pathway by examining more species. The 3 major chordate branches extant today are the urochordates (includes tunicates), the cephalochordates (includes amphioxus), and the vertebrates (Bishopric 2005). In the vertebrate lineage, the heart had evolved from a single layered tube in the urochordates and cephalochordates, to a two-chambered heart in fish, to a three-chambered heart in the amphibians, and eventually to a four-chambered heart in the reptiles, birds, and mammals. The urochordates and cephalochordates have a tubular heart that lacks chambers or valves, but the urochordates have two myogenic pacemakers at each end of the heart that allows for reversible blood flow (Solc 2007). It would be interesting to see if the sAC-mediated cardiac control pathway appeared earlier than the cyclostomes in the chordate lineage. 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Effects of bicarbonate on cardiac function in fish
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