Haverinen, Jaakko. Effects of temperature on the electrical excitability of fish

cardiac myocytes

– University of Joensuu, 2008, 159 pp.

University of Joensuu, PhD Dissertations in Biology, No. 54. ISSN 1795-7257

(printed), ISSN 1457-2486 (PDF)

ISBN 978-952-219-145-8 (printed), 978-952-219-144-1 (PDF)

Keywords: fish heart, atrium, ventricle, cardiac myocytes, temperature acclimation,

resting membrane potential (RMP), action potential (AP), sodium current (INa), rapid

component of the delayed rectifier potassium current (IKr), inward rectifier potassium

current (IK1), tetrodotoxin (TTX), rainbow trout, Oncorhynchus mykiss, crucian carp,

Carassius carassius, burbot, Lota lota, pike, Esox lucius, roach, Rutilus rutilus, perch,

Perca fluviatilis.

ABSTRACT

Introduction Fish can be subject to great and sometimes rapid changes in ambient

temperature which directly affect the rate of body functions. As a counterreaction to

chronic temperature changes, several fish show thermal compensation in metabolic

rate and body functions including electrical activity of the heart. Electrical excitation

of the fish heart originates from the sinoatrial pacemaker where a small number of

pacemaker cells generate spontaneous action potential (APs) and determines the rate

and rhythm of the heart. Cardiac AP is an outcome of the concerted action of several

ion channels in the plasma membrane of cardiac myocytes. When AP arrives at the

atrial and ventricular tissues, opening of the voltage-dependent Na+ channels causes

an abrupt Na+ influx into the cell, depolarization of membrane potential and rapid

impulse spread over the heart. After a long plateau phase of the cardiac AP, K+

currents repolarize the membrane back to the resting membrane potential (RMP)

(about -80 mV) and thereby adjust the duration of AP to comply with heart rate at

each temperature. The aim of this study was to examine how temperature acclimation

affects excitability of the fish heart through modification of sodium (INa), the delayed

rectifier potassium (IKr) and inward rectifier potassium (IK1) currents of cardiac

myocytes in species that have different temperature preferences and tolerances. The

exact anatomical site of the fish cardiac pacemaker and the effects of thermal

acclimation on the activity of pacemaker cells were examined in rainbow trout. The

electrophysiological properties of the cardiac INa in three teleost species (rainbow

trout, crucian carp and burbot) acclimated at two different temperatures were

examined, and the molecular composition of the trout cardiac Na+ channels was

clarified. Temperature-dependent regulation of AP duration by K+ currents was

studies in six different teleost species.

Materials and methods The fish were acclimated for four weeks at two different

temperatures within their normal thermal tolerance range. Atrial and ventricular APs

were recorded with intracellular microelectrodes from the spontaneously beating

hearts, and sarcolemmal ion currents were measured from enzymatically isolated

cardiac myocytes by the whole-cell patch-clamp method. Molecular cloning was used

to examine the α-subunit composition of the cardiac Na+ channels and their

tetrodotoxin (TTX) binding sites. Sinoatrial pacemaker tissue of the rainbow trout

heart was localized by conventional histological methods and by microelectrode

recordings.

Results The spontaneous rhythm of the trout heart originates from a small circular

tissue in the junctional area between sinus venosus, atrium and sinoatrial valve. The

discharge rate of this pacemaker was enhanced by cold-acclimation (4°C), possibly

due to the increased density of the IKr. Three different types of pacemaker cells

(spindle, spider and elongated spindle cells) were recognized on morphological

grounds and two categories (primary and secondary pacemaker cells) on the basis of

AP characteristics. Temperature acclimation modified atrial and ventricular INa in a

species-specific manner: the density of INa was increased in rainbow trout and burbot,

but depressed in crucian carp. The main Na+ channel isoform of the trout heart is the

ortholog of the mammalian cardiac skeletal muscle, omNav1.4, which unlike the

mammalian cardiac isoform, Nav1.5, is highly sensitive to TTX. Acclimation to cold

decreased the duration of cardiac AP in all six species examined and this response

was associated with increased density of the IK1 and/or the IKr.

Conclusion Chronic temperature changes have a profound effect on ion currents of

fish cardiac myocytes and provide explanations for temperature induced changes in

heart rate and AP duration. Compensatory shortening of AP duration seems to be an

almost ubiquitous response to cold-acclimation in fish, as is the up-regulation of the

IKr. By contrast, the IK1 and INa show more often species-specific responses. The

depression of INa in crucian carp may be associated with their winter dormancy in

anoxic winter waters, while different responses of the IK1 to temperature acclimation

may partly be a phylogenetically determined trait.

Jaakko Haverinen, Faculty of Biosciences, University of Joensuu, P.O. Box 111, FI-

80100 Joensuu, Finland

ACKNOWLEDGEMENTS

This study was supported by the Academy of Finland (project no. 210400). I would

like to express my warmest gratitude to my supervisor and mentor Professor Matti

Vornanen. His encouragement has been the key that unlocked the doors to the world

of scientific thinking and writing. While working with Matti, I have learned a great

deal about many aspects of science and life.

I am also grateful to the reviewers of this thesis, Dr. Holly A. Shiels and Docent

Tomi Streng.

I wish to thank my colleagues in the electrophysiological research group and all

who have assisted me in my research. Dr. Vesa Paajanen helped me to solve many

technical problems and advised me on the analysis of electrophysiological data. I am

grateful to Ms Minna Hassinen, MSc., for making all the molecular work of my

thesis. Ms Hanna Korajoki, MSc., advised me on many scientific and computing

questions, for which I wish to express my gratitude to her. Laboratory technician

Anita Kervinen prepared countless numbers of saline solutions for me, always quickly

and reliably. Research technician Harri Kirjavainen caught the fish for my

experiments and with Harri I took part in an adventurous fishing trip in the fishery of

Juuka. Animal attendant Leena Koponen took care of the welfare of the fish in the lab.

All your help has been invaluable!

I wish to acknowledge the whole staff of the Faculty of Biosciences at the

University of Joensuu and all of my numerous friends including Pentti, now sadly

deceased, for their support and encouragement during this work. Riikka-Liisa, I first

saw you on the first of May six years ago and since then your joy in life and success at

school have been stimulants for me during this work.

I warmly thank my parents, Mirjami and Juhani. With the love and the wisdom

of experience of life, you have always advised and motivated me to go forward in my

studies, work and in life. Father, through your heritage and guidance I have received

an inextinguishable spark of interest in fishing, hunting and nature. The inspiration I

received from these activities has helped me to complete this work. My sisters and

brothers, Jukka, Päivi, Jouni, Hanna, Kaisa, Janne and Sanna and their families,

thanks that you are all present in my life. In common moments and remembrances

have been the important resources for me. Special thanks to the men in my family

(also the small “men”) for unforgettable moments in fishing waters and in the

wilderness of Kainuu.

Finally, my dearest and greatest thanks belong to Tuija. I cannot hope to cover

the past years in these few sentences, I have not words enough, but they are

unnecessary - you understand. Thanks.

ABBREVIATIONS

AP action potential

APD action potential duration

RMP resting membrane potential

Vmax the maximum rate of AP upstroke

INa sodium current

IKr rapid component of the delayed rectifier potassium current

IKs slow component of the delayed rectifier potassium current

IK1 inward rectifier potassium current

Ito transient outward current

If or Ih hyperpolarization-activated pacemaker current

ICa, L L-type calcium current

ICa, T T-type calcium current

NCX sodium-calcium exchanger protein

Nav1.1-1.9 sodium channel isoforms

omNaV sodium channel of rainbow trout

TTX tetrodotoxin

IC50 half maximal inhibition of current

SR sarcoplasmic reticulum

Ry ryanodine

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following papers, which are referred to in the text by

chapter numbers:

Chapter 4 Haverinen, J. and Vornanen, M. 2007. Temperature acclimation

modifies sinoatrial pacemaker mechanism of the rainbow trout

heart. Am. J. Physiol. 292, R1023-R1032.

Chapter 5 Haverinen, J. and Vornanen, M. 2004. Temperature acclimation

modifies Na+ current in fish cardiac myocytes. J. Exp. Biol. 207,

2823-2833.

Chapter 6 Haverinen, J. and Vornanen, M. 2006. Significance of Na+

current in the excitability of atrial and ventricular myocardium of

the fish heart. J. Exp. Biol. 209, 549-557.

Chapter 7 Haverinen, J., Hassinen, M. and Vornanen, M. 2007. Fish cardiac

sodium channels are tetrodotoxin sensitive. Acta Physiol. 191,

197-204.

Chapter 8 Haverinen, J. and Vornanen, M. 2008. Responses of action

potential and K+ currents to temperature acclimation in fish

hearts. Phylogeny or thermal preferences? Physiol. Biochem.

Zool. 000, 000-000 (under revision).

Articles 4-7 were reprinted with permission from the publishers. The copyright for

publication 4 is held by the American Physiological Society, for publications 5-7 by

the Company of Biologists and for publication 5 by the Blackwell Synergy.

I participated in the design of the studies and conducted most of the

electrophysiological experiments. I analyzed all the electrophysiological data and

carried out the statistical analyses. I prepared the first version of the papers and

processed the articles in collaboration with the co-authors.

CONTENTS CHAPTER 1 General introduction 15 1.1 Thermal tolerance of fish 15 1.2 Fish heart 17 1.3 Initiation of the heartbeat 17 1.4 Cardiac action potential of atrial and ventricular myocytes 19 1.5 Sodium channels 20 1.6 Cardiac potassium channels 25 1.7 Importance of cardiac function in thermal tolerance of fish 26 1.8 Aims of the study 28 CHAPTER 2 Synopsis of methods 31 2.1 Whole-cell patch-clamp of ion currents 31 2.2 Microelectrode recording of action potentials 32 CHAPTER 3 Synopsis of results 35 3.1 Cardiac pacemaker of the rainbow trout heart (Chapter 4) 35 3.2 Regulation of cardiac excitability by sodium channels (Chapters 5-7) 35 3.3 Potassium currents and thermal action potential duration (Chapter 8) 37 References 39 CHAPTER 4 Temperature acclimation modifies sinoatrial pacemaker of the rainbow trout heart 53 CHAPTER 5 Temperature acclimation modifies Na+ current in fish cardiac myocytes 65 CHAPTER 6 Significance of Na+ current in the excitability of atrial and ventricular myocardium of the fish heart 79 CHAPTER 7 Fish cardiac sodium channels are tetrodotoxin sensitive 91 CHAPTER 8 Responses of action potential and K+ currents to temperature acclimation in fish hearts: Phylogeny or thermal preferences? 101 CHAPTER 9 General discussion 141 9.1 Rate and rhythm of the heart 141 9.2 Significance of sodium current in excitation of fish heart 145 9.3 Potassium currents and action potential duration 148 9.4 Conclusion 151

References 153

CHAPTER 1

GENERAL INTRODUCTION

GENERAL INTRODUCTION

Fish evolved around 500 million years ago and currently they form the oldest and

most diverse vertebrate group which includes more species than all other vertebrate

groups combined. The evolutionary position of fish and their ability to adapt to wide

variety of environments have made them frequently used models essentially in every

biological discipline including neurobiology, physiology, toxicology, endocrinology,

developmental biology and environmental research (Powers 1989).

1.1 Thermal tolerance of fish

Most fish are ectothermic and they have successfully occupied the most diverse

thermal habitats of aquatic ecosystems, from the subzero temperatures of the Southern

Ocean to the hot waters of desert caves (Brett 1956, Eastman 1997, Beitinger &

Bennett 2000). Indeed, the thermal tolerance range of fish extends from -2 to +44°C

(Beitinger & Bennett 2000). In north-temperate climates fish are exposed to great

seasonal changes of temperature which often require physiological plasticity, i.e. the

ability to change the physiological phenotype according to the seasonal temperature

conditions. The process of phenotype change in an individual or in a tissue occurs

within the framework of the same genome and is called temperature

acclimation/acclimatization: it is used to adjust physiology and performance in

response to an imposed chronic change of ambient temperature (Hazel & Prosser

1974, Bennet 1984). Thermal acclimation is often based on the ability to express

different proteins in response to changing temperature or to produce proteins that are

relatively insensitive to temperature (Somero 1975, Johnston & Temple 2002, Watabe

2002, Gracey et al. 2004, Vornanen et al. 2005).

Even in the changing temperature regimes of north-temperate latitudes fish show

quite high variability in their temperature tolerance. Some species, e.g. burbot (Lota

lota), prefer cold waters and probably tolerate only relative small temperature

changes, and they are therefore called temperate stenotherms (Bernard et al. 1993).

For temperate fish, rainbow trout have a relatively low upper thermal tolerance limit

(25°C) and are therefore often regarded as stenothermic (Hokanson 1977). By

contrast, eurythermic fish tolerate great temperature changes and are likely to show a

high physiological plasticity. Cyprinid fish, crucian carp (Carassius carassius L.)

among them, are well-known for their eurythermicity (Horoszevicz 1973). Fish with

intermediate thermal tolerance are called mesotherms and include such species as pike

(Esox lucius), perch (Perca fluciatilis) and roach (Rutilus rutilus) (Black 1953,

Horoszevicz 1973, Currie et al. 1998).

Physiological responses to chronic temperature changes are often such that they

compensate, partially or completely, for the effects of temperature on the rate of body

functions i.e. they tend to maintain metabolism, locomotion and cardiac function

relatively independent of temperature changes (Rome et al. 1985, Guderley 1990,

Driedzic et al. 1996, Aho & Vornanen 1999, Tiitu & Vornanen 2001). Under some

specific environmental conditions, e.g. in anoxic winter waters, it may be more

meaningful to allow temperature to exert its total impact to reduce the metabolic rate

and thereby diminish the use of limited energy reserves. Sometimes animals even

actively exaggerate the effects of temperature by suppressing the rate of physiological

processes more than would be caused by a simple temperature effect (Q10). This

process is called inverse acclimation and usually leads to metabolic depression under

harsh environmental conditions and thereby extends the survival time of the

individual (Crawshaw 1984, Driedzic & Gesser 1994, Vornanen 1994, Jackson 2000).

1.2 Fish heart

The heart of teleost fish consists of four compartments, two contracting muscular

chambers, the atrium and the ventricle, which propel blood into the vasculature and

two collecting cavities, the sinus venosus and the bulbus arteriosus which connect the

heart to the vasculature. The sinus venosus receives oxygen-poor venous blood from

the body and directs it to the atrium through an opening guarded by the sinoatrial

valve, while the bulbus arteriosus provides a blood pressure stabilizing and compliant

exit route for blood from the ventricle to the ventral aorta and further to the gills

(Santer 1985, Olson & Farrell 2006).

1.3 Initiation of the heartbeat

The function of the heart is to maintain continuous circulation of blood to active

tissues and provide them with oxygen and nutrients. The fish heart pumps blood via

the ventral aorta to the gills and further into the dorsal aorta and all body tissues

(Santer 1985). Each heartbeat is initiated by a pulse of electrical excitation or AP that

begins in a group of spontaneously active pacemaker cells in an anatomically and

functionally specialized cardiac tissue and subsequently spreads to the atrial and

ventricular muscles, eliciting sequential contraction of the muscular chambers. In the

mammalian heart, this center is located in the wall of the right atrium and is called the

sinoatrial node (Keith & Flack 1907, Bleeker et al. 1980, Bouman & Jongsma 1986).

In the fish heart, the pacemaker area is assumed to be located in the sinus venosus or

sinoatrial junction, but this has been experimentally verified in only a few species

(Mackenzie 1913, Saito 1969, Yamauchi et al. 1973).

Rhythmic activity of the cardiac pacemaker is due to the presence of a

spontaneous diastolic depolarization or pacemaker AP that is driven by a net inward

current through the sarcolemma of the pacemaker cells. The pacemaker AP is an

outcome of a complicated interaction between several ion channels and ion

transporters. The net inward current results from deactivation of an outward current,

i.e. the delayed rectifier K+ currents (IKs and IKr), and the activation of inwards

currents, including a Na+-dependent background current (Ib,Na), a hyperpolarization-

activated pacemaker current (If or Ih) and T- and L-type Ca2+ currents (ICa,T, ICa,L)

(Brown 1982, Irisawa et al. 1993, Boyett et al. 2000). Of the other cell membrane

mechanisms the sodium pump, sodium-calcium exchanger (NCX) and intracellular

Ca2+ stores of the sarcoplasmic reticulum (SR) seem to be involved in cardiac

pacemaking, too (Zhou & Lipsius 1993, Ju & Allen 1999). Although large number of

ion channels and transporters are known to be involved in pacemaker function, there

is no consensus about the pacemaker mechanism of the vertebrate heart. In some

models deactivation of the delayed rectifier current(s) drives the diastolic

depolarization (Irisawa et al. 1993, Ono & Ito 1995, Verhecjk et al. 1995), in another

the hyperpolarization-activated “pacemaker” current is in the central position

(DiFransesco et al. 1995), while in still others spontaneous Ca2+ release from the SR

and subsequent activation of the sarcolemmal NCX play the dominant roles (Maltsev

et al. 2006).

Thus far, the pacemaker mechanism of the fish heart has not been studied at

cellular and molecular level. The heart rate in fish is strongly modified by thermal

acclimation and this effect seems to be largely mediated by temperature-dependent

changes in the activity of the autonomic nervous system (von Skramlik 1935, Farrell

& Jones 1992). In particular, inhibitory cholinergic innervation seems to be involved

and is up-regulated in warm environments (Seibert 1979, Axelsson et al. 1987). Thus,

the current paradigm assumes that the pacemaker mechanism itself does not

contribute to temperature-induced changes in heart rate. However, this has not been

directly tested.

1.4 Cardiac action potential of atrial and ventricular myocytes

Although molecular composition and functional characteristics of the fish heart are

quite different from those of the mammalian heart (Santer 1985), its electrical

properties obey the same biophysical principles and are in many respects similar to

those of humans and other mammals (Sedmera et al. 2003, Kopp et al. 2005, Milan et

al. 2006, Arnaout et al. 2007). The normal electrical excitability of cardiac myocyte

results from an elaborate balance between depolarizing (inward) and repolarizing

(outward) currents. Depolarization is mediated by channels or transporters that enable

positively charged Na+ or Ca2+ ions to enter the cell (or negatively charged Cl- ions to

exit) and repolarization results when the K+ efflux exceeds the sum of the Na+ and

Ca2+ influx (Hodgkin & Huxley 1952a). When the ion channels open, they drive the

membrane potential of the cell towards the equilibrium potential of the ion in

question. The opening of the K+ channels drives the cell towards -90 mV and the

opening of the Na+ and Ca2+ channels forces the membrane potential towards positive

levels, +60 mV or more. The resulting electrical signal or cardiac AP consists of five

phases, numbered 0-4 (Figure 1), which are produced by the opening and closing of

the Na+, K+, Ca2+ and Cl- specific ion channels. Six major ion currents govern the

vertebrate cardiac AP. INa generates the rapid upstroke (phase 0). Transient outward

current (Ito, carried by K+ and Cl- ions) causes the small and rapid repolarization

following the AP upstroke (phase 1). Potassium currents (IKr, IKs, IK1) regulate APD

(phases 2 and 3) together with ICa and maintain the negative resting membrane

potential (IK1) (phase 4) (Opie 1998, Hille 2001).

Figure 1. Action potential (AP) of the rainbow trout atrium and schematic presentation of different phases of cardiac AP and the underlying ion currents. The rapid phase 0 depolarization is mediated by Na+ entry into the cells due to a marked increase in the number of open Na+ channels in the cell membrane. In phase 1 the transient outward current (Ito) is due to the movement of K+ and Cl- ions in opposite directions. Note that this phase is poorly developed in the AP of the fish heart. Phase 2 of the cardiac action potential is sustained by a balance between inward movement of Ca2+ through L-type calcium channels (ICa, L) and outward movement of K+ through potassium channels (IKs, IKr, IK1). During phase 3 of the action potential, the L-type Ca2+ channels close and net outward, positive current causes the cell to repolarize. IKs and IKr disappear when the membrane potential is restored to about -80 to -85 mV, while IK1 remains conducting throughout the phase 4. The cell remains at the resting membrane potential (RMP) until it is stimulated by an AP spreading from the cardiac pacemaker over the entire heart. Modified from Opie (1998).

1.5 Sodium channels

The Amazonian electric eel (Electrophorus electricus) and electric ray (Torpedo

californica) have electroplax tissue which they use to generate powerful electrical

shocks to repel predators and to stun prey animals (Powers 1989). Because the electric

organ is a rich source of Na+ channels, the first vertebrate Na+ channels were cloned

and purified from this fish tissue (Agnew et al. 1978, Noda et al. 1984).

Subsequently, several Na+ channel isoforms have been found in various tissues of

mammals and fish, including the heart.

Voltage-gated Na+ channels are composed of a pore-forming α subunit and 1 or

more auxiliary β subunits (Catterall 1992, 1996). During the past two decades,

altogether ten genes encoding Na+ channel α subunits have been identified and nine

orthologs of these channels have been found in different mammalian tissues (Goldin

2001) and eight orthologs in fish tissues (Lopreato et al. 2001). The α subunit consists

of four repeat domains (I-IV), each of them having six transmembrane segments (S1-

S6) (Figure 2). The S4 segments form positively charged voltage sensors, which

coordinate the opening of the Na+ channel in a voltage-dependent manner. The short

intracellular loop connecting domains III and IV in channel forms “the plug”, that

closes the channel upon inactivation (Catterall 1992).

Figure 2. Structures of three ion channel types of the vertebrate cardiac muscle examined in the present study. Kir channels (top) are tetramers of proteins with two-transmembrane segments (S1 and S2) and they generate the cardiac IK1 current. Voltage-gated potassium channels (middle) are tetramers of four identical proteins each having six transmembrane segments (S1-S6). This type of channel produces e.g. the IKr current. The voltage-sensitive sodium channels (bottom) consist of one large protein which has four similar membrane-spanning domains (I-IV), each containing six transmembrane segments (S1-S6). Segments and/or domains are connected by intracellular and extracellular loops in each channel type. In the sodium channel, TTX binding sites in the pore loop region between S5 and S6 of the domains I-IV are marked with gray circles. Modified from Goldin (2001).

The concentration of Na+ ions in extracellular and intracellular compartments is

about 140 and 10 mM, respectively, creating an electrochemical gradient of +60 mV

across the plasma membrane, towards which membrane potential is driven by opening

of the Na+ channels (Hille 2001, Bers et al. 2003). The functional cycle of the Na+

channel (and other voltage-gated channels) involves three states (resting, open and

inactivated) and four gating transitions (activation, deactivation, inactivation and

recovery from inactivation) (Figure 3). In the resting state, the inactivation gate (h) is

in the open position and the activation gate (m) in the closed position, and

consequently the channel is non-conducting. Depolarization of atrial and ventricular

myocytes by a pacemaker potential or an external stimulus over the threshold value

(positive to -60 mV) changes channels from the resting state to the open state

(activation), i.e. both gates are open and Na+ ions flow into the cell. Depolarization of

the cell membrane causes movement of the inactivation gate to the closed position

(inactivation) and switches off the Na+ conductance. The activation gate has to

recover its open position (deactivation) and the inactivation gate its closed position

(recovery from inactivation) before a new cycle can be elicited, i.e. the channel cannot

be opened from the inactivated state (Hodgkin & Huxley 1952 b, Hille 1978, 2001,

Grant 2001).

Figure 3. Scheme of sodium channel gating. A depolarizing voltage step (top), an associated INa of a rainbow trout atrial myocyte (centre) and a physical model to account for the transient INa are shown. Sodium channel proteins form a water-filled pore with two voltage-sensitive gates in the lipid membrane of the cardiac myocyte. In the resting state, the activation (m) gate is in the closed position and the inactivation (h) gate is in the open position. When the threshold voltage for sodium channel opening is exceeded, the m gate assumes an open position (activation) and with both gates open, sodium ions diffuse into the cell. The h gate then moves into the closed position (inactivation), blocking the channel. When membrane potential returns to the resting level (-80 mV), the m gate moves into the closed position (deactivation). In a time- and voltage-dependent process termed recovery from inactivation, the h gate moves into the open position (not shown). A similar scheme applies to other voltage-gated ion channels. Modified from Grant (2001).

Na+ channels are effectively blocked by guanidium compounds, most notably

saxitoxin and TTX, produced by the glands of some poisonous animals. TTX is a very

potent toxin with a lethal dose (LD50) value of 0.1 mg/kg in mouse, and it is

abundantly present in the ovaries and liver and in lesser amounts in the intestine and

skin of the puffer fish (fugu, Tetradon viridis) and its relatives from the family

Tetraodontidae as well as in some newts and marine invertebrates (Denac et al. 2000).

TTX blocks the ion permeation of Na+ channels from an extracellular site, but has no

effect on channel gating (Lipkind & Fozzard 1994). Based on their sensitivity to TTX,

Na+ channels are categorized either as TTX-sensitive (Kd=1-1000 nM) or TTX-

resistant (Kd=1000-30000 nM). Among mammalian Na+ channels, Nav1.5, Nav1.8 and

Nav1.9 are TTX-resistant, whereas Nav1.1, Nav1.2, Nav1.3, Nav1.4 and Nav1.6 are

inhibited by nanomolar concentrations of TTX, as is the isoform expressed in skeletal

muscle (Nav1.4) (Fozzard & Hanck 1996, Goldin 2001, Lei et al. 2004). TTX-

resistant Nav1.5 is the main cardiac isoform in mammalian hearts, even though TTX-

sensitive isoforms Nav1.4, Nav1.1, Nav1.2 and Nav1.6 are expressed in small amounts

in different parts of the heart (Maier et al. 2002). Thus, the mammalian cardiac INa is

quite generally termed TTX-resistant.

1.6 Cardiac potassium channels

Voltage-gated K+ channels (Kv) (Figure 2) are tetramers of proteins which have six

membrane spanning sequences, segments 1-4 being associated with voltage-sensing

and -gating, while the loops between segments 5 and 6 form the K+ selective pore of

the channel (Warmke & Ganetsky 1994, Jan & Jan 1997). The inward rectifier K+

channels (Kir) are much simpler in structure than voltage-gated K+ channels since they

lack the voltage-sensing and -gating structures. They are hetero- or homotetramers of

proteins having only two membrane spanning segments. In the absence of the voltage-

sensitive sequences, the characteristic voltage-dependence of the inward rectifier

current is produced by a current-dependent blocking of the channel by intracellular

Mg2+ and polyamines (spermine and spermidine) (Nichols & Lopatin 1997).

Until recently, one voltage-gated K+ current (IKr) (Vornanen et al. 2002 a) and

three inward rectifier K+ currents (ATP-sensitive K current, IK,ATP; background inward

rectifier current, IK1; and acetylcholine-activated inward rectifier, IK,Ach) (Aho &

Vornanen 2002, Paajanen & Vornanen 2002, 2004, Vornanen et al. 2002 a) have been

found in fish hearts. The inward rectifier K+ channels of the vertebrate heart are

tetramers of Kir2.1, Kir2.2 and Kir2.3 proteins, the Kir2.1 being the main isoform in

mammals (Wang et al. 1998, Dhamoon et al. 2004). The inward rectifier of the

rainbow trout heart is formed by Kir2.1 and Kir2.2 (Hassinen et al. 2007), while

crucian carp heart expresses three Kir2 channels (Kir2.1, Kir2.2 and Kir2.5) (Hassinen

et al. 2008).

1.7 Importance of cardiac function in thermal tolerance of fish

The function of the heart is intimately associated with the thermal preferences of the

fish. In both stenothermic (narrow thermal tolerance) and eurythermic (wide thermal

tolerance) species the optimum temperature for heart function is close to the preferred

body temperature of the fish, and the activity of the heart is depressed towards both

low and high extremes of thermal tolerance (Brett 1971, Matikainen & Vornanen

1992). Due to thermal limitation of the heart at both low and high extreme of

temperatures, the animal is likely to suffer from systemic hypoxia much below its

acute thermal tolerance limits (Pörtner et al. 2004), and may eventually succumb to

hypoxia.

The hypothesis that a mismatch between the demand for oxygen and the

capacity of oxygen supply to tissues restricts whole-animal tolerance to thermal

extremes has recently been presented (Pörtner et al. 2006). It is proposed that when

the temperature approaches the upper or lower limits of thermal tolerance, oxygen

delivery systems may fail to provide enough oxygen to the tissues and the animal will

die to hypoxia or anoxia, mainly due to temperature-dependent limitation of the

circulation. Central to this hypothesis is the assumption that heart function is

compromised at extremes of low and high temperatures and therefore the heart fails to

nourish the tissues with oxygen. This hypothesis is made particularly fascinating by a

recent finding that changes in the distribution of fish populations due to global

warming are caused by limitation of cardiac function (Pörtner & Knust 2007).

Understanding thermal tolerance of the heart function in ectothermic vertebrates may

thus improve our knowledge of the impact of global warming on fish populations.

Cardiac muscle is an electrically excitable tissue, where ion channel function

plays a vital role in setting the rate and rhythm of the heart, initiating contraction and

regulating the force production of cardiac myocytes. Electrical excitation or cardiac

AP is produced by delicate and complex interaction between several ion channels in

the plasma membrane of the cardiac myocyte (Opie 1998, Schram et al. 2002). As a

complex phenomenon, cardiac AP is sensitive to disturbances in ion channel function,

which may generate arrhythmias, cause conduction failures and compromise the force

of cardiac contraction (Wang & Rudy 2000). Relatively small changes in body

temperature can elicit severe malfunction of the ion channels, as exemplified by fatal

cardiac arrhythmias in hypothermic humans (Walpoth et al. 1997, Marban 2002). Yet,

many ectothermic vertebrates, including several fish species, routinely experience

seasonal temperature changes of 20-25°C. In these animals, the cardiac ion channels

must have high resistance to temperature changes. On the other hand, such an

extension of thermal tolerance may have brought ion channel function close to the

limits of temperature-dependent failure. As electrical excitation of myocyte plasma

membrane governs all vital aspects of heart function (rate, rhythm, impulse

conduction and force), ion channels could be critical for the adaptation and

acclimation of ectothermic hearts to temperature. However, relatively little is known

about the effects of temperature on cardiac ion currents and the molecular

composition of the cardiac ion channels. Most studies have dealt with L-type Ca2+

current (Hove-Madsen & Tort 1998, Hove-Madsen et al. 2000, Vornanen 1997, 1998,

Shiels et al. 2000, 2002), and some data also exists for ATP-sensitive K+ current and

the delayed rectifier K+ current (Paajanen & Vornanen 2002, 2004). However,

practically nothing (Warren et al. 2001) is known about the fish cardiac Na+ current,

especially regarding its molecular background.

1.8 Aims of the study

In this thesis my aim was to examine how temperature acclimation affects the

electrical excitability of the fish heart in species living in different aquatic habitats and

having different thermal preferences and tolerances. The location of the cardiac

pacemaker tissue and the effects of thermal acclimation on initiation of heart beat was

investigated in rainbow trout sinoatrial tissue and isolated pacemaker cells (Chapter

4). The electrophysiological characteristics and molecular composition of cardiac Na+

channels in the atrial and ventricular myocytes of three teleost species were studied

with regard to acute and chronic temperature changes (Chapters 5, 6 and 7). Finally,

temperature-dependent regulation of APD by repolarising K+ currents was

investigated in six different teleost species in order to observe species-specific

differences in electrical excitability (Chapter 8).

CHAPTER 2

SYNOPSIS OF METHODS

SYNOPSIS OF METHODS

Established methods of electrophysiology were used to measure the electrical activity

of the fish heart in multicellular cardiac preparations and single cardiac myocytes

(Chapters 4-8). The sodium channel composition of the trout heart and the amino acid

sequence of the tetrodotoxin (TTX) binding site of the Na+ channels were obtained by

molecular cloning and sequencing (Chapter 7).

2.1 Whole-cell patch-clamp of ion currents

The whole-cell patch-clamp method (Hamill et all. 1981) was used to measure the

density and kinetics of Na+ and K+ currents from enzymatically isolated cardiac

myocytes of the fish heart. Atrial and ventricular myocytes were isolated by

enzymatic digestion of the intercellular connective tissue of the heart. To this end, the

heart was cannulated and perfused with a saline solution containing trypsin (10

mg/ml) and collagenase (15 mg/ml) for 10-15 minutes (Vornanen 1997). Small pieces

of softened atrial and ventricular muscle were agitated through the opening of a

Pasteur pipette to release single cardiac myocytes. Single cells were “patched” in a

small flow-through chamber with continuous temperature control of the saline

solution. Unwanted ion currents were prevented from using specific blockers (TTX,

nifedipine and E-4031 for the Na+ current, L-type Ca2+ current and rapid component

of the delayed rectifier K+ current, respectively) in the external saline solution and/or

from replacing external and internal K+ with Cs+ to block K+ currents. Capacitive cell

surface area was routinely determined and amplitudes of ion currents were always

given as current densities (pA/pF).

2.2 Microelectrode recording of action potentials

The action potentials (APs) of ventricle, atrium and pacemaker tissue were measured

from multicellular cardiac preparations using sharp microelectrodes (Ling & Gerard

1949). A temperature controlled tissue chamber (10 ml) was filled with a

physiological saline solution and a sinoatrial preparation, or a small whole-heart was

mounted on the bottom of the chamber with small needles. Myocytes of the selected

heart area were impaled on electrodes filled with 3M KCl solution (resistance 10-20

MΩ) and APs were recorded through a voltage amplifier and a digitizer on the

computer. Resting membrane potential and characteristics of the APs (amplitude,

overshoot, duration and rate of the upstroke) were measured off-line using Clampfit

software.

CHAPTER 3

SYNOPSIS OF RESULTS

SYNOPSIS OF RESULTS

3.1 Cardiac pacemaker of the rainbow trout heart (Chapter 4)

Using electrophysiological and histological methods, the pacemaker of the trout heart

was located in a small ring-form tissue in the border area between the sinus venosus

and the atrium. Two categories of pacemaker cells (primary and secondary) were

characterized from this tissue on the basis of action potential (AP), while three types

of pacemaker cells (spindle, spider and elongated spindle cells) were recognized on

the basis of myocyte shape. Cold acclimation (+4°C) increased AP rate (heart rate)

and decreased the duration of pacemaker AP. These differences were attributed to the

increased density of the delayed rectifier potassium current (IKr) in the cold-acclimated

trout hearts. By contrast, inhibition of sarcoplasmic reticulum (SR) function with

ryanodine (Ry) and thapsigargin did not contribute to acclimation-dependent changes

in AP rate.

3.2 Regulation of cardiac excitability by sodium channels (Chapters 5-7)

Sodium current (INa) is considered to determine the rate of the AP upstroke and

conduction velocity of AP over the heart. Temperature acclimation modified INa of the

fish cardiac myocytes differently, depending on the animal’s lifestyle and/or thermal

preferences (Table 1). In cold-active species (rainbow trout and burbot) cold-

acclimation increased the density of INa, whereas in the cold-dormant crucian carp, the

density of INa was depressed in the cold-acclimated winter fish, suggesting that

increased INa is needed to maintain high electrical excitability and conductivity in the

cold.

The INa of atrial and ventricular myocytes was compared in the cold-acclimated

rainbow trout. Although the maximum rate of action potential upstroke was 21%

greater in atrial than in ventricular tissue, the density of INa did not differ between

atrial and ventricular myocytes. This contradiction might be due to the more extensive

resistive coupling with myocytes and fibroblasts in the ventricle than in the atrium,

since fibroblasts can function as current sinks for INa and thereby reduce the effect of

INa on the AP upstroke. Thus, in atrial muscle the high velocity of impulse conduction

would be attributable to weak electric coupling of myocytes to fibroblasts, and not to

INa. The voltage-dependence of INa activation was more negative in atrial than in

ventricular myocytes. This together with much higher input resistance of atrial

myocytes makes atrial muscle readily excitable by weak depolarizing voltages from

pacemaker cells.

The INa of the trout heart is about 1000 times more sensitive to tetrodotoxin

(TTX) (IC50 = 1.8–2 nM) than the mammalian cardiac INa and it is produced by three

sodium channels (Nav) α-subunits which are orthologs to mammalian skeletal muscle

Nav1.4, cardiac Nav1.5 and peripheral nervous system Nav1.6 isoforms respectively.

OmNav1.4a is the predominant isoform of the trout heart accounting for over 80% of

the Nav transcripts, while omNav1.5a forms about 18% and omNav1.6a only 0.1% of

the transcripts. Thus, the ortholog of the mammalian skeletal muscle isoform,

omNav1.4a, is the main cardiac isoform of fish hearts. Unlike the mammalian cardiac

Nav1.5a, all trout cardiac Na+ channels, including the omNav1.5a have aromatic

amino acids (phenylalanine or tyrosine) instead of non-aromatic cysteine or serine in

the critical TTX binding position of the domain I and this confers their high TTX

sensitivity. It is likely that the residues involved in TTX binding close to the

selectivity filter of the channel are functionally important, and thus phylogenetic

sequence differences in TTX binding site reflect the adaptation to variable internal

body conditions and functional requirements of cardiac INa in different vertebrate

groups.

Table 1. Effects of cold-acclimation (+4°C) on the properties of ventricular INa of the fish heart at 11°C.

INa

(pA/pF) SSAct. (mV)

SSInact. (mV)

Recovery from

inactivation

Rested-state inactivation

Kinetics of inactivation

Burbot ↑ - - ↓ - ↓ Rainbow trout ↑ - - - - ↑ Crucian carp ↓ - - ↓ - -

SSAct. voltage-dependence (V0.5) of steady-state activation; SSInact. voltage-dependence (V0.5) of steady-state inactivation. Upward (↑) and downward (↓) arrows indicate increase and decreases in the value, respectively. A hyphen (-) signifies the absence of thermal effects.

3.3 Potassium currents and thermal action potential duration (Chapter 8)

The responses of AP and K+ currents to temperature acclimation were examined in six

different teleost fish species. Cold-acclimation shortened action potential duration

(APD) in all species regardless of the thermal tolerances, lifestyles or phylogenies of

the fish, suggesting that this response is a common characteristic of the teleost heart

(Table 2). However, the strength of the response differed among the phylogenetic

groups Salmoniformes fish showing the greatest and the perch (Perciformes) the

weakest acclimation capacity of APD. The underlying ionic mechanisms were also

partly phylogeny-dependent. Modification of the delayed rectifier potassium current

(IKr) current was almost ubiquitously involved in the acclimation response of fish

cardiac myocytes to temperature, while the ability to change the inward rectifier

potassium current (IK1) under chronic thermal stress was absent or showed inverse

compensation in Salmoniformes species. Thus, in Salmoniformes fish the thermal

plasticity of APD is strongly based towards IKr, while other fish groups rely on both

IKr and IK1.

Table 2. Effect of cold acclimation on the delayed rectifier potassium current (IKr), inward rectifier potassium current (IK1) and duration of action potential at 90% of repolarization (APD90) in six different teleost fish atrial and ventricular myocytes. IKr (pA/pF) IK1 (pA/pF) APD90 (ms)

A V A V A V Burbot ↑ ↑ - ↑ ↓ - Rainbow trout ↑ ↑ - - ↓ ↓ Crucian carp ↑ ↑ ↑ ↑ ↓ - Roach ↑ ↑ ↑ ↑ - - Perch ↑ - - ↑ - - Pike - - - - ↓ ↓

Upward (↑) and downward (↓) arrows indicate increases and decreases in value, respectively. The hyphen (-) signifies the absence of thermal effects. The letter A refers to the atrial and V to the ventricular cell.

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CHAPTER 4

TEMPERATURE ACCLIMATION MODIFIES SINOATRIAL PACEMAKER

MECHANISM OF THE RAINBOW TROUT HEART

Haverinen J. and Vornanen, M (2007)

Am. J. Physiol. 292: R1023-R1032

CHAPTER 5

TEMPERATURE ACCLIMATION MODIFIES NA+ CURRENT IN FISH

CARDIAC MYOCYTES

Haverinen J. and Vornanen, M (2004)

J. Exp. Biol. 207: 2823-2833

CHAPTER 6

SIGNIFICANCE OF NA+ CURRENT IN THE EXCITABILITY OF ATRIAL AND

VENTRICULAR MYOCARDIUM OF THE FISH HEART

Haverinen J. and Vornanen, M (2006)

J. Exp. Biol. 209: 549-55

CHAPTER 7

FISH CARDIAC SODIUM CHANNELS ARE TETRODOTOXIN SENSITIVE

Haverinen J., Hassinen, M. and Vornanen, M (2007)

Acta Physiol. 191: 197-204

CHAPTER 8

RESPONSES OF ACTION POTENTIAL AND K+ CURRENTS TO

TEMPERATURE ACCLIMATION IN FISH HEARTS.

PHYLOGENY OR THERMAL PREFERENCES?

Haverinen J. and Vornanen, M (2008)

Physiol. Biochem. Zool. 000: 000-000 (under revision)

CHAPTER 9

GENERAL DISCUSSION

GENERAL DISCUSSION

The vertebrate heart is a muscle pump which propels blood through the closed

circulatory system. The volume of blood circulation is determined by cardiac output

which is the product of heart rate and stroke volume. Temperature has a strong effect

on the rate of all biological processes including the rate functions of the heart. The

heart rate, rate of impulse conduction through the heart and shortening velocity of the

cardiac muscle are strongly retarded by low temperatures and accelerated by high

temperatures (Farrell & Jones 1992, Opie 1998). As a consequence, the cardiac

function of ectothermic fish is strongly modulated by chronic temperature changes in

different seasons and by acute temperature changes e.g. when fish make transitions

through the thermocline. Since the whole body of the fish (with exception of some

partially endothermic species) is ectothermic, ambient temperature will similarly

affect the metabolic rate of the fish and function of the heart, and therefore a relative

close match will prevail between the metabolic need for oxygen and the circulatory

supply of oxygen (Johnston & Temple 2002, Pörtner et al. 2004, Pörtner et al. 2006).

Correspondingly, when physiology of the animal responds to chronic temperature

changes by acclimation/acclimatization, similar changes are expected to happen in the

metabolism and pump function of the heart in order to satisfy the metabolic needs of

different tissues, i.e. the cardiac function should reflect temperature-dependent

changes in the metabolism of the fish.

9.1 Rate and rhythm of the heart

The excised heart of vertebrate animals beats in physiological saline solution with a

rate determined by its own pacemaker center (intrinsic rate). In the intact animal body,

heart rate is under the constant control of the autonomic nervous system through

acceleratory adrenergic nerves and inhibitory cholinergic nerves, using adrenaline and

acetylcholine, respectively, as neurotransmitters. Furthermore, circulating

catecholamines may also affect the heart rate in fish (Laurent et al. 1983, Farrell and

Jones 1992). In particular, the inhibitory cholinergic tone has a significant modulating

effect on the heart rate in fish, while the acceleratory influences of the adrenergic

nerves are often much weaker (von Skramlik 1932, Cameron 1979, Lillywhite et al.

1999). Several studies have shown that acclimation-induced changes in the heart rate

of fish are due to changes in the cholinergic control of the heart; in cold conditions,

inhibitory cholinergic tone is decreased, resulting in a compensatory increase in heart

rate (Seibert 1979, Sureau et al. 1989). Thus, the prevailing paradigm on temperature-

induced changes in the heart rate of fish maintains that they are solely produced by the

autonomic control of the heart. Contrary to this hypothesis, the effect of temperature

acclimation on heart rate in rainbow trout is still evident in excised hearts, indicating

that autonomic nerves cannot be totally responsible for the temperature-induced

changes in heart rate (Aho & Vornanen 2001). Consistent with the latter finding, it

was shown that the effect of temperature acclimation is present in individual

pacemaker cells of the trout heart (Chapter 4). Thus the hypothesis of temperature-

induced changes in heart rate must be reformulated to include temperature-dependent

modulation of the pacemaker mechanism, i.e. ion regulation of the diastolic

depolarization of the primary pacemaker cells. Since temperature-induced changes in

heart rate are common among teleost fish (Chapter 8), it remains to be shown what the

relative significance of autonomic nervous control and cellular pacemaker mechanism

is in thermal acclimation of the heart rate.

Several ion currents contribute to the diastolic depolarization of the cardiac

pacemaker cells (Irisawa 1978, Hagiwara et al. 1988, Campbell et al. 1992). There are

several ionic models which try to explain the initiation of pacemaker depolarization

and the steady rhythm of the heart beat. Three major hypotheses on the pacemaker

mechanism of the vertebrate heart have been worked out using mammalian and frog

pacemaker tissues and cells, which emphasize different ionic mechanisms as the

primary cause of diastolic depolarization. In the earliest model of the cardiac

pacemaker mechanism, deactivation of the delayed rectifier K+ current (the fast and

slow components of the delayed rectifier were not yet known) was considered to be

crucial for the initiation of the pacemaker action potential (AP) (Irisawa et al. 1993).

The pacemaker current (If or Ih) of the mammalian heart was described for the first

time in 1980 and a hypothesis proposing a master role for to this current has

subsequently elaborated, mainly by Difransesco and his colleagues (Brown &

Difrancesco 1980, Difrancesco 1981, Difransesco et al. 1986). In this hypothesis Na+

and K+ influx through the pacemaker channels initiates diastolic depolarization and

determines the heart rate. The activity of the pacemaker channel is regulated by

adrenergic and cholinergic agonists, thereby providing an explanation for autonomic

control of the heart rate. An alternative hypothesis to that offered by the pacemaker

current is based on the interaction of sarcoplasmic reticulum (SR) Ca2+ release and

sodium-calcium exchanger (NCX) in the sarcolemma (Li et al. 1997, Ju & Allen

1999, Bogdanov et al. 2001, Mangoni et al. 2003, Lakatta et al. 2006). In this model,

a small spontaneous release of Ca2+ from the subsarcolemmal SR activates an

extrusion of Ca2+ out of the cell through the NCX and as an electrogenic system the

NCX generates a small inward current that primes the rate of the pacemaking.

Until the present, the pacemaker mechanism has not been examined in fish

hearts. Two of the prevailing hypotheses on pacemaker mechanisms of the heart were

tested with regard to temperature induced changes in the heart rate of the rainbow

trout. Although the Ih current has been demonstrated in embryonic cardiac cells of

zebrafish (Baker et al. 1997, Warren et al. 2001), it could not be found in trout atrial

or pacemaker cells (unpublished results). The first hypothesis was therefore

abandoned as a relevant pacemaker mechanism of the trout heart. The two other

hypotheses were tested by using ryanodine (Ry) and E-4031, specific blockers of the

SR Ca2+ release channel and the delayed rectifier potassium current (IKr) to inhibit the

prime molecules of the two hypotheses. It could be demonstrated that the effect of E-

4031 differed between warm-acclimated and cold-acclimated trout sinoatrial

preparations from warm-acclimated and cold-acclimated trout, in that the beating rate

of warm-acclimated hearts is more strongly depressed by E-401 in comparison to that

of cold-acclimated hearts (Chapter 4). This result together with the finding that IKr

current is greater in cold-acclimated than in warm-acclimated myocytes (Vornanen et

al. 2002 a), suggests that the IKr current contributes at least partly to the higher heart

rate of the cold-acclimated fish. In situ hybridization indicates the presence of the

delayed rectifier potassium channels (ERG) in the pacemaker tissue of the rainbow

trout (Hassinen et al. 2008 b). On the other hand, Ry had only a minor effect on the

beating rate of the trout heart, suggesting that SR Ca2+ release and NCX are not

central operators in trout pacemaker tissue (Chapter 4). It should be noted, however,

that the heart rate of warm-acclimated trout was slightly inhibited by Ry, suggesting

that the third hypothesis might have some bearing at high temperatures.

The ion currents of the fish cardiac pacemaker have not yet been examined, and

this is an interesting open area of research. Careful examination of ion currents, ion

exchangers and the function of the SR are needed in order to clarify the pacemaker

mechanism of the fish heart and its modification by temperature and other

environmental factors.

9.2 Significance of sodium current in excitation of fish heart

In every heart beat, voltage-sensitive Na+ channels initiate atrial and ventricular

excitation by generating the rapid upstroke (phase 0) of the cardiac AP, and therefore

are responsible for fast impulse conduction through the heart (Grant 1991, Catterall

1992). The generation of APs and the coordinated spread of excitation are essential

for the orderly pumping action of the heart and depend largely on the density and

kinetics of the sodium current (INa) (Furue et al. 1998). Without any temperature-

induced compensation in the Na+ channel function, excitation and impulse conduction

of the atrial and ventricular muscle would vary according to ambient temperature

changes which might compromise heart function or make the heart vulnerable to

arrhythmias. In many fish species the frequency of cardiac beating increases when the

animals are exposed to chronic cold (Seibert 1979, Sureau et al. 1989, Bowler & Tirri

1990, Aho & Vornanen 2001), which is expected to require compensatory changes in

the density of INa and/or its kinetics. Consistent with this, increases in the density of

cardiac INa were found in cold-acclimated individuals of rainbow trout and burbot, i.e.

species which show positive thermal compensation in the heart rate. Cold acclimation

depressed INa density in the heart of crucian carp, a species which, it is suggested to

become inactive in the cold and anoxic winter waters (Table 1, Chapters 5).

There were prominent species-specific differences in the density of INa, burbot

having clearly the highest current and crucian carp the lowest current, while the INa of

the rainbow trout heart was intermediate between the two (Table 1, Chapter 5). This

suggests that excitability and rate of impulse conduction varies in the order

burbot>rainbow trout>crucian carp. In particular, high INa density may be needed in

burbot and rainbow trout at low temperatures to support cardiac function at relatively

high heart rates (at 4°C 19 and 30 beats/min for burbot and rainbow trout,

respectively). Such demands are much smaller in crucian carp, in which the heart

beats only about 10 times per min at 4°C (Chapter 8).

In both mammals and fish, the contractile properties and AP characteristics of

the atrial and ventricular muscle differ in that the shortening velocity of contraction is

higher and the duration of AP less in the atrium than in the ventricle (Hume & Uehara

1985). It might therefore be hypothesized that the properties of the INa would also

differ between atrial and ventricular myocytes. This hypothesis was tested in the

rainbow trout heart. The maximum rate of AP upstroke (Vmax) was indeed

significantly higher in the atrial than in the ventricular muscle, suggesting that INa

density might be greater in the atrial than in the ventricular myocytes. Surprisingly, no

such difference was found in the density of INa. However, slight differences were

evident in the voltage-dependence of steady-state activation, steady-state inactivation

and inactivation kinetics between the atrial and the ventricle in that the values of atrial

myocytes shifted towards more negative voltages in comparison to those of

ventricular myocytes (Chapter 6). These findings agree well with similar experiments

in the zebrafish heart (Warren et al. 2001) and values obtained from the rabbit heart

(Li et al. 2002).

Tetrodotoxin (TTX) is a specific blocker of the voltage-gated Na+ channels and

on the basis of TTX sensitivity Na+ channels can be categorized as either TTX-

sensitive or TTX-resistant. The TTX binding affinity is mainly determined by the

amino acid in position 401 of domain I, where an aromatic residue (fenylalanine or

tyrosine) confers high TTX binding sensitivity and a nonaromatic serine or cysteine a

gives a low TTX binding sensitivity (Satin et al. 1992, Plummer & Meisler 1999).

The mammalian cardiac INa is well-known for its low sensitivity to TTX, which is due

to the major cardiac Na+ channel isoform, Nav1.5, having a non-aromatic cysteine in

the critical position 401 (Gellens et al. 1992, Heinemann et al. 1992). On the basis of

the generally accepted paradigm about the TTX resistance of the cardiac INa in

mammals, this concept seems to be expected also to apply the fish heart, but without

adequate evidence (Venkatesh et al. 2005, Novak et al. 2006 a, b). In the some

previous studies transcripts of the Nav1.4 and Nav1.5 isoforms have been found in the

cardiac muscle of Takifugu rupripes (fugu) and zebrafish, but little attention has been

paid to on their relative amounts and the TTX sensitivity of the INa (Venkatesh et al.

2005, Novak et al. 2006 a, Soong & Venkatesh 2006). The TTX sensitivity of the

fish cardiac INa and Na+ channel composition of the fish heart are thus still unresolved.

Interestingly, the present findings indicate that the INa of the rainbow trout heart

is about 1000 times as sensitive (Kd = 1.8-2 nM) to TTX as the mammalian INa (Kd =

1-10 µM) (Chapter 7), which raised a question of its molecular basis (the main

isoform of the mammalian heart, Nav1.5, is TTX-resistant). Three different isoforms

of the Na+ channels (omNav1.5, omNav1.4 and omNav1.6) were found in the trout

heart and surprisingly, the ortholog of the mammalian skeletal muscle isoform,

omNav1.4, was clearly the main isoform (over 84 % of all Na+ channel transcripts) in

the trout heart. This isoform is TTX-sensitive in mammals, and therefore in consistent

with high TTX sensitivity of the trout cardiac INa. Quite surprisingly, it was also found

that the omNav1.5 (forming 16% of Na+ channel transcripts), which is orthologous to

the mammalian cardiac isoform, has the sequence structure of a TTX-sensitive Na+

channel: an aromatic phenylalanine instead of a non-aromatic cysteine or serine in the

TTX binding site of Na+ channel (Chapter 7). Thus, in the rainbow trout heart both

isoforms, omNav1.5, omNav1.4, are TTX-sensitive. This seems to apply to other

teleost fish species as well (unpublished results).

The present findings show that the composition of the cardiac Na+ channels has

radically diverged in evolution so that Nav1.4 has taken on a dominating role in fish

hearts in contrast to the Nav1.5 of mammalian hearts (Chapter 7). What physiological

advantages could the dominance of Nav1.4 provide for the trout heart? Previous

studies have shown that Nav1.4 has faster activation and inactivation kinetics than

Nav1.5 (Zimmer et al. 2002). The fast kinetics of the Nav1.4 might therefore have

provided a selective advantage in ectothermic animals, which have relatively low

body temperature, i.e. the Nav1.4 could better support the cardiac function in the cold

than the kinetically slower Nav1.5. In addition, it is known that the critical amino acid

residue for TTX binding (position 401) also contributes to the pH dependence of

Nav1.4 and Nav1.5 generated currents (Khan et al. 2006). Specifically, cysteine in

position 401 of mammalian Nav1.5 makes this isoform more sensitive to protons than

channels having tyrosine, phenylalanine or serine in this position. Because fish hearts

are mainly supplied by venous blood, which can have a relatively low pH due to the

lactic acid released by the muscle tissue, the less pH-sensitive Nav1.4 might provide

more stable Na+ currents than the pH-sensitive Nav1.5 isoform.

9.3 Potassium currents and action potential duration

Under physiological conditions potassium channels mediate K+ efflux from the

cardiac myocyte and thereby tend to bring the membrane potential to the K+

equilibrium potential of -80 -90 mV. In other words, K+ currents are repolarizing and

regulate the duration of cardiac AP which is important for cardiac excitability and

contractility (Giles & Imaizumi 1988, Sanguinetti & Jurkiewicz 1990, Barry &

Nerbonne 1996). Action potential duration (APD) modulates the electrical and

mechanical refractory periods of the atrial and ventricular muscle, i.e. the ability of

the heart to generate APs and to produce force at short diastolic intervals. APD also

affects the duration of cardiac contraction (Opie 1998). Similar to mammalian hearts,

in each species of fish examined, atrial AP was much shorter than ventricular AP,

which probably contributes to the shorter duration of atrial contraction in comparison

to ventricular twitch: AP characteristics are tuned to the contractile properties in each

cardiac compartment (Chapter 8).

As heart rate increases, the total time available for each cardiac cycle decreases,

i.e. the diastolic and systolic phases of the cycle must take place faster. However,

proper cardiac function requires a definite time for diastolic filling which should be

approximately 2/3 of the cardiac cycle (Opie 1998). Therefore, at high heart rates, the

duration of AP must shorten in order to prevent excessive shortening of the diastolic

period. In many teleost species there is a compensatory increase in the heart rate

following cold-acclimation, and the inevitable shortening of the cardiac cycle is

expected to require compensatory shortening of APD (Aho & Vornanen 2001). In

agreement with this hypothesis, shortening of APD in both cardiac chambers was

noticed in all six species of fish (Table 2, Chapter 8). Taken together, the present

findings show that, regardless of the different thermal tolerances, lifestyles and

phylogenies of the fish, the electrical excitability of the fish heart is increased

following cold-acclimation, taking the form of shortened durations of atrial and

ventricular APs (Chapter 8).

The AP plateau results from zero net current flow through the sarcolemma when

inward (mainly Na+ and Ca2+) and outward (mainly K+) currents balance each other

(Hille 2001). As shown above, in cold-acclimation cardiac INa was increased in

rainbow trout and burbot and depressed in crucian carp (Table 1, Chapter 5). Since

Na+ channels inactivate very rapidly, the contribution of INa to APD is probably

minor. On the other hand, temperature acclimation has little effect on ICa,L of rainbow

trout and crucian carp ventricular myocytes (Vornanen 1998). It should be noted,

however, that acclimatization to winter has been shown to depress the ICa,L in the

heart of crucian carp. This study does not take into account the effects of thermal

acclimation on inward currents which could also contribute to APD in addition to the

K+ currents that were objects of this study. In particular, this applies to the pike heart,

where no changes in K+ currents were noticed (Chapter 8).

Studies on mammalian cardiac myocytes indicate that K+ currents are important

regulators of APD and flexible entities in many pathophysiological situations

(Nerbonne 2000). Several K+ channels are known to exist in the heart (see chapter 1)

including the delayed rectifier K+ currents, IKs and IKr, and the inward rectifier K+

current, IK1. Previous studies suggest that IKr and IK1 are the major K+ currents of fish

cardiac myocytes (Vornanen et al. 2002 a), and therefore they were also chosen as the

targets of this study. IKr activates earlier in the plateau phase than IK1 and is a stronger

repolarizing current than IK1, which is mainly responsible for the rapid repolarization

of AP phase 3 (Zeng et al. 1995; Sakmann & Trube 1984).

The IKr of fish cardiac myocytes seems to be mainly responsible for shortening

of APD in the cold conditions, since this current increased strongly in all fish species

except the pike (Table 2, Chapter 8). The outward charge transfer by IK1 was not

changed by temperature acclimation in the pike heart and the current was depressed

by chronic cold in the trout heart. This suggests that unlike the other teleost species

the Salmoniformes fish are unable to upregulate the cardiac IK1 in the cold. An

explanation for the absence of positive thermal compensation in the density of IK1 in

Salmoniformes fish may lie in the molecular composition of the Kir2 channels. Three

different isoforms of the Kir2 channels have been found in the fish heart, Kir2.1,

Kir2.2 and Kir2.5, which are variably present in different fish species and differently

affected by temperature acclimation. It is evident that Kir2.2 channels are upregulated

at high temperatures, while expression of the Kir2.5 is enhanced in cold conditions.

Kir2.1 seems to be only weakly temperature-responsive. Crucian carp express Kir2.2

and Kir2.5, while rainbow trout express Kir2.1 and Kir2.2, but not Kir2.5 in the heart.

Thus the absence of positive thermal compensation of the IK1 in the rainbow trout

heart may due to the absence of the cold isoform, Kir2.5, in this species (Hassinen et

al. 2007, Hassinen et al. 2008 a). Examination of Kir2 gene expression in other teleost

species would provide useful information on the molecular basis of temperature

responses of cardiac IK1, its constraints and phylogenetic origin.

9.4 Conclusions

Thermal acclimation modifies the cardiac pacemaker mechanism of the fish heart by

increasing the intrinsic AP discharge rate of the primary pacemaker cells in cold

conditions, which indicates that part of the temperature-induced response in heart rate

is independent of autonomic nervous control and intrinsic to the pacemaker process

proper. This is associated with shorter duration of the pacemaker AP and is likely to

involve increased density of IKr, which allows a higher rate of diastolic depolarization.

Thermal acclimation induced compensatory and non-compensatory changes in

the properties of fish cardiac INa. In cold-active species, rainbow trout and burbot, the

density of INa increased and in cold-dormant crucian carp INa decreased in the cold.

These responses suggest that the rate of impulse conduction is changed differently,

depending on lifestyle and environment conditions in the habitat of the fish.

Furthermore, it seems that the properties of INa support higher excitability of the atrial

muscle in comparison to the ventricular myocardium. Na+ channel composition and

TTX sensitivity of the fish heart are notably different from those of the mammalian

heart, which may be associated with the different functional demands of INa in

ectotherms and endotherms, e.g. as regards temperature and pH.

Positive thermal compensation is evident in electrical activity (APD) of the heart

in all fish species with the exception of the perch atrium. The shortening of APD is

due to cold-induced increase in the density of IKr and/or IK1 with the exception of the

pike heart, where AP shortening may be mediated by inward currents. In contrast to

other fish species, temperature compensation of IK1 was absent or inverse in

Salmoniformes fish, suggesting phylogenetic differences in the underlying molecular

mechanisms. IKr seems to be temperature-responsive in most fish species, the pike

being an exception among the studied species.

The results of the present study indicate that the ion channels of the fish heart

are plastic entities which respond to chronic temperature changes and enable different

cardiac electrical phenotypes depending on the environmental conditions and

lifestyles of the fish.

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