Na regulation of the cardiac excitation‐contraction‐relaxation coupling +

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Na ⁺ regulation of the cardiac excitation‐

contraction‐relaxation coupling

Nils Tovsrud 2016

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© Nils Tovsrud, 2016

Series of dissertations submitted to the Faculty of Medicine, University of Oslo

ISBN 978-82-8333-257-5

Cover: Hanne Baadsgaard Utigard

Printed in Norway: 07 Media AS – www.07.no

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Acknowledgements

The work leading to this thesis has been performed at the Institute for Experimental Medical Research, Ullevål University Hospital.

I started in 2004 as a Medical Research Curriculum student (Forskerlinjen) with research work one year full‐time and two years part‐time. I am thankful to The Research Council of Norway for granting me in that period. After medical school, I was awarded a stipendium from the Norwegian Health Association and the South‐Eastern Regional Health Authority, allowing me to work full time with research from 2008‐11. Since 2011, I have been working part time with research.

During these 12 years, a lot of people have contributed to the work leading to this thesis in many ways. First of all, a special thank to Fredrik Swift and Jon Arne Kro Birkeland, who guided me into the Medical Research Curriculum in the first place and introduced me to the methods. Fredrik became my main supervisor. I thank him and the cosupervisors Ivar Sjaastad and Ole Mathias Sejersted, also head of the institute, for guidance through all these years and for giving me the opportunity to get into the interesting world of basic heart science.

Thanks to the coauthors in the papers forming the basis of this thesis, which in addition to the supervisors are: Ulla Helene Enger, Jonas Skogestad, Jan Magnus Aronsen, Pimthanya

Wanichawan, Karina Hougen, Mathis Korseberg Stokke, Cathrine Rein Carlson, William Edward Louch and Leif Øyehaug. Excellent technical assistance has been offered by Roy Trondsen, Per Andreas Norseng, Vidar Magne Skulberg, Marita Martinsen, Heidi Kvaløy, Bjørg Austbø and Hilde Dishington.

All the nice people at the Institute for Experimental Medical Research deserve an extra thank for providing an inspiring work environment. Jan Magnus Aronsen deserves to be mentioned in particular. Without his enthusiasm, encouragement and help during the last years with research, it is likely that this thesis would not have been completed. I am very grateful for his contribution.

12 years is a long time. I remember my dear friend and mentor Karl Henrik Midtskogen, who encouraged me to do research, but died in 2004, short after I started the work finally leading to this thesis. During these 12 years, the institute has developed and expanded much. My life has changed a lot too ‐ from medical student to MD and family father. The final thanks should be passed to my beloved family, my sons Jonathan and Thomas, my parents and my dearest Ingrid.

Oslo, August 2016 Nils Tovsrud

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Supported by

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List of abbreviations

AP action potential AKAP A‐kinase anchor protein AnkB ankyrin B

AnkB^+/‐ heterozygous for a null mutation in ankyrin B AnkB^‐/‐ homozygous for a null mutation in ankyrin B AV‐node atrioventricular node

Ca²⁺c cytosolic Ca²⁺concentration CaMKII CaM kinase II

CCB Ca²⁺ channel blockers CD2 cytoplasmic domain 2 CD3 cytoplasmic domain 3 CICR Ca²⁺ induced Ca²⁺ release

CPVT catecholaminergic polymorphic ventricular tachycardia CSQ calsequestrin

DAD delayed afterdepolarization EAD early afterdepolarization Em membrane potential ECa equilibrium potential for Ca²⁺

ENa equilibrium potential for Na⁺

ENa/Ca equilibrium potential for Na⁺/Ca²⁺ exchange ECG electrocardiogram

ECR‐cycle excitation‐contraction‐relaxation cycle ICaL Ca²⁺current through L‐type Ca²⁺ channels IKr delayed rectifier K⁺current

IK1 inward rectifier K⁺ current INCX NCX current

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INKA NKA current

INa Na⁺ current

IP3R inositol trisphosphate receptor Iti transient inward current Ito transient outward K⁺current LTCC L‐type Ca²⁺ channel LQTS4 long QT‐syndrome type 4 MAB minimal ankyrin binding

Na⁺c cytosolic Na⁺concentration NCX Na⁺/Ca²⁺exchanger NKA Na⁺/K⁺ATPase PKA protein kinase A PKC protein kinase C PLB phospholamban

PMCA plasmalemmal Ca²⁺ ATPase RyR ryanodine receptor SA‐node sinoatrial node

SERCA2 sarco‐/endoplasmic reticulum Ca²⁺ ATPase 2 SR sarcoplasmic reticulum

t‐tubules transverse tubules

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Papers included in this thesis

1) The Na⁺/K⁺‐ATPase alpha2‐isoform regulates cardiac contractility in rat cardiomyocytes Swift F, Tovsrud N, Enger UH, Sjaastad I, Sejersted OM.

Cardiovasc Res. 2007 Jul 1;75(1):109‐17.

2) Coupling of the Na⁺/K⁺‐ATPase to ankyrin B controls Na⁺/Ca²⁺ exchange activity in cardiomyocytes

Tovsrud N, Skogestad J, Aronsen JM, Wanichawan P, Hougen K, Stokke MK, Carlson CR, Sjaastad I, Sejersted OM, Swift F

Manuscript

3) ICaL inhibition prevents arrhythmogenic Ca²⁺ waves caused by abnormal Ca²⁺sensitivity of RyR or SR Ca²⁺accumulation

Stokke MK*, Tovsrud N*, Louch WE, Øyehaug L, Hougen K, Sejersted OM, Swift F, Sjaastad I Cardiovasc Res. 2013 May 1;98(2):315‐25. ^*Equal contribution to the manuscript.

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1. Introduction

1.1 The heart

In 1628, William Harvey published “De motu cordis” [1]. This book made him the first to give a detailed description of the heart and the circulation. One of his findings was that the heart muscle pumps blood in a pulsatile manner, with a cycling between contraction (systole) and relaxation (diastole). Some duration of diastole is necessary to secure a sufficient filling of blood into the chamber to be expelled at the next systole. The duration of diastole is also important for efficient perfusion of the coronary arteries. The coordinated contraction of the heart muscle relies on spread of electrical activity, first described by Galvani [2]. The frequency of action potentials (APs) in the sinoatrial node (SA‐node), localized in the right atrium, determines the heart rate. From the SA‐node, the AP spreads to the right and left atrium via gap junctions between the atrial cardiomyocytes and to the atrioventricular node (the AV‐node), which slows conduction to allow filling of the ventricles.

Subsequently, the ventricles are activated via AP propagation through the bundle of His and Purkinje fibers, and the left ventricle is normally activated from the endocardium towards the epicardium and from apex to base to allow expulsion of blood through the aorta and the pulmonary artery [3, 4].

Repolarization occurs in the opposite direction, from base to apex. This is a fine‐tuned process, and altered depolarization‐repolarization sequence can lead to arrhythmias, and in some cases, cardiac arrest. Arrhythmias may disturb and impair the cardiac pump function, and in cardiac arrest, the pump function ceases due to electrical chaos in the heart.

To understand cardiac pump function, it is necessary to understand the mechanisms regulating contraction in single cardiomyocytes. Ca²⁺is necessary for heart contraction, as described by Ringer in 1883 [5]. Although clinical use of digitalis to treat heart failure patients was described already 100 years before that [6], it took many years to understand that cardiac glycosides inhibit the Na⁺/ K⁺ ATPase (NKA), and that the Na⁺/ Ca²⁺ exchanger (NCX) is a link between cytosolic Na⁺‐ and Ca²⁺

homeostasis, as reviewed in [7]. However, despite extensive research over decades, many controversies still exist in the role of Na⁺ dependent regulation of cardiac function. Improved understanding of Na⁺ dependent control of cardiac function is necessary for development of better therapies, especially for arrhythmias where disturbed or altered Na⁺ fluxes directly or indirectly contribute to arrhythmogenesis.

The aim of this thesis is to investigate the mechanisms by which cytosolic Na⁺and NKA control cardiomyocyte function through modulation of the NCX and the excitation‐contraction‐relaxation cycle (ECR‐cycle), and to explore possible antiarrhythmic approaches in selected clinical arrhythmias.

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1.2 The excitation‐contraction‐relaxation cycle in cardiomyocytes The ECR‐cycle is a fine‐tuned process that in the normal situation is the physiological basis for cardiomyocyte contraction. The ECR‐cycle at the single cell level can be described in a sequence of processes where the electrical activation (the AP) leads to Ca²⁺influx through the sarcolemma, which then triggers a greater Ca²⁺ release from the sarcoplasmic reticulum (SR). The resulting transient rise in cytosolic Ca²⁺concentration (Ca²⁺c), the Ca²⁺transient, triggers contraction of the cardiomyocyte, as Ca²⁺ binds to troponin C in the myofilaments and causes a conformational change inducing myofilament movement. Relaxation occurs when Ca²⁺ dissociates from the myofilaments and is removed from the cytosol. The shape and the amplitude of the Ca²⁺ transients determine the contraction force and kinetics of the regular heartbeat, and the Ca²⁺ transient is tightly regulated to avoid Ca²⁺ overload and induction of arrhythmias, as later discussed. The ECR‐cycle will in the following be discussed with special focus on selected factors of key importance for this thesis.

1.2.1 The excitation – the action potential

To ensure a synchronized and efficient contraction, the shape of the AP is different throughout the various regions of the heart. Here, only the APs of the left ventricular cardiomyocytes will be described, as this thesis is based on results from these cells. The AP in ventricular myocytes has five phases (phase 0‐4 as illustrated in figure 1).

 Phase 0: During the first phase, phase 0, the cell is depolarized by Na⁺ influx (INa) through voltage gated Na⁺ channels. This Na⁺ influx rapidly increases the membrane potential from about ‐70‐90 mV to +35‐50 mV (depending on species). The depolarization of the membrane potential activates voltage gated Ca²⁺‐ and K⁺ channels in the remaining phases of the AP.

 Phase 1: During phase 1, opening of the L‐type voltage gated Ca²⁺ channels (LTCCs) provides entry of Ca²⁺ into the cytosol, and by this initiates the Ca²⁺ transient as later discussed. In addition, a repolarizing transient outward K⁺current (Ito) counteracts the inward current through the LTCCs.

 Phase 2: The plateau during phase 2 evolves due to balance between Ca²⁺ influx mediated by LTCCs and NCXs, and K⁺efflux via delayed rectifier channels.

 Phase 3: The repolarization constitutes phase 3 and is due to outward K⁺ current, mainly in the inward rectifier and delayed rectifier K⁺ channels.

 Phase 4: During rest (phase 4), the membrane potential is kept at about ‐70‐90 mV due to high conductance for K⁺ in the IK1 channels and low permeability for other ions.

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Figure 1: The action potential in ventricular cardiomyocytes. For details, see text.

1.2.2. The contraction ‐ cytosolic Ca²⁺ release

In a resting cardiomyocyte, Ca²⁺c is about 0.1 µM, increasing to 0.6‐1 µM during contraction [8, 9].

The extracellular Ca²⁺ concentration is about 1.5 mM. The steep concentration gradient across the cell membrane and regulated influx and efflux of Ca²⁺ allow rapid changes of Ca²⁺c. Together with the transient and regular changes of Ca²⁺c between diastole and systole, this makes Ca²⁺ an efficient messenger [10]. The increase in Ca²⁺c comes from sarcolemmal Ca²⁺ influx during the AP and Ca²⁺

release from the SR.

1.2.2.1 Sarcolemmal Ca²⁺ influx

The Ca²⁺ transient is initiated by transsarcolemmal Ca²⁺ influx through the LTCCs (ICaL) [11]. ICaL is voltage dependent, and there is a bell‐shaped relationship between ICaL and membrane potential (Em) [12]. The LTCCs are open to allow Ca²⁺ influx at potentials between ‐ 40 and +40 mV, with a maximum current density at 0 mV, and the peak current is reached rapidly (within 2‐7 ms) after opening in phase 1 of the AP [13]. Inactivation of LTCCs is determined primarily by repolarization of the membrane potential and Ca²⁺ itself, and Ca²⁺ dependent inactivation is the key mechanism leading to closure of LTCCs at physiological conditions [12]. Ca²⁺ dependent inactivation is a negative feedback mechanism, where Ca²⁺ on the cytosolic site (from the rising Ca²⁺ transient) leads to closure of LTCCs [14‐16]. Regulation of ICaL is a main determinant of Ca²⁺ transients, and LTCC channel kinetics is both under physiological regulation by β‐adrenergic stimulation and serves as a pharmacological target for

Ca²⁺ channel blockers (CCBs). One main question in paper 3 of this thesis is whether Ca²⁺ channel

blockade represents a potential therapy for certain arrhythmias.

Ca²⁺ influx via NCX happens during the peak of the AP [17], when the cardiomyocyte is

depolarized, the cytosolic Na⁺ concentration (Na⁺c) is high and before ICaL causes local cytosolic Ca²⁺

elevation [18]. Whether Ca²⁺ influx via NCX can trigger SR Ca²⁺ release is controversial. The role of

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Ca²⁺ influx via NCX in ECR‐regulation might be more indirect, by priming the dyadic cleft with Ca²⁺

prior to LTCC openings in order to facilitate triggering of ryanodine receptors (RyRs) [19].

1.2.2.2 Ca²⁺ release from the sarcoplasmic reticulum

Sarcolemmal Ca²⁺ influx triggers Ca²⁺ release from the SR. The principal SR Ca²⁺ release channel is the RyR. The RyRs open upon binding of Ca²⁺ to the cytosolic site, releasing Ca²⁺from the SR. This process is often referred to as Ca²⁺ induced Ca²⁺ release (CICR) [20, 21].

A ventricular cardiomyocyte contains many junctions between the sarcolemma and the SR.

These junctions are localized mainly in the t‐tubules, invaginations of the sarcolemma. These junctions are called dyads, and the two membranes are separated by only 10‐15 nm [22]. This dyadic cleft provides a short distance for diffusion of Ca²⁺ entering the cell through LTCCs to the RyRs and allows rapid CICR within a small subcellular domain. The abundance of LTCCs is higher in the t‐

tubules than in the surface sarcolemma [23], consistent with a special role for the t‐tubules in the ECR‐cycle. The coupling of LTCCs and RyRs was first described in skeletal muscle [24], and is called a calcium release unit (CRU) or couplon [25]. A couplon in cardiomyocytes typically contains 10 LTCCs and 100 RyRs [13]. This organized structure allows independent events of SR Ca²⁺ release to be triggered by the Ca²⁺ flowing through a few LTCCs, and not by the bulk cytosolic Ca²⁺concentration.

The sarcolemmal Ca²⁺ influx through LTCCs triggers Ca²⁺ release (named a Ca²⁺ spark) from the corresponding RyRs in a couplon [8, 26]. A single Ca²⁺ spark leads to release of only a minor amount of Ca²⁺ from the SR, which is not sufficient to produce a detectable increase in the average Ca²⁺i. During a regular heartbeat, the LTCCs open within few milliseconds due to rapid AP‐propagation along the sarcolemma. This leads to generation of synchronized Ca²⁺ sparks throughout the cell, which together induce the rise in average Ca²⁺c and thus provide the Ca²⁺ necessary for myofilament movement. Spontaneous Ca²⁺ sparks, not elicited by CICR, may occur in settings with high SR Ca²⁺ content or increased Ca²⁺ conductance, and may trigger certain cardiac arrhythmias.

How SR Ca²⁺ overload might evolve due to disturbances in Na⁺ fluxes and potentially be treated with CCBs, will be discussed further in later sections.

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1.2.3 The relaxation – cytosolic Ca²⁺ removal

To achieve steady state Ca²⁺ transients and contractions, a Ca²⁺ amount equal to the Ca²⁺ released from the SR and the Ca²⁺ that entered over the sarcolemma, has to be removed from the cytosol. The sarco‐/endoplasmic Ca²⁺ATPase 2 (SERCA2) uses ATP to pump Ca²⁺ ions against a concentration gradient from the cytosol and into the SR. It is a key regulator of cardiac contractility since it determines the SR Ca²⁺ content and the rate of removal of cytosolic Ca²⁺. SERCA2‐activity is regulated by the short protein phospholamban (PLB), which in its phosphorylated form inhibits SERCA2‐activity.

PLB‐phosphorylation by PKA or CaMKII relieves the inhibitory effect of PLB on SERCA2, increasing the SERCA2‐activity [27].

Besides SERCA2, the other main transport mechanism for cytosolic Ca²⁺ is the NCX, which mediates Ca²⁺ efflux over the sarcolemma. The relative contribution of NCX and SERCA to cytosolic Ca²⁺ removal varies between species, as the ratio of Ca²⁺ transport via SERCA:NCX is close to 7:3 in humans and rabbits, and 9:1 in rodents [28]. The balance between SERCA2 and NCX mediated Ca²⁺

extrusion is a main regulator of cardiac contractility because Ca²⁺ extrusion by the NCX would tend to limit the SR Ca²⁺ concentration and Ca²⁺ availability for the subsequent CICR and vice versa.

Regulation of NCX activity is a central aspect in this thesis and will be discussed further in later sections.

In addition to SERCA2 and NCX mediated Ca²⁺ extrusion, slow Ca²⁺ transporters including the plasmalemmal Ca²⁺ ATPase (PMCA) [29] and the mitochondrial Ca²⁺ uniporter [30] contribute to the cytosolic Ca²⁺ removal. The contribution of these transporters appears to be minor on a beat‐to‐beat basis and will not be further discussed in this thesis.

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Figure 2: The proteins involved in Na⁺ and Ca²⁺ homeostasis of cardiomyocyte ECR‐coupling. See text for discussion.

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1.3. Na⁺ as determinant of Ca²⁺ transients in cardiomyocytes 1.3.1 Na⁺ balance in cardiomyocytes

Cardiac ECR‐coupling and Ca²⁺ transients are tightly regulated by Na⁺c, and even small alterations in

Na⁺c have a large impact on cardiac contractility [31, 32]. Cardiomyocytes have a large

electrochemical Na⁺ gradient, which controls the membrane transport of a variety of other molecules by secondary active transport, including Ca²⁺(NCX) and H⁺(Na⁺/H⁺exchanger) [33]. The Na⁺c is determined by the balance between Na⁺ influx and efflux, where voltage gated Na⁺ channels and the NCX are the two main Na⁺ influx pathways in beating cardiomyocytes. The NKA represents the main Na⁺ extrusion mechanism in cardiomyocytes, and Na⁺c is thus set by the balance between Na⁺ influx and NKA activity.

1.3.2 Voltage gated Na⁺ channels

INa induces the first phase in the AP and flows through voltage gated Na⁺ channels. The main Na⁺ channel is the Nav1.5, a cardiospecific channel [34], mediating 80‐90% of total INa during the AP [35, 36]. Brain type (NaV1.1‐1.3, 1.6) and skeletal muscle type (NaV1.4) Na⁺ channels are expressed in the heart and constitute the remaining INa, and these channels are enriched in the t‐tubules [35, 37, 38].

Whether the brain‐type and skeletal muscle type Na⁺ channels play a special role in the ECR‐cycle, is not clear.

1.3.3 The Na⁺/Ca²⁺ exchanger

NCX exists in three isoforms (NCX1‐3), but only NCX1 is expressed in the heart. NCX exchanges 1 Ca²⁺

with 3 Na⁺ ions, and thus transports net electrical charge in each translocation movement [39]. NCX can operate in two modes with different roles during the ECR‐ cycle in cardiomyocytes:

 Forward mode NCX activity/Ca²⁺ extrusion mode: Forward mode NCX activity extrudes 1 Ca²⁺ ion of the cytosol in exchange for 3 Na⁺ ions. Forward mode NCX activity thus leads to net influx of 1 positive electrical charge during each translocation movement. Forward mode NCX activity is the main sarcolemmal Ca²⁺ extrusion mechanism in cardiomyocytes, and the balance in activity between SERCA2 and NCX is a key determinant of SR Ca²⁺ load and cardiac contractility.

 Reverse mode NCX activity/Ca²⁺ influx mode: Reverse mode NCX activity extrudes 3 Na⁺ ions in exchange for influx of 1 Ca²⁺ ion, thus leading to net transport of 1 positive electrical charge out of the cell. Reverse mode NCX activity might contribute directly or indirectly to CICR as later discussed, but the exact role of reverse mode exchange in the ECR‐cycle has yet to be fully understood.

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The NCX operating mode is determined by Na⁺c , extracellular concentration of Na⁺ and Ca²⁺ and the membrane potential (Em), where the equilibrium potential for Na⁺/Ca²⁺ exchange, ENa/Ca = 3ENa – 2ECa (ENa and ECa are the equilibrium potentials for Na⁺ and Ca²⁺). During the regular ECR‐cycle, Em < ENa/Ca

and NCX operates in forward mode. During a short period in early depolarization of the AP, when

Em > ENa/Ca because of high Na⁺c due to opening of the voltage gated Na⁺ channels and low Ca²⁺c

before CICR starts, reverse mode NCX‐activity is favored [18, 40]. Two important factors in NCX‐

mediated control of ECR yet to be fully determined are:

 NCX localization: NCX are clustered in t‐tubules [41, 42], but the relative placement of NCX versus the dyadic cleft and the LTCC‐RyR couplon is not known in detail. Immunocytochemistry data has indicated that a fraction of NCX‐molecules in the t‐tubules are colocalized with RyRs [41, 43, 44], which might affect the ability of reverse mode NCX to induce CICR. Most NCX‐molecules, however, are likely to be localized outside of the dyad. Further, the molecular determinants of NCX localization are not known, but anchoring to the scaffolding molecule ankB might be an important factor, as later discussed

 Global or local regulation: Whether NCX‐function is under the control of localized pools of Na⁺ and Ca²⁺, or is controlled by the average cytosolic concentration of these ions, has remained a debated topic since the first report on the “fuzzy space” in 1990 [45], see section 1.4.2. Whether NCX resides close to the dyad and senses the high Ca²⁺ in the dyadic cleft during CICR and whether NCX is affected by the Na⁺ entering the cells during the AP, is not clear.

1.3.4 The Na⁺/K⁺ATPase

The NKA utilizes the energy of 1 ATP to pump 3 Na⁺ ions out of the cell and 2 K⁺ions into the cell against the concentration gradient for both ions [46]. NKA consists of two subunits: alpha () and beta (). The  subunit contains binding sites for Na⁺, K⁺, ATP and cardiac glycosides, and is expressed in three different isoforms in the heart (1‐3). All three isoforms are present in the human heart [47], while only 1 and 2 are expressed in adult rodent hearts. The  subunit is a regulatory subunit and exists in two isoforms, 1 and 2. The  subunit is important for correct insertion of the  subunit in the cell membrane [48].

NKA‐activity is primarily determined by the Na⁺c and the extracellular K⁺ concentration, in addition to ATP availability, the membrane potential and the regulatory protein phospholemman. K0.5 for cytosolic Na⁺ is between 10‐20 mM [46, 49, 50], close to the normal resting Na⁺c. Hence, the NKA‐activity is sensitive for small alterations in Na⁺c. The K0.5 for extracellular K⁺ is about 1.5 mM for

NKA1 and about 3 mM for NKA2 [51]. NKA‐activity is also voltage dependent and has its highest

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activity at depolarized potentials and decreases at negative membrane potentials [52, 53], allowing efficient extrusion of Na⁺ during the AP.

The different NKAα‐isoforms have different roles in controlling cardiac ECR‐coupling.

Heterozygous knockout mice lacking 1 are hypo‐contractile with reduced Ca²⁺ transients, whereas the heterozygous 2 knockouts are hyper‐contractile with increased Ca²⁺ transients [54], coupling

NKA2 to control of Ca²⁺fluxes and the ECR‐coupling. The underlying mechanism is not known, but

might involve interaction with NCX. This is further explored in paper 1 of this thesis.

Phospholemman, a short regulatory peptide coupled to NKA, reduces the Na⁺‐affinity and to a smaller extent K⁺‐affinity of the NKA [55, 56]. Phospholemman modulates NKA in a manner similar to the modulation of SERCA by PLB: the inhibition exerted by phospholemman on NKA is relieved by phosphorylation of PKC and PKA [55]. Phosphorylation of phospholemman increases NKA‐activity during β‐adrenergic activation. This is suggested to be a physiological adaptation during sympathetic activity leading to increased Na⁺‐extrusion, thus counteracting the concomitant increase in Ca²⁺c by promoting forward mode NCX‐activity [57].

NKA has been a central pharmacological target for treatment of cardiac disease, and various cardiac glycosides have been used to treat heart failure and arrhythmias for more than 200 years, first described by Withering in 1785 [6]. Cardiac glycosides bind reversibly to the extracellular side of the  subunit and inhibit NKA mediated ATP hydrolysis. The different sensitivity to the cardiac glycoside ouabain observed between rodent NKA1‐ and 2‐isoforms [58] provides an important experimental tool because this allows functional separation between the two isoforms using a low

dose of ouabain to inhibit the 2‐isoform, as applied in paper 1 in this thesis.

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1.4 Subcellular regulation of Na⁺ fluxes in cardiomyocytes

Sarcolemmal ion transporters exert specific physiological roles determined by their subcellular distribution. For example, most ion transporters involved in the ECR‐coupling are clustered in the t‐

tubules [42]. Specific anchoring molecules are important for determining this subcellular distribution, as they anchor the various ion transporters to specific subcellular domains by coupling to the cytoskeleton [59]. In addition, anchoring proteins serve to bring together two or more transporters or signaling proteins to create subcellular domains with localized signaling. An example is the A‐

kinase anchoring proteins (AKAPs), which regulate many Ca²⁺ transporters in clusters with various signaling proteins such as kinases and phosphatases [60].

1.4.1 The ankyrins

The ankyrins are a central group of anchoring molecules linking Na⁺‐transporters, including voltage gated Na⁺‐channels, NCX and NKA to the cytoskeleton [61]. Ankyrins consist of a membrane binding domain, spectrin binding domain, death domain and C‐terminal regulatory domain [62]. Three different genes (ANK1‐3) encode three main ankyrin polypeptides:

 Ankyrin R: Ankyrin R (ANK1 gene) was the first ankyrin to be described in the late 1970s as an important link between various anion exchangers and beta‐spectrin in erythrocytes [63‐65], and is also expressed in the heart [66].

 Ankyrin G: Ankyrin G (ANK3 gene) is ubiquitously expressed [67‐69], and anchors voltage gated

Na⁺ channels in the heart. A missense mutation in the ankyrin binding motif of the cardiac

isoform of voltage gated Na⁺ channels (Nav1.5) disrupts the interaction between ankyrin G and NaV1.5 [70]. This mutation has been linked to Brugada syndrome, a clinical arrhythmia syndrome with increased risk of sudden cardiac death due to ventricular fibrillation [71].

 Ankyrin B (ankB): AnkB (ANK2 gene) is ubiquitously expressed and present in the heart. AnkB is localized to both the M‐line and the Z‐line in adult ventricular cardiomyocytes [72] and scaffolds NCX [73, 74], NKA [75], IP3R [76] and a potassium channel, Kir6.2 [77]. Loss of function of‐ and dysfunctional ankB has been implicated in various arrhythmias in humans, see section 5.2, and the phenotype of ankB^+/‐ mice closely resembles the phenotype in patients with ank2 mutations [78].

Ankyrin binds to the cytoplasmic domain 2 and 3 (CD2 and CD3) of the ‐subunit of the NKA [75], where the CD2‐domain has the greatest affinity for ankyrin [79]. A 25 amino acid residue within this domain has been shown to constitute the minimal ankyrin‐binding (MAB) sequence (amino acid 144‐

166) of the NKAα isoform [79]. The MAB sequence represents a key experimental tool in paper 2 in this thesis, where we have synthesized this peptide and used it to disrupt the coupling of NKA to

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ankB and studied the functional role of the protein‐protein interaction between NKA and ankB in ventricular myocytes.

Figure 3: Proposed model for ankB‐dependent coupling of NCX and NKA in cardiomyocytes (left part) and mechanism for disruption of NKA from the proposed macromolecule by the MAB peptide as explained in the text (right part).

1.4.2 Localized subdomains for Na⁺ in cardiomyocytes

A key question in Na⁺ dependent regulation of ECR‐coupling in cardiomyocytes is if there are subcellular pockets close to either of the Na⁺ transporters where the ion concentration differs from the bulk Na⁺c. The concept of subcellular pockets is well established for Ca²⁺, where the Ca²⁺

concentration in the dyadic cleft is many times higher than the average cytosolic Ca²⁺ concentration [80], and by this regulates specific transport proteins. An example is the Ca²⁺ dependent termination of the Ca²⁺ entry through LTCCs, which is believed to be mediated by the Ca²⁺ concentration in the dyadic cleft and not the average Ca²⁺c [16].

The nature of potential subcellular microdomains for Na⁺, on the other hand, is still debated and remains to be directly demonstrated. Leblanc and Hume found that the Na⁺ influx through voltage gated Na⁺ channels caused a sufficient elevation in Na⁺ concentration at the cytosolic site of NCX to cause CICR in cells with inhibited ICaL [81]. The calculated Na⁺ influx in this setting was not sufficient to cause a detectable increase in average Na⁺c. In light of this, Lederer et al [45]

concluded that this finding would require a restricted subsarcolemmal space shared by voltage gated Na⁺ channels, NCX and RyRs, where Na⁺c increases sufficiently by Na⁺ influx to induce reverse mode NCX. This domain was due to its unknown nature coined the “fuzzy space”, and was postulated to constitute a larger intracellular volume than the dyadic cleft. Since these original findings, the possibility for Na⁺ microdomains in cardiomyocytes has been extensively examined by several groups. Main later findings in this field have been:

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Figure 4: Potential subcellular Na⁺and Ca²⁺ domains in cardiomyocytes. Figure from [7] with permission.

 Physiological role of the originally proposed “fuzzy space”: The idea of the original fuzzy space fits with the later demonstration that at least a subset of NCX transporters co‐localize with LTCCs and RyRs in cardiomyocytes [41, 43]. However, the original idea of a physiologically relevant role of the fuzzy space has been questioned, as the time to achieve sufficient Ca²⁺ influx via NCX to induce CICR in a setting with active LTCCs might be too short [17, 82‐84]. Data from NCX deficient cardiomyocytes support a role for INa in reverse mode NCX mediated control of CICR, but through a more indirect mechanism including priming of the dyadic cleft with Ca²⁺ [19].

 Sub‐sarcolemmal Na⁺ gradients: Functional and imaging based studies have found evidence for a potential gradient of Na⁺ between a little confined compartment just beneath the sarcolemma (the sub‐sarcolemmal space) and the rest of the cytosol [85‐87]. Studies comparing the measured Na⁺c with the theoretica Na⁺ sensed by various transporters, have concluded that the Na⁺ concentration sensed by the ion transporters are several times higher than the bulk

Na⁺c [40, 88]. Supportive of this idea, imaging based approaches suggest that Na⁺c is increased close to the sarcolemma, possibly both in systole and diastole [87]. This could indicate that there is a standing Na⁺ gradient between the bulk cytosol and the sub‐sarcolemmal space. The size of this sub‐sarcolemmal space has been estimated to account for 0,5‐14% of the total cell volume and to be confined to a space with a diameter around 10 nm on the intracellular side of the sarcolemma [85].

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 Na⁺ hotspot and coldspots: A subsarcolemmal Na⁺ gradient imposes an imbalance between Na⁺ leak (influx) and extrusion near the cell membrane. The diffusion speed of Na⁺ in the cytosol has been a central issue in modelling subcellular Na⁺ gradients [40, 89, 90]. Slow diffusion rate of Na⁺ and fast NKA Na⁺ extrusion kinetics would both favor the setup of localized Na⁺ domains. An important factor is the relative localization of Na⁺ influx pathways and the NKA, where a close localization between these will support the setup of a physiologically relevant Na⁺ gradient, analogous to localized Ca²⁺ regulation in the dyad. This idea could be viewed as an extension of the sub‐sarcolemmal Na⁺ gradient, where small localized subdomains (“nanodomains”) for Na⁺ might be set up in immediate vicinity to Na⁺ transporters such as the NCX. For example, co‐

localization of voltage gated Na⁺ channels and NCX could lead to a rapid rise in Na⁺ close to NCX on the cytosolic site before reaching the rest of the cytosol [45, 81], and thus generate a Na⁺ hotspot. Comparably, if NCX and NKA are colocalized, NKA activity could deplete the Na⁺c

sensed by the NCX and thus create a Na⁺ coldspot, as schematically illustrated in figure 4 [7, 85].

Supportive of the idea of Na⁺ coldspots and hotspots, Wendt‐Gallitelli et al used imaging studies to detect microheterogeneity within the subsarcolemmal space, where specific areas exerting higher and lower Na⁺ than the neighboring areas were identified [86, 91].

1.4.3 Do Na⁺ hotspots and coldspots exist in cardiomyocytes?

As stated above, several studies have explored the presence and physiological role of Na⁺ hotpots in cardiomyocytes. The role of Na⁺ coldspots is less studied. Such a localized domain can be of great physiological importance, exemplified by the regulatory role of NKAα2 on cardiac contractility as previously discussed [54]. As NKA‐NCX interactions remain to be directly demonstrated, a main aim of this thesis is to investigate whether NKA‐mediated Na⁺ extrusion controls NCX activity on a subcellular level. This is the main aim for paper 1 in this thesis, where we used a combination of immunocytochemistry and functional experiments to explore whether NKAα2 controls the Na⁺ sensed by the NCX independently of the bulk Na⁺c and whether this controls Ca²⁺ transients and cellular contractility, in line with the initial findings by James et al [54].

Further, the understanding of the molecular basis for colocalization of Na⁺ transporters has been extended by the detection of the ankB as a scaffolding protein, anchoring NKA and NCX [78, 92]. The ankB complex stands out as a possible structural basis for Na⁺ coldspots, as ankB clusters together NKA and NCX. In theory, this could lead to NKA‐mediated Na⁺ depletion close to the NCX, such that the ENa/Ca would be determined by the Na⁺c close to in the ankB‐directed complex rather than the bulk cytosol. This working hypothesis is the main focus for paper 2 in this thesis, while the role of the ankB complex in cellular arrhythmias is partly explored in paper 3 of this thesis.

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1.5 Arrhythmias due to Ca²⁺ overload in cardiomyocytes 1.5.1 Afterdepolarizations and Ca²⁺ waves

Ca²⁺ transients are tightly regulated to maintain cardiac function, and at the same time avoid Ca²⁺

overload in cardiomyocytes. High Ca²⁺c increases cardiac contractility and also the risk of cellular arrhythmias linked to Ca²⁺ overload by inducing afterdepolarizations. Afterdepolarizations in cardiomyocytes is a common trigger mechanism in clinical tachyarrhythmias such as atrial fibrillation, ventricular tachycardia and fibrillation [93]. Afterdepolarizations are divided into early

afterdepolarizations (EADs) and delayed afterdepolarizations (DADs) after the timing of the spontaneous depolarization of the cell membrane in relation to the regular AP. DADs occur in phase 4 of the AP [93, 94], and are closely linked to intracellular Ca²⁺ fluxes. DADs develop in three phases:

 Spontaneous SR Ca²⁺ release: While SR Ca²⁺ release normally is induced by CICR during a regular heartbeat, spontaneous SR Ca²⁺ release under certain conditions occurs between two APs in resting cells. Two factors increase the probability of spontaneous SR Ca²⁺ release, namely increased opening probability of RyRs and high SR Ca²⁺ content:

o SR Ca²⁺ content: The SR Ca²⁺ content will change during a temporary imbalance between Ca²⁺ influx and Ca²⁺ efflux. A new steady state will be reached rapidly so that the two fluxes again are matched [95]. The SERCA2‐NCX ratio is an important determinant of SR Ca²⁺ load. A high ratio favors SR Ca²⁺ reuptake and increases SR Ca²⁺ load, a situation relevant in clinical use of cardiac glycosides which reduces forward mode exchange through an increase of Na⁺c.

o RyR conductance: RyR conductance is regulated by luminal Ca²⁺, the amount of Ca²⁺ on the SR side of the channel and its posttranslational state [96, 97]. RyR conductance is increased in specific inheritable arrhythmias including catecholaminergic polymorphic ventricular tachycardia (CPVT) [98], characterized by “leaky” RyRs that increase the propensity for spontaneous SR Ca²⁺ release events at a given SR Ca²⁺ load. Of relevance for this thesis is ankB, which anchors NCX in relation to the dyadic cleft and thus determines RyR conductance by determination of the dyadic cleft [Ca²⁺].

 Ca²⁺ wave propagation: Spontaneous Ca²⁺ release in resting cells occurs as single Ca²⁺ sparks, which translate into a propagating Ca²⁺ wave along the SR membrane. The most accepted model for Ca²⁺ wave propagation is diffusion of the Ca²⁺ spontaneously released from the SR from one cluster of RyRs to the next along the SR membrane, thus increasing Ca²⁺c locally enough to be detected with fluorescence microscopy [99]. The process of Ca²⁺ wave propagation might be more complicated, as SERCA‐dependent reuptake of Ca²⁺ from the Ca²⁺wave could sensitize the RyR (via increased luminal Ca²⁺) and increase its open probability [100, 101].

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 Depolarization of the resting Em: The released Ca²⁺ ions during a Ca²⁺ wave can either be pumped into the SR by SERCA2, or be extruded by the NCX generating a inward current, Iti, due to the inward movement of Na⁺ ions and thus depolarization of Em [102]. If sufficient amounts of Ca²⁺

are released during the Ca²⁺ wave, the resulting depolarizing NCX current can induce a spontaneous AP that can propagate to neighboring cells and possibly trigger a tachyarrhythmia.

Clinically, betablockers are often used to prevent afterdepolarizations by lowering the SR Ca²⁺

content in addition to other effects [103].

1.5.2 Ankyrin B syndrome

Inherited long QT syndrome (LQTS) is a group of inherited diseases where prolonged ventricular repolarization leads to increased risk of ventricular tachyarrhythmias, and is typically caused by mutations in specific genes constituting ion channels in cardiomyocytes. In 2003, Mohler et al [78]

demonstrated that a loss‐of‐function point mutation (E1425G in most cases) in the ank2 gene is the underlying cause of inherited long QT‐syndrome type 4 (LQTS4) [104]. Patients suffering from this disease exert sinus node dysfunction causing bradycardia, atrial fibrillation, syncope and sudden cardiac death. ECG‐recordings typically showed a biphasic T‐wave morphology, ventricular arrhythmias and prolonged QTc‐interval [104]. Later studies have identified other loss of function mutations in ank2 associated with varying severity of sinus node dysfunction, atrial fibrillation and ventricular arrhythmias, but prolonged QTc‐interval has not been a consistent feature [105, 106]. The arrhythmias associated with ank2‐mutations are collectively referred to as the ankyrin B syndrome [105, 107‐109]. Patients with the ankyrin B syndrome have usually been treated with betablockers and/or pacemakers [104], but with limited clinical efficiency, as betablockers often fail to prevent arrhythmias in these patients [110].

1.5.3 Ca²⁺ channel blockers ‐ a new treatment option for ankyrin B syndrome?

Mice with heterozygous knockout of ankB (ankB^+/‐) closely resemble the clinical characteristics of the ankyrin B syndrome in humans. Similar to patients with the ankyrin B syndrome, these mice exhibit sinus node dysfunction [108], ventricular arrhythmias and sudden cardiac death during stress [78], and the latter two phenotypes have been linked to increased propensity for DADs. AnkB^+/‐ ventricular cardiomyocytes display increased SR Ca²⁺ content, afterdepolarizations and Ca²⁺ waves [78, 111].

Importantly, these cellular characteristics have been rescued by expression of exogenous wild type ankB, but not by expression of exogenous ankB containing the E1425G mutation, confirming that this mutation causes the cellular phenotype leading to the ankyrin B syndrome [78]. AnkB^+/‐ mice exert

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both increased SR Ca²⁺ load and an increased propensity towards arrhythmogenic SR Ca²⁺ release by RyRs independent of SR Ca²⁺ load [111]. Thus, the main mechanism leading to DADs in ankyrin B syndrome might be linked to either or both of factors increased SR Ca²⁺ load and increased RyR propensity for Ca²⁺ release. Nevertheless, both mechanisms could in theory be counteracted by reducing [Ca²⁺]c and/or SR Ca²⁺ content. A main hypothesis in paper 3 of this thesis is that CCBs might reduce SR Ca²⁺ load and Ca²⁺ wave propensity in ankB^+/‐ myocytes more directly than betablockers, providing a new and more efficient antiarrhythmic therapeutic strategy in patients with ankyrin B syndrome.

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2. Main aims

The main aim of this thesis is to

Investigate Na⁺ regulation of the cardiac excitation‐contraction‐relaxation coupling with a special focus on regulation of the Na⁺/Ca²⁺‐exchanger and antiarrhythmic approaches.

Specific aims:

1) Study the role of the Na⁺/K⁺ ATPase 2isoform as a regulator of the Na⁺/Ca²⁺‐exchanger activity in cardiomyocytes

2) Explore whether Na⁺/K⁺ ATPase coupling to ankyrin B regulates the activity of the Na⁺/Ca²⁺‐ exchanger in cardiomyocytes

3) Investigate whether inhibition of ICaL can reduce SR Ca²⁺ content and prevent development of

Ca²⁺ waves with special focus on the ankyrin B syndrome

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3. Methods

3.1 Animal models

In paper 1, we aimed to study the role of the NKAα2‐isoform in the regulation of NCX and their localization in the t‐tubules. We therefore chose the rat as a model since rats present a double advantage: 1) the α1 and α2 isoform can be functionally separated using ouabain (see section 1.3.4 in introduction), and 2) rats have a well‐developed t‐tubule network [112]. Rats were also used for the peptide experiments in paper 2.

In paper 2 and 3, we wanted to study the role of ankB. As mentioned in section 1.5.3, ankB^+/‐

mice display stress induced arrhythmias and altered Ca²⁺ handling, consistent with the phenotype observed in humans with ank2 mutations. Since it was not conceivable to study cardiomyocytes from patients with ank2 mutations, we used ankB^+/‐ mice to study the role of ankB. Mice homozygous for a null mutation in ankB (ankB ^‐/‐) die prenatally or within days after birth from central nervous system defects [113]. AnkB^+/‐ mice have a shorter expected lifespan (around 90 weeks, compared to around 120 weeks in WT) and premature ageing compared to WT mice [109].

A similar phenotype in the ankB^+/‐ mouse and patients with ank2 mutations indicates that the

ankB^+/‐ mouse is a good model to study the role of ankB in patients. However, there are some general

differences between cardiomyocytes from rodents and humans that need to be addressed:

 The AP in rodents in much shorter and lacks a plateau phase. This is mainly due to differences in Ito expression [114].

 The NCX/SERCA balance in cytosolic Ca²⁺ removal is more shifted in favor of SERCA in rodents (for details, see section 1.2.3).

 Whereas resting heart rate in humans is around 60 beats/min, the values in rats and mice are around 300 beats/min and 600 beats/min, respectively.

 Na⁺c is 10‐15 mM in rodents, and 4‐8 mM in mammals, including humans [33].

Despite the differences between human and rodent cardiac function, results obtained in rodent cardiomyocytes can still increase our general understanding of cardiomyocytes’ function.

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3.2 Isolated cardiomyocytes

The main aim of this thesis was to examine cellular function, and the majority of experiments in all three studies were performed in isolated left ventricular cardiomyocytes from rats and mice. Cell isolation of cardiomyocytes from rodent hearts are thus critical for the experiments described in the further sections. Cardiomyocytes comprise only a fraction of the heart’s cell population numberwise.

Neurons, smooth muscle cells, fibroblasts (which constitute more than 50% of the heart’s cell number [115]) and epithelial cells constitute a high fraction of the cell number in the heart. Hence enzymatic digestion of the heart is necessary to isolate single cardiomyocytes. We prepared fresh isolated cardiomyocytes for each day of experiments, as adult cardiomyocytes change properties rapidly, such as t‐tubule density, in primary cultures [116].

To prepare isolated cardiomyocytes, we used an enzymatic perfusion method with a modified Langendorff setup. The aorta was cannulated above the aortic valve and was perfused by gravity (80 cm column height) at 37 degrees C with a preoxygenated Tyrode solution containing 1g/l collagenase Type II (Worthington) for 8‐13 minutes until the aortic valve was digested (attested by the increased outflow of perfusate). Atrias and the right ventricle tissue were removed to obtain only left ventricular cardiomyocytes.

Cardiomyocytes from different wall layers of the ventricle display different properties. In rabbit ventricle, higher Na⁺c was found in cardiomyocytes from the epicardium versus endocardium [117] despite similar NKA expression in the two cell populations [118]. In canine heart, Na⁺c is higher in endocardial than epicardial cells, possibly due to differences in NKA‐current density [119].

Such differences could also exist in mice and rat cardiomyocytes. In our experiments, we used cardiomyocytes from the whole left ventricle without separating between wall layers. Two aspects of the cell isolation procedure are important to secure sufficient cell quality:

 Sufficient perfusion of the coronary arteries. Sufficient perfusion of the preoxygenated solution with collagenase is necessary to achieve tissue degradation and isolation of single cells. Careful mounting of the heart to the modified Langendorff setup and heparinization of rats before cardiac excision are empirical factors that improve perfusion of the coronary arteries.

 The type of collagenase. Of many collagenase types, type II from Worthington is recommended for heart tissue digestion. This is a crude enzyme preparation, containing not only collagenase, but also various proteases, and the content varies between lots. Different batches of collagenase have different enzyme activity level. Thus, we optimized the perfusion time for each batch to yield isolated cardiomyocytes with optimal quality. In general, we used lots with a collagenase activity close to 200U/ml and had specific batches of collagenase for each set of experiments.

Na regulation of the cardiac excitation‐contraction‐relaxation coupling +